Residual biomass from surfactin production is a source of arginase and adsorbed surfactin that is useful for environmental remediation.

Lipopeptides are important secondary metabolites produced by microbes. They find applications in environmental decontamination and in the chemical, pharmaceutical and food industries. However, their production is expensive. In the present work we propose three strategies to lower the production costs of surfactin. First, the coproduction of surfactin and arginase in a single growth. Second, extract the fraction of surfactin that adsorbs to the biomass and is removed from the growth medium through centrifugation. Third, use microbial biomass for the remediation of organic and inorganic contaminants. The coproduction of surfactin and arginase was evaluated by factorial design experiments using the LB medium supplemented with arginine. The best conditions for surfactin production were 22 h of growth at 37 °C using LB supplemented with arginine 7.3 g/L. Almost similar conditions were found to produce highest levels of arginase, 24 h and 6.45 g/L arginine. Decontamination of phenol and copper from artificial samples was attained by treatment with residues from lipopeptide production. Thus, cell suspensions and wash-waters used to extract surfactin from the biomass. Cell suspensions were used to successfully remove hydroquinone. Cell suspensions and wash-waters containing surfactin were successfully used to recover copper from solution. Specific monitoring methods were used for phenol and metal solutions, respectively a biosensor based on tyrosinase and either atomic absorption flame ionization spectrometry or absorbance coupled to the Arduino™ platform. Therefore, we report three alternative strategies to lower the production costs in lipopeptide production, which include the effective recovery of copper and phenol from contaminated waters using residues from surfactin production. Sustainable and profitable production of surfactin can be achieved by a coproduction strategy of lipopeptides and enzymes. Lipopeptides are collected in the supernatant and enzymes in the biomass. In addition, lipopeptides that coprecipitate with biomass can be recovered by washing. Lipopeptide wash-waters find applications in remediation and cells can also be used for environmental decontamination.

Microparticles enhance the formation of seven major classes of natural products in native and metabolically engineered actinobacteria through accelerated morphological development.

Actinobacteria provide a rich spectrum of bioactive natural products and therefore display an invaluable source towards commercially valuable pharmaceuticals and agrochemicals. Here, we studied the use of inorganic talc microparticles (hydrous magnesium silicate, 3MgO·4SiO2 ·H2 O, 10 µm) as a general supplement to enhance natural product formation in this important class of bacteria. Added to cultures of recombinant Streptomyces lividans, talc enhanced production of the macrocyclic peptide antibiotic bottromycin A2 and its methylated derivative Met-bottromycin A2 up to 109 mg L-1 , the highest titer reported so far. Hereby, the microparticles fundamentally affected metabolism. With 10 g L-1 talc, S. lividans grew to 40% smaller pellets and, using RNA sequencing, revealed accelerated morphogenesis and aging, indicated by early upregulation of developmental regulator genes such as ssgA, ssgB, wblA, sigN, and bldN. Furthermore, the microparticles re-balanced the expression of individual bottromycin cluster genes, resulting in a higher macrocyclization efficiency at the level of BotAH and correspondingly lower levels of non-cyclized shunt by-products, driving the production of mature bottromycin. Testing a variety of Streptomyces species, talc addition resulted in up to 13-fold higher titers for the RiPPs bottromycin and cinnamycin, the alkaloid undecylprodigiosin, the polyketide pamamycin, the tetracycline-type oxytetracycline, and the anthramycin-analogs usabamycins. Moreover, talc addition boosted production in other actinobacteria, outside of the genus of Streptomyces: vancomycin (Amycolatopsis japonicum DSM 44213), teicoplanin (Actinoplanes teichomyceticus ATCC 31121), and the angucyclinone-type antibiotic simocyclinone (Kitasatospora sp.). For teicoplanin, the microparticles were even crucial to activate production. Taken together, the use of talc was beneficial in 75% of all tested cases and optimized natural and heterologous hosts forming the substance of interest with clusters under native and synthetic control. Given its simplicity and broad benefits, microparticle-supplementation appears as an enabling technology in natural product research of these most important microbes.

Construction and description of a constitutive plipastatin mono-producing Bacillus subtilis.

