Improved ethanol production at high temperature by consolidated bioprocessing using Saccharomyces cerevisiae strain engineered with artificial zinc finger protein

Khatun, M. Mahfuza; Yu, Xiaohui; Kondo, Akihiko; Bai, Feng‐Wu; Zhao, Xin‐Qing

doi:10.1016/j.biortech.2017.05.088

Cited by 35 publications

(17 citation statements)

References 30 publications

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“…Of particular importance is cellobiohydrolase I (CBHI), an essential cellulase in fungal cellulase systems [ 9 , 10 ]. Previous research in developing yeast CBP organisms has focused on S. cerevisiae [ 1 , 11 , 12 ], Y. lipolytica, [ 13 – 15 ], and L. starkeyi [ 16 ]. However, these previous studies of the expression of fungal CBHIs in yeast have encountered major challenges, such as low secretion, yield, and activity of the recombinant proteins which directly discourages the application of these yeast to CBP [ 17 , 18 ].…”

Section: Introductionmentioning

confidence: 99%

Expression of an endoglucanase–cellobiohydrolase fusion protein in Saccharomyces cerevisiae, Yarrowia lipolytica, and Lipomyces starkeyi

Alahuhta

Wei

et al. 2018

Biotechnol Biofuels

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The low secretion levels of cellobiohydrolase I (CBHI) in yeasts are one of the key barriers preventing yeast from directly degrading and utilizing lignocellulose. To overcome this obstacle, we have explored the approach of genetically linking an easily secreted protein to CBHI, with CBHI being the last to be folded. The Trichoderma reesei eg2 (TrEGII) gene was selected as the leading gene due to its previously demonstrated outstanding secretion in yeast. To comprehensively characterize the effects of this fusion protein, we tested this hypothesis in three industrially relevant yeasts: Saccharomyces cerevisiae, Yarrowia lipolytica, and Lipomyces starkeyi. Our initial assays with the L. starkeyi secretome expressing differing TrEGII domains fused to a chimeric Talaromyces emersonii–T. reesei CBHI (TeTrCBHI) showed that the complete TrEGII enzyme, including the glycoside hydrolase (GH) 5 domain is required for increased expression level of the fusion protein when linked to CBHI. We found that this new construct (TrEGII–TeTrCBHI, Fusion 3) had an increased secretion level of at least threefold in L. starkeyi compared to the expression level of the chimeric TeTrCBHI. However, the same improvements were not observed when Fusion 3 construct was expressed in S. cerevisiae and Y. lipolytica. Digestion of pretreated corn stover with the secretomes of Y. lipolytica and L. starkeyi showed that conversion was much better using Y. lipolytica secretomes (50% versus 29%, respectively). In Y. lipolytica, TeTrCBHI performed better than the fusion construct. Furthermore, S. cerevisiae expression of Fusion 3 construct was poor and only minimal activity was observed when acting on the substrate, pNP-cellobiose. No activity was observed for the pNP-lactose substrate. Clearly, this approach is not universally applicable to all yeasts, but works in specific cases. With purified protein and soluble substrates, the exoglucanase activity of the GH7 domain embedded in the Fusion 3 construct in L. starkeyi was significantly higher than that of the GH7 domain in TeTrCBHI expressed alone. It is probable that a higher fraction of fusion construct CBHI is in an active form in Fusion 3 compared to just TeTrCBHI. We conclude that the strategy of leading TeTrCBHI expression with a linked TrEGII module significantly improved the expression of active CBHI in L. starkeyi.Electronic supplementary materialThe online version of this article (10.1186/s13068-018-1301-y) contains supplementary material, which is available to authorized users.

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Section: Introductionmentioning

confidence: 99%

Expression of an endoglucanase–cellobiohydrolase fusion protein in Saccharomyces cerevisiae, Yarrowia lipolytica, and Lipomyces starkeyi

Alahuhta

Wei

et al. 2018

Biotechnol Biofuels

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“…However, ethanol resistance is also known to be temperature dependent (Casey and Ingledew 1986). Prior studies have either focused on genetic variation in either ethanol or temperature stress but not both (Khatun et al 2017; Kitichantaropas et al 2016; Nuanpeng et al 2016; Benjaphokee et al 2012; Peter et al 2018). In this study we mapped ethanol tolerance at high temperature and identified two large effect amino acid substitutions in SEC24 .…”

Section: Discussionmentioning

confidence: 99%

Genetic Basis of Variation in Heat and Ethanol Tolerance inSaccharomyces cerevisiae

Riles

Fay

2019

G3 Genes|Genomes|Genetics

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Saccharomyces cerevisiae has the capability of fermenting sugar to produce concentrations of ethanol that are toxic to most organisms. Other Saccharomyces species also have a strong fermentative capacity, but some are specialized to low temperatures, whereas S. cerevisiae is the most thermotolerant. Although S. cerevisiae has been extensively used to study the genetic basis of ethanol tolerance, much less is known about temperature dependent ethanol tolerance. In this study, we examined the genetic basis of ethanol tolerance at high temperature among strains of S. cerevisiae. We identified two amino acid polymorphisms in SEC24 that cause strong sensitivity to ethanol at high temperature and more limited sensitivity to temperature in the absence of ethanol. We also identified a single amino acid polymorphism in PSD1 that causes sensitivity to high temperature in a strain dependent fashion. The genes we identified provide further insight into genetic variation in ethanol and temperature tolerance and the interdependent nature of these two traits in S. cerevisiae.

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“…Khatun et al (2017) applied a completely different strategy through the use of artificial zinc finger proteins (AZFP) to improve thermotolerance in S. cerevisiae . AZFP are synthetized transcription factors based on zinc finger proteins used in the development of desired phenotypes through metabolic reprogramming of microorganisms [ 39 ]. AZFP libraries contain AZFPs with random zinc motifs that carry a DNA binding domain that activates or represses gene transcription [ 40 , 41 ].…”

Section: Improvement Of Process-related Traitsmentioning

confidence: 99%

Engineered Saccharomyces cerevisiae for lignocellulosic valorization: a review and perspectives on bioethanol production

et al. 2020

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The biorefinery concept, consisting in using renewable biomass with economical and energy goals, appeared in response to the ongoing exhaustion of fossil reserves. Bioethanol is the most prominent biofuel and has been considered one of the top chemicals to be obtained from biomass. Saccharomyces cerevisiae, the preferred microorganism for ethanol production, has been the target of extensive genetic modifications to improve the production of this alcohol from renewable biomasses. Additionally, S. cerevisiae strains from harsh industrial environments have been exploited due to their robust traits and improved fermentative capacity. Nevertheless, there is still not an optimized strain capable of turning second generation bioprocesses economically viable. Considering this, and aiming to facilitate and guide the future development of effective S. cerevisiae strains, this work reviews genetic engineering strategies envisioning improvements in 2 nd generation bioethanol production, with special focus in process-related traits, xylose consumption, and consolidated bioprocessing. Altogether, the genetic toolbox described proves S. cerevisiae to be a key microorganism for the establishment of a bioeconomy, not only for the production of lignocellulosic bioethanol, but also having potential as a cell factory platform for overall valorization of renewable biomasses.

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Improved ethanol production at high temperature by consolidated bioprocessing using Saccharomyces cerevisiae strain engineered with artificial zinc finger protein

Cited by 35 publications

References 30 publications

Expression of an endoglucanase–cellobiohydrolase fusion protein in Saccharomyces cerevisiae, Yarrowia lipolytica, and Lipomyces starkeyi

Expression of an endoglucanase–cellobiohydrolase fusion protein in Saccharomyces cerevisiae, Yarrowia lipolytica, and Lipomyces starkeyi

Genetic Basis of Variation in Heat and Ethanol Tolerance inSaccharomyces cerevisiae

Engineered Saccharomyces cerevisiae for lignocellulosic valorization: a review and perspectives on bioethanol production

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