Advancing Metabolic Engineering of <i>Saccharomyces cerevisiae</i> Using the CRISPR/Cas System

Lian, Jiazhang; HamediRad, Mohammad; Zhao, Huimin

doi:10.1002/biot.201700601

Cited by 45 publications

(38 citation statements)

References 74 publications

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“…Deriving from CRISPR/Cas9, the CRISPR interference (CRISPRi) system containing a nuclease‐deficient Cas9 (dCas9) coexpressed with a sgRNA, has also been established for efficient gene repression . This CRISPRi platform has now been widely used for identifying functional genes as well as for metabolic engineering in microbes, such as Escherichia coli , Corynebacterium glutamicum , Saccharomyces cerevisiae , Clostridium , Synechocystis , and Mycobacteria …”

Section: Introductionmentioning

confidence: 99%

CRISPR/dCas9‐Mediated Multiplex Gene Repression in Streptomyces

Zhao

Lei

Zheng

et al. 2018

Biotechnology Journal

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Streptomycetes are Gram-positive bacteria with the capacity to produce copious bioactive secondary metabolites, which are the main source of medically and industrially relevant drugs. However, genetic manipulation of Streptomyces strains is much more difficult than other model microorganisms like Escherichia coli and Saccharomyces cerevisiae. Recently, CRISPR/Cas9 or dCas9-mediated genetic manipulation tools have been developed and facilitated Streptomyces genome editing. However, till now, CRISPR/dCas9-based interference system (CRISPRi) is only designed to repress single gene expression. Herein, the authors developed a novel CRISPRi system for multiplex gene repression in the model strain Streptomyces coelicolor. In this system, the integrative plasmid pSET152 is used as the backbone for the expression of the dCas9/sgRNA complex and both dCas9 and sgRNAs are designed to be under the control of constitutive promoters. Using the integrative CRISPRi system, the authors achieved efficient repression of multiple genes simultaneously; the mRNA levels of four targets are reduced to 2-32% of the control. Furthermore, it is successfully employed for functional gene screening, and an orphan response regulator (RR) (encoded by SCO2013) containing an RNA-binding ANTAR domain is identified being involved in bacterial growth. Collectively, this integrative CRISPRi system is very effective for multiplex gene repression in S. coelicolor, which could be extended to other Streptomyces strains for functional gene screening as well as for metabolic engineering.

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Section: Introductionmentioning

confidence: 99%

CRISPR/dCas9‐Mediated Multiplex Gene Repression in Streptomyces

Zhao

Lei

Zheng

et al. 2018

Biotechnology Journal

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“…Plasmids and dDNAs were introduced to yeasts by lithium acetate/single strand carrier DNA/polyethylene glycol method . PFK mutagenesis and promoter substitutions were performed using CRISPR‐Cas9 genome editing . To construct the PFK mutant strain, two gRNA‐expressing plasmids (pRS42K‐P1M and pRS42H‐P2M) and two dDNAs (dDNA‐mPFK1 and dDNA‐mPFK2) were simultaneously transformed into the D452‐2 strain carrying Cas9‐NAT plasmid (Addgene #64329) (Figure A; Figure S1A, Supporting Information).…”

Section: Methodsmentioning

confidence: 99%

Redirection of the Glycolytic Flux Enhances Isoprenoid Production in Saccharomyces cerevisiae

