Metabolically engineered methanotrophic, phototrophic microorganisms
This disclosure relates to the engineering of phototrophic microorganisms for conversion of alkanes into higher-value products. Recombinant phototrophic organisms such as cyanobacteria can be engineered, optionally in a modular format, to express enzymes involved in converting methane to methanol, methanol to formaldehyde, formaldehyde to central metabolic pathway intermediates, and such intermediates to n-butanol.
1. A recombinant Synechocystis sp. PCC 6803, comprising:
(i) a gene encoding a soluble methane monooxygenase (sMMO);
(ii) a gene encoding a methanol dehydrogenase (MDH);
(iii) a gene encoding a hexulose-6-phosphate synthase (HPS); and
(iv) a gene encoding a 6-phosphate-3-hexuloisomerase (PHI).
2. The recombinant Synechocystis sp. PCC 6803 of claim 1 , wherein the gene encoding the sMMO is a group of genes that together encode: an MmoX polypeptide, an MmoY polypeptide, an MmoB polypeptide, an MmoZ polypeptide, an MmoD polypeptide, and an MmoC polypeptide.
3. The recombinant Synechocystis sp. PCC 6803 of claim 1 , wherein the gene encoding the MDH encodes a polypeptide having the amino acid sequence of an NAD-dependent MDH from methylotrophic Bacillus methanolicus.
4. The recombinant Synechocystis sp. PCC 6803 of claim 1 , wherein the gene encoding the HPS encodes a polypeptide having the amino acid sequence of a HPS from Methylococcus capsulatus, Bacillus methanolicus , or Pyrococcus horikoshii.
5. The recombinant Synechocystis sp. PCC 6803 of claim 1 , wherein the gene encoding the PHI encodes a polypeptide having the amino acid sequence of a PHI from Methylococcus capsulatus, Bacillus methanolicus , or Pyrococcus horikoshii.
6. The recombinant Synechocystis sp. PCC 6803 of claim 2 , wherein:
the MmoX polypeptide, the MmoY polypeptide, the MmoB polypeptide, the MmoZ polypeptide, and the MmoC polypeptide are from Methylococcus capsulatus;
the MDH is a NAD-dependent MDH from methylotrophic Bacillus methanolicus;
the HPS is a HPS from Methylococcus capsulatus ; and
the PHI is a PHI from Methylococcus capsulatus.
7. The recombinant Synechocystis sp. PCC 6803 of claim 1 , wherein the Synechocystis sp. PCC 6803 is capable of growth in media containing up to 2% (v/v) methanol, or capable of growth in media containing up to 15 mM formaldehyde.
8. The recombinant Synechocystis sp. PCC 6803 of claim 1 , wherein expression of the genes of (i) to (iv) in the Synechocystis sp. PCC 6803 results in the oxidation of an alkane and the production of a product selected from the group consisting of: an alcohol, an amino acid, a dicarboxylic acid, a fatty acid, and an intermediate of a central metabolic pathway, when the Synechocystis sp. PCC 6803 is cultured in the presence of light and O 2 in a medium comprising the alkane.