基于关键酶分子改造定量调控大肠杆菌胞外分泌水平的机制研究

Escherichia coli is one of the important hosts heterologously expressing recombinant proteins, and it can efficiently express recombinant proteins. However, it was found that recombinant proteins expressed in E. coli were mainly distributed into the periplasm instead of efficiently secreted into the extracellular. Therefore, improving extracellular secretion level is vital for the production of recombinant proteins in E. coli. In this work, in order to secrete recombinant proteins with a high efficiency, we seek to quantitatively regulate the synthesis and stability of E. coli cytoderm peptidoglycan structure via protein and metabolic engineering approaches. Based on bioinformatics analysis, the key amino acid residues of four different D-alanyl-D-alanine carboxypeptidases will be identified, which guide for the construction of mutant libraries with gradient lower catalytic efficiency via saturation mutagenesis. The D-alanyl-D-alanine carboxypeptidase genes in E. coli genome are replaced by genes in the mutant libraries to obtain four E. coli mutant libraries via gene substitution approach, and E. coli mutant libraries with different secretion efficiency are constructed. Then recombinant vectors with protein gene are transformed into the E. coli mutants to express recombinant protein. The E. coli mutants with efficient extracellular secretion level are selected to determine the key D-alanyl-D-alanine carboxypeptidase. The influence and mechanism of the change in key enzyme in E. coli on recombinant protein secretion efficiency is analyzed and revealed based on multilevel. The pipeline and result of the present work are also useful for quantitative control of the other hosts’ secretion level.

大肠杆菌作为重组蛋白异源表达的重要宿主之一，可高效表达蛋白。但在大肠杆菌中，重组蛋白主要通过分泌系统被运输至周质空间，而不能高效分泌至胞外。提高大肠杆菌胞外分泌水平，对重组蛋白生产具有重要意义。本课题希望利用蛋白质工程与代谢工程方式对大肠杆菌细胞壁肽聚糖的合成与结构稳定性进行定量调控，实现重组蛋白高效胞外分泌。基于生物信息学解析，通过对4种不同D-丙氨酰-D-丙氨酸羧肽酶的关键氨基酸残基分别进行饱和突变，构建催化效率梯度降低的酶突变体文库。通过基因替换，构建具有不同分泌水平的大肠杆菌突变体文库。分别转入含外源蛋白基因的重组质粒，筛选高效胞外分泌型大肠杆菌突变体，确定影响胞外分泌效率的关键D-丙氨酰-D-丙氨酸羧肽酶。基于多水平分析，揭示关键酶的催化效率变化对大肠杆菌重组蛋白胞外分泌水平影响的关系机理。本课题研究方法与结果将为定量调控其他宿主的胞外分泌水平提供具有普遍意义的理论参考。

大肠杆菌作为重组蛋白异源表达的重要宿主之一，可高效表达蛋白，而不能高效分泌蛋白至胞外。提高大肠杆菌胞外分泌水平，对重组蛋白生产具有重要意义。本课题利用蛋白质工程与代谢工程方式对大肠杆菌细胞壁肽聚糖的合成与结构稳定性进行扰动与调控，实现了重组蛋白高效胞外分泌，并对相关机理进行了解析。方法及结果如下：1）在大肠杆菌中分别实现了4种D-丙氨酰-D-丙氨酸羧肽酶的过量表达。羧肽酶DacA、DacB的过量表达对大肠杆菌胞外蛋白的生产具有显著促进作用。当DacA或DacB过量表达时，绿色荧光蛋白（GFP）和淀粉酶的胞外酶活力分别对应提高至对照的1.7倍和2.3倍、4.5倍和11.9倍。2）采用基因敲除的方式，分别将4种羧肽酶基因进行敲除，DacA、DacB基因敲除突变株的胞外蛋白生产水平显著提高。DacA、DacB基因敲除突变株的淀粉酶胞外酶活力分别提高了96.8%和88.7%；突变体的GFP胞外生产水平分别提高至对照的2.2倍和2.1倍。通过上述研究结果，确定了影响胞外分泌效率的关键羧肽酶为DacA和DacB。（3）基于生物信息学解析，通过对关键酶——羧肽酶DacA的结构进行分析，确定了关键残基及多种突变方式。其中突变体K113G的催化效率提高最为显著，其kcat值由对照的6543.4 s-1提高至9428.1 s-1。将突变体K113G、K113R、F274G替换GFP绿色荧光蛋白和淀粉酶胞外生产水平均显著提高，最多提高了3.7倍。同时，研究发现，蛋白分泌能力增强的菌株其胞内可溶性肽聚糖含量、内外膜透性等均有增加趋势，其细胞形态等也发生了相应变化，这应为对细胞壁肽聚糖的合成与结构稳定性进行扰动与调控的结果，其促进了蛋白的胞外生产。本课题研究方法与结果将为定量调控宿主的胞外分泌水平提供具有普遍意义的理论参考。