Escherichia coli ethanologen. Escherichia coli strain:GLBRCE1

An ethanologenic E. coli K-12 with the same ethanol pathway as the E. coli W ethanologen strain KO11 was constructed by PCR amplification of the Z. mobilis PET cassette genes and flanking pflB sequence from KO11 and subsequent insertion into the pflB locus of MG1655 using lambda Red-mediated recombination. This yielded an insertion of pdc, adhB, and cat between nucleotides 396 and 400 of pflB (3 nucleotides [nt] of pflB were eliminated; pdc and adhB were in the same orientation as pflB and thus transcribed from pflB promoters). To enhance ethanol production, the lactate dehydrogenase (ldhA) and acetate kinase (ackA) genes responsible for the production of alternate fermentation end products were deleted, and an additional copy of the PET cassette was constitutively expressed from the low-copy-number plasmid pJGG2. The resulting strain was identified as GLBRCE1. Resequencing of GLBRCE1 by the Joint Genome Institute confirmed the ldhA and ack deletions and the PET insertion in pflB. In addition, variant alleles of crl, ylbE, glpR, and gatC recently reported to be in many “wild-type” E coli strains and single-nucleotide polymorphisms in yodD and gltB were identified. Thus, the genotype of GLBRCE1 relative to GenBank accession number U00096.2 is ldhA(4_969del 3insFRT) ackA(4_1182del 3insFRT) pflB[395ins(pdcZmo adhBZmo cat)] crl(70insIS1) ylbE(253insG) gltB(G3384A) yodD(A85T) glpR(150delG) gatC(916insCC) pJGG2.