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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2022, Vol. 42 Issue (1/2): 37-45    DOI: 10.13523/j.cb.2110003
工业微生物的设计、改造与应用专题     
丁酸梭菌及产丁酸代谢改造*
傅云扉1,2,魏琦麟2,袁明贵2,康桦华2,田雅2,向蓉2,3,**(),徐志宏2,3,**()
1 仲恺农业工程学院动物科技学院 广州 510225
2 广东省农业科学院动物卫生研究所 农业农村部兽用药物与诊断技术广东科学观测实验站广东省畜禽疫病防治研究重点实验室 广州 510640
3 岭南现代农业科学与技术广东省实验室肇庆分中心 肇庆 526238
Research Progress of Clostridium butyricum and Metabolic Modification of Butyric Acid Production
FU Yun-fei1,2,WEI Qi-lin2,YUAN Ming-gui2,KANG Hua-hua2,TIAN Ya2,XIANG Rong2,3,**(),XU Zhi-hong2,3,**()
1 Zhongkai University of Agriculture and Engineering, Guangzhou 510225, China
2 Guangdong Provincial Key Laboratory of Animal Disease Prevention, Scientific Observing and Experimental Station of Veterinary Drugs and Diagnostic of Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China
3 Zhaoqing Branch Center of Guangdong Laboratory for Lingnan Modern Agricultural Science and Technology, Zhaoqing 526238, China
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摘要：

丁酸梭菌(Clostridium butyricum)是一种专性厌氧菌,可通过多基因过表达、同源重组、基于非复制型质粒和非复制型质粒的同源重组等多种遗传操作方式对其进行改造。丁酸是丁酸梭菌发酵的产物之一,丁酸用途广泛,用于饲料添加剂,可提高动物抵抗力,减少抗生素的使用。用丁酸梭菌发酵产丁酸,其产量仍然较低,不利于工业化生产,有必要通过代谢工程对丁酸梭菌产丁酸的途径进行优化。对丁酸梭菌的主要代谢途径、遗传操作体系及丁酸合成途径的优化等研究进展进行了综述。在此基础上,对丁酸梭菌进一步改造的思路和想法进行了展望。
关键词： 丁酸梭菌;  代谢途径;  丁酸;  改造    
Abstract:

Clostridium butyricum is an obligate anaerobic bacterium, which can be modified by multiple genetic manipulation methods such as multi-gene overexpression, homologous recombination, homologous recombination based on non-replicating plasmids and non-replicating plasmids synthesis direction. Butyric acid is one of the fermentation products of Clostridium butyricum. It has a wide range of uses. It is used as a feed additive to improve animal resistance and reduce the use of antibiotics.The yield of butyric acid in the fermentation of Clostridium butyricum is still low, which is not conducive to industrial production. It is necessary to optimize the way of producing butyric acid by Clostridium butyricum through metabolic engineering.This article reviews the research progress of Clostridium butyricum’s main metabolic pathways, genetic manipulation systems, and the optimization of butyric acid synthesis pathways. On this basis, the thoughts and ideas for further transformation of Clostridium butyricum were prospected.
Key words: Clostridium butyricum    Metabolic pathway    Butyric acid    Modification
收稿日期: 2021-10-05 出版日期: 2022-03-03
ZTFLH:  Q812  
基金资助: * 广东省重点领域研发计划资助项目(2020B0202080004)
通讯作者: 向蓉,徐志宏     E-mail: 23753842@qq.com;13922162272@139.com
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引用本文:

傅云扉,魏琦麟,袁明贵,康桦华,田雅,向蓉,徐志宏. 丁酸梭菌及产丁酸代谢改造*[J]. 中国生物工程杂志, 2022, 42(1/2): 37-45.

