Dataset Information

Molecular evolutionary engineering of xylose isomerase to improve its catalytic activity and performance of micro-aerobic glucose/xylose co-fermentation in Saccharomyces cerevisiae.

ABSTRACT: Background:Expression of d-xylose isomerase having high catalytic activity in Saccharomyces cerevisiae (S. cerevisiae) is a prerequisite for efficient and economical production of bioethanol from cellulosic biomass. Although previous studies demonstrated functional expression of several xylose isomerases (XI) in S. cerevisiae, identification of XIs having higher catalytic activity is needed. Here, we report a new strategy to improve xylose fermentation in the S. cerevisiae strain IR-2 that involves an evolutionary engineering to select top-performing XIs from eight previously reported XIs derived from various species. Results:Eight XI genes shown to have good expression in S. cerevisiae were introduced into the strain IR-2 having a deletion of GRE3 and XKS1 overexpression that allows use of d-xylose as a carbon source. Each transformant was evaluated under aerobic and micro-aerobic culture conditions. The strain expressing XI from Lachnoclostridium phytofermentans ISDg (LpXI) had the highest d-xylose consumption rate after 72 h of micro-aerobic fermentation on d-glucose and d-xylose mixed medium. To enhance LpXI catalytic activity, we performed random mutagenesis using error-prone polymerase chain reaction (PCR), which yielded two LpXI candidates, SS82 and SS92, that showed markedly improved fermentation performance. The LpXI genes in these clones carried either T63I or V162A/N303T point mutations. The SS120 strain expressing LpXI with the double mutation of T63I/V162A assimilated nearly 85 g/L d-glucose and 35 g/L d-xylose to produce 53.3 g/L ethanol in 72 h with an ethanol yield of approximately 0.44 (g/g-input sugars). An in vitro enzyme assay showed that, compared to wild-type, the LpXI double mutant in SS120 had a considerably higher V max (0.107 µmol/mg protein/min) and lower K m (37.1 mM). Conclusions:This study demonstrated that LpXI has the highest d-xylose consumption rate among the XIs expressed in IR-2 under micro-aerobic co-fermentation conditions. A combination of novel mutations (T63I and V162A) significantly improved the enzymatic activity of LpXI, indicating that LpXI-T63I/V162A would be a potential construct for highly efficient production of cellulosic ethanol.

SUBMITTER: Seike T

PROVIDER: S-EPMC6551904 | biostudies-other | 2019

REPOSITORIES: biostudies-other

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Publications

Molecular evolutionary engineering of xylose isomerase to improve its catalytic activity and performance of micro-aerobic glucose/xylose co-fermentation in <i>Saccharomyces cerevisiae</i>.

Seike Taisuke T Kobayashi Yosuke Y Sahara Takehiko T Ohgiya Satoru S Kamagata Yoichi Y Fujimori Kazuhiro E KE

Biotechnology for biofuels 20190606

<h4>Background</h4>Expression of d-xylose isomerase having high catalytic activity in <i>Saccharomyces cerevisiae</i> (<i>S. cerevisiae</i>) is a prerequisite for efficient and economical production of bioethanol from cellulosic biomass. Although previous studies demonstrated functional expression of several xylose isomerases (XI) in <i>S. cerevisiae</i>, identification of XIs having higher catalytic activity is needed. Here, we report a new strategy to improve xylose fermentation in the <i>S. c ...[more]

PMID: 31178927

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Project description:BACKGROUND:The yeast Saccharomyces cerevisiae, a promising host for lignocellulosic bioethanol production, is unable to metabolize xylose. In attempts to confer xylose utilization ability in S. cerevisiae, a number of xylose isomerase (XI) genes have been expressed heterologously in this yeast. Although several of these XI encoding genes were functionally expressed in S. cerevisiae, the need still exists for a S. cerevisiae strain with improved xylose utilization ability for use in the commercial production of bioethanol. Although currently much effort has been devoted to achieve the objective, one of the solutions is to search for a new XI gene that would confer superior xylose utilization in S. cerevisiae. Here, we searched for novel XI genes from the protists residing in the hindgut of the termite Reticulitermes speratus. RESULTS:Eight novel XI genes were obtained from a cDNA library, prepared from the protists of the R. speratus hindgut, by PCR amplification using degenerated primers based on highly conserved regions of amino acid sequences of different XIs. Phylogenetic analysis classified these cloned XIs into two groups, one showed relatively high similarities to Bacteroidetes and the other was comparatively similar to Firmicutes. The growth rate and the xylose consumption rate of the S. cerevisiae strain expressing the novel XI, which exhibited highest XI activity among the eight XIs, were superior to those exhibited by the strain expressing the XI gene from Piromyces sp. E2. Substitution of the asparagine residue at position 337 of the novel XI with a cysteine further improved the xylose utilization ability of the yeast strain. Interestingly, introducing point mutations in the corresponding asparagine residues in XIs originated from other organisms, such as Piromyces sp. E2 or Clostridium phytofermentans, similarly improved xylose utilization in S. cerevisiae. CONCLUSIONS:A novel XI gene conferring superior xylose utilization in S. cerevisiae was successfully isolated from the protists in the termite hindgut. Isolation of this XI gene and identification of the point mutation described in this study might contribute to improving the productivity of industrial bioethanol.