Kwak

Yun

Lane

et al. 2019

Biotechnology Journal

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Sufficient supply of reduced nicotinamide adenine dinucleotide phosphate (NADPH) is a prerequisite of the overproduction of isoprenoids and related bioproducts in Saccharomyces cerevisiae. Although S. cerevisiae highly depends on the oxidative pentose phosphate (PP) pathway to produce NADPH, its metabolic flux toward the oxidative PP pathway is limited due to the rigid glycolysis flux. To maximize NADPH supply for the isoprenoid production in yeast, upper glycolytic metabolic fluxes are reduced by introducing mutations into phosphofructokinase (PFK) along with overexpression of ZWF1 encoding glucose-6-phosphate (G6P) dehydrogenase. The PFK mutations (Pfk1 S724D and Pfk2 S718D) result in less glycerol production and more accumulation of G6P, which is a gateway metabolite toward the oxidative PP pathway. When combined with the PFK mutations, overexpression of ZWF1 caused substantial increases of [NADPH]/[NADP + ] ratios whereas the effect of ZWF1 overexpression alone in the wild-type strain is not noticeable. Also, the introduction of ZWF1 overexpression and the PFK mutations into engineered yeast overexpressing acetyl-CoA C-acetyltransferase (ERG10), truncated HMG-CoA reductase isozyme 1 (tHMG1), and amorphadiene synthase (ADS) leads to a titer of 497 mg L -1 of amorphadiene (3.7-fold over the parental strain). These results suggest that perturbation of upper glycolytic fluxes, in addition to ZWF1 overexpression, is necessary for efficient NADPH supply through the oxidative PP pathway and enhanced production of isoprenoids by engineered S. cerevisiae.

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“…The mutants of BY4741 and HDL were constructed via the CRISPR technology (Lian, HamediRad, & Zhao, ; Lian, HamediRad, Hu, & Zhao, ). Guide RNAs (gRNAs) were designed in E‐CRISP website (http://www.e-crisp.org; Heigwer, Kerr, & Boutros, ).…”

Section: Methodsmentioning

confidence: 99%

Efficient production of S‐adenosyl‐l‐methionine from dl‐methionine in metabolic engineered Saccharomyces cerevisiae

Liu

Tang

Shi

et al. 2019

Biotech & Bioengineering

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S-Adenosyl-L-methionine (SAM) is an important small molecule compound widely used in treating various diseases. Although L-methionine is generally used, the low-cost DLmethionine is more suitable as the substrate for industrial production of SAM. However, Dmethionine is inefficient for SAM formation due to the substrate-specificity of SAM synthetase. In order to increase the utilization efficiency of DL-methionine, intracellular conversion of D-methionine to L-methionine was investigated in the type strain Saccharomyces cerevisiae BY4741 and an industrial strain S. cerevisiae HDL. Firstly, via disruption of HPA3 encoding D-amino acid-N-acetyltransferase, D-methionine was accumulated in vivo and no N-acetyl-D-methionine production was observed. Further, codon-optimized D-amino acid oxidase (DAAO) gene from Trigonopsis variabilis (Genbank MK280686) and L-phenylalanine dehydrogenase gene (L-PheDH) from Rhodococcus jostii (Genbank MK280687) were introduced to convert D-methionine to L-methionine, SAM concentration and content was increased by 110% and 72.1% in BY4741 (plasmid borne) and increased by 38.2% and 34.1% in HDL (genome integrated), by feeding 0.5 g/L Dmethionine. Using the recently developed CRISPR tools, the DAAO and L-PheDH expression cassettes were integrated into the HPA3 and SAH1 loci while SAM2 expression was integrated into the SPE2 and GLC3 loci of HDL, and the resultant strain HDL-R2 accumulated 289% and 192% more SAM concentration and content, respectively, by feeding 0.5 g/L DL-methionine. Further, in a 10 L fed-batch fermentation process, 10.3 g/L SAM were accumulated with the SAM content of 242 mg/g dry cell weight by feeding 16 g/L DL-methionine. The strategies used here provided a promising approach to enhance SAM production using low-cost DL-methionine. K E Y W O R D S

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Advancing Metabolic Engineering of Saccharomyces cerevisiae Using the CRISPR/Cas System

Cited by 45 publications

References 74 publications

CRISPR/dCas9‐Mediated Multiplex Gene Repression in Streptomyces

CRISPR/dCas9‐Mediated Multiplex Gene Repression in Streptomyces

Redirection of the Glycolytic Flux Enhances Isoprenoid Production in Saccharomyces cerevisiae

Efficient production of S‐adenosyl‐l‐methionine from dl‐methionine in metabolic engineered Saccharomyces cerevisiae

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