Increasing the production of (R)-3-hydroxybutyrate in recombinant Escherichia coli by improved cofactor supply
BACKGROUND Halomonas boliviensis is a halophilic bacterium that is included in the γ-Proteobacteria sub-group, and is able to assimilate different types of carbohydrates. H. boliviensis is also able to produce poly(3-hydroxybutyrate) (PHB) in high yields using glucose as the carbon precursor. Accumulation of PHB by microorganisms is induced by excess of intracellular NADH.The genome sequences and organization in microorganisms should be the result of evolution and adaptation influenced by mutation, gene duplication, horizontal gen transfer (HGT) and recombination. Furthermore, the nearly neutral theory of evolution sustains that genetic modification of DNA could be neutral or selected, albeit most mutations should be at the border between neutrality and selection, i.e. slightly deleterious base substitutions in DNA are followed by a slightly advantageous substitutions. RESULTS This article reports the genome sequence of H. boliviensis. The chromosome size of H. boliviensis was 4 119 979 bp, and contained 3 863 genes. A total of 160 genes of H. boliviensis were related to carbohydrate transport and metabolism, and were organized as: 70 genes for metabolism of carbohydrates; 47 genes for ABC transport systems and 43 genes for TRAP-type C4-dicarboxylate transport systems. Protein sequences of H. boliviensis related to carbohydrate transport and metabolism were selected from clusters of orthologous proteins (COGs). Similar proteins derived from the genome sequences of other 41 archaea and 59 bacteria were used as reference. We found that most of the 160 genes in H. boliviensis, c.a. 44%, were obtained from other bacteria by horizontal gene transfer, while 13% of the genes were acquired from haloarchaea and thermophilic archaea, only 34% of the genes evolved among Proteobacteria and the remaining genes encoded proteins that did not cluster with any of the proteins obtained from the reference strains. Furthermore, the diversity of the enzymes derived from these genes led to polymorphism in glycolysis and gluconeogenesis. We found further that an optimum ratio of glucose and sucrose in the culture medium of H. boliviensis favored cell growth and PHB production. CONCLUSIONS Results obtained in this article depict that most genetic modifications and enzyme polymorphism in the genome of H. boliviensis were mainly influenced by HGT rather than nearly neutral mutations. Molecular adaptation and evolution experienced by H. boliviensis were also a response to environmental conditions such as the type and amount of carbohydrates in its ecological niche. Consequently, the genome evolution of H. boliviensis showed to be strongly influenced by the type of microorganisms, genetic interaction among microbial species and its environment. Such trend should also be experienced by other prokaryotes. A system for PHB production by H. boliviensis that takes into account the evolutionary adaptation of this bacterium to the assimilation of combinations of carbohydrates suggests the feasibility of a bioprocess economically viable and environmentally friendly.