Tunable expression rate control of a growth-decoupled T7 expression system by l-arabinose only

Abstract

Background Precise regulation of gene expression is of utmost importance for the production of complex membrane proteins (MP), enzymes or other proteins toxic to the host cell. In this article we show that genes under control of a normally Isopropyl β-d-1-thiogalactopyranoside (IPTG)-inducible PT7-lacO promoter can be induced solely with l-arabinose in a newly constructed Escherichia coli expression host BL21-AI<gp2>, a strain based on the recently published approach of bacteriophage inspired growth-decoupled recombinant protein production. Results Here, we show that BL21-AI<gp2> is able to precisely regulate protein production rates on a cellular level in an l-arabinose concentration-dependent manner and simultaneously allows for reallocation of metabolic resources due to l-arabinose induced growth decoupling by the phage derived inhibitor peptide Gp2. We have successfully characterized the system under relevant fed-batch like conditions in microscale cultivation (800 µL) and generated data proofing a relevant increase in specific yields for 6 different Escherichia coli derived MP-GFP fusion proteins by using online-GFP signals, FACS analysis, SDS-PAGE and western blotting. Conclusions In all cases tested, BL21-AI<gp2> outperformed the parental strain BL21-AI, operated in growth-associated production mode. Specific MP-GFP fusion proteins yields have been improved up to 2.7-fold. Therefore, this approach allows for fine tuning of MP production or expression of multi-enzyme pathways where e.g. particular stoichiometries have to be met to optimize product flux.

Regulating the T7 RNA Polymerase Expression in E. coli BL21 (DE3) to Provide More Host Options for Recombinant Protein Production

“…The three inducible promoters selected in this article, the inducers such as L-arabinose [9,26], Lrhamnose [15,27], anhydrotetracycline [28] had all been studied to control gene expression. Chou et al developed the L-arabinose-induced pET system in the strain JM109 (DE3), and the production of penicillin acylase (PAC) was signi cantly improved, which proved that arabinose was more effective as an inducer than IPTG [29].…”

“…Chou et al developed the L-arabinose-induced pET system in the strain JM109 (DE3), and the production of penicillin acylase (PAC) was signi cantly improved, which proved that arabinose was more effective as an inducer than IPTG [29]. There was also study to prove that induction with L-arabinose can serve as a substitution to combined induction with 1 mM IPTG [9]. mLacI was placed under the P rhaBAD , which can strictly control the target gene and do not affect the growth of the strain, and L-rhamnose-inducible promoter had been widely used in the production of membrane proteins and secreted proteins of E. coli [27].…”

“…Based on this, researchers had developed many methods to balance intracellular resource allocation, such as decoupling protein production from cell growth and changing promoter intensity. Stargardt et al realized the decoupling of cell growth and protein expression by introducing the gene GP2 (an inhibitor of E. coli RNA polymerase), the resources in the cell owed to the expression of T7 RNAP to produce the target protein when there was enough biomass [9,10]. Recently, a gene growth switch based on CRISPRi was developed to achieve e cient production of target proteins by targeting genes related to cell growth and DNA replication in E. coli [11].…”

“…Furthermore, there is a disadvantage that the addition of the inducer IPTG will cause toxicity to the cells [13,14]. To eliminate the toxicity of IPTG to cells, the BL21-AI<gp2> strain using arabinose promoter to drive transcription of the T7 RNAP was developed and used to successfully overexpress toxic proteins [9].…”

Production of full-length SARS-CoV-2 nucleocapsid protein from Escherichia coli optimized by native hydrophobic interaction chromatography hyphenated to multi-angle light scattering detection

“…The SARS-CoV-2 NP sequence was amplified via PCR using the qPCR control plasmid (2019-nCoV_N) from Integrated DNA Technologies (Coralville, Iowa, USA). The cDNA encoding the fusion proteins (Supporting Information) were cloned into the pET30a cer or pET30a cer -CASPON expression vector [ 26 , 35 ] with unique restriction sites. The POI is under the control of a T7 promoter.…”