Thermo-resistant enzyme-producing microorganisms isolated from composting / Microrganismos produtores de enzimas termo-resistentes isolados de compostagem

Organo-mineral fertilizers supplemented with biological additives are an alternative to chemical fertilizers. In this study, thermoresistant microorganisms from composting mass were isolated by two-step procedures. First, samples taken at different time points and temperatures (33 days at 52 ºC, 60 days at 63 ºC, and over 365 days at 26 ºC) were pre-incubated at 80 oC for 30 minutes. Second, the microbial selection by in vitro culture-based methods and heat shock at 60 oC and 100 oC for 2h and 4h. Forty-one isolates were able to grow at 60 °C for 4h; twenty-seven at 100 °C for 2h, and two at 100 °C for 4h. The molecular identification by partial sequencing of the 16S ribosomal gene using universal primers revealed that thirty-five isolates were from eight Bacillus species, one Brevibacillus borstelensis, three Streptomyces thermogriseus, and two fungi (Thermomyces lanuginosus and T. dupontii). Data from amylase, phytase, and cellulase activity assays and the enzymatic index (EI) showed that 38 of 41 thermo-resistant isolates produce at least one enzyme. For amylase activity, the highest EI value was observed in Bacillus licheniformis (isolate 21C2, EI= 4.11), followed by Brevibacillus borstelensis (isolate 6C2, EI= 3.66), Bacillus cereus (isolate 18C2, EI= 3.52), and Bacillus paralicheniformis (isolate 20C2, EI= 3.34). For phytase, the highest EI values were observed for Bacillus cereus (isolate 18C2, EI= 2.30) and Bacillus licheniformis (isolate 3C1, EI= 2.15). Concerning cellulose production, B. altitudinis (isolate 6C1) was the most efficient (EI= 6.40), followed by three Bacillus subtilis (isolates 9C1, 16C2, and 19C2) with EI values of 5.66, 5.84, and 5.88, respectively, and one B. pumilus (isolate 27C2, EI= 5.78). The selected microorganisms are potentially useful as a biological additive in organo-mineral fertilizers and other biotechnological processes.

Os fertilizantes organo-minerais suplementados com aditivos biológicos são uma alternativa aos adubos químicos. Neste estudo, microrganismos termoresistentes foram isolados de compostagem por procedimentos de duas etapas. Inicialmente, as amostras tomadas em diferentes períodos e temperaturas (33 dias a 52 ºC, 60 dias a 63 ºC e mais de 365 dias a 26 ºC) foram pré-incubadas a 80 oC por 30 minutos. Posteriormente, a seleção microbiana foi conduzida por métodos baseados em cultura in vitro e choque térmico a 60 oC e 100 oC por 2h e 4h. Quarenta e um isolados foram capazes de crescer a 60 °C por 4h; vinte e sete a 100 °C por 2h e dois a 100 °C por 4h. A identificação molecular por sequenciamento parcial do gene ribossomal 16S usando primers universais revelou que trinta e cinco isolados eram de oito espécies de Bacillus, um Brevibacillus borstelensis, três Streptomyces thermogriseus e dois fungos (Thermomyces lanuginosus e T. dupontii). Os dados dos ensaios de atividade de amilase, fitase e celulase e o índice enzimático (IE) mostraram que 38 dos 41 isolados termorresistentes produziram pelo menos uma enzima. Para a atividade da amilase, o maior valor de IE foi observado em Bacillus licheniformis (isolado 21C2, IE = 4,11), seguido por Brevibacillus borstelensis (isolado 6C2, IE = 3,66), Bacillus cereus (isolado 18C2, IE = 3,52) e Bacillus paralicheniformis (isolado 20C2, IE = 3,34). Para a fitase, os maiores valores de IE foram observados para B. cereus (isolado 18C2, IE = 2,30) e B. licheniformis (isolado 3C1, IE = 2,15). Em relação à produção de celulose, B. altitudinis (isolado 6C1) foi o mais eficiente (IE = 6,40), seguido por três Bacillus subtilis (isolados 9C1, 16C2 e 19C2) com valores de IE de 5,66, 5,84 e 5,88, respectivamente, e um B. pumilus (isolado 27C2, IE = 5,78). Pode-se inferir que os microrganismos selecionados são potencialmente úteis como aditivos biológicos em fertilizantes organo-minerais e outros processos biotecnológicos.

