1 Engineering the thermotolerant industrial yeast Kluyveromyces marxianus for anaerobic growth 1 Wijbrand J. C. Dekker, Raúl A. Ortiz-Merino, Astrid Kaljouw, Julius Battjes, Frank W. Wiering, Christiaan 2 Mooiman, Pilar de la Torre, and Jack T. Pronk* 3 Department of Biotechnology, Delft University of Technology, van der Maasweg 9, 2629 HZ Delft, The 4 Netherlands 5 *Corresponding author: Department of Biotechnology, Delft University of Technology, van der Maasweg 6 9, 2629 HZ Delft, The Netherlands, E-mail: j.t.pronk@tudelft.nl, Tel: +31 15 2783214. 7 Wijbrand J.C. Dekker w.j.c.dekker@tudelft.nl 8 Raúl A. Ortiz-Merino raul.ortiz@tudelft.nl https://orcid.org/0000-0003-4186-8941 9 Astrid Kaljouw astridk20@gmail.com 10 Julius Battjes juliusbattjes@hotmail.com 11 Frank Willem Wiering frank.wiering@gmail.com 12 Christiaan Mooiman c.mooiman@tudelft.nl 13 Pilar de la Torre pilartocortes@gmail.com 14 Jack T. Pronk j.t.pronk@tudelft.nl https://orcid.org/0000-0002-5617-4611 15 Manuscript for submission in Nature Biotechnology, section: Article. 16 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.07.425723doi: bioRxiv preprint mailto:j.t.pronk@tudelft.nl mailto:w.j.c.dekker@tudelft.nl mailto:raul.ortiz@tudelft.nl https://orcid.org/0000-0003-4186-8941 mailto:c.mooiman@tudelft.nl mailto:j.t.pronk@tudelft.nl https://orcid.org/0000-0002-5617-4611 https://doi.org/10.1101/2021.01.07.425723 http://creativecommons.org/licenses/by-nc-nd/4.0/ 2 Abstract 17 Current large-scale, anaerobic industrial processes for ethanol production from renewable 18 carbohydrates predominantly rely on the mesophilic yeast Saccharomyces cerevisiae. Use of 19 thermotolerant, facultatively fermentative yeasts such as Kluyveromyces marxianus could confer 20 significant economic benefits. However, in contrast to S. cerevisiae, these yeasts cannot grow in the 21 absence of oxygen. Response of K. marxianus and S. cerevisiae to different oxygen-limitation regimes 22 were analyzed in chemostats. Genome and transcriptome analysis, physiological responses to sterol 23 supplementation and sterol-uptake measurements identified absence of a functional sterol-uptake 24 mechanism as a key factor underlying the oxygen requirement of K. marxianus. Heterologous expression 25 of a squalene-tetrahymanol cyclase enabled oxygen-independent synthesis of the sterol surrogate 26 tetrahymanol in K. marxianus. After a brief adaptation under oxygen-limited conditions, tetrahymanol-27 expressing K. marxianus strains grew anaerobically on glucose at temperatures of up to 45 °C. These 28 results open up new directions in the development of thermotolerant yeast strains for anaerobic 29 industrial applications. 30 Keywords: Ergosterol, tetrahymanol, anaerobic metabolism, thermotolerance, ethanol production, 31 yeast biotechnology, metabolic engineering 32 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.07.425723doi: bioRxiv preprint https://doi.org/10.1101/2021.01.07.425723 http://creativecommons.org/licenses/by-nc-nd/4.0/ 3 In terms of product volume (87 Mton y-1)1,2, anaerobic conversion of carbohydrates into ethanol by the 33 yeast Saccharomyces cerevisiae is the single largest process in industrial biotechnology. For 34 fermentation products such as ethanol, anaerobic process conditions are required to maximize product 35 yields and to minimize both cooling costs and complexity of bioreactors3. While S. cerevisiae is applied in 36 many large-scale processes and is readily accessible to modern genome-editing techniques4,5, several 37 non-Saccharomyces yeasts have traits that are attractive for industrial application. In particular, the high 38 maximum growth temperature of thermotolerant yeasts, such as Kluyveromyces marxianus (up to 50 °C 39 as opposed to 39 °C for S. cerevisiae), could enable lower cooling costs6–8. Moreover, it could reduce the 40 required dosage of fungal polysaccharide hydrolases during simultaneous saccharification and 41 fermentation (SSF) processes9,10. However, as yet unidentified oxygen requirements hamper 42 implementation of K. marxianus in large-scale anaerobic processes11–13. 43 In S. cerevisiae, fast anaerobic growth on synthetic media requires supplementation with a source of 44 unsaturated fatty acids (UFA), sterols, as well as several vitamins14–17. These nutritional requirements 45 reflect well-characterized, oxygen-dependent biosynthetic reactions. UFA synthesis involves the oxygen-46 dependent acyl-CoA desaturase Ole1, NAD+ synthesis depends on the oxygenases Bna2, Bna4, and Bna1, 47 while synthesis of ergosterol, the main yeast sterol, even requires 12 moles of oxygen per mole. 48 Oxygen-dependent reactions in NAD+ synthesis can be bypassed by nutritional supplementation of 49 nicotinic acid, which is a standard ingredient of synthetic media for cultivation of S. cerevisiae17,18. 50 Ergosterol and the UFA source Tween 80 (polyethoxylated sorbitan oleate) are routinely included in 51 media for anaerobic cultivation as ‘anaerobic growth factors’ (AGF)15,17,19. Under anaerobic conditions, S. 52 cerevisiae imports exogenous sterols via the ABC transporters Aus1 and Pdr1120. Mechanisms for uptake 53 and hydrolysis of Tween 80 by S. cerevisiae are unknown but, after its release, oleate is activated by the 54 acyl-CoA synthetases Faa1 and Faa421,22. 55 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.07.425723doi: bioRxiv preprint https://doi.org/10.1101/2021.01.07.425723 http://creativecommons.org/licenses/by-nc-nd/4.0/ 4 Outside the whole-genome duplicated (WGD) clade of Saccharomycotina yeasts, only few yeasts 56 (including Candida albicans and Brettanomyces bruxellensis) are capable of anaerobic growth in 57 synthetic media supplemented with vitamins, ergosterol and Tween 8012,13,23,24. However, most currently 58 known yeast species readily ferment glucose to ethanol and carbon dioxide when exposed to oxygen-59 limited growth conditions13,25,26, indicating that they do not depend on respiration for energy 60 conservation. The inability of the large majority of facultatively fermentative yeast species to grow 61 under strictly anaerobic conditions is therefore commonly attributed to incompletely understood 62 oxygen requirements for biosynthetic processes11. Several oxygen-requiring processes have been 63 proposed including involvement of a respiration-coupled dihydroorotate dehydrogenase in pyrimidine 64 biosynthesis, limitations in uptake and/or metabolism of anaerobic growth factors, and redox-cofactor 65 balancing constraints11,13,27. 66 Quantitation, identification and elimination of oxygen requirements in non-Saccharomyces yeasts is 67 hampered by the very small amounts of oxygen required for non-dissimilatory purposes. For example, 68 preventing entry of the small amounts of oxygen required for sterol and UFA synthesis in laboratory-69 scale bioreactor cultures of S. cerevisiae requires extreme measures, such as sparging with ultra-pure 70 nitrogen gas and use of tubing and seals that are resistant to oxygen diffusion25,28. This technical 71 challenge contributes to conflicting reports on the ability of non-Saccharomyces yeasts to grow 72 anaerobically, as exemplified by studies on the thermotolerant yeast K. marxianus29–31. Paradoxically, 73 the same small oxygen requirements can represent a real challenge in large-scale bioreactors, in which 74 oxygen availability is limited by low surface-to-volume ratios and vigorous carbon-dioxide production. 75 Identification of the non-dissimilatory oxygen requirements of non-conventional yeast species is 76 required to eliminate a key bottleneck for their application in industrial anaerobic processes and, on a 77 fundamental level, can shed light on the roles of oxygen in eukaryotic metabolism. The goal of this study 78 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.07.425723doi: bioRxiv preprint https://doi.org/10.1101/2021.01.07.425723 http://creativecommons.org/licenses/by-nc-nd/4.0/ 5 was to identify and eliminate the non-dissimilatory oxygen requirements of the facultatively 79 fermentative, thermotolerant yeast K. marxianus. To this end, we analyzed and compared physiological 80 and transcriptional responses of K. marxianus and S. cerevisiae to different oxygen- and anaerobic-81 growth factor limitation regimes in chemostat cultures. Based on the outcome of this comparative 82 analysis, subsequent experiments focused on characterization and engineering of sterol metabolism and 83 yielded K. marxianus strains that grew anaerobically at 45 °C. 84 Results 85 K. marxianus and S. cerevisiae show different physiological responses to extreme oxygen limitation 86 To investigate oxygen requirements of K. marxianus, physiological responses of strain CBS6556 were 87 studied in glucose-grown chemostat cultures operated at a dilution rate of 0.10 h-1 and subjected to 88 different oxygenation and AGF limitation regimes (Fig. 1a). Physiological parameters of K. marxianus in 89 these cultures were compared to those of S. cerevisiae CEN.PK113-7D subjected to the same cultivation 90 regimes. 91 In glucose-limited, aerobic chemostat cultures (supplied with 0.5 L air·min-1, corresponding to 54 mmol 92 O2 h-1), the Crabtree-negative yeast K. marxianus32 and the Crabtree-positive yeast S. cerevisiae33 both 93 exhibited a fully respiratory dissimilation of glucose, as evident from absence of ethanol production and 94 a respiratory quotient (RQ) close to 1 (Table 1). Apparent biomass yields on glucose of both yeasts 95 exceeded 0.5 g biomass (g glucose)-1 and were approximately 10 % higher than previously reported due 96 to co-consumption of ethanol, which was used as solvent for the anaerobic growth factor ergosterol32,34. 97 At a reduced oxygen-supply rate of 0.4 mmol O2 h-1 , both yeasts exhibited a mixed respiro-fermentative 98 glucose metabolism. RQ values close to 50 and biomass-specific ethanol-production rates of 11.5 ± 0.6 99 mmol·g·h-1 for K. marxianus and 7.5 ± 0.1 mmol·g·h-1 for S. cerevisiae (Table 1), indicated that glucose 100 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.07.425723doi: bioRxiv preprint https://doi.org/10.1101/2021.01.07.425723 http://creativecommons.org/licenses/by-nc-nd/4.0/ 6 dissimilation in these cultures was predominantly fermentative. Biomass-specific rates of glycerol 101 production which, under oxygen-limited conditions, enables re-oxidation of NADH generated in 102 biosynthetic reactions35, were approximately 2.5-fold higher (p = 2.3·10-4) in K. marxianus than in S. 103 cerevisiae. Glycerol production showed that the reduced oxygen-supply rate constrained mitochondrial 104 respiration. However, low residual glucose concentrations (Table 1) indicated that sufficient oxygen was 105 provided to meet most or all of the biosynthetic oxygen requirements of K. marxianus. 106 To explore growth of K. marxianus under an even more stringent oxygen-limitation, we exploited 107 previously documented challenges in achieving complete anaerobiosis in laboratory bioreactors19,28. 108 Even in chemostats sparged with pure nitrogen, S. cerevisiae grew on synthetic medium lacking Tween 109 80 and ergosterol, albeit at an increased residual glucose concentration (Fig. 1, Table 1). In contrast, K. 110 marxianus cultures sparged with pure N2 and supplemented with both AGFs consumed only 20 % of the 111 glucose fed to the cultures. These severely oxygen-limited cultures showed a residual glucose 112 concentration of 15.9 ± 0.3 g·L-1 and a low but constant biomass concentration of 0.4 ± 0.0 g·L-1. This 113 pronounced response of K. marxianus to extreme oxygen-limitation provided an experimental context 114 for further analyzing its unknown oxygen requirements. 