Strain improvement of the pentose-fermenting yeast Pichia stipitis by genome shuffling
Strain improvement of the pentose-fermenting yeast Pichia stipitis by genome shuffling
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DOI：
10.1016/j.mimet.2010.03.009
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发表时间：
2010-05-01
影响因子：
2.2
通讯作者：
Lee, Hung
中科院分区：
文献类型：
--
作者：
Bajwa, Paramjit K.;Pinel, Dominic;Lee, Hung
采用基于杂交的基因组改组技术提高戊糖发酵酵母树干毕赤酵母（Pichia stipitis）对硬木亚硫酸废液（HW SSL）的耐受性。以树干毕赤酵母的6株紫外诱变突变株为出发菌株，对其进行4轮基因组改组。在每轮之后，基于它们在HW SSL梯度板上的生长来选择改良的菌株。在每轮之后建立突变体文库，并且这些改进的突变体菌株用作下一轮改组的起始池。梯度板上对HW SSL的表观耐受性随着每轮改组逐渐增加，最多4轮。进一步测试选择的改良突变体对液体HW SSL的耐受性。经过4轮洗牌，4个变种人，两个来自第三轮（指定为GS 301和GS 302）和第四轮的两个（指定为GS 401和GS 402），其可以在80%（v/v）HW SSL中生长，GS 301和GS 302也可以在85%（v/v）HW SSL中生长。尽管没有观察到细胞数量的增加。树干毕赤酵母野生型菌株（WT）不能在HW SSL上生长，除非将其稀释至65%（v/v）或更低。对HW SSL耐受性提高的基因组改组菌株保留了其发酵能力。在限定培养基和HW SSL中评估了对液体HW SSL表现出最佳耐受性的2种菌株GS 301和GS 302的发酵性能。两种菌株都比WT更有效地利用4%（w/v）的木糖或葡萄糖，并产生更多的乙醇。它们还利用了4%（w/v）的甘露糖或半乳糖，并产生与WT相同程度的乙醇。GS 301和GS 302能够在未稀释的HW SSL中产生低水平的乙醇。(C)2010 Elsevier BV保留所有权利。
Genome shuffling based on cross mating was used to improve the tolerance of the pentose-fermenting yeast Pichia stipitis towards hardwood spent sulphite liquor (HW SSL). Six UV-induced mutants of P. stipitis were used as the starting strains, and they were subjected to 4 rounds of genome shuffling. After each round, improved strains were selected based on their growth on HW SSL gradient plates. Mutant libraries were established after each round and these improved mutant strains served as the starting pool for the next round of shuffling. Apparent tolerance to HW SSL on the gradient plate increased progressively with each round of shuffling up to 4 rounds. Selected improved mutants were further tested for tolerance to liquid HW SSL. After 4 rounds of shuffling, 4 mutants, two from the third round (designated as GS301 and GS302) and two from the fourth round (designated as GS401 and GS402), were selected that could grow in 80% (v/v) HW SSL GS301 and GS302 grew also in 85% (v/v) HW SSL GS301 was viable in 90% (v/v) HW SSL, although no increase in cell number was seen. The P. stipitis wild type strain (WT) could not grow on HW SSL unless it was diluted to 65% (v/v) or lower. Genome-shuffled strains with improved tolerance to HW SSL retained their fermentation ability. Fermentation performance of GS301 and GS302, the 2 strains that exhibited the best tolerance to liquid HW SSL, was assessed in defined media and in HW SSL Both strains utilized 4% (w/v) of xylose or glucose more efficiently and produced more ethanol than the WT. They also utilized 4% (w/v) of mannose or galactose and produced ethanol to the same extent as the WT. GS301 and GS302 were able to produce low levels of ethanol in undiluted HW SSL. (C) 2010 Elsevier B.V. All rights reserved.