Study On The Biosynthesis And Heterogeneous Expression Of The ?-Poly-L-Lysine And The Stable Expression Vector Construction

?-poly-L-lysine(?-PL)is a natural amino acid homopolymer which is constitutive of the L-lysine residues by the condensation reaction of the ?-carboxyl groups and ?-amino groups.It is characterized by broad-spectrum bacteriostat,good water solubility,thermal stability and good biosafety.Therefore,?-poly-L-lysine has been approved for using as a natural biological preservative in some countries.Besides,the different polymerization degree of ?-PL has advantages and disadvantages in the biological characteristics.It can also be used in many aspects such as drug carrier,food therapy agent and biological material.Therefore,the research on the biosynthesis mechanism and the efficient expression vector of the ?-PL is of great practical significance and application value.In this study,a new stable expression system of ?-poly-L-lysine synthesize without the condition of antibiotic selection pressure was constructed by a type II toxin-antitoxin system relBE2 sca which was found in S.cattleya DSM 46488.This new stable expression system was designed to replace the traditional Streptomyces expression system with resistance makers.The anti-toxin gene relB2 sca and S.diastatochromogenes CGMCC3145 pls gene were cloned together into the expression vector,and then the expression vector was transferred to S.diastatochromogenes CGMCC3145 pls-deletion mutant.The toxin gene relE2 sca was integrated into the chromosome of the mutant strain.In this way,a new Streptomyces expression system under the condition of no antibiotic selection pressure was constructed.At the same time,the experimental results showed that the TA system can indeed delay the loss of ?-PL expression plasmid.In view of the difficulty in genetic manipulation of S.cattleya DSM 46488 and the low level of fermentation of the short-chain ?-PL producing strain,this study screened the commonly used efficient Streptomyces expression hosts,in the hope of finding the efficient host that can carry out the heterogeneous expression of ?-PL,and constructed a new stable Streptomyces expression system of ?-poly-L-lysine synthesize in the host.In this way,we can build an efficient and stable short-chain ?-PL engineering fungus.However,the results showed that the pls genes of both the long-chain ?-PL bacteria and the short-chain ?-PL bacteria could not be expressed in our selected heterologous expression host.To explore why pls genes can not express in different source host,the pls genes of S.cattleya DSM 46488 and S.diastatochromogenes CGMCC3145 were expressed in the S.cattleya DSM 46488 pls double-deletion mutant strains.The experimental results showed that covering the s.cattleya DSM 46488 plasmid pls gene mutations restored the short-chain ?-PL production capacity but production is far lower than the wild type.However,S.diastatochromogenes CGMCC3145 pls gene could not be expressed in S.cattleya DSM 46488 pls double-deletion mutant strains,which suggested that besides pls genes,the synthesis of ?-PL requires the assistance of other factors,which may also be the cause of the failure of heterogenous expression in commonly used Streptomyces expression hosts.We chose S.diastatochromogenes CGMCC3145 and S.cattleya DSM 46488 as the main research objectives.Through gene knockout and backfilling,it is further determined that the pls gene is the key factor to control the ?-PL synthesis,which lays the foundation for further research on the synthesis mechanism of the ?-PL.At the same time,the results of a new type of Streptomyces ?-poly-L-lysine synthesize stable expression system under the condition of no selective pressure of antibiotics provides a train of thought in building a food safety level of Streptomyces expression system and highly efficient and convenient without antibiotic selection pressure Streptomyces expression system construction based on relBE2 sca.