115 S. cerevisiae can import exogenous sterols under severely oxygen-limited or anaerobic conditions20. If 116 the latter were also true for K. marxianus, omission of ergosterol from the growth medium of severely 117 oxygen-limited cultures would increase biomass-specific oxygen requirements and lead to an even lower 118 biomass concentration. In practice however, omission of ergosterol led to a small increase of the 119 biomass concentration and a corresponding decrease of the residual glucose concentration in severely 120 oxygen-limited chemostat cultures (Fig. 1b, Table 1). This observation suggested that, in contrast to S. 121 cerevisiae, K. marxianus cannot replace de novo oxygen-dependent sterol synthesis by uptake of 122 exogenous sterols. 123 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.07.425723doi: bioRxiv preprint https://doi.org/10.1101/2021.01.07.425723 http://creativecommons.org/licenses/by-nc-nd/4.0/ 7 Fig. 1 | Chemostat cultivation of S. cerevisiae CEN.PK113-7D and K. marxianus CBS6556 under 124 different aeration and anaerobic-growth-factor (AGF) supplementation regimes. The ingoing gas flow 125 of all cultures was 500 mL·min-1, with oxygen partial pressures of 21·104 ppm (O21·104), 840 ppm 126 (O840), or < 0.5 ppm (O0.5). The AGFs Ergosterol (E) and/or Tween 80 (T) were added to media as 127 indicated. a, Schematic representation of experimental set-up. Data for each cultivation regime were 128 obtained from independent replicate chemostat cultures. b, Residual glucose concentrations and 129 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.07.425723doi: bioRxiv preprint https://doi.org/10.1101/2021.01.07.425723 http://creativecommons.org/licenses/by-nc-nd/4.0/ 8 biomass-specific oxygen consumption rates (qO2) under different aeration and AGF-supplementation 130 regimes. Data represent mean and standard deviation of independent replicate chemostat cultures. c, 131 Distribution of consumed glucose over biomass and products in chemostat cultures of S. cerevisiae (left 132 column) and K. marxianus (right column), normalized to a glucose uptake rate of 1.00 mol·h-1. Numbers 133 in boxes indicate averages of measured metabolite formation rates (mol·h-1) and biomass production 134 rates (g dry weight·h-1) for each aeration and AGF supplementation regime. 135 Table 1 | Physiology of S. cerevisiae CEN.PK113-7D and K. marxianus CBS6556 in glucose-grown 136 chemostat cultures with different aeration and anaerobic-growth-factor (AGF) supplementation 137 regimes. Cultures were grown at pH 6.0 on synthetic medium with urea as nitrogen source and 7.5 g·L-1 138 glucose (aerobic cultures) or 20 g·L-1 glucose (oxygen-limited cultures) as carbon and energy source. 139 Data are represented as mean ± SE of data from independent chemostat cultures for each condition. 140 The AGFs ergosterol (E) and Tween 80 (T) were added to the media as indicated. Cultures were aerated 141 at 500 mL·min-1 with gas mixtures containing 21·104 ppm O2 (O21·104), 840 ppm O2 (O840) or < 0.5 ppm 142 O2 (O0.5). Tween 80 was omitted from media used for aerobic cultivation to prevent excessive foaming. 143 Ethanol measurements were corrected for evaporation (Supplementary Fig. 1). Positive and negative 144 biomass-specific conversion rates (q) represent consumption and production rates, respectively. 145 S. cerevisiae CEN.PK113-7D K. marxianus CBS6556 Condition 1 2 3 4 5 1 2 3 4 Aeration regime O21·1 04 O840 O0.5 O0.5 O0.5 O21·1 04 O840 O0.5 O0.5 AGF E TE TE T - E TE TE T Replicates 3 3 2 5 2 2 5 2 2 D (h-1) 0.10 ± 0.00 0.10 ± 0.00 0.10 ± 0.00 0.10 ± 0.00 0.10 ± 0.00 0.10 ± 0.00 0.11 ± 0.01 0.12 ± 0.01 0.12 ± 0.01 Biomass (g·L-1) 4.22 ± 0.06 2.29 ± 0.04 1.98 ± 0.01 1.56 ± 0.03 1.12 ± 0.02 3.79 ± 0.02 1.57 ± 0.10 0.35 ± 0.02 0.50 ± 0.04 Residual glucose (g·L-1) 0.00 ± 0.00 0.07 ± 0.00 0.06 ± 0.02 0.23 ± 0.04 1.47 ± 0.01 0.00 ± 0.00 0.10 ± 0.02 15.92 ± 0.26 13.67 ± 0.16 Y biomass/glucose (g·g-1) 0.57 ± 0.01 0.12 ± 0.00 0.10 ± 0.00 0.08 ± 0.00 0.06 ± 0.00 0.53 ± 0.00 0.08 ± 0.00 0.09 ± 0.00 0.09 ± 0.01 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.07.425723doi: bioRxiv preprint https://doi.org/10.1101/2021.01.07.425723 http://creativecommons.org/licenses/by-nc-nd/4.0/ 9 Y ethanol/glucose (g·g-1) - 1.67 ± 0.06 1.63 ± 0.02 1.65 ± 0.02 1.68 ± 0.02 - 1.53 ± 0.03 1.31 ± 0.05 1.40 ± 0.02 qglucose (mmol·g·h-1) -0.95 ± 0.03 -4.59 ± 0.10 -5.25 ± 0.04 -6.77 ± 0.27 -9.06 ± 0.15 -1.05 ± 0.00 -7.46 ± 0.30 -7.30 ± 0.81 -8.53 ± 0.00 qethanol (mmol·g·h-1) -0.44 ± 0.03 7.48 ± 0.10 8.40 ± 0.02 10.96 ± 0.56 15.03 ± 0.47 -0.52 ± 0.00 11.49 ± 0.44 10.25 ± 0.66 12.69 ± 0.11 RQ 1.08 ± 0.02 52.2 ± 2.4 - - - 1.06 ± 0.01 49.3 ± 7.5 - - Glycerol/biomass (mmol·(g biomass)-1) 0.00 ± 0.00 3.67 ± 0.05 5.58 ± 0.02 6.73 ± 0.25 11.26 ± 0.40 0.00 ± 0.00 9.51 ± 0.46 16.90 ± 0.76 18.45 ± 2.09 Carbon recovery (%) 99.9 ± 0.7 101.2 ± 3.3 100.4 ± 0.1 100.1 ± 1.3 104.0 ± 0.2 100.5 ± 0.1 91.1 ± 2.0 101.6 ± 6.5 99.7 ± 3.9 Degree of reduction recovery (%) 98.4 ± 0.7 100.9 ± 0.8 100.1 ± 0.9 98.1 ± 0.6 100.1 ± 1.8 98.8 ± 0.1 94.5 ± 0.4 97.8 ± 6.2 99.1 ± 3.5 146 Transcriptional responses of K. marxianus to oxygen limitation involve ergosterol metabolism 147 To further investigate the non-dissimilatory oxygen requirements of K. marxianus, transcriptome 148 analyses were performed on cultures of S. cerevisiae and K. marxianus grown under the aeration and 149 anaerobic-growth-factor supplementation regimes discussed above. The genome sequence of K. 150 marxianus CBS6556 was only available as draft assembly and was not annotated36. Therefore, long-read 151 genome sequencing, assembly and de novo genome annotation were performed, the annotation was 152 refined by using transcriptome assemblies (Data availability). Comparative transcriptome analysis of S. 153 cerevisiae and K. marxianus focused on orthologous genes with divergent expression patterns that 154 revealed a strikingly different transcriptional response to growth limitation by oxygen and/or anaerobic-155 growth-factor availability (Fig. 2). 156 In S. cerevisiae, import of exogenous sterols by Aus1 and Pdr11 can alleviate the impact of oxygen 157 limitation on sterol biosynthesis20. Consistent with this role of sterol uptake, sterol biosynthetic genes in 158 S. cerevisiae were only highly upregulated in severely oxygen-limited cultures when ergosterol was 159 omitted from the growth medium (Fig. 3b, Supplementary Fig. 6, contrast 43). Also the mevalonate 160 pathway for synthesis of the sterol precursor squalene, which does not require oxygen, was upregulated 161 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.07.425723doi: bioRxiv preprint https://doi.org/10.1101/2021.01.07.425723 http://creativecommons.org/licenses/by-nc-nd/4.0/ 10 (contrast 43), reflecting a relief of feedback regulation by ergosterol37. In contrast, K. marxianus showed 162 a pronounced upregulation of genes involved in sterol, isoprenoid and fatty-acid metabolism (Fig. 2ab, 163 Fig. 3, contrast 31) in severely oxygen-limited cultures supplemented with ergosterol and Tween 80. No 164 further increase of the expression levels of sterol biosynthetic genes was observed upon omission of 165 these anaerobic growth factors from the medium of these cultures (Supplementary Fig. 6, contrast 43). 166 These observations suggested that K. marxianus may be unable to import ergosterol when sterol 167 synthesis is compromised. Consistent with this hypothesis, co-orthology prediction with Proteinortho38 168 revealed no orthologs of the S. cerevisiae sterol transporters Aus1 and Pdr11 in K. marxianus. 169 K. marxianus harbors two dihydroorotate dehydrogenases, a cytosolic fumarate-dependent enzyme 170 (KmUra1) and a mitochondrial quinone-dependent enzyme (KmUra9). In vivo activity of the latter 171 requires oxygen because the reduced quinone is reoxidized by the mitochondrial respiratory chain39. 172 Consistent with these different oxygen requirements, KmURA9 was down-regulated under severely 173 oxygen-limited conditions, while KmURA1 was upregulated (Fig. 2b, contrast 31). Upregulation of 174 KmURA1 coincided with increased production of succinate (Table 1). 175 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.07.425723doi: bioRxiv preprint https://doi.org/10.1101/2021.01.07.425723 http://creativecommons.org/licenses/by-nc-nd/4.0/ 11 Fig. 2 | Transcriptional response of K. marxianus and S. cerevisiae to oxygen limitation and sterol, 176 Tween 80 supplementation. Transcriptome analyses were performed for each cultivation regime (1 to 177 5) of S. cerevisiae CEN.PK113-7D (scer) and K. marxianus CBS6556 (kmar). Data for each regime were 178 obtained from independent replicate chemostat cultures (Fig. 1). a, Comparison of GO-term gene-set 179 enrichment analysis of biological processes in contrast 31 of S. cerevisiae and K. marxianus with short 180 description of GO-terms (Supplementary Fig. 2-5). GO-terms were vertically ordered based on their 181 distinct directionality calculated with Piano40 with GO-terms enriched solely with up-regulated genes 182 (blue) at the top, GO-terms with mixed- or no-directionality in the middle (white) and GO-terms with 183 solely down-regulated genes at the bottom (brown). b, c, d, Subsets of differentially expressed 184 orthologous genes obtained from the gene-set analyses for both yeasts in contrasts 31 and 43, and with 185 genes without orthologs depicted with logFC value of 0 in the respective yeast. b, S. cerevisiae genes 186 previously shown as consistently upregulated under anaerobic conditions in four different nutrient-187 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.07.425723doi: bioRxiv preprint https://doi.org/10.1101/2021.01.07.425723 http://creativecommons.org/licenses/by-nc-nd/4.0/ 12 limitations41. c, As described for panel b but for downregulated genes. d, Differentially expressed genes 188 uniquely found in this study. e, f, g, h, Highlighted gene-sets showing divergent expression patterns 189 across the two yeasts. e, S. cerevisiae genes upregulated in contrast 31 but downregulated in K. 190 marxianus. f, S. cerevisiae genes downregulated in contrast 31 but upregulated in K. marxianus. g, h, 191 Similar to e and f but for contrast 43. 192 Fig. 3 | Different transcriptional regulation of ergosterol-biosynthesis in K. marxianus and S. 193 cerevisiae. a, RNAseq was performed on independent replicate chemostat cultures of S. cerevisiae 194 CEN.PK113-7D and K. marxianus CBS6556 for each aeration and anaerobic-growth-factor 195 supplementation regime (1 to 5; Fig. 1). b, Transcriptional differences in the mevalonate- and 196 ergosterol-pathway genes of S. cerevisiae and K. marxianus for contrasts 21 (O2 840 TE |O 21·104 E), 31 197 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.07.425723doi: bioRxiv preprint https://doi.org/10.1101/2021.01.07.425723 http://creativecommons.org/licenses/by-nc-nd/4.0/ 13 (O2 0.5 TE | O 21·104 E), 32 (O2 0.5 TE | O2 840 TE), 43 (O2 0.5 T | O2 0.5 TE), 54 (O2 0.5 | O2 0.5 T). 198 Lumped biochemical reactions are represented by arrows. Colors indicate up- (blue) or down-regulation 199 (brown) with color intensity indicating the log 2 fold change with color range capped to a maximum of 4. 