Genetically engineered cyanobacteria for growth in unsterilized conditions using antibiotic-free selection
AssigneeNanyang Technological University
InventorsTiago de Sousa Jorge Toscano SELÃO, Inger Birgitta NORLING, Peter Julian NIXON
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Abstract
The present invention relates to methods of metabolic engineering cells to increase their ability to compete with contaminating microorganisms without the need for antibiotics. More particularly, the invention provides methods to engineer cyanobacteria to utilize melamine as nitrogen source, phosphite as phosphorous source, optionally also utilizing NADP+ over NAD+, and also provides genetically engineered cells made using such methods.
Claims29
An isolated genetically engineered cyanobacterium, wherein the cyanobacterium has been transformed by at least one polynucleotide molecule;
- the at least one polynucleotide molecule comprising heterologous melamine utilization pathway genes, atzD, trzE, DUR1,2, trzC, guaD and triA operably linked to at least one promoter, wherein;
- i) the triA gene comprises one or more mutations which encode amino acid substitutions, wherein the amino acid substitutions are at positions selected from the group comprising Leu88Phe, His254Tyr, Glu317Lys, Ala355Val, Trp471Stop and the combination of Thr218Asn and Val278Met;
- and/or
- ii) the triA gene has a ribosome binding site (RBS) comprising a AGGAGA to AGAAGA mutation,
- wherein said genetically engineered cyanobacterium has no heterologous antibiotic resistance genes.
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An isolated genetically engineered cyanobacterium, wherein the cyanobacterium has been transformed by at least one polynucleotide molecule;
- the at least one polynucleotide molecule comprising a heterologous phosphite dehydrogenase (ptxD) gene operably linked to a promoter, wherein the ptxD gene comprises a polynucleotide sequence set forth in SEQ ID NO:
- 90, or SEQ ID NO:
- 91,
- wherein said genetically engineered cyanobacterium has no heterologous antibiotic resistance genes.
A recombinant vector comprising melamine pathway genes triA, DUR1,2, atzD, trzC, trzE, and guaD, operably linked to at least one promoter, wherein
- i) the triA gene comprises one or more mutations which encode amino acid substitutions, wherein the amino acid substitutions are at positions selected from the group comprising Leu88Phe, His254Tyr, Glu317Lys, Ala355Val, Trp471Stop, and the combination of Thr218Asn and Val278Met;
- and/or
- ii) the triA gene has a ribosome binding site (RBS) comprising a AGGAGA to AGAAGA mutation,
- wherein the vector lacks antibiotic resistance genes.
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Dependencies are inferred from the claim text.