基因组重排技术选育乙醇高产菌株

doi:10.13523/j.cb.20140709

中国生物工程杂志

2014, Vol. 34

Issue (7): 56-62 DOI: 10.13523/j.cb.20140709

技术与方法

基因组重排技术选育乙醇高产菌株

黄俊^1,2,3, 黎贞崇³, 吴仁智³, 陈英^1,2,3, 陈东^1,2,3, 黄日波^1,2,3

1. 广西大学亚热带农业生物资源保护与利用国家重点实验室南宁 530005;
2. 广西大学生命科学与技术学院南宁 530005;
3. 广西科学院国家非粮生物质能源研究工程中心南宁 530007

Screening and Breeding of high Ethanol-producing Strains by Genome Shuffling

HUANG Jun^1,2, WU Ren-zhi³, CHEN Ying^1,2,3, LU Zhi-long³, CHEN Xiao-ling³, CHEN Dong^1,2,3, HUANG Ri-bo^1,2,3

1. State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Nanning 530005, China;
2. College of Science and Technology, Guangxi University, Nanning 530005, China;
3. National Non-grain Bio-energy Engineering Research Center, Nanning 530007, China

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摘要：

里氏木霉（Trichoderma reesei）被认为是最合适联合生物加工（consolidated bioprocessing）的微生物之一。原始里氏木霉菌株产乙醇能力太低，需要进一步提高其产酒量。我们通过基因组重排技术提高了里氏木霉菌株产乙醇能力和乙醇耐受力。首先对CICC40360菌株孢子进行NTG诱变得到正向突变菌株，再以此为出发菌株进行基因组重排。进行基因组重排后，重组菌株在含不同乙醇浓度的原生质体再生培养基上进行筛选。突变菌株和原始菌株一起做摇瓶发酵实验进行比较以确定产乙醇能力的提高。经过两轮基因组重排后，筛选获得表现最优异的重组菌S2-254。该菌株能在利用50g/l葡萄糖发酵出6.2g/l乙醇，同时能耐受3.5% （v/v）浓度乙醇。上述结果表明，本实验采用的基因组重排技术能够有效而且快速获得具有目的性状的优良菌株。

关键词： 里氏木霉; 基因组重排; 联合生物加工

Abstract:

Trichoderma reesei can be considered a candidate for consolidated bioprocessing (CBP) microorganism. However, its ethanol yield needs to be improved significantly. Here we improved the ethanol tolerance of a wild-type strain CICC40360 by genome shuffling while simultaneously enhancing the ethanol productivity. The starting population was generated by nitrosoguanidine treatment of the spores, and then subjected for the recursive genome shuffling. The positive colonies from the mutant library, created by genome shuffling were screened for growth on regeneration of protoplasts medium plates containing different concentrations of ethanol. Characterization of all mutants and wild-type strain in the shake-flask indicated the compatibility of ethanol yields enhancement. After two rounds of genome shuffling, the best performing strain, S2-254, was obtained. It was found capable of completely utilizing 50g/l glucose, producing 6.2g/l ethanol, and tolerating 3.5% (v/v) ethanol stress. The result demonstrates that this method was effective and easy to operate for the construction a recombinant strain with desired phenotypes in a short time.

Key words: Trichoderma reesei Genome shuffling Consolidated bioprocessing

收稿日期: 2014-05-26 出版日期: 2014-07-25

ZTFLH:

Q93

基金资助:

纤维素乙醇清洁生产关键技术的引进与合作研究（2011DFA61910）

通讯作者: 黄日波 E-mail: rbhuang@gxas.ac.cn

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引用本文:

黄俊, 黎贞崇, 吴仁智, 陈英, 陈东, 黄日波. 基因组重排技术选育乙醇高产菌株[J]. 中国生物工程杂志, 2014, 34(7): 56-62.

