Bioproduction of 3-hydroxypropionaldehyde and 3-methylcatechol by organic solvent-tolerant bacteria

Abstract

In the present day, biotechnological production of industrial chemicals has become significantly important and has several advantages over the chemical processes. Nevertheless, the toxicity of substrates and/or hydrocarbon products may limit the survival and catalytic activity of biocatalyst. Organic solvent-tolerant (OST) bacteria are bacteria that can thrive in the presence of high concentrations of hydrocarbon and solvent as a result of their tolerance mechanisms. Therefore, OST bacteria and a suitable bioproduction system were explored for hydrophobic and hydrophilic chemical bioproduction in this study. Firstly, for bioproduction of 3-methylcatechol (3MC) as a hydrophobic chemical, it was carried out by a mutant OST Pseudomonas putida T-57 strain TODE1 under a two-phase system, where 1-decanol was used as a second phase. The supplementation with either glycerol, Mn²⁺ or Fe²⁺ significantly enhanced the 3MC productivity. Based upon the response surface methodology-based central composite design optimized conditions, the overall 3MC production was produced up to 34 mM, which is the highest yield reported so far. Secondly, for bioproduction of 3-hydroxypropionaldehyde (3-HPA) as a hydrophilic chemical, it was explored by hydrophilic OST bacteria under a single-phase system. Seven hydrophilic OST bacteria were isolated based on their tolerance capability to various types of toxic hydrophilic organic solvent, including acetonitrile and propionaldehyde. To apply these bacteria as a bioproduction host, the transformation system and genetic manipulation of hydrophilic OST bacteria were established for 3-HPA bioproduction. Despite the fact that effect of promoter, ribosome binding site and source of structural gene were taken into consideration and modified, a low titer of 3-HPA was obtained when either Bacillus subtilis BKS2-P2/3 or P. putida T-57 was used as a bacterial host. Because the information of tolerance mechanisms of hydrophilic OST bacteria is scarce, but it is important for further applications. The mechanisms study was conducted with the bacterium with high tolerance to acetonitrile, Exiguobacterium sp. SBH81. The results may suggest that its tolerance mechanisms rely on increase of cell size to lower solvent partitioning into cells and the extrusion of solvents through the efflux system (RND and MATE system). In conclusion, this study provides examples of the development of bacterial platforms for hydrophobic and hydrophilic chemical production using genetically engineered OST bacteria. The important roles of OST mechanism of the biocatalytic host, molecular mechanism for gene expression as well as bioproduction process described in this study would be important information for further development of chemical production by biotechnological processes.