CA Biosynthesis Research Articles

Clavulanic Acid Overproduction: A Review of Environmental Conditions, Metabolic Fluxes, and Strain Engineering in Streptomyces clavuligerus

Clavulanic acid is a potent β-lactamase inhibitor produced by Streptomyces clavuligerus, widely used in combination with β-lactam antibiotics to combat antimicrobial resistance. This systematic review analyzes the most successful methodologies for clavulanic acid overproduction, focusing on the highest yields reported in bench-scale and bioreactor-scale fermentations. Studies have demonstrated that glycerol is the preferred carbon source for clavulanic acid production over other sources like starch and dextrins. The optimization of feeding strategies, especially in fed-batch operations, has improved glycerol utilization and extended the clavulanic acid production phase. Organic nitrogen sources, particularly soybean protein isolates and amino acid supplements such as L-arginine, L-threonine, and L-glutamate, have been proven effective at increasing CA yields both in batch and fed-batch cultures, especially when balanced with appropriate carbon sources. Strain engineering approaches, including mutagenesis and targeted genetic modifications, have allowed for the obtainment of overproducer S. clavuligerus strains. Specifically, engineering efforts that overexpress key regulatory genes such as ccaR and claR, or that disrupt competing pathways, redirect the metabolic flux towards CA biosynthesis, leading to high clavulanic acid titers. The fed-batch operation at the bioreactor scale emerges as the most feasible alternative for prolonged clavulanic acid production with both wild-type and mutant strains, allowing for the attainment of high titers during cultivations.

Integrated Metabolomics and Transcriptomics Analyses of the Biosynthesis of Arbutin and 6'-O-Caffeoylarbutin in Vaccinium dunalianum Cell Suspension Cultures Fed with Hydroquinone.

Arbutin and 6'-O-caffeoylarbutin (CA) from Vaccinium dunalianum Wight are known for their ability to inhibit melanin synthesis. To boost the production of arbutin and CA, precursor feeding with hydroquinone (HQ) was studied in V. dunalianum suspension cells. The effect of HQ on the biosynthesis of arbutin and CA in the suspension cells was investigated using high-performance liquid chromatography (HPLC), and possible molecular mechanisms were analyzed using metabolomics and transcriptomics analyses. HPLC analysis only showed that the addition of HQ significantly enhanced arbutin synthesis in cells, peaking at 15.52 ± 0.28 mg·g-1 after 0.5 mmol·L-1 HQ treatment for 12 h. Subsequently, metabolomics identified 78 differential expression metabolites (DEMs), of which arbutin and CA were significantly up-regulated metabolites. Moreover, transcriptomics found a total of 10,628 differential expression genes (DEGs). The integrated transcriptomics and metabolomics revealed that HQ significantly enhanced the expression of two arbutin synthase (AS) genes (Unigene0063512 and Unigene0063513), boosting arbutin synthesis. Additionally, it is speculated that CA was generated from arbutin and 3,4,5-tricaffeoylquinic acid catalyzed by caffeoyl transferase, with Unigene0044545, Unigene0043539, and Unigene0017356 as potentially associated genes with CA synthesis. These findings indicate that the precursor feeding strategy offers a promising approach for the mass production of arbutin and CA in V. dunalianum suspension cells and provides new insights for CA biosynthesis in V. dunalianum.

Alternative Pathway to the Formation of trans-Cinnamic Acid Derived from l-Phenylalanine in Tea (Camellia sinensis) Plants and Other Plants.

trans-Cinnamic acid (CA) is a precursor of many phenylpropanoid compounds, including catechins and aroma compounds, in tea (Camellia sinensis) leaves and is derived from l-phenylalanine (l-Phe) deamination. We have discovered an alternative CA formation pathway from l-Phe via phenylpyruvic acid (PPA) and phenyllactic acid (PAA) in tea leaves through stable isotope-labeled precursor tracing and enzyme reaction evidence. Both PPA reductase genes (CsPPARs) involved in the PPA-to-PAA pathway were isolated from tea leaves and functionally characterized in vitro and in vivo. CsPPAR1 and CsPPAR2 transformed PPA into PAA and were both localized in the leaf cell cytoplasm. Rosa hybrida flowers (economic crop flower), Lycopersicon esculentum Mill. fruits (economic crop fruit), and Arabidopsis thaliana leaves (leaf model plant) also contained this alternative CA formation pathway, suggesting that it occurred in most plants, regardless of different tissues and species. These results improve our understanding of CA biosynthesis in tea plants and other plants.

