Dicarboxylic acids from xylose, using natural and engineered hosts

Abstract: Chemical building blocks for plastics can be produced from renewable biomass feedstocks using microbial production organisms, such as yeast or bacteria, in a biorefinery. One class of chemical building blocks that are suitable for production of biobased and biodegradable plastics are dicarboxylic acids, e.g. succinic acid. In order to avoid competition with food and feed production it is desirable to use hydrolysates of lignocellulosic feedstocks which often not only contain hexose sugars but also pentoses, out of which xylose is the most common. One example of such a feedstock is spent sulphite liquor (SSL), a side stream from sulphite pulping of Eucalyptus, which is rich in xylose. In this thesis, microbial production of dicarboxylic acids from xylose-rich feedstocks has been studied using different host organisms. The natural succinic acid producing bacterium Actinobacillus succinogenes was found able to produce succinate from a xylose rich synthetic model medium mimicking sugar com- position in SSL, at a titer of 31 g L-1 and yield of 0.71 g g-1. In addition, A. succinogenes was tested for tolerance towards inhibiting by-products along with a related succinate producer, Basfia succiniciproducens. Of the by-products, both organisms were found to be most sensitive to formate (18-22 g L-1), while high concentration of acetate (38 g L-1) and succinate (55 g L-1) were tolerated. Succinate production with A. succinogenes was also tested in SSL, and titers above 22 g L-1 of succinate were obtained in fed-batch cultivations. A strain of Saccharomyces cerevisiae engineered for xylose utilization and formation of dicarboxylic acids was assessed and found rather tolerant to SSL even at acidic condi- tions. The relative distribution between malate and succinate was affected by cultivation conditions, with succinate strongly favoured at carboxylating conditions at high pH. Genes encoding enzymes of the Weimberg pathway, an orthogonal xylose degradation pathway, were introduced in S. cerevisiae. The complete pathway was not functional and growth on xylose was not obtained. However, the intermediate compound xylonate was formed at close to stoichiometric yields. In addition, Caulobacter crescentus, the natural host of the Weimberg pathway, was characterised. Activity of the Weimberg pathway was found during growth on both xylose and arabinose, but not on glucose. Interestingly, high yields of α-ketoglutarate (up to 0.43 g g-1) were formed during growth on xylose.