Non-ribosomal peptide synthetases catalyze the biosynthesis of structurally and functionally diverse non-ribosomal peptide natural products, which have broad applications in pharmaceutical, agricultural, and industrial sectors. Engineered non-ribosomal peptide synthetases can be used to produce novel non-ribosomal peptides through combinatorial biosynthesis. This conforms to the concept of green chemistry, thus attracts increasing attention across the world. Herein, three different engineering strategies were summarized, and recent advances in this field were reviewed.
Biological Products ; Peptide Synthases/genetics* ; Peptides ; Protein Engineering

Previously, we demonstrated that galangin (3,5,7-trihydroxyflavone) protects human keratinocytes against ultraviolet B (UVB)-induced oxidative damage. In this study, we investigated the effect of galangin on induction of antioxidant enzymes involved in synthesis of reduced glutathione (GSH), and investigated the associated upstream signaling cascades. By activating nuclear factor-erythroid 2-related factor (Nrf2), galangin treatment significantly increased expression of glutamate-cysteine ligase catalytic subunit (GCLC) and glutathione synthetase (GSS). This activation of Nrf2 depended on extracellular signal-regulated kinases (ERKs) and protein kinase B (AKT) signaling. Inhibition of GSH in galangin-treated cells attenuated the protective effect of galangin against the deleterious effects of UVB. Our results reveal that galangin protects human keratinocytes by activating ERK/AKT-Nrf2, leading to elevated expression of GSH-synthesizing enzymes.
Catalytic Domain ; Extracellular Signal-Regulated MAP Kinases ; Glutamate-Cysteine Ligase ; Glutathione Synthase ; Glutathione* ; Humans* ; Keratinocytes* ; Proto-Oncogene Proteins c-akt

Based on polyketide syntheses gene (PKS) and non-ribosomal peptide synthetases gene (NRPS), one strain with high anti-pathogenic activity was screened from 77 strains isolated from Arctic marine sediments and identified. By optimizing the composition of culture medium and fermentation conditions, the production of this strain's active metabolites was improved and the main metabolites were identified by HRMS, ¹H NMR and ¹³C NMR. The antibacterial spectrum of the main metabolites and the effect of the metabolites on cucumber Fusarium wilt were also determined. The results showed that the strain was Bacillus velezensis and it showed growth promoting effect on plants. When the strain was cultured in 5 g/L maltose, 10 g/L tryptone, 10 g/L sodium chloride, at 30 ℃, 150 r/min for 60 h, the diameter of the inhibition zone increased from (16.23±0.42) to (24.42±0.57) mm. The metabolites of this strain mainly contain macrolide compound macrolactin A, which has antagonistic effect on a variety of pathogenic bacteria and fungi. Cucumber seedling experiments showed that the metabolites of this strain had a protective effect on cucumber Fusarium wilt, and showed a good potential for development and application as a biocontrol agent.
Polyketides/pharmacology* ; Fungi ; Bacteria ; Fusarium/genetics* ; Anti-Bacterial Agents/pharmacology* ; Peptide Synthases/genetics*

Novel macrolides epothilones, produced by cellulolytic myxobacterium Sorangium cellulosum, have the activity to promote microtubule assembly, and are considered to be a potential successor to the famous antitumor drug taxol. The biosynthetic genes leading to the epothilones are clustered into a large operon. The multi-enzyme complex is a hetero-gene cluster of polyketide synthase (PKS) and non-ribosomal peptide synthetases (NRPS) and contains several functional modules, i.e. a loading module, one NRPS module, eight PKS modules, and a P450 epoxidase. The former ten modules biosynthesize desoxyepothilone (epothilones C and D), which is then epoxidized at C12 and C13 and converted into epothilones (epothilones A and B) by the P450 epoxidase. The NRPS module is responsible for the formation of the thiazole side chain from cysteine. The biosynthesis procedure of epothilones can be divided into 5 stages, i.e. formation of holo-ACP/PCP, chain initiation and thiazole ring formation, chain elongation, termination and epoxidation, and post-modification. The analysis of the gene cluster and the biosynthetic pathway reveals that novel epothilone analogs could not only be produced by chemical synthesis/modification, tranditional microbial technologies, but also can be genetically manipulated through combinatiorial biosynthesis approaches.
Bacterial Proteins ; genetics ; metabolism ; Epothilones ; chemistry ; metabolism ; Molecular Structure ; Multigene Family ; genetics ; physiology ; Myxococcales ; enzymology ; genetics ; metabolism ; Peptide Synthases ; genetics ; metabolism ; Polyketide Synthases ; genetics ; metabolism

