Metabolic engineering of Escherichia coli for de novo biosynthesis of vitamin B12

Fang, Huan; Li, Dong; Kang, Jie; Jiang, Pingtao; Sun, Jibin; Zhang, Dawei

doi:10.1038/s41467-018-07412-6

Cited by 119 publications

(86 citation statements)

References 44 publications

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“…Some types of Co-dependent enzymes have been overexpressed in E. coli, as well as the high-level synthesis of vitamin B12 in E. coli through metabolic engineering (Wu et al, 2017;Fang et al, 2018;Moon et al, 2018). In most cases of expressing and characterizing Co-dependent enzymes, the enzymes were first overexpressed and purified, then cobalt ions were added in vitro.…”

Section: Resultsmentioning

confidence: 99%

Efficient Overproduction of Active Nitrile Hydratase by Coupling Expression Induction and Enzyme Maturation via Programming a Controllable Cobalt-Responsive Gene Circuit

Han

Cui

Lin

et al. 2020

Front. Bioeng. Biotechnol.

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A robust and portable expression system is of great importance in enzyme production, metabolic engineering, and synthetic biology, which maximizes the performance of the engineered system. In this study, a tailor-made cobalt-induced expression system (CIES) was developed for low-cost and eco-friendly nitrile hydratase (NHase) production. First, the strong promoter P veg from Bacillus subtilis, the Ni(II)/Co(II) responsive repressor RcnR, and its operator were reorganized to construct a CIES. In this system, the expression of reporter green fluorescent protein (GFP) was specifically triggered by Co(II) over a broad range of concentration. The performance of the cobalt-induced system was evolved to version 2.0 (CIES 2.0) for adaptation to different concentrations of Co(II) through programming a homeostasis system that rebalances cobalt efflux and influx with RcnA and NiCoT, respectively. Harnessing these synthetic platforms, the induced expression of NHase was coupled with enzyme maturation by Co(II) in a synchronizable manner without requiring additional inducers, which is a unique feature relative to other induced systems for production of NHase. The yield of NHase was 111.2 ± 17.9 U/ml using CIES and 114.9 ± 1.4 U/ml using CIES 2.0, which has a producing capability equivalent to that of commonly used isopropyl thiogalactoside (IPTG)-induced systems. In a scale-up system using a 5-L fermenter, the yielded enzymatic activity reached 542.2 ± 42.8 U/ml, suggesting that the designer platform for NHase is readily applied to the industry. The design of CIES in this study not only provided a low-cost and eco-friendly platform to overproduce NHase but also proposed a promising pipeline for development of synthetic platforms for expression of metalloenzymes.

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Section: Resultsmentioning

confidence: 99%

Efficient Overproduction of Active Nitrile Hydratase by Coupling Expression Induction and Enzyme Maturation via Programming a Controllable Cobalt-Responsive Gene Circuit

Han

Cui

Lin

et al. 2020

Front. Bioeng. Biotechnol.

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“…The DSBs produced by Cas9 can be repaired by the non-homologous end joining (NHEJ) pathway or the homology-directed repair (HDR) system. A combination of Cas9 protein with DNA repair pathways can be used for gene deletion or insertion in the bacterial genome [16].…”

Section: Applications Of Type II Crispr/cas Systems In Bacteriamentioning

confidence: 99%

Application of different types of CRISPR/Cas-based systems in bacteria

et al. 2020

Self Cite

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As important genome editing tools, CRISPR/Cas systems, especially those based on type II Cas9 and type V Cas12a, are widely used in genetic and metabolic engineering of bacteria. However, the intrinsic toxicity of Cas9 and Cas12amediated CRISPR/Cas tools can lead to cell death in some strains, which led to the development of endogenous type I and III CRISPR/Cas systems. However, these systems are hindered by complicated development and limited applications. Thus, further development and optimization of CRISPR/Cas systems is needed. Here, we briefly summarize the mechanisms of different types of CRISPR/Cas systems as genetic manipulation tools and compare their features to provide a reference for selecting different CRISPR/Cas tools. Then, we show the use of CRISPR/Cas technology for bacterial strain evolution and metabolic engineering, including genome editing, gene expression regulation and the base editor tool. Finally, we offer a view of future directions for bacterial CRISPR/Cas technology.

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“…The reconstitution of genetic clusters in heterologous hosts has often been used to improve the production performance or diversify the product spectrum over the native producers . Recently, Escherichia coli ( E. coli ) has successfully overexpressed nearly 30 genes to produce vitamin B 12 via an engineered de novo aerobic biosynthetic pathway . To optimize the flux through metabolic pathways, the modulation of enzyme expression using a synthetic protein scaffold to improve the pathway flux has been demonstrated .…”

Section: Current Industrial Biotechnologymentioning

confidence: 99%

Next‐Generation Industrial Biotechnology‐Transforming the Current Industrial Biotechnology into Competitive Processes

Chen

2019

Biotechnology Journal

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The chemical industry has made a contribution to modern society by providing cost‐competitive products for our daily use. However, it now faces a serious challenge regarding environmental pollutions and greenhouse gas emission. With the rapid development of molecular biology, biochemistry, and synthetic biology, industrial biotechnology has evolved to become more efficient for production of chemicals and materials. However, in contrast to chemical industries, current industrial biotechnology (CIB) is still not competitive for production of chemicals, materials, and biofuels due to their low efficiency and complicated sterilization processes as well as high‐energy consumption. It must be further developed into “next‐generation industrial biotechnology” (NGIB), which is low‐cost mixed substrates based on less freshwater consumption, energy‐saving, and long‐lasting open continuous intelligent processing, overcoming the shortcomings of CIB and transforming the CIB into competitive processes. Contamination‐resistant microorganism as chassis is the key to a successful NGIB, which requires resistance to microbial or phage contaminations, and available tools and methods for metabolic or synthetic biology engineering. This review proposes a list of contamination‐resistant bacteria and takes Halomonas spp. as an example for the production of a variety of products, including polyhydroxyalkanoates under open‐ and continuous‐processing conditions proposed for NGIB.

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Metabolic engineering of Escherichia coli for de novo biosynthesis of vitamin B12

Cited by 119 publications

References 44 publications

Efficient Overproduction of Active Nitrile Hydratase by Coupling Expression Induction and Enzyme Maturation via Programming a Controllable Cobalt-Responsive Gene Circuit

Efficient Overproduction of Active Nitrile Hydratase by Coupling Expression Induction and Enzyme Maturation via Programming a Controllable Cobalt-Responsive Gene Circuit

Application of different types of CRISPR/Cas-based systems in bacteria

Next‐Generation Industrial Biotechnology‐Transforming the Current Industrial Biotechnology into Competitive Processes

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