Using transcriptional control to increase titers of secreted heterologous proteins by the type III secretion system

Abstract

The type III secretion system (T3SS) encoded at the Salmonella pathogenicity island 1 (SPI-1) locus secretes protein directly from the cytosol to the culture media in a concerted, one-step process, bypassing the periplasm. While this approach is attractive for heterologous protein production, product titers are too low for many applications. In addition, the expression of the SPI-1 gene cluster is subject to native regulation, which requires culturing conditions that are not ideal for high-density growth. We used transcriptional control to increase the amount of protein that is secreted into the extracellular space by the T3SS of Salmonella enterica. The controlled expression of the gene encoding SPI-1 transcription factor HilA circumvents the requirement of endogenous induction conditions and allows for synthetic induction of the secretion system. This strategy increases the number of cells that express SPI-1 genes, as measured by promoter activity. In addition, protein secretion titer is sensitive to the time of addition and the concentration of inducer for the protein to be secreted and SPI-1 gene cluster. Overexpression of hilA increases secreted protein titer by >10-fold and enables recovery of up to 28±9 mg/liter of secreted protein from an 8-h culture. We also demonstrate that the protein beta-lactamase is able to adopt an active conformation after secretion, and the increase in secreted titer from hilA overexpression also correlates to increased enzyme activity in the culture supernatant.

FIG 1

Effect of hilA overexpression on secretion and cell growth. Cultures carrying the upregulation vector were induced with 100 μM IPTG at the time of subculture and denoted “+hilA.” Cultures that did not carry the upregulation vector are denoted “−hilA.” (A) Western blot of soluble cell fraction (“C”) and supernatant (“S”) samples, with sample load amounts normalized according to the cell harvest OD₆₀₀. (B) Quantification of SptP-DH-2xF-6xH secreted protein titer using the P_sic DH export plasmid with various growth conditions and hilA overexpression. (C) Western blot of soluble cell fraction and supernatant samples of wild-type and prgI deletion strains, with sample load amounts normalized according to the cell harvest OD₆₀₀, from cultures grown in the “−T3SS” condition. (D) Growth of S. enterica. Cultures grown in the “−T3SS” and “+T3SS” condition are denoted with a solid and dashed black line, respectively. Cultures without or with hilA overexpression are marked with a circle or a rectangle, respectively. The experiment was performed on different days in biological triplicate. Error bars represent one standard deviation.

FIG 2

Plot of fraction of cells exhibiting P_sic activity from culture with or without hilA overexpression in different growth conditions. Cultures carrying the upregulation vector were induced with 100 μM IPTG at the time of subculture and denoted “+hilA.” Cultures that did not carry the upregulation vector are denoted “−hilA.” Cultures grown in the “−T3SS” condition without or with hilA overexpression are denoted with solid squares, and a dashed line or a solid line, respectively. Cultures grown in the “+T3SS” condition without or with hilA overexpression are denoted with open black circles, and a dashed line or a solid line, respectively.

FIG 3

Effect of hilA overexpression on secreted protein titer when controlling SptP-DH-2xF-6xH and SPI-1 production orthogonally. (A) Quantification of secreted protein titer for the P_tet DH construct using a quantitative Western blot. Cultures carrying the upregulation vector were induced with 100 μM IPTG at the time of subculture and denoted “+hilA.” Cultures that did not carry the upregulation vector are denoted “−hilA.” (B to D) All samples are “−T3SS” growth condition. Samples denoted “C” are soluble cell pellet samples, and samples denoted “S” are supernatant samples. Sample load amounts are normalized according to the cell harvest OD₆₀₀. (B) Western blot of culture fractions with various times of addition of inducer (100 ng/ml aTc) for SptP-DH-2xF-6xH, the protein of interest (POI), relative to subculture and induction of the upregulation vector (100 μM IPTG) at the time of the subculture. (C) Western blot of culture fractions with various amounts of POI inducer (aTc) added 3 h after subculture and induction of the upregulation vector (100 μM IPTG) at the time of the subculture. (D) Western blot of culture fractions with various amounts of hilA inducer (IPTG) added at the time of subculture. POI in this experiment is under P_sic control, which is hilA dependent. The sample denoted “−hilA” did not carry the upregulation vector.

FIG 4

Quantification of secreted protein titers for different POIs by quantitative Western blotting. Samples were grown in the “−T3SS” condition and with induction of the upregulation vector (100 μM IPTG) at the time of the subculture.

FIG 5

Plot of the initial reaction velocity (V₀) for culture supernatant samples. Samples denoted “−bla” are cultures carrying the P_sic gfp plasmid. Samples were grown in the “−T3SS” condition. Cultures carrying the upregulation vector were induced with 100 μM IPTG at the time of subculture and denoted “+hilA.” Cultures that did not carry the upregulation vector are denoted “−hilA.”