Construction And Optimization Of Synthetic Pathway Of 3,4-dihydrobutyric Acid In Recombinant Escherichia Coli

3,4-Dihydroxybutyric acid(3,4-DHBA)is a multi-functional C4 platform compound with applications in the synthesis of pharmaceuticals and materials.Until now,few studies were on 3,4-DHBA.3,4-DHBA is mainly produced by chemical methods,but most of them have disadvantages such as low yield,by-products,harsh reaction conditions,high cost,and environmental pollution.Therefore,it is urgent to establish a green and efficient synthetic pathway of 3,4-DHBA.A biosynthetic pathway to produce 3,4-DHBA from xylose was established in E.coli,but the yield is still low.Inspired by this study,a four-step biosynthetic pathway of 3,4-DHBA was constructed in E.coli in our study.Strategies such as screening of enzymes with high activities,disruption of shunt metabolism,fusion expression of rate-limiting enzymes and optimization of catalytic reaction conditions were used for improving the 3,4-DHBA production of the engineered E.coli.The main contents of this study are as follows:1.Screening of enzymes and construction of biosynthetic pathway of 3,4-DHBA in E.coli:Based on the literatures and NCBI database,the enzymes catalyzed D-xylose to 3,4-DHBA were screened according to their expression in E.coli BL21(DE3)and catalytic activities.The glucose dehydrogenase(GDH)from B.subtilis,D-xylonate dehydratase(YagF)from E.coli,benzoylformate decarboxylase(PpMdlC)from P.putida and putative NAD+-dependent aldehyde dehydrogenase(Gox0499)from G.oxydans were selected to catalyze the dehydrogenation of D-xylose,dehydration of D-xylonate,decarboxylation of 2-keto-3-deoxy-D-xylonate and dehydrogenation of 3,4-dihydroxybutanal,respectively.GDH,YagF,PpMdlC,and Gox0499 were co-expressed in E.coli BL21(DE3),and the obtained recombinant strain named as E-C4 could produce 3,4-DHBA with titer of 3.03±0.22 g/L and the space-time yield of 1.224 g/(L*d).In this study,dihydroxy-acid dehydratase(CfXylD)from C.flavus and dihydroxy-acid dehydratase(CsXylD)from C.segnis were firstly demonstrated capable of catalyzing D-xylonate to 2-keto-3-deoxy-D-xylonate.The putative NAD+-dependent aldehyde dehydrogenase(Gox0499 and Gox1122)from G.oxydans showed the oxidative activity toward 3,4-dihydroxybutanal.2.Optimization of the 3,4-DHBA production in recombinant E.coli by metabolic engineering strategies:The xylose isomerase gene(xylA),glyoxylate reductase genes(ghrA and ghrB),alcohol dehydrogenase gene(adhP)were deleted in the recombinant E-C4 using CRISPR-Cas9 system in order to improve the metabolic flux in synthesis of 3,4-DHBA.Then,the rate-limiting enzymes(PpMdlC)and YagF were fusion-expressed in E.coli to establish a shorter substrate tunnel via the spatial proximity effects,and the engineered strain named as E-05-F4 was obtained,which produced 7.71±0.18 g/L of 3,4-DHBA,affording the space-time yield of 3.864 g/(L*d).3.Optimization of culture and catalytic conditions for the engineered strain E-05-F4:The culture and catalytic conditions,such as the IPTG concentration and induction temperature,as well as the substrate concentration,cofactor(NAD+,TPP)concentration,Mg2+ concentration and reaction pH,were optimized.Titer and the space-time yield of 3,4-DHBA were increased to 10.16±0.32 g/L and 5.208 g/(L*d),respectively.