scholarly journals Multiplex genome editing by natural transformation (MuGENT) for synthetic biology inVibrio natriegens

2017 ◽  
Author(s):  
Triana N. Dalia ◽  
Chelsea A. Hayes ◽  
Sergey Stolyar ◽  
Christopher J. Marx ◽  
James B. McKinlay ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Metabolic Engineering ◽  
Genome Editing ◽  
High Efficiency ◽  
Natural Transformation ◽  
Value Added ◽  
Industrial Applications ◽  
Proof Of Concept ◽  
Natural Competence ◽  
Low Efficiency

ABSTRACTVibrio natriegenshas recently emerged as an alternative toEscherichia colifor molecular biology and biotechnology, but low-efficiency genetic tools hamper its development. Here, we uncover how to induce natural competence inV. natriegensand describe methods for multiplex genome editing by natural transformation (MuGENT). MuGENT promotes integration of multiple genome edits at high-efficiency on unprecedented timescales. Also, this method allows for generating highly complex mutant populations, which can be exploited for metabolic engineering efforts. As a proof-of-concept, we attempted to enhance production of the value added chemical poly-β-hydroxybutyrate (PHB) inV. natriegensby targeting the expression of nine genes involved in PHB biosynthesis via MuGENT. Within 1 week, we isolated edited strains that produced ~100 times more PHB than the parent isolate and ~3.3 times more than a rationally designed strain. Thus, the methods described here should extend the utility of this species for diverse academic and industrial applications.

scholarly journals An improved method for precise genome editing in zebrafish using CRISPR-Cas9 technique

2021 ◽  
Author(s):  
Eugene V. Gasanov ◽  
Justyna Jędrychowska ◽  
Michal Pastor ◽  
Malgorzata Wiweger ◽  
Axel Methner ◽  
...  
Keyword(s):  
Genome Editing ◽  
High Efficiency ◽  
Target Site ◽  
Proof Of Concept ◽  
Intervention Site ◽  
End Joining ◽  
Guide Rna ◽  
Guide Rnas ◽  
Cas9 Nuclease ◽  
Non Homologous End Joining

AbstractCurrent methods of CRISPR-Cas9-mediated site-specific mutagenesis create deletions and small insertions at the target site which are repaired by imprecise non-homologous end-joining. Targeting of the Cas9 nuclease relies on a short guide RNA (gRNA) corresponding to the genome sequence approximately at the intended site of intervention. We here propose an improved version of CRISPR-Cas9 genome editing that relies on two complementary guide RNAs instead of one. Two guide RNAs delimit the intervention site and allow the precise deletion of several nucleotides at the target site. As proof of concept, we generated heterozygous deletion mutants of the kcng4b, gdap1, and ghitm genes in the zebrafish Danio rerio using this method. A further analysis by high-resolution DNA melting demonstrated a high efficiency and a low background of unpredicted mutations. The use of two complementary gRNAs improves CRISPR-Cas9 specificity and allows the creation of predictable and precise mutations in the genome of D. rerio.

scholarly journals Advances and opportunities in gene editing and gene regulation technology for Yarrowia lipolytica

2019 ◽  
Vol 18 (1) ◽  
Author(s):  
Vijaydev Ganesan ◽  
Michael Spagnuolo ◽  
Ayushi Agrawal ◽  
Spencer Smith ◽  
Difeng Gao ◽  
...  
Keyword(s):  
Metabolic Engineering ◽  
Genome Editing ◽  
Yarrowia Lipolytica ◽  
Gene Editing ◽  
Molecular Genetic ◽  
Industrial Applications ◽  
Cell Factory ◽  
Fuel Feed ◽  
Renewable Chemicals ◽  
Pharmaceutical Applications

AbstractYarrowia lipolytica has emerged as a biomanufacturing platform for a variety of industrial applications. It has been demonstrated to be a robust cell factory for the production of renewable chemicals and enzymes for fuel, feed, oleochemical, nutraceutical and pharmaceutical applications. Metabolic engineering of this non-conventional yeast started through conventional molecular genetic engineering tools; however, recent advances in gene/genome editing systems, such as CRISPR–Cas9, transposons, and TALENs, has greatly expanded the applications of synthetic biology, metabolic engineering and functional genomics of Y. lipolytica. In this review we summarize the work to develop these tools and their demonstrated uses in engineering Y. lipolytica, discuss important subtleties and challenges to using these tools, and give our perspective on important gaps in gene/genome editing tools in Y. lipolytica.

