Construction And Systematical Engineering Of L-phenylglycine Biosynthsis In E. Coli

The aproteinogenic amino acid L-phenylglycine (L-Phg) is an important side chainbuilding block for the preparation of pristinamycin I, virginiamycin S and other antibiotics. Itwas also used in the preparation of the protease inhibitor of AIDS virus and the antitumorcompound taxol. L-Phg is currently produced by chemical resolution of a racemic mixturederived from petrochemical feedstock. Renewable sourcing chemicals have become attractiveas the margins for commodity products from synthetic organic chemistry are decreasing dueto increasing petroleum prices. It would be of commercial and environmental values tospecifcally synthesize chiral L-Phg by fermentation from renewable and sustainableresources. In this study, we frstly described the biosynthetic pathway of L-Phg based on anengineered L-phenylalanine (L-Phe) producing chassis.1. Screening of mandelate oxidase and L-Phg aminotransferase to form the cassetterecycle synthesis of L-PhgMandelate oxidase (MO) belongs to the family of homologous FMN-dependentalpha-hydroxyacid oxidizing enzymes. The extensively studied members in this family areglycolate oxidase (GOX), mandelate dehydrogenase (MDH) and flavocytochrome b2. Thesequence features of the proper cytosolic mandelate oxidase were summarized, and were usedin the screening process. Structural-based screening (sequence alignment, homologousmodeling, and molecular docking) was carried out, and the cytosolic MO that convertedL-mandelate to phenylglyoxylate using oxygen as the final electron acceptor was found. MOfrom Streptomyces coelicolor (HmoSC) and Amycolatopsis orientalis (HmoAO) wereheterologously expressed and characterized. The HmoSCshowed higher thermal stability,substrate affinity and yield.An L-Phg aminotransferase (HpgT) was found, then it was expressed and purified. Theresults of homologous modeling, molecular docking and kinetic analysis demonstrated thatHpgTAOused L-Phe as the amino donor. The use of L-Phe as an amino donor in the reductiveamination of phenylglyoxylate to L-Phg made an economical pathway: as phenylglyoxylatewas transaminated to L-Phg, the L-Phe yielded one molecular of phenylpyruvate to serve as asubstrate for HmaS and primed the three-enzyme cycle of HmaS, Hmo and HpgT for anotherturn. This synthetic pathway possessed the highest efficiency of carbon conversion, which isthe ideal way to L-Phg from L-Phe. While no L-Phg was accumulated in the fermentationbroth of E. coli W3110expressing the L-Phg biosynthetic genes, due to the feedbackregulation in L-Phe synthetic pathway. As L-Phe was the supplier for phenylpyruvate andamino donor, the engineering of L-Phe synthetic pathway was necessary for L-Phgaccumulation.2. Systematical level engineering E. coli to be an L-Phe producing chassisThe biosynthesis of L-Phe is one of the most complicated amino acid synthetic pathways.In this study, the engineering of L-Phe producer, E. coli W14(pR15BABKG), was carried outat a systematical level to construct an L-Phe producing chassis.(1) Genetic switch on or off the expression of thermostable mutants phefbrand aroG15, as well as ydiB, aroK, and tyrB to increase the supply of precursors. The mutant AroG15wasdemonstrated to be a high efficient and thermostable mutant. The PheAfbrin this study sharedthe same catalytic characteristics (thermostability, acitivity and Kcat) with its native form PheAexcept a higher Kmvalue. This decreased substrate binding ability of PheAfbrcould beneutralized by the overexpression of pheAfbrand the high intracellular substrate concentrationsachieved by overexpressions of aroG15, ydiB, aroK. This is the first time to use the thermalstabile and efficient mutants aroG15and pheAfbrto produce L-Phe. The coding sequence oftyrB contained a strong TyrR Box which was artificially changed to enhance the expression oftyrB.(2) Decreasing the glucose uptake speed to reduce the “overfow metabolism”. Theglucose specifc phosphoenolpyruvate-carbohydrate phosphotransferase system (PTS) wasinactivated by deleting the genes ptsG, ptsI and crr. The specific glucose consumption andspecific acetic acid production of Escherichia coli W2(△crr) were the lowest with the highestcell concentration and the shortest lag phase.(3) Employing a tyrA mutant strain to reduce carbon diversion away from tyrosine and toresult in non-growing cells. The utilizaiton of tyrA mutant strain made it possible toartificially control the biomass and to produce the non-growing cells. The yield of L-Phe onglucose of E. coli W14(pR15ABK) increased to25%from18.8%.(4) Enhancing the efflux of Phe by overexpression of yddG to shift equilibrium towardsL-Phe synthesis and to release the feedback regulation. The expression of yddG increased thesecretion of L-Phe and decreased the intracellular concentration. The terminal intracellularPhe concentration decreased from0.4g L-1(2.5mM) to0.3g L-1(1.8mM) which couldrelease the inhibition of the synthetic pathway.After engineering, the L-Phe producing chassis E. coli W14(pR15BABKG) produced47g L-1L-Phe with a yield of25.2%(the theoretical yield is27%), which is industrial valuable.The strain E. coli W14(pR15BABKG) can be easily engineered to produce L-Phg and theother aromatic compounds. The fnal L-Phe concentration and yield in this study was thehighest under the non-optimized fermentation condition.3. Functional expression of L-Phg biosynthetic genes, and engineering the L-Pheproducing chassis to produce L-PhgThe hmaSSC, hmoSCand hpgTAOwere respectively connected with thetemperature-controllable promoter PR, then they were ligated together to synchronouslyexpress L-Phg biosynthetic genes in L-Phe producing chassis. E. coli could automaticallyexport the L-Phg without engineering the transport system. Deletion of tyrB and aspCobviously decreased L-Phe formation. The double mutant (E. coli BC) increased the L-Phgyield by12.6-fold comparing with E. coli B.The intracellular HmaS activity is only3.8%of Hmo. Then the expression of HmaSSCwas increased, reasonably, the production of L-Phg was significantly increased by174-foldand the yield of L-Phe and phenylpyruvate markedly decreased. Optimizing expression ofhmaS, hmo and hpgT and attenuation of L-Phe transamination increased L-Phg yield by224-fold, achieving48.7mg DCW-1(16mg L-1).In conclusion, the construction and engineering of cassette recycle synthetic pathway of L-Phg were firstly reported. This pathway which used L-Phe as the precursor possessed thehighest efficiency of carbon conversion, which is the ideal way to L-Phg. Then, an L-Pheproducing chassis was engineered at the systematical level. This chassis was potentialindustrial valuable. At last, L-Phg biosynthetic pathway was constructed in the L-Pheproducing chassis to biosynthesis L-Phg. The effect of TyrB, AspC, IlvE and HmaS was alsdisscussed. This reach lay the foundation for L-Phg biosynthesis.