Dataset Information

Genetic Engineering of Escherichia coli BL21 (DE3) with a codon-optimized insecticidal toxin complex gene tccZ.

ABSTRACT: A toxin complex consists of a high-molecular-weight group of toxins that exhibits insecticidal activity against insect pests. These toxins are a promising alternative to Bacillus thuringiensis (Bt) toxins that have been extensively utilized in insect pest control. Herein, a codon-optimized insecticidal gene (tccZ) (381 bp) identified in Pantoea ananatis strain MHSD5 (a bacterial endophyte previously isolated from Pellaea calomelanos) was ligated into the pET SUMO expression vector and expressed in Escherichia coli BL21 (DE3). We report the success of cloning the tccZ gene into the pET SUMO vector and ultimately the transformation into E. coli BL21 (DE3) competent cells. However, despite conducting a time course of expression as well as isopropyl β-d-1-thiogalactopyranoside (IPTG) dosage optimization to determine optimal conditions for expression, TccZ protein expression could not be detected on Stain-Free and Coomassie-stained SDS-PAGE gels.

SUBMITTER: Malomane MT

PROVIDER: S-EPMC9968953 | biostudies-literature | 2023

REPOSITORIES: biostudies-literature

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Genetic Engineering of Escherichia coli BL21 (DE3) with a codon-optimized insecticidal toxin complex gene tccZ.

Malomane Mosibudi Thabiki MT Kondiah Kulsum K Serepa-Dlamini Mahloro Hope MH

Access microbiology 20230124 1

A toxin complex consists of a high-molecular-weight group of toxins that exhibits insecticidal activity against insect pests. These toxins are a promising alternative to Bacillus thuringiensis (Bt) toxins that have been extensively utilized in insect pest control. Herein, a codon-optimized insecticidal gene (tccZ) (381 bp) identified in Pantoea ananatis strain MHSD5 (a bacterial endophyte previously isolated from Pellaea calomelanos) was ligated into the pET SU ...[more]

PMID: 36860507

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Project description:The Escherichia coli B strain BL21(DE3) has had a profound impact on biotechnology through its use in the production of recombinant proteins. Little is understood, however, regarding the physiology of this important E. coli strain. We show here that BL21(DE3) totally lacks activity of the four [NiFe]-hydrogenases, the three molybdenum- and selenium-containing formate dehydrogenases and molybdenum-dependent nitrate reductase. Nevertheless, all of the structural genes necessary for the synthesis of the respective anaerobic metalloenzymes are present in the genome. However, the genes encoding the high-affinity molybdate transport system and the molybdenum-responsive transcriptional regulator ModE are absent from the genome. Moreover, BL21(DE3) has a nonsense mutation in the gene encoding the global oxygen-responsive transcriptional regulator FNR. The activities of the two hydrogen-oxidizing hydrogenases, therefore, could be restored to BL21(DE3) by supplementing the growth medium with high concentrations of Ni²⁺ (Ni²⁺-transport is FNR-dependent) or by introducing a wild-type copy of the fnr gene. Only combined addition of plasmid-encoded fnr and high concentrations of MoO₄²⁻ ions could restore hydrogen production to BL21(DE3); however, to only 25-30% of a K-12 wildtype. We could show that limited hydrogen production from the enzyme complex responsible for formate-dependent hydrogen evolution was due solely to reduced activity of the formate dehydrogenase (FDH-H), not the hydrogenase component. The activity of the FNR-dependent formate dehydrogenase, FDH-N, could not be restored, even when the fnr gene and MoO₄²⁻ were supplied; however, nitrate reductase activity could be recovered by combined addition of MoO₄²⁻ and the fnr gene. This suggested that a further component specific for biosynthesis or activity of formate dehydrogenases H and N was missing. Re-introduction of the gene encoding ModE could only partially restore the activities of both enzymes. Taken together these results demonstrate that BL21(DE3) has major defects in anaerobic metabolism, metal ion transport and metalloprotein biosynthesis.

Project description:BackgroundEscherichia coli is one of the most widely used hosts for recombinant protein production in academia and industry. Strain BL21(DE3) is frequently employed due to its advantageous feature of lacking proteases which avoids degradation of target protein. Usually it is used in combination with the T7-pET system where induction is performed by one point addition of IPTG. We recently published a few studies regarding lactose induction in BL21(DE3) strains. BL21(DE3) can only take up the glucose-part of the disaccharide when fed with lactose. However, initially additional glucose has to be supplied as otherwise the ATP-related lactose uptake barely happens. Yet, as lactose is an inexpensive compound compared to glucose and IPTG, a new induction strategy by a lactose-only feed during induction seems attractive. Thus, we investigated this idea in the galactose metabolizing strain HMS174(DE3).ResultsWe show that strain HMS174(DE3) can be cultivated on lactose as sole carbon source during induction. We demonstrate that strain HMS174(DE3) exhibits higher product and biomass yields compared to BL21(DE3) when cultivated in a lactose fed-batch. More importantly, HMS174(DE3) cultivated on lactose even expresses more product than BL21(DE3) in a standard IPTG induced glucose fed-batch at the same growth rate. Finally, we demonstrate that productivity in HMS174(DE3) lactose-fed batch cultivations can easily be influenced by the specific lactose uptake rate (qs,lac). This is shown for two model proteins, one expressed in soluble form and one as inclusion body.ConclusionsAs strain HMS174(DE3) expresses even slightly higher amounts of target protein in a lactose fed-batch than BL21(DE3) in a standard cultivation, it seems a striking alternative for recombinant protein production. Especially for large scale production of industrial enzymes cheap substrates are essential. Besides cost factors, the strategy allows straight forward adjustment of specific product titers by variation of the lactose feed rate.

Dataset Information

Genetic Engineering of Escherichia coli BL21 (DE3) with a codon-optimized insecticidal toxin complex gene tccZ.

Publications

Genetic Engineering of <i>Escherichia coli</i> BL21 (DE3) with a codon-optimized insecticidal toxin complex gene <i>tccZ</i>.

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