Construction Of 1-Deoxynojirimycin High-producing Strains And Optimization On The Fermentation Conditions

1-deoxynojirimycin（DNJ）is a piperidine polyhydroxy alkaloid,which can inhibitα-glucosidase,and it is considered as a potential drug for diabetes mellitus.DNJ can be obtained from a number of sources.As microorganisms have advantages of fast growth and easy culture,and produce DNJ,more researchers have focused on DNJ production through fermentation by microorganisms.However,the production of DNJ by Bacillus amyloliquefaciens HZ-12 is not efficient with low yield,thus it is necessary to engineer this strain for high DNJ yield.In this study,B.amyloliquefaciens strains were engineered by targeting the DNJ biosynthetic pathway and glucose transport pathway,and fermentation optimization of those engineered strains was performed.First,three key genes（gab T1,ykt C1 and gut B1）involved in DNJ synthesis were expressed in B.amyloliquefaciens HZ-12 respectively.The results showed those strains（HZ-12/p HY-gab T1,HZ-12/p HY-ykt C1 and HZ-12/p HY-gut B1）had higher DNJ production than the control strain HZ-12/p HY300（but not significantly higher than HZ-12）.Furthermore,the integrative expression strains（HZ-12ΔXkd G::gab T1,HZ-12ΔXkd E::ykt C1and HZ-12ΔXtm A::gut B1）were recombined,and the DNJ concentration was 19.1%,13.4%and 16.7%higher than the control strain HZ-12.Second,the glucose transport pathway（with key genes glc P1,glc U and pts G）was metabolically engineered that the glucose transporters were deleted in the B.amyloliquefaciens HZ-12 genome.HZ-12△glc P1,HZ-12△glc U and HZ-12△pts G were obtained,and the DNJ concentration of all those gene knockout strains was decimated.Then the gene over expression strains HZ-12/p HY-glc P1,HZ-12/p HY-glc U and HZ-12/p HY-pts G were gained.The DNJ concentration of strain HZ-12/p HY-glc P1increased 19.5%than the control strain HZ-12,while the over expression of glc U and pts G also increased the DNJ concentration compared with strain HZ-12/p HY300.Third,to further improve the DNJ production,the polygenic integration strains HZ-12/TB（Integrating gab T1 and gut B1 into the HZ-12 genome）,HZ-12/TCB（Integrating gab T1,ykt C1 and gut B1 into the HZ-12 genome）and HZ-12/TBP（Integrating gab T1,gut B1 and glc P1 into the HZ-12 genome）were generated,and the results showed that the combination of gab T1,gut B1 and glc P1 on HZ-12 genome led to an increase of 32.3%。In addition,the fermentation medium composition and culture conditions of HZ-12/TBP were optimized,and The optimized medium was as follows（g/L）:malt extract 70,soluble starch 30,K₂HPO₄·3H₂O 1.0,Mg SO₄·7H₂O 0.2,Fe SO₄·7H₂O 0.075,Mn SO₄·H₂O 0.075,culture temperature 37℃,inoculum age 9 h,the inoculation amount2%（v/v）,the broth content 30 m L/250 m L flask,the initial fermentation p H 7.5.the optimized fermentation conditions,the DNJ concentration of HZ-12/TBP reached 315.2mg/L,which increased 26.8%compared to HZ-12 and 88.4%compared to the initial medium.Meanwhile,when 2%lactose and 1%malt extract were added in soybean solid medium,the maximal DNJ concentration reached 1135.6 mg/kg,showing great prospects for DNJ production.