Our newly engineered cyanobacterial factories are capable of producing fumarate directly from CO2 using the energy of (sun)light. However, experts in the fields pointed out to us that it is only the fumarate that is excreted from the cells that is readily available in a downstream process of a “real-world” scenario. Furthermore, if fumarate is accumulated intracellularly, it may prove to inhibit its own production, and potentially even growth. We therefore anticipate that ensuring an effective fumarate transport system is an important engineering target for our cell factories. In this module of the project the goal is to: (i) study the native fumarate transport system of Synechocystis; and (ii) overexpress heterologous transport systems that have been shown to improve fumarate extracellular production in chemoheterotrophic organisms.
A native fumarate transport system in Synechocystis is yet to be identified. However, we have successfully measured extracellular fumarate production in our engineered strains, suggesting a transport mechanism of some kind must to be present. But which?
We have carried a thorough bioinformatics homology search using sequences from known fumarate transporters from other organisms. Amongst the roughly 900 candidate genes of Synechocystis predicted to encode putative membrane proteins, we found two suitable targets that may encode components of a dicarboxylic acid transporter (such as fumarate). To test this in silico prediction we constructed single knock-out mutants of these putative genes of unknown function. Furthermore, we have also exploited the possibility of increasing fumarate production in our engineered strains through the expression of known heterologous fumarate transporters. This strategy has led to an increase in fumarate yields in E. Coli of 53%1 . Two versatile BioBricks (Bba_K2385001 and Bba_K2385000) have been constructed that encode fumarate transporters from Escherichia coli and Nodularia Spumigena. Finally, we performed extensive phenotyping of all the strains constructed, with particular emphasis on the impact of the genetic interventions on fumarate import and export.
- Identification of two genes in Synechocystis, sll1103 and sll1314, encoding putative dicarboxylate transporters
- Experimental validation that sll1314 encodes (part of) a transporter system involved in fumarate export
- Experimental validation that Synechocystis does not uptake fumarate from the environment
- Expression of neither a known fumarate transporter from E. Coli, nor one from N. Spumigena, leads to fumarate excretion in wild type Synechocystis
- IG Janausch, E Zientz, QH Tran, A Kröger, and G Unden. C 4-dicarboxylate carriers and sensors in bacteria. Biochimica et Biophysica Acta (BBA)-Bioenergetics, 1553(1):39-56, 2002.
- Paulsen, I. T., Nguyen, L., Sliwinski, M. K., Rabus, R., & Saier, M. H. (2000). Microbial genome analyses: comparative transport capabilities in eighteen prokaryotes. Journal of molecular biology, 301(1), 75-100.
- Rost, B. (1999). Twilight zone of protein sequence alignments. Protein engineering, 12(2), 85-94.
- Pirovano, W., Feenstra, K. A., & Heringa, J. (2008). PRALINE™: a strategy for improved multiple alignment of transmembrane proteins. Bioinformatics, 24(4), 492-497.
- Söding, J. (2004). Protein homology detection by HMM-HMM comparison. Bioinformatics, 21(7), 951-960.
- Altschul, S. F., Madden, T. L., Schäffer, A. A., Zhang, J., Zhang, Z., Miller, W., & Lipman, D. J. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic acids research, 25(17), 3389-3402.
- Thompson, J. D., Gibson, T., & Higgins, D. G. (2002). Multiple sequence alignment using ClustalW and ClustalX. Current protocols in bioinformatics, 2-3.
- Marchler-Bauer, A., Derbyshire, M. K., Gonzales, N. R., Lu, S., Chitsaz, F., Geer, L. Y., ... & Lanczycki, C. J. (2014). CDD: NCBI's conserved domain database. Nucleic acids research, 43(D1), D222-D226.
- Bairoch, A., Bucher, P., & Hofmann, K. (1997). The PROSITE database, its status in 1997. Nucleic Acids Research, 25(1), 217-221.
- Forward, J. A., Behrendt, M. C., Wyborn, N. R., Cross, R., & Kelly, D. J. (1997). TRAP transporters: a new family of periplasmic solute transport systems encoded by the dctPQM genes of Rhodobacter capsulatus and by homologs in diverse gram-negative bacteria. Journal of bacteriology, 179(17), 5482-5493.
- Kaneko, T., Sato, S., Kotani, H., Tanaka, A., Asamizu, E., Nakamura, Y., ... & Kimura, T. (1996). Sequence analysis of the genome of the unicellular cyanobacterium Synechocystis sp. strain PCC6803. II. Sequence determination of the entire genome and assignment of potential protein-coding regions. DNA research, 3(3), 109-136.
- Quintero, M. J., Montesinos, M. L., Herrero, A., & Flores, E. (2001). Identification of genes encoding amino acid permeases by inactivation of selected ORFs from the Synechocystis genomic sequence. Genome Research, 11(12), 2034-2040.
- Du., W. unpublished.
- Savakis, P. E., Angermayr, S. A., & Hellingwerf, K. J. (2013). Synthesis of 2, 3-butanediol by Synechocystis sp. PCC6803 via heterologous expression of a catabolic pathway from lactic acid-and enterobacteria. Metabolic engineering, 20, 121-130.
- Choi, K. H., & Dobbs, F. C. (1999). Comparison of two kinds of Biolog microplates (GN and ECO) in their ability to distinguish among aquatic microbial communities. Journal of Microbiological Methods, 36(3), 203-213.