To eliminate the dependence of KO11 and LY01 on costly nutritional supple­mentation, a new ethanologenic E. coli strain was constructed. The starting strain SZ110, a derivative of KO11 modified for production of lactic acid in mineral salts medium (see Sect. 5.1), was re-engineered for ethanol produc­tion.

2.2.1

Conversion of SZ110 to LY168

Strain SZ110, a derivative of KO11, was engineered and metabolically evolved to produce lactic acid, as described in detail below [39]. Evolved derivatives of SZ110 produced D-lactate at 92% yield from 100 gL-1 glucose in inexpen­sive mineral salts media. Since this cheap and efficient utilization of large amounts of sugar is the desired biocatalyst behavior, strain SZ110 was chosen as the starting point for re-engineering of ethanologenic E. coli(Yomano et al., submitted). Conversion of this strain from lactic acid production to ethanol production involved several steps, beginning with deletion of the lactic acid production gene IdhA. The Z. mobilis PET operon, inserted at the pfl locus in KO11, was removed during engineering of SZ110 for lactic acid production by deletion of the entire focA-pflB region [39]. Since elimination of ackA and adhE prevents undesirable carbon loss, deletion of pflB is unnecessary and possibly limits acetyl-CoA levels. Therefore, the native pfl gene was restored in the re-engineered ethanologenic E. coli. To select for optimal integration of the Z. mobilis homoethanol pathway, a promoterless operon containing pdc, adhA, and adhB was randomly inserted by transposon.

Specific growth requirements of both the donor and recipient strains en­abled direct functional selection in minimal medium without antibiotics. Candidate ethanologenic strains were enriched by serial transfers in mineral salts medium. One clone was selected and designated LY160. Further evolu­tion of strain LY160 by serially subculturing into fresh mineral salts medium every 24 h for 32 days led to strain LY160im, an intermediate strain with con­tinued improvement in performance. It was determined that the Z. mobilis ethanol pathway in LY160im was integrated within rrlE, a 23S ribosomal RNA subunit, concurrent with the direction of transcription. The complex regu­lation of ribosomal RNA transcription is reviewed in [40,41]; the presence of two promoters results in high expression at high growth rates and basal expression at low growth rates and during stationary phase, making rrlE an excellent site for PET integration. The Pseudomonas putida short chain es­terase estZ gene was also integrated into the microbial biocatalyst to lower ethyl acetate levels in the broth. The final strain was designated LY168.

2.2.2