Metabolic engineering strategies for caffeic acid production in Escherichia coli

Caffeic acid (CA; 3,4-dihydroxycinnamic acid) is an aromatic compound obtained by the phenylpropanoid pathway. This natural product has antioxidant, antitumor, antiviral, and anti-inflammatory activities. It is also a precursor of CA phenethyl ester (CAPE), a compound with potential as an antidiabetic and liver-protective agent. CA can be found at low concentrations in plant tissues, and hence, its purification is difficult and expensive. Knowledge regarding the pathways, enzymes, and genes involved in CA biosynthesis has paved the way for enabling the design and construction of microbial strains with the capacity of synthesizing this metabolite. In this review, metabolic engineering strategies for the generation of Escherichia coli strains for the biotechnological production of CA are presented and discussed.

Screening of medium constituents for clavulanic acid production by Streptomyces clavuligerus.

Clavulanic acid is a β-lactam compound with potent inhibitory activity against β-lactamases. Studies have shown that certain amino acids play essential roles in CA biosynthesis. However, quantitative evaluations of the effects of these amino acids are still needed in order to improve CA production. Here, we report a study of the nutritional requirements of Streptomyces clavuligerus for CA production. Firstly, the influence of the primary nitrogen source and the salts composition was investigated. Subsequently, soybean protein isolate was supplemented with arginine (0.0–3.20 g L−1), threonine (0.0–1.44 g L−1), ornithine (0.0–4.08 g L−1), and glutamate (0.0–8.16 g L−1), according to a two-level central composite rotatable design. A medium containing ferrous sulfate yielded CA production of 437 mg L−1, while a formulation without this salt produced only 41 mg L−1 of CA. This substantial difference suggested that Fe2+ is important for CA biosynthesis. The experimental design showed that glutamate and ornithine negatively influenced CA production while arginine and threonine had no influence. The soybean protein isolate provided sufficient C5 precursor for CA biosynthesis, so that supplementation was unnecessary. Screening of medium components, together with experimental design tools, could be a valuable way of enhancing CA titers and reducing the process costs.

Optimisation of the glycerol-to-ornithine molar ratio in the feed medium for the continuous production of clavulanic acid by Streptomyces clavuligerus

Amino acids are well metabolized by Streptomyces clavuligerus during the production of clavulanic acid using glycerol as main carbon and energy source. However, only a few amino acids such as arginine and ornithine are favorable for CA biosynthesis. The aim of this work was to optimize the glycerol:ornithine molar ratio in the feed medium containing only these compounds to maximize CA production in continuous cultivation. A minimum number of experiments were performed by means of a simple two-level full-factorial central composite design to investigate the combined effect of glycerol and ornithine feeding on the CA concentration during the intermittent and continuous process in shake-flasks. Statistical analysis of the experimental data using the response surface methodology showed that a glycerol-to-ornithine molar ratio of approximately 40:1 in the feed medium resulted in the highest CA concentration when fermentation was stopped. Under these optimized conditions, in bench-scale fermentor runs, the CA concentration reached more than double the concentration obtained in shake-flasks runs.

