Study On The Heterologous Extension Pathway Of L-proline In Bacillus Subtilis

Microorganism can produce trans-4-hydroxyproline（HYP）from free L-proline by introducing exogenous proline-4-hydroxylase gene（p4h）.However,the precursor L-proline provided by host is limited,and may not be able to meet the requirments of HYP synthesis stably.Although exogenous L-proline supplementation is beneficial to synthesis for HYP,it is not conducive to industrial production.In this paper,we constructed the HYP biosynthesis pathway in Bacillus subtilis.On the basis of this,we did some investigations on related genes of L-proline synthesis pathway,and the productivity succeed improved,which laid the foundation for the synthesis of HYP more efficiency.The main contents and conclusions are as follows:（1）After transforming constitutive plasmid p MA5-P4H and inducible plasmid p HY-Bs.xyl-P4H into WB600,we got recombinant Bacillus subtilis WB600/p MA5-P4H and WB600/p HY-Bs.xyl-P4H,respectively.After fermentation for 48 h,the HYP content of WB600/p MA5-P4H in fermentation broth was 24.13 mg·L^-1.With final concentration of 1.5%xylcose supplementation,WB600/p HY-Bs.xyl-P4H accumulated 80.59 mg·L^-1 and 125.51mg·L^-1 HYP,respectively,when fermentated 48 h and 96 h.（2）According to the proline sysnthesis pathway with glutamic acid as precursor of Bacillus subtilis,metabolic engineered strains were constructed to enhance proline synthesis and reduce glutamate decompose flux,and to investigate the effects of different modification methods on intracellular and extracellular proline accumulation.Results showed,the measures of efficiently improving extracellular and intracelluar proline accumulation including the synthesis gene overexpression or the glumate shunt gene deletion,respectively.Then recombinant Bacillus subtilis WB604[Δgln A,gln A::（P43-pro B-Neo）]was constructed,which based on the results of single gene modification.The extracellular proline content and unit cell yield were 7.15 and10.41 times higher than that of the original strain,and the intracellular free proline content was4.86 times of the original strain.Further study showed that,under 5%Na Cl stress,the intracellular free proline concentration of the experimental strains all increased compared with non stress,and reached a relative balance.The extracellular proline content and unit cell yield of WB604 were 5.51 and 7.69 times of the original strain,respectively,and its biomass was1.26 times of recombinant strain WB603.In addition,the biomass of recombinant strain WB601with pro B overexpressing and WB602 with pro A overexpressing were 1.40 and 2.05 times higher than that under non stress conditions,respectively.These results indicated that overexpression of pro B and pro A could enhance salt tolerance of cell.（3）The HYP producing strain WB604/p HY-Bs.xyl-P4H was constructed based on recombinant strain WB604.The results of shake flask fermentation showed,with 10 m M glutamine（Gln）as the initial supplementation,the content of extracellular L-proline and unit cell yield were 698.54 mg·L^-1 and 233.70 mg·g^-1 DCW at 12 h,which were 4.17 and 4.23 times of the control WB600/p HY-Bs.xyl-P4H,respectively.In addition,the HYP concentration reached 143.02 mg·L^-1 after incubation for 96 h,which was increased by 30.41%compared with no Gln supplementation.After which,we increased the supplementation of Gln,the HYP concentration of supernatant without significant change.Compared with control,the concentration of HYP were increased by 13.95%and 31.09%,which with Gln supplementation and none,respectively.（4）The optimization of carbon sources and organic nitrogen sources in the shake flask fermentation of engineered strain WB604/p HY-Bs.xyl-P4H was carried out.When the initial carbon source was 15 g·L^-1 glucose,and peptone:yeast extract was 1:2,the concentration of HYP in fermentation broth reached 448.57 mg·L^-1,which was 3.14 times higher than that before optimization.