key: cord-102555-vnmc9ii8 authors: Araki, Takuma; Tanatani, Kenta; Kamimura, Naofumi; Otsuka, Yuichiro; Yamaguchi, Muneyoshi; Nakamura, Masaya; Masai, Eiji title: Sphingobium sp. SYK-6 syringate O-demethylase gene is regulated by DesX, unlike other vanillate and syringate catabolic genes regulated by DesR date: 2020-07-28 journal: bioRxiv DOI: 10.1101/2020.07.27.224295 sha: doc_id: 102555 cord_uid: vnmc9ii8 Syringate and vanillate are the major metabolites of lignin biodegradation. In Sphingobium sp. strain SYK-6, syringate is O demethylated to gallate by consecutive reactions catalyzed by DesA and LigM, and vanillate is O demethylated to protocatechuate by a reaction catalyzed by LigM. The gallate ring is cleaved by DesB, and protocatechuate is catabolized via the protocatechuate 4,5-cleavage pathway. The transcriptions of desA, ligM, and desB are induced by syringate and vanillate, while that of ligM and desB are negatively regulated by the MarR-type transcriptional regulator DesR, which is not involved in desA regulation. Here we clarified the regulatory system for desA transcription by analyzing the IclR-type transcriptional regulator desX, located downstream of desA. Quantitative reverse transcription (RT)-PCR analyses of a desX mutant indicates that the transcription of desA was negatively regulated by DesX. In contrast, DesX was not involved in the regulation of ligM and desB. The ferulate catabolic genes (ferBA) under the control of a MarR-type transcriptional regulator FerC are located upstream of desA. RT-PCR analyses suggest that the ferB-ferA-SLG_25010-desA gene cluster consists of the ferBA operon and the SLG_25010-desA operon. Promoter assays reveal that a syringate- and vanillate-inducible promoter is located upstream of SLG_25010. Purified DesX bound to this promoter region, which overlaps with an 18-bp-inverted repeat sequence that appears to be essential for the DNA binding of DesX. Syringate and vanillate inhibited the DNA binding of DesX, indicating that these compounds are effector molecules of DesX. IMPORTANCE Syringate is a major degradation product in the microbial and chemical degradation of syringyl lignin. Along with other low-molecular-weight aromatic compounds, syringate is produced by chemical lignin depolymerization. Converting this mixture into value-added chemicals using bacterial metabolism (i.e., biological funneling) is a promising option for lignin valorization. To construct an efficient microbial lignin conversion system, it is necessary to identify and characterize the genes involved in the uptake and catabolism of lignin-derived aromatic compounds and elucidate their transcriptional regulation. In this study, we found that the transcription of desA, encoding syringate O-demethylase in SYK-6, is regulated by an IclR-type of transcriptional regulator, DesX. The findings of this study, combined with our previous results on desR (a MarR transcriptional regulator that controls the transcription of ligM and desB), provide an overall picture of the transcriptional regulatory systems for syringate and vanillate catabolism in SYK-6. phenylcoumaran, and diarylpropane) and monoaryls (e.g., ferulate, vanillin, and 82 syringaldehyde) as its sole carbon and energy source (7, 8). These aromatic compounds, 99 crescentus, and C. glutamicum ATCC 13032, the transcriptional regulation of vanAB is 100 negatively regulated by a GntR-type, GntR-type, and PadR-like transcriptional regulator, 101 respectively, all of which are called VanR (17, 21, 22) . VA was determined to be an 102 effector molecule for the latter two systems (17, 22) . The degradation of SA by bacteria other than SYK-6 has recently been reported in Novosphingobium aromaticivorans 104 DSM 12444 (23), Microbacterium sp. strain RG1 (24), and Pseudomonas sp. strain 105 NGC7 (25). While the SA catabolic pathway genes have been identified in DSM 12444 106 and predicted in RG1, the regulatory system of SA catabolism has not been studied in 107 any bacterium. 