HUANG Jun, WU Ren-zhi, CHEN Ying, LU Zhi-long, CHEN Xiao-ling, CHEN Dong, HUANG Ri-bo. Screening and Breeding of high Ethanol-producing Strains by Genome Shuffling. China Biotechnology, 2014, 34(7): 56-62.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20140709 或 https://manu60.magtech.com.cn/biotech/CN/Y2014/V34/I7/56

[1] 黄俊, 陈东, 黄日波. 纤维小体在燃料乙醇中的应用. 中国生物工程杂志, 2011, 31(1): 103-108. Huang J, Chen D, Huang R B. Research progress in cellulosome application in bio-ethanol. China biotechnology, 2011,31(1):103-108.

[2] 陈雅娟,董园,芦志龙,等. 碱性纤维素酶产生菌株的筛选及其酶学性质研究. 广西科学,2014,21(1): 28-33. Chen Y J, Dong Y, Lu Z L, et al. Screening of alkaline cellulose-producing strains and research on enzymatic characteristics. Guangxi sciences, 2014,21(1): 28-33.

[3] 蒋西然, 李文利. 纤维素乙醇基因工程研究进展. 中国生物工程杂志, 2009, 29(7): 127-133. Jiang X R, Li W L. Research progress in genetic engineering for cellulosic ethanol. China biotechnology, 2009, 29(7): 127-133.

[4] Lynd L R, Weimer PJ, Van Zyl W H, et al. Microbial cellulose utilization: fundamentals and biotechnology. Microbiology and molecular biology reviews, 2002, 66(3): 506-577.

[5] Xu Q, Singh A, Himmel M E. Perspectives and new directions for the production of bioethanol using consolidated bioprocessing of lignocellulose. Current Opinion in Biotechnology, 2009, 20(3): 364-371.

[6] Zhang Y X, Perry K, Vinci V A, et al. Genome shuffling leads to rapid phenotypic improvement in bacteria. Nature, 2002, 415, 644-646.

[7] Hida H, Yamada T, and Yamada Y, Genome shuffling of Streptomyces sp. U121 for improved production of hydroxycitric acid. Appl. Microbiol. Biotechnol., 2007,73, 1387-1393.

[8] Patnaik R, Louie S, Gavrilovic V, et al. Genome shuffling of lactobacillus for improved acid tolerance. Nat. Biotechnol. 2002, 20, 707-712.

[9] Gruber F, Visser J, Kubicek C P, et al. The development of a heterologous transformation system for the cellulolytic fungus Trichoderma reesei based on a pyrGnegative mutant strain. Curr. Genet. 1990, 18, 71-76.

[10] Knapp J E, Chandlee J M. RNA/DNA mini-prep from a single sample of orchid tissue. Biotechniques, 1996, 21, 54-56.

[11] Xu F, Jin H, Li H, et al. Genome shuffling of Trichoderma viride for enhanced cellulase production. Ann Microbiol, 2012, 62(2): 509-515.

[12] Okamoto K, Imashiro K, Akizawa Y, et al. Production of ethanol by the white-rot basidiomycetes Peniophora cinerea and Trametes suaveolens. Biotechnol. Lett. 2010, 32, 909-913.

[13] de Almeida M N, Guimarães V M, Falkoski D L, et al. Direct ethanol production from glucose, xylose and sugarcane bagasse by the corn endophytic fungi Fusarium verticillioides and Acremonium zeae. J. Biotechnol., 2013,168, 71-77.

[14] Okamoto K, Nitta Y, Maekawa N, et al. Direct ethanol production from starch, wheat bran and rice straw by the white rot fungus Trametes hirsuta. Enzyme and microbial technology, 2011, 48(3): 273-277.

[15] Hedayatkhah A, Motamedi H, Najafzadeh V, Ghezelbash G et al. Improvement of hydrolysis and fermentation of sugarcane bagasse by soaking in aqueous ammonia and methanolic ammonia. Biosci. Biotechnol. Biochem, 2013, 77, 1379-1383.

[16] Imada C, Ikemoto Y, Kobayashi T, et al. Isolation and characterization of the interspecific fusants from Streptomycetes obtained using a liquid regeneration method. Fish. Sci. 2002,68,395-402.

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