Characterization of an omega-class glutathione S-transferase in the stress response of the silkmoth.

The glutathione S-transferase (GST) superfamily is involved in detoxification of various xenobiotics. Using real-time PCR, mRNA encoding an omega-class GST of Bombyx mori (bmGSTO) was shown to be induced after exposure to various environmental stresses. A soluble form of recombinant protein (rbmGSTO) was functionally overexpressed in Escherichia coli cells and purified to homogeneity. Cys 38 and Pro 39 were found to be highly conserved in omega-class GSTs, and their roles were investigated by site-directed mutagenesis/kinetic analysis. Mutations of Cys 38 and Pro 39 residues affected the catalytic efficiency of enzymes, indicating that the presence of Cys 38 and Pro 39 residues is important for bmGSTO activity. Thus, bmGSTO could contribute to increasing the environmental stress resistance of lepidopteran insects.

Production of riboflavin by metabolically engineered Corynebacterium ammoniagenes.

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.

Construction of a plasmid carrying both CTP synthetase and a fused gene formed from cholinephosphate cytidylyltransferase and choline kinase genes and its application to industrial CDP-choline production: enzymatic production of CDP-choline from orotic acid (Part II).

A new method for enzymatic production of cytidine diphosphate choline (CDP-choline) from orotic acid and choline chloride was developed. To establish an industrial manufacturing process, we constructed a plasmid, pCKG55, which simultaneously expressed in Escherichia coli the three following enzymes; CTP synthetase (encoded by the pyrG gene from E. coli), cholinephosphate cytidylyltransferase (encoded by the CCT gene from Saccharomyces cerevisiae), and choline kinase (encoded by the CKI gene from S. cerevisiae). CCT and CKI genes on pCKG55 were designed to be expressed as a single CCT/CKI fused protein. This CCT/CKI fused protein retained both activities and the thermal stability of its cholinephosphate cytidylyltransferase activity was nearly the same as the native CCT enzyme. Corynebacterium ammoniagenes KY13505 and E. coli MM294/pCKG55 were cultured in 5-liter jar fermentor independently. Equal volumes of each broth were mixed in a 2-liter jar fermentor, and then the enzymatic reaction was done using 47 mM orotic acid and 60 mM choline chloride as substrates. After 23 h of the reaction at 32 degrees C, 21.5 mM (11 g/liter) of CDP-choline was accumulated.

A novel process of inosine 5'-monophosphate production using overexpressed guanosine/inosine kinase.

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.

EI-1511-3, -5 and EI-1625-2, novel interleukin-1 beta converting enzyme inhibitors produced by Streptomyces sp. E-1511 and E-1625. I. Taxonomy of producing strain, fermentation and isolation.

EI-1511-3, -5 and EI-1625-2, novel interleukin-1 beta converting enzyme (ICE) inhibitors, were isolated from the culture broths of Streptomyces sp. E-1511 and E-1625. EI-1511-3, -5 and EI-1625-2 selectively inhibited the recombinant human ICE activity with IC50 values of 0.09, 0.38 and 0.2 microM, respectively. Taxonomy, fermentation of the producing strain and isolation of EI-1511-3, -5 and EI-1625-2 are described.

EI-1507-1 and -2, novel interleukin-1 beta converting enzyme inhibitors produced by Streptomyces sp. E-1507.

EI-1507-1 and -2, novel interleukin-1 beta converting enzyme (ICE) inhibitors, were isolated from the culture broths of Streptomyces sp. E-1507. EI-1507-1 and EI-1507-2 selectively inhibited the recombinant human ICE activity with IC50 values of 0.23 and 0.42 microM, respectively. EI-1507-1 and EI-1507-2 also inhibited mature interleukin-1 beta secretion from THP-1 cells with IC50 values of 1.1 and 1.4 microM, respectively.

Construction of catalase deficient Escherichia coli strains for the production of uricase.

To produce catalase-free uricase preparations, we constructed catalase-deficient strains from Escherichai coli MC1000 and MM294 and used them as recombinant host strains. The parent strains and catalase-deficient strains showed no differences in the growth characteristics by shaking culture in Erlenmeyer flasks. The catalase deficient strain derived from MC1000 transformed with the uricase expression plasmid pUT118 (strain SN0037) had growth characteristics and the uricase productivity comparable to those of the parent host strain MC1000 in fed-batch culture in a jar fermentor and no catalase activity was detected in cell-free extracts. However, the katG disrupted strains from MM294 carrying pUT118 had poor growth and their uricase productivities were low compared to those of the parent strain MM294. Using the strain SN0037, a catalase-free uricase preparation was obtained with fewer purification procedures and the final recovery of uricase activity was improved. The catalase-deficient E. coli host strain will be a suitable host for the production of the uricase, free of catalase activity, in high yield.

