基因组重排技术选育乙醇高产菌株

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。

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

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.