Team:Aalto-Helsinki/Design
PROJECT DESIGN
The main objective of our iGEM project was to investigate if Vibrio natriegens could be a capable chassis for therapeutic protein production. But what exactly has to be taken into account for a competent solution?
THE IDEA
Speed, titer and yield are three competitive features for an industrial protein expression system. Research has shown that Vibrio natriegens can produce large amounts of protein quickly, thanks to its high rates of substrate uptake and growth. (Hoffart et al., 2017). Therefore our aim was to create a tool to advance therapeutic protein production and development processes.
We sought to utilize tunable promoters to achieve dose-dependent induction on a single-cell level (Marschall et al., 2017). This would allow optimizing the ratio of product to transporter, improving the yield by decreasing cytoplasmic degradation and inclusion body formation. Our plan was to overexpress the Tat pathway components by adding a Ptac promoter upstream of the chromosomal TatAB operon, and a weaker Plac promoter upstream of the tatC gene. In addition, the integration of the lacI gene into the chromosome would be necessary to repress the new promoters. As an expression plasmid for the product, we used a rhamnose-inducible PC203 based on the P15A ori and pRhaB promoter.
VIBXPRESSO STRAIN
To meet the objective of competent solution, our aim was to create a modified Vibrio natriegens strain with an inducible Tat-pathway system in the spirit of previously described E. coli “TatExpress” strains (Browning et al., 2017). This allows rapid and agile production of different recombinant proteins in increased secretion titer.
TWIN ARGININE TRANSLOCATION
(TAT) PATHWAY
The twin-arginine translocation (Tat) pathway is a multimeric transmembrane protein export system responsible for translocating proteins up to 150 kDa in size through the inner lipid membrane in several bacteria, archaea and plants (reviewed by Lee et al., 2006). The Tat pathway has been demonstrated to be capable of secreting various heterologous proteins into the bacterial periplasm (Alanen et al., 2015; Browning et al., 2017). Therefore various therapeutic protein compounds could potentially be expressed and easily purified with our VibXPresso strain. This is a desired feature in an industrial organism, since production workflows and equipment may vary between organisms.
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Vibxpresso strain
We sought to utilize tunable promoters to achieve dose-dependent induction on a single-cell level (Marschall et al., 2017). This would allow optimizing the ratio of product to transporter, improving the yield by decreasing cytoplasmic degradation and inclusion body formation. Our plan was to overexpress the Tat pathway components by adding a Ptac promoter upstream of the chromosomal TatAB operon, and a weaker Plac promoter upstream of the tatC gene. In addition, the integration of the lacI gene into the chromosome would be necessary to repress the new promoters. As an expression plasmid for the product, we used a rhamnose-inducible PC203 based on the P15A ori and pRhaB promoter.
Tunable induction requires that the uptake of an inducer is not positively regulated by said inducer, i. e. there must be no positive feedback loop (Afroz et al., 2014). Due to lack of lactose transporters, IPTG-induction should be tunable by default in V. natriegens.
We also attempted deleting an extracellular nuclease, Dns, to improve the transformability of our strain (Blokesch & Schoolnik, 2004).
Protein expression &
secretion
Our aim was to test the expression and translocation of Tat signal peptide including proteins YGFP and human growth hormone (hGH) in Vibrio natriegens. Naturally Tat complexes are present in low numbers so overexpression of the system components may lead to increased protein yield – one of the most important features of a protein expression system (Browning et al., 2017). This lead us to design our own Vibrio natriegens strain (VibXPresso) for protein production purposes, with the idea of relatively easy and tunable protein expression and harvest. In our final design, the Tat pathway genes (tatAB and tatC) would be overexpressed from the bacterium’s chromosome by inducible promoters, whilst the protein of interest fused with a Tat signal peptide is expressed from an expression plasmid.
The Tat system exports fully folded proteins, and has the potential to export complex molecules while retaining their activity. The system has been demonstrated to exhibit a “quality control” mechanism with several proteins, which means that it rejects the translocation of incorrectly folded proteins (Alanen et al., 2015; Lee et al., 2006). Incorrectly folded proteins stay in the cytoplasm where they are degraded or incorporated into inclusion bodies. This eases the purification process of the expression product while assuring secretion of only high quality proteins. Protein purification from the periplasm is more efficient compared to purification from the cytoplasm. Thus lowering downstream processing costs, which centrally contributes to the total production costs of therapeutic proteins.
