异源表达透明颤菌血红蛋白结合调控策略提高全细胞催化合成苯乳酸产量的研究OA

[Objective]This study aims to improve the oxygen(O2)utilization rate of strains in the whole-cell catalytic reaction system and efficiently catalyze the synthesis of phenyllactic acid(PLA)from phenylalanine(PHE).[Methods]Based on the effect of heterologous expression of Vitreoscilla hemoglobin(VHb)on the growth of host Escherichia coli,genetic engineering technology was used to construct a multi-enzyme coupled expression system including VHb,L-amino acid deaminase(L-AAD)and lactate dehydrogenase(LDH).Subsequently,the expression regulation strategies of key enzymes were optimized,and the genetic stability of recombinant strains was evaluated by continuous passage experiments.[Results]Heterologous expression of VHb could improve the oxygen utilization rate of strains and significantly promote the growth rate and maximum biomass of host E.coli.In shake flask culture,the time for the strain to enter the logarithmic growth phase during the scale-up culture stage was shortened by 1.5 h,and the maximum biomass was increased by 46%when VHb expression was induced alone.The dominant recombinant strain E.coli BL21(pRSFDuet-aad-ldh-T7-rbs2-2vhb)was obtained by optimizing the VHb expression regulation strategy.Furthermore,the whole-cell catalytic reaction system was optimized,and the optimal reaction conditions were determined as follows:0.010 mol/L PBS buffer,pH 7.0,temperature 37℃,and rotation speed 250 r/min.Under these conditions,the maximum PLA yield of the dominant recombinant strain was 53.7 g/L;The dominant recombinant strain had good stability;after 10 consecutive passages,the change rate of PLA yield in each generation was less than 5%,showing potential to become an industrial strain.[Conclusion]Heterologous co-expression of VHb can promote the growth of E.coli and improve the yield of PLA via whole-cell catalysis.