Overview of Regulatory Strategies and Molecular Elements in Metabolic Engineering of Bacteria

Wang, Tianwen; Ma, Xiaochun; Du, Guocheng; Chen, Jian

doi:10.1007/s12033-012-9514-y

Cited by 20 publications

(24 citation statements)

References 81 publications

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“…The relative copy number of a pCD034-1derivative, pCDLbu-1, was estimated to exceed 200 copies per chromosome [27]. Besides transcription and gene dosage, also translation can be a major bottle neck and design of the ribosomal binding site strongly influences protein production levels [28,29].…”

Section: Introductionmentioning

confidence: 99%

Tuning constitutive recombinant gene expression in Lactobacillus plantarum

et al. 2014

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BackgroundLactobacillus plantarum constitutes a well-recognized food-grade system for the expression of recombinant proteins in the field of industrial and medical biotechnology. For applications in vivo or in biotechnological processes, the level of expression of e.g. antigens or enzymes is often critical, as expression levels should be of a certain effectiveness, yet, without putting too much strain to the overall system. The key factors that control gene expression are promoter strength, gene copy number and translation efficiency. In order to estimate the impact of these adjusting screws in L. plantarum CD033, we have tested several constitutive promoters in combination with high and low copy number plasmid backbones and varying space between the Shine-Dalgarno sequence and the start-codon.ResultsBy combining strong promoters, such as transcription elongation factor promoters, isolated from L. plantarum CD033 and L. buchneri CD034, a synthetic promoter, originally derived from L. plantarum WCSF1 and a heterologous promoter derived from L. buchneri CD034 with a high and a low copy number origin of replication we demonstrated various expression levels of the model protein mCherry. All promoters were feasible for protein expression and in all cases, the high copy number origin of replication increased expression twofold. We found that the optimal spacer between the Shine-Dalgarno sequence and the start codon in L. plantarum consists of 8 nucleotides and elongation as well as shortening this sequence gradually down-regulates gene expression.ConclusionsWe have evaluated the effects of a set of gene regulatory tools to fine tune recombinant gene expression in L. plantarum CD033. We have thus, provided potential expression vectors useful for constitutive protein expression in lactic acid bacteria ranging from moderate to strong production levels.

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Section: Introductionmentioning

confidence: 99%

Tuning constitutive recombinant gene expression in Lactobacillus plantarum

et al. 2014

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“…Inducible gene expression systems are useful tools in metabolic engineering as well as studies of gene function (19). They are commonly employed for genetic complementation studies, for finetuning protein expression for physiological studies, to express toxic genes for counterselection, and/or to express a synthetic pathway for synthesis of desired products.…”

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confidence: 99%

Lactose-Inducible System for Metabolic Engineering of Clostridium ljungdahlii

Banerjee

Leang

Ueki

et al. 2014

Appl Environ Microbiol

103

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The development of tools for genetic manipulation of Clostridium ljungdahlii has increased its attractiveness as a chassis for autotrophic production of organic commodities and biofuels from syngas and microbial electrosynthesis and established it as a model organism for the study of the basic physiology of acetogenesis. In an attempt to expand the genetic toolbox for C. ljungdahlii, the possibility of adapting a lactose-inducible system for gene expression, previously reported for Clostridium perfringens, was investigated. The plasmid pAH2, originally developed for C. perfringens with a gusA reporter gene, functioned as an effective lactose-inducible system in C. ljungdahlii. Lactose induction of C. ljungdahlii containing pB1, in which the gene for the aldehyde/alcohol dehydrogenase AdhE1 was downstream of the lactose-inducible promoter, increased expression of adhE1 30-fold over the wild-type level, increasing ethanol production 1.5-fold, with a corresponding decrease in acetate production. Lactose-inducible expression of adhE1 in a strain in which adhE1 and the adhE1 homolog adhE2 had been deleted from the chromosome restored ethanol production to levels comparable to those in the wild-type strain. Inducing expression of adhE2 similarly failed to restore ethanol production, suggesting that adhE1 is the homolog responsible for ethanol production. Lactose-inducible expression of the four heterologous genes necessary to convert acetyl coenzyme A (acetyl-CoA) to acetone diverted ca. 60% of carbon flow to acetone production during growth on fructose, and 25% of carbon flow went to acetone when carbon monoxide was the electron donor. These studies demonstrate that the lactose-inducible system described here will be useful for redirecting carbon and electron flow for the biosynthesis of products more valuable than acetate. Furthermore, this tool should aid in optimizing microbial electrosynthesis and for basic studies on the physiology of acetogenesis.

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“…Besides selecting appropriate sources of enzymes, another approach available is to create enzymes with the desired functions through site-directed mutagenesis (Wang et al 2012;Tee and Wong 2013). Protein engineering and mutagenesis of 4CL and STS have indeed been applied to improve resveratrol production capabilities in microorganisms.…”

Section: Enzyme Selection and Protein Engineeringmentioning

confidence: 99%

Heterologous production of resveratrol in bacterial hosts: current status and perspectives

Braga

Ferreira

Oliveira

et al. 2018

World J Microbiol Biotechnol

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The polyphenol resveratrol (3,5,4'-trihydroxystilbene) is a well-known plant secondary metabolite, commonly used as a medical ingredient and a nutritional supplement. Due to its health-promoting properties, the demand for resveratrol is expected to continue growing. This stilbene can be found in different plants, including grapes, berries (blackberries, blueberries and raspberries), peanuts and their derived food products, such as wine and juice. The commercially available resveratrol is usually extracted from plants, however this procedure has several drawbacks such as low concentration of the product of interest, seasonal variation, risk of plant diseases and product stability. Alternative production processes are being developed to enable the biotechnological production of resveratrol by genetically engineering several microbial hosts, such as Escherichia coli, Corynebacterium glutamicum, Lactococcus lactis, among others. However, these bacterial species are not able to naturally synthetize resveratrol and therefore genetic modifications have been performed. The application of emerging metabolic engineering offers new possibilities for strain and process optimization. This mini-review will discuss the recent progress on resveratrol biosynthesis in engineered bacteria, with a special focus on the metabolic engineering modifications, as well as the optimization of the production process. These strategies offer new tools to overcome the limitations and challenges for microbial production of resveratrol in industry.

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Overview of Regulatory Strategies and Molecular Elements in Metabolic Engineering of Bacteria

Cited by 20 publications

References 81 publications

Tuning constitutive recombinant gene expression in Lactobacillus plantarum

Tuning constitutive recombinant gene expression in Lactobacillus plantarum

Lactose-Inducible System for Metabolic Engineering of Clostridium ljungdahlii

Heterologous production of resveratrol in bacterial hosts: current status and perspectives

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