Metabolic Engineering Of Bacillus Subtilis For 3-hydroxypropionic Acid Production

3-Hydroxypropionic acid(3-HP),a commercially valuable platform chemical,has a wide range of industrial applications.Biosynthesis of 3-HP is highly efficient,green and sustainable.In this work,the heterologous mcr gene(encode malonyl-CoA reductase)and accADBC gene(encode acetyl-CoA carboxylase)were introduced into Bacillus subtilis,successfully built the malonyl-CoA pathway to product 3-hydroxypropionic acid.This is the first report successfully introduce the malonyl-CoA pathway in Bacillus subtilis.The low-copy plasmids pHP13 and middle-plasmid pEBS were chosen to overexpress the mcr gene of the Chloroflexus aurantiacus,however no 3-HP was detected in recombined B.subtilis.The enzyme assay showed that MCR was successfully expressed.Then the accADBC gene of E.coli K-12 MG1655 was integrated into the chromosomal alsS locus,however,3-HP could neither be detected.To integrate one copy mcr gene at the chromosomal amyE locus and introduce the plasmid pHP13 harboring the accADBC gene into BSF1(B.subtilis 168△upp).The resulted strain BSF10 could produce trace 3-HP(1 mg/L),which was further improved to 84.7 mg/L after optimization of the culture medium.Based on the optimized culture medium,the 3-HP fermentation was re-evaluated and the results showed that the introduction of the mcr gene alone could activate the malonyl-CoA pathway to produce 3-HP.Moreover,the malonyl-CoA production was the speed limit step for the route.