Dissecting difference in heterologous protein secretion titer by Type III secretion system between strains of Salmonella enterica

Abstract

Type III secretion systems (T3SS) are complex macromolecular machines found in Gram‐negative bacteria. These bacteria utilize T3SS to facilitate microbe‐host interactions by translocating proteins, known as effectors, into the host cells. This provides an interesting system for engineering the selective translocation of proteins out of the bacterial cell. Secretion strategies have been useful in exporting proteins of biotechnological interest in systems such as yeast, insect cells and mammalian cells. Moreover, bacteria fermentations are faster and cheaper.In our lab, we make use of the SPI‐1 T3SS in Salmonella enterica serovar Typhimurium for protein export. Because the Salmonella SPI‐1 is not an essential metabolic pathway, we can harness it for heterologous protein secretion without affecting cell viability. Using secretion assays based on western blotting and a split GFP system, we were able to determine that the DW01 strain secretes ~20% more proteins than the LT2 strain.In order to determine the genetic basis for the differences in secretion titer, we sequenced the genomes of both strains using next generation sequencing to determine single nucleotide variants and indels unique to DW01. Some of the mutations were then further verified by Sanger sequencing. The alleles for each locus are then exhaustively swapped in both strain using the recombineering method established by the Court lab. This allows us to determine the contribution of each alleles at every locus to the increased secretion observed in DW01. Other phenotypic differences are also characterized.Through this work, we also identify genes outside of the SPI‐1 pathogenicity island that can modulate the heterologous protein secretion capability of S. enterica. Taken together, these experiments provide us with more tools to further increase the titer and achieve the industrial standards of >1g/L of protein.Support or Funding InformationH.T.W. is funded by A*STAR. L.B. is funded by NSF GRFP. This work is funded by NSF CBET grant number 1706125.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.