200 Reactions are annotated with corresponding gene, K. marxianus genes are indicated with the name of 201 the S. cerevisiae orthologs. Ergosterol uptake by S. cerevisiae requires additional factors beyond the 202 membrane transporters Aus1 and Pdr1142. No orthologs of the sterol-transporters or Hmg2 were 203 identified for K. marxianus and low read counts for Erg3, Erg9 and Erg20 precluded differential gene 204 expression analysis across all conditions (dark grey). Enzyme abbreviations: Erg10 acetyl-CoA 205 acetyltransferase, Erg13 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase, Hmg1/Hmg2 HMG-CoA 206 reductase, Erg12 mevalonate kinase, Erg8 phosphomevalonate kinase, Mvd1 mevalonate 207 pyrophosphate decarboxylase, Idi1 isopentenyl diphosphate:dimethylallyl diphosphate (IPP) isomerase, 208 Erg20 farnesyl pyrophosphate synthetase, Erg9 farnesyl-diphosphate transferase (squalene synthase), 209 Erg7 lanosterol synthase, Erg11 lanosterol 14α-demethylase, Cyb5 cytochrome b5 (electron donor for 210 sterol C5-6 desaturation), Ncp1 NADP-cytochrome P450 reductase, Erg24 C-14 sterol reductase, Erg25 C-211 4 methyl sterol oxidase, Erg26 C-3 sterol dehydrogenase, Erg27 3-keto-sterol reductase, Erg28 212 endoplasmic reticulum membrane protein (may facilitate protein-protein interactions between Erg26 213 and Erg27, or tether these to the ER), Erg6 Δ24-sterol C-methyltransferase, Erg2 Δ24-sterol C-214 methyltransferase, Erg3 C-5 sterol desaturase, Erg5 C-22 sterol desaturase, Erg4 C24/28 sterol 215 reductase, Aus1/Pdr11 plasma-membrane sterol transporter. 216 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.07.425723doi: bioRxiv preprint https://doi.org/10.1101/2021.01.07.425723 http://creativecommons.org/licenses/by-nc-nd/4.0/ 14 Absence of sterol import in K. marxianus 217 To test the hypothesis that K. marxianus lacks a functional sterol-uptake mechanism, uptake of 218 fluorescent sterol derivative 25-NBD-cholesterol (NBDC) was measured by flow cytometry43. Since S. 219 cerevisiae sterol transporters are not expressed in aerobic conditions20 and to avoid interference of 220 sterol synthesis, NBDC uptake was analysed in anaerobic cell suspensions (Fig. 4a). Four hours after 221 NBDC addition to cell suspensions of the reference strain S. cerevisiae IMX585, median single-cell 222 fluorescence increased by 66-fold (Fig. 4bc). In contrast, the congenic sterol-transporter-deficient strain 223 IMK809 (aus1Δ pdr11Δ) only showed a 6-fold increase of fluorescence, probably reflected detergent-224 resistant binding of NBDC to S. cerevisiae cell-wall proteins43,44. K. marxianus strains CBS6556 and 225 NBRC1777 did not show increased fluorescence, neither after 4 h nor after 23 h of incubation with NBDC 226 (< 2-fold, Fig. 4bc, Supplementary Fig. 7). 227 Fig. 4 | Uptake of the fluorescent sterol derivative NBDC by S. cerevisiae and K. marxianus strains. a, 228 Experimental approach. S. cerevisiae strains IMX585 (reference) and IMK809 (aus1Δ pdr11Δ), and K. 229 marxianus strains NBRC1777 and CBS6556 were each anaerobically incubated in four replicate shake-230 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.07.425723doi: bioRxiv preprint https://doi.org/10.1101/2021.01.07.425723 http://creativecommons.org/licenses/by-nc-nd/4.0/ 15 flask cultures. NBDC and Tween 80 (NBDC T) were added to two cultures, while only Tween 80 (T) was 231 added to the other two. After 4 h incubation, cells were stained with propidium iodide (PI) and analysed 232 by flow cytometry. PI staining was used to eliminate cells with compromised membrane integrity from 233 analysis of NBDC fluorescence. Cultivation conditions and flow cytometry gating are described in 234 Methods and in Supplementary Fig. 8, Supplementary Data set 1 and 2. b, Median and pooled standard 235 deviation of fluorescence intensity (λex 488 nm | λem 533/30 nm, FL1-A) of PI-negative cells with variance 236 of biological replicates after 4 h exposure to Tween 80 (white bars) or Tween 80 and NBDC (blue bars). 237 Variance was pooled for the strains IMX585, CBS6556 and NBRC1777 by repeating the experiment. c, 238 NBDC fluorescence-intensity distribution of cells in a sample from a single culture for each strain, shown 239 as modal-scaled density function. Dashed lines represent background fluorescence of unstained cells of 240 S. cerevisiae and K. marxianus. Fluorescence data for 23-h incubations with NBDC are shown in 241 Supplementary Fig. 7. 242 Engineering K. marxianus for oxygen-independent growth 243 Sterol uptake by S. cerevisiae, which requires cell wall proteins as well as a membrane transporter, has 244 not yet been fully resolved42,43. Instead of expressing a heterologous sterol-import system in K. 245 marxianus, we therefore explored production of tetrahymanol, which acts as a sterol surrogate in 246 strictly anaerobic fungi 45. Expression of a squalene-tetrahymanol cyclase from Tetrahymena 247 thermophila (TtSTC1), which catalyzes the single-step oxygen-independent conversion of squalene into 248 tetrahymanol (Fig. 5a), was recently shown to enable sterol-independent growth of S. cerevisiae46. 249 TtSTC1 was expressed in K. marxianus NBRC1777, which is more genetically amenable than strain 250 CBS655647. After 40 h of anaerobic incubation, the resulting strain contained 2.4 ± 0.4 mg·(g biomass)-1 251 tetrahymanol, 0.4 ± 0.1 mg·g-1 ergosterol and no detectable squalene, while strain NBRC1777 contained 252 3.5 ± 0.1 mg·g-1 squalene and 3.4 ± 0.2 mg·g-1 ergosterol (Fig. 5b). In strictly anaerobic cultures on sterol-253 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.07.425723doi: bioRxiv preprint https://doi.org/10.1101/2021.01.07.425723 http://creativecommons.org/licenses/by-nc-nd/4.0/ 16 free medium, strain NBRC1777 grew immediately after inoculation but not after transfer to a second 254 anaerobic culture (Fig. 5c), consistent with ‘carry-over’ of ergosterol from the aerobic preculture19. The 255 tetrahymanol-producing strain did not grow under these conditions (Fig. 5c) but showed sustained 256 growth under severely oxygen-limited conditions that did not support growth of strain NBRC1777 (Fig. 257 5de). Single-cell isolates derived from these oxygen-limited cultures (IMS1111, IMS1131, IMS1132, 258 IMS1133) showed instantaneous as well as sustained growth under strictly anaerobic conditions (Figure 259 5f and 5g). Tetrahymanol contents in the first, second and third cycle of anaerobic cultivation of isolate 260 IMS1111 were 7.6 ± 0.0 mg·g-1, 28.0 ± 13.0 mg·g-1 and 11.5 ± 0.1 mg·g-1, respectively (Fig. 5b), while no 261 ergosterol was detected. 262 To identify whether adaptation of the tetrahymanol-producing strain IMX2323 to anaerobic growth 263 involved genetic changes, its genome and those of the four adapted isolates were sequenced 264 (Supplementary Table 1). No copy number variations were detected in any of the four adapted isolates. 265 Only strain IMS1111 showed two non-conservative mutations in coding regions: a single-nucleotide 266 insertion in a transposon-borne gene and a stop codon at position 350 (of 496 bp) in KmCLN3, which 267 encodes for a G1 cyclin48. The apparent absence of mutations in the three other, independently adapted 268 strains indicated that their ability to grow anaerobically reflected a non-genetic adaptation. 269 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.07.425723doi: bioRxiv preprint https://doi.org/10.1101/2021.01.07.425723 http://creativecommons.org/licenses/by-nc-nd/4.0/ 17 Fig. 5 | Sterol-independent anaerobic growth of K. marxianus strains expressing TtSTC1. a, Oxygen-270 dependent sterol synthesis and cyclisation of squalene to tetrahymanol by TtStc1. b, Squalene, 271 ergosterol, and tetrahymanol contents with mean and standard error of the mean of (left panel) S. 272 cerevisiae strains IMX585 (reference), IMX1438 (sga1Δ::TtSTC1), and K. marxianus strains NBRC1777 273 (reference), IMX2323 (TtSTC1). Lipid composition of single-cell isolate IMS1111 (TtSTC1) (right panel) 274 over 3 serial transfers (C1-C3). Data from replicate cultures grown in strictly anaerobic (c, f, g) or 275 severely oxygen-limited shake-flask cultures (d, e). Aerobic grown pre-cultures were used to inoculate 276 the first anaerobic culture on SMG-urea and Tween 80, when the optical density started to stabilize the 277 cultures were transferred to new media. Data depicted are of each replicate culture (points) and the 278 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.07.425723doi: bioRxiv preprint https://doi.org/10.1101/2021.01.07.425723 http://creativecommons.org/licenses/by-nc-nd/4.0/ 18 mean (dotted line) from independent biological duplicate cultures, serial transfers cultures are 279 represented with C1-C5. Strains NBRC1777 (wild-type, upward red triangles), IMX2323 (TtSTC1, cyan 280 downward triangle), and the single-cell isolates IMS1111 (TtSTC1, orange circles), IMS1131 (TtSTC1, blue 281 circles), IMS1132 (TtSTC1, yellow circles), IMS1133 (TtSTC1, purple circles). S. cerevisiae IMX585 282 (reference, purple circle) and IMX1438 (TtSTC1, orange circles). c, Extended data with double inoculum 283 size is available in Supplementary Fig. 10. d, Extended data is available in Supplementary Fig. 9a. 284 Test of anaerobic thermotolerance and selection for fast growing anaerobes 285 One of the attractive phenotypes of K. marxianus for industrial application is its high thermotolerance 286 with reported maximum growth temperatures of 46-52 °C49,50. To test if anaerobically growing 287 tetrahymanol-producing strains retained thermotolerance, strain IMS1111 was grown in anaerobic 288 sequential-batch-reactor (SBR) cultures (Fig. 6) in which, after an initial growth cycle at 30 °C, the growth 289 temperature was shifted to 42 °C. Specific growth at 42 °C progressively accelerated from 0.06 h-1 to 290 0.13 h-1 over 17 SBR cycles (corresponding to ca. 290 generations; Fig. 6b). A subsequent temperature 291 increase to 45 °C led to a strong decrease of the specific growth rate which, after approximately 1000 292 generations of selective growth, stabilized at approximately 0.08 h-1. Whole-population genome 293 sequencing of the evolved populations revealed no common mutations or chromosomal copy number 294 variations (Supplementary Table 1). These data show that TtSTC1-expressing K. marxianus can grow 295 anaerobically at temperatures up to at least 45 °C. 296 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.07.425723doi: bioRxiv preprint https://doi.org/10.1101/2021.01.07.425723 http://creativecommons.org/licenses/by-nc-nd/4.0/ 19 Fig. 6 | Thermotolerance and anaerobic growth of tetrahymanol-producing K. marxianus strain. The 297 strain IMS1111 was grown in triplicate sequential batch bioreactor cultivations in synthetic media 298 supplemented with 20 g·L-1 glucose and 420 mg·L-1 Tween 80 at pH 5.0. a, Experimental design of 299 sequential batch fermentation with cycles at step-wise increasing temperatures to select for faster 300 growing mutants, each cycle consisted of three phases; (i) (re)filling of the bioreactor with fresh media 301 up to 100 mL and adjustment of temperature to a new set-point, (ii) anaerobic batch fermentation at a 302 fixed culture temperature with continuous N2 sparging for monitoring of CO2 in the culture off-gas, and 303 (iii) fast broth withdrawal leaving 7 mL (14.3 fold dilution) to inoculate the next batch. b, Maximum 304 specific estimated growth rate (circles) of each batch cycle for the three independent bioreactor 305 cultivations (M3R blue, M5R orange, M6L grey) with the estimated number of generations. The growth 306 rate was calculated from the CO2 production as measured in the off-gas and should be interpreted as an 307 estimate and in some cases could not be calculated. The culture temperature profile (dotted line) for 308 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.07.425723doi: bioRxiv preprint https://doi.org/10.1101/2021.01.07.425723 http://creativecommons.org/licenses/by-nc-nd/4.0/ 20 each independent bioreactor cultivation (blue, grey, orange) consisted of a step-wise increment of the 309 temperature at the onset of the fermentation phase in each batch cycle. c, Representative section of 310 CO2 off-gas profiles of the individual bioreactor (M5R) cultivation over time with CO2 fraction (orange 311 line) and culture temperature (grey dotted line), data of the entire experiment is available in 312 Supplementary Fig. 11 (Data availability). 