Study On Biosynthesis Of (L)-β-Hydroxyisobutyric Acid

(L)-β-hydroxyisobutyric acid is an important precursor for the synthesis of vitamin E and other drugs.At present,chemical synthesis is the main method for the synthesis of(L)-β-hydroxyisobutyric acid.Its product is a racemate of both(L)-β-hydroxyisobutyric acid and(D)-β-hydroxyisobutyric acid,making the later separation of the product extremely difficult.It has been reported that(L)-β-hydroxyisobutyric acid is an intermediate in the metabolic pathway of valine in certain microorganisms,and due to the necessity of metabolic synthesis of microorganisms,the resulting β-hydroxyisobutyric acid has a single configuration,which can be greatly simplify the downstream separation process.This study explored the method for the detection of(L)-β-hydroxyisobutyric acid and the synthesis of(L)-β-hydroxyisobutyric acid was carried out in two aspects.The first was the screening of natural strains capable of synthesizing(L)-β-hydroxyisobutyric acid,and(L)-β-hydroxyisobutyric acid was produced by microbial fermentation.The second is to synthesize(L)-β-hydroxyisobutyric acid by constructing engineering bacteria.In this study,isobutyric acid was used as the sole carbon source,and strains capable of synthesizing(L)-β-hydroxyisobutyric acid were preliminary screened by a single variable method.In microbial fermentation,isobutyric acid was used as a substrate and the product was detected by GC.It was found that two strains of Pseudomonas putida and Yeast indeed synthesize(L)-β-hydroxyisobutyric acid.On the other hand,the genes of the genus of the two strains were compared with the genes in the gene bank,and the enzymes required for the synthesis of(L)-β-hydroxyisobutyric acid with isobutyric acid as the substrate were cloned,They are isobutyrate-CoA ligase,acyl-CoA dehydrogenase,enoyl-CoA hydratase,and 3-hydroxyisobutyryl-CoA hydrolase,respectively.Then the four enzymes were ligated to the expression vector pET28 a harbouring His-tag.And the four enzymes were successfully expressed under suitable induction conditions.In the process of biocatalysis,isobutyric acid as a substrate,Yeast and Pseudomonas putida as catalysts respectively.The yield of(L)-β-hydroxyisobutyric acid was maximized at 18 th hour.Among them,the product accumulation of Yeast at the 18 th hour was 4.97 g/L,and that of Pseudomonas putida at the 18 th hour was 7.26 g/L,the conversion rate is 19.9% and 29.0% respectively.The yield of(L)-β-hydroxyisobutyric acid of Pseudomonas putida is higher than that of the Yeast.