|
|
|
| Screening and Breeding of high Ethanol-producing Strains by Genome Shuffling |
| HUANG Jun1,2, WU Ren-zhi3, CHEN Ying1,2,3, LU Zhi-long3, CHEN Xiao-ling3, CHEN Dong1,2,3, HUANG Ri-bo1,2,3 |
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 |
|
|
|
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.
|
|
Received: 26 May 2014
Published: 25 July 2014
|
|
|
|
|
|
[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.
|
|
Viewed |
|
|
|
Full text
|
|
|
|
|
Abstract
|
|
|
|
|
Cited |
|
|
|
|
| |
Shared |
|
|
|
|
| |
Discussed |
|
|
|
|
