Applications of CRISPR/Cas System to Bacterial Metabolic Engineering

Cho, Suhyung; Shin, Jisoo; Cho, Byung-Kwan

doi:10.3390/ijms19041089

Cited by 127 publications

(95 citation statements)

References 148 publications

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“…In CRISPRi, Cas9 has its endonuclease function inactivated (dCas9), but it still binds to a target genome loci directed by the sgRNA. Gene transcription can be blocked by the binding of dCas9-sgRNA complex to the upstream region of the gene (Cho et al, 2018). Escherichia coli was engineered to express the genes from C. necator PHA operon and orfZ, sucD, and 4hbD from C. kluyreri.…”

Section: Monomer Composition and Microstructure Control Of P(3hb-co-4hb)mentioning

confidence: 99%

Poly(4-Hydroxybutyrate): Current State and Perspectives

Utsunomia

Ren

Zinn

2020

Front. Bioeng. Biotechnol.

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By the end of 1980s, for the first time polyhydroxyalkanoate (PHA) copolymers with incorporated 4-hydroxybutyrate (4HB) units were produced in the bacterium Cupriavidus necator (formally Ralstonia eutropha) from structurally related carbon sources. After that, production of PHA copolymers composed of 3-hydroxybutyrate (3HB) and 4HB [P(3HB-co-4HB)] was demonstrated in diverse wild-type bacteria. The P4HB homopolymer, however, was hardly synthesized because existing bacterial metabolism on 4HB precursors also generate and incorporate 3HB. The resulting material assumes the properties of thermoplastics and elastomers depending on the 4HB fraction in the copolyester. Given the fact that P4HB is biodegradable and yield 4HB, which is a normal compound in the human body and proven to be biocompatible, P4HB has become a prospective material for medical applications, which is the only FDA approved PHA for medical applications since 2007. Different from other materials used in similar applications, high molecular weight P4HB cannot be produced via chemical synthesis. Thus, aiming at the commercial production of this type of PHA, genetic engineering was extensively applied resulting in various production strains, with the ability to convert unrelated carbon sources (e.g., sugars) to 4HB, and capable of producing homopolymeric P4HB. In 2001, Metabolix Inc. filed a patent concerning genetically modified and stable organisms, e.g., Escherichia coli, producing P4HB and copolymers from inexpensive carbon sources. The patent is currently hold by Tepha Inc., the only worldwide producer of commercial P4HB. To date, numerous patents on various applications of P4HB in the medical field have been filed. This review will comprehensively cover the historical evolution and the most recent publications on P4HB biosynthesis, material properties, and industrial and medical applications. Finally, perspectives for the research and commercialization of P4HB will be presented.

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Section: Monomer Composition and Microstructure Control Of P(3hb-co-4hb)mentioning

confidence: 99%

Poly(4-Hydroxybutyrate): Current State and Perspectives

Utsunomia

Ren

Zinn

2020

Front. Bioeng. Biotechnol.

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“…The system also has the advantages of strong applicability and no obvious offtarget phenomenon. These characteristics mean CRISPRi is widely used in the eld of metabolic engineering [28,29]. After CRISPRi was applied, we obtained a titer of 8.53 g/L MVA and a yield of 68.7% (mol/mol).…”

Section: Introductionmentioning

confidence: 98%

Fine and dynamic tuning the glycolytic flux ratio of an artificial carbon saving pathway for high yield of mevalonate in Escherichia coli

Liu¹,

Xian²,

Xu³

et al. 2020

Preprint

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Background In natural Escherichia coli, glucose is mainly metabolized via the Embden-Meyerhoff-Parnas (EMP) pathway. However, in the metabolic process of conversion of pyruvate to acetyl-CoA, one-third of the carbon is lost at CO2. To decrease the loss of glucose in the metabolic process and enhance the carbon conversion efficiency production of desired products by E. coli, we constructed a carbon saving pathway, EP-bifido pathway. As the balance of energy and reducing power was not optimal, we use synthetic biology methods to precisely and dynamically adjust the EMP pathway and pentose phosphate pathway (PPP) flux to improve the production of mevalonate (MVA) via the EP-bifido pathway. ResultHere, we enhanced the MVA titer and yield in E. coli in two ways. First, the promoter of the first gene of the PPP, zwf, was replaced with a set of promoters of different strength to enhance PPP flux for NADPH supply. Compared with the previous EP-bifido strains, the zwf-modified strains showed obvious differences in NADPH, NADH, and ATP synthesis levels and production routes. Among them, strain BP10BF accumulated 11.2 g/L of MVA after 72 h of fermentation and the molar conversion rate from glucose reached 62.2%. Second, the expression of pfkA was suppressed at a certain time by the clustered regularly interspaced short palindromic repeats interference (CRISPRi) system to avoid the growth defect caused by pfkA direct knock-out. The resulting MVA yield of strain BiB1F was 8.53 g/L, and the conversion rate from glucose reached 68.7%. ConclusionThis is the highest MVA conversion rate reported in shaken flask fermentation. The CRISPRi and promoter fine-tuning provided an effective strategy for metabolic flux redistribution in many metabolic pathways and promotes the chemicals production.

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“…Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas), which are adaptive immune systems conserved in prokaryotes (Cho et al, 2018;Makarova et al, 2015), are widely used as tools for genome editing (Jiang et al, 2015;Liu et al, 2017;McAllister et al, 2017;Song et al, 2017). ered biotechnologically important because of its potential utility in bioelectrochemical systems (Hirose et al, 2019;Sydow et al, 2014).…”

Section: Introductionmentioning

confidence: 99%

CRISPR/Cas9-mediated genome editing of <i>Shewanella oneidensis</i> MR-1 using a broad host-range pBBR1-based plasmid

Suzuki

Kouzuma

Watanabe

2020

J. Gen. Appl. Microbiol.

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Commonly used CRISPR/Cas9 systems such as the type-II system from Streptococcus pyogenes are composed of an RNA-guided endonuclease (Cas9) and a single-guide RNA (sgRNA) comprising a CRISPR RNA (crRNA) complementary to a 20-nt target DNA sequence as well as a transactivating crRNA (tracrRNA) that recruits the crRNA to Cas9 (Doudna and Charpentier, 2014). The 20-nt target DNA sequence is followed by a 5¢-NGG-3¢ protospacer adjacent motif (PAM) (Choudhary et al., 2015; Nayak and Metcalf, 2017). The Cas9 complex, guided by the sgRNA, cleaves the target sequence approximately three base pairs upstream of the PAM to generate a double-strand DNA break (DSB), which triggers mutations through nonhomologous end joining (NHEJ) repair or homologydirected repair (HDR) (Cho et al., 2018; Ran et al., 2013). Despite extensive use of genome-editing technologies in eukaryotes, their use in bacteria is limited because of the lack of vectors for CRISPR/Cas9 systems. Several cas9-expressing plasmids applicable to genome editing of bacteria are available (

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Applications of CRISPR/Cas System to Bacterial Metabolic Engineering

Cited by 127 publications

References 148 publications

Poly(4-Hydroxybutyrate): Current State and Perspectives

Poly(4-Hydroxybutyrate): Current State and Perspectives

Fine and dynamic tuning the glycolytic flux ratio of an artificial carbon saving pathway for high yield of mevalonate in Escherichia coli

CRISPR/Cas9-mediated genome editing of <i>Shewanella oneidensis</i> MR-1 using a broad host-range pBBR1-based plasmid

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