There exists in Escherichia coli a known set of enzymes that were shown to function in an efficient and concerted way to convert threonine to serine. The sequence of reactions catalyzed by these enzymes is designated the Tut cycle (threonine utilization). To demQnstrate that the relevant genes and their protein products play essential roles in serine biosynthesis, a number of mutants were analyzed. Strains of E. coli with lesions in serA, serB, serC, or glyA grew readily on minimal medium supplemented with elevated levels of leucine, arginine, lysine, threonine, and methionine. No growth on this medium was observed upon testing double mutants with lesions in one of the known ser genes plus a second lesion in glyA (serine hydroxymethyltransferase), gcv (the glycine cleavage system), or tdh (threonine dehydrogenase). Pseudorevertants of ser mutants capable of growth on either unsupplemented minimal medium or medium supplemented with low levels of leucine, arginine, lysine, threonine, and methionine were isolated. At least two unlinked mutations were associated with such phenotypes.

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The productivity of stably transfected cell lines is of critical importance for the manufacturing of therapeutic proteins. Various methods have been successfully implemented to increase the production output of mammalian cell cultures. Increasing evidence suggests that optimization of the gene coding sequences of an expression vector can improve specific cell line yield of the recombinant protein. Here we demonstrate that gene optimization substantially enhances antibody production in Chinese hamster ovary cells. When gene optimization was applied to the heavy and light chain genes of a therapeutic antibody, we observed increased antibody production in transient transfection. Elevated heavy chain mRNA level was associated with the increase of antibody production. Further analysis suggested that the increased antibody expression is attributable to enhanced mRNA stability resulting from gene optimization. Gene optimization also led to increased antibody production in stable clones.

A 3563 bp EcoRl fragment that contains the genes for E. coli threonine dehydrogenase and 2amino-3-ketobutyrate coenzyme A ligase (AKB ligase; EC 2.3.1.29) (1) was sequenced using the dideoxy chain termination method. These genes lie at coordinate 81.2 of the standard E. coli genetic map (2). The nucleotide sequence of the AKB ligase gene (designated kbl) and its promoter is presented. AKB ligase contains 398 amino acids. The predicted N-terminal sequence, MW and amino acid composition are in agreement with the known properties of the purified enzyfie (3). The pattern of

The plasmid pDR121 is a pBR322 derivative that contains a 3.7-kilobase-pair EcoRI fragment of DNA from the 81.2-min region of the Escherichia coli chromosome. The genomic insert encodes threonine dehydrogenase and at least one other protein. Several physical and kinetic properties of threonine dehydrogenase, overproduced in cells harboring pDR121, are identical to those of pure threonine dehydrogenase from a haploid mutant of E. coli K-12 that produces this enzyme constitutively. Tester strains with serB or glyA mutations harboring pDR121 are prototrophs. The ability to confer prototrophy on such tester strains is associated with elevated levels of threonine dehydrogenase. The functional roles of various segments of the 3.7-kilobase-pair insert of pDR121 were analyzed by constructing specific deletions and insertions. Certain subclones retained the ability to specify threonine dehydrogenase without conferring prototrophy on tester strains. This suggests that at least one other protein encoded within pDR121 plays an essential role in the conversion of threonine to serine.

The threonine dehydrogenase (tdh) gene of Escherichia coli, cloned within the plasmid pDR121, was inactivated in vitro by inserting a segment of DNA carrying the chloramphenicol acetyltransferase (cat) gene. The insertionally inactivated tdh gene was then transferred by homologous recombination into the E. coli chromosome by the procedure of Winans et al. (J. Bacteriol. 161:1219(J. Bacteriol. 161: -1221(J. Bacteriol. 161: , 1985. Mating experiments, followed by Pl-mediated two-and three-point crosses, enabled us to localize tdh near min 81.2. The order with respect to known markers is mtl-cysE-tdh-pyrE.

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