Idengtificationg Of Key Genes In ε-polylysine Biosynthesis And Enhancing Its Production Through Genes Combinatorial Overexpression Strategy In Streptomyces Albulus

ε-Poly-L-lysine（ε-PL）is a secondary metabolite produced by Streptomyces albulus.It has a wide range of antibacterial activity,which makes it a higher safe new natural food preservative widely used in the food industry.Therefore,it is particularly important to obtain an excellentε-PL producing strain to improve fermentation efficiency and reduce production cost ofε-PL.Breedingε-polylysine producing strains via traditional methods has encountered some bottlenecks.Therefore,metabolic engineering has been recognized an effective strategy to improve the fermentation efficiency and reduce the cost ofε-PL production.However,the key metabolic nodes and regulatory mechanism ofε-PL biosynthesis have rarely been explored.In this study,S.albulus WG-608 was selected as the research object.Based on the result of metabolic flux analysis between high and low-producing strains,the potential key genes ofε-PL biosynthesis were identified.Three effective genes,ppc,pyc and pls,were overexpressed in double or triple tandem,and then metabolic flow analysis of the triple tandem strain was performed to evaluate the potency of the overexpressed genes.The main research contents and results are as follows:（1）Metabolic flow analysis was performed onε-PL producing strain Streptomyces albulus M-Z18 and high-producing strain Streptomyces albulus WG-608 to strengthen the understanding of key metabolic nodes and regulatory mechanisms ofε-PL biosynthesis.Compared with M-Z18,the flux ofε-PL synthesis in WG-608 was increased by 27.8%,and the flux of pentose phosphate pathway,compensation pathway（PP pathway）,tricarboxylic acid cycle pathway（TCA pathway）and L-lysine synthesis pathway increased by 11.1%,29.3%,5.5%and 16.7%,respectively.In this study,five key genes involved in L-lysine biosynthesis pathway and theε-PL synthase gene were picked out based on metabolic flow analysis,i.e.pyruvate carboxylase gene（pyc）,phosphoenolpyruvate carboxylase gene（ppc）,glucose-6-phosphate dehydrogenase gene（zwf）,dihydropyridine dicarboxylate synthase gene（dap A）,diaminopelate decarboxylase gene（lys A）andε-PL synthase gene（pls）.（2）The ppc,zwf,dap A,lys A,pyc and pls were overexpressed respectively in S.albulus WG-608.The results showed that the overexpression of ppc,pyc and pls has promoted the synthesis ofε-PL,and the overexpression of pls combined with L-lysine addition led to a significant increaseε-PL production.In fed-batch fermentation,theε-PL productions of the overexpression strains OE-ppc,OE-pyc and OE-pls were 2.8%,9.9%and 16.3%higher than those of the wild-type strain S.albulus WG-608,respectively;the unit cell synthesis capacity was increased by 12.7%,21.8%and 56.4%,and the glucose conversion rate was increased by12.3%,10.2%and 24.1%,respectively.The fed-batch fermentation of OE-pls strain was carried out using a L-lysine addition strategy in a 5 L fermenter.After 192 h of fed-batch fermentation,theε-PL production reached 61.3 g·L^-1,was 35.4%higher than the wild-type strain S.albulus WG-608.（3）Double or triple overexpression of ppc,pyc and pls was conducted in S.albulus WG-608,and the result showed that both double or triple overexpression could promoteε-PL synthesis.After 192 h of fed-batch fermentation,theε-PL productions of the tandem overexpression strains OE-ppc-pyc,OE-pls-pyc and OE-ppc-pyc-pls were 14.1%,12.1%and11.3%higher than those of the wild-type,while the unit cell synthesis capacity was increased by 39.4%,25.2%and 26.1%,respectively.Therefore,it can be seen that the multiple gene tandem overexpression can improveε-PL production and unit bacterial synthesis ability.However,the multi-gene tandem overexpression strains showed a decrease in cell mass and an increase in the rate of glucose consumption,which may be due to the fact that the insertion of long gene fragments brought a larger load to cell growth,and strain needed to consume more carbon source to maintain its normal growth.The metabolic flux of the high-yielding engineered strain OE-ppc-pyc-pls was compared with that of the starting strain WG-608,and the results showed that overexpression indeed achieved the effect of changing the metabolic flux of the strain.