Production of trans-cinnamic acid by whole-cell bioconversion from L-phenylalanine in engineered Corynebacterium glutamicum.

BACKGROUND: trans-cinnamic acid (t-CA) is a phenylpropanoid with a broad spectrum of biological activities including antioxidant and antibacterial activities, and it also has high potential in food and cosmetic applications. Although significant progress has been made in the production of t-CA using microorganisms, its relatively low product titers still need to be improved. In this study, we engineered Corynebacterium glutamicum as a whole-cell catalyst for the bioconversion of L-phenylalanine (L-Phe) into t-CA and developed a repeated bioconversion process. RESULTS: An expression module based on a phenylalanine ammonia lyase-encoding gene from Streptomyces maritimus (SmPAL), which mediates the conversion of L-Phe into t-CA, was constructed in C. glutamicum. Using the strong promoter PH36 and ribosome binding site (RBS) (in front of gene 10 of the T7 phage), and a high-copy number plasmid, SmPAL could be expressed to levels as high as 39.1% of the total proteins in C. glutamicum. Next, to improve t-CA production at an industrial scale, reaction conditions including temperature and pH were optimized; t-CA production reached up to 6.7 mM/h in a bioreactor under optimal conditions (50 °C and pH 8.5, using NaOH as base solution). Finally, a recycling system was developed by coupling membrane filtration with the bioreactor, and the engineered C. glutamicum successfully produced 13.7 mM of t-CA (24.3 g) from 18.2 mM of L-Phe (36 g) and thus with a yield of 75% (0.75 mol/mol) through repetitive supplementation. CONCLUSIONS: We developed a highly efficient bioconversion process using C. glutamicum as a biocatalyst and a micromembrane-based cell recycling system. To the best of our knowledge, this is the first report on t-CA production in C. glutamicum, and this robust platform will contribute to the development of an industrially relevant platform for the production of t-CA using microorganisms.

Discovery of anti-infective adipostatins through bioactivity-guided isolation and heterologous expression of a type III polyketide synthase.

Antibiotic resistance and emerging viral pandemics have posed an urgent need for new anti-infective drugs. By screening our microbial extract library against the main protease of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the notorious ESKAPE pathogens, an active fraction was identified and purified, leading to an initial isolation of adipostatins A (1) and B (2). In order to diversify the chemical structures of adipostatins toward enhanced biological activities, a type III polyketide synthase was identified from the native producer, Streptomyces davawensis DSM101723, and was subsequently expressed in an E. coli host, resulting in the isolation of nine additional adipostatins 3-11, including two new analogs (9 and 11). The structures of 1-11 were established by HRMS, NMR, and chemical derivatization, including using a microgram-scale meta-chloroperoxybenzoic acid epoxidation-MS/MS analysis to unambiguously determine the double bond position in the alkyl chain. The present study discovered SARS-CoV-2 main protease inhibitory activity for the class of adipostatins for the first time. Several of the adipostatins isolated also exhibited antimicrobial activity against selected ESKAPE pathogens.

Enzymatic reactions in teleocidin B biosynthesis.

