High yield of mannosylglycerate production by upshock fermentation and bacterial milking of trehalose-deficient mutant Thermus thermophilus RQ-1

Abstract A production process, using upshock fermentation and osmotic downshock, for the effective production/excretion of mannosylglycerate (MG) by the trehalose-deficient mutant of the strain Thermus thermophilus RQ-1 has been developed. In the first phase of fed-batch fermentation, the knockout mutant was grown at 70°C on a NaCl-free medium. After the culture reached the end of the exponential growth phase, upshift in temperature and NaCl concentration was applied. The temperature was increased to 77°C, and NaCl was added up to 3.0% and kept constant during the second phase of fermentation. Although this shift in cultivation parameters caused a dramatic drop of cell density, a significant improvement in accumulation of MG up to 0.64 µmol/mg protein compared to batch fermentations (0.31 µmol/mg protein) was achieved. A total yield of 4.6 g MG/l of fermentation broth was obtained in the dialysis bioreactor with a productivity of 0.29 g MG l-1 h-1. The solute was released from the harvested biomass by osmotic downshock using demineralized water at 70°C. More than 90% of the intracellularly accumulated solute was recovered from the water fraction. The process was very efficient, as hyperosmotic shock, release of the solute, and reiterative fed-batch fermentation could be repeated at least four times

Mannosylglycerate is essential for osmotic adjustment in Thermus thermophilus strains HB27 and RQ-1

Abstract We disrupted the mpgS encoding mannosyl-3-phosphoglycerate synthase (MpgS) of Thermus thermophilus strains HB27 and RQ-1, by homologous recombination, to assess the role of the compatible solute mannosylglycerate (MG) in osmoadaptation of the mutants, to examine their ability to grow in NaCl-containing medium and to identify the intracellular organic solutes. Strain HB27 accumulated only MG when grown in defined medium containing 2% NaCl; mutant HB27M9 did not grow in the same medium containing more than 1% NaCl. When trehalose or MG was added, the mutant was able to grow up to 2% of NaCl and accumulated trehalose or MG, respectively, plus amino acids. T. thermophilus RQ-1 grew in medium containing up to 5% NaCl, accumulated trehalose and lower amounts of MG. Mutant RQ-1M1 lost the ability to grow in medium containing more than 3% NaCl and accumulated trehalose and moderate levels of amino acids. Exogenous MG did not improve the ability of the organism to grow above 3% NaCl, but caused a decrease in the levels of amino acids. Our results show that MG serves as a compatible solute primarily during osmoadaptation at low levels of NaCl while trehalose is primarily involved in osmoadaptation during growth at higher NaCl levels