Utilization of ornithine and arginine as specific precursors of clavulanic acid.

Ornithine and arginine (5 to 20 mM), but not glutamic acid or proline, exerted a concentration-dependent stimulatory effect on the biosynthesis of clavulanic acid in both resting-cell cultures and long-term fermentations of Streptomyces clavuligerus. Ornithine strongly inhibited cephamycin biosynthesis in the same strain. [1-14C]-, [5-14C]-, or [U-14 C] ornithine was efficiently incorporated into clavulanic acid, whereas the incorporation of uniformly labeled glutamic acid was very poor. [U-14C] citrulline were not incorporated at all. Mutant nca-1, a strain that is blocked in clavulanic acid biosynthesis, did not incorporate arginine into clavulanic acid. S. clavuligerus showed arginase activity, converting arginine into ornithine, but not amidinotransferase activity. Both arginase activity and clavulanic acid formation were enhanced simultaneously by supplementing the production medium with 10 mM arginine.     

Utilization of ornithine and arginine as specific precursors of clavulanic acid

Ornithine and arginine (5 to 20 mM), but not glutamic acid or proline, exerted a concentration-dependent stimulatory effect on the biosynthesis of clavulanic acid in both resting-cell cultures and long-term fermentations of Streptomyces clavuligerus. Ornithine strongly inhibited cephamycin biosynthesis in the same strain. [1-14C]-, [5-14C]-, or [U-14 C] ornithine was efficiently incorporated into clavulanic acid, whereas the incorporation of uniformly labeled glutamic acid was very poor. [U-14C] citrulline were not incorporated at all. Mutant nca-1, a strain that is blocked in clavulanic acid biosynthesis, did not incorporate arginine into clavulanic acid. S. clavuligerus showed arginase activity, converting arginine into ornithine, but not amidinotransferase activity. Both arginase activity and clavulanic acid formation were enhanced simultaneously by supplementing the production medium with 10 mM arginine.     

Clavulanic acid biosynthesis and genetic manipulation for its overproduction

Clavulanic acid, a β-lactamase inhibitor, is used together with β-lactam antibiotics to create drug mixtures possessing potent antimicrobial activity. In view of the clinical and industrial importance of clavulanic acid, identification of the clavulanic acid biosynthetic pathway and the associated gene cluster(s) in the main producer species, Streptomyces clavuligerus, has been an intriguing research question. Clavulanic acid biosynthesis was revealed to involve an interesting mechanism common to all of the clavam metabolites produced by the organism, but different from that of other β-lactam compounds. Gene clusters involved in clavulanic acid biosynthesis in S. clavuligerus occupy large regions of nucleotide sequence in three loci of its genome. In this review, clavulanic acid biosynthesis and the associated gene clusters are discussed, and clavulanic acid improvement through genetic manipulation is explained.     

The involvement of superoxide anions in the nitro blue tetrazolium chloride reduction mediated by NADH and phenazine methosulfate

Oxygen inhibits competitively the reduction of nitro blue tetrazolium chloride (nitro BT) by NADH and phenazine methosulfate (PMS). The oxygen-dependent inhibition is stronger in the presence of superoxide dismutase, whereas cyanide counteracts the oxygen interference. On the other hand, the oxidation of NADH mediated by PMS and dioxygen is affected only marginally by superoxide dismutase and cyanide. Therefore, it is concluded that the involvement of superoxide anions occurs at the level of nitro BT reduction via a nitro blue tetrazolinyl radical, as has been suggested by Picker and Fridovich [1984) Arch. Biochem. Biophys. 228, 155-158) and not at the level of PMS oxidation. The inhibition of the oxygen interference in the nitro BT reduction by cyanide is dependent on the cyanide concentration, whereas in nitrogen cyanide has no effect on the reduction. It is caused by competition between cyanide and oxygen to reduce or oxidize the nitro BT radical to either formazan with concomitant cyanogen production or nitro BT, respectively. For the histochemical localization and analysis of electropherograms of NAD(P)+-dependent dehydrogenase activities, the interference of oxygen can be avoided by anaerobic incubations or by the use of 5 mM nitro BT when incubating aerobically.     

