Construction Of Recombinant Bacillus Subtilis As Catalyst For Preparing D-p-hydroxyphenylglycine

D-p-Hydroxyphenylglycine?D-HPG?is mainly used for semi-synthetic antibiotics such as amoxycillin,cefadroxil and cefoperazone,and has a large market demand.The hydantoinase process refers to the D-p-hydroxyphenhydantoin?D-HPH?is catalyzed by D-hydantoinase and D-carbamoylase to generate D-HPG via intermediate substance N-carbamoyl-D-p-hydroxyphenylglycine,which is the most potential method.In this study,we focus on the construction of recombinant Bacillus subtilis expressing the heterologous D-hydantionase and D-carbamoylas,which is used as the whole cell catalyst to producing of D-HPG.The hyd_a gene from Bacillus stearothermophilus SD-1,was integrated and expressed under the control of an acetoin-induced promoter in Bacillus subtilis 168N,resulting in strain LS10.Furthermore,expression plasmids pHPS and pUBS were constructed,then strains 168N/pHPS and 168N/pUBS with D-hydantionase activity were obtained.It is Mn²⁺that significantly enhanced the D-hydantionase activity of168N/pUBS strain,which reached 956 U/gDCW when 0.16g/L MnCl₂·4H₂O was added into LB medium.The acoR gene was overexpressed in different degree under the background of constitutive expression of sigL gene.The results showed that when the transcriptional level of acoR gene was increased by 302.33-fold,the D-hydantionase activity of LSL13strain with single copy of hyd_a gene was seriously decreased;When the transcriptional level of acoR gene was increased by 2.11-fold,the D-hydantionase activity of LSL01/pHPS strain reached 1230 U/gDCW;when the transcriptional level of acoR gene was increased by 63.56-fold,D-hydantionase activity of LSL02/pUBS reached1470 U/gDCW.The above results showed that the intracellular level of AcoR affected the expression of high-copy of hyd_a gene.In Bacillus subtilis,the D-carbamoylas?adc gene?from the Agrobacterium sp.KNK712 was heterologously expressed.The results of promoter screening and optimization showed that the expression level of promoter P_AE2 was the highest.After culture medium optimization,the D-carbamoylas activity of of strain LSL04s with adc_a2 gene reached 22.1 U/gDCW,while it decreased significantly in mutant adc_a2.The co-expression plasmid pUBSC with high-copy number of adc_a2-hyd_a gene was constructed and LSL02/pUBSC strain with two-enzymes activity was obtained.The whole-cell catalytic activity of LSL02/pUBSC reached 64.6 U/gDCW after induced and cultured for 22 h when soy powder and yeast powder were used as delayed nitrogen sources.Under the optimum conditions of pH 8.0 and 40?,initial substrate concentration of 20 g/L,the catalytic activity of LSL02/pUBSC could last for 12 h to generate 14.32 g/L of D-HPG with the conversion rate of 95%,yield of 82.4%and productivity of 1.19 g/L/h.