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Heterologous xylose isomerase pathway and evolutionary engineering improve xylose utilization in Saccharomyces cerevisiae.

ABSTRACT: Xylose utilization is one key issue for the bioconversion of lignocelluloses. It is a promising approach to engineering heterologous pathway for xylose utilization in Saccharomyces cerevisiae. Here, we constructed a xylose-fermenting yeast SyBE001 through combinatorial fine-tuning the expression of XylA and endogenous XKS1. Additional overexpression of genes RKI1, RPE1, TKL1, and TAL1 in the non-oxidative pentose phosphate pathway (PPP) in SyBE001 increased the xylose consumption rate by 1.19-fold. By repetitive adaptation, the xylose utilization rate was further increased by ?10-fold in the resultant strain SyBE003. Gene expression analysis identified a variety of genes with significantly changed expression in the PPP, glycolysis and the tricarboxylic acid cycle in SyBE003.

SUBMITTER: Qi X

PROVIDER: S-EPMC4612707 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Heterologous xylose isomerase pathway and evolutionary engineering improve xylose utilization in Saccharomyces cerevisiae.

Qi Xin X Zha Jian J Liu Gao-Gang GG Zhang Weiwen W Li Bing-Zhi BZ Yuan Ying-Jin YJ

Frontiers in microbiology 20151021

Xylose utilization is one key issue for the bioconversion of lignocelluloses. It is a promising approach to engineering heterologous pathway for xylose utilization in Saccharomyces cerevisiae. Here, we constructed a xylose-fermenting yeast SyBE001 through combinatorial fine-tuning the expression of XylA and endogenous XKS1. Additional overexpression of genes RKI1, RPE1, TKL1, and TAL1 in the non-oxidative pentose phosphate pathway (PPP) in SyBE001 increased the xylose consumption rate by 1.19-fo ...[more]

PMID: 26539187

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Project description: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.

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

Heterologous xylose isomerase pathway and evolutionary engineering improve xylose utilization in Saccharomyces cerevisiae.

Publications

Heterologous xylose isomerase pathway and evolutionary engineering improve xylose utilization in Saccharomyces cerevisiae.

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