scholarly journals PCR-Based Seamless Genome Editing with High Efficiency and Fidelity in Escherichia coli

PLoS ONE ◽  
2016 ◽  
Vol 11 (3) ◽  
pp. e0149762 ◽  
Author(s):  
Yilan Liu ◽  
Maohua Yang ◽  
Jinjin Chen ◽  
Daojiang Yan ◽  
Wanwan Cheng ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Genome Editing ◽  
High Efficiency

scholarly journals Efficient production of myo-inositol in Escherichia coli through metabolic engineering

2020 ◽  
Author(s):  
Ran You ◽  
Lei Wang ◽  
Congrong Shi ◽  
Hao Chen ◽  
Shasha Zhang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Metabolic Engineering ◽  
Carbon Flux ◽  
Value Added ◽  
Efficient Production ◽  
Wild Type ◽  
Food Industries ◽  
E Coli ◽  
Mixed Carbon Source

Abstract Background: The biosynthesis of high value-added compounds using metabolically engineered strains has received wide attention in recent years. Myo-inositol (inositol), an important compound in the pharmaceutics, cosmetics and food industries, is usually produced from phytate via a harsh set of chemical reactions. Recombinant Escherichia coli strains have been constructed by metabolic engineering strategies to produce inositol, but with a low yield. The proper distribution of carbon flux between cell growth and inositol production is a major challenge for constructing an efficient inositol-synthesis pathway in bacteria. Construction of metabolically engineered E. coli strains with high stoichiometric yield of inositol is desirable.Results: In the present study, we designed an inositol-synthesis pathway from glucose with a theoretical stoichiometric yield of 1 mol inositol/mol glucose. Recombinant E. coli strains with high stoichiometric yield (>0.7 mol inositol/mol glucose) were obtained. Inositol was successfully biosynthesized after introducing two crucial enzymes: inositol-3-phosphate synthase (IPS) from Trypanosoma brucei, and inositol monophosphatase (IMP) from E. coli. Based on starting strains E. coli BW25113 (wild-type) and SG104 (ΔptsG::glk, ΔgalR::zglf, ΔpoxB::acs), a series of engineered strains for inositol production was constructed by deleting the key genes pgi, pfkA and pykF. Plasmid-based expression systems for IPS and IMP were optimized, and expression of the gene zwf was regulated to enhance the stoichiometric yield of inositol. The highest stoichiometric yield (0.96 mol inositol/mol glucose) was achieved from recombinant strain R15 (SG104, Δpgi, Δpgm, and RBSL5-zwf). Strain R04 (SG104 and Δpgi) reached high-density in a 1-L fermenter when using glucose and glycerol as a mixed carbon source. In scaled-up fed-batch bioconversion in situ using strain R04, 0.82 mol inositol/mol glucose was produced within 23 h, corresponding to a titer of 106.3 g/L (590.5 mM) inositol.Conclusions: The biosynthesis of inositol from glucose in recombinant E. coli was optimized by metabolic engineering strategies. The metabolically engineered E. coli strains represent a promising method for future inositol production. This study provides an essential reference to obtain a suitable distribution of carbon flux between glycolysis and inositol synthesis.

scholarly journals Kinetic compartmentalization by unnatural reaction for itaconate production

2021 ◽  
Author(s):  
Daeyeol Ye ◽  
Myung Hyun Noh ◽  
Jo Hyun Moon ◽  
Alfonsina Milito ◽  
Minsun Kim ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Metabolic Engineering ◽  
Carbon Source ◽  
Metabolic Pathways ◽  
Mitochondrial Membrane ◽  
Membrane System ◽  
Proof Of Concept ◽  
Substrate Availability ◽  
Competitive Reactions ◽  
Metabolic Reactions

Abstract Physical compartmentalization of metabolisms using membranous organelles in eukaryotes is helpful for chemical biosynthesis to ensure the availability of substrates from competitive metabolic reactions. Bacterial hosts lack such a membranous system, which is one of the major limitations for efficient metabolic engineering. Here, we introduced kinetic compartmentalization as an alternative strategy to enable substrate availability from competitive reactions. This method utilizes a non-natural biochemical reaction performed by an engineered enzyme to kinetically isolate the metabolic pathways and ensure substrate availability for the desired reaction. As a proof of concept, we could successfully demonstrate kinetic separation for efficient itaconate production from acetate in Escherichia coli, mimicking the native mitochondrial membrane system in Aspergillus species. Despite the utilization of the non-preferred carbon source, kinetic compartmentalization could lead to substantial increases of itaconate in both yield and titer, suggesting enough potential of our strategy for broad applications in diverse engineering.

