L-malate production by metabolically engineered escherichia coli

An isolated bacterial cell, said bacterial cell lacking fumarate reductase (FRD) activity and having increased levels of phosphoenol pyruvate carboxykinase (PCK) activity as compared to endogenous PCK activity of the parental bacterial strain, wherein FRD activity is inactivated by genetic modification of said bacterial cell via genetic modification of a gene selected from frdA, frdB, fdrC, frdD, frdAB, frdAC, frdAD, frdBC, frdBD, frdCD, frdABC, frdCBD, frdACD, frdABD, frdABCD or combinations thereof and said genetic modification comprises complete or partial deletion of a coding region of the gene, introducing frame shift mutations within the coding region of the gene, insertions of sequences that disrupt the activities of the proteins encoded by the genes, introduction of stop codons in the coding region of the gene or any combination thereof, the bacterial cell further comprising inactivation of genes encoding lactate dehydrogenase (ldhA), alcohol dehydrogenase (adhE), acetate kinase (ackA), and pyruvate-formate lyase (pflB), said inactivation comprises complete or partial deletion of a coding region of the gene, introducing frame shift mutations within the coding region of the gene, insertions of sequences that disrupt the activities of the proteins encoded by the genes, introduction of stop codons in the coding region of the gene or any combination thereof;

• and said increased levels of PCK activity arising from:
• a) replacement of a native promoter sequence either with a constitutive or inducible heterologous promoter operably linked to the endogenous pck gene;
• b) expression of a multicopy plasmid with a native promoter or an exogenous promoter sequence operably linked to a pck gene;
• c) integration of additional copies of the pck gene into the bacterial cell genome;
• or
• d) replacement of nucleotide G with nucleotide A at position 64 up stream of the endogenous pck gene start codon when the bacterial cell is an E. coli cell.