Flux balance analysis in the production of clavulanic acid by Streptomyces clavuligerus

In this work, in silico flux balance analysis is used for predicting the metabolic behavior of Streptomyces clavuligerus during clavulanic acid production. To choose the best objective function for use in the analysis, three different optimization problems are evaluated inside the flux balance analysis formulation: (i) maximization of the specific growth rate, (ii) maximization of the ATP yield, and (iii) maximization of clavulanic acid production. Maximization of ATP yield showed the best predictions for the cellular behavior. Therefore, flux balance analysis using ATP as objective function was used for analyzing different scenarios of nutrient limitations toward establishing the effect of limiting the carbon, nitrogen, phosphorous, and oxygen sources on the growth and clavulanic acid production rates. Obtained results showed that ammonia and phosphate limitations are the ones most strongly affecting clavulanic acid biosynthesis. Furthermore, it was possible to identify the ornithine flux from the urea cycle and the α‐ketoglutarate flux from the TCA cycle as the most determinant internal fluxes for promoting clavulanic acid production. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1226–1236, 2015     

Heterologous production of clavulanic acid intermediates in <Emphasis Type="Italic">Streptomyces venezuelae</Emphasis>

Heterologous expression can enhance production of diverse secondary metabolites by redirecting precursor pools towards compound of interest. In this study, Streptomyces venezuelae YJ028 was utilized as the heterologous host for the expression of four structural clavulanic acid biosynthesis genes, which encode carboxyethylarginine synthase (ceas2), β-lactam synthetase (bls2), clavaminate synthase (cas2), and proclavaminate amidinohydrolase (pah2). These genes were cloned into pIBR25 expression vector containing ermE* promoter to generate pBS4. The cas2 gene was also cloned into pSET152 to generate pCas2. It was then integrated into the genome of S. venezuelae YJ028. Upon metabolite profiling of recombinant strains by ultra-pressure liquid chromatography-photodiode array (UPLC-PDA) and high resolution liquid chromatography quadruple time-offlight electrospray ionization mass spectrometry (HR-LC-QTOF-ESI/MS), the production of following clavulanic acid intermediates in S. venezuelae recombinant were confirmed: deoxygaunidinoproclavaminic acid, guanidinoproclavaminic acid, and dihydroclavaminic acid. This work demonstrates the production of β-lactam intermediates of the clavulanic acid pathway by heterologous expression in S. venezuelae YJ028.     

Dissociation of cephamycin and clavulanic acid biosynthesis in Streptomyces clavuligerus

Summary Streptomyces clavuligerus produced simultaneously cephamycin C and clavulanic acid in defined medium in long-term fermentations and in resting-cell cultures. Biosynthesis of cephamycin by phosphate-limited resting cells was dissociated from clavulanic acid formation by removing either glycerol or sulphate from the culture medium. In absence of glycerol no clavulanic acid was formed but cephamycin production occurred, whereas in absence of sulphate no cephamycin was synthesized but clavulanic biosynthesis took place. Sulphate, sulphite and thiosulphate were excellent sulphur sources for cephamycin biosynthesis while l-methionine and l-cysteine were poor precursors of this antibiotic. Increasing concentrations of sulphate also stimulated clavulanic acid formation. The biosynthesis of clavulanic acid was much more sensitive to phosphate (10–100 mM) regulation than that of cephamycin. Therefore, the formation of both metabolites was pertially dissociated at 25 mM phosphate. By contrast, nitrogen regulation by ammonium salts or glutamic acid strongly reduced the biosynthesis of both cephamycin and clavulanic acid.     