Metabolic engineering of Escherichia coli using CRISPR–Cas9 meditated genome editing

2015 ◽  
Vol 31 ◽  
pp. 13-21 ◽  
Author(s):  
Yifan Li ◽  
Zhenquan Lin ◽  
Can Huang ◽  
Yan Zhang ◽  
Zhiwen Wang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Metabolic Engineering ◽  
Genome Editing

scholarly journals Efficient CRISPR/Cas12a-based genome editing toolbox for metabolic engineering in Methanococcus maripaludis

2021 ◽  
Author(s):  
Jichen Bao ◽  
Silvan Scheller
Keyword(s):  
Metabolic Engineering ◽  
Genome Editing ◽  
Value Added ◽  
Primary Metabolism ◽  
Methanococcus Maripaludis ◽  
Knock Out ◽  
Homology Directed Repair ◽  
A Genome ◽  
Product Scope ◽  
Positive Rate

Methanococcus maripaludis is a fast-growing and genetically tractable methanogen. To become a useful host organism for the biotechnological conversion of CO2 and renewable hydrogen to fuels and value-added products, its product scope needs to be extended. Metabolic engineering requires reliable and efficient genetic tools, in particular for genome editing related to the primary metabolism that may affect cell growth. We have constructed a genome editing toolbox by utilizing Cas12a from Lachnospiraceae bacterium ND2006 (LbCas12a) in combination with the homology-directed repair machinery natively present in M. maripaludis. The toolbox enables gene knock-out with a positive rate typically above 89%, despite M. maripaludis being hyper-polyploid. We have replaced the flagellum operon (around 8.9kb) by a beta-glucuronidase gene to demonstrate a larger deletion, and to enable quantification of promotor strengths. The CRISPR/LbCas12a toolbox presented here is currently perhaps the most reliable and fastest method for genome editing in a methanogen.

scholarly journals Bioproduction of Isoprenoids and Other Secondary Metabolites Using Methanotrophic Bacteria as an Alternative Microbial Cell Factory Option: Current Stage and Future Aspects

Catalysts ◽  
2019 ◽  
Vol 9 (11) ◽  
pp. 883 ◽  
Author(s):  
Young Chan Jeon ◽  
Anh Duc Nguyen ◽  
Eun Yeol Lee
Keyword(s):  
Metabolic Engineering ◽  
Secondary Metabolites ◽  
Value Added ◽  
Industrial Applications ◽  
Cell Factory ◽  
Methanotrophic Bacteria ◽  
Microbial Cell Factory ◽  
Future Outlook ◽  
Metabolites Production ◽  
Current Stage

Methane is a promising carbon feedstock for industrial biomanufacturing because of its low price and high abundance. Recent advances in metabolic engineering and systems biology in methanotrophs have made it possible to produce a variety of value-added compounds from methane, including secondary metabolites. Isoprenoids are one of the largest family of secondary metabolites and have many useful industrial applications. In this review, we highlight the current efforts invested to methanotrophs for the production of isoprenoids and other secondary metabolites, including riboflavin and ectoine. The future outlook for improving secondary metabolites production (especially of isoprenoids) using metabolic engineering of methanotrophs is also discussed.

scholarly journals Cloning, expression and characterization of a chitinase from Paenibacillus chitinolyticus strain UMBR 0002

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e8964 ◽  
Author(s):  
Cong Liu ◽  
Naikun Shen ◽  
Jiafa Wu ◽  
Mingguo Jiang ◽  
Songbiao Shi ◽  
...  
Keyword(s):  
Genomic Library ◽  
Specific Activity ◽  
Sodium Dodecyl ◽  
Value Added ◽  
Industrial Applications ◽  
Degree Of Polymerization ◽  
Colloidal Chitin ◽  
Target Sequence ◽  
E Coli ◽  
Low Efficiency