313 Discussion 314 Industrial production of ethanol from carbohydrates relies on S. cerevisiae, due to its capacity for 315 efficient, fast alcoholic fermentation and growth under strictly anaerobic process conditions. Many 316 facultatively fermentative yeast species outside the Saccharomycotina WGD-clade also rapidly ferment 317 sugars to ethanol under oxygen-limited conditions26, but cannot grow and ferment in the complete 318 absence of oxygen11,13,25. Identifying and eliminating oxygen requirements of these yeasts is essential to 319 unlock their industrially relevant traits for application. Here, this challenge was addressed for the 320 thermotolerant yeast K. marxianus, using a systematic approach based on chemostat-based quantitative 321 physiology, genome and transcriptome analysis, sterol-uptake assays and genetic modification. S. 322 cerevisiae, which was used as a reference in this study, shows strongly different genome-wide 323 expression profiles under aerobic and anaerobic or oxygen-limited conditions51. Although only a small 324 fraction of these differences were conserved in K. marxianus (Fig. 2), we were able to identify absence 325 of a functional sterol import system as the critical cause for its inability to grow anaerobically. Enabling 326 synthesis of the sterol surrogate tetrahymanol yielded strains that grew anaerobically at temperatures 327 above the permissive temperature range of S. cerevisiae. 328 A short adaptation phase of tetrahymanol-producing K. marxianus strains under oxygen-limited 329 conditions reproducibly enabled strictly anaerobic growth. Although this ability was retained after 330 aerobic isolation of single-cell lines, we were unable to attribute this adaptation to mutations. In 331 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.07.425723doi: bioRxiv preprint https://doi.org/10.1101/2021.01.07.425723 http://creativecommons.org/licenses/by-nc-nd/4.0/ 21 contrast to wild-type K. marxianus, a non-adapted tetrahymanol-producing strain did not show ‘carry-332 over growth’ after transfer from aerobic to strictly anaerobic conditions and adapted cultures showed 333 reduced squalene contents (Fig. 5). These observations suggest that interactions between tetrahymanol, 334 ergosterol and/or squalene influence the onset of anaerobic growth and that oxygen-limited growth 335 results in a stable balance between these lipids that is permissive for anaerobic growth. 336 Comparative genomic studies in Saccharomycotina yeasts have previously led to the hypothesis that 337 sterol transporters are absent from pre-WGD yeast species11,52. While our observations on K. marxianus 338 reinforce this hypothesis, which was hitherto not experimentally tested, they do not exclude 339 involvement of additional oxygen-requiring reactions in other non-Saccharomyces yeasts. For example, 340 pyrimidine biosynthesis is often cited as a key oxygen-requiring process in non-Saccharomyces yeasts, 341 due to involvement of a respiratory-chain-linked dihydroorotate dehydrogenase (DHOD)53,54. K. 342 marxianus, is among a small number of yeast species that, in addition to this respiration dependent 343 enzyme (KmUra9), also harbors a fumarate-dependent DHOD (KmUra1)55. In K. marxianus the activation 344 of this oxygen-independent KmUra1 is a crucial adaptation for anaerobic pyrimidine biosynthesis. The 345 experimental approach followed in the present study should be applicable to resolve the role of 346 pyrimidine biosynthesis and other oxygen-requiring reactions in additional yeast species. 347 Enabling K. marxianus to grow anaerobically represents an important step towards application of this 348 thermotolerant yeast in large-scale anaerobic bioprocesses. However, specific growth rates and biomass 349 yields of tetrahymanol-expressing K. marxianus in anaerobic cultures were lower than those of wild-type 350 S. cerevisiae strains. A similar phenotype of tetrahymanol-producing S. cerevisiae was proposed to 351 reflect an increased membrane permeability46. Additional membrane engineering or expression of a 352 functional sterol transport system is therefore required for further development of robust, anaerobically 353 growing industrial strains of K. marxianus56. 354 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.07.425723doi: bioRxiv preprint https://doi.org/10.1101/2021.01.07.425723 http://creativecommons.org/licenses/by-nc-nd/4.0/ 22 online Methods 355 Yeast strains, maintenance and shake-flask cultivation 356 Saccharomyces cerevisiae CEN.PK113-7D57,58 (MATa MAL2-8c SUC2) was obtained from Dr. Peter Kötter, 357 J.W. Goethe University, Frankfurt. Kluyveromyces marxianus strains CBS 6556 (ATCC 26548; NCYC 2597; 358 NRRL Y-7571) and NBRC 1777 (IFO 1777) were obtained from the Westerdijk Fungal Biodiversity 359 Institute (Utrecht, The Netherlands) and the Biological Resource Center, NITE (NBRC) (Chiba, Japan), 360 respectively. Stock cultures of S. cerevisiae were grown at 30 °C in an orbital shaker set at 200 rpm, in 361 500 mL shake flasks containing 100 mL YPD (10 g·L-1 Bacto yeast extract, 20 g·L-1 Bacto peptone, 20 g·L-1 362 glucose). For cultures of K. marxianus, the glucose concentration was reduced to 7.5 g·L-1. After addition 363 of glycerol to early stationary-phase cultures, to a concentration of 30 % (v/v), 2 mL aliquots were stored 364 at -80 °C. Shake-flask precultures for bioreactor experiments were grown in 100 mL synthetic medium 365 (SM) with glucose as carbon source and urea as nitrogen source (SMG-urea)17,59. For anaerobic 366 cultivation, synthetic medium was supplemented with ergosterol (10 mg·L-1) and Tween 80 (420 mg·L-1) 367 as described previously14,17,19. 368 Expression cassette and plasmid construction 369 Plasmids used in this study are described in (Table 4). To construct plasmids pUDE659 (gRNAAUS1) and 370 pUDE663 (gRNAPDR11), the pROS11 plasmid-backbone was PCR amplified using Phusion HF polymerase 371 (Thermo Scientific, Waltham, MA) with the double-binding primer 6005. PCR amplifications were 372 performed with desalted or PAGE-purified oligonucleotide primers (Sigma-Aldrich, St Louis, MO) 373 according to manufacturer’s instructions. To introduce the gRNA-encoding nucleotide sequences into 374 gRNA-expression plasmids, a 2μm fragment was first amplified with primers 11228 and 11232 375 containing the specific sequence as primer overhang using pROS11 as template. PCR products were 376 purified with genElutePCR Clean-Up Kit (Sigma-Aldrich) or Gel DNA Recovery Kit (Zymo Research, Irvine, 377 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.07.425723doi: bioRxiv preprint https://doi.org/10.1101/2021.01.07.425723 http://creativecommons.org/licenses/by-nc-nd/4.0/ 23 CA). The two DNA fragments were then assembled by Gibson Assembly (New England Biolabs, Ipswich, 378 MA) according to the manufacturer’s instructions. Gibson assembly reaction volumes were downscaled 379 to 10 µL and 0.01 pmol·µL-1 DNA fragments at 1:1 molar ratio for 1 h at 50 °C. Chemically competent E. 380 coli XL1-Blue was transformed with the Gibson assembly mix via a 5 min incubation on ice followed by a 381 40 s heat shock at 42 °C and 1 h recovery in non-selective LB medium. Transformants were selected on 382 LB agar containing the appropriate antibiotic. Golden Gate assembly with the yeast tool kit60 was 383 performed in 20 µL reaction mixtures containing 0.75 µL BsaI HF V2 (NEB, #R3733), 2 µL DNA ligase 384 buffer with ATP (New England Biolabs), 0.5 µL T7-ligase (NEB) with 20 fmol DNA donor fragments and 385 MilliQ water. Before ligation at 16 °C was initiated by addition of T7 DNA ligase, an initial BsaI digestion 386 (30 min at 37 °C) was performed. Then 30 cycles of digestion and ligation at 37 °C and 16 °C, 387 respectively, were performed, with 5 min incubation times for each reaction. Thermocycling was 388 terminated with a 5 min final digestion step at 60 °C. 389 To construct a TtSTC1 expression vector, the coding sequence of TtSTC1 (pUD696) was PCR amplified 390 with primer pair 16096/16097 and Golden gate assembled with the donor plasmids pGGkd015 (ori 391 ampR), pP2 (KmPDC1p), pYTK053 (ScADH1t) resulting in pUDE909 (ori ampR KmPDC1p-TtSTC1-392 ScADH1t). For integration of TtSTC1 cassette into the lac4 locus both upstream and downstream flanks 393 (877/878 bps) of the lac4 locus were PCR amplified with the primer pairs 14197/14198 and 394 14199/14200, respectively. An empty integration vector, pGGKd068, was constructed by BsaI golden 395 gate cloning of pYTK047 (GFP-dropout), pYTK079 (hygB), pYTK090 (kanR), pYTK073 (ConRE’), pYTK008 396 (ConLS’) together with the two lac4 homologous nucleotide sequences. Plasmid assembly was verified 397 by PCR amplification with primers 15210, 9335, 16274 and 16275 and by digestion with BsmBI (New 398 England Biolabs, #R0580). The integration vector pUDI246 with the TtSTC1 expression cassette was 399 constructed by Gibson assembly of the PCR amplified pGGKd068 and pUDE909 with primer pairs 400 16274/16275 and 16272/16273, thereby adding 20 bp overlaps for assembly. For this step, the 401 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.07.425723doi: bioRxiv preprint https://doi.org/10.1101/2021.01.07.425723 http://creativecommons.org/licenses/by-nc-nd/4.0/ 24 incubation time of the Gibson assembly was increased to 90 min. Plasmid assembly was verified by 402 diagnostic PCR amplification using DreamTaq polymerase (Thermo Scientific) with primers 5941, 8442, 403 15216 and subsequent Illumina short-read sequencing. 404 Table 2 | Strains used in this study. Abbreviations: Saccharomyces cerevisiae (Sc), Kluyveromyces 405 marxianus (Km), Tetrahymena thermophila (Tt). 406 Genus Strain Relevant genotype Reference S. cerevisiae CEN.PK113-7D MATa URA3 HIS3 LEU2 TRP1 MAL2-8c SUC2 Entian and Kötter, 2007 57 S. cerevisiae IMX585 CEN.PK113-7D can1Δ::cas9-natNT2 Mans et al., 2015 61 S. cerevisiae IMX1438 IMX585 sga1Δ::TtSTC1 Wiersma et al., 2020 46 S. cerevisiae IMK802 IMX585 aus1Δ This study S. cerevisiae IMK806 IMX585 pdr11Δ This study S. cerevisiae IMK809 IMX585 aus1Δ pdr11Δ This study K. marxianus CBS6556 URA3 HIS3 LEU2 TRP1 CBS-KNAW* K. marxianus NBRC1777 URA3 HIS3 LEU2 TRP1 NBRC** K. marxianus IMX2323 KmPDC1p-TtSTC1-ScADH1t-hygB This study K. marxianus IMS1111 KmPDC1p-TtSTC1-ScADH1t-hygB This study K. marxianus IMS1112 KmPDC1p-TtSTC1-ScADH1t-hygB This study K. marxianus IMS1113 KmPDC1p-TtSTC1-ScADH1t-hygB This study K. marxianus IMS1131 KmPDC1p-TtSTC1-ScADH1t-hygB This study K. marxianus IMS1132 KmPDC1p-TtSTC1-ScADH1t-hygB This study K. marxianus IMS1133 KmPDC1p-TtSTC1-ScADH1t-hygB This study 407 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.07.425723doi: bioRxiv preprint https://doi.org/10.1101/2021.01.07.425723 http://creativecommons.org/licenses/by-nc-nd/4.0/ 25 Table 3 | CRISPR gRNA target sequences used in this study. gRNA target sequences are shown with 408 PAM sequences underlined. Position in ORF indicates the base pair after which the Cas9-mediated 409 double-strand break is introduced. AT score indicates the AT content of the 20-bp target sequence and 410 RNA score indicates the fraction of unpaired nucleotides of the 20-bp target sequence, predicted with 411 the complete gRNA sequence using a minimum free energy prediction by the RNAfold algorithm62. 412 Locus Target sequence (5'-3') Position in ORF (bp) AT score RNA score AUS1 CATTATTGTAAATGATTTGGTGG 320/4184 0.75 1 PDR11 ATCTTTCATATAAATAACATAGG 1627/4235 0.85 1 413 Table 4 | Plasmids used in this study. Restriction enzyme recognition sites are indicated in superscript. 414 US/DS represent upstream and downstream homologous recombination sequences used for genomic 415 integration into the K. marxianus lac4 locus. Abbreviations: Saccharomyces cerevisiae (Sc), 416 Kluyveromyces marxianus (Km), Tetrahymena thermophila (Tt). 