Association between Expression Levels of CA 19-9 and N-Acetylglucosamine-β1,3-Galactosyltransferase 5 Gene in Human Pancreatic Cancer Tissue

Objective: CA 19-9, equivalent to Sialyl Lewis^a antigen, is a well-known tumor marker in pancreatic cancer. At the initial step of the biosynthesis of CA 19-9, N-acetylglucosamine-β1,3-galactosyltransferase (β3Gal-T) transfers galactose to N-acetylglucosamine (GlcNAc). Recently, β3Gal-T5 has been presumed to be related to the formation of the type 1 chain in an in vitro experiment in terms of kinetic enzyme characterization. The purpose of this study was to investigate which β3Gal-T is related to the synthesis of CA 19-9 in human pancreatic cancer tissues. Methods: We examined β3Gal-T1, T2, T3, T4, and β3Gal-T5 mRNA expressions in 13 noncancerous and cancerous tissues of the human pancreas using real-time polymerase chain reaction, and compared those gene expression levels with the immunoreactivity of CA 19-9 and its precursor DUPAN-2 in cancerous tissues. Results:β3Gal-T5 gene expression significantly augmented in cancerous tissues, when compared with the adjacent noncancerous tissues. Additionally, there was a good correlation between β3Gal-T5 gene transcription levels and immunohistochemical grades of CA 19-9 or its precursor DUPAN-2 in cancerous tissues. However, no correlation was observed between β3Gal-T1, T2, T3, and β3Gal-T4 gene expression levels and CA 19-9 or DUPAN-2 immunoreactive grades in cancerous tissue. Conclusion: β3Gal-T5 is presumed to be responsible for the synthesis of CA 19-9 in pancreatic cancer tissue.

Differential modulation of catecholamines by chlorotriazine herbicides in pheochromocytoma (PC12) cells in vitro.

Epidemiological, wildlife, and laboratory studies have pointed to the possible adverse health effects of chlorotriazine herbicide (i.e. , atrazine, simazine, and cyanazine) exposure. However, the cellular mechanism(s) of action of these compounds remains unknown. Recently, it was reported by Cooper et al. (2000, Toxicol. Sci. 53, 297-307) that atrazine disrupts ovarian function by altering hypothalamic catecholamine concentrations and subsequently the regulation of luteinizing hormone (LH) and prolactin (PRL) secretion by the pituitary. In this study, we examined the effect of three chlorotriazines on catecholamine metabolism in vitro using PC12 cells. Intracellular norepinephrine (NE) and dopamine (DA) concentrations and spontaneous NE release were measured following treatment with different concentrations of atrazine, simazine (0, 12. 5, 25, 50, 100, and 200 microM) and cyanazine (0, 25, 50, 100, and 400 microM) for 6, 12, 18, 24, and 48 h. Atrazine and simazine significantly decreased intracellular DA concentration in a concentration-dependent manner. Intracellular NE concentration was also significantly decreased by 100 and 200 microM atrazine and 200 microM simazine. Similarly, there was a dose-dependent inhibition of NE release with 100 and 200 microM concentrations of both compounds. Although 100 and 400 microM cyanazine increased intracellular NE concentration, 50, 100, and 400 microM cyanazine significantly increased NE release at 24 and 36 h. In contrast, intracellular DA concentration was decreased by cyanazine, but only at 400 microM. The GABA(A)-receptor agonist, muscimol (0, 0.01, 0.1, and 1.0 microM) had no effect on either the release or on intracellular catecholamine concentrations from 6 through 24 h of treatment. Cell viability was somewhat lower in the groups exposed to 100 and 200 microM atrazine and simazine. However, the reduction in viability was significant only in the highest dose of atrazine used (200 microM) at 24 h. Cyanazine did not have an effect on the viability at any of the doses tested, and the cells were functional, even up to 48 h of exposure. These data indicate that both atrazine and simazine inhibit the cellular synthesis of DA mediated by the tyrosine hydroxylase (TH), and NE mediated by dopamine beta-hydroxylase (DbetaH), and, as a result, there is a partial or significant inhibition of NE release. Cyanazine, on the other hand, stimulated the synthesis of intracellular NE, and not DA. Thus, chlorotriazine compounds presumably act at the enzymatic steps or sites of CA biosynthesis to modulate monoaminergic activity in PC12 cells.