108 In terms of the transcriptional regulation involved in VA and SA catabolism in 109 SYK-6, we have reported that the PCA 4,5-cleavage pathway genes are positively 110 regulated by LigR, a LysR-type transcriptional regulator that recognizes PCA and GA as 111 effectors ( Fig. 1 ) (26). Moreover, we have shown that a MarR-type transcriptional 112 regulator, DesR, negatively regulates the transcription of ligM and desB, and VA and SA 113 are effectors that release the repression by DesR (Fig. 1) (27) . Although the 114 transcription of desA is also induced by VA and SA, DesR does not participate in the 115 transcriptional regulation of desA; thus, its regulatory system remains unknown. 116 In this study, we clarified the regulatory system of SA catabolism in SYK-6 by 117 identifying and characterizing an IclR-type transcriptional regulator, DesX, which 118 regulates desA transcription. Downstream of desA, there is SLG_24970, which is similar to the IclR-type 137 transcriptional regulator (ITTR) (Fig 2A; Table S1 ). To clarify whether the gene product 138 of ferC or SLG_24970 is involved in the binding of the desAp2 probe, we conducted 139 EMSAs using cell extracts of a ferC mutant (ferC) and an SLG_24970 mutant 140 (24970). ferC was obtained in our previous study (28), while 24970 was 141 constructed through homologous recombination in this study (Fig. S1 ). EMSAs using a 142 cell extract of ferC grown in Wx-SEMP show a band shift similar to that of the wild 143 type; however, no such band shift occurs in EMSA using a cell extract of 24970 grown 144 in the same medium (Fig. 2C) . These results strongly suggest that the band shift was 145 due to the binding of the SLG_24970 gene product to the desAp2 region. 146 To determine whether the disruption of ferC and SLG_24970 affects SA and VA 147 catabolism in SYK-6, we measured the growth of ferC and 24970 on 5 mM SA and 148 VA. 24970 grew on SA and VA somewhat faster than the wild type, while ferC grew 149 on SA and VA as well as the wild type ( Fig. 2D and E) . These results suggest that the 150 SLG_24970 gene product negatively regulates the transcription of desA by binding to 151 the desAp2 region. Introduction of a plasmid carrying SLG_24970 (pJB24970) into 152 24970 and SYK-6 caused a substantial delay in their growth on SA compared to 153 24970 and SYK-6 harboring the vector (pJB866) (Fig. S2) . These results indicate that 154 the disruption of SLG_24970 caused the changed phenotype of 24970. Thus, we 155 designated SLG_24970 as desX. (Fig. 6) , desA is transcribed at higher rate in vivo under VA-inducing conditions compared to SA-inducing conditions (Fig. 3A) . The reason for this discrepancy is still 315 unclear, however, the in vivo results may not completely reflect in vitro results, as the 316 former is affected by other factors, such as the level of substrate uptake. 