FU Yun-fei,WEI Qi-lin,YUAN Ming-gui,KANG Hua-hua,TIAN Ya,XIANG Rong,XU Zhi-hong. Research Progress of Clostridium butyricum and Metabolic Modification of Butyric Acid Production. China Biotechnology, 2022, 42(1/2): 37-45.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2110003    或    https://manu60.magtech.com.cn/biotech/CN/Y2022/V42/I1/2/37

图1  丁酸梭菌的丁酸代谢途径
图2  丁酸梭菌对葡萄糖和木糖的分解代谢
菌株名称 改造策略 改造基因 目的
C.tyrobutyricum ATCC 25755 敲除 磷酸转乙酰酶基因(pta)和乙酸激酶基因(ack) 增加丁酸合成方向的代谢流
过表达 6-磷酸果糖激酶基因(pfkA)和丙酮酸激酶基因(pykA)
替换 丁酸关键酶基因thl替换了乙酸关键酶基因pta
过表达 Class I 型热激蛋白基因groESL、htpG 增加丁酸耐受性
过表达 醛酮还原酶基因(akr),短链脱氢酶/还原酶基因(sdr) 提高糠醛耐受性
克隆表达 丙酮丁醇梭菌ATCC 824中的scrA、scrB和scrK 提高对蔗糖的利用
丙酮丁醇梭菌ATCC 824中的galKET 提高对半乳糖的利用
丁酸杆菌Clostridium tyrobutyricum ATCC25755、大肠杆菌Escherichia coli BL21(DE3)、噬热厌氧杆菌Escherichia coli BL21(DE3)及嗜热厌氧杆菌Thermoanaerobacterium aotearoense SCUT27中编码转醛醇酶的tal基因 提高对木糖的利用
C.tyrobutyricum ATCC 25755/ ket 过表达 galKET,半乳糖非磷酸化转运蛋白基因(galP) 提高对半乳糖的利用
表1  丁酸梭菌改造策略
[1] Jiang L, Wang J F, Liang S Z, et al. Production of butyric acid from glucose and xylose with immobilized cells of Clostridium tyrobutyricum in a fibrous-bed bioreactor. Applied Biochemistry and Biotechnology, 2010, 160(2):350-359.
doi: 10.1007/s12010-008-8305-1 pmid: 18651247
[2] Hudcovic T, Kolinska J, Klepetar J, et al. Protective effect of Clostridium tyrobutyricum in acute dextran sodium sulphate-induced colitis: differential regulation of tumour necrosis factor-α and interleukin-18 in BALB/c and severe combined immunodeficiency mice. Clinical and Experimental Immunology, 2012, 167(2):356-365.
doi: 10.1111/j.1365-2249.2011.04498.x pmid: 22236013
[3] Luo H Z, Yang R L, Zhao Y P, et al. Recent advances and strategies in process and strain engineering for the production of butyric acid by microbial fermentation. Bioresource Technology, 2018, 253:343-354.
doi: 10.1016/j.biortech.2018.01.007
[4] 唐万. 酪丁酸梭菌代谢机理及其应用的探索研究. 杭州: 浙江工业大学, 2018.
Tang W. Study on metabolic mechanism of Clostridium tyrobutyricum and its application for butyric acid production. Hangzhou: Zhejiang University of Technology, 2018.
[5] Zhang L, Cao G T, Zeng X F, et al. Effects of Clostridium butyricum on growth performance, immune function, and cecal microflora in broiler chickens challenged with Escherichia coli K88. Poultry Science, 2014, 93(1):46-53.
doi: 10.3382/ps.2013-03412 pmid: 24570422
[6] Zhang L, Zhang L L, Zhan X A, et al. Effects of dietary supplementation of probiotic, Clostridium butyricum, on growth performance, immune response, intestinal barrier function, and digestive enzyme activity in broiler chickens challenged with Escherichia coli K88. Journal of Animal Science and Biotechnology, 2016, 7:3.
doi: 10.1186/s40104-016-0061-4 pmid: 26819705
[7] Takahashi M, McCartney E, Knox A, et al. Effects of the butyric acid-producing strain Clostridium butyricum MIYAIRI 588 on broiler and piglet zootechnical performance and prevention of necrotic enteritis. Animal Science Journal = Nihon Chikusan Gakkaiho, 2018, 89(6):895-905.
[8] Huang T, Peng X Y, Gao B, et al. The effect of Clostridium butyricum on gut microbiota, immune response and intestinal barrier function during the development of necrotic enteritis in chickens. Frontiers in Microbiology, 2019, 10:2309.
doi: 10.3389/fmicb.2019.02309 pmid: 31681193
[9] 魏琦麟, 向蓉, 袁明贵, 等. 顶空-气相色谱法测定丁酸梭菌发酵液中短链脂肪酸含量. 中国油脂, 2019, 44(10):147-151, 155.