The teleocidin B family members are terpene indole compounds isolated from Streptomyces bacteria, and they strongly activate protein kinase C (PKC). Their unique structures have attracted many researchers in the natural product chemistry and pharmacology fields, and numerous isolation and bioactivity studies have been conducted. The accumulated information has facilitated the identification of the enzymatic reactions in teleocidin biosynthesis, and new developments in structural biology have strongly aided efforts to clarify the finer points of these reactions. This review describes the recent biochemical and structural biological studies to reveal their reaction mechanisms, with a primary focus on the terpene cyclization triggered by the C-N bond formation by P450 oxygenase (TleB), the prenyltransferase (TleC), and the methyltransferase (TleD). This new knowledge will benefit future engineering studies to create unnatural PKC activators.

Discovery and Biosynthetic Investigation of a New Antibacterial Dehydrated Non-Ribosomal Tripeptide.

Dehydroalanine (Dha) and dehydrobutyrine (Dhb) display considerable flexibility in a variety of chemical and biological reactions. Natural products containing Dha and/or Dhb residues are often found to display diverse biological activities. While the (Z) geometry is predominant in nature, only a handful of metabolites containing (E)-Dhb have been found thus far. Here we report discovery of a new antimicrobial peptide, albopeptide, through NMR analysis and chemical synthesis, which contains two contiguous unsaturated residues, Dha-(E)-Dhb. It displays narrow-spectrum activity against vancomycin-resistant Enterococcus faecium. In-vitro biochemical assays show that albopeptide originates from a noncanonical NRPS pathway featuring dehydration processes and catalysed by unusual condensation domains. Finally, we provide evidence of the occurrence of a previously untapped group of short unsaturated peptides in the bacterial kingdom, suggesting an important biological function in bacteria.

In Vivo Studies of Inoculated Plants and In Vitro Studies Utilizing Methanolic Extracts of Endophytic Streptomyces sp. Strain DBT34 Obtained from Mirabilis jalapa L. Exhibit ROS-Scavenging and Other Bioactive Properties.

Reactive oxygen species (ROS) and other free radicals cause oxidative damage in cells under biotic and abiotic stress. Endophytic microorganisms reside in the internal tissues of plants and contribute to the mitigation of such stresses by the production of antioxidant enzymes and compounds. We hypothesized that the endophytic actinobacterium Streptomyces sp. strain DBT34, which was previously demonstrated to have plant growth-promoting (PGP) and antimicrobial properties, may also have a role in protecting plants against several stresses through the production of antioxidants. The present study was designed to characterize catalase and superoxide dismutase (SOD), two enzymes involved in the detoxification of ROS, in methanolic extracts derived from six endophytic actinobacterial isolates obtained from the traditional medicinal plant Mirabilis jalapa. The results of a preliminary screen indicated that Streptomyces sp. strain DBT34 was the best overall strain and was therefore used in subsequent detailed analyses. A methanolic extract of DBT34 exhibited significant antioxidant potential in 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS) assays. The cytotoxicity of DBT34 against liver hepatocellular cells (HepG2) was also determined. Results indicated that methanolic extract of Streptomyces sp. strain DBT34 exhibited significant catalase and SOD-like activity with 158.21 U resulting in a 55.15% reduction in ROS. The IC50 values of a crude methanolic extract of strain DBT34 on DPPH radical scavenging and ABTS radical cation decolorization were 41.5 µg/mL and 47.8 µg/mL, respectively. Volatile compounds (VOC) were also detected in the methanolic extract of Streptomyces sp. strain DBT34 using GC-MS analysis to correlate their presence with bioactive potential. Treatments of rats with DBT34 extract and sitagliptin resulted in a significant (p ≤ 0.001) reduction in total cholesterol, LDL-cholesterol, and VLDL-cholesterol, relative to the vehicle control and a standard diabetic medicine. The pancreatic histoarchitecture of vehicle control rats exhibited a compact volume of isolated clusters of Langerhans cells surrounded by acinies with proper vaculation. An in-vivo study of Streptomyces sp. strain DBT34 on chickpea seedlings revealed an enhancement in its antioxidant potential as denoted by lower IC50 values for DPPH and ABTS radical scavenging activity under greenhouse conditions in relative comparison to control plants. Results of the study indicate that strain DBT34 provides a defense mechanism to its host through the production of antioxidant therapeutic agents that mitigate ROS in hosts subjected to biotic and abiotic stresses.

