US 8,993,303 to South Dakota State University: Genetically engineered cyanobacteria

The first claim of US 8,993,303:

A composition comprising an Anabaena spp. genetically engineered with at least one recombinant polynucleotide expression construct, wherein the at least one recombinant polynucleotide expression construct comprises a nucleotide sequence encoding at least one enzyme, wherein the at least one enzyme increases production of a carbon based product of interest by the genetically engineered Anabaena spp. following expression of the polynucleotide expression construct, wherein said Anabaena spp. is ethanol producing Anabaena sp. PCC7120 (pZR672) strain deposited under ATCC accession number PTA-12833 or is linalool producing Anabaena sp. PCC7120 (pZR808) strain deposited under ATCC accession number PTA-12832.


From within the specification:

In one embodiment, the host cell is genetically engineered to increase production of ethanol through transformation with an expression vector containing polynucleotides encoding ethanol producing enzymes. As used herein, an ethanol producing enzyme is an enzyme active in the end production of ethanol from a precursor molecule in a metabolic pathway. The polynucleotide encodes pyruvate decarboxylase (SEQ ID NO. 5) and/or alcohol dehydrogenase (SEQ ID NO. 6) in exemplary embodiments. Embodiments also include enzymes having sequence identity of about 76%, 80%, 85%, at least about 90%, and at least about 95%, 96%, 97%, 98% or 99% to SEQ ID NO. 5 and SEQ ID NO. 6. The host is genetically engineered with polynucleotides encoding one or both enzymes. In many embodiments, host cells are engineered to express both enzymes. Known sources of polynucleotides encoding pyruvate decarboxylase and alcohol dehydrogenase exist. For example, the nucleic acid encoding the enzymes may be from organisms such as Zymomonas mobilis, Zymobacter paimae, or Saccharomyces cerevisciae (Ingram L O, Conway T, Clark D P, Sewell G W, Preston J F. 1987. Genetic engineering of ethanol production in Escherichia coli. Appl Environ Microbiol. 53(10):2420-5). Any pyruvate decarboxylase (pdc) gene capable of expression in the host may be used in with the disclosed embodiments. In some embodiments, the pdc gene is the Zymomonas mobilis pdc gene. In these embodiments, the pdc gene is often obtained from the Zymomonas mobilis plasmid pLOI295. In other embodiments, the pdc gene is from Zymobacter paimae. The NCBI accession number for the complete pdc protein sequence from Zymobacter paimae is AF474145. Similarly, any alcohol dehydrogenase (adh) gene capable expression in the host may be used with the disclosed embodiments. In some embodiments, the adh gene is the Zymomonas mobilis adhII gene. In these embodiments, the adh gene is often obtained from the Zymomonas mobilis plasmid pLOI295.

Polynucleotides encoding genes such as omrA, lmrA, and lmrCD, which increase the ability of the host to handle commercially relevant amounts of ethanol, may be included on the same or a different vector as the polynucleotides encoding the pdc and adh genes. Bourdineaud J P, Nehme B, Tesse S, Lonvaud-Funel A. 2004. A bacterial gene homologous to ABC transporters protect Oenococcus oeni from ethanol and other stress factors in wine. Int. J. Food Microbiol. 92(1):1-14. For example, in some embodiments, the expression vector comprising the pdc/adh genes further comprises the omrA gene. In other embodiments, the expression vector comprising the pdc/adh genes further comprises the lmrA gene. In yet other embodiments, the expression vector comprising the pdc/adh genes further comprises the lmrCD gene. And in still further embodiments, the expression vector comprising the pdc/adh genes further comprises polynucleotides encoding the omrA, lmrA, and lmrCD genes.