Carbon dioxide fixation via bypassing feedback regulation

Abstract

Genetically engineered cells and methods are presented that allow for the production of various value products from CO 2 . Contemplated cells have a CBB cycle that is genetically modified such that two molecules of CO 2 fixed in the CBB cycle can be withdrawn from the modified CBB cycle as a single C2 compound. In contemplated aspects a CBB cycle includes an enzymatic activity that generates the single C2 compound from a compound of the CBB cycle, while further modifications to the CBB cycle will not introduce additional recombinant enzymatic activity/activities outside the already existing catalytic activities in the CBB cycle.

What is claimed is: 
     
       1. A metabolically engineered cell having a native Calvin-Benson-Bassham (CBB) cycle, comprising:
 a recombinant nucleic acid comprising a nucleic acid sequence encoding a first enzyme and a second enzyme;
 wherein the first enzyme is a phosphoketolase enzyme belonging to EC 4.1.2.9, that utilizes an intermediate of the CBB pathway as a substrate, and generates a first acetyl phosphate product; and the second enzyme is a phosphoribulokinase enzyme belonging to EC2.7.1.19 that is overexpressed in the genetically modified organism in an amount to achieve a phosphoribulokinase activity level that is higher than the native phosphoribulokinase activity level of the organism, and the second enzyme utilizes ribulose-5-phosphate to produce ribulose-1,5-bisphosphate; and 
 wherein production of phosphoenolpyruvate (PEP) in the genetically modified organism, when grown under nitrogen depletion, results in a PEP concentration below a feedback inhibitory concentration for the CBB cycle. 
 
 
     
     
       2. The metabolically engineered cell of  claim 1 , wherein the cell is a bacterial cell. 
     
     
       3. The metabolically engineered cell of  claim 2 , wherein the bacterial cell belongs to the genus  Ralstonia.    
     
     
       4. The metabolically engineered cell of  claim 3 , that comprises a plasmid comprising an alsS-ilvC-ilvD operon. 
     
     
       5. The metabolically engineered cell of  claim 3 , that comprises a plasmid comprising a kivd-yqhD operon. 
     
     
       6. The metabolically engineered cell of  claim 3 , that comprises:
 a) a plasmid comprising a genomic template encoding phosphoketolase (F/XPK) obtained from  B. adolescentis  (ATCC 15703); and 
 b) a plasmid comprising a genomic template encoding phosphoribulokinase (PRK) obtained from  Synechocystis  sp. PCC 6803; 
 c) a plasmid comprising an alsS, ilvC, and ilvD operon; and 
 d) a plasmid comprising an kivd-yqhD operon. 
 
     
     
       7. The metabolically engineered cell of  claim 1 , wherein the concentration of PEP in the genetically modified organism is maintained below a concentration of 0.2 mM.