Identification of a novel type of polyunsaturated fatty acid synthase involved in arachidonic acid biosynthesis

AbstractArachidonic acid (ARA) is a polyunsaturated fatty acid (PUFA) and an essential component of membrane lipids. However, the PUFA synthase required for ARA biosynthesis has not been identified in any organism. To identify the PUFA synthase producing ARA, we determined the draft genome sequence of the marine bacterium Aureispira marina, which produces a high level of ARA, and found a gene cluster encoding a putative PUFA synthase for ARA production. Expression of the gene cluster in Escherichia coli induced production of ARA, demonstrating that the gene cluster encodes a PUFA synthase required for ARA biosynthesis

Significance of the Cgl1427 gene encoding cytidylate kinase in microaerobic growth of Corynebacterium glutamicum

The Cgl1427 gene was previously found to be relevant to the microaerobic growth of Corynebacterium glutamicum (Ikeda et al. Biosci Biotechnol Biochem 73:2806-2808, 2009). In the present work, Cgl1427 was identified as a cytidylate kinase gene (cmk) by homology analysis of its deduced amino acid sequence with that of other bacterial cytidylate kinases (CMP kinases) and on the basis of findings that deletion of Cgl1427 results in loss of CMP kinase activity. Deletion of the cmk gene significantly impaired the growth of C. glutamicum in oxygen-limiting static culture, and the impaired growth was restored by introducing a plasmid containing the cmk gene, suggesting that this gene plays an important role in the microaerobic growth of C. glutamicum. On the other hand, in the main culture with aerobic shaking, a prolonged lag phase was observed in the cmk disruptant, despite an unchanged growth rate, compared to the behavior of the wild-type strain. The prolongation was observed when using seed culture grown to later growth stages in which oxygen limitation occurred, but it was not observed when using seed culture grown to an earlier growth stage in which oxygen remained relatively plentiful. Since nucleotide biosynthesis in C. glutamicum requires oxygen, we hypothesized that the ability of the cmk disruptant to synthesize nucleotides was influenced by oxygen limitation in the later growth stages of the seed culture, which caused the prolongation of the lag phase in the following shaken culture. To verify this hypothesis, a plasmid containing genes encoding all components of a homologous ribonucleotide reductase, a key enzyme for nucleotide synthesis that requires oxygen for its reaction, was introduced into the cmk disruptant, which significantly ameliorated the lag phase prolongation. Furthermore, this experimental setup almost completely restored the growth of the cmk disruptant in the oxygen-limiting static culture. These results indicate that CMP kinase plays an important role in normal nucleotide biosynthesis under an oxygen-limiting environment.ArticleAPPLIED MICROBIOLOGY AND BIOTECHNOLOGY. 97(3):1259-1267 (2013)journal articl

A third glucose uptake bypass in Corynebacterium glutamicum ATCC 31833

In Corynebacterium glutamicum, the phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS) has long been the only known glucose uptake system, but we recently found suppressor mutants emerging from a PTS-negative strain of C. glutamicum ATCC 31833 on glucose agar plates, and identified two alternative potential glucose uptake systems, the myo-inositol transporters encoded by iolT1 and iolT2. The expression of either gene renders the PTS-negative strain WT Delta ptsH capable of growing on glucose. In the present study, we found a suppressor strain that still grew on glucose even after the iolT1 and iolT2 genes were both disrupted under the PTS-negative background. Whole-genome sequencing of the suppressor strain SPH1 identified a G-to-T exchange at 134 bp upstream of the bglF gene encoding an EII component of the beta-glucoside-PTS, which is found in limited wild-type strains of C. glutamicum. Introduction of the mutation into strain WT Delta ptsH allowed the PTS-negative strain to grow on glucose. Reverse transcription-quantitative PCR analysis revealed that the mutation upregulates the bglF gene by approximately 11-fold. Overexpression of bglF under the gapA promoter in strain WT Delta ptsH rendered the strain capable of growing on glucose, and deletion of bglF in strain SPH1 abolished the growth again, proving that bglF is responsible for glucose uptake in the suppressor strain. Simultaneous disruption of three glucokinase genes, glk (Cgl2185, NCgl2105), ppgK (Cgl1910, NCgl1835), and Cgl2647 (NCgl2558), in strain SPH1 resulted in no growth on glucose. Plasmid-mediated expression of any of the three genes in the triple-knockout mutant restored the growth on glucose. These results indicate that C. glutamicum ATCC 31833 has an additional non-PTS glucose uptake route consisting of the bglF-specified EII permease and native glucokinases.ArticleAPPLIED MICROBIOLOGY AND BIOTECHNOLOGY. 99(6):2741-2750 (2015)journal articl

