摘要
本文主要对发酵木糖的酵母工程菌的构建进行了研究，并对获得的理想融合菌株进行了发酵条件的优化研究，为纤维素类生物质的全面利用打下了菌种方面的基础。通过对大量菌株的筛选，得到了高温酿酒酵母G47。该菌株在45℃能良好生长，并能较好地发酵葡萄糖，但不能发酵木糖。当底物浓度为10％葡萄糖时，41℃和45℃下发酵，发酵液中酒精终浓度为分别为3.9％(w／w)和2.0％(w／w)，转化率分别为75％和39％。
本研究以高温下发酵葡萄糖的酿酒酵母G47和木糖发酵菌株休哈塔假丝酵母1766作为出发菌株，通过单倍体诱导生成及分离，得到G47-12(a)和1766-115(a)等9株单倍体菌株。单一营养缺陷型为稳定可靠的遗传选择标志，通过UV和DES诱变对所挑选出的G47-2(a)和1766-4(a)两株单倍体进行诱变。通过对突变株的筛选鉴定得到G47-2 Lys'(a)和1766-4 Ura'(a)两突变株，实验证明两突变株为正突变株。本研究以G47-2Lys'(a)和1766-4 Ura'(a)作为构建高温木糖发酵菌株的融合亲株。
通过聚乙二醇(PEG)和电诱导融合等方法，实现了发酵爪糖产酒精的休哈塔假丝酵母和高温酿酒酵母之间的融合。融合子经DNA含量、细胞体积测定和稳定性能试验证明为稳定的融合子。融合子F-71能在45℃下发酵木糖产酒精，其在45℃发酵木糖所产生的酒精体积百分数为1.67％(v／v)。与亲株相比，F-71的酒精耐受能力也提高了14.3％。
通过对融合子发酵培养基优化以及系列发酵条件的研究，使融合子发酵木糖产酒精的终产量提高到1.98％(v／v)，转化率为81％。
本论文还进行了体外重组构建发酵木糖产酒精的酵母工程的初探工作。
Construction of Xylose co-fermentation yeast engineering strains and the optimal fermentation conditions were studied in this research. These studies have important meaning to the strains in making full use of lignocellulose biomass. S.cerevisiae G47 was gained by a great mount of screen work. This strain was characterized by growing at high temperature (45 ℃) and can ferment glucose well at this temperature. With the same substrate sugar concentration of 10%, the final ethanol concentration of the broth is 3.9% (w/w) and 2.0%(w/w) when fermenting at 41℃ and 45℃ separately. Their transform rate are 75% and 39% correspondingly.
S.cerevisiae G47 and Candida shehatea 1766 which can produce ethanol by xylose-fermenting were used as the parents strains. Nine halpolids were got through halploid inducing formation and separation. The auxotrophic mutants with only one selectable marker were stable and dependable. So both of G47-2 (a) and 1766-4 (a) were induced by DES and UV in this research. It was proved that auxotrophic mutants G47-2 Lys-1(a) and 1766-4 Ura-1(a) which used as parent strains were positive mutants.
We got ideal fusants between S.cerevisiae and Candida shehatea through inter-generic protoplast fusion induced by PEG and electroporation. Fusants were testified to be stable by its DNA content, the volume of cell and the stable experiment. Fusant F-71 can produce ethanol by xylose-fermenting at 45℃ and the final ethanol concentration of broth was 1.67%(v/v). Compared with parents strains, the ethanol tolerance ability of fusants were improved by 14.3%.
The optimal fermentation medium composition and fermentation conditions about the fusants were studied. And the final ethanol productivity through xylose-fermenting with our fusants was 1.98%(v/v). Its transform rate was 81 %.
The construction of xylose-fermenting yeasts by gene recombinant was studied briefly also.
引文
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