BACKGROUND: Plipastatin is a potent Bacillus antimicrobial lipopeptide with the prospect to replace conventional antifungal chemicals for controlling plant pathogens. However, the application of this lipopeptide has so far been investigated in a few cases, principally because of the yield in low concentration and unknown regulation of biosynthesis pathways. B. subtilis synthesizes plipastatin by a non-ribosomal peptide synthetase encoded by the ppsABCDE operon. In this study, B. subtilis 3NA (a non-sporulation strain) was engineered to gain more insights about plipastatin mono-production. RESULTS: The 4-phosphopantetheinyl transferase Sfp posttranslationally converts non-ribosomal peptide synthetases from inactive apoforms into their active holoforms. In case of 3NA strain, sfp gene is inactive. Accordingly, the first step was an integration of a repaired sfp version in 3NA to construct strain BMV9. Subsequently, plipastatin production was doubled after integration of a fully expressed degQ version from B. subtilis DSM10T strain (strain BMV10), ensuring stimulation of DegU-P regulatory pathway that positively controls the ppsABSDE operon. Moreover, markerless substitution of the comparably weak native plipastatin promoter (Ppps) against the strong constitutive promoter Pveg led to approximately fivefold enhancement of plipastatin production in BMV11 compared to BMV9. Intriguingly, combination of both repaired degQ expression and promoter exchange (Ppps::Pveg) did not increase the plipastatin yield. Afterwards, deletion of surfactin (srfAA-AD) operon by the retaining the regulatory comS which is located within srfAB and is involved in natural competence development, resulted in the loss of plipastatin production in BMV9 and significantly decreased the plipastatin production of BMV11. We also observed that supplementation of ornithine as a precursor for plipastatin formation caused higher production of plipastatin in mono-producer strains, albeit with a modified pattern of plipastatin composition. CONCLUSIONS: This study provides evidence that degQ stimulates the native plipastatin production. Moreover, a full plipastatin production requires surfactin synthetase or some of its components. Furthermore, as another conclusion of this study, results point towards ornithine provision being an indispensable constituent for a plipastatin mono-producer B. subtilis strain. Therefore, targeting the ornithine metabolic flux might be a promising strategy to further investigate and enhance plipastatin production by B. subtilis plipastatin mono-producer strains.

Simultaneous hydrolysis with lipase and fermentation of rapeseed cake for iturin A production by Bacillus amyloliquefaciens CX-20.

BACKGROUND: Rapeseed cake (RSC), as the intermediate by-product of oil extraction from the seeds of Brassica napus, can be converted into rapeseed meal (RSM) by solvent extraction to remove oil. However, compared with RSM, RSC has been rarely used as a raw material for microbial fermentation, although both RSC and RSM are mainly composed of proteins, carbohydrates and minerals. In this study, we investigated the feasibility of using untreated low-cost RSC as nitrogen source to produce the valuable cyclic lipopeptide antibiotic iturin A using Bacillus amyloliquefaciens CX-20 in submerged fermentation. Especially, the effect of oil in RSC on iturin A production and the possibility of using lipases to improve the iturin A production were analyzed in batch fermentation. RESULTS: The maximum production of iturin A was 0.82 g/L at the optimal initial RSC and glucose concentrations of 90 and 60 g/L, respectively. When RSC was substituted with RSM as nitrogen source based on equal protein content, the final concentration of iturin A was improved to 0.95 g/L. The production of iturin A was further increased by the addition of different lipase concentrations from 0.1 to 5 U/mL into the RSC medium for simultaneous hydrolysis and fermentation. At the optimal lipase concentration of 0.5 U/mL, the maximal production of iturin A reached 1.14 g/L, which was 38.15% higher than that without any lipase supplement. Although rapeseed oil and lipase were firstly shown to have negative effects on iturin A production, and the effect would be greater if the concentration of either was increased, their respective negative effects were reduced when used together. CONCLUSIONS: Appropriate relative concentrations of lipase and rapeseed oil were demonstrated to support optimal iturin A production. And simultaneous hydrolysis with lipase and fermentation was an effective way to produce iturin A from RSC using B. amyloliquefaciens CX-20.

A comprehensive genomic and growth proteomic analysis of antitumor lipopeptide bacillomycin Lb biosynthesis in Bacillus amyloliquefaciens X030.

