基于比较基因组学的Clostridium kluyveri己酸代谢途径关键酶生物信息学分析
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摘要
对3株白酒来源重要的己酸生产菌株Clostridium kluyveri(NBRC 12016、JZZ和DSM 555)全基因组信息进行比较基因组学分析,聚焦于己酸代谢途径核心催化酶,发现NBRC 12016的己酸代谢途径未得到有效注释,JZZ的己酸代谢途径核心催化酶酰基辅酶A脱氢酶存在注释错误。进一步对C. kluyveri模式菌株DSM 555己酸代谢途径的关键酶硫解酶ThlA进行生物信息学分析,发现DSM 555携带3拷贝的ThlA,且具有序列多态性,可能与催化脂肪酸代谢链延长的底物特异性相关。结构分析和分子对接表明,硫解酶Thl A1的底物催化属于氧化还原开关调控机制,并预测了酶的关键催化位点和具体的催化过程。上述分析结果有助于为后续进一步改进菌株的己酸生产性能和更好地应用于白酒酿造提供依据。
In this study, the genomic information of three Clostridium kluyveri strains(NBRC 12016, JZZ and DSM 555)were analyzed by comparative genomics with focus on the core enzymes of the hexanoic acid metabolism pathway. It was found that the metabolic pathway of hexanoic acid in NBRC 12016 was not annotated in details and there was misannotated information on the core enzyme of the hexanoic acid metabolic pathway in JZZ, acyl coenzyme A dehydrogenase.Bioinformatic analysis was carried out for the key enzyme thiolase(ThlA) of hexanoic acid metabolism in DSM 555. Also,we found that DSM 555 carried three copies of the ThlA gene with sequence polymorphism, which might be related to the substrate specificity of fatty acid chain elongation enzymes. The structural analysis and molecular docking indicated that the catalytic characteristics of ThlA1 contributed to the redox-switch regulatory mechanism. The key catalytic sites and the catalytic process were predicted. The above analysis will be helpful for further improving the hexanoic acid-producing ability of C. kluyveri for application in the brewing of Baijiu.
In this study, the genomic information of three Clostridium kluyveri strains(NBRC 12016, JZZ and DSM 555)were analyzed by comparative genomics with focus on the core enzymes of the hexanoic acid metabolism pathway. It was found that the metabolic pathway of hexanoic acid in NBRC 12016 was not annotated in details and there was misannotated information on the core enzyme of the hexanoic acid metabolic pathway in JZZ, acyl coenzyme A dehydrogenase.Bioinformatic analysis was carried out for the key enzyme thiolase(ThlA) of hexanoic acid metabolism in DSM 555. Also,we found that DSM 555 carried three copies of the ThlA gene with sequence polymorphism, which might be related to the substrate specificity of fatty acid chain elongation enzymes. The structural analysis and molecular docking indicated that the catalytic characteristics of ThlA1 contributed to the redox-switch regulatory mechanism. The key catalytic sites and the catalytic process were predicted. The above analysis will be helpful for further improving the hexanoic acid-producing ability of C. kluyveri for application in the brewing of Baijiu.
引文
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[2]孙宝国,吴继红,黄明泉,等.白酒风味化学研究进展[J].中国食品学报,2015,15(9):1-8.DOI:10.16429/j.1009-7848.2015.09.001.
[3]康永璞,郭新光,刘凤翔,等.浓香型白酒:GB/T 10781.1-2006[S].北京:中国标准出版社,2006.
[4]王晓丹,胥思霞,班世栋,等.红曲酯化酶粗酶制剂在浓香型青酒大曲酒生产中的应用研究[J].酿酒科技,2014(7):57-60.DOI:10.13746/j.njkj.2014.0093.
[5]李绍亮,李亚凤,李学思.酿酒大曲中酯化红曲的分离及应用[J].酿酒,2014,41(2):32-39.DOI:10.3969/j.issn.1002-8110.2014.02.010.
