Developing Gram-negative bacteria for the secretion of heterologous proteins

Abstract

Gram-negative bacteria are attractive hosts for recombinant protein production because they are fast growing, easy to manipulate, and genetically stable in large cultures. However, the utility of these microbes would expand if they also could secrete the product at commercial scales. Secretion of biotechnologically relevant proteins into the extracellular medium increases product purity from cell culture, decreases downstream processing requirements, and reduces overall cost. Thus, researchers are devoting significant attention to engineering Gram-negative bacteria to secrete recombinant proteins to the extracellular medium. Secretion from these bacteria operates through highly specialized systems, which are able to translocate proteins from the cytosol to the extracellular medium in either one or two steps. Building on past successes, researchers continue to increase the secretion efficiency and titer through these systems in an effort to make them viable for industrial production. Efforts include modifying the secretion tags required for recombinant protein secretion, developing methods to screen or select rapidly for clones with higher titer or efficiency, and improving reliability and robustness of high titer secretion through genetic manipulations. An additional focus is the expression of secretion machineries from pathogenic bacteria in the "workhorse" of biotechnology, Escherichia coli, to reduce handling of pathogenic strains. This review will cover recent advances toward the development of high-expressing, high-secreting Gram-negative production strains.

Keywords: Bacterial secretion systems; Protein secretion; Recombinant protein.

Fig. 1

One-step secretion systems. Proteins (dark blue) are translocated directly from the cytosol to the extracellular space in an unfolded state, bypassing the inner and outer membranes (IM and OM, respectively). A C-terminal (T1SS, a) or an N-terminal (T3SS, b) secretion tag is required for translocation and remains attached to the cargo upon exiting the secretion apparatus. A list of engineering features for each secretion system is listed below the diagrams, and those highlighted in green are considered advantages of each system. “Substrate range” and the level of characterization refer specifically to heterologous protein secretion, and “complexity” describes the secretion machinery

Fig. 2

Two-step secretion systems. a Proteins (dark blue) are exported across the inner membrane (IM) via either Sec or Tat before passively diffusing into the extracellular space. b An example of transport via a fusion partner. Export pathway specificity is unknown for many fusion partners, but YebF (purple box) is secreted only when it is exported through Sec. It is believed to translocate the outer membrane (OM) via a porin (orange). c Proteins are exported through either Sec or Tat before entering the pseudopilus apparatus (pink) that transports cargo across the OM. d The translocation domain-passenger domain fusion is exported through the Sec pathway. The translocation domain (yellow) inserts in the outer membrane and the passenger domain (green) is secreted through the pore. An autocleavage event releases the passenger domain in the class of T5SS discussed here. e Proteins fused to the curli subunit (teal) are exported through Sec and are thought to traverse the outer membrane via an entropy gradient in a chaperonin-like structure (magenta). In the absence of the protein that anchors curli subunits to the outer membrane, fibers spontaneously polymerize and aggregate into networks in the extracellular space. A list of engineering features for each secretion system is listed below the diagrams, and those highlighted in green are considered advantages of each system. “Substrate range” and the level of characterization refer specifically to heterologous protein secretion, and “complexity” describes the secretion machinery