Yeast extract mediated autoinduction of lacUV5 promoter: an insight

Nair, Rahul Ravindran; Salvi, Pankaj; Banerjee, Sampali; Raiker, Veena A.; Bandyopadhyay, Suman; Soorapaneni, Sudheerbabu; Kotwal, Prakash; Padmanabhan, Sriram

doi:10.1016/j.nbt.2009.08.002

Cited by 16 publications

(7 citation statements)

References 16 publications

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“…The complex unrefined nature of the media ingredients used (namely yeast extract and tryptone peptone) results in an inherent variation in the composition of these according to the commercial source and production batch and we have previously noted unintended induction for various proteins as a result of these disparities. Indeed others have also noted this and indicated that low variable levels of lactose contamination in yeast extract and to a lesser extent also tryptone peptone may be responsible for this unintended induction in rich media [24,41]. …”

Section: Resultsmentioning

confidence: 92%

Batch production of a silk-elastin-like protein in E. coli BL21(DE3): key parameters for optimisation

et al. 2013

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BackgroundSilk-elastin-like proteins (SELPs) combining the physicochemical and biological properties of silk and elastin have a high potential for use in the pharmaceutical, regenerative medicine and materials fields. Their development for use is however restrained by their production levels. Here we describe the batch production optimisation for a novel recently described SELP in the pET-E. coli BL21(DE3) expression system. Both a comprehensive empirical approach examining all process variables (media, induction time and period, temperature, pH, aeration and agitation) and a detailed characterisation of the bioprocess were carried out in an attempt to maximise production with this system.ResultsThis study shows that maximum SELP volumetric production is achieved at 37°C using terrific broth at pH 6–7.5, a shake flask volume to medium volume ratio of 10:1 and an agitation speed of 200 rpm. Maximum induction is attained at the beginning of the stationary phase with 0.5 mM IPTG and an induction period of at least 4 hours. We show that the selection agents ampicillin and carbenicillin are rapidly degraded early in the cultivation and that plasmid stability decreases dramatically on induction. Furthermore, acetate accumulates during the bioprocess to levels which are shown to be inhibitory to the host cells. Using our optimised conditions, 500 mg/L of purified SELP was obtained.ConclusionsWe have identified the optimal conditions for the shake flask production of a novel SELP with the final production levels obtained being the highest reported to date. While this study is focused on SELPs, we believe that it could also be of general interest to any study where the pET (ampicillin selective marker)-E. coli BL21(DE3) expression system is used. In particular, we show that induction time is critical in this system with, in contrast to that which is generally believed, optimal production being obtained by induction at the beginning of the stationary phase. Furthermore, we believe that we are at or near the maximum productivity for the system used, with rapid degradation of the selective agent by plasmid encoded β-lactamase, plasmid instability on induction and high acetate production levels being the principal limiting factors for further improved production.

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Section: Resultsmentioning

confidence: 92%

Batch production of a silk-elastin-like protein in E. coli BL21(DE3): key parameters for optimisation

et al. 2013

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“…6c ). The presence of lactose in medium containing yeast extract was previously described and could explain the effectiveness of SILEX in these media 25 . A reasonable hypothesis may be that the interaction between E. coli GAPDH and hHsp70 decreases glucose metabolism, thereby favoring the lactose energy source; hence, lactose induction with either a low amount of lactose or residual glucose in the medium is favored.…”

Section: Discussionmentioning

confidence: 91%

A self-inducible heterologous protein expression system in Escherichia coli

Briand

Marcion

Kriznik

et al. 2016

Sci Rep

106

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Escherichia coli is an important experimental, medical and industrial cell factory for recombinant protein production. The inducible lac promoter is one of the most commonly used promoters for heterologous protein expression in E. coli. Isopropyl-β-D-thiogalactoside (IPTG) is currently the most efficient molecular inducer for regulating this promoter’s transcriptional activity. However, limitations have been observed in large-scale and microplate production, including toxicity, cost and culture monitoring. Here, we report the novel SILEX (Self-InducibLe Expression) system, which is a convenient, cost-effective alternative that does not require cell density monitoring or IPTG induction. We demonstrate the broad utility of the presented self-inducible method for a panel of diverse proteins produced in large amounts. The SILEX system is compatible with all classical culture media and growth temperatures and allows protein expression modulation. Importantly, the SILEX system is proven to be efficient for protein expression screening on a microplate scale.

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“…Supplementation of vinasse with yeast extract at 5 g L −1 showed the greatest improvement in maximum biomass concentration and CV2025 ω‐TAm specific activity, increasing 1.7 and 3.7‐fold, respectively compared with the non‐supplemented culture. The presence of vitamins, minerals and transcription enhancers, such as cyclic adenosine monophosphate (cAMP), in the yeast extract may also facilitate recombinant protein expression as previously reported …”

Section: Resultsmentioning

confidence: 99%

Potential of sugar beet vinasse as a feedstock for biocatalyst production within an integrated biorefinery context

Suhaili

Cárdenas-Fernández

Ward

et al. 2018

J of Chemical Tech & Biotech

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BACKGROUND: This work explores the feasibility of vinasse as an inexpensive feedstock for industrial biocatalyst production within the context of an integrated sugar beet biorefinery. As an exemplar, production of CV2025 -Transaminase ( -TAm) in Escherichia coli BL21 was studied. RESULTS:Characterisation of vinasse showed that it comprised mainly of glycerol along with several reducing sugars, sugar alcohols, acetate, polyphenols and protein. Preliminary results showed E. coli BL21 cell growth and CV2025 -TAm production were feasible in cultures using 17% to 25% (v/v) vinasse with higher concentrations demonstrating inhibitory effects. The D-galactose present in vinasse facilitated auto-induction of the pQR801 plasmid enabling CV2025 -TAm expression without addition of expensive Isopropyl--D-thiogalactopyranoside (IPTG). Assessment of different vinasse pre-processing options confirmed simple dilution of the vinasse was sufficient to reduce the concentration of polyphenols to below inhibitory levels. Optimisation experiments, carried out using a controlled, 24-well microbioreactor platform, showed supplementation of diluted vinasse medium with 10 g L −1 yeast extract enabled enhancements of 2.8, 2.5, 5.4 and 3-fold in specific growth rate, maximum biomass concentration, CV2025 -TAm volumetric and specific activity, respectively. Investigation into the metabolic preferences of E. coli BL21 when grown in vinasse showed a preference for D-mannitol utilisation before simultaneous metabolism of glycerol, D-xylitol, D-dulcitol and acetate. Scale-up of optimised conditions for batch CV2025 -TAm production to a 7.5 L stirred tank reactor (STR) was demonstrated based on matched volumetric mass transfer coefficient (k L a). The results showed good comparability with respect to cell growth, substrate consumption and CV2025 -TAm production representing over a 700-fold volumetric scale translation. Further enhancements in CV2025 -TAm production were possible in the STR when operated at higher k L a values. CONCLUSION: This work describes the promising application of vinasse for production of microbial enzymes and insights into carbon source utilisation in complex feedstocks. Exploitation of vinasse as a fermentation feedstock could be further extended to other processes involving different microorganisms and target enzymes.

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Yeast extract mediated autoinduction of lacUV5 promoter: an insight

Cited by 16 publications

References 16 publications

Batch production of a silk-elastin-like protein in E. coli BL21(DE3): key parameters for optimisation

Batch production of a silk-elastin-like protein in E. coli BL21(DE3): key parameters for optimisation

A self-inducible heterologous protein expression system in Escherichia coli

Potential of sugar beet vinasse as a feedstock for biocatalyst production within an integrated biorefinery context

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