Streptomyces from traditional medicine: sources of new innovations in antibiotic discovery.

Given the increased reporting of multi-resistant bacteria and the shortage of newly approved medicines, researchers have been looking towards extreme and unusual environments as a new source of antibiotics. Streptomyces currently provides many of the world's clinical antibiotics, so it comes as no surprise that these bacteria have recently been isolated from traditional medicine. Given the wide array of traditional medicines, it is hoped that these discoveries can provide the much sought after core structure diversity that will be required of a new generation of antibiotics. This review discusses the contribution of Streptomyces to antibiotics and the potential of newly discovered species in traditional medicine. We also explore how knowledge of traditional medicines can aid current initiatives in sourcing new and chemically diverse antibiotics.

Enzymatic Degradation of p-Nitrophenyl Esters, Polyethylene Terephthalate, Cutin, and Suberin by Sub1, a Suberinase Encoded by the Plant Pathogen Streptomyces scabies.

The genome of Streptomyces scabies, the predominant causal agent of potato common scab, encodes a potential cutinase, the protein Sub1, which was previously shown to be specifically induced in the presence of suberin. The sub1 gene was expressed in Escherichia coli and the recombinant protein Sub1 was purified and characterized. The enzyme was shown to be versatile because it hydrolyzes a number of natural and synthetic substrates. Sub1 hydrolyzed p-nitrophenyl esters, with the hydrolysis of those harboring short carbon chains being the most effective. The Vmax and Km values of Sub1 for p-nitrophenyl butyrate were 2.36 mol g-1 min-1 and 5.7 10-4 M, respectively. Sub1 hydrolyzed the recalcitrant polymers cutin and suberin because the release of fatty acids from these substrates was observed following the incubation of the enzyme with these polymers. Furthermore, the hydrolyzing activity of the esterase Sub1 on the synthetic polymer polyethylene terephthalate (PET) was demonstrated by the release of terephthalic acid (TA). Sub1 activity on PET was markedly enhanced by the addition of Triton and was shown to be stable at 37°C for at least 20 d.

Impact of Phospholipid-Tocopherol Combinations and Enzyme-Modified Lecithin on the Oxidative Stability of Bulk Oil.

Phosphatidylethanolamine (PE) and phosphatidylserine (PS) have been shown to increase the antioxidant activity of α-tocopherol. This study investigated the ability of PE or PS to increase the antioxidant activity of different tocopherol homologues in bulk oil. In addition, the ability of a phospholipase-D-modified lecithin (high in PE) to increase the activity of α-tocopherol was determined. Results showed that PE was much more effective than PS at increasing the activity of the tocopherol homologues. The combination of mixed tocopherols with PE presented the greatest increase in antioxidant activity, with hydroperoxides and hexanal lag phases increasing 54 and 53 days compared to the mixed tocopherols alone. Phospholipase-D-modified lecithin increased the antioxidant activity of α-tocopherol in stripped bulk oil as well as a commercially refined oil with no added tocopherols. The study indicates that PE is a powerful tool to increase the antioxidant activity of tocopherols in bulk oil and that modification of lecithin to increase the PE concentration could be a commercially viable option to functionalize lecithin, so that its ability to inhibit lipid oxidation increases in bulk oils.

Bioengineering of microbial transglutaminase for biomedical applications.

Microbial transglutaminase (mTGase) is commonly known in the food industry as meat glue due to its incredible ability to "glue" meat proteins together. Aside from being widely exploited in the meat processing industries, mTGase is also widely applied in other food and textile industries by catalysing the formation of isopeptide bonds between peptides or protein substrates. The advancement of technology has opened up new avenues for mTGase in the field of biomedical engineering. Efforts have been made to study the structural properties of mTGase in order to gain an in-depth understanding of the structure-function relationship. This review highlights the developments in mTGase engineering together with its role in biomedical applications including biomaterial fabrication for tissue engineering and biotherapeutics.

