The Study Of Microbial Producing Of 2-Hydroxyisobutyric Acid (2-HIBA)

2-Hydroxyisobutyric acid (2-HIBA) is an important hydroxy carboxylic acid containing tertiary carbon atom.2-HIBA could be converted to a variety of pharmaceutical intermediates and polymers, thus drawing much industrial attentions for its potential applications in many fields. Due to it rare structure, no large scale industrial production of 2-HIBA was reported so far. The chemical synthesis has lots of disadvantages including high production cost, harsh producing conditions, and environmental pollutions. Based on the approach of synthetic biology, molecular biology and metabolic engineering, we may re-build a biological producing pathway using microorganisms.Here we studied the possibility of microbial producing of 2-hydroxyisobutyrate from renewable carbon sources. Plasmid pEnBHTI was constructed to expressing bktB?hbd?icmA?icmB and tesB genes in Escherichia coli, which can help to construct a 2-HIBA producing pathway from acetyl-CoA as precursor through four steps. We found that the recombinant E. coli harboring plasmid pEnBHTI produced 3-hydroxybutyrate from glucose as carbon source in shake flasks. However, no 2-HIBA was detected in the cultures. The results showed here indicated that the expression of bktB?hbd and tesB was successful to drive the carbon metabolism to 3-hydroxybutyrate production, but the expression of icmAB might be too low to convert 3-hydroxybutyryl-CoA to 2-hydroxyisobutyryl-CoA, or the tesB activity towards 2-hydroxyisobutyryl-CoA is too low to convert it into the final product.Then we proposed another 2-hydroxyisobutyrate producing pathway based on the fatty acid metabolism in Aeromonas hydrophila. Plasmid pMCSV12 was constructed to express icmA and icmB in A.hydrophila, thus build a 2-hydroxyisobutyrate producing pathway from the intermediates of fatty acid oxidation pathway. The recombinate A. hydrophila harboring plasmid pMCSV12 could produce trace amounts of 2-HIBA from lauric acid. Based on the results, we have further optimized the carbon sources, the cultivation process in shake flasks, and finally 46 mg/L 2-HIBA could be produced.Our results provide a feasible approach for the biosynthesis of 2-hydroxyisobutyrate from remewable carbon sources.