Genomic and transcriptomic analysis of Candida intermedia reveals important genes for xylose utilization
Conference contribution, 2018
The urgency to reduce carbon emissions and to lower our dependence on oil makes it necessary to strive towards a more sustainable bio-based economy, where energy, chemicals, materials and food are produced from renewable resources. Lignocellulosic biomass constitutes a great source of raw material for such a future bio-based economy since it is widely available, relatively inexpensive and do not compete with food and feed production. The pentose D-xylose, the second most prevalent sugar in lignocellulose after glucose, is an underutilized resource, in large due to the inefficient fermentation of this sugar by the most industrially relevant microorganisms (e.g. Saccharomyces cerevisiae). Thus, development of microorganisms that can ferment all lignocellulosic sugars is of foremost importance for economically viable production processes.
Native xylose-utilizing yeasts represent a major source of knowledge and genes for xylose uptake and assimilation that can be transferred to S. cerevisiae. The yeast Candida intermedia is an interesting candidate to characterize further, as it displays a high xylose transport capacity and multiple xylose reductases, of which one appears to prefer NADH over NAPDH. Furthermore, the C. intermedia strain CBS 141442, isolated in the liquid fraction of wheat straw hydrolysate in our laboratory as a contaminant of a xylose fermenting population of S. cerevisiae, is capable of glucose and xylose co-fermentation under certain conditions.
The aim of this study was to elucidate the genetic features that are the basis of the xylose utilization capacity of C. intermedia CBS141442. PacBio sequencing and de novo assembly of the genome revealed a haploid yeast with a genome size of 13.2 Mb and a total of 5936 protein-coding genes spread over seven chromosomes. In order to gain insight on the genes involved in the utilization of xylose, we analysed the changes in the transcriptome of C. intermedia CBS141442 during growth in xylose and glucose (as reference condition). Cells were collected in mid-exponential phase at the maximum growth rate when no metabolites were accumulating. The total RNA was extracted and cDNA libraries were prepared after polyA selection. Each sample was sequenced in an Illumina HiSeq2500 system with an average cover of 5-20 million reads. The analysis of the differential expression data lead to the identification of two new genes potentially encoding xylose transporters and no less than three xylose reductases genes with different expression patterns. The xylose reductase genes were heterologously expressed in S. cerevisiae to determine their co-factor preferences and substrate specificities. Whereas two of them are strictly NADPH-dependent, the third can use both co-factors and shows preference for NADH. The heterologous expression of this gene can improve the capacity of S. cerevisiae to ferment xylose, and thus contribute to a more efficient use of lignocellulosic biomass.