University of Groningen
Engineering endogenous hexose transporters in Saccharomyces cerevisiae for efficient
D-xylose transport
Nijland, Jeroen
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Publication date: 2019
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Nijland, J. (2019). Engineering endogenous hexose transporters in Saccharomyces cerevisiae for efficient D-xylose transport. University of Groningen.
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193
SUMMARY
192 BIBLOGRAPHY
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Treha-lose Metabolism by Saccharomyces cerevisiae: NTH2 Encodes a Func-tional Cytosolic Trehalase, and Deletion of TPS1 Reveals Ath1p-Depen-dent Trehalose Mobilization. Appl Environ Microbiol 74:605–614. 175. De Virgilio C, Bürckert N, Bell W, Jenö P, Boller T, Wiemken A. 1993.
Disruption of TPS2, the gene encoding the 100-kDa subunit of the treha-lose-6-phosphate synthase/phosphatase complex in Saccharomyces cer-evisiae, causes accumulation of trehalose-6-phosphate and loss of treha-lose-6-phosphate phosphatase activity. Eur J Biochem 212:315–23. 176. Reinders A, Bürckert N, Hohmann S, Thevelein JM, Boller T, Wiemken
A, De Virgilio C. 1997. Structural analysis of the subunits of the
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Regula-tion of the yeast trehalose–synthase complex by cyclic AMP-dependent phosphorylation. Biochim Biophys Acta - Gen Subj 1840:1646–1650. 178. Thevelein JM, Hohmann S. 1995. Trehalose synthase: guard to the gate
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SUMMARY
For 1st and 2nd generation bioethanol formation, the yeast
Saccharo-myces cerevisiae is the premier organism for fermentation. However, S. cerevisiae cannot naturally ferment pentose sugars like D-arabinose
and D-xylose which are main products, next to D-glucose, of lignocel-lulosic biomass conversion in the 2nd generation bioethanol production process. Therefore, a xylose pathway was introduced via the expression of a fungal xylose isomerase in order for D-xylose to enter the pentose phosphate pathway (Figure 1). Although this resulted in the desired D-xylose fermentation, the consumption of D-xylose in the presence of high concentrations of D-glucose present in the lignocellulosic hy-drolysate remains difficult. In general, xylose-fermenting S. cerevisiae strains first consume D-glucose, before D-xylose is metabolized. The
Figure 1. D-glucose and D-xylose metabolism in DS71054-evoB-Hxk2 (A) and
DS71054-evo6-Hxk2 (B). The D-glucose transport flux via the hexose trans-porters Hxt1, Hxt2, Hxt4, Hxt5 and Hxt7 in DS71054-evoB-Hxk2 is substan-tial as compared to the D-xylose flux through Hxt36 N367I. In DS71054-evo6-Hxk2 the remaining expression of only Hxt2 and Hxt4 decreased the D-glucose flux while the flux of D-xylose in to the cell was increased via the increased expression of Hxt37 N367I. Increased intracellular D-xylose inhib-its the conversion of D-glucose via Hxk2 and causes accumulation of treha-lose-6-phosphate, which also inhibits Hxk2 activity.
194 SUMMARY SUMMARY 195
first hurdle for efficient D-xylose consumption is the uptake of D-xylose. Since this uptake occurs via the extended family of endogenous hexose transporters, D-xylose is normally competed out by D-glucose and only once the D-glucose is exhausted to low levels, uptake of D-xylose can occur at appreciable rates. To overcome the inability of S. cerevisiae to efficiently transport D-xylose, specific D-xylose transporters derived from other organisms have been introduced into yeast. However, these heterologous systems in general only support low rates of D-xylose transport, often because they are high affinity and low capacity trans-porters or are only poorly expressed in yeast. The goal of this thesis work was to overcome the D-xylose transport issue by engineering endogenous hexose transporters and alter their specificity from hexose to pentose sugars. Such re-wiring has the advantage that these trans-porters remain well integrated in the regulatory systems of yeast and thus will be well expressed under relevant conditions.
