Establish And Optimization Of The System For L-ter-leucine Microbial Transformation

L-tert-leucine is an unnatural amino acid.It can be used for templates in asymmetric synthesis and as building blocks for pharmaceutically active compounds.At present,the synthesis of L-tert-leucine mainly include two kinds of synthesis methods:chemical method and biological method.When biocatalytic methods is used for the production of L-tert-leucine,the L-tert-Leucine synthesis can be performed continuously by the collaboration of leucine dehydrogenase(LeuDH)and formate dehydrogenase(FDH)with trimethylpyruvate as substrate.In this study,the synthesis of L-tert-leucine has been explored by biocatalytic methods to reseach reaction condition of the system.First,LeuDH from Bacillus cereus and FDH from Candida boidinii were amplified.And then the two key enzymes were constructed in Escherichia coli BL21(DE3)for expression.The recombinant strains pET-28a-LeuDH and pET-28a-FDH were obtained.The trimethylpyruvate was used as substrate,whole cells and cell extraction of strains were both used to synthesize L-tert-Leucine.Aftr the optimal reaction conditions,1 mL whole cells could produce 1.05 mg L-tert-Leucine,while 1 mL cells extract could produce 4.5 mg L-ttert-Leucine.Furthermore,LeuDH and FDH were co-expressed in Escherichia coli-BL21(DE3),recombination strains pET-28a-LeuDH-FDH and pET-28a-FDH-LeuDH were obtained.And L-tert-Leucine was successfully synthesized by the two series expression of strains respectively.Aftr the optimal reaction conditions,1 mL cells extract of strain pET-28a-LeuDH-FDH could produce 3.375 mg L-tert-Leucine,while the yield fall to 1.635 mg/mL when the whole cell was used.The results also show that,the barrier of the cell membrane for substate and cofactors is one of the important limit reaction.That is why the yield of cells extract is better than the whole cell of strains.When the whole cell of strains were used to produce L-tert-Leucine,it is better to use strains of co-expressed the two enzymes than the the strains of expressed respectively.In order to obtain high conversion efficiency and avoid the ultrasonic broken recombination strains,LeuDH and FDH were constructed into Pichia GS115 for secretory expression.The expressed-vector pPIC-9K-LeuDH and pPIC-9K-FDH were constructed,and guided into Pichia GS115 for inducible expression to obtain recombination strains GS115/pPIC9K-LeuDH and GS115/pPIC9K-FDH.Protein electrophoresis showed that two enzymes were successfully constructed and expressed the soluble protein.But the enzyme activity of fermentation supernatant and concentrate active was low,unable to produce L-tert-Leucine successfully.In sumary,the L-tert-leucine had been successfully composited by biocatalytic methods,the highest yield of L-tert-Leucine could achieve 4.5 mg/mL.The Escherichia coli expression system and Pichia expression system had been constructed for LeuDH and FDH.The two key enzymes were install in series in Escherichia coli and the L-tert-leucine was obtained using the whole cell of those recombinant strains.These results for the biosynthesis of L-tert-leucine provide certain theory basis and practical value.