417 Plasmid Characteristics Source pGGkd015 ori ampR ConLS GFP ConR1 Hassing et al., 2019 63 pGGKd068 ori kanR NotIKmlac4US BsmBIConRE’BsaIsfGFPBsaI ConLS’BsmBI hygB Kmlac4DSNotI This study pP2 ori camR KmPDC1p Rajkumar et al., 2019 47 pROS11 ori ampR 2μm amdSYM pSNR52-gRNACAN1 pRSNR52-gRNAADE2 Mans et al., 2015 61 pUD696 ori kanR TtSTC1 Wiersma et al., 2020 46 pUDE659 ori ampR 2μm amdSYM pSNR52-gRNAAUS1 pRSNR52-gRNAAUS1 This study pUDE663 ori ampR 2μm amdSYM pSNR52-gRNAPDR11 pRSNR52-gRNAPDR11 This study pUDE909 ori ampR KmPDC1p-TtSTC1-ScADH1t This study pUDI246 ori kanR NotIKmlac4US KmPDC1p-TtSTC1-ScADH1t hygB Kmlac4DSNotI This study pYTK008 ori camR ConLS’ Lee et al., 2015 60 pYTK047 ori camR GFP dropout Lee et al., 2015 60 pYTK053 ori camR ScADH1t Lee et al., 2015 60 pYTK073 ori camR ConRE' Lee et al., 2015 60 pYTK079 ori camR hygB Lee et al., 2015 60 418 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.07.425723doi: bioRxiv preprint https://doi.org/10.1101/2021.01.07.425723 http://creativecommons.org/licenses/by-nc-nd/4.0/ 26 Table 5 | Oligonucleotide primers used in this study. 419 Primer Sequence (5'->3') 11228 TGCGCATGTTTCGGCGTTCGAAACTTCTCCGCAGTGAAAGATAAATGATCCATTATTGTAAATGATTTGGGTTTTA GAGCTAGAAATAGCAAGTTAAAATAAG 11232 TGCGCATGTTTCGGCGTTCGAAACTTCTCCGCAGTGAAAGATAAATGATCATCTTTCATATAAATAACATGTTTTA GAGCTAGAAATAGCAAGTTAAAATAAG 11233 TAGTAAAGACTGCTGTAATTCATCTCTCAGTCCTTGCAGTCTGCTTTTTCTGGAATTAATTACCATTTTTAAATAT ATTTCTACTTTCTACTTAATAGCAATTTTAATTAATCTAATTAT 11234 ATAATTAGATTAATTAAAATTGCTATTAAGTAGAAAGTAGAAATATATTTAAAAATGGTAATTAATTCCAGAAAAA GCAGACTGCAAGGACTGAGAGATGAATTACAGCAGTCTTTACTA 11241 TAGCAAAAAAATTCACAACTAAACACGATAGAGTAAAATTAGAGAAGCAACGCCTCGCGGTCAGTGAATAGCGTTC CGTTAGAAAACATTCAAAATTACCTAATACTATTCAACAGTTCT 11242 AGAACTGTTGAATAGTATTAGGTAATTTTGAATGTTTTCTAACGGAACGCTATTCACTGACCGCGAGGCGTTGCTT CTCTAATTTTACTCTATCGTGTTTAGTTGTGAATTTTTTTGCTA 11243 TGTCACTACAGCCACAGCAG 11244 TTGGTAAGGCGCCACACTAG 11251 AGAGAAGCGCCACATAGACG 11252 TGCATATGCTACGGGTGACG 11897 CACCCAAGTATGGTGGGTAG 14148 AAGCATCGTCTCATCGGTCTCATATGTCAATTTCAAAGTACTTCACTCCCGTTGCTGAC 14149 TTATGCCGTCTCAGGTCTCAGGATTTAGTTCTGTACAGGCTTCTTC 14150 TTATGCCGTCTCAGGTCTCAAGAATTAGTTCTGTACAGGCTTCTTC 14151 AAGCATCGTCTCATCGGTCTCATATGTCTTTCACTAAAATCGCTGCCTTATTAG 14152 TTATGCCGTCTCAGGTCTCAGGATATCATAAGAGCATAGCAGCGGCACCGGCAATAG 14197 AAGCATCGTCTCATCGGTCTCACAATGAAAGTGATTGAAGAACCCTCAAAC 14198 TTATGCCGTCTCAGGTCTCAAGGGTTAAGCAATTGGATCCTACC 14199 AAGCATCGTCTCATCGGTCTCAGAGTTGCTTAATTAGCTTGTACATGGCTTTG 14200 TTATGCCGTCTCAGGTCTCATCGGGAAGGCCCATATTGAAGACG 14339 CCCAAATCATTTACAATAATGGATCATTTATC 14340 CATGTTATTTATATGAAAGATGATCATTTATC 16366 GTCCCTAGGTTCGTCATT 16367 CAAGATCAATGGTGGCTCTC 420 Strain construction 421 The lithium-acetate/polyethylene-glycol method was used for yeast transformation64. Homologous 422 repair (HR) DNA fragments for markerless CRISPR-Cas9-mediated gene deletions in S. cerevisiae were 423 constructed by annealing two 120 bp primers, using primer pairs 11241/11242 and 11233/11234 for 424 deletion of PDR11 and AUS1, respectively. After transformation of S. cerevisiae IMX585 with gRNA 425 plasmids pUDE659 and pUDE663 and double-stranded repair fragments, transformants were selected 426 on synthetic medium with acetamide as sole nitrogen source65. Deletion of AUS1 and PDR11 was 427 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.07.425723doi: bioRxiv preprint https://doi.org/10.1101/2021.01.07.425723 http://creativecommons.org/licenses/by-nc-nd/4.0/ 27 confirmed by PCR amplification with primer pairs 11243/11244 and 11251/11252, respectively. Loss of 428 gRNA plasmids was induced by cultivation of single-colony isolates on YPD, after which plasmid loss was 429 assessed by absence of growth of single-cell isolates on synthetic medium with acetamide as nitrogen 430 source. An aus1Δ pdr11Δ double-deletion strain was similarly constructed by chemical transformation of 431 S. cerevisiae IMK802 with pUDE663 and repair DNA. To integrate a TtSTC1 expression cassette into the 432 K. marxianus lac4 locus, K. marxianus NBRC1777 was transformed with 2 μg DNA NotI-digested 433 pUDI246. After centrifugation, cells were resuspended in YPD and incubated at 30 °C for 3 h. Cells were 434 then again centrifuged, resuspended in demineralized water and plated on 200 µg·L-1 hygromycin B 435 (InvivoGen, Toulouse, France) containing agar with 40 µg·L-1 X-gal, 5-bromo-4-chloro-3-indolyl-β-D-436 galactopyranoside (Fermentas, Waltham, MA). Colonies that could not convert X-gal were analyzed for 437 correct genomic integration of the TtSTC1 by diagnostic PCR with primers 16366, 16367 and 11897. 438 Genomic integration of TtSTC1 into the chromosome outside the lac4 locus was confirmed by short-read 439 Illumina sequencing. 440 Chemostat cultivation 441 Chemostat cultures were grown at 30 °C in 2 L bioreactors (Applikon, Delft, the Netherlands) with a 442 stirrer speed of 800 rpm. The dilution rate was set at 0.10 h-1 and a constant working volume of 1.2 L 443 was maintained by connecting the effluent pump to a level sensor. Cultures were grown on synthetic 444 medium with vitamins17. Concentrated glucose solutions were autoclaved separately at 110 °C for 20 445 min and added at the concentrations indicated, along with sterile antifoam pluronic 6100 PE (BASF, 446 Ludwigshafen, Germany; final concentration 0.2 g·L-1). Before autoclaving, bioreactors were tested for 447 gas leakage by submerging them in water while applying a 0.3 bar overpressure. 448 Anaerobic conditions of bioreactor cultivations were maintained by continuous reactor headspace 449 aeration with pure nitrogen gas (≤ 0.5 ppm O2, HiQ Nitrogen 6.0, Linde AG, Schiedam, the Netherlands) 450 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.07.425723doi: bioRxiv preprint https://doi.org/10.1101/2021.01.07.425723 http://creativecommons.org/licenses/by-nc-nd/4.0/ 28 at a flowrate of 500 mL N2 min-1 (2.4 vvm). Gas pressure of 1.2 bar of the reactor headspace was set with 451 a reduction valve (Tescom Europe, Hannover, Germany) and remained constant during cultivation. To 452 prevent oxygen diffusion into the cultivation the bioreactor was equipped with Fluran tubing (14 Barrer 453 O2, F-5500-A, Saint-Gobain, Courbevoie, France), Viton O-rings (Eriks, Alkmaar, the Netherlands), and no 454 pH probes were mounted. The medium reservoir was deoxygenated by sparge aeration with nitrogen 455 gas (≤ 1 ppm O2, HiQ Nitrogen 5.0, Linde AG). 456 For aerobic cultivation the reactor was sparged continuously with dried air at a flowrate of 500 mL air 457 min-1 (2.4 vvm). Dissolved oxygen levels were analyzed by Clark electrodes (AppliSens, Applikon) and 458 remained above 40% during the cultivation. For micro-aerobic cultivations nitrogen (≤ 1 ppm O2, HiQ 459 Nitrogen 5.0, Linde AG) and air were mixed continuously by controlling the fractions of mass flow rate of 460 the dry gas to a total flow of 500 mL min-1 per bioreactor. The mixed gas was distributed to each 461 bioreactor and analyzed separately in real-time. Continuous cultures were assumed to be in steady state 462 when after at least 5 volumes changes, culture dry weight and the specific carbon dioxide production 463 rates changed by less than 10%. 464 Cell density was routinely measured at a wavelength of 660 nm with spectrophotometer Jenway 7200 465 (Cole Palmer, Staffordshire, UK). Cell dry weight of the cultures were determined by filtering exactly 10 466 mL of culture broth over pre-dried and weighed membrane filters (0.45 µm, Thermo Fisher Scientific), 467 which were subsequently washed with demineralized water, dried in a microwave oven (20 min, 350 W) 468 and weighed again66. 469 Metabolite analysis 470 For determination of substrate and extracellular metabolite concentrations, culture supernatants were 471 obtained by centrifugation of culture samples (5 min at 13000 rpm) and analyzed by high-performance 472 liquid chromatography (HPLC) on a Waters Alliance 2690 HPLC (Waters, MA, USA) equipped with a Bio-473 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.07.425723doi: bioRxiv preprint https://doi.org/10.1101/2021.01.07.425723 http://creativecommons.org/licenses/by-nc-nd/4.0/ 29 Rad HPX-87H ion exchange column (BioRad, Veenendaal, the Netherlands) operated at 60 °C with a 474 mobile phase of 5 mM H2SO4 at a flowrate of 0.6 mL·min-1. Compounds were detected by means of a 475 dual-wavelength absorbance detector (Waters 2487) and a refractive index detector (Waters 2410) and 476 compared to reference compounds (Sigma-Aldrich). Residual glucose concentrations in continuous 477 cultivations were determined by HPLC analysis from rapid quenched culture samples with cold steel 478 beads67. 479 Gas analysis 480 The off-gas from bioreactor cultures was cooled with a condenser (2 °C) and dried with PermaPure Dryer 481 (Inacom Instruments, Veenendaal, the Netherlands) prior to analysis of the carbon dioxide and oxygen 482 fraction with a Rosemount NGA 2000 Analyser (Baar, Switzerland). The Rosemount gas analyzer was 483 calibrated with defined mixtures of 1.98 % O2, 3.01 % CO2 and high quality nitrogen gas N6 (Linde AG). 484 Ethanol evaporation rate 485 To correct for ethanol evaporation in the continuous bioreactor cultivations the ethanol evaporation 486 rate was determined in the same experimental bioreactor set-up without the yeast. To SM glucose 487 media with urea 400 mM of ethanol was added after which the decrease in the ethanol concentration 488 was measured over time by periodic measurements and quantification by HPLC analysis over the course 489 of at least 140 hours. To reflect the media composition used for the different oxygen regimes and 490 anaerobic growth factor supplementation, the ethanol evaporation was measured for bioreactor sparge 491 aeration with Tween 80, bioreactor head-space aeration both with and without Tween 80. The ethanol 492 evaporation rate was measured for each condition in triplicate. 493 Lipid extractions & GC analysis 494 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.07.425723doi: bioRxiv preprint https://doi.org/10.1101/2021.01.07.425723 http://creativecommons.org/licenses/by-nc-nd/4.0/ 30 For analysis of triterpene and triterpenoid cell contents biomass was harvested, washed once with 495 demineralized water and stored as pellet at -80 °C before freeze-drying the pellets using an Alpha 1-4 LD 496 Plus (Martin Christ, Osterode am Harz, Germany) at -60 °C and 0.05 mbar. Freeze-dried biomass was 497 saponificated with 2.0 M NaOH (Bio-Ultra, Sigma-Aldrich) in methylation glass tubes (PYREXTM 498 Boroslicate glass, Thermo Fisher Scientific) at 70 °C. As internal standard 5α-cholestane (Sigma-Aldrich) 499 was added to the saponified biomass suspension. Subsequently tert-butyl-methyl-ether (tBME, Sigma-500 Aldrich) was added for organic phase extraction. Samples were extracted twice using tBME and dried 501 with sodium-sulfate (Merck, Darmstadt, Germany) to remove remaining traces of water. The organic 502 phase was either concentrated by evaporation with N2 gas aeration or transferred directly to an 503 injection vial (VWR International, Amsterdam, the Netherlands). The contents were measured by GC-FID 504 using Agilent 7890A Gas Chromatograph (Agilent Technologies, Santa Clara, CA) equipped with an 505 Agilent CP9013 column (Agilent). The oven was programmed to start at 80 °C for 1 min, ramp first to 280 506 °C with 60 °C·min-1 and secondly to 320 °C with a rate of 10 °C·min-1 with a final temperature hold of 15 507 min. Spectra were compared to separate calibration lines of squalene, ergosterol, α-cholestane, 508 cholesterol and tetrahymanol as described previously46. 