Construction Of Ε-polylysine High-yielding Strains Based On DAP Pathway Enhancement Strategy

ε-poly-L-lysine(ε-PL)is widely used in many fields such as food,medicine and biomaterials because of its advantages such as wide inhibition spectrum,high stability and good water solubility.In April 2014,China officially approved ε-PL and its hydrochloride(ε-PL.HCL)as a new variety of food additives.At present,the demand for ε-PL is gradually increasing,but the research on ε-PL in China started late,and related research on ε-PL production still needs to be carried out to reduce the application cost of ε-PL.In the early stage of our laboratory,we obtained a high-yielding strain of Streptomyces diastatochromogenes 6#-7 by ARTP and NTG mutagenesis with S.diastatochromogenes TUST as the starting strain,but its high-yielding mechanism is unclear Therefore,in this paper,we compared the transcript levels of key enzymes of the diaminoheptanoate pathway(DAP)between the two strains at different fermentation stages using q RTPCR.Subsequently,to investigate the effect of DAP pathway enhancement on ε-PL synthesis,succinyl-diaminopimelate desuccinylase(dap E),diaminopimelate epimerase(dap F)and diaminopimelate decarboxylase(lys A)were overexpressed in strain 6#-7 by genetic engineering,The specific research results are as follows:(1)In this paper,four expression vectors were successfully constructed using the E.coli-Streptomyces shuttle plasmid p IMEP as the starting vector : p IMEP-dap E,p IMEP-dap F,p IMEP-lys A,p IMEP-dap F-lys A.(2)Four different genetically engineered strains were obtained by E.coli-Streptomyces splice transfer method,respectively,and the genetically engineered strains with excellent yield of ε-PL were screened by preliminary screening in methylene blue plates,shake flask screening,and fermentation verification by batch replenishment in 5L fermenters.The results showed that the engineered strains S.diastatochromogenes dap E,S.diastatochromogenes dap F,S.diastatochromogenes lys A and S.diastatochromogenes dap F-lys A were better than their starting strains S.diastatochromogenes dap F-lys A and S.diastatochromogenes dap F-lys A.The ε-PL yields of S.diastatochromogenes 6#-7 were increased by 22.38 %,27.43 %,30.39 % and 55.00 %,respectively.This indicates that the enhanced DAP pathway has a positive effect on the synthesis of ε-PL in amylase-producing Streptomyces diastatochromogenes.(3)In order to study the high-yielding mechanism of S.diastatochromogenes dap F-lys A,q RT-PCR was used to analyze the differences in the transcription levels of the key enzyme genes EMP pathway,TCA cycle,DAP pathway and pls.The results showed that during the fermentation process,the central carbon metabolism pathway,DAP pathway and pls of the strain dap F-lys A were intensified as a whole,which led to the accumulation of more ε-PL presubstrate L-lysine during the fermentation process.The cells provide more energy,and these two aspects increase the ε-PL production of the strain dap F-lys A.