Study On The Derivation,Conversion And Technology Of Bulk Chemical Lysine

Lysine is the first restrictive amino acid which could not been synthesized by human and animal.It is widely used as a nutrition supplement in feed,food and beverage industries as well as a chemical intermediate.The calculated global lysine market is about 2.3 million tons in 2016,and is still growing at a rate of about 7%per year.It is estimated that lysine production will exceed 3 million tons by 2020.Under the current situation of excess lysine production capacity,the cost of lysine could be further decreased by strain improvement and fermentation optimization.Furthermore,the conversion of lysine into other high value-added chemicals for active pharmaceutical ingredients,drugs or materials can maintain the lysine market and expand the lysine industry chain.In this paper,we used L-lysine as raw material,novel derivatives from lysine were synthesized by utilizing metabolic engineering and synthetic biology.Fermentation,enzymatic catalysis or whole-cell catalysis of L-lysine to produce a series of new bio-based polymer monomers,such as 5-aminovalerate,6-aminocaproate and 7-aminoheptanoate,and L-pipecolic acid as a biomedical intermediate,are conducive to maintaining the lysine market and further expanding the lysine industry.These green biomanufacturing strategies could also be applied to enhance the competitiveness of other amino acid industry.The main results are as follows:?1?A method for the determination of L-lysine,5-aminovalerate,6-aminocaproate and 7-aminoheptanoate by pre-column derivatization high performance liquid chromatography was established.A chiral non-derivative high performance liquid chromatography method for the determination of L-pipecolic acid was established.The separation and determination of L-and R-pipecolic acid were achieved by using copper sulfate as mobile phase.?2?The fermentation process of L-pipecolic acid as a biomedical intermediate was established.A metabolic pathway for the one-pot production of L-pipecolic acid was established by Overexpression of L-lysine?-oxidase RaiP,?¹-piperideine-2-carboxylase reductase DpkA,glucose dehydrogenase GDH and L-lysine transporter LysP.By studying the effect of substrate on the production of L-pipecolic acid,L-lysine HCl was identified as a better substrate for the production of L-pipecolic acid than L-lysine,which could increase the production of L-pipecolic acid by 21.28%and reduce the fermentation cost by 16.67%.The knock out of key enzyme CadA in lysine degradation pathway and overespression of LysP improved the L-pipecolic acid titer about 52.52%and 49.88,respectively.This engineered microbial factory achieved the L-pipicolic acid production of 46.7 g/L with a yield of 0.89 g/g in 5-L fermentor for 40 h.?3?A bio-chemical coupling process for the production of 5-aminovalerate as a biomaterial monomer was established.Based on the production of 5-aminovalerate by fermentation,a bio-chemical coupling production process of 5-aminovalerate was established by coupling chemical oxidation process.The effects of L-lysine and L-lysine HCl on the production of 5-aminovalerate were discussed.L-lysine HCl was chosen as a better substrate.The recombinant Escherichia coli CJ02 strain was cultured in a medium containing 4%?v/v?ethanol,10 mM H₂O₂ and 10 g/L-lysine HCl,producing 5.61 g/L 5-aminovalerate with a yield of 0.56 g/g.Compared with the original strain,the titer represents an 18-fold increase.It was revealed that ethanol enhanced the soluble protein expression of an exogenous gene L-lysine?-oxidase RaiP in Escherichia coli.Excellently,29.12 g/L 5-aminovalerate could be achived with a yield of 0.44 g/g in a 5-L fermenter.?4?A whole-cell catalytic process for the production of 5-aminovalerate by ethanol pretreatment whole-cell catalyst CJ02RaiP was established.Under conditions of 37 ^oC,pH 8.0,200 rpm,3 mM Mg²⁺,10 mM H₂O₂,100 g/L-lysine HCl,50.62 g/L of5-aminovalerate could be produced by 400 mL whole-cell catalytic system for 48 h.?5?Here,we show the construction of an artificial iterative carbon-chain-extension cycle-multi-enzyme cascade catalytic system in Escherichia coli for simultaneous production of a series of non-natural amino acids,such as 5-aminovalerate,6-aminocaproate and 7-aminoheptanoate.The rate-limiting enzyme LeuA was identified and analyzed,and the saturated mutant libraries of key loci H97 and S139 were established.The effects of coenzyme NAD⁺and ThDP on the production and distribution of nonnatural straight-chain amino acids were investigated.The engineered system demonstrated simultaneous in vitro production of 99.16 mg/L of5-aminovalerate,46.96 mg/L of 6-aminocaproate and 4.78 mg/L aminoheptanoate after8 h of enzyme catalysis.