Engineering a Cyanobacterial Cell Factory for Production of Lactic Acid

Abstract

Metabolic engineering of microorganisms has become a versatile tool to facilitate production of bulk chemicals, fuels, etc. Accordingly, CO 2 has been exploited via cyanobacterial metabolism as a sustainable carbon source of biofuel and bioplastic precursors. Here we extended these observations by showing that integration of an ldh gene from Bacillus subtilis (encoding an L-lactate dehydrogenase) into the genome of Synechocystis sp. strain PCC6803 leads to L-lactic acid production, a phenotype which is shown to be stable for prolonged batch culturing. Coexpression of a heterologous soluble transhydrogenase leads to an even higher lactate production rate and yield (lactic acid accumulating up to a several-millimolar concentration in the extracellular medium) than those for the single ldh mutant. The expression of a transhydrogenase alone, however, appears to be harmful to the cells, and a mutant carrying such a gene is rapidly outcompeted by a revertant(s) with a wild-type growth phenotype. Furthermore, our results indicate that the introduction of a lactate dehydrogenase rescues this phenotype by preventing the reversion.C oncerns about shrinking supplies of fossil fuel and about climate change have been a strong incentive during the past decade for the development of sustainable alternatives to provide fuel and chemical feedstock. Because of these concerns, several different ways to produce solar biofuel have been proposed. Initially the proposed procedures were based on the conversion of agricultural crops into short-chain alcohols. This unfortunately results in a direct competition for resources between the energy sector and food supply. Therefore, second-generation types of processes were proposed, in which one aims at the conversion of the triglyceride fraction from, e.g., green algae into biodiesel, which relaxes the competition between energy and the food supply. Nevertheless, the conversion of CO 2 into biofuel, driven by solar energy, is still rather indirect even in this second-generation approach: CO 2 is first converted into the complex building blocks of biomass, from which subsequently the triglyceride fraction must be extracted and converted into biodiesel.For these reasons, a third-generation type of process has been proposed, in which CO 2 is converted into biofuel directly, accomplished by the introduction of genes encoding a metabolic-e.g., fermentative-pathway into a cyanobacterium, resulting in the emergence of a "photofermentative" chimera. For several biofuel and chemical feedstock products, including ethanol, ethylene, isoprene, D-lactic acid, glucose, sucrose, isobutyraldehyde, and 1-butanol, the proof of principle of this approach has been provided (see references 3,8,10,24,25,28, and 39 and references therein). Ideally, this process would be carried out in such a way that the majority of the CO 2 that is fixed in the cyanobacterial CalvinBenson cycle is converted into the selected product. This implies that the central metabolism in the selected chimera should be largely redirected tow...

Expression of holo-proteorhodopsin in Synechocystis sp. PCC 6803

“…450-550 nm), a range that is well covered by the absorption spectrum of many PRs (29,30). Introduction of PRs into such a system could lead to increased CO 2 fixation, which could ultimately increase production rates of interesting compounds by cyanobacterial cell factories (31). Hence, it has previously been suggested that PRs could supply additional free energy to oxyphototrophic organisms (29,30), even more so when it turns out to be possible to shift the window of absorption of this pigmented protein outside that of the PAR region (32).…”

Culturing Synechocystis sp. Strain PCC 6803 with N 2 and CO 2 in a Diel Regime Reveals Multiphase Glycogen Dynamics with Low Maintenance Costs

“…Consequently, there is an interest in the large-scale growth of these organisms, in the compounds contained in their biomass, and in compounds excreted into the growth medium (11). So far, excreted products, synthesized via heterologous pathways, typically are precursors to bioplastics (e.g., butane-diol and lactic acid) (12,13), biofuels (e.g., ethanol and butanol) (14), and secondary metabolites and flavor compounds, such as vitamins and terpenes (15,16).…”

Chirality Matters: Synthesis and Consumption of the d -Enantiomer of Lactic Acid by Synechocystis sp. Strain PCC6803

“…Employing a photosynthetic microorganism as the production host has the advantage of enabling the direct conversion of CO 2 into (poly)lactic acid (6)(7)(8)(9). Such production, which is dependent on cyanobacterial cell factories, allows compound formation without the need to generate complex (plant) biomass first, only to break it down again later for its utilization by a chemotrophic fermentative microorganism (10).…”