Dataset Information

CRISPR-Cas9 Approach Constructing Cellulase sestc-Engineered Saccharomyces cerevisiae for the Production of Orange Peel Ethanol.

ABSTRACT: The development of lignocellulosic bioethanol plays an important role in the substitution of petrochemical energy and high-value utilization of agricultural wastes. The safe and stable expression of cellulase gene sestc was achieved by applying the clustered regularly interspaced short palindromic repeats-Cas9 approach to the integration of sestc expression cassette containing Agaricus biporus glyceraldehyde-3-phosphate-dehydrogenase gene (gpd) promoter in the Saccharomyces cerevisiae chromosome. The target insertion site was found to be located in the S. cerevisiae hexokinase 2 by designing a gRNA expression vector. The recombinant SESTC protein exhibited a size of approximately 44 kDa in the engineered S. cerevisiae. By using orange peel as the fermentation substrate, the filter paper, endo-1,4-?-glucanase, exo-1,4-?-glucanase activities of the transformants were 1.06, 337.42, and 1.36 U/mL, which were 35.3-fold, 23.03-fold, and 17-fold higher than those from wild-type S. cerevisiae, respectively. After 6 h treatment, approximately 20 g/L glucose was obtained. Under anaerobic conditions the highest ethanol concentration reached 7.53 g/L after 48 h fermentation and was 37.7-fold higher than that of wild-type S. cerevisiae (0.2 g/L). The engineered strains may provide a valuable material for the development of lignocellulosic ethanol.

SUBMITTER: Yang P

PROVIDER: S-EPMC6191481 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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CRISPR-Cas9 Approach Constructing Cellulase sestc-Engineered Saccharomyces cerevisiae for the Production of Orange Peel Ethanol.

Yang Peizhou P Wu Yun Y Zheng Zhi Z Cao Lili L Zhu Xingxing X Mu Dongdong D Jiang Shaotong S

Frontiers in microbiology 20181010

The development of lignocellulosic bioethanol plays an important role in the substitution of petrochemical energy and high-value utilization of agricultural wastes. The safe and stable expression of cellulase gene sestc was achieved by applying the clustered regularly interspaced short palindromic repeats-Cas9 approach to the integration of sestc expression cassette containing Agaricus biporus glyceraldehyde-3-phosphate-dehydrogenase gene (gpd) promoter in the Sacc ...[more]

PMID: 30364071

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Project description:Glycerol is an eco-friendly solvent that enhances plant biomass decomposition via glycerolysis in many pretreatment methods. Nonetheless, inefficient conversion of glycerol to ethanol by natural Saccharomyces cerevisiae limits its use in these processes. In this study, we have developed an efficient glycerol-converting yeast strain by genetically modifying the oxidation of cytosolic NAD (NADH) by an O2-dependent dynamic shuttle and abolishing both glycerol phosphorylation and biosynthesis in S. cerevisiae strain D452-2, as well as by vigorous expression of whole genes in the dihydroxyacetone (DHA) pathway (Candida utilis glycerol facilitator, Ogataea polymorpha glycerol dehydrogenase, endogenous dihydroxyacetone kinase, and triosephosphate isomerase). The engineered strain showed conversion efficiencies (CE) up to 0.49 g ethanol/g glycerol (98% of theoretical CE), with a production rate of >1 g liter-1 h-1 when glycerol was supplemented in a single fed-batch fermentation in a rich medium. Furthermore, the engineered strain converted a mixture of glycerol and glucose into bioethanol (>86 g/liter) with 92.8% CE. To the best of our knowledge, this is the highest reported titer of bioethanol produced from glycerol and glucose. Notably, we developed a glycerol-utilizing transformant from a parent strain which cannot utilize glycerol as a sole carbon source. The developed strain converted glycerol to ethanol with a productivity of 0.44 g liter-1 h-1 on minimal medium under semiaerobic conditions. Our findings will promote the utilization of glycerol in eco-friendly biorefineries and integrate bioethanol and plant oil industries. IMPORTANCE With the development of efficient lignocellulosic biorefineries, glycerol has attracted attention as an eco-friendly biomass-derived solvent that can enhance the dissociation of lignin and cell wall polysaccharides during the pretreatment process. Coconversion of glycerol with the sugars released from biomass after glycerolysis increases the resources for ethanol production and lowers the burden of component separation. However, low conversion efficiency from glycerol and sugars limits the industrial application of this process. Therefore, the generation of an efficient glycerol-fermenting yeast will promote the applicability of integrated biorefineries. Hence, metabolic flux control in yeast grown on glycerol will lead to the generation of cell factories that produce chemicals, which will boost biodiesel and bioethanol industries. Additionally, the use of glycerol-fermenting yeast will reduce global warming and generation of agricultural waste, leading to the establishment of a sustainable society.

Project description:BackgroundThe abundance of glucuronoxylan (GX) in agricultural and forestry residual side streams positions it as a promising feedstock for microbial conversion into valuable compounds. By engineering strains of the widely employed cell factory Saccharomyces cerevisiae with the ability to directly hydrolyze and ferment GX polymers, we can avoid the need for harsh chemical pretreatments and costly enzymatic hydrolysis steps prior to fermentation. However, for an economically viable bioproduction process, the engineered strains must efficiently express and secrete enzymes that act in synergy to hydrolyze the targeted polymers.ResultsThe aim of this study was to equip the xylose-fermenting S. cerevisiae strain CEN.PK XXX with xylanolytic enzymes targeting beechwood GX. Using a targeted enzyme approach, we matched hydrolytic enzyme activities to the chemical features of the GX substrate and determined that besides endo-1,4-β-xylanase and β-xylosidase activities, α-methyl-glucuronidase activity was of great importance for GX hydrolysis and yeast growth. We also created a library of strains expressing different combinations of enzymes, and screened for yeast strains that could express and secrete the enzymes and metabolize the GX hydrolysis products efficiently. While strains engineered with BmXyn11A xylanase and XylA β-xylosidase could grow relatively well in beechwood GX, strains further engineered with Agu115 α-methyl-glucuronidase did not display an additional growth benefit, likely due to inefficient expression and secretion of this enzyme. Co-cultures of strains expressing complementary enzymes as well as external enzyme supplementation boosted yeast growth and ethanol fermentation of GX, and ethanol titers reached a maximum of 1.33 g L- 1 after 48 h under oxygen limited condition in bioreactor fermentations.ConclusionThis work underscored the importance of identifying an optimal enzyme combination for successful engineering of S. cerevisiae strains that can hydrolyze and assimilate GX. The enzymes must exhibit high and balanced activities, be compatible with the yeast's expression and secretion system, and the nature of the hydrolysis products must be such that they can be taken up and metabolized by the yeast. The engineered strains, particularly when co-cultivated, display robust growth and fermentation of GX, and represent a significant step forward towards a sustainable and cost-effective bioprocessing of GX-rich biomass. They also provide valuable insights for future strain and process development targets.

Dataset Information

CRISPR-Cas9 Approach Constructing Cellulase sestc-Engineered Saccharomyces cerevisiae for the Production of Orange Peel Ethanol.

Publications

CRISPR-Cas9 Approach Constructing Cellulase <i>sestc-</i>Engineered <i>Saccharomyces cerevisiae</i> for the Production of Orange Peel Ethanol.

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