[6]蒋育萌,吴成全.酯化红曲在浓香型白酒生产中的应用[J].酿酒科技,2011(12):70-72.DOI:10.13746/j.njkj.2011.12.031.
[7]ZHU X,TAO Y,LIANG C,et al.The synthesis of n-caproate from lactate:a new efficient process for medium-chain carboxylates production[J].Scientific Reports,2015,5:14360.DOI:10.1038/srep14360.
[8]SEEDORF H,FRICKE W F,VEITH B,et al.The genome of Clostridium kluyveri,a strict anaerobe with unique metabolic features[J].Proceedings of the National Academy of Sciences,2008,105(6):2128-2133.DOI:10.1073/pnas.0711093105.
[9]VASUDEVAN D,RICHTER H,ANGENENT L T.Upgrading dilute ethanol from syngas fermentation to n-caproate with reactor microbiomes[J].Bioresource Technology,2014,151:378-382.DOI:10.1016/j.biortech.2013.09.105.
[10]TAO Y,ZHU X,WANG H,et al.Complete genome sequence of Ruminococcaceae bacterium CPB6:a newly isolated culture for efficient n-caproic acid production from lactate[J].Journal of Biotechnology,2017,259:91-94.DOI:10.1016/j.jbiotec.2017.07.036.
[11]CHEON Y,KIM J S,PARK J B,et al.A biosynthetic pathway for hexanoic acid production in Kluyveromyces marxianus[J].Journal of Biotechnology,2014,182:30-36.DOI:10.1016/j.jbiotec.2014.04.010.
[12]KIM S G,JANG S,LIM J H,et al.Optimization of hexanoic acid production in recombinant Escherichia coli by precise flux rebalancing[J].Bioresource Technology,2018,247:1253-1257.DOI:10.1016/j.biortech.2017.10.014.
[13]XIAO Y,FRANCKE C,ABEE T,et al.Clostridial spore germination versus bacilli:genome mining and current insights[J].Food Microbiology,2011,28(2):266-274.DOI:10.1016/j.fm.2010.03.016.
[14]DARLING A C E,MAU B,BLATTNER F R,et al.Mauve:multiple alignment of conserved genomic sequence with rearrangements[J].Genome Research,2004,14(7):1394-1403.DOI:10.1101/gr.2289704.
[15]GASTEIGER E,HOOGLAND C,GATTIKER A,et al.Protein identification and analysis tools on the ExPASy server[M]//WALKERJ M.The proteomics protocols handbook.Clifton:Humana Press,2005:571-607.DOI:10.1385/1-59259-890-0:571.
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[17]NIELSEN H.Predicting secretory proteins with signal P[J].Methods in Molecular Biology,2017,1611:59-73.DOI:10.1007/978-1-4939-7015-5_6.
[18]GEOURJON C,DELEAGE G.SOPMA:significant improvements in protein secondary structure prediction by consensus prediction from multiple alignments[J].Bioinformatics,1995,11(6):681-684.DOI:10.1093/bioinformatics/11.6.681.
[19]ARNOLD K,BORDOLI L,KOPP J,et al.The SWISS-MODELworkspace:a web-based environment for protein structure homology modelling[J].Bioinformatics,2006,22(2):195-201.DOI:10.1093/bioinformatics/bti770.
[20]GAO Y D,HUANG J F.An extension strategy of Discovery Studio 2.0for non-bonded interaction energy automatic calculation at the residue level[J].Zoological Research,2011,32(3):262-266.DOI:10.3724/SP.J.1141.2011.03262.
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[27]HARTL F U,BRACHER A,HAYER-HARTL M.Molecular chaperones in protein folding and proteostasis[J].Nature,2011,475:324-332.DOI:10.1038/nature10317.
[28]KIM S,JANG Y S,HA S C,et al.Redox-switch regulatory mechanism of thiolase from Clostridium acetobutylicum[J].Nature Communications,2015,6:8410.DOI:10.1038/ncomms9410.
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