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

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

Proteins adopt functionally active conformations after type III secretion

Additional file 1. Supplemental calculations and tables

High-salinity growth conditions promote tat-independent secretion of tat substrates in Bacillus subtilis

The Gram-positive bacterium Bacillus subtilis contains two Tat translocases, which can facilitate transport of folded proteins across the plasma membrane. Previous research has shown that Tat-dependent protein secretion in B. subtilis is a highly selective process and that heterologous proteins, such as the green fluorescent protein (GFP), are poor Tat substrates in this organism. Nevertheless, when expressed in Escherichia coli, both B. subtilis Tat translocases facilitated exclusively Tat-dependent export of folded GFP when the twin-arginine (RR) signal peptides of the E. coli AmiA, DmsA, or MdoD proteins were attached. Therefore, the present studies were aimed at determining whether the same RR signal peptide-GFP precursors would also be exported Tat dependently in B. subtilis. In addition, we investigated the secretion of GFP fused to the full-length YwbN protein, a strict Tat substrate in B. subtilis. Several investigated GFP fusion proteins were indeed secreted in B. subtilis, but this secretion was shown to be completely Tat independent. At high-salinity growth conditions, the Tat-independent secretion of GFP as directed by the RR signal peptides from the E. coli AmiA, DmsA, or MdoD proteins was significantly enhanced, and this effect was strongest in strains lacking the TatAy-TatCy translocase. This implies that high environmental salinity has a negative influence on the avoidance of Tat-independent secretion of AmiA-GFP, DmsA-GFP, and MdoD-GFP. We conclude that as-yet-unidentified control mechanisms reject the investigated GFP fusion proteins for translocation by the B. subtilis Tat machinery and, at the same time, set limits to their Tat-independent secretion, presumably via the Sec pathway

Evaluation of the Salmonella type 3 secretion system (T3SS) as part of a protein production platform for space biology applications

As interest in space exploration and in situ resource utilization grows, the potential to leverage synthetic biology and engineered microorganisms has garnered significant attention. Microorganisms provide a robust and efficient biological chassis to demonstrate the human blueprint for advancing space biology. However, progress toward these applications is hindered by the limited access to space-like environments and a lack of knowledge about how unique environmental factors affect relevant microbial systems. To address these issues, we evaluated the Salmonella Pathogenicity Island 1 (SPI-1) type Ⅲ secretion system (T3SS) as a protein production platform for space applications. Using a NASA-designed microgravity-simulating bioreactor system, we investigated the effects of simulated microgravity on cell growth, stress response, and protein secretion via SPI-1 T3SS. Our results demonstrated increased stress responses in cells grown under simulated microgravity. However, the SPI-1 T3SS maintained its ability to secrete proteins directly into the extracellular space in a single step under simulated microgravity, simplifying downstream purification processes. These findings suggest that the SPI-1 T3SS is a viable candidate for future space biology applications

Export of functional Streptomyces coelicolor alditol oxidase to the periplasm or cell surface of Escherichia coli and its application in whole-cell biocatalysis

Streptomyces coelicolor A3(2) alditol oxidase (AldO) is a soluble monomeric flavoprotein in which the flavin cofactor is covalently linked to the polypeptide chain. AldO displays high reactivity towards different polyols such as xylitol and sorbitol. These characteristics make AldO industrially relevant, but full biotechnological exploitation of this enzyme is at present restricted by laborious and costly purification steps. To eliminate the need for enzyme purification, this study describes a whole-cell AldO biocatalyst system. To this end, we have directed AldO to the periplasm or cell surface of Escherichia coli. For periplasmic export, AldO was fused to endogenous E. coli signal sequences known to direct their passenger proteins into the SecB, signal recognition particle (SRP), or Twin-arginine translocation (Tat) pathway. In addition, AldO was fused to an ice nucleation protein (INP)-based anchoring motif for surface display. The results show that Tat-exported AldO and INP-surface-displayed AldO are active. The Tat-based system was successfully employed in converting xylitol by whole cells, whereas the use of the INP-based system was most likely restricted by lipopolysaccharide LPS in wild-type cells. It is anticipated that these whole-cell systems will be a valuable tool for further biological and industrial exploitation of AldO and other cofactor-containing enzymes.

A unifying mechanism for the biogenesis of membrane proteins co-operatively integrated by the Sec and Tat pathways

The majority of multi-spanning membrane proteins are co-translationally inserted into the bilayer by the Sec pathway. An important subset of membrane proteins have globular, cofactor-containing extracytoplasmic domains requiring the dual action of the co-translational Sec and post-translational Tat pathways for integration. Here, we identify further unexplored families of membrane proteins that are dual Sec-Tat-targeted. We establish that a predicted heme-molybdenum cofactor-containing protein, and a complex polyferredoxin, each require the concerted action of two translocases for their assembly. We determine that the mechanism of handover from Sec to Tat pathway requires the relatively low hydrophobicity of the Tat-dependent transmembrane domain. This, coupled with the presence of C-terminal positive charges, results in abortive insertion of this transmembrane domain by the Sec pathway and its subsequent release at the cytoplasmic side of the membrane. Together, our data points to a simple unifying mechanism governing the assembly of dual targeted membrane proteins.</jats:p

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

Deconstructing synthetic biology across scales: A conceptual approach for training synthetic biologists

Synthetic biology allows us to reuse, repurpose, and reconfigure biological systems to address society’s most pressing challenges. Developing biotechnologies in this way requires integrating concepts across disciplines, posing challenges to educating students with diverse expertise. We created a framework for synthetic biology training that deconstructs biotechnologies across scales—molecular, circuit/network, cell/cell-free systems, biological communities, and societal—giving students a holistic toolkit to integrate cross-disciplinary concepts towards responsible innovation of successful biotechnologies. We present this framework, lessons learned, and inclusive teaching materials to allow its adaption to train the next generation of synthetic biologists