Elevation of serum CA 19-9 in parallel with HbA 1c in a diabetic female with the Lewis(a + b −) blood group

We report a 66-yr-old diabetic female with her serum CA 19-9 levels elevated in parallel with her hemoglobin A 1c (HbA 1c) levels. She was initially suspected of having pancreatic cancer, but this was later proved to be false. Her serum CA 19-9 levels varied between 58 and 235 U/ml over a 3-yr period, and correlated significantly ( r = 0.68, P < 0.01) with her HbA 1c levels (7.2–13.3%). She belonged to the Lewis (a + b −) blood group. Serum CA 19-9 levels were also assessed in 73 diabetic patients (36 males and 37 females) in relation to sex and Lewis blood group. The mean level of serum CA 19-9 was higher in females than in males in each Lewis blood group, and it was the highest in patients with Le(a + b −) and the lowest in Le(a − b −). Patients with a higher level of HbA 1c showed a higher level of serum CA 19-9 in Le(a + b −), but patients with Le (a − b −) showed a low level of serum CA 19-9 irrespective of HbA 1c level. Patients with Le(a − b +) appeared to intermediate between the patients with Le(a + b −) Le(a − b −). Thus, the elevation of serum CA 19-9 level due to hyperglycemia was indicated to be affected by sex as well as Lewis blood group. The biosynthesis of CA 19-9 seemed to be accelerated in hyperglycemic diabetic patients, especially in females. Consequently, it was considered that the present case was a diabetic female with the Le(a + b −) blood group, which led to showing a relatively high level of serum CA 19-9 correlated with HbA 1c level.

CDNA cloning, sequence, and expression of carbonic anhydrase in Chlamydomonas reinhardtii: regulation by environmental CO2 concentration.

cDNA clones for the periplasmic carbonic anhydrase (CA; carbonate hydro-lyase, EC 4.2.1.1) of Chlamydomonas reinhardtii cells were isolated and characterized. The fact that the cloned cDNA fragments encoded a 377-amino acid polypeptide (41,626 Da) consisting of an NH2-terminal hydrophobic signal peptide of 20 amino acids, a large (35,603 Da) subunit polypeptide, and a small (4144 Da) subunit polypeptide indicates that the two subunits are cotranslated as a precursor polypeptide. The amino acid sequence of mature subunits deduced from the nucleotide sequence showed 20-22% homology with human CA isozymes (CAI, CAII, and CAIII). Three zinc-liganded histidine residues and those forming a hydrogen-bond network to zinc-bound solvent molecules were highly conserved in the plant and animal enzymes. Three possible asparagine-linked glycosylation sites were found in the large subunit. Northern blot analysis was carried out using the cDNA fragment as a probe. The level of 2.0-kilobase CA mRNA increased within 1 hr when CO2 concentration of the bubbling gas was changed from 5% to 0.04% (air level) in the presence of light. On the other hand, CA mRNA did not accumulate when CO2 concentration was lowered in the dark. Experiments using 3-(3,4-dichlorophenyl)-1,1-dimethylurea showed that photosynthesis is absolutely required for the accumulation of CA mRNA. These results indicate that CA biosynthesis is regulated by changes in environmental CO2 concentration as well as light at the level of mRNA abundance.