Background Chitinases are enzymes which degrade β-1,4-glycosidid linkages in chitin. The enzymatic degradation of shellfish waste (containing chitin) to chitooligosaccharides is used in industrial applications to generate high-value-added products from such waste. However, chitinases are currently produced with low efficiency and poor tolerance, limiting the industrial utility. Therefore, identifying chitinases with higher enzymatic activity and tolerance is of great importance. Methods Primers were designed using the genomic database of Paenibacillus chitinolyticus NBRC 15660. An exochitinase (CHI) was cloned into the recombinant plasmid pET-22b (+) to form pET-22b (+)-CHI, which was transformed into Escherichia coli TOP10 to construct a genomic library. Transformation was confirmed by colony-polymerase chain reaction and electrophoresis. The target sequence was verified by sequencing. Recombinant pET-22b (+)-CHI was transformed into E. coli Rosetta-gami B (DE3) for expression of chitinase. Recombinant protein was purified by Ni-NTA affinity chromatography and enzymatic analysis was carried out. Results The exochitinase CHI from P. chitinolyticus strain UMBR 0002 was successfully cloned and heterologously expressed in E. coli Rosetta-gami B (DE3). Purification yielded a 13.36-fold enrichment and recovery yield of 72.20%. The purified enzyme had a specific activity of 750.64 mU mg−1. The optimum pH and temperature for degradation of colloidal chitin were 5.0 and 45 °C, respectively. The enzyme showed high stability, retaining >70% activity at pH 4.0–10.0 and 25–45 °C (maximum of 90 min). The activity of CHI strongly increased with the addition of Ca2+, Mn2+, Tween 80 and urea. Conversely, Cu2+, Fe3+, acetic acid, isoamyl alcohol, sodium dodecyl sulfate and β-mercaptoethanol significantly inhibited enzyme activity. The oligosaccharides produced by CHI from colloidal chitin exhibited a degree of polymerization, forming N-acetylglucosamine (GlcNAc) and (GlcNAc)2 as products. Conclusions This is the first report of the cloning, heterologous expression and purification of a chitinase from P. chitinolyticus strain UMBR 0002. The results highlight CHI as a good candidate enzyme for green degradation of chitinous waste.

scholarly journals Reversed paired-gRNA plasmid cloning strategy for efficient genome editing in Escherichia coli

2019 ◽  
Author(s):  
Tingting Ding ◽  
Chaoyong Huang ◽  
Zeyu Liang ◽  
Xiaoyan Ma ◽  
Ning Wang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Genome Editing ◽  
High Efficiency ◽  
Replication Slippage ◽  
High Frequencies ◽  
Guide Rnas ◽  
Large Deletions ◽  
Plasmid Recombination ◽  
Cloning Strategy ◽  
Chromosome Fragments

Abstract BackgroundThe CRISPR-Cas9 system is a powerful tool for genome editing in various organisms. Several of its applications, including the generation of large deletions, require co-expression of two distinct guide RNAs (gRNAs). However, the instability of paired-gRNA plasmids prevents these applications from being scalable in Escherichia coli. Coexpressing paired gRNAs under the driving of independent but identical promoters in the same direction triggers plasmid recombination, due to the presence of direct repeats (DRs). ResultsIn this study, plasmid deletion between DRs occurred with high frequencies during plasmid construction and subsequent duplication processes, when three DRs-involved paired-gRNA plasmids cloning strategies were tested. This recombination phenomenon was RecA-independent, in agreement with the replication slippage model. To completely eliminate the DRs-induced plasmid instability, a reversed paired-gRNA plasmids (RPGPs) cloning strategy was developed by converting DRs to the more stable invert repeats (IRs). ConclusionsUsing RPGPs, we achieved a rapid deletion of chromosome fragments up to 100 kb with high efficiency of 83.33% in Escherichia coli. This study provides general solutions to construct stable plasmids containing short DRs, which can improve the performances of CRISPR systems that rely on paired gRNAs, and also facilitate other applications involving repeated genetic parts.

Sign in / Sign up

Export Citation Format

Share Document