Microbial transglutaminase in noodle and pasta processing.

Nowadays, there is an aggressive rate in consumption of noodles and pasta products throughout the world. Consumer acceptability and preference of these functional products can be promoted by the discovery of novel knowledge to improve their formulation and quality. The development of fortified-formulations for noodles and pasta products based on microbial transglutaminase (MTGase) can guarantee the shelf life extension with minimum quality losses. The current review focuses on recent trends and future prospects of MTGase utilization in the structural matrix of noodles and pasta products and represents the quality changes of cooking loss, texture, microstructure, color and sensory attributes of the MTGase-incorporated products. Digestibility, nutritional and health aspects of the MTGase-enriched formulations are also reviewed with a vision toward physical functions and safety outcomes of MTGases isolated from new microbial sources. The high potential of MTGase in developing commercial noodles and pasta products is successfully demonstrated. MTGase by modifying the crystallinity or molecular structure via covalent crosslinks between protein molecules strengthens the doughs stability and the textural characteristics of final products with the low- or high-protein flour. Compared with the control samples, the MTGase-supplemented products indicate slower digestion rates and better sensory and cooking properties without any remarkable color instability.

Report- Antioxidant, cytotoxicity, protein kinase inhibition and antibacterial activities of Fragaria x ananassa leaves.

Fragaria × ananassa leaves extracts prepared in different solvents were subject for antioxidative, cytotoxicity, protein kinase inhibition and antibacterial activities. The extracts showed varying activities depending upon solvent used for extraction. Combined effect of methanol and ethyl acetate showed maximum antioxidant and reducing power potential (207.65±6µg AAE/mg and 88.58±20µg AAE/mg, respectively). Maximum DPPH (2,2-diphenyl-1-picryl hydrazyl) free radical scavenging activity was calculated by when methanol: chloroform and acetate fractions were used (87.68% and 86.88% inhibition, respectively). Total phenolics varied from 186 to 1.91µg AAE/mg while total flavonoids also significantly varied among the extracts. The extracts also showed significant activities against brine shrimp larvae and bacterial strains tested. The study concludes that Fragaria × ananassa leaves can be a good source for isolation of active phytochemicals to be used in different industries.

Effects of AttM lactonase on the pathogenicity of Streptomyces scabies.

The biosynthesis of phytotoxin thaxtomin A (TXT) constitutes the major pathogenicity determinant in Streptomyces scabies, the most widely studied phytopathogen causing scab disease in potato and other root crops. It is recognized that S. scabies regulates its pathogenicity via γ-butyrolactone (GBL)-dependent quorum sensing (QS) signalling. AttM, from Agrobacterium tumefaciens C58 strain, has recently been proposed to have GBL-assimilative capacity. Here, we presented the introduction of A. tumefaciens-derived attM gene into S. scabies using the Escherichia coli-Streptomyces shuttle vector pIJ8600 via intergeneric conjugation, followed by the investigation of secondary metabolism (mycelium growth, TXT production and pathogenicity) in S. scabies attM exconjugants (S.s/attM) in comparison with their wild-type parent strain (S.s/WT). Among the resultant S.s/attM exconjugants, attM was found to be integrated into S. scabies chromosome as analysed by Southern blotting. Moreover, S.s/attM failed to evoke the disease symptoms in planta and displayed altered morphological differentiation in contrast to S.s/WT. The abolishment of TXT production in S.s/attM substantiated the loss of pathogenicity and also implied that attM, when constitutively expressed in S. scabies, could paralyse its GBL signalling pathway. Altogether, lactonase-coding gene attM would be useful in a quorum quenching strategy for plant protection via suppressing TXT production and pathogenicity of S. scabies. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides an efficient means to introduce the lactonase gene attM from Agrobacterium tumefaciens into Streptomyces scabies for evaluating the role of γ-butyrolactone (GBL) in thaxtomin A production and pathogenicity, etc. Our results showed that pathogenicity was abrogated in attM-expressing S. scabies exconjugants. Although there are gene knockout approaches to inactivating GBL signalling and thus pathogenicity in S. scabies, they are not only time consuming due to refractory host but also possibly incomplete in view of gene redundancy. Our work is the first report for a kind of lactonase affecting pathogenicity and/or virulence of scab-causing Streptomyces species.