BACKGROUND: Exposure to ethanol abuse and severe oxidative stress are risk factors for hepatocarcinoma. The aim of this study was to evaluate the effects of S-adenosylmethionine (SAMe) and its combinations with taurine and/or betaine on the level of glutathione (GSH), a powerful antioxidant in the liver, in acute hepatotoxicity induced by ethanol. METHODS: To examine the effects of SAMe and its combinations with taurine and/or betaine on ethanol-induced hepatotoxicity, AML12 cells and C57BL/6 mice were pretreated with SAMe, taurine, and/or betaine, followed by ethanol challenge. Cell viability was detected with an MTT assay. GSH concentration and mRNA levels of GSH synthetic enzymes were measured using GSH reductase and quantitative real-time reverse transcriptase-PCR. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were measured with commercially available kits. RESULTS: Pretreatment of SAMe, with or without taurine and/or betaine, attenuated decreases in GSH levels and mRNA expression of the catalytic subunit of glutamate-cysteine ligase (GCL), the rate-limiting enzyme for GSH synthesis, in ethanol-treated cells and mice. mRNA levels of the modifier subunit of GCL and glutathione synthetase were increased in mice treated with SAMe combinations. SAMe, taurine, and/or betaine pretreatment restored serum ALT and AST levels to control levels in the ethanol-treated group. CONCLUSIONS: Combinations of SAMe with taurine and/or betaine have a hepatoprotective effect against ethanol-induced liver injury by maintaining GSH homeostasis.
Alanine Transaminase ; Animals ; Aspartate Aminotransferases ; Betaine* ; Catalytic Domain ; Cell Survival ; Ethanol ; Glutamate-Cysteine Ligase ; Glutathione Synthase ; Glutathione* ; Homeostasis* ; Liver ; Mice ; Oxidative Stress ; Oxidoreductases ; Risk Factors ; RNA, Messenger ; S-Adenosylmethionine* ; Taurine*

Recombinant plasmid pICG was constructed by replacing the internal fragment of a-acetohydroxyacid synthase (AHAS) gene (ILV2) with a copy of gamma-glutamylcysteine synthetase gene (GSH1) and copper chelatin gene (CUP1) from the industrial brewing yeast strain YSF31. YSF31 was transformed with plasmid pICG linearized by Kpn I and Pst I. A recombinant strain with high-glutathione and low-diacetyl production was selected. The results of fermentation in 100-L bioreactor showed that the lagering time of beer produced for recombinant strain T2 was shortened by 3 days and the shelf life of the beer was prolonged about 50%. It may be more acceptable for the commercial application, as it does not contain foreign DNA.
Acetolactate Synthase ; genetics ; metabolism ; Beer ; microbiology ; Cloning, Molecular ; Diacetyl ; metabolism ; Fermentation ; Gene Expression Regulation, Fungal ; Glutamate-Cysteine Ligase ; genetics ; metabolism ; Glutathione ; biosynthesis ; Metallothionein ; genetics ; metabolism ; Organisms, Genetically Modified ; genetics ; Saccharomyces cerevisiae ; genetics ; metabolism ; Saccharomyces cerevisiae Proteins ; genetics ; metabolism

Aims: Glutamate cysteine ligase (GCL) enzyme is involved in the synthesis of glutathione, which functions as an antioxidant. Polymorphisms in the sequence of amino acids making up the gene GCLC will cause differences in enzyme expression and GCLC activity. Gene expression that is influenced by oxidative stress can be used to measure markers such as F2-isoprostanes. This study aims to examine the association between the polymorphism in the GCLC gene with glutathione plasma level and F2-isoprostanes in contacts of person with infectious tuberculosis (TB). Methodology and results: Samples are taken from the family members of pulmonary TB patients who seeks treatment at the Pulmonary Centre (Lung Health Center for Public = BBKPM) and Policlinic of Dr Wahidin Sudirohusodo Hospital, Makassar. Total of approximately 4 mL of venous blood are taken from each person with pulmonary TB contacts and furtherly analyzed using genomic PCR-RFLP method and ELISA. Our results described that contacts of person with infectious TB for approximately 6 months have polymorphism C/C genotype at 80.3%, C/T of 18.3% and T/T for 1.4% of the total 71 samples with high levels of glutathione from 0.167 to 0.548 mM/mL and F2-isoprostanes level 72.4 - 1343.9 pg/mL. Conclusion, significance and impact of study: There are no significant association between GCLC gene polymorphism with glutathione and F2-isoprostanes levels of individual who had contacted infection TB. In this study the elevation of F2-isoprostanes equal to the decrease levels of glutathione.
Glutamate-Cysteine Ligase

