【作者】 余心宇；

【导师】 刘秀霞；

【作者基本信息】 江南大学 ， 发酵工程， 2022， 硕士

【摘要】 微生物蛋白细胞工厂主要采用表达元件优化和底盘菌株改造相结合的构建流程。然而,由于生物系统的多层次网络结构及复杂性,特别是缺乏系统理解基因组未知位点对重组蛋白合成及分泌的影响机制,现有的底盘菌株改造仍主要依赖于“试错”方法,造成其开发周期长、成本高、产品生产效率低等问题。因此,如何快速高效地挖掘基因组范围内的未知关联基因是提高微生物蛋白细胞工厂设计效率的重要方向。为此,本研究工作提出了“CRISPR-微流控筛选”策略,通过建立全基因组规模CRISPRi文库,结合高通量的分泌蛋白表征技术和液滴微流控筛选平台,在新兴的重组药物蛋白表达宿主——谷氨酸棒杆菌中绘制了蛋白分泌的基因型-表型关联图谱,系统地揭示了其基因组中能够用于改善分泌蛋白生产的基因位点,并最终指导构建了高效分泌重组蛋白的底盘菌株。具体的研究内容如下:（1）以双砷-四半胱氨酸反应为基础,通过反应体系和操作流程的优化,成功在皮升级液滴水平建立了非酶依赖的分泌蛋白产量表征方法,设计了“单细胞液滴生成-离线培养-双砷底物注入-低温孵育-荧光激活液滴分选”的分泌蛋白高产菌株的高通量筛选平台,处理通量达到>10⁵个单细胞/天。（2）构建了All-in-one CRISPRi系统,并用于乙醇诱导启动子P_A256活性关联基因的筛选,揭示了该启动子的转录激活是乙醇代谢进入TCA循环的结果;设计构建了谷氨酸棒杆菌中的第一个全基因组规模CRISPRi文库,以46,549条sg RNA实现对基因组99.71%蛋白质编码基因和85.36%非编码基因的遗传扰动;以纳米抗体VHH作为模式分泌蛋白,使用建立的液滴微流控平台对文库中超过50万个单细胞进行了筛选,成功富集了VHH产量显著提高的菌株。（3）通过三轮分选文库的基因型分析,绘制了谷氨酸棒杆菌蛋白分泌的基因型-表型关联图谱,揭示了包括氧化还原调控、氨基酸代谢、ABC转运体等生物过程中存在大量提高分泌蛋白产量的潜在靶点;通过重构实验与表型性能评价明确了24个可以提高VHH产量的有效基因靶点,其中,敲低8个氨基酸代谢途径中的基因靶点使VHH产量提高了8.8%～48.1%,敲低8个ABC转运体相关基因使VHH产量提高了4.3～48.1%;揭示了氧化还原相关转录因子是影响谷氨酸棒杆菌分泌蛋白产量的重要基因位点,并通过氧还转录因子CosR和RshA的组合敲除构建了VHH产量提高2.78倍的底盘菌株,并用于I型骨胶原前肽和单链可变区片段的高效分泌生产。更多还原

【Abstract】 The microbial protein cell factory mainly adopts the construction process combining expression element optimization and chassis strain engineering.However,due to the multi-layer network structure and complexity of biological systems,especially the lack of systematic understanding of the mechanism of unknown loci in the genome affecting the synthesis and secretion of recombinant proteins,the chassis strains engineering is still mainly dependent on"trial and error"methods,resulting in long development cycle,high cost and low production efficiency.Therefore,how to quickly and efficiently mine unknown associated genes within the genome is an important direction to improve the efficiency of designing microbial protein cell factories.To address this,our study proposed the"CRISPR-microfluidic screen"strategy.We combined CRISPRi for trackable genetic perturbations with a droplet microfluidics system for high-throughput phenotypic screening to systematically identify cellular determinants that limit the production of secreted proteins from the entirety of the C.glutamicum genome.The following are the particular research contents:（1）Based on the FlAsH-tetracysteine assay,we created an enzyme-independent approach for assessing the yield of secreted proteins in picoliter droplets.And the high-throughput screening platform of"Generation-offline culture-substrate injection-offline incubation-droplet sorting"was designed,with a throughput of>10⁵ single-cell/day.（2）The all-in-one CRISPRi system was built and used to screen for genes linked with the activity of the ethanol-induced promoter P_A256,indicating that transcriptional activation of this promoter is caused by ethanol metabolism entering the TCA cycle.The first genome-wide CRISPRi library of Corynebacterium glutamate was designed and constructed,containing46,549 sg RNAs to perturb 99.71%protein-coding genes and 85.36%non-coding genes.More than 0.5 million single cells in the library were screened by droplet microfluidic platform with the nanobody VHH as the model secretory protein,and the strains with significantly increased VHH production were successfully enriched.（3）Genotype-phenotype mapping of protein secretion in Corynebacterium glutamate was generated by NGS analysis of sorting libraries,revealing a large number of beneficial targets with functions in various biological processes,including redox regulation,amino acid metabolism and ABC transporters;among them,24 effective gene targets were demonstrated by reconstruction experiments and phenotypic performance evaluation,in which transcriptional inhibition of 8 amino acid metabolism-related genes increased VHH yield by 8.8%～48.1%,and that of 8 ABC transporter related genes increased VHH yield by 4.3～48.1%;redox-transcription factors were proved to be important factors affecting protein production;through engineering on CosR and RshA,we constructed a chassis strain with 2.78-fold increase in VHH production,and it was used to efficiently secrete collagen and scFV.更多还原