Mining of a phospholipase D and its application in enzymatic preparation of phosphatidylserine.

Phosphatidylserine (PS) is useful as the additive in industries for memory improvement, mood enhancement and drug delivery. Conventionally, PS was extracted from soybeans, vegetable oils, egg yolk, and biomass; however, their low availability and high extraction cost were limiting factors. Phospholipase D (PLD) is a promising tool for enzymatic synthesis of PS due to its transphosphatidylation activity. In this contribution, a new and uncharacterized PLD was first obtained from GenBank database via genome mining strategy. The open reading frame consisted of 1614 bp and potentially encoded a protein of 538-amino-acid with a theoretical molecular mass of 60 kDa. The gene was successfully cloned and expressed in Escherichia coli. Its enzymatic properties were experimentally characterized. The temperature and pH optima of PLD were determined to be 60°C and 7.5, respectively. Its hydrolytic activity was improved by addition of Ca2+ at 5 mM as compared with the control. The enzyme displayed suitable transphosphatidylation activity and PS could be synthesized with L-serine and soybean lecithin as substrates under the catalysis of PLD. This PLD enzyme might be a potential candidate for industrial applications in PS production. To the best of our knowledge, this is the first report on genome mining of PLDs from GenBank database.

Structure and specificity of a new class of Ca2+-independent housekeeping sortase from Streptomyces avermitilis provide insights into its non-canonical substrate preference.

Surface proteins in Gram-positive bacteria are incorporated into the cell wall through a peptide ligation reaction catalyzed by transpeptidase sortase. Six main classes (A-F) of sortase have been identified of which class A sortase is meant for housekeeping functions. The prototypic housekeeping sortase A (SaSrtA) from Staphylococcus aureus cleaves LPXTG-containing proteins at the scissile T-G peptide bond and ligates protein-LPXT to the terminal Gly residue of the nascent cross-bridge of peptidoglycan lipid II precursor. Sortase-mediated ligation ("sortagging") of LPXTG-containing substrates and Gly-terminated nucleophiles occurs in vitro as well as in cellulo in the presence of Ca2+ and has been applied extensively for protein conjugations. Although the majority of applications emanate from SaSrtA, low catalytic efficiency, LPXTG specificity restriction, and Ca2+ requirement (particularly for in cellulo applications) remain a drawback. Given that Gram-positive bacteria genomes encode a variety of sortases, natural sortase mining can be a viable complementary approach akin to engineering of wild-type SaSrtA. Here, we describe the structure and specificity of a new class E sortase (SavSrtE) annotated to perform housekeeping roles in Streptomyces avermitilis Biochemical experiments define the attributes of an optimum peptide substrate, demonstrate Ca2+-independent activity, and provide insights about contrasting functional characteristics of SavSrtE and SaSrtA. Crystal structure, substrate docking, and mutagenesis experiments have identified a critical residue that dictates the preference for a non-canonical LAXTG recognition motif over LPXTG. These results have implications for rational tailoring of substrate tolerance in sortases. Besides, Ca2+-independent orthogonal specificity of SavSrtE is likely to expand the sortagging toolkit.

Biosynthesis, purification and characterization of ß-1,4-xylanase from a novel mangrove associated actinobacterium Streptomyces olivaceus (MSU3) and its applications.