Cloning of a guanosine-inosine kinase gene of Escherichia coli and characterization of the purified gene product.

We attempted to clone an inosine kinase gene of Escherichia coli. A mutant strain which grows slowly with inosine as the sole purine source was used as a host for cloning. A cloned 2.8-kbp DNA fragment can accelerate the growth of the mutant with inosine. The fragment was sequenced, and one protein of 434 amino acids long was found. This protein was overexpressed. The overexpressed protein was purified and characterized. The enzyme had both inosine and guanosine kinase activity. The Vmaxs for guanosine and inosine were 2.9 and 4.9 mumol/min/mg of protein, respectively. The Kms for guanosine and inosine were 6.1 microM and 2.1 mM, respectively. This enzyme accepted ATP and dATP as a phosphate donor but not p-nitrophenyl phosphate. These results show clearly that this enzyme is not a phosphotransferase but a guanosine kinase having low (Vmax/Km) activity with inosine. The sequence of the gene we have cloned is almost identical to that of the gsk gene (K.W. Harlow, P. Nygaard, and B. Hove-Jensen, J. Bacteriol. 177:2236-2240, 1995).

Effects of the amplification of the genes coding for the L-threonine biosynthetic enzymes on the L-threonine production from methanol by a gram-negative obligate methylotroph, Methylobacillus glycogenes.

We constructed recombinant plasmids carrying the genes coding for the L-threonine biosynthetic enzymes, the hom gene, the hom-thrC genes, and the thrB genes, of a gram-negative obligate methylotroph, Methylobacillus glycogenes, and examined the effects of them on the production of L-threonine from methanol. The hom gene, which encodes the homoserine dehydrogenase, and the hom-thrC genes, containing the gene coding for threonine synthase together with the hom gene, were cloned from a wild-type strain, and the thrB gene encoding the desensitized homoserine kinase was cloned from an L-threonine-producing mutant, ATR80. The recombinant plasmids were transferred into ATR80 and its L-isoleucine auxotroph, A513, by conjugation. Amplification of the genes coding for the L-threonine biosynthetic enzymes elevated the activities of the L-threonine biosynthetic enzymes of the transconjugants 10- to 30-fold over those of the strains containing only vectors. The L-threonine production from methanol in test-tube cultivation was increased about 30% and 40% by the amplification of the hom gene and the hom-thrC gene respectively, and it was slightly increased by that of the thrB gene. The effects of gene amplification were confirmed by the cultivation in 5-1 jar fermentors. The best producer, an A513 transconjugant containing the plasmid carrying the hom-thrC genes, produced 16.3 g/l L-threonine for 72 h.

Cloning and nucleotide sequences of the homoserine dehydrogenase genes (hom) and the threonine synthase genes (thrC) of the gram-negative obligate methylotroph Methylobacillus glycogenes.

We have cloned the homoserine dehydrogenase genes (hom) from the gram-negative obligate methylotrophs Methylobacillus glycogenes ATCC 21276 and ATCC 21371 by complementation of an Escherichia coli homoserine dehydrogenase-deficient mutant. The 4.15-kb DNA fragment cloned from M. glycogenes ATCC 21371 also complemented an E. coli threonine synthase-deficient mutant, suggesting the DNA fragment contained the thrC gene in addition to the hom gene. The homoserine dehydrogenases expressed in the E. coli recombinants were hardly inhibited by L-threonine, L-phenylalanine, or L-methionine. However, they became sensitive to the amino acids after storage at 4 degrees C for 4 days as in M. glycogenes. The structures of the homoserine dehydrogenases overexpressed in E. coli were thought to be different from those in M. glycogenes, probably in subunit numbers of the enzyme, and were thought to have converted to the correct structures during the storage. The nucleotide sequences of the hom and thrC genes were determined. The hom genes of M. glycogenes ATCC 21276 and ATCC 21371 encode peptides with M(r)s of 48,225 and 44,815, respectively. The thrC genes were located 50 bp downstream of the hom genes. The thrC gene of ATCC 21371 encodes a peptide with an M(r) of 52,111, and the gene product of ATCC 21276 was truncated. Northern (RNA) blot analysis suggests that the hom and thrC genes are organized in an operon. Significant homology between the predicted amino acid sequences of the hom and thrC genes and those from other microorganisms was found.

Identification and characterization of the tktB gene encoding a second transketolase in Escherichia coli K-12.

We isolated a transposon Tn10 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.

Amino Acid Production from Methanol by Methylobacillus glycogenes Mutants: Isolation of L-Glutamic Acid Hyper-producing Mutants from M. glycogenes Strains, and Derivation of L-Threonine and L-Lysine-producing Mutants from Them.

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.

Cloning of the ARO3 gene of Saccharomyces cerevisiae and its regulation.

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.