509 Sterol uptake assay 510 Sterol uptake was monitored by the uptake of fluorescently labelled 25-NBD-cholesterol (Avanti Polar 511 Lipids, Alabaster, AL). A stock solution of 25-NBD-cholesterol (NBDC) was prepared in ethanol under an 512 argon atmosphere and stored at -20 °C. Shake flasks with 10 mL SM glucose media were inoculated with 513 yeast strains from a cryo-stock and cultivated aerobically at 200 rpm at 30 °C overnight. The yeast 514 cultures were subsequently diluted to an OD660 of 0.2 in 400 mL SM glucose media in 500 mL shake 515 flasks to gradually reduce the availability of oxygen and incubated overnight. Yeast cultures were 516 transferred to fresh SM media with 40 g·L-1 glucose and incubated under anaerobic conditions at 30 °C 517 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.07.425723doi: bioRxiv preprint https://doi.org/10.1101/2021.01.07.425723 http://creativecommons.org/licenses/by-nc-nd/4.0/ 31 at 200 rpm. After 22 hours of anaerobic incubation 4 µg·L-1 NBD-cholesterol with 420 mg·L-1 Tween 80 518 were pulsed to the cultures. Samples were taken and washed with PBS 5 mL·L-1 Tergitol NP-40 pH 7.0 519 (Sigma-Aldrich) twice before resuspension in PBS and subsequent analysis. Propidium Iodide (PI) 520 (Invitrogen) was added to the sample (20 µM) and stained according to the manufacturer’s 521 instructions68. PI intercalates with DNA in cells with a compromised cell membrane, which results in red 522 fluorescence. Samples both unstained and stained with PI were analyzed with Accuri C6 flow cytometer 523 (BD Biosciences, Franklin Lakes, NJ) with a 488 nm laser and fluorescence was measured with emission 524 filter of 533/30 nm (FL1) for NBD-cholesterol and > 670 nm (FL3) for PI. Cell gating and median 525 fluorescence of cells were determined using FlowJo (v10, BD Bioscience). Cells were gated based on 526 forward side scatter (FSC) and side-scatter (SSC) to exclude potential artifacts or clumping cells. Within 527 this gated population PI positive and negatively stained cells were differentiated based on the cell 528 fluorescence across a FL3 FL1 dimension. Flow cytometric gates were drafted for each yeast species and 529 used for all samples. The gating strategy is given in Supplementary Fig. 8. Fluorescence of a strain was 530 determined by a sample of cells from independent shake-flask cultures and compared to cells from 531 identical unstained cultures of cells with the exact same chronological age. The staining experiment of 532 the strains IMX585, CBS6556 and NBRC1777 samples was repeated twice for reproducibility, the mean 533 and pooled variance was subsequently calculated from the biological duplicates of the two experiments. 534 The NBDC intensity and cell counts obtained from the NBDC experiments are available for re-analysis in 535 Supplementary Data set 1, and raw flow cytometry plots are depicted in Supplementary Data set 2. 536 Long read sequencing, assembly, and annotation 537 Cells were grown overnight in 500-mL shake flasks containing 100 mL liquid YPD medium at 30 °C in an 538 orbital shaker at 200 rpm. After reaching stationary phase the cells were harvested for a total OD660 of 539 600 by centrifugation for 5 min at 4000 g. Genomic DNA of CBS6556 and NBRC1777 was isolated using 540 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.07.425723doi: bioRxiv preprint https://doi.org/10.1101/2021.01.07.425723 http://creativecommons.org/licenses/by-nc-nd/4.0/ 32 the Qiagen genomic DNA 100/G kit (Qiagen, Hilden, Germany) according to the manufacturer’s 541 instructions. MinION genomic libraries were prepared using the 1D Genomic DNA by ligation (SQK-542 LSK108) for CBS6556, and the 1D native barcoding Genomic DNA (EXP-NBD103 & LSK108) for NBRC1777 543 according to the manufacturer’s instructions with the exception of using 80% EtOH during the ‘End 544 Repair/dA-tailing module’ step. Flow cell quality was tested by running the MinKNOW platform QC 545 (Oxford Nanopore Technology, Oxford, UK). Flow cells were prepared by removing 20 μL buffer and 546 subsequently primed with priming buffer. The DNA library was loaded dropwise into the flow cell for 547 sequencing. The SQK-LSK108 library was sequenced on a R9 chemistry flow cell (FLO-MIN106) for 48 h. 548 Base-calling was performed using Albacore (v2.3.1, Oxford Nanopore Technologies) for CBS6556, and for 549 NBRC1777 with Guppy (v2.1.3, Oxford Nanopore Technologies) using dna_r9.4.1_450bps_flipflop.cfg. 550 CBS6556 reads were assembled using Canu (v1.8)69, and NBRC1777 reads were assembled using Flye 551 (v2.7.1-b1673)70. Assemblies were polished with Pilon (v1.18)71 using Illumina data available at the 552 Sequence Read Archive under accessions SRX3637961 and SRX3541357. Both de novo genome 553 assemblies were annotated using Funannotate (v1.7.1)72, trained and refined using de novo 554 transcriptome assemblies (see below), adding functional annotation with Interproscan (v5.25-64.0)73. 555 Illumina sequencing 556 Plasmids were sequenced on a MiniSeq (Illumina, San Diego, CA) platform. Library preparation was 557 performed with Nextera XT DNA library preparation according to the manufacturer’s instructions 558 (Illumina). The library preparation included the MiniSeq Mid Output kit (300 cycles) and the input & final 559 DNA was quantified with the Qubit HS dsDNA kit (Life Technologies, Thermo Fisher Scientific). 560 Nucleotide sequences were assembled with SPAdes74 and compared to the intended in silico DNA 561 construct. For whole-genome sequencing, yeast cells were harvested from overnight cultures and DNA 562 was isolated with the Qiagen genomic DNA 100/G kit (Qiagen) as described earlier. DNA quantity was 563 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.07.425723doi: bioRxiv preprint https://doi.org/10.1101/2021.01.07.425723 http://creativecommons.org/licenses/by-nc-nd/4.0/ 33 measured with the QuBit BR dsDNA kit (Thermo Fisher Scientific). 300 bp paired-end libraries were 564 prepared with the TruSeq DNA PCR-free library prep kit (Illumina) according to the manufacturer’s 565 instructions. Short read whole-genome sequencing was performed on a MiSeq platform (Illumina). 566 RNA isolation, sequencing and transcriptome analysis 567 Culture broth from chemostat cultures was directly sampled into liquid nitrogen to prevent mRNA 568 turnover. The cell cultures were stored at -80 °C and processed within 10 days after sampling. After 569 thawing on ice, cells were harvested by centrifugation. Total RNA was extracted by a 5 min heatshock at 570 65 °C with a mix of isoamyl alcohol, phenol and chloroform at a ratio of 125:24:1, respectively 571 (Invitrogen). RNA was extracted from the organic phase with Tris-HCl and subsequently precipitated by 572 the addition of 3 M Nac-acetate and 40 % (v/v) ethanol at -20 °C. Precipitated RNA was washed with 573 ethanol, collected and after drying resuspended in RNAse free water. The quantity of total RNA was 574 determined with a Qubit RNA BR assay kit (Thermo Fisher Scientific). RNA quality was determined by the 575 RNA integrity number with RNA screen tape using a Tapestation (Agilent). RNA libraries were prepared 576 with the TruSeq Stranded mRNA LT protocol (Illumina, #15031047) and subjected to paired-end 577 sequencing (151 bp read length, NovaSeq Illumina) by Macrogen (Macrogen Europe, Amsterdam, the 578 Netherlands). 579 Pooled RNAseq libraries were used to perform de novo transcriptome assembly using Trinity (v2.8.3)75 580 which was subsequently used as evidence for both CBS6556 and NBRC1777 genome annotations. 581 RNAseq libraries were mapped into the CBS6556 genome assembly described above, using bowtie 582 (v1.2.1.1)76 with parameters (-v 0 -k 10 --best -M 1) to allow no mismatches, select the best out of 10 583 possible alignments per read, and for reads having more than one possible alignment randomly report 584 only one. Alignments were filtered and sorted using samtools (v1.3.1)77. Read counts were obtained 585 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.07.425723doi: bioRxiv preprint https://doi.org/10.1101/2021.01.07.425723 http://creativecommons.org/licenses/by-nc-nd/4.0/ 34 with featureCounts (v1.6.0)78 using parameters (-B -C) to only count reads for which both pairs are 586 aligned into the same chromosome. 587 Differential gene expression (DGE) analysis was performed using edgeR (v3.28.1)79. Genes with 0 read 588 counts in all conditions were filtered out from the analysis, same as genes with less than 10 counts per 589 million. Counts were normalized using the trimmed mean of M values (TMM) method80, and dispersion 590 was estimated using generalized linear models. Differentially expressed genes were then calculated 591 using a log ratio test adjusted with the Benjamini-Hochberg method. Absolute log2 fold-change values > 592 2, false discovery rate < 0.5, and P value < 0.05 were used as significance cutoffs. 593 Gene set analysis (GSA) based on gene ontology (GO) terms was used to get a functional interpretation 594 of the DGE analysis. For this purpose, GO terms were first obtained for the S. cerevisiae CEN.PK113-7D 595 (GCA_002571405.2) and K. marxianus CBS6556 genome annotations using Funannotate and 596 Interproscan as described above. Afterwards, Funannotate compare was used to get (co)ortholog 597 groups of genes generated with ProteinOrtho538 using the following public genome annotations S. 598 cerevisiae S288C (GCF_000146045.2), K. marxianus NBRC1777 (GCA_001417835.1), K. marxianus 599 DMKU3-1042 (GCF_001417885.1), in addition to the new genome annotations generated here for S. 600 cerevisiae CEN.PK113-7D, and K. marxianus CBS6556 and NBRC1777. Predicted GO terms for S. 601 cerevisiae CEN.PK113-7D and K. marxianus CBS6556 were kept, and merged with those from 602 corresponding (co)orthologs from S. cerevisiae S288C. Genes with term GO:0005840 (ribosome) were 603 not considered for further analyses. GSA was then performed with Piano (v2.4.0)40. Gene set statistics 604 were first calculated with the Stouffer, Wilcoxon rank-sum test, and reporter methods implemented in 605 Piano. Afterwards, consensus results were derived by p-value and rank aggregation, considered 606 significant if absolute Fold Change values > 1. ComplexHeatmap (v2.4.3)81 was used to draw GSA results 607 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.07.425723doi: bioRxiv preprint https://doi.org/10.1101/2021.01.07.425723 http://creativecommons.org/licenses/by-nc-nd/4.0/ 35 into Fig. 2, highlighting differentially expressed genes found in a previous study51. DGE and GSA were 608 performed using R (v4.0.2)82. 609 Anaerobic growth experiments 610 Anaerobic shake-flask experiments were performed in a Bactron anaerobic workstation (BACTRON300-611 2, Sheldon Manufacturing, Cornelius, OR) at 30 °C. The gas atmosphere consisted of 85% N2, 10% CO2 612 and 5% H2 and was maintained anaerobic by a Pd catalyst. The catalyst was re-generated by heating till 613 160 °C every week and interchanged by placing it in the airlock whenever the pass-box was used. 50-mL 614 Shake flasks were filled with 40 mL (80 % volumetric) media and placed on an orbital shaker (KS 130 615 basic, IKA, Staufen, Germany) set at 240 rpm inside the anaerobic chamber. Sterile growth media was 616 placed inside the anaerobic chamber 24 h prior to inoculation to ensure complete removal of traces of 617 oxygen. 618 The anaerobic growth ability of the yeast strains was tested on SMG-urea with 50 g·L-1 glucose at pH 6.0 619 with Tween 80 prepared as described earlier. The growth experiments were started from aerobic pre-620 cultures on SMG-urea media and the anaerobic shake flasks were inoculated at an OD660 of 0.2 621 (corresponding to an OD600 of 0.14). In order to minimize opening the anaerobic chamber, culture 622 growth was monitored by optical density measurements inside the chamber using an Ultrospec 10 cell 623 density meter (Biochrom, Cambridge, UK) at a 600 nm wavelength. When the optical density of culture 624 no longer increased or decreased new shake-flask cultures were inoculated by serial transfer at an initial 625 OD600 of 0.2. 