The present study was undertaken to evaluate the xylanolytic properties of an actinobacterium Streptomyces olivaceus (MSU3) isolated from the sediment sample of mangrove environment. It showed highest xylanase activity on initial screening in Breg's mineral salts medium supplemented with 0.5% xylan. Further the organism expressed maximum xylanase production at optimized culture conditions of pH 7, temperature 30 °C with 2.5% of inoculum size at 72 h of incubation, the nutrient sources like sucrose (2%) and yeast extract (3%) have observed as best carbon and nitrogen sources respectively. In purification, the xylanase resulted 4.27fold increase with the yield of 15.57% at the final step using sephadex G-75 chromatography. The molecular weight of the purified xylanase was observed as 42 kDa on 10% SDS-PAGE and further identified by MALDI-TOF-MS analysis. It belongs to GH43 family encoded 485 amino acid residues and the xylanase gene isolated using PCR amplification was partially sequenced. It showed 98% sequence similarity to the xylanase gene of S. olivaceus. The maximum activity of purified xylanase was observed at pH 8, temperature 40 °C and also the production medium substituted with Fe3+ metal ion, 2.0% xylan and 1.5% NaCl along with Km and Vmax values of 8.16 mg/ml and 250.01 µg/min/mg, respectively. In xylanolytic hydrolysis of pretreated agro-wastes, especially the sugarcane juice substituted medium yielded maximum (52.19%) reducing sugar, followed by bioethanol production (4.19  g/L) at 72 h of incubation. Based on the results, it could be confirmed that the selected isolate is a potent strain and it can able to produce xylanase through fermentation process and also it can able to convert the pretreated agro-wastes into economically important byproduct like bioethanol.

Significance of oxygen carriers and role of liquid paraffin in improving validamycin A production.

Validamycin A (Val-A) synthesized by Streptomyces hygroscopicus 5008 is widely used as a high-efficient antibiotic to protect plants from sheath blight disease. A novel fermentation strategy was introduced to stimulate Val-A production by adding oxygen carriers. About 58 % increase in Val-A production was achieved using liquid paraffin. Further, biomass, carbon source, metabolic genes, and metabolic enzymes were studied. It was also found that the supplementation of liquid paraffin increased the medium dissolved oxygen and intracellular oxidative stress level. The expression of the global regulators afsR and soxR sensitive to ROS, ugp catalyzing synthesis of Val-A precursor, and Val-A structural genes was enhanced. The change of the activities of glucose-6-phosphate dehydrogenase and glyceraldehyde 3-phosphate dehydrogenase was observed, which reflected the redirection of carbon metabolic flux. Based on these results, liquid paraffin addition as an oxygen carrier could be a useful technique in industrial production of Val-A and our study revealed a redox-based secondary metabolic regulation in S. hygroscopicus 5008, which provided a new insight into the regulation of the biosynthesis of secondary metabolites.

Evaluation of antagonistic and plant growth promoting activities of chitinolytic endophytic actinomycetes associated with medicinal plants against Sclerotium rolfsii in chickpea.

AIMS: To evaluate the potential of chitinolytic endophytic Actinomycetes isolated from medicinal plants in order to diminish the collar rot infestation induced by Sclerotium rolfsii in chickpea. METHODS AND RESULTS: Sixty-eight chitinolytic endophytic Actinomycetes were recovered from various medicinal plants and evaluated for their chitinase activity. Among these isolates, 12 were screened for their plant growth promoting abilities and antagonistic potential against Sc. rolfsii. Further, these isolates were validated in vivo for their ability to protect chickpea against Sc. rolfsii infestation under greenhouse conditions. The isolates significantly (P < 0·05) increased the biomass (1·2-2·0 fold) and reduced plant mortality (42-75%) of chickpea. On the basis of 16S rDNA profiling, the selected antagonistic strains were identified as Streptomyces diastaticus, Streptomyces fradiae, Streptomyces olivochromogenes, Streptomyces collinus, Streptomyces ossamyceticus and Streptomyces griseus. CONCLUSION: This study is the first report of the isolation of endophytic Actinomycetes from various medicinal plants having antagonistic and plant growth promoting abilities. The isolated species showed potential for controlling collar rot disease on chickpea and could be useful in integrated control against diverse soil borne plant pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: Our investigation suggests that endophytic Actinomycetes associated with medicinal plants can be used as bioinoculants for developing safe, efficacious and environment-friendly biocontrol strategies in the near future.