IMPULSE ACTIVITY DIFFERENTIALLY REGULATES LEUENKEPHALIN AND CATECHOLAMINE (CA) CHARACTERS IN THE ADRENAL MEDULLA

Trans-synaptic impulse activity has long been known to increase CA biosynthesis and CA synthetic enzymes in the adrenal medulla. Since opiate peptides are co-stored and co-released with CA's in medullary cells, and since medullary opiate peptides may be important modulators of the sympathoadrenal stress response, we examined trans-synaptic regulation of the putative peptide neurohumour, leucine-enkephalin (ENK) and the CA enzymes, tyrosine hydroxylase (TH) and phenyl-ethanolamine-N-methyl transferase (PNMT) in the rat adrenal medulla in vivo and in vitro. Surgical denervation of the adrenal or pharmacologic blockade of synaptic transmission, treatments known to decrease CA traits, increased ENK-like immunoreactivity to 150% of control. Medullae explanted to culture exhibited a 50 fold rise in ENK in 4 days, whereas TH remained constant, and PNMT decreased to a new baseline. Depolarizing concentrations of K+ prevented the accumulation of ENK while not affecting TH or PNMT. Our studies suggest that enkephalinergic and catecholamine characters are differentially regulated by impulse activity and depolarization in the adrenal medulla. Independent regulation of adrenal neuropeptides and CA enzymes may allow new therapeutic approaches to shock, hypoxemia, or stress-induced analgesia through alteration of specific neurohumoral mechanisms. (Supported by NIH Grants HL00756, NS10259, and HD12108).

Metabolic development and mitochondrial changes during cyclopiazonic acid production in Penicillium cyclopium.

During a study of cyclopiazonic acid (total α- and β-cyclopiazonic acids, CA) production by Penicillium cyclopium Westling, it was found that nitrogen depletion controlled the advent of secondary metabolism and that nitrate reduction was not rate-limiting. In concert with N depletion both tryptophan (Trp) biosynthesis and glucose catabolism fell. The rate of Trp production dropped to match the rate of CA biosynthesis. Glucose utilization varied between an early and late preference for the pentose cycle, with a mixture of pentose and Emden–Meyerhof pathways at an intermediate time. This pattern may well reflect the need for erythrose-4-phosphate in Trp biosynthesis. Additions of Trp caused a drop in CA production possibly because of repression of Trp synthetase. Exogeneous dimethylallylpyrophosphate (DMAPP) increased CA biosynthesis, while mevalonic acid had no effect which suggested a rate-limiting step between these two metabolites. These changes probably best fit Bu'Lock's hypothesis that secondary metabolism links fermentative and aerobic metabolism (ATP generation). In this case protein and polyisoprenoid biosynthesis, sugar utilization, and energy requirements would all be interlocked with the advent of secondary metabolism. The possible relationship between the observed metabolic changes and energy control were supported by electron-microscopic examination of the mitochondria in situ, when these ATP-generating organelles underwent major changes. Addition of ATP to the culture depressed CA production, as did excessive aeration. The metabolic regulator cAMP had no effect other than to increase autocatalysis.

Genetic variation in the carbonic anhydrase isozymes of macaque monkeys: III. Biosynthesis of carbonic anhydrases in bone marrow erythroid cells and peripheral blood reticulocytes of Macaco nemestrina

The carbonic anhydrase isozymes (CA I and CA II) of the pig-tailed macaque, Macaca nemestrina, have been chosen to study the regulation of enzyme levels in red blood cells. Two quantitative variants of CA I that are ideal for studies of enzyme regulation exist in this species. One variant is one of four known electrophoretic types of CA I, designated CA Ia, which is present at levels about 30% of those of the other electrophoretic types. The other is a deficiency variant of CA I which, in homozygotes, reduces the product of the CA I locus about 5000-fold and reduces the product of the CA II locus by about 60%. l-[ 14C]Serine was used to study the biosynthesis of CA I and CA II isozymes in the reticulocytes of animals carrying these CA I variants. Specific radioactivity and total incorporation data from bone marrow erythroid cells, and peripheral blood reticulocytes indicate that the reduced CA Ia concentration is probably the result of degradation. This degradation appears to occur for only a short time before the reticulocytes enter the peripheral blood. It was not possible to determine whether the 5000-fold reduction of CA I in the CA I-deficient animals is due to reduced transcription, reduced translation, or degradation. The effect of the CA I-deficiency mutation on the synthesis of CA II was also studied. For each dose of CA I-deficiency gene, there appears to be a 30% reduction in the rate of l-[ 14C]serine incorporation into CA II, thereby accounting for the reduced CA II concentration in CA I-deficient animals.