Wei Q L, Xiang R, Yuan M G, et al. Determination of short chain fatty acids in Clostridium butyricum fermentation broth by headspace-gas chromatography. China Oils and Fats, 2019, 44(10):147-151, 155.
[10] 易宏波, 唐青松, 侯磊, 等. 断奶仔猪肠道健康分级及其无抗营养策略. 动物营养学报, 2020, 32(10):4501-4517.
Yi H B, Tang Q S, Hou L, et al. Antibiotic-free nutritional strategies based on intestinal health grade classification of weaned piglets. Chinese Journal of Animal Nutrition, 2020, 32(10):4501-4517.
[11] Liu X G. Production of butyric acid and hydrogen by metabolically engineered mutants of Clostridium tyrobutyricum. Columbus:The Ohio State University, 2005.
[12] Lee J, Jang Y S, Han M J, et al. Deciphering Clostridium tyrobutyricum metabolism based on the whole-genome sequence and proteome analyses. mBio, 2016, 7(3):e00743-e00716.
[13] Moreno M S, Schneider B L, Maile R R, et al. Catabolite repression mediated by the CcpA protein in Bacillus subtilis: novel modes of regulation revealed by whole-genome analyses. Molecular Microbiology, 2001, 39(5):1366-1381.
pmid: 11251851
[14] Zhu Y, Wu Z T, Yang S T. Butyric acid production from acid hydrolysate of corn fibre by Clostridium tyrobutyricum in a fibrous-bed bioreactor. Process Biochemistry, 2002, 38(5):657-666.
doi: 10.1016/S0032-9592(02)00162-0
[15] 索玉凯. 代谢工程改造酪丁酸梭菌(Clostridium tyrobutyricum)强化丁酸生产及木质纤维素利用. 广州: 华南理工大学, 2018.
Suo Y K. Metabolic engineering of Clostridium tyrobutyricum for enhanced butyric acid production and lignocellulosic utilization. Guangzhou: South China University of Technology, 2018.
[16] Fu H X, Yu L, Lin M, et al. Metabolic engineering of Clostridium tyrobutyricum for enhanced butyric acid production from glucose and xylose. Metabolic Engineering, 2017, 40:50-58.
doi: 10.1016/j.ymben.2016.12.014
[17] Gu Y, Jiang Y, Yang S, et al. Utilization of economical substrate-derived carbohydrates by solventogenic clostridia: pathway dissection, regulation and engineering. Current Opinion in Biotechnology, 2014, 29:124-131.
doi: 10.1016/j.copbio.2014.04.004 pmid: 24769507
[18] Rodionov D A, Mironov A A, Gelfand M S. Transcriptional regulation of pentose utilisation systems in the Bacillus/Clostridium group of bacteria. FEMS Microbiology Letters, 2001, 205(2):305-314.
pmid: 11750820
[19] Jiang L, Zhu L Y, Xu X, et al. Genome sequence of Clostridium tyrobutyricum ATCC 25755, a butyric acid-overproducing strain. Genome Announcements, 2013, 1(3):e00308-e00313.
[20] Lorca G L, Chung Y J, Barabote R D, et al. Catabolite repression and activation in Bacillus subtilis: dependency on CcpA, HPr, and HprK. Journal of Bacteriology, 2005, 187(22):7826-7839.
doi: 10.1128/JB.187.22.7826-7839.2005
[21] Tangney M, Mitchell W J. Characterisation of a glucose phosphotransferase system in Clostridium acetobutylicum ATCC 824. Applied Microbiology and Biotechnology, 2007, 74(2):398-405.
pmid: 17096120
[22] Xiao H, Gu Y, Ning Y Y, et al. Confirmation and elimination of xylose metabolism bottlenecks in glucose phosphoenolpyruvate-dependent phosphotransferase system-deficient Clostridium acetobutylicum for simultaneous utilization of glucose, xylose, and arabinose. Applied and Environmental Microbiology, 2011, 77(22):7886-7895.
doi: 10.1128/AEM.00644-11 pmid: 21926197
[23] Zhu Y, Liu X G, Yang S T. Construction and characterization of pta gene-deleted mutant of Clostridium tyrobutyricum for enhanced butyric acid fermentation. Biotechnology and Bioengineering, 2005, 90(2):154-166.
