Characterization and engineering of sugar transporters in the yeast Kluyveromyces marxianus for biotechnological applications

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Donzella, Lorena
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University College Cork
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Sustainable and efficient use of lignocellulosic hydrolysates in yeast-based biorefineries requires the simultaneous consumption of all the sugars present in this raw material, hexoses and pentoses. This represents a key bottleneck because of the low number of specific pentose transporters identified and the slow rate of pentose uptake into the yeast cell when glucose is present. Kluyveromyces marxianus is a non-conventional yeast that has attracted considerable attention due to its traits of industrial interest, like the ability of naturally assimilating pentoses, high thermotolerance and fast growth rate. In this thesis, the main goal was to investigate and engineer pentose uptake in K. marxianus to obtain a transporter that would allow co-consumption of glucose and xylose for the application in biorefineries. In order to identify novel pentose transporters, a K. marxianus screening platform (ΔPT) was constructed by deleting 12 genes that encoded transporters of the major facilitator superfamily (MFS). Using this ΔPT platform, which was unable to grow on pentoses, we identified several native K. marxianus transporters that recovered the native strain’s ability to grow on either xylose and/or arabinose. Kinetic studies on the most promising candidates using 14C-labelled sugars were carried on. One member of these transporters showed high affinity for both xylose and glucose, though the affinity for glucose was still 150-fold higher than for xylose. Using in silico analysis we selected target amino acids of this transporter for site-directed mutagenesis in an attempt to convert it into a specific xylose transporter. Several mutants were constructed and characterised by radioactive uptake and growth assays in bioreactors on mixture of sugars. One of these mutants decreased its ability to transport glucose but still transported xylose with even higher affinity kinetics. The successful reprogramming of this transporter to increase its preference for xylose over glucose was used to facilitate efficient co-consumption of hexose and pentose sugars in a novel K. marxianus strain, significantly shortening the time of the whole process. Keywords: yeast, sugar transporters, xylose, biorefineries, Kluyveromyces spp.
Yeast , Sugar transporters , Xylose , Biorefineries , Kluyveromyces spp.
Donzella, L. 2022. Characterization and engineering of sugar transporters in the yeast Kluyveromyces marxianus for biotechnological applications. PhD Thesis, University College Cork.