Opioid and non-opioid receptor-mediated immunoregulatory role of Leucine-enkephalin in teleost Channa punctatus

Leucine-enkephalin (Leu-enk) is an endogenous opioid peptide and highly conserved throughout the vertebrates. Despite its conserved nature, the immunoregulatory property of Leu-enk is explored only in mammals. The present study describes the immunomodulatory role of Leu-enk in a lower vertebrate, spotted murrel Channa punctatus. Leu-enk increased the percentage phagocytosis and phagocytic index, though its stimulatory effect on phagocytosis markedly decreased at concentrations higher than 10^?9 M. Moreover, it had bell-shaped stimulatory effect also on the superoxide production by phagocytes. On the other hand, Leu-enk showed bimodal effects on nitrite release. The lower concentrations of Leu-enk produced inhibitory effect, while higher concentrations had stimulatory effect on nitrite release. Interestingly, the Leu-enk-induced increase in nitrite release was unaltered by non-selective opioid receptor antagonist though the same completely antagonized the inhibitory effect of Leu-enk on nitrite release and the stimulatory effect on phagocytosis and superoxide production. This suggests that the stimulatory effect of Leu-enk on nitrite production is mediated by the non-opioid receptor. Further, δ-opioid receptor was precisely seen involved in mediating the stimulatory effect of Leu-enk on phagocytosis and superoxide production, or inhibitory effect on nitrite release. It can be concluded that Leu-enk regulates the innate immune response of splenic phagocytes acting via both opioid and non-opioid receptor in the fish C. punctatus.     

Deletion of the pyc gene blocks clavulanic acid biosynthesis except in glycerol-containing medium: evidence for two different genes in formation of the C3 unit

The beta-lactamase inhibitor clavulanic acid is formed by condensation of a pyruvate-derived C3 unit with a molecule of arginine. A gene (pyc, for pyruvate converting) located upstream of the bls gene in the clavulanic acid gene cluster of Streptomyces clavuligerus encodes a 582-amino-acid protein with domains recognizing pyruvate and thiamine pyrophosphate that shows 29.9% identity to acetohydroxyacid synthases. Amplification of the pyc gene resulted in an earlier onset and higher production of clavulanic acid. Replacement of the pyc gene with the aph gene did not cause isoleucine-valine auxotrophy in the mutant. The pyc replacement mutant did not produce clavulanic acid in starch-asparagine (SA) or in Trypticase soy broth (TSB) complex medium, suggesting that the pyc gene product is involved in the conversion of pyruvate into the C3 unit of clavulanic acid. However, the beta-lactamase inhibitor was still formed at the same level as in the wild-type strain in defined medium containing D-glycerol, glutamic acid, and proline (GSPG medium) as confirmed by high-pressure liquid chromatography and paper chromatography. The production of clavulanic acid by the replacement mutant was dependent on addition of glycerol to the medium, and glycerol-free GSPG medium did not support clavulanic acid biosynthesis, suggesting that an alternative gene product catalyzes the incorporation of glycerol into clavulanic acid in the absence of the Pyc protein. The pyc replacement mutant overproduces cephamycin.     

Applications of Gene Replacement Technology to Streptomyces clavuligerus Strain Development for Clavulanic Acid Production

Cephamycin C production was blocked in wild-type cultures of the clavulanic acid-producing organism Streptomyces clavuligerus by targeted disruption of the gene (lat) encoding lysine -aminotransferase. Specific production of clavulanic acid increased in the lat mutants derived from the wild-type strain by 2- to 2.5-fold. Similar beneficial effects on clavulanic acid production were noted in previous studies when gene disruption was used to block the production of the non-clavulanic acid clavams produced by S. clavuligerus. Therefore, mutations in lat and in cvm1, a gene involved in clavam production, were introduced into a high-titer industrial strain of S. clavuligerus to create a double mutant with defects in production of both cephamycin C and clavams. Production of both cephamycin C and non-clavulanic acid clavams was eliminated in the double mutant, and clavulanic acid titers increased about 10% relative to those of the parental strain. This represents the first report of the successful use of genetic engineering to eliminate undesirable metabolic pathways in an industrial strain used for the production of an antibiotic important in human medicine.     