L-Lysine production independent of the oxidative pentose phosphate pathway by Corynebacterium glutamicum with the Streptococcus mutans gapN gene

Published online 29 March 2016We have recently developed a Corynebacterium glutamicum strain that generates NADPH via the glycolytic pathway by replacing endogenous NAD-dependent glyceraldehyde 3-phosphate dehydrogenase (GapA) with a nonphosphorylating NADP-dependent glyceraldehyde 3-phosphate dehydrogenase (GapN) from Streptococcus mutans. Strain RE2, a suppressor mutant spontaneously isolated for its improved growth on glucose from the engineered strain, was proven to be a high-potential host for l-lysine production (Takeno et al., 2010). In this study, the suppressor mutation was identified to be a point mutation in rho encoding the transcription termination factor Rho. Strain RE2 still showed retarded growth despite the mutation rho696. Our strategy for reconciling improved growth with a high level of l-lysine production was to use GapA together with GapN only in the early growth phase, and subsequently shift this combination-type glycolysis to one that depends only on GapN in the rest of the growth phase. To achieve this, we expressed gapA under the myo-inositol-inducible promoter of iolT1 encoding a myo-inositol transporter in strain RE2. The resulting strain RE2Aiol was engineered into an l-lysine producer by introduction of a plasmid carrying the desensitized lysC, followed by examination for culture conditions with myo-inositol supplementation. We found that as a higher concentration of myo-inositol was added to the seed culture, the following fermentation period became shorter while maintaining a high level of l-lysine production. This finally reached a fermentation period comparable to that of the control GapA strain, and yielded a 1.5-fold higher production rate compared with strain RE2. The transcript level of gapA, as well as the GapA activity, in the early growth phase increased in proportion to the myo-inositol concentration and then fell to low levels in the subsequent growth phase, indicating that improved growth was a result of increased GapA activity, especially in the early growth phase. Moreover, blockade of the pentose phosphate pathway through a defect in glucose 6-phosphate dehydrogenase did not significantly affect l-lysine production in the engineered GapN strains, while a drastic decrease in l-lysine production was observed for the control GapA strain. Determination of the intracellular NADPH/NADP+ ratios revealed that the ratios in the engineered strains were significantly higher than the ratio of the control GapA strain irrespective of the pentose phosphate pathway. These results demonstrate that our strain engineering strategy allows efficient l-lysine production independent of the oxidative pentose phosphate pathway.ArticleMETABOLIC ENGINEERING. 37:1-10 (2016)journal articl

Development of Fatty Acid-Producing Corynebacterium glutamicum Strains

To date, no information has been made available on the genetic traits that lead to increased carbon flow into the fatty acid biosynthetic pathway of Corynebacterium glutamicum. To develop basic technologies for engineering, we employed an approach that begins by isolating a fatty acid-secreting mutant without depending on mutagenic treatment. This was followed by genome analysis to characterize its genetic background. The selection of spontaneous mutants resistant to the palmitic acid ester surfactant Tween 40 resulted in the isolation of a desired mutant that produced oleic acid, suggesting that a single mutation would cause increased carbon flow down the pathway and subsequent excretion of the oversupplied fatty acid into the medium. Two additional rounds of selection of spontaneous cerulenin-resistant mutants led to increased production of the fatty acid in a stepwise manner. Whole-genome sequencing of the resulting best strain identified three specific mutations (fasR20, fasA63(up), and fasA2623). Allele-specific PCR analysis showed that the mutations arose in that order. Reconstitution experiments with these mutations revealed that only fasR20 gave rise to oleic acid production in the wild-type strain. The other two mutations contributed to an increase in oleic acid production. Deletion of fasR from the wild-type strain led to oleic acid production as well. Reverse transcription-quantitative PCR analysis revealed that the fasR20 mutation brought about upregulation of the fasA and fasB genes encoding fatty acid synthases IA and IB, respectively, by 1.31-fold +/- 0.11-fold and 1.29-fold +/- 0.12-fold, respectively, and of the accD1 gene encoding the beta-subunit of acetyl-CoA carboxylase by 3.56-fold +/- 0.97-fold. On the other hand, the fasA63(up) mutation upregulated the fasA gene by 2.67-fold +/- 0.16-fold. In flask cultivation with 1% glucose, the fasR20 fasA63(up) fasA2623 triple mutant produced approximately 280 mg of fatty acids/liter, which consisted mainly of oleic acid (208 mg/liter) and palmitic acid (47 mg/liter).ArticleAPPLIED AND ENVIRONMENTAL MICROBIOLOGY. 79(21):6776-6783 (2013)journal articl

