Engineering high titer heterologous protein secretion in bacteria

The commercial-scale production of proteins in eukaryotic cells traditionally includes a secretion step to separate the product from the cellular milieu. Including such a step in bacterial processes is a well-known yet elusive biotechnological goal that would enable similarly efficient protein production at both research and industrial scales. The type III secretion system (T3SS) in Salmonella enterica is an ideal path to protein secretion because it is nonessential for bacterial metabolism and allows for target proteins to cross both bacterial membranes in one step, via characteristic needle-like protein structures (1). We took several important steps to engineer this system for biotechnology applications, including i) altering the regulation of the system1, ii) protein engineering of the secretion machinery structural proteins2, iii) manipulating the genome to eliminate native secreted proteins, and iv) optimizing media composition. The resulting platform now enables high-titer production of a variety of biochemically challenging heterologous proteins, such as degradation-prone biopolymer proteins, antibodies, and toxic antimicrobial peptides3 at titers of up to 400 mg/l – over 400-fold improvement on wild type levels. The purity of the secreted proteins of interest are routinely \u3e80% after a single chromatography step, with minimal truncation products or other contaminants common to cytosolically produced proteins3. Moreover, secretion into the relatively dilute extracellular space permits folding into functional forms and disulfide formation for a significant fraction of the products4. This presentation will explore the details of each engineering step and the implications for the production of enzymes, biomaterials, and antibodies. 1．Metcalf K.J., Finnerty C., Azam A., Valdivia E., Tullman-Ercek D. (2014) “Using transcriptional control to increase titer of secreted heterologous proteins by the type III secretion system.” Appl. Environ. Microbiol. 80(19):5927-34. 2．Glasgow A.A., Wong H.T., Tullman-Ercek D. “Identifying a dual role for Salmonella protein SipD in increasing protein secretion.” (In revision). 3．Azam A., Metcalf K.J., Li C., Tullman-Ercek D. (2016) “Type III secretion as a generalizable strategy for the development of peptide-based biomaterials.” Biotechnol. Bioeng. 113(11):2313-20. 4．Metcalf K.J., Bevington J.L., Rosales S.L., Burdette L.A., Valdivia E., Tullman-Ercek D. (2016) “Proteins adapt a functionally active conformations in the media following type III secretion.” Microb. Cell Fact. 15(1):213

Quantitative characterization of all single amino acid variants of a viral capsid-based delivery vehicle

Self-assembling protein containers are promising delivery vehicles for cellular and gene therapy applications, but the ability to predict how mutations alter self-assembly and other particle properties remains a significant challenge. Here, we combine comprehensive codon mutagenesis with high-throughput sequencing to characterize the assembly-competency of all single amino acid variants of a virus-like particle. The coat protein (CP) of MS2 bacteriophage was chosen because of its potential in targeted delivery and imaging. An assembly selection revealed a high-resolution fitness landscape that challenged several conventional protein engineering assumptions. Using the same approach with additional comprehensive mutagenesis strategies and selective pressures identified several other previously-uncharacterized variants for enabling efficient chemical and post-translational modifications as well as altered stability features. For example, the wild-type CP is acid tolerant down to pH 2, but we identified a variant with a single point mutation that confers stability at neutral pH but acid-triggered disassembly. Acid sensitivity is highly desirable in targeted delivery to improve the efficiency of endosomal release. In addition to providing a blueprint of how to tune the chemical and physical properties of the MS2 CP and other structurally-related virus-like particles, these techniques can readily be applied to the systematic study of other self-assembling proteins and protein-based delivery vehicles

A Pseudomonas putida efflux pump acts on short-chain alcohols

Abstract Background The microbial production of biofuels is complicated by a tradeoff between yield and toxicity of many fuels. Efflux pumps enable bacteria to tolerate toxic substances by their removal from the cells while bypassing the periplasm. Their use for the microbial production of biofuels can help to improve cell survival, product recovery, and productivity. However, no native efflux pump is known to act on the class of short-chain alcohols, important next-generation biofuels, and it was considered unlikely that such an efflux pump exists. Results We report that controlled expression of the RND-type efflux pump TtgABC from Pseudomonas putida DOT-T1E strongly improved cell survival in highly toxic levels of the next-generation biofuels n-butanol, isobutanol, isoprenol, and isopentanol. GC-FID measurements indicated active efflux of n-butanol when the pump is expressed. Conversely, pump expression did not lead to faster growth in media supplemented with low concentrations of n-butanol and isopentanol. Conclusions TtgABC is the first native efflux pump shown to act on multiple short-chain alcohols. Its controlled expression can be used to improve cell survival and increase production of biofuels as an orthogonal approach to metabolic engineering. Together with the increased interest in P. putida for metabolic engineering due to its flexible metabolism, high native tolerance to toxic substances, and various applications of engineering its metabolism, our findings endorse the strain as an excellent biocatalyst for the high-yield production of next-generation biofuels