Lipopeptides (such as iturin, fengycin, and surfactin) from Bacillus possess antibacterial, antifungal, and antiviral activities and have important application in agriculture and pharmaceuticals. Although unremitting efforts have been devoted to improve lipopeptide production by designing gene regulatory circuits or optimizing fermentation process, little attention has been paid to utilizing multi-omics for systematically mining core genes and proteins during the bacterial growth cycle. Here, lipopeptide bacillomycin Lb from new Bacillus amyloliquefaciens X030 was isolated and first found to have anticancer activity in various cancer cells (such as SMMC-7721 and MDA-MB-231). A comprehensive genomic and growth proteomic analysis of X030 revealed bacillomycin Lb biosynthetic gene cluster, key enzymes and potential regulatory proteins (PerR, PhoP, CcpA, and CsfB), and novel links between primary metabolism and bacillomycin Lb production in X030. The antitumor activity of the fermentation supernatant supplemented with amino acids (such as glutamic acid) and sucrose was significantly increased, verifying the role of key metabolic switches in the metabolic regulatory network. Quantitative real-time PCR analysis confirmed that 7 differential expressed genes exhibited a positive correlation between changes at transcriptional and translational levels. The study not only will stimulate the deeper and wider antitumor study of lipopeptides but also provide a comprehensive database, which promotes an in-depth analysis of pathways and networks for complex events in lipopeptide biosynthesis and regulation and gives great help in improving the yield of bacillomycin Lb (media optimization, genetic modification, or pathway engineering).

Astin C Production by the Endophytic Fungus Cyanodermella asteris in Planktonic and Immobilized Culture Conditions.

The fungal endophyte Cyanodermella asteris (C. asteris) has been recently isolated from the medicinal plant Aster tataricus (A. tataricus). This fungus produces astin C, a cyclic pentapeptide with anticancer and anti-inflammatory properties. The production of this secondary metabolite is compared in immobilized and planktonic conditions. For immobilized cultures, a stainless steel packing immersed in the culture broth is used as a support. In these conditions, the fungus exclusively grows on the packing, which provides a considerable advantage for astin C recovery and purification. C. asteris metabolism is different according to the culture conditions in terms of substrate consumption rate, cell growth, and astin C production. Immobilized-cell cultures yield a 30% increase of astin C production, associated with a 39% increase in biomass. The inoculum type as spores rather than hyphae, and a pre-inoculation washing procedure with sodium hydroxide, turns out to be beneficial both for astin C production and fungus development onto the support. Finally, the influence of culture parameters such as pH and medium composition on astin C production is evaluated. With optimized culture conditions, astin C yield is further improved reaching a five times higher final specific yield compared to the value reported with astin C extraction from A. tataricus (0.89 mg g-1 and 0.16 mg g-1 respectively).

Cyclic lipopeptide signature as fingerprinting for the screening of halotolerant Bacillus strains towards microbial enhanced oil recovery.

Cyclic lipopeptides (CLPs) are non-ribosomal biosurfactants produced by Bacillus species that exhibit outstanding interfacial activity. The synthesis of CLPs is under genetic and environmental influence, and representatives from different families are generally co-produced, generating isoforms that differ in chemical structure and biological activities. This study to evaluate the effect of low and high NaCl concentrations on the composition and surface activity of CLPs produced by Bacillus strains TIM27, TIM49, TIM68, and ICA13 towards microbial enhanced oil recovery (MEOR). The strains were evaluated in mineral medium containing NaCl 2.7, 66, or 100 g L-1 and growth, surface tension and emulsification activity were monitored. Based on the analysis of 16S rDNA, gyrB and rpoB sequences TIM27 and TIM49 were assigned to Bacillus subtilis, TIM68 to Bacillus vallismortis, and ICA13 to Bacillus amyloliquefaciens. All strains tolerated up to 100-g L-1 NaCl, but only TIM49 and TIM68 were able to reduce surface tension at this concentration. TIM49 also showed emulsification activity at concentrations up to 66-g L-1 NaCl. ESI-MS analysis showed that the strains produced a mixture of CLPs, which presented distinct CLP profiles at low and high NaCl concentrations. High NaCl concentration favored the synthesis of surfactins and/or fengycins that correlated with the surface activities of TIM49 and TIM68, whereas low concentration favored the synthesis of iturins. Taken together, these findings suggest that the determination of CLP signatures under the expected condition of oil reservoirs can be useful in the guidance for choosing well-suited strains to MEOR.

Lichenysin production is improved in codY null Bacillus licheniformis by addition of precursor amino acids.

Lichenysin is categorized into the family of lipopeptide biosurfactants and has a variety of applications in the petroleum industry, bioremediation, pharmaceuticals, and the food industry. Currently, large-scale production is limited due to the low yield. This study found that lichenysin production was repressed by supplementation of extracellular amino acids. The global transcriptional factor CodY was hypothesized to prevent lichenysin biosynthesis under an amino acid-rich condition in Bacillus licheniformis. Thus, the codY null strain was constructed, and lichenysin production was increased by 31.0% to 2356 mg/L with the addition of precursor amino acids, and the lichenysin production efficiency was improved by 42.8% to 98.2 mg/L• h. Correspondingly, the transcription levels of the lichenysin synthetase gene lchAA, and its corresponding regulator genes comA, degQ, and degU, were upregulated. Also, the codY deletion enhanced biosynthesis of lichenysin precursor amino acids (Gln, Ile, Leu, and Val) and reduced the formation of byproducts, acetate, acetoin, and 2,3-butanediol. This study firstly reported that lichenysin biosynthesis was negatively regulated by CodY and lichenysin production could be further improved with the precursor amino acid amendment in the codY null strain.