doi: 10.1002/(ISSN)1097-0290
[24] Liu X G, Yang S T. Kinetics of butyric acid fermentation of glucose and xylose by Clostridium tyrobutyricum wild type and mutant. Process Biochemistry, 2006, 41(4):801-808.
doi: 10.1016/j.procbio.2005.10.009
[25] Liu X, Zhu Y, Yang S T. Construction and characterization of ack deleted mutant of Clostridium tyrobutyricum for enhanced butyric acid and hydrogen production. Biotechnology Progress, 2006, 22(5):1265-1275.
doi: 10.1021/(ISSN)1520-6033
[26] Zhang Y L, Yu M R, Yang S T. Effects of ptb knockout on butyric acid fermentation by Clostridium tyrobutyricum. Biotechnology Progress, 2012, 28(1):52-59.
doi: 10.1002/btpr.730 pmid: 22038864
[27] 谢丽静. 丁酸梭菌优良菌株的诱变选育及其发酵工艺研究. 阿拉尔: 塔里木大学, 2018.
Xie L J. Mutation breeding and fermentation technology of Clostridium butyricum superior strains. Ala’er: Tarim University, 2018.
[28] 吴茜. 代谢工程定向改造酪丁酸梭菌以提高抗氧化性能的研究. 南京: 南京工业大学, 2017.
Wu X. Metabolic Engineering Oriented modification of Clostridium tyrobutyricum to improve antioxidant performance. Nanjing: Nanjing University of Technology, 2017.
[29] 严丽玉. 基因工程菌Clostridium tyrobutyrium/TAL的构建及发酵小麦秸秆酸水解液产丁酸的研究. 广州: 华南理工大学, 2016.
Yan L Y. Construction and characterization of Clostridium tyrobutyrium/TAL+ for enhanced butyric acid fermentation in wheat straw hydrolyzate. Guangzhou: South China University of Technology, 2016.
[30] 张杰, 周秀清, 黄火清. 一种基于内源CRISPR-Cas系统的丁酸梭菌基因编辑系统及其应用:中国,CN112029699B. 2020-10-05[2021-06-08]. https://kns.cnki.net/kcms/detail/detail.aspx?dbcode=SCPD&dbname=SCPD2021&filename=CN112029699B&uniplatform=NZKPT&v=dvhDNd6zf7vLuo6vPRWlXTfIOyptpVpzrr1sxrMa4brnr2GUolfuAOaKHRHlTG-Y.
Zhang J, Zhou X Q, Huang H Q, et al. 2020-10-05[2021-06-08]. https://kns.cnki.net/kcms/detail/detail.aspx?dbcode=SCPD&dbname=SCPD2021&filename=CN112029699B&uniplatform=NZKPT&v=dvhDNd6zf7vLuo6vPRWlXTfIOyptpVpzrr1sxrMa4brnr2GUolfuAOaKHRHlTG-Y.
[31] 杨贵清, 刘刚, 杨长得. 酪丁酸梭菌代谢工程菌的构建及其发酵特性. 生物工程学报, 2010, 26(2):170-176.
Yang G Q, Liu G, Yang C D. Engineering and metabolic characteristics of a Clostridium tyrobutyricum strain. Chinese Journal of Biotechnology, 2010, 26(2):170-176.
[32] Ezeji T, Qureshi N, Blaschek H P. Butanol production from agricultural residues: impact of degradation products on Clostridium beijerinckii growth and butanol fermentation. Biotechnology and Bioengineering, 2007, 97(6):1460-1469.
doi: 10.1002/(ISSN)1097-0290
[33] Mills T Y, Sandoval N R, Gill R T. Cellulosic hydrolysate toxicity and tolerance mechanisms in Escherichia coli. Biotechnology for Biofuels, 2009, 2:26.
doi: 10.1186/1754-6834-2-26
[34] Zhang Y, Ezeji T C. Elucidating and alleviating impacts of lignocellulose-derived microbial inhibitors on Clostridium beijerinckii during fermentation of Miscanthus giganteus to butanol. Journal of Industrial Microbiology and Biotechnology, 2014, 41(10):1505-1516.
doi: 10.1007/s10295-014-1493-5 pmid: 25085743
[35] Ai B L, Chi X, Meng J, et al. Consolidated bioprocessing for butyric acid production from rice straw with undefined mixed culture. Frontiers in Microbiology, 2016, 7:1648.