Chapter 2 describes a gene shuffling approach of hexose transport-ers and selection method for improved growth on low concentrations of D-xylose. Gene shuffling was used to screen for endogenous Hxt transporters with an increased affinity of D-xylose. Various libraries of shuffled HXT genes were transformed to a hexose transporter deletion strain (ΔHXT1-7 and ΔGAL2). This strain is unable to grow on xylose as the main hexose transporters have been deleted from the genome. Shuf-fled genes were selected via growth on low concentrations of D-xylose. This screening yielded two homologous fusion proteins (fusion 9,4 and 9,6) both consisting of the major central part of Hxt2 and various smaller parts of other Hxt proteins. Both chimeric proteins showed a similar increase in D-xylose affinity (~ 8 mM) as compared to Hxt2 (~ 24 mM). The increased D-xylose affinity could be related to the replacement of the C-terminus of Hxt2, more specifically to a cysteine to proline mutation at position 505 in Hxt2. The Hxt2C505P mutation could provide a way to increase the D-xylose transport flux at low D-xylose concen-tration in e.g., at the end of an industrial fermentation when D-xylose concentration is nearly zero. However, these chimeric Hxt transporters are still capable of transporting D-glucose and thus the chimers do not provide a solution for the D-glucose inhibition of D-xylose transport.
In chapter 3, therefore another approach was followed. Herein, a quadruple hexokinase deletion mutant of the D-xylose fermenting
S. cerevisiae strain DS69473 was used in an in vivo engineering approach.
The quadruple hexokinase deletion abolishes D-glucose consumption therefore these cells will only grow on D-xylose or an alternative car-bon source like ethanol. Due to the preference of the endogenous Hxt transporters for D-glucose, growth of the hexokinase deletion strain on D-xylose in the presence of high(er) concentrations of D-glucose is poor. The evolutionary engineering was performed by growing the cells in the presence of D-xylose and gradually increasing the D-glucose concentration in the chemostat. Eventually, this yielded a strain that efficiently utilized D-xylose in the presence of high D-glucose concen-trations. Genome sequence analysis of the evolved strain revealed a mutation (Y353C) in the general co-repressor Cyc8/Ssn6, which was found to be responsible for the phenotype when introduced individu-ally in the non-evolved strain. The Y353C mutation in the CYC8 gene is located in the before last (number 9 out of 10) tetratricopeptide (TPRs) domain. These are functional domains required for the interaction with Tup1. A distinct subset of TPR motifs is needed for the repression of different classes of genes affected by the Cyc8-Tup1 co-repressor complex, especially TPRs 8 and 9, and possibly 10. These are shown to be critical for glucose repression. During glucose repression, the Cyc8-Tup1 co-repressor complex interacts with Mig1 and inhibits Mig1 activation. Transcriptome analysis revealed the altered expression of in total 95 genes, including genes involved in hexose transport, maltose metabolism, cell wall function, and unknown functions (e.g., Seripau-perin multigene family). Out of the 18 known HXT transporters, 9 were significantly upregulated, especially the low or non-expressed HXT10,
HXT13, HXT15 and HXT16 genes. The evolved strain showed
increas-ing uptake rates of D-xylose in the presence of D-glucose, as well as an elevated Vmax for both D-glucose and D-xylose transport. The data
suggests that the increased expression of multiple hexose transporters renders D-xylose metabolism less sensitive to D-glucose inhibition due an elevated transport rate of D-xylose into the cell.
Since the aforementioned evolutionary approach did not lead to an altered specificity of endogenous hexose transporters for D-xylose, a more stringent evolutionary engineering approach was initiated. In chapter 4, a similar but optimized quadruple hexokinase deletion strain that was generated at DSM was used to increase D-xylose transport in the xylose-fermenting S. cerevisiae DS71054 strain. Since S. cerevisiae lacks specific pentose transporters it depends on endogenous hexose
196 SUMMARY SUMMARY 197
transporters for low affinity pentose uptake. To improve the specificity towards D-xylose the aforementioned DS71054 strain was used in an evolutionary engineering experiment to select for growth on D- xylose in the presence of high D-glucose concentrations. This resulted in D-glu-cose-tolerant growth of the yeast on D-xylose. This phenomenon, in DS71054-evoB, could be attributed to a mutation at N367 in the en-dogenous chimeric Hxt36 transporter causing a defect in D-glucose transport while still allowing uptake of D-xylose (Figure 1A). The Hxt36 N367I mutation created a specific D-xylose binding site, albeit with a slightly reduced affinity and lowered Vmax. Specifically, the bulky
isoleu-cine group prevents the aldehyde group of D-glucose to bind efficiently in the binding pocket, and hence the affinity for D-glucose is reduced significantly magnitude. Saturation mutagenesis of the N367 position yielded the variant Hxt36 N367A that transports D-xylose with a high rate and improved affinity. In fermentation trials, this mutant allowed the efficient co-consumption of D-glucose and D-xylose when provided in equimolar concentration. Therefore, this transporter can potentially be used for efficient lignocellulosic bioethanol production.