Most mammalian embryos implant on the uterine endometrium after hatching fromzona pellucida of the expanded blastocyst and pregnancy takes place. The blastocysts produceand control a variety of prostaglandins which activate a few proteolytic enzymes thatdissolve the zona pellucida of the embryos. In the present study, the goal was to investigatethe indomethacin and aspirin(inhibitors of cyclooxygenase pathway which regulates thepathway from arachidonic acid to prostaglandins), which can affect the hatching and implantationof the mouse embryos by the treatment of the different dose level of indomethacinand aspirin in the culture of mouse embryos.The female and male ICR mice, 6~8 weeks and were used for superovulation andmating and M16 was used as a basic culture medium.The above results can be summarized as following:1. Indomethacin seemed to inhibit the development of the mouse embryos and hatchingprocess because of inhibiting or blocking the activation of hatching related enzymes andhigh dose of indomethacin inhibited implantation.2. Aspirin had no effect on the hatching of the embryos at the dose of 0.16 mg/ml.3. FBS seemed to contain a factor which induced outgrowth of the embryo whereasBSA did not and outgrowth did not take place in the BSA contained medium.4. Blastocysts produced enough prostaglandins F2alpha which was needed for thehatching whereas they needed a factor for implantation which might be produced in theendometrium or exuded from the blood.In conclusion the concentration of indomethacin used in the present study inhibithatching of the blastocysts. This seems to be caused by inhibiting the synthesis of theproteins need for hatching. The factor that induce the outgrowth of the blastocysts doesnot seem to be produced in the blastocysts themselves but seem to be present in the FBS.
Animals ; Arachidonic Acid ; Aspirin ; Blastocyst ; Embryo, Mammalian ; Embryonic Structures ; Endometrium ; Female ; Humans ; Indomethacin ; Male ; Mice* ; Mice, Inbred ICR ; Peptide Hydrolases ; Pregnancy ; Prostaglandin-Endoperoxide Synthases ; Prostaglandins ; Superovulation ; Zona Pellucida

OBJECTIVETo examine allelic frequencies of coding single nucleotide polymorphisms (cSNPs) of folypolyglutamate synthetase (FPGS) gene in Chinese Han children with acute leukemia (AL), in order to provide a basis for detecting the relationship between FPGS genetic polymorphisms and tumor individualized chemotherapy.

METHODScSNPs of exon 5 were detected with polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) in 91 children with AL and 124 children with upper respiratory infection as controls. Genotypes and allelic frequencies were examined.

RESULTSA novel missense mutation, 502/490 T>C (L151/101P), was found in exon 5 of FPGS from control children. The novel mutation was found in mitochondrial variants in two cases and cytosolic variants in three cases. The cytosolic and mitochondrial variants displayed allelic frequencies of 0.70 % and 0.47 % respectively. The novel mutation was not associated with susceptibility to AL.

CONCLUSIONSA novel missense mutation 502/490 T>C (L151/101P) in exon 5 of FPGS gene is firstly found in Chinese Han children, and the cytosolic and mitochondrial variants display allelic frequencies of 0.70 % and 0.47 % respectively.

Child ; Child, Preschool ; Denaturing Gradient Gel Electrophoresis ; Exons ; Female ; Humans ; Infant ; Male ; Methotrexate ; pharmacology ; Mutation, Missense ; Peptide Synthases ; genetics ; Polymorphism, Single Nucleotide ; Sequence Analysis, DNA

Patent ductus arteriosus (PDA) is a major morbidity in preterm infants, especially in extremely premature infants less than 28 weeks. Early diagnosis of hemodynamically significant PDA (hs-PDA) is not easy because the symptoms of PDA in preterm infants are non-specific. Echocardiography is a good diagnostic tool for early detection of PDA. Clinical investigation has been continued to establish a criteria for selecting an infant who needs early targeted treatment of PDA by echocardiography. The biomarkers such as brain natriuretic peptide (BNP) and N-terminal pro-BNP (NTpBNP) are currently under research as a diagnostic and prognostic marker of PDA. Cyclooxygenase (COX) inhibitor is the treatment of choice and highly effective for PDA closure in preterm infants. Oral ibuprofen is emerging as a better alternative because it is as effective as indomethacin with fewer side effects. PDA ligation is a treatment option for hs-PDA when medical treatment is failed. There is lack of long term benefits of such treatments to induce ductal closure. Thus, it is prudent to treat an infant with clinically significant PDA on the basis of gestational age, birth weight, clinical status, and echocardiographic findings. Better diagnostic tools to identify infants who might benefit from ductal closure are needed.
Biomarkers ; Birth Weight ; Ductus Arteriosus, Patent ; Early Diagnosis ; Echocardiography ; Gestational Age ; Humans ; Ibuprofen ; Indomethacin ; Infant ; Infant, Extremely Premature ; Infant, Newborn ; Infant, Premature ; Ligation ; Natriuretic Peptide, Brain ; Peptide Fragments ; Prostaglandin-Endoperoxide Synthases