Biological production of organic compounds

#This application contains a Sequence Listing submitted as an electronic text file entitled “11-18_ST25.txt,” having a size in bytes of 20 kb and created on Jul. 27, 2012. Pursuant to 37 CFR §1.52(e)(5), the information contained in the above electronic file is hereby incorporated by reference in its entirety.

#Using algae or cyanobacteria to produce carbon compounds photosynthetically from CO2 and water has theoretical potential but has yet to be realized on an industrial scale. One major limitation is that these cells are naturally high in protein, especially under conditions that lead to maximum growth rates. Thus a large fraction of carbon from photosynthesis is used to produce nitrogen containing amino acids rather than other products containing only carbon, hydrogen, and oxygen. When nitrogen sources are removed from cultures of non-diazotrophic cyanobacteria, cells accumulate high concentrations of glycogen, but large-scale harvesting of microbial oxygenic phototrophs is difficult and expensive with currently available technologies.

#Alpha ketoglutarate (AKG) is used as an organic synthesis intermediate, a medicine ingredient, a biochemical reagent, and as a nutritional additive in food and sport drinks Currently AKG is produced by chemical synthesis using triethyl oxalosuccinic ester derived from petroleum and concentrated hydrochloric acid, or by fermentation using sugar as feedstock. Photosynthetic production of AKG could therefore replace petroleum or sugar as the feedstock and eliminate the use of corrosive acid.

#Exemplary embodiments are illustrated in referenced figures of the drawings. It is intended that the embodiments and figures disclosed herein are to be considered illustrative rather than limiting.

#FIG. 1 illustrates the major carbon biosynthetic pathways in cyanobacteria such as Synechocystis 6803 under nitrogen-deprived conditions.

#FIG. 2 shows growth curves of batch cultures of Synechocystis sp. PCC 6803 cultured in full BG11 media (A) and after suspension in BG11-N media (B). Cultures were under 24 hour light at ˜50 μEm−2 s−1 light flux. Panel (C) shows growth curves for wild type and mutant strains under nitrogen deprivation conditions.

#Disclosed herein are strains of cyanobacteria that produce high levels of alpha ketoglutarate (AKG) and pyruvate. One example is a mutant strain of Synechocystis lacking the glgC gene (FIG. 12A), which does not produce a functional AGP protein (FIG. 12B) or glycogen in detectable levels but over-produces AKG and pyruvate. Methods of culturing these cyanobacteria to produce AKG and pyruvate are also described herein.

#Synechocystis, a non-diazotrophic cyanobacterium, accumulates large amounts of glycogen when starved of nitrogen. While not wishing to be bound to any particular theory, it is believed that carbon flux from photosynthesis can be rerouted away from a storage product (e.g., glycogen) and toward an excreted product (e.g., AKG or pyruvate) by creating stable mutational strains of cyanobacterial cells that are incapable of synthesizing storage products such as glycogen or glucosylglycerol. Such strains include those with disruptions or deletions in the gene that encodes an ADP-glucose pyrophosphorylase (AGP), which catalyzes the conversion of alpha-d-glucose-1-phosphate to adenosine diphosphoglucose (ADP-glucose), the immediate precursor to glycogen. Such AGP-deficient (AGP−) strains may not make detectable amounts of glycogen under nitrogen starvation, but continue to fix carbon photosynthetically at a rate similar to wild-type cells. Yields of AKG and pyruvate in such mutant strains may exceed 100% of the initial cell dry weight of cultures incubated under continuous light in a nitrogen-free growth medium. In Synechocystis sp. PCC 6803, a glgC gene (slr1176) encodes an AGP enzyme.

#Suitable AGP− strains include those with a genetic mutation to interrupt expression of glgC yet maintain the ability to grow and produce metabolites such as AKG or pyruvate under nitrogen starvation conditions. Additional mutations or deletions in genes in the glycogen synthesis pathway (e.g., glycogen synthase) may also result in the increased production of AKG or pyruvate. Under nitrogen depletion conditions, the mutant strains may produce high levels of AKG, up to or greater than 30% of the cell dry weight, which is on the order of a 10,000-fold increase over the wild type. In certain embodiments, the mutant strains may exhibit at least a 100, 200, 300, 400, 500, 1000, 2500, 5000, 7500, or 10,000-fold increase in AKG production when compared to the wild type strain. In some embodiments, the mutant strains may exhibit at least a 100, 200, 300, 400, 500, 1000, 2500, 5000, 7500, or 10,000-fold increase in pyruvate production when compared to the wild type strain.