317 The transcription start site of the SLG_25010-desA operon is located 10 bp 318 upstream from the initiation codon of SLG_25010, and a weak Shine-Dalgarno 319 sequence is 8-to 6-bp upstream of the initiation codon (Fig. S5) . According to the RBS 320 calculator (31), the translation initiation rate of SLG_25010 from the putative mRNA 321 sequence of the SLG_25010-desA transcript is 1.77, a rate that is markedly lower than to OMA by a hydrolase whose gene has not yet been identified (Fig. 1) (32) . Recently, 332 the methylesterase (DesC) and the cis-trans isomerase (DesD) genes were reported to 333 be involved in the conversion of CHMOD to OMA during SA catabolism in 334 Novosphingobium aromaticivorans DSM12444 (23). In the SYK-6 genome, the 335 SLG_12720 and SLG_07230 amino acid sequences are 45% and 40% similar to those 336 of desC and desD, respectively. However, there is no SLG_25010 ortholog in the DSM 337 12444 genome. It will be necessary to investigate the involvement of these genes in the 338 conversion of CHMOD in SYK-6 in the future. 339 In N. aromaticivorans DSM 12444, SA is converted to 3MGA by the Saro_2404 340 gene product (DesA NA ), whose amino acid sequence is 71% similar to that of SYK-6 341 DesA. The resulting 3MGA is metabolized via CHMOD as described above (23). In the 342 DSM 12444 genome, a BLAST search reveals Saro_2407, encoding a product with 343 51% amino acid sequence identity with DesX. Because Saro_2404 (desA NA ) and 344 Saro_2407 (desX ortholog) are closely located (Fig. S9) , desA NA is probably regulated catabolism. This information is essential for creating engineered bacteria that can 364 efficiently produce value-added chemicals from lignin. Bacterial strains, plasmids, culture conditions, primers, and chemicals. The 368 bacterial strains and plasmids used in this study are listed in Table 1 , and PCR primers 369 are listed in Table 2 . Sphingobium sp. SYK-6 and its mutants were grown at 30C with (Table 460 2), and Q5 Hot Start High-Fidelity DNA Polymerase (New England Biolabs). PCR 461 products were electrophoresed on a 0.8% agarose gel. pSDA1_F pSDA1_R pSDA2_F pSDA2a_F pSDA2b_F pSDA2c_F pSDA2_R pSDA3_F pSDA3_R pSDA4_F pSDA4_R p16bdX_F p16bdX_R desAp1_F desAp1_R desAp2_F desAp2_R desAp3_F desAp3_R desAp4_F desAp4_R desAp5_F desAp5_R desAp6_F desAp7_F desAp7_R desAp8_F desAp8_R GCGCAGAACCTTACCAACGT AGCCATGCAGCACCTGTCA GCCTTCGCCTTCCTCAACTA CACCGGAACCCACTGCTT GCTCTCCGACACGATGATCA ACGTACTGCTTCGCCTTGTTG TTTCGAGCATTATTCGCATTTC TCCGCAGGCGAATATTCCT CCGGTGGAACGGGAAGA CCACGCCACGTTGTTCAC GACATGCTGTGGCAGATGTG CGCATCTGCCGCTCATAC CAGAAGGTGGACTCGTCGT AGGATATCGAGCGTGCG TGACGTACGACAATGCGGAA TGACGCCTCCATCATTCTCG ATGATCCGCGTCTTCTCGTC ATGAGTCACTCGCCTTCCA TCAGCACCGGCATTCACTT GCATCGATGAGGGCATCCAT CCGACGAGGTTGAACTGGTT TCATAATCCGCCCAGGGAC GACGTCACCATGGGAAGCTTGACACGATCTACCTGCGCA CCTGCAGGATATCTGGATCCTCCTCGTGGGACTGGTCAT Carbon balance in terrestrial detritus Lignins: natural polymers from oxidative coupling of 518 4-hydroxyphenyl-propanoids Lignin biosynthesis Lignin biosynthesis and structure A field of dreams: Lignin valorization into chemicals, materials, fuels, 523 and health-care products Opportunities and challenges 525 in biological lignin valorization Genetic and biochemical investigations on bacterial catabolic 527 pathways for lignin-derived aromatic compounds Bacterial 529 catabolism of lignin-derived aromatics: new findings in a recent decade: update on bacterial lignin 530 catabolism Engineered microbial production of 2-pyrone-4,6-dicarboxylic acid from 533 lignin residues for use as an industrial platform chemical Glucose-free cis,cis-muconic acid production via new metabolic designs corresponding to the 536 heterogeneity of lignin Development of the production of 2-pyrone-4,6-dicarboxylic acid from 539 lignin extracts, which are industrially formed as by-products, as raw materials The protocatechuate 4,5-cleavage pathway: overview and new findings Genetic and biochemical 613 characterization of a 2-pyrone-4,6-dicarboxylic acid hydrolase involved in the protocatechuate 614 4,5-cleavage pathway of Sphingomonas paucimobilis SYK-6 Identification of three alcohol dehydrogenase genes involved in the 617 stereospecific catabolism