CRISPR/Cas9编辑大肠杆菌工程菌生产氨基葡萄糖

CRISPR/Cas9-mediated genome editing of Escherichia coli for the production of glucosamine

【背景】氨基葡萄糖(glucosamine,GlcN)及其衍生物N-乙酰氨基葡萄糖(N-acetylglucosamine,GlcNAc)是合成糖胺聚糖的重要前体物质,在医药、化妆品和保健品领域具有广泛的应用价值。传统的生产方式存在诸多弊端,如环境污染、原料限制、不适于海鲜易过敏人群等问题,因此利用微生物发酵法生产GlcN和GlcNAc越来越受到青睐。【目的】利用微生物发酵生产并提高N-乙酰氨基葡萄糖的产量,探索分子改造及发酵条件优化策略。【方法】以大肠杆菌MG1655为出发菌株,首先利用表达载体共表达大肠杆菌来源的glmS和酿酒酵母来源的gna1,构建GlcNAc的生物合成路径,然后利用CRISPR/Cas9技术敲除GlcNAc的分解代谢与转运途径,以提高GlcNAc的产量,最后结合发酵条件优化使GlcNAc的产量得到进一步提升。【结果】通过分子改造得到一株产GlcNAc菌株RY-5,发酵20 h后GlcNAc的产量达到了2.36 g/L,相较于初始构建的菌株RY-1提高了29倍,进一步对装液量和诱导剂IPTG的添加时间等条件进行发酵优化,GlcNAc产量达到了7.74 g/L,与优化前相比提高了2.3倍。【结论】成功构建一株生产GlcN和GlcNAc的重组大肠杆菌工程菌株,并为微生物合成GlcN和GlcNAc的工业化生产奠定了基础。

[Background]Glucosamine(GlcN)and its derivative N-acetylglucosamine(GlcNAc)are important precursors for the synthesis of glycosaminoglycans,which have a wide range of applications in the fields of medicine,cosmetics,and healthcare products.The traditional production methods of GlcN and GlcNAc have many drawbacks,such as environmental pollution,raw material limit,and unsuitability for people with seafood allergies.Therefore,the microbial fermentation for producing GlcN and GlcNAc has attracted increasing attention.[Objective]To explore the molecular modification for producing GlcNAc by microbial fermentation and optimize the fermentation conditions for increasing the production.[Methods]Firstly,the glmS from Escherichia coli MG1655 and gna1 from Saccharomyces cerevisiae were co-expressed in E.coli MG1655 by the pTrcHisA vector.CRISPR/Cas9 was then employed to knock out the genes responsible for GlcN and GlcNAc transport and metabolism to improve the production of GlcNAc.Finally,the fermentation conditions were optimized to further increase the production of GlcNAc.[Results]The RY-5 strain was constructed by the molecular modification.The production of GlcNAc by RY-5 reached 2.36 g/L after shake flask fermentation for 20 h,which was 29 times that by RY-1 strain.After optimization of the fermentation conditions,the production of GlcNAc reached 7.74 g/L,which increased by 2.3 times compared with that before optimization.[Conclusion]This study successfully developed a recombinant E.coli strain producing GlcN and GlcNAc,which launched a foundation for the industrial production of GlcN and GlcNAc by microbial fermentation.