Metabolic Synthesis Of D-phenylalanine In E. Coli

D-phenylalanine(D-Phe) is an important side chain building block for semi-synthetic polypeptide antibiotics such as bacitracin, gramicidin and polymyxin B, and a significant component of antitumor drug and HIV protease inhibitors. D-Phe is currently produced by chemical resolution of a racemic mixture derived from petrochemical feedstock. In contrast to present method, many chemicals produced by microorganisms are usually chiral pure and the sources are renewable without utilization of environmentally unfriendly organic solvents. The fermentative production of chemicals based on green chemistry and renewable resources makes microbial production of D-Phe of great interest. Based on the reseaches of biosynthesis of L-phenylalanine(L-Phe) and L-phenylglycine(L-Phg) in our laboratory, we constructed a biosynthetic pathway of D-Phe based on an engineered L-Phe producing chassis in this study.1. Deleting tyrB, aspC, ilvE and dad A to weaken the branched metabolic pathwayDeleted D-alanine dehydrogenase gene dadA by λ Red recombination system to construct mutants E. coli BCA and E. coli BCEA. E. coli BCA and E. coli BCEA and genes mutants E. coli W14 B, E. coli BC and E. coli BCE were employed as D-Phe synthetic strains. The growth condition of mutants E. coli W14 B, E. coli BC, E. coli BCE, E. coli BCA and E. coli BCEA were detected by shake flask fermentation, found that the deletion tyr B, ilv E and dad A had no remarkable influence to the growth of strains. However, E. coli BC, E. coli BCE, E. coli BCA and E. coli BCEA showed obvious defects, and the conditions were changed after supplement 1 g×L-1 L-aspartate, this phenomenon demonstrated the mutants E. coli BC, E. coli BCE, E. coli BCA and E. coli BCEA were L-aspartate auxotroph.2. Heterologous expression of D-amino acid aminotransferase to enhance D-Phe biosynthesisCloned D-amino acid aminotransferase gene(datBS, datBL and datBA) from Bacillus subtilis, B. licheniformis, and B. amyloliquefaciens to realize the heterologous expression in E. coli BL21(DE3). Product configuration identification by HPLC showed all the Dat had the ability of D-Phe synthesis. The optimum temperature of all the Dat was 45 oC; Dat showed higher thermal stability. The optimum p H was about 8.0, and all the Dat were stable at alkalescent environment. The specific activity of DatBA(466 U×g-1) was highest, but the DatBS showed the highest activity in the crude enzyme, so Dat from B. subtilis showed a better potential for D-Phe synthesis than those from B. licheniformis and B. amyloliquefaciens.3. Temperature induction expression of D-amino acid aminotransferase and alanine racemase to enhance the supply of precursors and construct D-Phe biosynthetic strainThe gene datBS, datBL, datBA were respectively connected with the temperature-controllable promoter PR, then ligated to p R15 ABK. To ensure the abundant supply of amino donor D-alanine, gene alr coded alanine racemase was cloned from E. coliW3110 and connected with p R15 ABK to construct the recombinant plasmids p R15 ABKAp Rdat. Then the recombinant plasmids were transformed into all the mutants and fed-batch fermentation was performed. Among all the engineered strains, E. coli BCEA(p R15 ABKAp RdatBS) accumulated 1.72 g×L-1 D-Phe in a 15-L jar fermenter after 60 h which is the highest fermentation concentration of them.In conclusion, this research continued to extend the biosynthetic pathway of L-phenylalanine based on the study of L-phenylalanine and L-phenyglycine in our laboratory. We built a D-Phe biosynthetic pathway by weakening the branch metabolic pathway, heterologous expression of D-amino acid aminotransferase and temperature control expression of D-amino acid aminotransferase and alanine racemase. D-Phe yield was reached the highest level without implement any precursors currently. Although the D-Phe yield did not meet the requirements of introdustrial production, but this reseach still lay the foundation for other D-amino acids.