Identification of superoxide production by Arabidopsis thaliana aldehyde oxidases AAO1 and AAO3

Plant aldehyde oxidases (AOs) have gained great attention during the last years as they catalyze the last step in the biosynthesis of the phytohormone abscisic acid by oxidation of abscisic aldehyde. Furthermore, oxidation of indole-3-acetaldehyde by AOs is likely to represent one route to produce another phytohormone, indole-3-acetic acid, and thus, AOs play important roles in many aspects of plant growth and development. In the present work we demonstrate that heterologously expressed AAO1 and AAO3, two prominent members of the AO family from Arabidopsis thaliana, do not only generate hydrogen peroxide but also superoxide anions by transferring aldehyde-derived electrons to molecular oxygen. In support of this, superoxide production has also been found for native AO proteins in Arabidopsis leaf extracts. In addition to their aldehyde oxidation activity, AAO1 and AAO3 were found to exhibit NADH oxidase activity, which likewise is associated with the production of superoxide anions. According to these results and due to the fact that molecular oxygen is the only known physiological electron acceptor of AOs, the production of hydrogen peroxide and/or superoxide has to be considered in any physiological condition in which aldehydes or NADH serve as substrate for AOs. In this respect, conditions such as natural senescence and stress-induced stomatal movement, which both require simultaneously elevated levels of abscisic acid and hydrogen peroxide/superoxide, are likely to benefit from AOs in two ways, namely by formation of abscisic acid and by concomitant formation of reactive oxygen species.     

PEG and ABA Trigger the Burst of Reactive Oxygen Species to Increase Tanshinone Production in Salvia miltiorrhiza Hairy Roots

Salvia miltiorrhiza is one of the most popular traditional Chinese medicinal plants because of its excellent performance in treating coronary heart disease. Tanshinones, a group of active compounds in S. miltiorrhiza, are derived from two biosynthetic pathways: the mevalonate (MVA) pathway in the cytosol and the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway in the plastids. Water stress is well known to stimulate the accumulation of secondary metabolites in plants. Reactive oxygen species (ROS) serve as important secondary messengers in water stress-induced signal transduction pathways. In this study, the effects of polyethylene glycol (PEG) and abscisic acid (ABA) on tanshinone production in S. miltiorrhiza hairy roots were investigated and the roles of ROS in PEG- and ABA-induced tanshinone production were further elucidated. The results showed that contents and yields of four tanshinones in S. miltiorrhiza hairy roots were significantly enhanced by 2 % PEG and 200?μM ABA. Simultaneously, the mRNA levels and activities of two key enzymes (3-hydroxy-3-methylglutaryl coenzyme A reductase and 1-deoxy-D-xylulose 5-phosphate synthase) involved in tanshinone biosynthesis were upregulated. Both PEG and ABA were able to trigger the burst of H₂O₂ and O₂ ^?. The PEG- and ABA-induced increases of tanshinone production, gene expression, and enzyme activity were all dramatically suppressed by two ROS scavengers, catalase and superoxide dismutase. In addition, ROS treatments resulted in a significant increase in tanshinone production. These results demonstrated that the MVA and MEP pathways were activated by PEG and ABA to stimulate tanshinone biosynthesis, and the increase of tanshinone production was probably via ROS signaling.     

Uncoupling Protein 1 Decreases Superoxide Production in Brown Adipose Tissue Mitochondria

In thermogenic brown adipose tissue, uncoupling protein 1 (UCP1) catalyzes the dissipation of mitochondrial proton motive force as heat. In a cellular environment of high oxidative capacity such as brown adipose tissue (BAT), mitochondrial uncoupling could also reduce deleterious reactive oxygen species, but the specific involvement of UCP1 in this process is disputed. By comparing brown adipose tissue mitochondria of wild type mice and UCP1-ablated litter mates, we show that UCP1 potently reduces mitochondrial superoxide production after cold acclimation and during fatty acid oxidation. We address the sites of superoxide production and suggest diminished probability of “reverse electron transport” facilitated by uncoupled respiration as the underlying mechanism of reactive oxygen species suppression in BAT. Furthermore, ablation of UCP1 represses the cold-stimulated increase of substrate oxidation normally seen in active BAT, resulting in lower superoxide production, presumably avoiding deleterious oxidative damage. We conclude that UCP1 allows high oxidative capacity without promoting oxidative damage by simultaneously lowering superoxide production.