Escherichia coli Can Adapt Its Protein Translocation Machinery for Enhanced Periplasmic Recombinant Protein Production

Abstract

Recently, we engineered a tunable rhamnose promoter-based setup for the production of recombinant proteins in E. coli. This setup enabled us to show that being able to precisely set the production rate of a secretory recombinant protein is critical to enhance protein production yields in the periplasm. It is assumed that precisely setting the production rate of a secretory recombinant protein is required to harmonize its production rate with the protein translocation capacity of the cell. Here, using proteome analysis we show that enhancing periplasmic production of human Growth Hormone (hGH) using the tunable rhamnose promoter-based setup is accompanied by increased accumulation levels of at least three key players in protein translocation; the peripheral motor of the Sec-translocon (SecA), leader peptidase (LepB), and the cytoplasmic membrane protein integrase/chaperone (YidC). Thus, enhancing periplasmic hGH production leads to increased Sec-translocon capacity, increased capacity to cleave signal peptides from secretory proteins and an increased capacity of an alternative membrane protein biogenesis pathway, which frees up Sec-translocon capacity for protein secretion. When cells with enhanced periplasmic hGH production yields were harvested and subsequently cultured in the absence of inducer, SecA, LepB, and YidC levels went down again. This indicates that when using the tunable rhamnose-promoter system to enhance the production of a protein in the periplasm, E. coli can adapt its protein translocation machinery for enhanced recombinant protein production in the periplasm.

Strategies to Enhance Periplasmic Recombinant Protein Production Yields in Escherichia coli

“…In the section “Tuning transcription and translation”, it was described how hGH was produced using the rhamnose promoter in a rha operon deletion strain background at varying concentrations of rhamnose and four different signal peptides. Cells producing hGH in the periplasm at the highest level, for each of the four signal peptides used, were analyzed using proteomics ( Karyolaimos et al, 2020 ). Irrespective of the signal peptide used, the accumulation levels of SecA, LepB and YidC were increased.…”

“…Furthering our knowledge in these areas may also open up new avenues for enhancing periplasmic protein production yields. Studying E. coli cells producing proteins in the periplasm using omics approaches, may help to identify components that can positively and also negatively affect periplasmic protein production yields ( e.g., ( Ceroni et al, 2015 ; Reis et al, 2019 ; Karyolaimos et al, 2020 ; Mateus et al, 2020 ; Tan et al, 2020 ; Rugbjerg et al, 2021 )). Such studies may extend the repertoire of components that can be co-produced and used for the isolation of variants as well as genes that can be inactivated to enhance periplasmic protein production yields.…”

“…However, the production of a protein in the periplasm is often cumbersome. Usually, a significant fraction of the produced protein accumulates in the unprocessed form in the cytoplasm ( Schlegel et al, 2013 ; Baumgarten et al, 2018 ; Karyolaimos et al, 2019 ; Karyolaimos et al, 2020 ). Periplasmic protein production can also have a negative effect on the fitness of the production host and consequently biomass formation as well as protein production yields ( Schlegel et al, 2013 ; Baumgarten et al, 2018 ).…”

Secretion and Periplasmic Activation of a Potent Endonuclease in E. coli

“…In addition to the commonly used E. coli BL21 (DE3) strain, we also evaluated a C43 (DE3) strain known as the Walker strain often used to provide higher membrane and secreted protein accumulation. This strain has multiple mutations that weaken the lacUV5 promoter used for T7 RNA polymerase expression [21,23,24], thereby slowing transcription initiation rates for target gene expression. Slower production can avoid saturating translocation capacity which otherwise could lead to inclusion body formation, translocon inhibition, and/or growth arrest [25][26][27][28].…”

“…This mechanism is believed to either co-or post-translationally translocate SecB-bound or SRP-directed polypeptides targeted for translocation by an N-terminal signal sequence [17][18][19][20]. This production mode is expected to avoid possible cytoplasmic SmEn toxicity and periplasmic protein folding can be facilitated after signal peptide removal by signal peptidase by the disulfide bond forming and isomerizing oxidoreductases DsbA and DsbC) [21,22].…”