Single cell and in vivo analyses elucidate the effect of xylC lactonase during production of D-xylonate in Saccharomyces cerevisiae.

D-xylonate is a potential platform chemical which can be produced by engineered Saccharomyces cerevisiae strains. In order to address production constraints in more detail, we analysed the role of lactone ring opening in single cells and populations. Both D-xylono-γ-lactone and D-xylonate were produced when the Caulobacter crescentus xylB (D-xylose dehydrogenase) was expressed in S. cerevisiae, with or without co-expression of xylC (D-xylonolactonase), as seen by (1)H NMR. XylC facilitated rapid opening of the lactone and more D-xylonate was initially produced than in its absence. Using in vivo(1)H NMR analysis of cell extracts, culture media and intact cells we observed that the lactone and linear forms of D-xylonic acid were produced, accumulated intracellularly, and partially exported within 15-60min of D-xylose provision. During single-cell analysis of cells expressing the pH sensitive fluorescent probe pHluorin, pHluorin fluorescence was gradually lost from the cells during D-xylonate production, as expected for cells with decreasing intracellular pH. However, in the presence of D-xylose, only 9% of cells expressing xylB lost pHluorin fluorescence within 4.5h, whereas 99% of cells co-expressing xylB and xylC lost fluorescence, a large proportion of which also lost vitality, during this interval. Loss of vitality in the presence of D-xylose was correlated to the extracellular pH, but fluorescence was lost from xylB and xylC expressing cells regardless of the extracellular condition.