Abstract
BackgroundOrthogonal T7 RNA polymerase (T7RNAP) and T7 promoter is a powerful genetic element to mediate protein expression in different cells. Among all, Escherichia coli possess advantages of fast growth rate, easy for culture and comprehensive elements for genetic engineering. As E. coli W3110 owns the benefits of more heat shock proteins and higher tolerance to toxic chemicals, further execution of T7-based system in W3110 as cell factory is a conceivable strategy.ResultsThree novel W3110 strains, i.e., W3110:IL5, W3110::L5 and W3110::pI, were accomplished by chromosome-equipped T7RNAP. At first, the LacZ and T7RNAP with isopropyl-β-D-thiogalactopyranoside (IPTG) induction showed higher expression levels in W3110 derivatives than that in BL21(DE3). The plasmids with and without lacI/lacO repression were used to investigate the protein expression of super-fold green fluorescence protein (sfGFP), carbonic anhydrase (CA) for carbon dioxide uptake and lysine decarboxylase (CadA) to produce a toxic chemical cadaverine (DAP). All the proteins showed better expression in W3110::L5 and W3110::pI, respectively. As a result, the highest cadaverine production of 36.9 g/L, lysine consumption of 43.8 g/L and up to 100% yield were obtained in W3110::pI(−) with plasmid pSU-T7-CadA constitutively.ConclusionEffect of IPTG and lacI/lacO regulator has been investigated in three chromosome-based T7RNAP E. coli strains. The newly engineered W3110 strains possessed similar protein expression compared to commercial BL21(DE3). Furthermore, W3110::pI displays higher production of sfGFP, CA and CadA, due to it having the highest sensitivity to IPTG, thus it represents the greatest potential as a cell factory.
Highlights
It is indispensable to develop the recombinant technology which enables enhanced enzyme expression to endorse high-value chemicals production in recent years
The E. coli DH5α was used for plasmid construction, and BL21(DE3) was applied for gene expression, while W3110 was engineered to equip with a chromosome-based T7 RNA polymerase (T7RNAP) by HK022 phage attack site
Engineered E. coli W3110 equipped with T7RNAP Three different T7RNAP cassettes were constructed and integrated into the chromosome of W3110 by traditional site-specific recombination at the attB sites of lambdoid coliphage HK022 (Fig. 1a)
Summary
It is indispensable to develop the recombinant technology which enables enhanced enzyme expression to endorse high-value chemicals production in recent years. The cellular engineering in E. coli involved plasmid construction (Datta et al 2006), pET system for overexpression under orthogonal T7RNAP and promoter (Studier and Moffatt 1986), homologous recombination (Yu et al 2000), and CRISPR/Cas technology for genome editing recently (Chung et al 2016; Jiang et al 2013; Ng et al 2016). Orthogonal T7 RNA polymerase (T7RNAP) and T7 promoter is a powerful genetic element to mediate protein expression in different cells. As E. coli W3110 owns the benefits of more heat shock proteins and higher tolerance to toxic chemicals, further execution of T7-based system in W3110 as cell factory is a conceivable strategy
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