In order to attain the desired high product titers, biocatalysts must be sup­plied with high levels of sugars. These high sugar levels in turn create osmotic stress, which is compounded by the desire to use simple mineral salts media. Osmolytes such as trehalose, betaine, proline, and glutamate help bacteria maintain appropriate cell turgor and volume despite changes in extracellu­lar osmolality; osmolyte uptake and synthesis are reviewed in [80]. Increased activity of the native trehalose synthesis pathway elevated the growth rate of E. coli W3110 in the presence of various osmotic stress agents [81], and betaine supplementation increased the production of D-lactic acid by E. coli SZ132 in NBS mineral salts media [42]. A combination of betaine supple­mentation and elevated trehalose synthesis increased the tolerance of W3110 to xylose, glucose, sodium lactate, and sodium chloride more than betaine supplementation or elevated trehalose synthesis alone [82].

As described above, the poor performance of ethanologenic E. coli strain KO11 in minimal media has been attributed to NADH-mediated inhibition of citrate synthase, limiting the availability of glutamate, a protective os — molyte [33]. Additionally, the increased performance of LY01 relative to KO11 can be partly attributed to increased osmolyte production and uptake [83]. NMR analysis confirmed that intracellular pools of glutamate, trehalose, and betaine are very low in KO11 during anaerobic growth relative to aerobic growth in the same medium [84]. Growth and ethanol production of KO11 was increased by supplementation with various osmolytes, demonstrating that the glutamate limitation is related to osmotic stress, not to a specific metabolic demand for glutamate [84].

4.3