626 Laboratory evolution in low oxygen atmosphere 627 Adaptive laboratory evolution for strict anaerobic growth was performed in a Bactron anaerobic 628 workstation (BACTRON BAC-X-2E, Sheldon Manufacturing) at 30 °C. 50-mL Shake flasks were filled with 629 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.07.425723doi: bioRxiv preprint https://doi.org/10.1101/2021.01.07.425723 http://creativecommons.org/licenses/by-nc-nd/4.0/ 36 40 mL SMG-urea with 50 g·L-1 glucose and including 420 mg·L-1 Tween 80. Subsequently the shake-flask 630 media were inoculated with IMX2323 from glycerol cryo-stock at OD660 < 0.01 and thereafter placed 631 inside the anaerobic chamber. Due to frequent opening of the pass-box and lack of catalyst inside the 632 pass-box oxygen entry was more permissive. After the optical density of the cultures no longer 633 increased, cultures were transferred to new media by 40-50x serial dilution. For IMS1111, IMS1112, 634 IMS1113 three and for IMS1131, IMS1132, IMS1133 four serial transfers in shake-flask media were 635 performed after which single colony isolates were made by plating on YPD agar media with hygromycin 636 antibiotic at 30 °C aerobically. Single colony isolates were subsequently restreaked sequentially for 637 three times on the same media before the isolates were propagated in SM glucose media and glycerol 638 cryo stocked. 639 To determine if an oxygen-limited pre-culture was required for the strict anaerobic growth of IMX2323 640 strain a cross-validation experiment was performed. In parallel, yeast strains were cultivated in 50-mL 641 shake-flask cultures with SMG-urea with 50 g·L-1 glucose at pH 6.0 with Tween 80 in both the Bactron 642 anaerobic workstation (BACTRON BAC-X-2E, Sheldon Manufacturing) with low levels of oxygen-643 contamination, and in the Bactron anaerobic workstation (BACTRON300-2, Sheldon Manufacturing) with 644 strict control of oxygen-contamination. After stagnation of growth was observed in the second serial 645 transfer of the shake-flask cultures a 1.5 mL sample of each culture was taken, sealed, and used to 646 inoculate fresh-media in the other Bactron anaerobic workstation. Simultaneously, the original culture 647 was used to inoculate fresh media in the same Bactron anaerobic workstation, thereby resulting in 4 648 parallel cultures of each strain of which halve were derived from the other Bactron anaerobic 649 workstation. 650 Laboratory evolution in sequential batch reactors 651 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.07.425723doi: bioRxiv preprint https://doi.org/10.1101/2021.01.07.425723 http://creativecommons.org/licenses/by-nc-nd/4.0/ 37 Laboratory evolution for selection of fast growth at high temperatures was performed in 400-mL 652 MultiFors (Infors Benelux, Velp, the Netherlands) bioreactors with a working volume of 100 mL for the 653 strain IMS1111 on SMG 20 g·L-1 glucose media with Tween 80 in triplicate. Anaerobic conditions were 654 created and maintained by continuous aeration of the cultures with 50 mL·min-1 (0.5 vvm) N2 gas and 655 continuous aeration of the media vessels with N2 gas. The pH was set at 5.0 and maintained by the 656 continuous addition of sterile 2 M KOH. Growth was monitored by analysis of the CO2 in the bioreactor 657 off-gas and a new empty-refill cycle was initiated when the batch time had at least elapsed 15 hours and 658 the CO2 signal dropped to 70% of the maximum reached in each batch. The dilution factor of each 659 empty-refill cycle was 14.3-fold (100 mL working volume, 7 mL residual volume). The first batch 660 fermentation was performed at 30 °C after which in the second batch the temperature was increased to 661 42 °C and maintained at for 18 consecutive sequential batches. After the 18 batch cycle at 42 °C the 662 culture temperature was again increased to 45 °C and maintained subsequently. Growth rate was 663 calculated based on the CO2 production as measured by the CO2 fraction in the culture off-gas in 664 essence as described previously83. In short, the CO2 fraction in the off-gas was converted to a CO2 665 evolution rate of mmol per hour and subsequently summed over time for each cycle. The corresponding 666 cumulative CO2 profile was transformed to natural log after which the stepwise slope of the log 667 transformed data was calculated. Subsequently an iterative exclusion of datapoints of the stepwise 668 slope of the log transformed cumulative CO2 profile was performed with exclusion criteria of more than 669 one standard deviation below the mean. 670 Variant calling 671 DNA sequencing reads were aligned into the NBRC1777 described above including an additional 672 sequence with TtSTC1 construct, and used to detect sequence variants using a method previously 673 reported84. Briefly, reads were aligned using BWA (v0.7.15-r1142-dirty)85, alignments were processed 674 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.07.425723doi: bioRxiv preprint https://doi.org/10.1101/2021.01.07.425723 http://creativecommons.org/licenses/by-nc-nd/4.0/ 38 using samtools (v1.3.1)77 and Picard tools (v2.20.2-SNAPSHOT) (http://broadinstitute.github.io/picard), 675 and variants were then called using the Genome Analysis Toolkit (v3.8-1-0-gf15c1c3ef)86 HaplotypeCaller 676 in DISCOVERY and GVCF modes. Variants were only called at sites with minimum variant confidence 677 normalized by unfiltered depth of variant samples (QD) of 20, read depth (DP) ≥ 5, and genotype quality 678 (GQ) > 20, excluding a 7.1 kb region in chromosome 5 containing rDNA. Variants were annotated using 679 the genome annotation described above, including the TtSTC1 construct, with SnpEff (v5.0)87 and 680 VCFannotator (http://vcfannotator.sourceforge.net). 681 Statistics 682 Statistical test performed are given as two sided with unequal variance t-test unless specifically stated 683 otherwise. We denote technical replicates as measurements derived from a single cell culture. Biological 684 replicates are measurements originating from independent cell cultures. Independent experiments are 685 two experiments identical in set-up separated by the difference in execution days. If possible variance 686 from independent experiments with identical setup were pooled together, but independent 687 experiments from time-course experiments (anaerobic growth studies) are reported separately. p-688 values were corrected for multiple-hypothesis testing which is specifically reported each time. No data 689 was excluded based on the resulting data out-come. 690 Data availability 691 Data supporting the findings of this work are available within the paper and source data for all figures in 692 this study are available at the www.data.4TU.nl repository with the doi:10.4121/13265552. 693 The raw RNA-sequencing data that supports the findings of this study are available from the Genome 694 Expression Omnibus (GEO) website (https://www.ncbi.nlm.nih.gov/geo/) with number GSE164344. 695 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.07.425723doi: bioRxiv preprint http://www.data.4tu.nl/ https://www.ncbi.nlm.nih.gov/geo/ https://doi.org/10.1101/2021.01.07.425723 http://creativecommons.org/licenses/by-nc-nd/4.0/ 39 Whole-genome sequencing data of the CBS6556, NBRC1777 and evolved strains were deposited at NCBI 696 (https://www.ncbi.nlm.nih.gov/) under BioProject accession number PRJNA679749. 697 Code availability 698 The code that were used to generate the results obtained in this study are archived in a Gitlab 699 repository (https://gitlab.tudelft.nl/rortizmerino/kmar_anaerobic). 700 Author’s contributions 701 WD and JTP designed the study and wrote the manuscript. WD performed molecular cloning, bioreactor 702 cultivation experiment, transcriptome analysis and sterol-uptake experiments. JB contributed to 703 bioreactor cultivation experiments and molecular cloning. FW contributed to the molecular cloning and 704 sterol-uptake experiments. AK and CM contributed to bioreactor experiments and transcriptome 705 studies. PdlT performed plasmid and genome sequencing. RO contributed to transcriptome analysis and 706 performed sequence annotation and assembly. 707 Acknowledgements 708 We thank Mark Bisschops and Hannes Jürgens for fruitful discussions. We thank Erik de Hulster for 709 fermentation support and Marcel van den Broek for input on the bioinformatics analyses. 710 Competing interest 711 WD and JTP are co-inventors on a patent application that covers aspects of this work. The authors 712 declare no conflict of interest. 713 Funding 714 This work was supported by Advanced Grant (grant #694633) of the European Research Council to JTP. 715 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. 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A program for annotating and predicting the effects of single nucleotide 914 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.07.425723doi: bioRxiv preprint https://doi.org/10.1101/2021.01.07.425723 http://creativecommons.org/licenses/by-nc-nd/4.0/ 45 polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-915 3. Fly (Austin). 6, 80–92 (2012). 916 917 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.07.425723doi: bioRxiv preprint https://doi.org/10.1101/2021.01.07.425723 http://creativecommons.org/licenses/by-nc-nd/4.0/ 46 Description of Additional Supplementary Files 918 Supplementary Data Set 1 | Overview of flow cytometry samples with meta-data. Meta-data Table of 919 file names, frequency of cells compared to parent, number of cells in each group, strain name, time 920 point of fluorescence measurement after 4 hours (1) or 23 hours (2), staining of cells with propidium-921 iodide (PI) with value (PI) or without PI staining (-), staining of cells with Tween 80 NBD-cholesterol (TN) 922 or with Tween 80 only (T), with species names abbreviated K. marxianus (Km) or S. cerevisiae (Sc). 923 [Example picture of file FlowCyto_Table.xlsx] 924 925 Supplementary Data set 2 | Flow cytometry non-gated data of FL3-A versus FL1-A of all samples. 926 Flow cytometry data of showing fluorescent NBDC uptake by K. marxianus, S. cerevisiae strains with for 927 each sample the intensity of counts (pseudo-colored) for 533/30 nm (FL1) for NBDC and > 670 nm (FL3) 928 for PI. 929 [Example of first row of FlowCyto_FL1_FL3.pdf] 930 Filename Strain Time point PI # Day Staining Cells/PI-ne Cells/PI-po Cells/PI-ne A09 CBS6556_T_A_PI_1.fcs CBS6556 1 PI A 1 T 576 411000 75590 B09 CBS6556_T_B_PI_1.fcs CBS6556 1 PI B 1 T 625 398024 88212 A01 IMX585_T_A___1.fcs IMX585 1 - A 2 T 1391 3 472000 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.07.425723doi: bioRxiv preprint https://doi.org/10.1101/2021.01.07.425723 http://creativecommons.org/licenses/by-nc-nd/4.0/ 47 931 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.07.425723doi: bioRxiv preprint https://doi.org/10.1101/2021.01.07.425723 http://creativecommons.org/licenses/by-nc-nd/4.0/ 50 Supplemental material for: 934 Engineering the thermotolerant industrial yeast Kluyveromyces marxianus for anaerobic growth 935 Wijbrand J. C. Dekker, Raúl A. Ortiz-Merino, Astrid Kaljouw, Julius Battjes, Frank Wiering, Christiaan 936 Mooiman, Pilar de la Torre, and Jack T. Pronk* 937 Department of Biotechnology, Delft University of Technology, van der Maasweg 9, 2629 HZ Delft, The 938 Netherlands 939 *Corresponding author: Department of Biotechnology, Delft University of Technology, Van der Maasweg 940 9, 2629 HZ Delft, The Netherlands, E-mail: j.t.pronk@tudelft.nl, Tel: +31 15 2783214. 