Metabolic Engineering Of Bacillus Amyloliquefa Ciens For High Production Of 1-Deoxynojirimycin

1-Deoxynojirimycin(DNJ)is an effective α-glucosidase inhibitor with several biological activities,such as hypoglycemia,anti-viral,and anti-inflammatory.Its derivative,miglitol,has been served as the clinical medicine for type Ⅱ diabetes.DNJ from microbes shows strong hypoglycemic effect and safety.In addition,it possesses the advantages of low production cost and short production cycle,which attracts many people’s attentions.At present,the studies are mainly focused on the strain selection and medium optimization for the efficient synthesis of DNJ by microorganism s.However,the low yield limits the further application of DNJ.In this study,a DNJ-producing Bacillus amyloliquefaciens HZ-12 in our laboratory was served as the original strain,and metabolic engineering breeding was applied to construct the engineered strain with high production of DNJ.This study explored from the following four parts:1.The effects of carbon regulatory factors CcpC and CcpN on DNJ synthesis was explored.2.For L-glucose is as the substrate for DNJ synthesis,optimizing glucose transport pathway was conducted to promote the synthetic capability of DNJ.3.The precursor supply was enhanced:6-phosphate fructose(6-P-F)is converted from L-glucose,which also provides the carbon backbone for DNJ synthesis.L-glutamic acid is an amino donor that required for DNJ synthesis.Enhancing the supplies of 6-P-F and L-glutamate benefited DNJ synthesis.4.The promoter was screened to improve the expression of DNJ synthase gene cluster and DNJ yield.Firstly,the single-gene of ccpC,and ccpN-deleted strains was separately constructed,and then their DNJ production was checked.The results showed that the DNJ yield of ccpC-deleted strain HZΔccpC reached 284.16 mg/L,and increased by 24.61%compared with the original strain HZ-12(228.03 mg/L).Moreover,the absence of ccpC was found to be beneficial for cell growth.Compared with HZ-12,the DNJ yield of ccpN-deleted strain HZΔccpN was decreased by 32.72%.Subsequently,to optimize the glucose transport pathway,the phosphotransferase systems(PTS)pathway of HZ-12 was inactivated,and then the PTS-independent transport system(NPTS)was strengthened simultaneously.The results showed that optimizing the glucose transport pathway could reduce the yields of by-products acetic acid,acetoin,and 2,3-butanediol,and DNJ yield was enhanced significantly.The engineerd strain HZΔptsG/pHY-glcP was constructed,and its DNJ yield was 20.83%higher than that of original strain HZ-12,and increased by 12.93%compared with HZ/pHY-glcP.In addition,the total amount of by-products of HZΔptsG/pHY-glcP decreased by 8.66%compared with that of HZ-12.Generally,6-P-F involves in multiple metabolic reactions,such as EMP pathway,gluconeogenic pathway,peptidoglycan synthesis,DNJ synthesis,et al.In order to make more 6-P-F into DNJ synthesis,the 6-P-F metabolic node of B.amyloliquefaciens had been modified.Based on strain HZΔccpC,the genes fructose 1,6-bisphosphatase gene glpX in gluconeogenic pathway and 6-phosphate glucose isomerase gene pgi in glycolysis were strengthened via promoter replacement,resulting strains HZΔccpC-PbacA-glpX and HZΔccpC-PbacA-pgi,and the DNJ yields of these strains reached 319.10 mg/L and 304.10 mg/L,which were higher 12.25%and 6.98%than that of the control strain HZΔccpC,respectively.Meantime,the nanR-deleted strain HZΔnanR was constructed by deleting the regulatory factor gene nanR in HZ-12,and the gene is related to peptidoglycan synthesis.DNJ fermentation result showed that the DNJ yield of HZΔnanR was increased by 14.11%,compared with that of the original strain HZ-12.In addition,exogenous addition of L-glutamic acid benefited DNJ synthesis,and the best production was attained at the condition of adding 1.5 g/L L-glutamic acid at 36 h of fermentation time,and DNJ yield was increased by 2.34 folds compared with that of the control.Finally,to improve the expression level of DNJ synthetase gene cluster,different strong promoters(PbacA,PykzA,P43,RBS6 and R5)in our laboratory were applied,and resulted in the engineered strains HZΔccpC/pHY-PbacA-TYB,HZΔccpC/pHY-PykzA-TYB,HZΔccpC/pHY-P43-TYB,HZΔccpC/pHY-RBS6-TYB,and HZΔccpC/pHY-R5-TYB.Among these strains,the DNJ synthetic gene cluster TYB over-expressing strains mediated by promoters P43,RBS6,and R5,showed the better performance,and DNJ yield of HZΔccpC/pHY-P43-TYB,HZΔccpC/pHY-RBS6-TYB,and HZΔccpC/pHY-R5-TYB reached 461.14 mg/L,503.57 mg/L,and 512.50 mg/L,respectively,which were increased by 170.63%,195.53%,and 200.77%compared with that of the control strain HZΔccpC/pHY300,respectively.Subsequently,the DNJ synthesis ability of HZΔccpC/pHY-R5-TYB strain was further explored.L-glutamate addition test was carried out at 36 h,and the DNJ yield of HZΔccpC/pHY-R5-TYB was increased to 1210.70 mg/L,which was the highest yield of DNJ that produced by Bacillus till now.In this study,an engineered strain of B.amyloliquefaciens with high yield of DNJ was constructed via microbial metabolic breeding,which provides a foundation for large-scale preparation of DNJ and its derivatives by microorganisms.In addition,it also is a reference for high-level production of other α-glucosidases.