Snapgene files of the genetic constructions for Morella thermoacetica used in Task 2.2 (Advanced GMO preparation for gas (CO2/CO/H2) fermentation process)

Part of WP2 these snapgene files of the genetic constructions  for Morella thermoacetica were used in Task 2.2 (Advanced GMO preparation for gas (CO2/CO/H2) fermentation process). The strains we were able to construct were:

1.- M. thermoacetica DSM 521 pK18ACSACassette complete2.- M. thermoacetica DSM 521 pK18ACSACassette AatA3.- M. thermoacetica DSM 2955 pK18ACSACassette PudL-AckA4.- M. thermoacetica DSM 2955 pK18ACSACassette AatA

To construct these strains CSIC used a modular synthetic construction carrying the regions up and down of the acsA gene to delete it. Within these regions, a gene cluster composed by the GmR, pduL, ackA and aatA genes, all of them controlled by the strong constitutional promoter of the glyceraldehyde 3-phosphate dehydrogenase gene from Moorella were inserted. The modular design of our recombinant cassette allows us to construct different cassettes with different gene combinations. In all cases, the acsA gene will be deleted and the acetate cannot be transformed back into acetyl-CoA:

Cassette 1.- ACSA-GmR-pduL-ackA-aatA-ACSA: this is a complete cassette which carries the genes encoding the phosphotransacetylase, the acetate kinase and the acetate exporter. This strain should accumulate a higher concentration of acetate inside the cell that should be exported to the medium.

Cassette 2.- ACSA-GmR-pduL-ackA-ACSA: this cassette lacks the transporter, so this strain should accumulate a higher concentration inside the cell. Then, the only way to prevent acetate toxicity is by increasing its own mechanisms to export this compound or by growing slower in order to manage acetate accumulation.

Cassette 3.- ACSA-GmR-aatA-ACSA: this cassette only carries the acetate exporter, so virtually all the acetate produced inside the cell should be exported. This is another strategy to increase the metabolic flux inside the cell towards acetate without overexpressing any other gene of the acetate pathway, and at the same time avoiding toxicity problems.

Cassette 4.- ACSA-GmR-ACSA: this cassette would generate an insertion mutant in which acsA is only replaced by the GmR encoding gene. This mutant should also generate a higher amount of acetate since it cannot be transformed back to acetyl-CoA.