Our newly engineered cyanobacterial factories are capable of producing fumarate directly from CO
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
; 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
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- Du., W. unpublished.
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