Metabolic engineering considerations for the heterologous expression of xylose-catabolic pathways in Saccharomyces cerevisiae

Jeong, Deokyeol; Oh, Eun‐Suok; Ko, Ja Kyong; Nam, Ju-Ock; Park, Hee-Soo; Liu, Jing Jing; Lee, Eun Jung; Kim, Soo Rin

doi:10.1371/journal.pone.0236294

Cited by 31 publications

(19 citation statements)

References 56 publications

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“…As previously reported, Δ pho13 improves the xylose fermentation capability of engineered strains of S. cerevisiae ( Ni et al, 2007 ; Van Vleet et al, 2008 ), and other studies have confirmed this with different strain backgrounds ( Fujitomi et al, 2012 ; Kim et al, 2013d ; Jeong et al, 2020 ). Although the detailed molecular mechanism remains unknown, Δ pho13 causes transcriptional activation of the genes involved in non-oxidative PPP ( Kim et al, 2015 ; Ye et al, 2019 ) and reduction of the dephosphorylation product of sedoheptulose-7-phosphate, suggesting the phosphatase activity of Pho13 ( Xu et al, 2016 ).…”

Section: Resultssupporting

confidence: 83%

“…Saccharomyces cerevisiae plays an essential role in the production of lignocellulosic biofuels by fermenting lignocellulosic sugars, mainly glucose and xylose, which requires engineering of the yeast via a heterologous xylose pathway (Kim et al, 2013c;Richa et al, 2019). Current efforts on the metabolic engineering of yeast remain focused on improving the xylose fermentation yield and productivity under multiple stress conditions of lignocellulosic biomass hydrolyzates (Jeong et al, 2020;Qin et al, 2020).…”

Section: Introductionmentioning

confidence: 99%

“…Previously, an efficient xylose-fermenting strain of S. cerevisiae (SR8) was developed through the introduction of a heterologous xylose pathway, optimization of its expression levels, and adaptive evolution, which resulted in a loss-of-function mutation in PHO13 ( Kim et al, 2013d ; Jeong et al, 2020 ). Continued efforts have discovered that the deletion of PHO13 (Δ pho13 ) resulted in transcriptional and metabolic changes favorable to xylose and other C5 sugar fermentation ( Kim et al, 2015 ; Xu et al, 2016 ; Ye et al, 2019 ).…”

Section: Introductionmentioning

confidence: 99%

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Transcriptomic Changes Induced by Deletion of Transcriptional Regulator GCR2 on Pentose Sugar Metabolism in Saccharomyces cerevisiae

Shin

Park

Kim

et al. 2021

Front. Bioeng. Biotechnol.

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Being a microbial host for lignocellulosic biofuel production, Saccharomyces cerevisiae needs to be engineered to express a heterologous xylose pathway; however, it has been challenging to optimize the engineered strain for efficient and rapid fermentation of xylose. Deletion of PHO13 (Δpho13) has been reported to be a crucial genetic perturbation in improving xylose fermentation. A confirmed mechanism of the Δpho13 effect on xylose fermentation is that the Δpho13 transcriptionally activates the genes in the non-oxidative pentose phosphate pathway (PPP). In the current study, we found a couple of engineered strains, of which phenotypes were not affected by Δpho13 (Δpho13-negative), among many others we examined. Genome resequencing of the Δpho13-negative strains revealed that a loss-of-function mutation in GCR2 was responsible for the phenotype. Gcr2 is a global transcriptional factor involved in glucose metabolism. The results of RNA-seq confirmed that the deletion of GCR2 (Δgcr2) led to the upregulation of PPP genes as well as downregulation of glycolytic genes, and changes were more significant under xylose conditions than those under glucose conditions. Although there was no synergistic effect between Δpho13 and Δgcr2 in improving xylose fermentation, these results suggested that GCR2 is a novel knockout target in improving lignocellulosic ethanol production.

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Section: Resultssupporting

confidence: 83%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Transcriptomic Changes Induced by Deletion of Transcriptional Regulator GCR2 on Pentose Sugar Metabolism in Saccharomyces cerevisiae

Shin

Park

Kim

et al. 2021

Front. Bioeng. Biotechnol.

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“…Cell quenching, intracellular metabolite extraction, and derivatization for intracellular metabolite analyses were performed as previously described [33,34]. Briefly, 5 mL of cell culture at the mid-exponential growth phase was quickly injected and quenched in 25 mL 60% (v/v) cold methanol with 10 mM HEPES (pH 7.1) at −40 • C. Quenched cells were centrifuged, and supernatants were discarded.…”

Section: Intracellular Metabolite Extraction and Derivatizationmentioning

confidence: 99%

l-Lactic Acid Production Using Engineered Saccharomyces cerevisiae with Improved Organic Acid Tolerance

Jang

Jeong

et al. 2021

JoF

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Lactic acid is mainly used to produce bio-based, bio-degradable polylactic acid. For industrial production of lactic acid, engineered Saccharomyces cerevisiae can be used. To avoid cellular toxicity caused by lactic acid accumulation, pH-neutralizing agents are used, leading to increased production costs. In this study, lactic acid-producing S. cerevisiae BK01 was developed with improved lactic acid tolerance through adaptive laboratory evolution (ALE) on 8% lactic acid. The genetic basis of BK01 could not be determined, suggesting complex mechanisms associated with lactic acid tolerance. However, BK01 had distinctive metabolomic traits clearly separated from the parental strain, and lactic acid production was improved by 17% (from 102 g/L to 119 g/L). To the best of our knowledge, this is the highest lactic acid titer produced by engineered S. cerevisiae without the use of pH neutralizers. Moreover, cellulosic lactic acid production by BK01 was demonstrated using acetate-rich buckwheat husk hydrolysates. Particularly, BK01 revealed improved tolerance against acetic acid of the hydrolysates, a major fermentation inhibitor of lignocellulosic biomass. In short, ALE with a high concentration of lactic acid improved lactic acid production as well as acetic acid tolerance of BK01, suggesting a potential for economically viable cellulosic lactic acid production.

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“…The product of both pathways, xylulose is phosphorylated by xylulose kinase (XK) and then enters into the pentose phosphate pathway for conversion to ethanol. Comparison of both pathways in different S. cerevisiae strains using various substrates [ 13 – 16 ] revealed that the XI pathway always gave a higher ethanol yield than the XR/XDH pathway. When both pathways were introduced into a single S. cerevisiae strain, a synergistic effect on xylose fermentation was observed [ 17 , 18 ].…”

Section: Introductionmentioning

confidence: 99%

Directed evolution and secretory expression of xylose isomerase for improved utilisation of xylose in Saccharomyces cerevisiae

Bae

Kim

Sung

et al. 2021

Biotechnol Biofuels

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Background Xylose 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. Results The 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. Conclusions Isomerisation 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.

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Metabolic engineering considerations for the heterologous expression of xylose-catabolic pathways in Saccharomyces cerevisiae

Cited by 31 publications

References 56 publications

Transcriptomic Changes Induced by Deletion of Transcriptional Regulator GCR2 on Pentose Sugar Metabolism in Saccharomyces cerevisiae

Transcriptomic Changes Induced by Deletion of Transcriptional Regulator GCR2 on Pentose Sugar Metabolism in Saccharomyces cerevisiae

l-Lactic Acid Production Using Engineered Saccharomyces cerevisiae with Improved Organic Acid Tolerance

Directed evolution and secretory expression of xylose isomerase for improved utilisation of xylose in Saccharomyces cerevisiae

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