Lipid composition in a strain of Bacillus subtilis, a producer of iturin A lipopeptides that are active against uropathogenic bacteria.

Urinary tract infections are a common disease in humans. Therefore, new methods are needed to destroy biofilms that are formed by uropathogens. Iturin A lipopeptides (LPs) C14 and C15 are potent biosurfactants synthetized by the Bacillus subtilis I'1a strain. The biological activity of extracted LPs was confirmed by examining extracts from I'1a cultures against uropathogenic bacteria that had been isolated from biofilms on urinary catheters. Compared with cultures of DSM 3257, which produce surfactin at a relatively low level, the extract obtained from strain I'1a exhibited a greater inhibitory effect against both planktonic and sessile forms of Escherichia coli, Serratia marcescens, Enterobacter cloacae, Proteus mirabilis, Citrobacter freundii and Enterococcus faecalis. Moreover, cyclic LP biosurfactants may disturb the integrity of cytoplasmic membranes; therefore, we investigated the effects of synthetized LPs on fatty acids and phospholipids of B. subtilis. LPs and lipids were analyzed using GC-MS, LC-MS/MS and MALDI-TOF/TOF techniques. Compared with B. subtilis DSM 3257, membranes of the I'1a strain were characterized by an increased amount of anteiso fatty acids and a ten-fold higher ratio of phosphatidylglycerol (PG)-to-phosphatidylethanolamine (PE). Interestingly, in cultures of B. subtilis DSM 3257 supplemented with LP extracts of the I'1a strain, the PG-to-PE ratio was fourfold higher, and the amount of anteiso fatty acids was also increased.

Simultaneous biosynthesis of putrebactin, avaroferrin and bisucaberin by Shewanella putrefaciens and characterisation of complexes with iron(III), molybdenum(VI) or chromium(V).

Cultures of Shewanella putrefaciens grown in medium containing 10mM 1,4-diamino-2-butanone (DBO) as an inhibitor of ornithine decarboxylase and 10mM 1,5-diaminopentane (cadaverine) showed the simultaneous biosynthesis of the macrocyclic dihydroxamic acids: putrebactin (pbH2), avaroferrin (avH2) and bisucaberin (bsH2). The level of DBO did not completely repress the production of endogenous 1,4-diaminobutane (putrescine) as the native diamine substrate of pbH2. The relative concentration of pbH2:avH2:bsH2 was 1:2:1, which correlated with the substrate selection of putrescine:cadaverine in a ratio of 1:1. The macrocycles were characterised using LC-MS as free ligands and as 1:1 complexes with Fe(III) of the form [Fe(pb)]+, [Fe(av)]+ or [Fe(bs)]+, with labile ancillary ligands in six-coordinate complexes displaced during ESI-MS acquisition; or with Mo(VI) of the form [Mo(O)2(pb)], [Mo(O)2(av)] or [Mo(O)2(bs)]. Chromium(V) complexes of the form [CrO(pb)]+ were detected from solutions of Cr(VI) and pbH2 in DMF using X-band EPR spectroscopy. Supplementation of S. putrefaciens medium with DBO and 1,3-diaminopropane, 1,6-diaminohexane or 1,4-diamino-2(Z)-butene (Z-DBE) resulted only in the biosynthesis of pbH2. The work has identified a native system for the simultaneous biosynthesis of a suite of three macrocyclic dihydroxamic acid siderophores and highlights both the utility of precursor-directed biosynthesis for expanding the structural diversity of siderophores, and the breadth of their coordination chemistry.

Rhamnolipid and surfactin production from olive oil mill waste as sole carbon source.

