We isolated a transposon TnlO insertion mutant of Escherichia coli K-12 which could not grow on MacConkey plates containing D-ribose. Characterization of the mutant revealed that the level of the transketolase activity was reduced to one-third of that of the wild type. The mutation was mapped at 63.5 min on the E. coli genetic map, in which the transketolase gene (tkt) had been mapped. A multicopy suppressor gene which complemented the tkt mutation was cloned on a 7.8-kb PstI fragment. The cloned gene was located at 53 min on the chromosome. Subcloning and sequencing of a 2.7-kb fragment containing the suppressor gene identified an open reading frame encoding a polypeptide of 667 amino acids with a calculated molecular weight of 72,973. Overexpression of the protein and determination of its N-terminal amino acid sequence defined unambiguously the translational start site of the gene. The deduced amino acid sequence showed similarity to sequences of transketolases from Saccharomyces cerevisiae and Rhodobacter sphaeroides. In addition, the level of the transketolase activity increased in strains carrying the gene in multicopy. Therefore, the gene encoding this transketolase was designated tktB and the gene formerly called tkt was renamed tktA. Analysis of the phenotypes of the strains containing tktA, tktB, or tktA tktB mutations indicated that tktA and tktB were responsible for major and minor activities, respectively, of transketolase in E. coli.Transketolase (EC 2.2.1.1) catalyzes transfer of the glycol aldehyde moiety from a ketose or its phosphate to an aldose or its phosphate. The enzyme is found in animals, plants, and bacteria and is involved in the pentose phosphate pathway responsible for production of NADPH and several sugar phosphate intermediates such as ribose 5-phosphate, erythrose 4-phosphate, and sedoheptulose 7-phosphate. Ribose 5-phosphate is utilized for the biosynthesis of purine and pyrimidine nucleotides and histidine, erythrose 4-phosphate is used for the biosynthesis of aromatic amino acids, and sedoheptulose 7-phosphate is used for the biosynthesis of cell wall components in gram-negative bacteria. The reaction is also important in CO2 fixation in photosynthetic organisms.In Escherichia coli Josephson and Fraenkel (18,19) first isolated mutants defective in transketolase activity. These mutants were unable to use L-arabinose or D-xylose as a sole carbon source and required shikimic acid or aromatic amino acids for growth on a minimal medium. The requirement for aromatic amino acids was shown to be slightly leaky, and the existence of low residual transketolase activity was suggested by the authors. The mutations were mapped around 62 min on the E. coli chromosome. A similar mutant was also isolated from Salmonella typhimurium, and the role of transketolase in supplying sedoheptulose 7-phosphate was examined (7). In this report, we describe isolation of a transposon TnlO insertion mutant of E. coli defective in transketolase as a ribose-sensitive mutant on MacConkey plates containing D-ri...

Regulation of the two isozymes of 3-deoxy-D-arabino-heptulosonate-7phosphate synthase (DAHP synthase; EC 4.1.2.15) encoded by the genes ARO3 and ARO4 of Saccharomyces cerevisiae was studied. Both genes were shown to respond equally well to the general control of amino acid biosynthesis. Strains with mutations in these two genes were obtained by selecting first for a single aro3 mutation and afterwards for a double aro3 aro4 mutation. Gene ARO3, coding for the phenylalanine-dependent isozyme of DAHP synthase was cloned on the 2 micron multicopy vector pJDB207 by complementation of mutation aro3-1 in yeast. The ARO3 gene, carried originally on a 9.6 kb BamHI fragment (plasmid pME541A), was subcloned on a 1.9 kb HindIII-XbaI fragment (plasmid pME543). A transcript of about 1.5 kb was shown to proceed from the HindIII towards the XbaI site. Expression from the 9.6 kb as well as from the 1.9 kb fragment was normal on a multicopy vector, since in both cases DAHP synthase levels of about 50-fold the wild-type level were observed.

A novel process for producing inosine 5'-monophosphate (5'-IMP) has been demonstrated. The process consists of two sequential bioreactions; the first is a fermentation of inosine by a mutant of Corynebacterium ammoniagenes, and the second is a unique phosphorylating reaction of inosine by guanosine/inosine kinase (GIKase). GIKase was produced by an Escherichia coli recombinant strain, MC1000(pIK75), which overexpressed the enzyme up to 50% of the total cellular protein. The overproducing plasmid, pIK75, which was randomly screened out from deletion plasmids with various lengths of intermediate sequence between the E. coli trpL Shine-Dalgarno sequence, derived from the vector plasmid, and the start codon of the GIKase structural gene. In pIK75, the start ATG was placed 16 bp downstream of the trpL Shine-Dalgarno sequence under the control of the E. coli trp promoter. Fermentation of inosine and its phosphorylation were sequentially performed in a 5-1 jar fermenter. At the end of inosine fermentation by C. ammoniagenes KY13761, culture broth of MC1000(pIK75) was mixed with that of KY13761 to start the phosphorylating reaction. Inosine in the reaction mixture was stoichiometrically phosphorylated, and 91 mM 5'-IMP accumulated in a 12-h reaction. This new biological process has advantages over traditional methods for producing 5'-IMP.

Improved strains for the production of riboflavin (vitamin B2) were constructed through metabolic engineering using recombinant DNA techniques in Corynebacterium ammoniagenes. A C. ammoniagenes strain harboring a plasmid containing its riboflavin biosynthetic genes accumulated 17-fold as much riboflavin as the host strain. In order to increase the expression of the biosynthetic genes, we isolated DNA fragments that had promoter activities in C. ammoniagenes. When the DNA fragment (P54-6) showing the strongest promoter activity in minimum medium was introduced into the upstream region of the riboflavin biosynthetic genes, the accumulation of riboflavin was 3-fold elevated. In that strain, the activity of guanosine 5'-triphosphate (GTP) cyclohydrolase II, the first enzyme in riboflavin biosynthesis, was 2.4-fold elevated whereas that of riboflavin synthase, the last enzyme in the biosynthesis, was 44.1-fold elevated. Changing the sequence containing the putative ribosome-binding sequence of 3,4-dihydroxy-2-butanone 4-phosphate synthase/GTP cyclohydrolase II gene led to higher GTP cyclohydrolase II activity and strong enhancement of riboflavin production. Throughout the strain improvement, the activity of GTP cyclohydrolase II correlated with the productivity of riboflavin. In the highest producer strain, riboflavin was produced at the level of 15.3 g l(-1) for 72 h in a 5-l jar fermentor without any end product inhibition.

L-Glutamic acid (Glu) hyper-producing mutants were isolated from Methylobacillus glycogenes ATCC 21276 and ATCC 21371 during the screening of amino acid auxotrophs. iA111, derived from ATCC 21276, and 1009 (Phe(-)), derived from ATCC 21371, produced about 10 times as much Glu as the wild type strains. The highest producer, RV3, a phenylalanine auxotrophic revertant of 1009, produced 38.8 g/liters of Glu in 84h in a 5-liter jar fermentor. iA111 and 1009 were mutagenized, and L-threonine (Thr) producing mutants were isolated among Thr and L-Iysine (Lys) analog-resistant strains. The highest Thr producer derived from iA111, AL119 (AHV + Lys(R)), produced 11.0g/liters of Thr in 72h in a 5-liter jar fermentor. AL119 was further mutagenized, and a Thr and Lys producer, DHL 122 (DHL(R)), was isolated. DHL 122 co-produced 3.1 g/liters of Lys and 5.6 g/liters of Thr in 72 h in a 5-liter jar fermentor.