Candida glycerinogenes基因组大片段缺失菌的构建及表征  

Deletion of large genomic DNA in Candida glycerinogenes by CRISPR/Cas9 system

作　　者：李沁 李海铭 陆信曜 宗红 诸葛斌[1] LI Qin;LI Haiming;LU Xinyao;ZONG Hong;ZHUGE Bin(The Key Laboratory of Industrial Biotechnology,Ministry of Education,Jiangnan University,School of Biotechnology,Research Centre of Industrial Microbiology,Wuxi 214122,China)

机构地区：[1]工业生物技术教育部重点实验室(江南大学),工业微生物研究中心,江苏无锡214122

出　　处：《食品与发酵工业》2023年第3期1-8,共8页Food and Fermentation Industries

基　　金：国家自然科学基金项目(31970033)。

摘　　要：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.glycerinogenesΔ7.8 kb at 30℃and C.glycerinogenesΔ7.8 kb andΔ25 kb at 42℃,indicating that the lack of repeat sequence on the genome of C.glycerinogenesΔ25 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.glycerinogenesΔ7.8 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 co

关 键 词：基因组精简 底盘细胞 产甘油假丝酵母 CRISPR/Cas9 基因组大片段编辑 生理表征 

分 类 号：TQ920.1[轻工技术与工程—发酵工程] Q78[生物学—分子生物学]