Dataset Information

Endogenous 2? Plasmid Editing for Pathway Engineering in Saccharomyces cerevisiae.

ABSTRACT: In Saccharomyces cerevisiae, conventional 2?-plasmid based plasmid (pC2?, such as pRS425) have been widely adopted in pathway engineering for multi-copy overexpression of key genes. However, the loss of partition and copy number control elements of yeast endogenous 2? plasmid (pE2?) brings the issues concerning plasmid stability and copy number of pC2?, especially in long-term fermentation. In this study, we developed a method based on CRISPR/Cas9 to edit pE2? and built the pE2? multi-copy system by insertion of the target DNA element and elimination of the original pE2? plasmid. The resulting plasmid pE2?RAF1 and pE2?REP2 demonstrated higher copy number and slower loss rate than a pC2? control plasmid pRS425RK, when carrying the same target gene. Then, moving the essential gene TPI1 (encoding triose phosphate isomerase) from chromosome to pE2?RAF1 could increase the plasmid viability to nearly 100% and further increase the plasmid copy number by 73.95%. The expression using pE2? multi-copy system demonstrated much smaller cell-to-cell variation comparing with pC2? multi-copy system. With auxotrophic complementation of TPI1, the resulting plasmid pE2?RT could undergo cultivation of 90 generations under non-selective conditions without loss. Applying pE2? multi-copy system for dihydroartemisinic acid (DHAA) biosynthesis, the production of DHAA was increased to 620.9 mg/L at shake-flask level in non-selective rich medium. This titer was 4.73-fold of the strain constructed based on pC2? due to the more stable pE2? plasmid system and with higher plasmid copy number. This study provides an improved expression system in yeast, and set a promising platform to construct biosynthesis pathway for valuable products.

SUBMITTER: Zeng BX

PROVIDER: S-EPMC7921170 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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Publications

Endogenous 2μ Plasmid Editing for Pathway Engineering in <i>Saccharomyces cerevisiae</i>.

Zeng Bo-Xuan BX Yao Ming-Dong MD Xiao Wen-Hai WH Luo Yun-Zi YZ Wang Ying Y Yuan Ying-Jin YJ

Frontiers in microbiology 20210216

In <i>Saccharomyces cerevisiae</i>, conventional 2μ-plasmid based plasmid (pC2μ, such as pRS425) have been widely adopted in pathway engineering for multi-copy overexpression of key genes. However, the loss of partition and copy number control elements of yeast endogenous 2μ plasmid (pE2μ) brings the issues concerning plasmid stability and copy number of pC2μ, especially in long-term fermentation. In this study, we developed a method based on CRISPR/Cas9 to edit pE2μ and built the pE2μ multi-cop ...[more]

PMID: 33664720

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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.

Project description:BackgroundGlycosylation of flavonoids is a promising approach to improve the pharmacokinetic properties and biological activities of flavonoids. Recently, many efforts such as enzymatic biocatalysis and the engineered Escherichia coli biotransformation have increased the production of flavonoid glucosides. However, the low yield of flavonoid glucosides can not meet the increasing demand for human medical and dietary needs. Saccharomyces cerevisiae is a generally regarded as safe (GRAS) organism that has several attractive characteristics as a metabolic engineering platform for the production of flavonoid glucosides. However, endogenous glucosidases of S. cerevisiae as a whole-cell biocatalyst reversibly hydrolyse the glucosidic bond and hinder the biosynthesis of the desired products. In this study, a model flavonoid, scutellarein, was used to exploit how to enhance the production of flavonoid glucosides in the engineered S. cerevisiae.ResultsTo produce flavonoid glucosides, three flavonoid glucosyltransferases (SbGTs) from Scutellaria baicalensis Georgi were successfully expressed in E. coli, and their biochemical characterizations were identified. In addition, to synthesize the flavonoid glucosides in whole-cell S. cerevisiae, SbGT34 was selected for constructing the engineering yeast. Three glucosidase genes (EXG1, SPR1, YIR007W) were knocked out using homologous integration, and the EXG1 gene was determined to be the decisive gene of S. cerevisiae in the process of hydrolysing flavonoid glucosides. To further enhance the potential glycosylation activity of S. cerevisiae, two genes encoding phosphoglucomutase and UTP-glucose-1-phosphate uridylyltransferase involved in the synthetic system of uridine diphosphate glucose were over-expressed in S. cerevisiae. Consequently, approximately 4.8 g (1.2 g/L) of scutellarein 7-O-glucoside (S7G) was produced in 4 L of medium after 54 h of incubation in a 10-L fermenter while being supplied with ~3.5 g of scutellarein.ConclusionsThe engineered yeast harbouring SbGT with a deletion of glucosidases produced more flavonoid glucosides than strains without a deletion of glucosidases. This platform without glucosidase activity could be used to modify a wide range of valued plant secondary metabolites and to explore of their biological functions using whole-cell S. cerevisiae as a biocatalyst.

Dataset Information

Endogenous 2? Plasmid Editing for Pathway Engineering in Saccharomyces cerevisiae.

Publications

Endogenous 2μ Plasmid Editing for Pathway Engineering in <i>Saccharomyces cerevisiae</i>.

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