Tunable recombinant protein expression in E. coli: enabler for continuous processing?

Marschall, Lukas; Sagmeister, Patrick; Herwig, Christoph

doi:10.1007/s00253-016-7550-4

Cited by 35 publications

(26 citation statements)

References 67 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…21 Therefore, for optimum production, the expression levels must be modulated and adjusted. 22,23 The enzymatic activity of the protein is indicative of proper folding and solubility. In this study, the conditions required to obtain the greatest amount of active protein in the most efficient manner were established.…”

Section: Resultsmentioning

confidence: 99%

Optimizing the expression of a Heterologous chitinase: A study of different promoters

et al. 2016

View full text Add to dashboard Cite

Many relevant applications have been demonstrated for chitinolytic enzymes. However, their successful exploitation depends upon the availability of strains and expression conditions that allow the production of active forms and large quantities of these enzymes. Escherichia coli has been commonly used to express and overproduce different proteins, among them chitinases. Improving the functional gene expression of chitinases is key to exploiting their potential. In a recent study, we described the effect of various parameters on the functional expression of 2 chitinases from different families, demonstrating that the effect of each of these parameters on the activity of both chitinases was specific to each enzyme. In this study, the expression of a Lactococcus lactis chitinase encoded by a new allele, ChiA1-2, was optimized. The results showed that not only the expression parameters seemed to influence protein production, solubility and activity but also the plasmid used for the expression. Herein, we describe the effect of 2 different promoters, tac and T7, on the expression of the active form of the chitinolytic enzyme.

show abstract

Section: Resultsmentioning

confidence: 99%

Optimizing the expression of a Heterologous chitinase: A study of different promoters

et al. 2016

View full text Add to dashboard Cite

show abstract

“…The highly efficient, robust, and tunable gene expression system is essential for heterologous protein production (Ahmad et al, 2014;Ito et al, 2015;Marschall et al, 2016). Various gene expression systems are induced by physical signals (e.g., light and temperature), and natural and synthetic molecules have been constructed in different organisms (Tabor and Richardson, 1985;Bhavsar et al, 2001;Phan and Schumann, 2007;Zhao et al, 2018;Castillo-Hair et al, 2019).…”

Section: Introductionmentioning

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.

View full text Add to dashboard Cite

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.

show abstract

“…In comparison, microbial conversion processes can be advantageous for their mild production conditions and direct conversion instead of step-wise chemical conversions (some steps can have low yield and high cost) [10]. Also, metabolic engineering and bioreactor engineering tools can be used to maximize the yield and selectivity of the desired product and thus minimize the concentrations of coproducts [11][12][13][14][15][16].…”

Section: Introductionmentioning

confidence: 99%

Synthesis and analysis of separation processes for extracellular chemicals generated from microbial conversions

Yenkie

Maravelias

2019

BMC Chem Eng

View full text Add to dashboard Cite

Recent advances in metabolic engineering have enabled the production of chemicals via bio-conversion using microbes. However, downstream separation accounts for 60-80% of the total production cost in many cases. Previous work on microbial production of extracellular chemicals has been mainly restricted to microbiology, biochemistry, metabolomics, or techno-economic analysis for specific product examples such as succinic acid, xanthan gum, lycopene, etc. In these studies, microbial production and separation technologies were selected apriori without considering any competing alternatives. However, technology selection in downstream separation and purification processes can have a major impact on the overall costs, product recovery, and purity. To this end, we apply a superstructure optimization based framework that enables the identification of critical technologies and their associated parameters in the synthesis and analysis of separation processes for extracellular chemicals generated from microbial conversions. We divide extracellular chemicals into three categories based on their physical properties, such as water solubility, physical state, relative density, volatility, etc. We analyze three major extracellular product categories (insoluble light, insoluble heavy and soluble) in detail and provide suggestions for additional product categories through extension of our analysis framework. The proposed analysis and results provide significant insights for technology selection and enable streamlined decision making when faced with any microbial product that is released extracellularly. The parameter variability analysis for the product as well as the associated technologies and comparison with novel alternatives is a key feature which forms the basis for designing better bioseparation strategies that have potential for commercial scalability and can compete with traditional chemical production methods.

show abstract

Tunable recombinant protein expression in E. coli: enabler for continuous processing?

Cited by 35 publications

References 67 publications

Optimizing the expression of a Heterologous chitinase: A study of different promoters

Optimizing the expression of a Heterologous chitinase: A study of different promoters

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

Synthesis and analysis of separation processes for extracellular chemicals generated from microbial conversions

Contact Info

Product

Resources

About