L-Alanine, used in the pharmaceutical industry [150] and as a food addi­tive [151], is commercially produced by enzymatic decarboxylation of L-aspartic acid with either immobilized cells or cells suspensions [152]. How­ever, recent attention has shifted to fermentative production [150,151].

Given our success in producing microbial biocatalysts by a combination of directed engineering and metabolic evolution, we modified lactic-acid produc­ing E. coli B derivative SZ194 for alanine production. The native IdhA gene was replaced with the ribosome binding site, coding region, and transcriptional terminator of the thermostable alanine dehydrogenase alaD from Geobacillus stearothermophilus XL-65-6 (Zhang et al., unpublished results). While the ini­tial microbial biocatalyst was capable of producing l-alanine as the primary fermentation product, long incubation times were required and the productiv­ity was low. As with other microbial biocatalysts designed in our laboratory, metabolic evolution was used for strain improvement. The strain was further engineered to reduce co-product formation (mgsA) and increase the chiral purity (dadX). The final microbial biocatalyst, XZ132, produced near 1.3 M l — alanine from 12%glucose within 48 h, a yield greater than 95%, in AM1 mineral salts media.

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