通过基因组精简可获得代谢背景降低、更适用于工业生物技术的底盘细胞。产甘油假丝酵母(Candida glycerinogenes)是具有优良抗逆性能的工业菌株,对其基因组进行精简以获得更优的底盘化细胞。以CRISPR/Cas9为工具进行C.glycerinogenes基因组非必需DNA大片段删除,实现了25、50 kb片段的敲除,并研究了缺失菌的耐受性和发酵特性。结果显示,片段删除菌株的胁迫耐受能力无明显改变,30 ℃下Δ7.8 kb菌生物量降低20%,42 ℃下Δ7.8 kb、Δ25 kb菌的生物量下降20%。而在发酵过程中各缺失菌均表现出生物量降低20%,其中Δ7.8 kb菌株表现出了发酵初生物量提高了14%、单位菌体甘油产量提高了22%,还原力相关基因G6PD、6PGDH分别上调10倍和29倍,NADPH/NADP+值提高了100%。结果表明,上述非必需DNA大片段删除对C.glycerinogenes耐受特性无明显影响,而会导致生物量降低和单位菌体甘油产量提高,缺失菌株可能因基因表达水平的差异而与野生菌株在性状上产生区别。该研究是对CRISPR/Cas9系统在C.glycerinogenes中的潜力挖掘,有助于推进该非模式工业菌株底盘化进程和应用。
Microbial genome reduction plays an important role in constructing chassis cells for synthetic biology. Genome reduction of strains with the potential to become robust industrial biological hosts in order to obtain better chassis cells has become a hotspot. Industrial chassis cells with lower metabolic background can be obtained through genome reduction. Genome reduction of Candida glycerinogenes, an industrial strain with robust stress resistance is to obtain its chassis cells. Unlike the previous application in target knockout, the CRISPR/Cas9 system with pairs of sgRNA discovered in this study can perform the ability to large-scale knockout on the C. glycerinogenes genome and several mutant strains with different degrees of deletion were obtained. In this study, large fragments without essential genes were identified as knockout regions and CRISPR/Cas9 system exhibited the deletion of large non-essential DNA fragments of 25 kb and 50 kb. We characterized tolerance and fermentation of the genomic deletion strains. Firstly, the effect of large DNA fragments on several tolerance was investigated, including high glucose tolerance, high salt, furfural and ethanol. The results showed that no significant difference existed in the several stress tolerance. While biomass showed 20% reduction of C. glycerinogenes Δ7.8 kb at 30℃ and C. glycerinogenes Δ7.8 kb andΔ25 kb at 42℃, indicating that the lack of repeat sequence on the genome of C. glycerinogenes Δ25 kb weakened the high temperature resistance. The biomass of the genomic deletion strains was reduced by 20% during fermentation. Compared with wild-type strain, C. glycerinogenes Δ7.8 kb selected as the investigation object showed a 14% higher log-phase biomass , the unit yield of glycerin increased by about 22% after 108 h of fermentation, a higher glucose consumption and ethanol accumulation ,the genes G6PD (gene coding glucose 6-phosphate dehydrogenase) and 6PGDH (gene coding 6-phosphogluconate dehydrogenase) related to reducing power were respectively up-regulated 10 and 29 times, and cytosolic coenzyme NADPH/NADP+ closely related to the pentose phosphate pathway increased by 100%. The increase of this destination to ethanol reflects that the carbon flow of C. glycerinogenes Δ7.8 kb into the glycolysis pathway has increased, which is manifested as an increase in glucose consumption. The results suggested that the deletion of non-essential DNA fragments has no effect on tolerance, causes to reduce biomass, increase the unit yield of glycerin and improve the level of cytosolic coenzyme NADPH. And mutants might differ from the wild-type strain in characterization because of the difference of key gene expression level related to carbon metabolism. Furthermore, the flux of pentose phosphate pathway was changed. This study is to explore the potential of CRISPR/Cas9 system, and to provide the reference for development and application in non-type industrial strain C. glycerinogenes. This study shows that the CRISPR/Cas9 system can be used for the deletion of large fragments in C. glycerinogenes and the study on genome reduction, which provides a reference for the subsequent research on the chassis of non-model diploid yeast. Subsequent studies on the function of the above-mentioned large fragments of the genome can further help to understand the regulatory mechanism of glycerol anabolism in this eukaryote.
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