| Fengycin is a compound composed of hydrophobicβ-fatty acid chain and hydrophilic peptide ring,which is fermented and produced by Bacillus spp.It has very significant antifungal activity.However,the wild-type Bacillus subtilis 168 does not have the ability to synthesize fengycin from glucose.In this paper,synthetic biology methods are used to create artificial cells that use glucose to synthesize fengycin,and a genome-scale metabolic network model is constructed for artificial cells,and the strain is modified to increase the production of fengycin under the guidance of model simulation prediction.First,in order to obtain a stable Bacillus subtilis fengycin synthetic strain,a gene expression module p HP13-P43-sfp-deg Q consisting of the P43 promoter gene,the deg Q gene of Bacillus subtilis 168,and the sfp gene of Bacillus amyloliquefaciens FZB42 was constructed in this study,verified the correctness of the expression module by measuring the size and direction of the gene sequence.Then it was introduced into the original strain of Bacillus subtilis 168 to construct an engineered strain of Bacillus subtilis BSP000 that can produce fengycin.The stability of the engineered strain was evaluated through experiments,and the results showed that it is a cell with high-quality performance.The shake flask yield of the engineered strain BSP000 reached 26.44±1.98 mg/L fengycin under micro-oxygen conditions.Secondly,the gene protein reaction list of Bacillus subtilis 168 was compiled from databases such as NCBI and KEGG.After manual refining,a genome-scale metabolic network model of the BSP000 strain was obtained.The specific sugar uptake rate and the synthesis rate of fengycin of the strain were determined experimentally and used as the constraint condition of the model.The biomass of the strain was taken as the objective function,and the maximum specific growth rate of the strain was calculated as 0.1281 h-1through simulation.Compared with the experimentally determined maximum specific growth rate of 0.1216 h-1,the error is within 6%,indicating that the reliability of the model is relatively high.At the same time,the predicted targets were sorted and analyzed according to the f PHvalue,and the target genes acc A,cyp C and gap A were screened out as amplification targets.Finally,the acc A,cyp C and gap A genes were overexpressed in the strain BSP000.Compared with the original strain,the yield of fengycin overexpressing acc A,cyp C and gap A genes increased by 56.4%,46.6%,and 20.46%,respectively.In order to further improve the synthetic ability of the strain fengycin,a comprehensive study on the co-expression of acc A and cyp C genes and the co-expression of acc A,cyp C and gap A genes was carried out on the strain BSP000.The acc A and cyp C gene co-expression strain BSP002 fengycin yield reached53.38 mg/L,an increase of 101.89%.By co-expressing the genes acc A,cyp C and gap A at the same time,the yield of the recombinant strain BSP003 fengycin reached59.87 mg/L,which was nearly 2.3 times that of the BSP000 strain fengycin. |
