Production of D-xylonate and organic acid tolerance in yeast: Dissertation

Various organic acids have huge potential as industrial platform chemicals. Biotechnological routes of organic acid production are currently being sought, so that fossil resources and petrochemistry could be replaced with renewable resources. Microbial production of organic acids imposes stresses on the organism and understanding the physiology of micro-organisms which have been genetically engineered to produce an organic acid, can make valuable contributions to the development of production organisms for biorefineries. Production of D-xylonate, an industrial platform chemical with high application potential, was successfully demonstrated in various yeast species. D-xylonate is produced from D-xylose via D-xylono-γ-lactone that can be hydrolysed to D-xylonate spontaneously or with the aid of a lactonase enzyme. Various ways to improve production of D-xylonate in the yeast Saccharomyces cerevisiae, Kluyveromyces lactis or Pichia kudriavzevii as production organisms were successfully applied. The best D-xylonate production was obtained by expression of the D-xylose dehydrogenase encoding gene xylB from Caulobacter crescentus and the highest D-xylonate titre was achieved with P. kudriavzevii that produced 171 and 146 g D-xylonate l-1, at a rate of 1.4 or 1.2 g l-1 h-1, at pH 5.5 and pH 3, respectively. The consequences of D-xylonate production on the physiology of S. cerevisiae were studied in detail, both at population and single-cell level. D-xylonate and D-xylono-γ-lactone were produced and also exported from the cells from the very start of cultivation in D-xylose, even in the presence of D-glucose. There was no apparent preference for export of either compound. However, great amounts of D-xylono-?-lactone and/or D-xylonate was accumulated inside the cells during the production. The D-xylonolactone lactonase encoding gene xylC was co-expressed with the D-xylose dehydrogenase encoding gene xylB (both genes from C. crescentus). This lead to a significant increase in the D-xylonate production rate compared to cells expressing only xylB and showed that accumulation of D-xylonate and protons releases during hydrolysis, was harmful for the cells. The accumulation of D-xylonate led to lost vitality and acidification of the cytosol, as determined by loss of pHluorin (a pH dependent fluorescent protein) fluorescence. This loss of fluorescence was faster in cells co-expressing xylC with xylB compared to cells expressing xylB alone. The decrease in vitality and challenges in export of D-xylonate are major obstacles for D-xylonate production by S. cerevisiae. The excellent D-xylonate producer, P. kudriavzevii also accumulated large amounts of D-xylonate and suffered decreased vitality, especially when D-xylonate was produced at low pH. The stress response to weak organic acids is highly dependent on the properties of the acids and the presence of high concentrations of weak organic acids may lead to lost viability. The role of Pdr12, a membrane transporter, in resistance to weak organic acids was studied and found to be highly dependent on the acid. Deletion of PDR12 led to improved tolerance to formic and acetic acids, a feature that makes this modification interesting for micro-organisms used in biorefining of lignocellulosic hydrolysates that commonly contain these acids. Biotechnological production of D-xylonic acid with yeast clearly has the potential of becoming an industrially applicable process. In order for biotechnological production processes to become economically feasible, biorefinery approaches in which lignocellulosic hydrolysates or other biomass side- or waste streams are used as raw materials need to be employed. This thesis provides new understanding on how production of an organic acid affects the production host and presents novel approaches for studying and increasing the production.