Strain Engineering
Unlike in E. coli, where the TatABC-proteins are expressed from a single operon, V. natriegens’ Tat-complex is encoded by two operons. One contains TatA and TatB, while the other contains only TatC. To induce the overexpression of the Tat-complex, we would have to insert two additional inducible promoters into the genome of our V. natriegens strain, one upstream of both operons. We decided to use sugar-inducible promoters because of their familiarity to most people in the field.
However, these sugar-inducible promoters have a major undesirable feature for our application: an all-or-nothing pattern of induction at low inducer concentrations (Afroz et al., 2014). This often ignored phenomenon is caused by the upregulation of sugar transporters, induced by the uptake of said sugar. Thus, a positive feedback loop is created, leading to a heterogenous induction profile in the cell culture. This makes the expression levels of genes under these promoters dose-dependent only on a whole-culture level, which is not desirable for our application of aiming to optimize the Tat-complex-to-product -ratio on a single-cell level.
To combat this phenomenon, a common approach is to decouple the sugar transporter from the positive feedback loop by moving it under a different promoter (Marschall et al., 2017). However, due to time constraints, we came up with a different approach. V. natriegens is known not to metabolize lactose (Hoffart et al., 2017), and a quick BLAST -search did not identify any lactose transporters in its genome. Lactose analog IPTG can however enter the cell through other means. Thus, we hypothesize that V. natriegens has no positive feedback loop for the uptake of IPTG, meaning that promoters induced by it should be tunable on a single-cell level.
Chromosomal Editing
Chromosomal edits were started using homologous recombination with linear vectors.
This was supported by expressing V. natriegens native TfoX from an inducible plasmid during the editing phase.
TfoX is a regulator of natural competence in V. natriegens (Dalia et al, 2017). This increases the efficiency of homologous recombination, making the editing easier.
The vectors were constructed with overlap extension PCR (OE-PCR).
Reference
Afroz, T., Biliouris, K., Kaznessis, Y., & Beisel, C. L. (2014). Bacterial sugar utilization gives rise to distinct single‐cell behaviours. Molecular microbiology, 93(6), 1093-1103.
Alanen, H. I., Walker, K. L., Suberbie, M. L. V., Matos, C. F., Bönisch, S., Freedman, R. B., ... & Robinson, C. (2015). Efficient export of human growth hormone, interferon α2b and antibody fragments to the periplasm by the Escherichia coli Tat pathway in the absence of prior disulfide bond formation. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research, 1853(3), 756-763.
Blokesch, M., & Schoolnik, G. K. (2008). The extracellular nuclease Dns and its role in natural transformation of Vibrio cholerae. Journal of bacteriology, 190(21), 7232-7240.
Browning, D. F., Richards, K. L., Peswani, A. R., Roobol, J., Busby, S. J., & Robinson, C. (2017). Escherichia coli “TatExpress” strains super‐secrete human growth hormone into the bacterial periplasm by the Tat pathway. Biotechnology and bioengineering, 114(12), 2828-2836.
Burdette, L.A., Leach S.A., Wong H.T. & Tullman-Ercek D. (2018). Developing Gram-negative bacteria for the secretion of heterologous proteins. Microbial Cell Factories 17:196.
Dalia, T. N., Hayes, C. A., Stolyar, S., Marx, C. J., McKinlay, J. B., & Dalia, A. B. (2017). Multiplex genome editing by natural transformation (MuGENT) for synthetic biology in Vibrio natriegens. ACS synthetic biology, 6(9), 1650-1655.
De Boer, H. A., Comstock, L. J., & Vasser, M. (1983). The tac promoter: a functional hybrid derived from the trp and lac promoters. Proceedings of the National Academy of Sciences, 80(1), 21-25.
Hoffart, E., Grenz, S., Lange, J., Nitschel, R., Müller, F., Schwentner, A., ... & Blombach, B. (2017). High substrate uptake rates empower Vibrio natriegens as production host for industrial biotechnology. Appl. Environ. Microbiol., 83(22), e01614-17.
Jack, R. L., Sargent, F., Berks, B. C., Sawers, G., & Palmer, T. (2001). Constitutive expression of Escherichia coli tat genes indicates an important role for the twin-arginine translocase during aerobic and anaerobic growth. Journal of bacteriology, 183(5), 1801-1804.
Lee, P. A., Tullman-Ercek, D., & Georgiou, G. (2006). The bacterial twin-arginine translocation pathway. Annu. Rev. Microbiol., 60, 373-395.
Marschall, L., Sagmeister, P., & Herwig, C. (2017). Tunable recombinant protein expression in E. coli: promoter systems and genetic constraints. Applied microbiology and biotechnology, 101(2), 501-512.