Although the endogenous hexose transporter Hxt36 was successfully engineered into a specific D-xylose transporter, Hxt36 is subjected to D-glucose-regulated protein degradation. Therefore, in chapter 5, protein degradation of hexose transporters was studied and optimized. In the absence of glucose or when the D-glucose is exhausted from the medium, some Hxt proteins with high D-xylose transport capacity are rapidly degraded and removed from the cytoplasmic membrane. Thus, turnover of such Hxt proteins may lead to poor growth on solely D-xylose. In contrast, Hxt11, which is normally not expressed in S.
cer-evisiae, is not subjected to protein degradation as not recognized by
the quality control mechanisms. It remains on the cytoplasmic mem-brane both at high and low D-glucose concentrations. At low D-glucose concentration protein degradation is a major issue for the low affinity hexose transporters Hxt1, Hxt36 (Hxt3 variant) and Hxt5 which are also subjected to catabolite degradation. This is evidenced by a loss of the aforementioned hexose transporters from the membrane upon D-glucose depletion as monitored with GFP fusions. The catabolite degradation occurs through ubiquitination, which is a major signaling pathway for turnover. N-terminal lysine residues of the aforementioned Hxt proteins predicted to be the target of ubiquitination were replaced
for arginine residues. The mutagenesis resulted in improved membrane localization when cells were grown on solely D-xylose concomitantly with significantly stimulated growth on D-xylose. The decreased ubiq-uitination also improved the late stages of sugar fermentation when cells are grown on both D-glucose and D-xylose.
Although the evolved hexokinase deletion DS71054-evoB strain bearing the Hxt36 N367I mutation from chapter 4 showed significantly improved growth on D-xylose in the presence of high concentrations of D-glucose, the mutant suffers from a reduced Vmax for xylose transport.
This was partially solved by the aforementioned saturation mutagen-esis of position N367, which yielded the Hxt36 N367A mutant with improved D-xylose transport properties, but this mutant also regained some D-glucose transport activity. Therefore, in chapter 6 a 2nd round of evolutionary engineering was performed in order to determine if the capacity of the Hxt36 N367I mutant to transport D-xylose can be im-proved by additional mutations while maintaining the strict specificity for D-xylose. The hexokinase deletion DS71054-evoB strain was evolved in a chemostat during three months on 1% D-xylose in the presence of 10% D-glucose. During the course of the experiment the dilution rate was gradually increased to select for faster growth. Whereas several intermediate evolved strains with improved growth rates were isolated, the final strain DS71054-evo6 shows equal growth rates on D-xylose only as compared to D-xylose in presence of an excess D-glucose. The improved growth rate could be attributed to improved D-xylose
uptake via increased expression levels of Hxt37 N367I, and an overall reduction in the expression of other Hxt transporters except for Hxt4. Re-introduction of the hexokinase Hxk2 (and other hexokinases) in the evolved DS71054-evoB mutant restored D-glucose consumption. However, the Hxk2 complemented evolutionary engineering lineage showed an increased D-xylose consumption that was paralleled by a decreased D-glucose consumption. Overall this led to a net reduction in sugar consumption and growth rate. On the other hand, consumption of D-glucose alone was restored to the levels of the parental and the non-evolved strain leading to the conclusion that glucose consumption per se was not altered in the evolved strains. When the Hxk2 com-plemented evolved strains were grown on a mixture of D-xylose and D-glucose, the progressive accumulation of trehalose-6-phosphate was observed. Trehalose-6-phosphate is an inhibitor of hexokinases and its