[36] Chi X, Li J Z, Wang X, et al. Bioaugmentation with Clostridium tyrobutyricum to improve butyric acid production through direct rice straw bioconversion. Bioresource Technology, 2018, 263:562-568.
doi: 10.1016/j.biortech.2018.04.120
[37] Suo Y K, Liao Z P, Qu C Y, et al. Metabolic engineering of Clostridium tyrobutyricum for enhanced butyric acid production from undetoxified corncob acid hydrolysate. Bioresource Technology, 2019, 271:266-273.
doi: 10.1016/j.biortech.2018.09.095
[38] Huang J, Cai J, Wang J, et al. Efficient production of butyric acid from Jerusalem artichoke by immobilized Clostridium tyrobutyricum in a fibrous-bed bioreactor. Bioresource Technology, 2011, 102(4):3923-3926.
doi: 10.1016/j.biortech.2010.11.112 pmid: 21169015
[39] Huang J, Zhu H, Tang W, et al. Butyric acid production from oilseed rape straw by Clostridium tyrobutyricum immobilized in a fibrous bed bioreactor. Process Biochemistry, 2016, 51(12):1930-1934.
doi: 10.1016/j.procbio.2016.08.019
[40] Kim M, Kim K Y, Lee K M, et al. Butyric acid production from softwood hydrolysate by acetate-consuming Clostridium sp. S1 with high butyric acid yield and selectivity. Bioresource Technology, 2016, 218:1208-1214.
doi: 10.1016/j.biortech.2016.07.073
[41] Xiao Z P, Cheng C, Bao T, et al. Production of butyric acid from acid hydrolysate of corn husk in fermentation by Clostridium tyrobutyricum: kinetics and process economic analysis. Biotechnology for Biofuels, 2018, 11:164.
doi: 10.1186/s13068-018-1165-1
[42] Rocky-Salimi K, Hashemi M, Safari M, et al. Valorisation of untreated cane molasses for enhanced phytase production by Bacillus subtilis K46b and its potential role in dephytinisation. Journal of the Science of Food and Agriculture, 2017, 97(1):222-229.
doi: 10.1002/jsfa.7716 pmid: 26991843
[43] Yin F W, Zhu S Y, Guo D S, et al. Development of a strategy for the production of docosahexaenoic acid by Schizochytrium sp. from cane molasses and algae-residue. Bioresource Technology, 2019, 271:118-124.
doi: 10.1016/j.biortech.2018.09.114
[44] Sjöblom M, Matsakas L, Christakopoulos P, et al. Production of butyric acid by Clostridium tyrobutyricum (ATCC25755) using sweet Sorghum stalks and beet molasses. Industrial Crops and Products, 2015, 74:535-544.
doi: 10.1016/j.indcrop.2015.05.041
[45] Zhou X L, Zhang Y, Shen Y B, et al. Economical production of androstenedione and 9α-hydroxyandrostenedione using untreated cane molasses by recombinant Mycobacteria. Bioresource Technology, 2019, 290:121750.
doi: 10.1016/j.biortech.2019.121750
[46] Guo X L, Fu H X, Feng J, et al. Direct conversion of untreated cane molasses into butyric acid by engineered Clostridium tyrobutyricum. Bioresource Technology, 2020, 301:122764.
doi: 10.1016/j.biortech.2020.122764
[47] He F F, Qin S W, Yang Z, et al. Butyric acid production from spent coffee grounds by engineered Clostridium tyrobutyricum overexpressing galactose catabolism genes. Bioresource Technology, 2020, 304:122977.
doi: 10.1016/j.biortech.2020.122977
[48] Cook G M, Janssen P H, Morgan H W. Simultaneous uptake and utilisation of glucose and xylose by Clostridium thermohydrosulfuricum. FEMS Microbiology Letters, 1993, 109(1):55-61.
doi: 10.1111/fml.1993.109.issue-1
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[15] 温赛, 刘怀然, 韩煦, 李天, 邢旋. 综述人工合成型抗菌肽及其药学应用研究进展[J]. 中国生物工程杂志, 2016, 36(8): 89-98.
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