Abstract
BackgroundThe production of biofuels in photosynthetic microalgae and cyanobacteria is a promising alternative to the generation of fuels from fossil resources. To be economically competitive, producer strains need to be established that synthesize the targeted product at high yield and over a long time. Engineering cyanobacteria into forced fuel producers should considerably interfere with overall cell homeostasis, which in turn might counteract productivity and sustainability of the process. Therefore, in-depth characterization of the cellular response upon long-term production is of high interest for the targeted improvement of a desired strain.ResultsThe transcriptome-wide response to continuous ethanol production was examined in Synechocystis sp. PCC6803 using high resolution microarrays. In two independent experiments, ethanol production rates of 0.0338% (v/v) ethanol d-1 and 0.0303% (v/v) ethanol d-1 were obtained over 18 consecutive days, measuring two sets of biological triplicates in fully automated photobioreactors. Ethanol production caused a significant (~40%) delay in biomass accumulation, the development of a bleaching phenotype and a down-regulation of light harvesting capacity. However, microarray analyses performed at day 4, 7, 11 and 18 of the experiment revealed only three mRNAs with a strongly modified accumulation level throughout the course of the experiment. In addition to the overexpressed adhA (slr1192) gene, this was an approximately 4 fold reduction in cpcB (sll1577) and 3 to 6 fold increase in rps8 (sll1809) mRNA levels. Much weaker modifications of expression level or modifications restricted to day 18 of the experiment were observed for genes involved in carbon assimilation (Ribulose bisphosphate carboxylase and Glutamate decarboxylase). Molecular analysis of the reduced cpcB levels revealed a post-transcriptional processing of the cpcBA operon mRNA leaving a truncated mRNA cpcA* likely not competent for translation. Moreover, western blots and zinc-enhanced bilin fluorescence blots confirmed a severe reduction in the amounts of both phycocyanin subunits, explaining the cause of the bleaching phenotype.ConclusionsChanges in gene expression upon induction of long-term ethanol production in Synechocystis sp. PCC6803 are highly specific. In particular, we did not observe a comprehensive stress response as might have been expected.
Highlights
The production of biofuels in photosynthetic microalgae and cyanobacteria is a promising alternative to the generation of fuels from fossil resources
Characterization of Synechocystis 6803 upon long-term ethanol production Engineering cyanobacteria to produce ethanol from pyruvate is accomplished by coupled overexpression of the cytosolic enzymes pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH)
That is to minimize the chance of detecting non-ethanol effects resulting from imbalances in, for example, nutrient availability or physical parameters that may arise upon long-term cultivation between producer and wild type
Summary
The production of biofuels in photosynthetic microalgae and cyanobacteria is a promising alternative to the generation of fuels from fossil resources. Producer strains need to be established that synthesize the targeted product at high yield and over a long time. To achieve economically feasible production rates, the following two goals need to be addressed: (i) the yield of the intended product is to be maximized, and (ii) the producer strains should be of considerable robustness to tolerate the product, which is frequently alien to their metabolism. A depressed growth rate and a yellow-green phenotype interpreted as severe metabolic stress was reported for an ethyleneproducing strain of Synechococcus sp. A substantial and unspecific general stress response was found upon the external application of ethanol both at proteome [8], as well as transcriptome level in Synechocystis 6803 [9]
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