941 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.07.425723doi: bioRxiv preprint mailto:j.t.pronk@tudelft.nl https://doi.org/10.1101/2021.01.07.425723 http://creativecommons.org/licenses/by-nc-nd/4.0/ 51 Supplementary Fig. 1 | Ethanol evaporation rate. Ethanol concentration over time with reactor volume 942 of 1200 mL SM glucose urea media maintained at 30 °C, stirred with 800 rpm and aerated with a 943 volumetric gas flow rate of 500 mL·min-1. The reactor off-gas was cooled by passing through a condenser 944 cooled at 2 °C. Circles and orange line represent the condition with sparge aeration and Tween 80 (T) 945 media supplementation, diamonds and blue line head-space aeration with Tween 80, triangle and red 946 line represent head space aeration and Tween 80 omission. Data represent mean with standard 947 deviation from three independent reactor experiments. 948 AGF Aeration type Ethanol evaporation (mmol·h-1) T Sparge 0.00578 ± 0.00062 T Head-space 0.00625 ± 0.00032 Head-space 0.00653 ± 0.00020 949 950 100 150 200 250 300 350 400 450 0 24 48 72 96 120 144 168 192 c e th an ol (m M ) Time (h) .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.07.425723doi: bioRxiv preprint https://doi.org/10.1101/2021.01.07.425723 http://creativecommons.org/licenses/by-nc-nd/4.0/ 52 Supplementary Fig. 2 | Consensus biological process GO term enrichment for K. marxianus contrast 951 31. GO terms are clustered according to their rank. See legend of Fig. 2 for experimental details. 952 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.07.425723doi: bioRxiv preprint https://doi.org/10.1101/2021.01.07.425723 http://creativecommons.org/licenses/by-nc-nd/4.0/ 53 Supplementary Fig. 3 | Consensus biological process GO term enrichment for K. marxianus contrast 953 43. GO terms are clustered according to their rank. See legend of Fig. 2 for experimental details. 954 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.07.425723doi: bioRxiv preprint https://doi.org/10.1101/2021.01.07.425723 http://creativecommons.org/licenses/by-nc-nd/4.0/ 54 955 Supplementary Fig. 4 | Consensus biological process GO term enrichment for S. cerevisiae contrast 31. 956 GO terms are clustered according to their rank. See legend of Fig. 2 for experimental details. 957 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.07.425723doi: bioRxiv preprint https://doi.org/10.1101/2021.01.07.425723 http://creativecommons.org/licenses/by-nc-nd/4.0/ 55 958 Supplementary Fig. 5 | Consensus biological process GO term enrichment for S. cerevisiae contrast 43. 959 GO terms are clustered according to their rank. See legend of Fig. 2 for experimental details. 960 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.07.425723doi: bioRxiv preprint https://doi.org/10.1101/2021.01.07.425723 http://creativecommons.org/licenses/by-nc-nd/4.0/ 56 Supplementary Fig. 6 | GO term enrichment comparison of biological process of K. marxianus (kmar) 961 to S. cerevisiae (scer) of contrast 43. GO terms were annotated with the color of distinct directionality 962 (up (blue) down (brown)) and the color intensity was determined by the magnitude of the inverse rank. 963 GO terms with significant mixed-directionality or non-directionality, as having no pronounced distinct 964 directionality, are colored white. Shared GO terms between K. marxianus and S. cerevisiae are 965 connected by a line.966 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.07.425723doi: bioRxiv preprint https://doi.org/10.1101/2021.01.07.425723 http://creativecommons.org/licenses/by-nc-nd/4.0/ 57 Supplementary Fig. 7 | Uptake of the fluorescent sterol derivative NBDC by S. cerevisiae and K. 967 marxianus strains after 23 h staining. 968 Flow cytometry data of Fig. 4 with prolonged staining after pulse-addition of NBD-cholesterol to the 969 shake-flask cultures for 23 h. Bar charts of the median and pooled standard deviation of the NBD-970 cholesterol fluorescence intensity of PI-negative cells with pooled variance from the biological replicate 971 cultures. See legend Fig. 4 for experimental details. 972 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.07.425723doi: bioRxiv preprint https://doi.org/10.1101/2021.01.07.425723 http://creativecommons.org/licenses/by-nc-nd/4.0/ 58 Supplementary Fig. 8 | Flow cytometry gating strategy of both K. marxianus (left panel) and S. 973 cerevisiae (right panel) samples. Gates were set per one species for all samples independent of NBDC 974 staining. Density of events were calculated by FlowJo software and represented in pseudo-color (blue 975 low density, red high-density). The gate between PI-negative and PI-positive was inside the “Cells” 976 gated-population. 977 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.07.425723doi: bioRxiv preprint https://doi.org/10.1101/2021.01.07.425723 http://creativecommons.org/licenses/by-nc-nd/4.0/ 59 Supplementary Fig. 9 | Cross-validation of oxygen-limited and anaerobic growth of K. marxianus 978 IMX2323. Strains were grown in shake-flask cultures in an oxygen-limited (a) and strict anaerobic 979 environment (b). To perform cross-validation between the two parallel running experiments, 1.5 mL 980 aliquot of each culture was sealed and transferred quickly between anaerobic chambers and used to 981 inoculate two shake-flask cultures, represented with crossed-arrows (⤮). The cultures from the strain 982 NBRC1777 (⤮) in the third transfer (C3) in the strict anaerobic environment (b) were hence inoculated 983 from an aliquot of the cultures of NBRC1777 (C2) grown in oxygen-limited environment (a). This resulted 984 in a serial transfer of 26.7 times dilution from transfer C2 to C3. Aerobic grown pre-cultures were used 985 to inoculate the first anaerobic culture on SMG-urea containing 50 g·L-1 glucose and Tween 80. Data 986 depicted are of each replicate culture (points) and the mean (dotted line) from independent biological 987 duplicate cultures, serial transfers cultures are represented with the number of respective transfer (C1-988 3) .989 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.07.425723doi: bioRxiv preprint https://doi.org/10.1101/2021.01.07.425723 http://creativecommons.org/licenses/by-nc-nd/4.0/ 60 Supplementary Fig. 10 | Sterol-independent anaerobic growth of S. cerevisiae IMX585 (reference), 990 IMX1438 (TtSTC1), K. marxianus NBRC1777 (reference) and IMX2323 (TtSTC1). Aerobic grown pre-991 cultures were used to inoculate shake-flask cultures with SMG-urea containing 50 g·L-1 glucose and 992 Tween 80 in a strict anaerobic environment at an OD600 of 0.1 for all strains, and both at OD600 of 0.1 and 993 0.6 for NBRC1777 and IMX2323. Data depicted are of each replicate culture (points) and the mean 994 (dotted line) from independent biological duplicate cultures, serial transfers cultures are represented 995 with the number of respective transfer (C1-2). 996 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.07.425723doi: bioRxiv preprint https://doi.org/10.1101/2021.01.07.425723 http://creativecommons.org/licenses/by-nc-nd/4.0/ 61 Supplementary Fig. 11 | CO2 fraction in the off-gas of K. marxianus IMS1111. Production of CO2 as 997 measured by the fraction of CO2 in the off-gas of the individual bioreactor cultivations of the K. 998 marxianus strain IMS1111 on SMG media pH 5.0 with 20 g·L-1 glucose, 420 mg·L-1 Tween 80 over time 999 (Left panels). The temperature profile was incrementally increased at the beginning of a new batch cycle 1000 (right panels). After 430 h the performance of the off-gas analyzer of replicate M3R deteriorated. 1001 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.07.425723doi: bioRxiv preprint https://doi.org/10.1101/2021.01.07.425723 http://creativecommons.org/licenses/by-nc-nd/4.0/ 62 Supplementary Table 1 | Mutations identified by whole-genome sequencing in comparison to the 1002 reference K. marxianus strain IMX2323. Overview of mutations detected in the strains after selected for 1003 strict anaerobic growth IMS1111, IMS1131, IMS1132, IMS1133 compared to the TtSTC1 engineered 1004 strain (IMX2323). Resequencing of IMS1111 after 4 transfers in strict anaerobic conditions is for clarity 1005 referred with the strain name IMS1115. Overview of mutations of the bioreactor populations after 1006 prolonged selection for anaerobic growth at elevated temperatures, represented by the bioreactor 1007 replicates (M3R, M5R, and M6L). Mutations in coding regions are annotated as synonymous (SYN), non-1008 synonymous (NSY), insertion or deletions. Mutations in non-coding regions are reported with the 1009 identifier of the neighboring gene, directionality and strand (+/-). For K. marxianus genes, corresponding 1010 S. cerevisiae orthologs with the S288C identifier are listed if applicable. QD refers to quality by depth 1011 calculated by GATK and genotyping overviews are given per strain using the GATK fields GT: 1/1 for 1012 homozygous alternative, 1/0 for heterozygous, AD: allelic depth (number of reads per reference and 1013 alternative alleles called), DP: approximate read depth at the corresponding genomic position, and GQ: 1014 genotype quality. NA indicates variants were not called in that position in the corresponding strain. 1015 Chro mos ome Po siti on Descri ption Type Kmar ID S28 8cSy stID G e n e Q D IM X2 32 3 IMS11 11 IMS 113 1 IMS1 132 IMS 113 3 IMS11 15 M3R M5R M6L Mutation spectra of IMX2323 derived single isolates after selection for strict anaerobic growth 3 89 78 44 Asp- 747- Asp CDS:(S YN) TPUv 2_00 2092 YDR 283 C G cn 2 3 2 NA 1/1:0, 120:1 20:99 NA NA NA 1/1:0, 105:1 05:99 1/1:0 ,99:9 9:99 1/1:0, 110:1 10:99 1/1:0, 118:1 18:99 8 59 15 6 codon: TCA CDS:IN SERTI ON[1] TPUv 2_00 4766 Tran spos on 2 7 NA 1/1:0, 7:7:21 NA 1/1:0 ,15:1 5:45 1/1: 0,9: 9:27 1/1:0, 9:9:27 1/1:0 ,12:1 2:36 1/1:0, 7:7:21 1/1:0, 7:7:21 8 55 04 50 Trp- 350- STP CDS:(N ON) TPUv 2_00 4999 YAL 040 C Cl n 3 2 3 NA 1/1:0, 119:1 19:99 NA NA NA 1/1:0, 143:1 43:99 1/1:0 ,89:8 9:99 1/1:0, 117:1 17:99 1/1:1, 98:99: 99 4 45 97 50 TPUv2 _0026 39-T1 p3UTR :+ TPUv 2_00 2639 YGR 156 W Pt i1 3 5 NA NA 1/1: 0,9: 9:29 1/1:0 ,9:11 :54 1/1: 0,9: 9:38 1/1:0, 4:6:24 1/1:0 ,10:1 0:35 1/1:0, 7:7:26 NA 5 17 74 29 TPUv2 _0031 61-T1 p5UTR :- TPUv 2_00 3161 YBR 283 C Ss h 1 2 7 NA NA 1/1: 0,9: 9:27 NA NA 0/1:1, 7:8:21 NA NA NA 5 90 94 77 UTP22 p5UTR :+ TPUv 2_00 3518 YGR 090 W U tp 2 2 3 5 NA 1/1:1, 11:12: 34 NA NA 1/1: 1,8: 9:24 1/1:0, 11:11: 36 NA NA NA Mutations in whole populations after selection for anaerobic growth at elevated temperatures .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.07.425723doi: bioRxiv preprint https://doi.org/10.1101/2021.01.07.425723 http://creativecommons.org/licenses/by-nc-nd/4.0/ 63 3 13 52 43 0 codon: AAT CDS:D ELETIO N[-3] TPUv 2_00 2327 YLR 352 W Lu g 1 2 2 NA NA NA NA NA NA NA NA 0/1:39 ,65:10 7:99 8 63 57 79 codon: CAG CDS:IN SERTI ON[9] TPUv 2_00 5049 No similarity 2 6 NA NA NA NA NA NA NA NA 0/1:25 ,49:74 :99 1016 .CC-BY-NC-ND 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.07.425723doi: bioRxiv preprint https://doi.org/10.1101/2021.01.07.425723 http://creativecommons.org/licenses/by-nc-nd/4.0/ Abstract Results K. marxianus and S. cerevisiae show different physiological responses to extreme oxygen limitation Transcriptional responses of K. marxianus to oxygen limitation involve ergosterol metabolism Absence of sterol import in K. marxianus Engineering K. marxianus for oxygen-independent growth Test of anaerobic thermotolerance and selection for fast growing anaerobes Discussion online Methods Yeast strains, maintenance and shake-flask cultivation Expression cassette and plasmid construction Strain construction Chemostat cultivation Metabolite analysis Gas analysis Ethanol evaporation rate Lipid extractions & GC analysis Sterol uptake assay Long read sequencing, assembly, and annotation Illumina sequencing RNA isolation, sequencing and transcriptome analysis Anaerobic growth experiments Laboratory evolution in low oxygen atmosphere Laboratory evolution in sequential batch reactors Statistics Data availability Code availability Author’s contributions Acknowledgements Competing interest Funding References Description of Additional Supplementary Files Reporting summary Supplemental material for: