Simultaneous saccharification and fermentation by engineered Saccharomyces cerevisiae without supplementing extracellular β-glucosidase

Lee, Won Heong; Nan, Hong; Kim, Hyo Jin; Jin, Yong Su

doi:10.1016/j.jbiotec.2013.06.016

Cited by 54 publications

(39 citation statements)

References 27 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Among many bottlenecks, glucose repression on utilizing nonglucose sugars is a significant problem for fermenting mixed sugars by engineered S. cerevisiae. Direct fermentation of cellobiose by engineered yeast opened the possibility of simultaneous utilization of cellobiose and nonglucose sugars (22,23), and it is proven that cellobiose fermentation by engineered yeast can be applied for fermenting pretreated cellulosic biomass (33,34).…”

Section: Discussionmentioning

confidence: 99%

Gene Amplification on Demand Accelerates Cellobiose Utilization in Engineered Saccharomyces cerevisiae

Skerker

Kim

et al. 2016

Appl Environ Microbiol

Self Cite

View full text Add to dashboard Cite

Efficient microbial utilization of cellulosic sugars is essential for the economic production of biofuels and chemicals. Although the yeast Saccharomyces cerevisiae is a robust microbial platform widely used in ethanol plants using sugar cane and corn starch in large-scale operations, glucose repression is one of the significant barriers to the efficient fermentation of cellulosic sugar mixtures. A recent study demonstrated that intracellular utilization of cellobiose by engineered yeast expressing a cellobiose transporter (encoded by cdt-1) and an intracellular ␤-glucosidase (encoded by gh1-1) can alleviate glucose repression, resulting in the simultaneous cofermentation of cellobiose and nonglucose sugars. Here we report enhanced cellobiose fermentation by engineered yeast expressing cdt-1 and gh1-1 through laboratory evolution. When cdt-1 and gh1-1 were integrated into the genome of yeast, the single copy integrant showed a low cellobiose consumption rate. However, cellobiose fermentation rates by engineered yeast increased gradually during serial subcultures on cellobiose. Finally, an evolved strain exhibited a 15-fold-higher cellobiose fermentation rate. To identify the responsible mutations in the evolved strain, genome sequencing was performed. Interestingly, no mutations affecting cellobiose fermentation were identified, but the evolved strain contained 9 copies of cdt-1 and 23 copies of gh1-1. We also traced the copy numbers of cdt-1 and gh1-1 of mixed populations during the serial subcultures. The copy numbers of cdt-1 and gh1-1 in the cultures increased gradually with similar ratios as cellobiose fermentation rates of the cultures increased. These results suggest that the cellobiose assimilation pathway (transport and hydrolysis) might be a rate-limiting step in engineered yeast and copies of genes coding for metabolic enzymes might be amplified in yeast if there is a growth advantage. This study indicates that on-demand gene amplification might be an efficient strategy for yeast metabolic engineering. IMPORTANCEIn order to enable rapid and efficient fermentation of cellulosic hydrolysates by engineered yeast, we delve into the limiting factors of cellobiose fermentation by engineered yeast expressing a cellobiose transporter (encoded by cdt-1) and an intracellular ␤-glucosidase (encoded by gh1-1). Through laboratory evolution, we isolated mutant strains capable of fermenting cellobiose much faster than a parental strain. Genome sequencing of the fast cellobiose-fermenting mutant reveals that there are massive amplifications of cdt-1 and gh1-1 in the yeast genome. We also found positive and quantitative relationships between the rates of cellobiose consumption and the copy numbers of cdt-1 and gh1-1 in the evolved strains. Our results suggest that the cellobiose assimilation pathway (transport and hydrolysis) might be a rate-limiting step for efficient cellobiose fermentation. We demonstrate the feasibility of optimizing not only heterologous metabolic pathways in yeast through laboratory evol...

show abstract

Section: Discussionmentioning

confidence: 99%

Gene Amplification on Demand Accelerates Cellobiose Utilization in Engineered Saccharomyces cerevisiae

Skerker

Kim

et al. 2016

Appl Environ Microbiol

Self Cite

View full text Add to dashboard Cite

show abstract

“…Entre esses açúcares não metabolizados por S. cerevisiae, estão a celobiose e a xilose. Desse modo, alguns pesquisadores veem a engenharia genética dessa levedura como opção para a viabilização da produção de etanol 2G (Stambuk et al, 2008;Lee et al, 2013). Por outro lado, há trabalhos recentes que demonstram a possibilidade do emprego de outras espécies de leveduras nas dornas de fermentação como alternativa à engenharia genética de S. cerevisiae (Reis et al, 2014;Wang et al, 2016).…”

Section: Ufscar -São Carlos -Sp 16 a 19 De Julho De 2017unclassified

Caracterização Dos Perfis De Crescimento Celular De Meyerozyma Caribbica Em Meios De Cultura Contendo Glicose, Xilose E Celobiose

Tadioto¹,

Milani²,

Barrilli³

et al. 2017

Blucher Chemical Engineering Proceedings

View full text Add to dashboard Cite

RESUMO -O etanol tradicional deixa para trás resíduos de bagaço e palha de cana-de-açúcar ricos em açúcares fermentáveis. Esses carboidratos podem, contudo, ser amplamente utilizados numa nova fermentação microbiana (etanol de segunda geração ou 2G). A Saccharomyces cerevisiae, levedura responsável pela produção do etanol de primeira geração, é muito bem adaptada às condições industriais de fermentação alcoólica, porém ela é incapaz de fermentar xilose e celobiose, razão essa que limita a produção de etanol de segunda geração. Levando em conta que a biomassa lignocelulósica do bagaço e da palha de gramíneas é decomposta com o auxílio de diferentes fungos filamentosos e leveduras que, ao contrário da S. cerevisiae, são capazes de utilizar esses carboidratos citados, o presente trabalho se propôs a analisar os perfis de crescimento celular de cinco linhagens de Meyerozyma caribbica isoladas de bagaço de milho em decomposição. Todas as linhagens analisadas foram capazes de metabolizar glicose, xilose e celobiose. A glicose foi o açúcar mais rapidamente assimilado pelas células, e os meios com celobiose foram os que proporcionaram maior crescimento celular. Diante dos resultados obtidos, é possível inferir que as leveduras aqui analisadas apresentam potencial para contribuir com a otimização da produção de etanol de segunda geração. INTRODUÇÃOO bagaço da cana-de-açúcar é um resíduo proveniente da produção de etanol de primeira geração, obtido a partir da fermentação do caldo dessa gramínea. Esse resíduo representa 150 milhões de toneladas anuais no país; de modo geral a maior parte do bagaço é utilizada para gerar calor e/ou energia elétrica na própria indústria (Stambuk et al., 2008; Silva et al., 2014).Com o cenário atual de uma crescente demanda de combustíveis no Brasil, nos próximos anos aumentará ainda mais a quantidade desse tipo resíduo. De outro modo, o bagaço e a palha representam uma biomassa lignocelulósica rica em açúcares fermentáveis que podem ser utilizados para a produção do etanol de segunda geração, que é uma alternativa viável no aumento da produção de etanol, sem a necessidade de haver uma maior área plantada dessa gramínea (Zanin et al., 2000;Stambuk et al., 2008;Soccol et al., 2010).

show abstract

“…Furthermore, addition of cellulase to the SSF process resulted in ethanol production from cellulose with more efficiency compared to the wild-type strain (Galazka et al 2010;Ha et al, 2013). Lee et al (2013) demonstrated efficient ethanol production without supplementation of β-glucosidase using an engineered S. cerevisiae strain expressing a cellodextrin transporter and an intracellular β-glucosidase from N. crassa. The engineered strain did not need an exogenous supplementation of β-glucosidase, and showed better ethanol productivity from 8% (w/v) pure Avicel (27.0 g/l ethanol) than the parental strain which required β-glucosidase supplementation.…”

Section: Cellodextrin Transportersmentioning

confidence: 99%

Advances in consolidated bioprocessing systems for bioethanol and butanol production from biomass: a comprehensive review

2015

View full text Add to dashboard Cite

HIGHLIGHTS Various CBP strategies have been discussed. Recently, lignocellulosic biomass as the most abundant renewable resource has been widely considered for bioalcohols production. However, the complex structure of lignocelluloses requires a multi-step process which is costly and time consuming. Although, several bioprocessing approaches have been developed for pretreatment, saccharification and fermentation, bioalcohols production from lignocelluloses is still limited because of the economic infeasibility of these technologies. This cost constraint could be overcome by designing and constructing robust cellulolytic and bioalcohols producing microbes and by using them in a consolidated bioprocessing (CBP) system. This paper comprehensively reviews potentials, recent advances and challenges faced in CBP systems for efficient bioalcohols (ethanol and butanol) production from lignocellulosic and starchy biomass. The CBP strategies include using native single strains with cellulytic and alcohol production activities, microbial co-cultures containing both cellulytic and ethanologenic microorganisms, and genetic engineering of cellulytic microorganisms to be alcohol-producing or alcohol producing microorganisms to be cellulytic. Moreover, high-throughput techniques, such as metagenomics, metatranscriptomics, next generation sequencing and synthetic biology developed to explore novel microorganisms and powerful enzymes with high activity, thermostability and pH stability are also discussed. Currently, the CBP technology is in its infant stage, and ideal microorganisms and/or conditions at industrial scale are yet to be introduced. So, it is essential to bring into attention all barriers faced and take advantage of all the experiences gained to achieve a high-yield and low-cost CBP process.

show abstract

Simultaneous saccharification and fermentation by engineered Saccharomyces cerevisiae without supplementing extracellular β-glucosidase

Cited by 54 publications

References 27 publications

Gene Amplification on Demand Accelerates Cellobiose Utilization in Engineered Saccharomyces cerevisiae

Gene Amplification on Demand Accelerates Cellobiose Utilization in Engineered Saccharomyces cerevisiae

Caracterização Dos Perfis De Crescimento Celular De Meyerozyma Caribbica Em Meios De Cultura Contendo Glicose, Xilose E Celobiose

Advances in consolidated bioprocessing systems for bioethanol and butanol production from biomass: a comprehensive review

Contact Info

Product

Resources

About