Project description:Economic bioconversion of plant cell wall hydrolysates into fuels and chemicals has been hampered mainly due to the inability of microorganisms to efficiently co-ferment pentose and hexose sugars, especially glucose and xylose, which are the most abundant sugars in cellulosic hydrolysates. Saccharomyces cerevisiae cannot metabolize xylose due to a lack of xylose-metabolizing enzymes. We developed a rapid and efficient xylose-fermenting S. cerevisiae through rational and inverse metabolic engineering strategies, comprising the optimization of a heterologous xylose-assimilating pathway and evolutionary engineering. Strong and balanced expression levels of the XYL1, XYL2, and XYL3 genes constituting the xylose-assimilating pathway increased ethanol yields and the xylose consumption rates from a mixture of glucose and xylose with little xylitol accumulation. The engineered strain, however, still exhibited a long lag time when metabolizing xylose above 10 g/l as a sole carbon source, defined here as xylose toxicity. Through serial-subcultures on xylose, we isolated evolved strains which exhibited a shorter lag time and improved xylose-fermenting capabilities than the parental strain. Genome sequencing of the evolved strains revealed that mutations in PHO13 causing loss of the Pho13p function are associated with the improved phenotypes of the evolved strains. Crude extracts of a PHO13-overexpressing strain showed a higher phosphatase activity on xylulose-5-phosphate (X-5-P), suggesting that the dephosphorylation of X-5-P by Pho13p might generate a futile cycle with xylulokinase overexpression. While xylose consumption rates by the evolved strains improved substantially as compared to the parental strain, xylose metabolism was interrupted by accumulated acetate. Deletion of ALD6 coding for acetaldehyde dehydrogenase not only prevented acetate accumulation, but also enabled complete and efficient fermentation of xylose as well as a mixture of glucose and xylose by the evolved strain. These findings provide direct guidance for developing industrial strains to produce cellulosic fuels and chemicals.

Project description:BACKGROUND:Saccharomyces cerevisiae strains expressing D-xylose isomerase (XI) produce some of the highest reported ethanol yields from D-xylose. Unfortunately, most bacterial XIs that have been expressed in S. cerevisiae are either not functional, require additional strain modification, or have low affinity for D-xylose. This study analyzed several XIs from rumen and intestinal microorganisms to identify enzymes with improved properties for engineering S. cerevisiae for D-xylose fermentation. RESULTS:Four XIs originating from rumen and intestinal bacteria were isolated and expressed in a S. cerevisiae CEN.PK2-1C parental strain primed for D-xylose metabolism by over expression of its native D-xylulokinase. Three of the XIs were functional in S. cerevisiae, based on the strain's ability to grow in D-xylose medium. The most promising strain, expressing the XI mined from Prevotella ruminicola TC2-24, was further adapted for aerobic and fermentative growth by serial transfers of D-xylose cultures under aerobic, and followed by microaerobic conditions. The evolved strain had a specific growth rate of 0.23 h-1 on D-xylose medium, which is comparable to the best reported results for analogous S. cerevisiae strains including those expressing the Piromyces sp. E2 XI. When used to ferment D-xylose, the adapted strain produced 13.6 g/L ethanol in 91 h with a metabolic yield of 83% of theoretical. From analysis of the P. ruminicola XI, it was determined the enzyme possessed a Vmax of 0.81 ?mole/min/mg protein and a Km of 34 mM. CONCLUSION:This study identifies a new xylose isomerase from the rumen bacterium Prevotella ruminicola TC2-24 that has one of the highest affinities and specific activities compared to other bacterial and fungal D-xylose isomerases expressed in yeast. When expressed in S. cerevisiae and used to ferment D-xylose, very high ethanol yield was obtained. This new XI should be a promising resource for constructing other D-xylose fermenting strains, including industrial yeast genetic backgrounds.

Project description:BackgroundXylose contained in lignocellulosic biomass is an attractive carbon substrate for economically viable conversion to bioethanol. Extensive research has been conducted on xylose fermentation using recombinant Saccharomyces cerevisiae expressing xylose isomerase (XI) and xylose reductase/xylitol dehydrogenase (XR/XDH) pathways along with the introduction of a xylose transporter and amplification of the downstream pathway. However, the low utilization of xylose in the presence of glucose, due to the varying preference for cellular uptake, is a lingering challenge. Studies so far have mainly focused on xylose utilization inside the cells, but there have been little trials on the conversion of xylose to xylulose by cell before uptake. We hypothesized that the extracellular conversion of xylose to xylulose before uptake would facilitate better utilization of xylose even in the presence of glucose. To verify this, XI from Piromyces sp. was engineered and hyper-secreted in S. cerevisiae for the extracellular conversion of xylose to xylulose.ResultsThe optimal pH of XI was lowered from 7.0 to 5.0 by directed evolution to ensure its high activity under the acidic conditions used for yeast fermentation, and hyper-secretion of an engineered XI-76 mutant (E56A and I252M) was accomplished by employing target protein-specific translational fusion partners. The purified XI-76 showed twofold higher activity than that of the wild type at pH 5. The secretory expression of XI-76 in the previously developed xylose utilizing yeast strain, SR8 increased xylose consumption and ethanol production by approximately 7-20% and 15-20% in xylose fermentation and glucose and xylose co-fermentation, respectively.ConclusionsIsomerisation of xylose to xylulose before uptake using extracellular XI was found to be effective in xylose fermentation or glucose/xylose co-fermentation. This suggested that glucose competed less with xylulose than with xylose for uptake by the cell. Consequently, the engineered XI secretion system constructed in this study can pave the way for simultaneous utilization of C5/C6 sugars from the sustainable lignocellulosic biomass.

Dataset Information

Molecular evolutionary engineering of xylose isomerase to improve its catalytic activity and performance of micro-aerobic glucose/xylose co-fermentation in Saccharomyces cerevisiae.

Publications

Molecular evolutionary engineering of xylose isomerase to improve its catalytic activity and performance of micro-aerobic glucose/xylose co-fermentation in <i>Saccharomyces cerevisiae</i>.

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