Identification of novel pentose transporters in Kluyveromyces marxianus using a new screening platform

Abstract

The capacity of yeasts to assimilate xylose or arabinose is strongly dependent on plasma membrane transport proteins. Because pentoses comprise a substantial proportion of available sugars in lignocellulosic hydrolysates, their utilisation is centrally important for the development of second generation biorefineries. Relatively few native pentose transporters have been studied and there is intense interest in expanding the repertoire. To aid the identification of novel transporters, we developed a screening platform in the native pentose-utilising yeast Kluyveromyces marxianus. This involved the targeted deletion of twelve transporters of the major facilitator superfamily (MFS) and application of a synthetic biology pipeline for rapid testing of candidate pentose transporters. Using this K. marxianus ΔPT platform, we identified several K. marxianus putative xylose or arabinose transporter proteins that recovered a null strain's ability to growth on these pentoses. Four proteins of the HGT-family were able to support growth in media with high or low concentrations of either xylose or arabinose, while six HXT-like proteins displayed growth only at high xylose concentrations, indicating solely low affinity transport activity. The study offers new insights into the evolution of sugar transporters in yeast and expands the set of native pentose transporters for future functional and biotechnological studies.

Keywords: biotechnology; evolution; major facilitator superfamily; pentose; screen; yeast.

Figure 4.

The K. marxianus STL1 protein, KMAR_60179, can mediate both glycerol and xylose transport. The two K. marxianus genes with homology to S. cerevisiae STL1, and Sc_STL1, were individually expressed on a plasmid in K. marxianus ΔPT. Following pre-growth on 2% lactose, strains were plated in serial dilution on agar plates containing 2% glycerol or 2% or 0.1% xylose as indicated. Plates were photographed after 96 h of growth at 30°C.

Figure 5.

Application of K. marxianus ΔPT to compare the performance of putative low and high affinity pentose transporters. Genes encoding the candidate high affinity (KMAR_10531) and low affinity (KMAR_60179) pentose transporters were expressed from the TEF1 promoter on the TU-DO-G418 plasmid in K. marxianus ΔPT. Following pre-growth on 2% lactose, the strains carrying KMAR_10531 (green squares), KMAR_60179 (orange circles), or empty plasmid (black diamonds) were inoculated in MM with 2% xylose (A) or arabinose (B) and grown at 30°C. Growth (solid symbols) and xylose or arabinose concentration (open symbols) were measured for 120 h.

Figure 1.

Phylogenetic comparison of potential MFS transporters between K. marxianus, K. lactis and S. cerevisiae. Bioinformatic analysis identified 27 predicted proteins in K. marxianus NBRC 1777 that satisfied the criteria of being potential sugar transporters (MFS family with 11 or 12 transmembrane domains) (in green). Homologous sequences in S. cerevisiae and K. lactis (in black and purple, respectively) were retrieved and phylogenetic analysis carried out using MEGA6/MUSCLE. The tree is derived from maximum-likelihood analysis with 600 times bootstrapping. The expanded KHT/HXT and HGT families in K. marxianus are highlighted with yellow and pink squares, respectively. Labelling is used to indicate the known or predicted function of proteins where data are available. The labels A—D mark Kluyveromyces proteins of unknown function that lack clear orthologues in S. cerevisiae. Sc_Fsy1 is a fructose transporter that was acquired by horizontal gene transfer in wine strains and is not a native S. cerevisiae protein.

Figure 2.

The K. marxianus platform strain ΔPT is unable to grow on the pentose sugars, xylose and arabinose. A. Growth of mutants on MM containing various sugars (2%). The ∆hgt mutant failed to grow on arabinose, indicating that all the functional arabinose transporters are encoded within the HGT cluster. The double mutant ∆hgt∆kht grew slowly on xylose indicating both the presence of at least one xylose transporter in the KHT/HXT cluster and at least one other xylose transporter elsewhere in the genome. The ΔPT strain, which also deleted KMAR_60179 in the ∆hgt∆kht background, failed to grow on either xylose or arabinose indicating the inactivation of all functional pentose transporters. B-CK. marxianus NBRC 1777 wild-type (green diamonds), ∆hgt (blue triangles), ∆kht (orange squares), ∆hgt∆kht (red circles) and ΔPT (black triangles) were pre-grown on lactose medium and inoculated into 2% xylose or arabinose and cultivated in flasks at 30°C for 96h.

Figure 3.

Identification of pentose transporters in K. marxianus ∆PT. Candidate native K. marxianus pentose transporters were individually expressed on a plasmid in the screening platform K. marxianus ΔPT. Strains were pre-grown on 2% lactose and then plated in serial dilution on agar plates containing either 2% or 0.1% xylose or arabinose as indicated. Plates were photographed after 96h of growth at 30°C.