Proteins adopt functionally active conformations after type III secretion

Additional file 1. Supplemental calculations and tables

Engineering the Salmonella type III secretion system to export spider silk monomers

The type III secretion system (T3SS) exports proteins from the cytoplasm, through both the inner and outer membranes, to the external environment. Here, a system is constructed to harness the T3SS encoded within Salmonella Pathogeneity Island 1 to export proteins of biotechnological interest. The system is composed of an operon containing the target protein fused to an N-terminal secretion tag and its cognate chaperone. Transcription is controlled by a genetic circuit that only turns on when the cell is actively secreting protein. The system is refined using a small human protein (DH domain) and demonstrated by exporting three silk monomers (ADF-1, -2, and -3), representative of different types of spider silk. Synthetic genes encoding silk monomers were designed to enhance genetic stability and codon usage, constructed by automated DNA synthesis, and cloned into the secretion control system. Secretion rates up to 1.8 mg l−1 h−1 are demonstrated with up to 14% of expressed protein secreted. This work introduces new parts to control protein secretion in Gram-negative bacteria, which will be broadly applicable to problems in biotechnology

Characterization and engineering of the twin-arginine translocation pathway of Escherichia coli

textThe twin-arginine translocation (Tat) pathway of Escherichia coli provides a novel method for the export of proteins from the cytoplasm to the periplasm. Remarkably, it allows for large, folded proteins to cross the inner membrane, with no apparent effect on cell viability. Protein export from the cytoplasm is employed in a variety of biotechnological applications including manufacturing and protein engineering. However, until the discovery of the Tat pathway, such applications relied on the Sec pathway, in which proteins transverse the lipid bilayer membrane in an extended form and protein folding takes place after export. Many proteins of biotechnological interest are not compatible with export via the “classical” Sec pathway. Thus, the export of such Sec-incompatible proteins via the Tat pathway could open the way for new biotechnology applications. This work explores several mechanistic, physiological and technology-related aspects of Tat export In all organisms, proteins are secreted by virtue of a peptide extension, or signal peptide, comprising 15-45 amino acids. The signal peptide serves as a “zip code” and is cleaved after export. E. coli contains 29 putative Tat-specific signal peptides but their ability to mediate export via the Tat pathway has not been confirmed experimentally. The export pathway (Tat or Sec) utilized by this set of 29 signal peptides was characterized using fusions to protein reporters. The reporter proteins chosen for this study are functional only when translocated across the membrane either via the Sec or Tat pathways. Surprisingly, it was found that while 11/29 signal peptides are Tat-specific and 2/29 are Sec-specific, a set of 16/29 signal peptides were able to direct export via both the Tat and Sec pathways. Interestingly, increasing the charge of the region surrounding the cleavage site – particularly the N-terminus of the mature protein – resulted in Tat specificity. In separate studies we showed that in addition to an appropriate signal sequence, proteins destined for export via the Tat pathway must complete their folding in the cytoplasm. Partially folded proteins are not competent for export via this pathway. The requirement for folding in the cytoplasm prior to export was demonstrated by using an elegant system whereby the conformation of the polypeptide chain in the cytoplasm could be controlled using conditions that supported or prevented the formation of disulfide bonds. These results led us to propose that the Tat pathway contains an intrinsic folding quality control mechanism, a concept that has since been widely adopted in the literature. Finally, a new methodology was developed for the engineering of proteins that require the cytoplasmic machinery to fold but must then be exported into the bacterial periplasmic space. Specifically, we created a Tat-based system to enable the display of proteins on filamentous phage, a prerequisite for the high-throughput screening of protein libraries. This system relies on the forced dimerization of the phage coat protein p3, which is exported into the periplasm via the Sec pathway, and the protein that is to be displayed, which is exported via the Tat pathway. Forced dimerization of p3 and the desired protein in the periplasm was mediated by coiled-coil interactions. We further demonstrated that this Tat-dependent display platform shows promise for use in engineering ligand-binding loops into green fluorescent protein (GFP) for sensor applications.Chemical Engineerin