Engineering Escherichia coli to convert acetic acid to ß-caryophyllene.

BACKGROUND: Under aerobic conditions, acetic acid is the major byproduct produced by E. coli during the fermentation. And acetic acid is detrimental to cell growth as it destroys transmembrane pH gradients. Hence, how to reduce the production of acetic acid and how to utilize it as a feedstock are of intriguing interest. In this study, we provided an evidence to produce ß-caryophyllene by the engineered E. coli using acetic acid as the only carbon source. RESULTS: Firstly, to construct the robust acetate-utilizing strain, acetyl-CoA synthases from three different sources were introduced and screened in the E. coli. Secondly, to establish the engineered strains converting acetic acid to ß-caryophyllene, acetyl-CoA synthase (ACS), ß-caryophyllene synthase (QHS1) and geranyl diphosphate synthase (GPPS2) were co-expressed in the E. coli cells. Thirdly, to further enhance ß-caryophyllene production from acetic acid, the heterologous MVA pathway was introduced into the cells. What's more, acetoacetyl-CoA synthase (AACS) was also expressed in the cells to increase the precursor acetoacetyl-CoA and accordingly resulted in the increase of ß-caryophyllene. The final genetically modified strain, YJM67, could accumulate the production of biomass and ß-caryophyllene up to 12.6 and 1.05 g/L during 72 h, respectively, with a specific productivity of 1.15 mg h(-1) g(-1) dry cells, and the conversion efficiency of acetic acid to ß-caryophyllene (gram to gram) reached 2.1%. The yield of ß-caryophyllene on acetic acid of this strain also reached approximately 5.6% of the theoretical yield. CONCLUSIONS: In the present study, a novel biosynthetic pathway for ß-caryophyllene has been investigated by means of conversion of acetic acid to ß-caryophyllene using an engineered Escherichia coli. This was the first successful attempt in ß-caryophyllene production by E. coli using acetic acid as the only carbon source. Therefore, we have provided a new metabolic engineering tool for ß-caryophyllene synthesis.

Production of the Streptomyces scabies coronafacoyl phytotoxins involves a novel biosynthetic pathway with an F420 -dependent oxidoreductase and a short-chain dehydrogenase/reductase.

Coronafacoyl phytotoxins are secondary metabolites that are produced by various phytopathogenic bacteria, including several pathovars of the Gram-negative bacterium Pseudomonas syringae as well as the Gram-positive potato scab pathogen Streptomyces scabies. The phytotoxins are composed of the polyketide coronafacic acid (CFA) linked via an amide bond to amino acids or amino acid derivatives, and their biosynthesis involves the cfa and cfa-like gene clusters that are found in P. syringae and S. scabies, respectively. The S. scabies cfa-like gene cluster was previously reported to contain several genes that are absent from the P. syringae cfa gene cluster, including one (oxr) encoding a putative F420 -dependent oxidoreductase, and another (sdr) encoding a predicted short-chain dehydrogenase/reductase. Using gene deletion analysis, we demonstrated that both oxr and sdr are required for normal production of the S. scabies coronafacoyl phytotoxins, and structural analysis of metabolites that accumulated in the Δsdr mutant cultures revealed that Sdr is directly involved in the biosynthesis of the CFA moiety. Our results suggest that S. scabies and P. syringae use distinct biosynthetic pathways for producing coronafacoyl phytotoxins, which are important mediators of host-pathogen interactions in various plant pathosystems.