Status of Nanosatellite Development for Mothership Daughtership Space Experiment by Japanese Universities

We have begun the feasibility study to build a small satellite in the 3kg mass range and in the power range less than 30W since October in 1998, sponsored by National Space Development Agency of Japan (NASDA). This satellite installs three reaction wheels for an attitude control and camera which will demonstrate optical navigation experiment by using image processing. The BBM design status is described in this paper. The components such as a small reaction wheel, electric flash and onboard computer were built. Mothership - daughtership technology and formation flying experiments are planned. New electric propulsion is being developed for small satellite

Peritoneal dissemination of prostate cancer due to laparoscopic radical prostatectomy: a case report

<p>Abstract</p> <p>Introduction</p> <p>Peritoneal dissemination with no further metastases of prostate cancer is very rare, with only three cases reported in the available literature. We report the first case of iatrogenic peritoneal dissemination due to laparoscopic radical prostatectomy.</p> <p>Case Presentation</p> <p>A 59-year-old Japanese man underwent laparoscopic radical prostatectomy for clinical T2bN0M0 prostate cancer, and the pathological diagnosis was pT3aN0 Gleason 3+4 adenocarcinoma with a negative surgical margin. Salvage radiation therapy was performed since his serum prostate-specific antigen remained at a measurable value. After the radiation, he underwent castration, followed by combined androgen blockade with estramustine phosphate and dexamethasone as each treatment was effective for only a few months to a year. Nine years after the laparoscopic radical prostatectomy, computed tomography revealed a peritoneal tumor, although no other organ metastasis had been identified until then. He died six months after the appearance of peritoneal metastasis. An autopsy demonstrated peritoneal dissemination of the prostate cancer without any other metastasis.</p> <p>Conclusion</p> <p>Physicians should take into account metastasis to unexpected sites. Furthermore, we suggest that meticulous care be taken not to disseminate cancer cells to the peritoneum during laparoscopic radical prostatectomy.</p

Development of Biotin-Prototrophic and -Hyperauxotrophic Corynebacterium glutamicum Strains

To develop the infrastructure for biotin production through naturally biotin-auxotrophic Corynebacterium glutamicum, we attempted to engineer the organism into a biotin prototroph and a biotin hyperauxotroph. To confer biotin prototrophy on the organism, the cotranscribed bioBF genes of Escherichia coli were introduced into the C. glutamicum genome, which originally lacked the bioF gene. The resulting strain still required biotin for growth, but it could be replaced by exogenous pimelic acid, a source of the biotin precursor pimelate thioester linked to either coenzyme A (CoA) or acyl carrier protein (ACP). To bridge the gap between the pimelate thioester and its dedicated precursor acyl-CoA (or -ACP), the bioI gene of Bacillus subtilis, which encoded a P450 protein that cleaves a carbon-carbon bond of an acyl-ACP to generate pimeloyl-ACP, was further expressed in the engineered strain by using a plasmid system. This resulted in a biotin prototroph that is capable of the de novo synthesis of biotin. On the other hand, the bioY gene responsible for biotin uptake was disrupted in wild-type C. glutamicum. Whereas the wildtype strain required approximately 1 mu g of biotin per liter for normal growth, the bioY disruptant (Delta bioY) required approximately 1 mg of biotin per liter, almost 3 orders of magnitude higher than the wild-type level. The Delta bioY strain showed a similar high requirement for the precursor dethiobiotin, a substrate for bioB-encoded biotin synthase. To eliminate the dependency on dethiobiotin, the bioB gene was further disrupted in both the wild-type strain and the Delta bioY strain. By selectively using the resulting two strains (Delta bioB and Delta bioBY) as indicator strains, we developed a practical biotin bioassay system that can quantify biotin in the seven-digit range, from approximately 0.1 mu g to 1 g per liter. This bioassay proved that the engineered biotin prototroph of C. glutamicum produced biotin directly from glucose, albeit at a marginally detectable level (approximately 0.3 mu g per liter).ArticleAPPLIED AND ENVIRONMENTAL MICROBIOLOGY. 79(15):4586-4594 (2013)journal articl