of arylglycerol-β-aryl ether by Sphingobium sp. strain SYK-6 DdvK, a novel major facilitator 620 superfamily transporter essential for 5,5'-dehydrodivanillate uptake by Sphingobium sp A rapid and sensitive method for the quantitation of microgram quantities of 623 protein utilizing the principle of protein-dye binding Cloning and expression of Pseudomonas paucimobilis SYK-6 genes involved in the degradation of 626 vanillate and protocatechuate in P. putida Use of bacteriophage T7 RNA polymerase to direct selective 628 high-level expression of cloned genes Improved M13 phage cloning vectors and host strains: 630 nucleotide sequences of the M13mp18 and pUC19 vectors ZAP: a bacteriophage λ expression vector with 632 in vivo excision properties Small mobilizable 634 multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection 635 of defined deletions in the chromosome of Corynebacterium glutamicum Improved 637 broad-host-range RK2 vectors useful for high and low regulated gene expression levels in 638 gram-negative bacteria D666-D675. enzyme genes involved in the conversion of an arylglycerol-β-aryl ether metabolite and their use in 645 generating a metabolic pathway for lignin valorization and the regulation of the PCA 4,5-cleavage genes by LigR (26) are highlighted in blue 660 background and black bold, respectively. The transcriptional regulation of desA by 661 DesX 666 Transcriptional regulators: DesX, IclR-type regulator Abbreviations: VA, vanillate; PCA, protocatechuate; CHMS, 668 4-carboxy-2-hydroxymuconate-6-semialdehyde PDC, 2-pyrone-4,6-dicarboxylate KCH, 2-keto-4-carboxy-3-hexenedioate CHA, 670 4-carboxy-4-hydroxy-2-oxoadipate GA, 671 gallate; CHMOD, 4-carboxy-2-hydroxy-6-methoxy-6-oxohexa-2,4-dienoate Black bars under the map show the DNA fragments used 679 for EMSA (desAp1desAp5 probes). (B) EMSAs of SYK-6 cell extracts using the 680 desAp1desAp5 probes EMSAs of ferC and 24970 cell extracts using the desAp2 probe. The 684 desAp2 probe (500 pM) was incubated in the presence (+) and absence () of the 685 extracts (0.4 g protein/l) of ferC and 24970 cells grown in Wx-SEMP Cells of SYK-6 (gray), ferC 687 (magenta), and 24970 (cyan) were incubated in Wx-5 mM SA (D) or Wx-5 mM VA 688 (E), and OD 660 was periodically monitored Total RNAs were isolated from the cells of SYK-6 and a desX 693 mutant (desX) grown in Wx-SEMP, Wx-5 mM SA, and Wx-5 mM VA. The relative 694 mRNA amounts of desA (A), ligM (B), desB (C), ferB (D), SLG_25010 (E), and desX 695 (F; measured only in the wild type) indicate the fold increases relative to the amount of 696 mRNA in SYK-6 cells grown in Wx-SEMP (level of 1.0). Values for each amount of 697 mRNA were normalized to the level of 16S rRNA This study This study This study This study This study This study This study This study This study This study This study This study This study This study ACGGTCATCGCAGATCAG ACCTGCAGGCATGCAAGCTTTCCATCATTCTCGACGGCG ATGTTTTTCCTCCTAAGCTTTCAGTCCACCAGCATCAGG ACCTGCAGGCATGCAAGCTTGCGCATCCAGGAACTCGAT ACCTGCAGGCATGCAAGCTTGCATGACCTTTCAATTGTGCG ACCTGCAGGCATGCAAGCTTCGTATATACGAAGAACATCGG ACCTGCAGGCATGCAAGCTTCGGATTTCCATGAGTCACTCG ATGTTTTTCCTCCTAAGCTTTTCCCCATAGCCCGCAA ACCTGCAGGCATGCAAGCTTCCTGATCTGCGATGACCGT ATGTTTTTCCTCCTAAGCTTCGCATCTGCCGCTCATAC ACCTGCAGGCATGCAAGCTTGACATGCTGTGGCAGATGTG ATGTTTTTCCTCCTAAGCTTTTCCGGCATTGTCCAGCA TATCGAAGGTCGTCATATGATCCAGAAGGTGGACTC CTTTGTTAGCAGCCGGATCCTCAGCGGTCGCCAAG CTGCAGGATGTGCGCC GTGAATGCCAGCCCCAAA GCATGACCTTTCAATTGTGCG AAGTGAATGCCGGTGCTGAT AGATCATTCGCCGCGCA ATGGCTTCATGCTGCACC ACCGGCGCCGATGATGC TGGTTGAAGAGCACGGCGG AACTGGCGCAACGAGCA CTGCAGCTGGAAGCGATAGT ATGAGTCACTCGCCTTCCA TTTCCCTCTGCACGACGT GTGCCCAGATACGGTCATC GCGCATCCAGGAACTCGAT TTTCATCTACTCGAAGACCGG