Olive mill waste (OMW) creates a major environmental problem due to the difficulty of further waste processing. In this work we present an approach to give OMW added value by using it for the production of biosurfactants. Two bacterial species, Pseudomonas aeruginosa and Bacillus subtilis, were grown with OMW as the sole carbon source. Glycerol and waste frying oil were used as comparative carbon sources. B. subtilis produced surfactin (a lipopeptide) at a maximum concentration of 3.12 mg/L with 2% w/v of OMW in the medium, dropping to 0.57 mg/L with 10% w/v of OMW. In contrast, P. aeruginosa produced 8.78 mg/L of rhamnolipid with 2% w/v OMW increasing to 191.46 mg/L with 10% w/v OMW. The use of solvent-extracted OMW reduced the biosurfactant production by 70.8% and 88.3% for B. subtilis and P. aeruginosa respectively. These results confirm that OMW is a potential substrate for biosurfactant production.

The self-assembly of a cyclic lipopeptides mixture secreted by a B. megaterium strain and its implications on activity against a sensitive Bacillus species.

Cyclic lipopeptides are produced by a soil Bacillus megaterium strain and several other Bacillus species. In this work, they are detected both in the Bacillus intact cells and the cells culture medium by MALDI-TOF mass spectrometry. The cyclic lipopeptides self-assemble in water media producing negatively charged and large aggregates (300-800 nm of mean hydrodynamic radius) as evaluated by dynamic light scattering and zeta-potential analysis. The aggregate size depends on pH and ionic strength. However, it is not affected by changes in the osmolarity of the outer medium suggesting the absence of an internal aqueous compartment despite the occurrence of low molecular weight phospholipids in their composition as determined from inorganic phosphorus analysis. The activity against a sensitive Bacillus cereus strain was evaluated from inhibition halos and B. cereus lysis. Essential features determining the antibiotic activity on susceptible Bacillus cereus cells are the preserved cyclic moiety conferring cyclic lipopeptides resistance to proteases and the medium pH. The aggregates are inactive per se at the pH of the culture medium which is around 6 or below. The knock out of the sensitive cells only takes place when the aggregates are disassembled due to a high negative charge at pH above 6.

The biosurfactant viscosin produced by Pseudomonas fluorescens SBW25 aids spreading motility and plant growth promotion.

Food security depends on enhancing production and reducing loss to pests and pathogens. A promising alternative to agrochemicals is the use of plant growth-promoting rhizobacteria (PGPR), which are commonly associated with many, if not all, plant species. However, exploiting the benefits of PGPRs requires knowledge of bacterial function and an in-depth understanding of plant-bacteria associations. Motility is important for colonization efficiency and microbial fitness in the plant environment, but the mechanisms employed by bacteria on and around plants are not well understood. We describe and investigate an atypical mode of motility in Pseudomonas fluorescens SBW25 that was revealed only after flagellum production was eliminated by deletion of the master regulator fleQ. Our results suggest that this 'spidery spreading' is a type of surface motility. Transposon mutagenesis of SBW25ΔfleQ (SBW25Q) produced mutants, defective in viscosin production, and surface spreading was also abolished. Genetic analysis indicated growth-dependency, production of viscosin, and several potential regulatory and secretory systems involved in the spidery spreading phenotype. Moreover, viscosin both increases efficiency of surface spreading over the plant root and protects germinating seedlings in soil infected with the plant pathogen Pythium. Thus, viscosin could be a useful target for biotechnological development of plant growth promotion agents.

Cyclic dipeptides produced by marine sponge-associated bacteria as quorum sensing signals.

Four bacterial strains belonging to the genera Vibrio, Pseudoalteromonas and Photobacterium were isolated from the marine sponges Dysidea avara and Geodia cynodium. A Bacillus strain was isolated from Ircinia variabilis. A screening of molecules involved in quorum sensing (QS) was carried out by TLC-overlay and a new "plate T-streak" test. To analyze quorum quenching (QQ), a plate T-streak was performed with Chromobacterium violaceum. Strains of Vibrio isolated from both marine sponges and a strain of Photobacterium isolated from G. cynodium, activated QS bioreporters. A strain of Pseudoalteromonas isolated from D. avara showed QQ activity. Finally, it is reported that cyclic dipeptides isolated from strains of Vibrio sp. and Bacillus sp. (isolated from D. avara and I. variabilis, respectively) were involved in the QS mechanism. The simultaneous presence of bacteria that showed contrasting responses in bioassays for QS signal molecule synthesis in marine sponges could add an interesting dimension to the signalling interactions which may be happening in sponges.

An enzymatic route to selenazolines.

Ringing the changes: Selenazolines have applications in medicinal chemistry, but their synthesis is challenging. We report a new convenient and less toxic route to these heterocycles that starts from commercially available selenocysteine. The new route depends on a heterocyclase enzyme that creates oxazolines and thiazolines from serines/threonines and cysteines.

Enhanced production of mosquitocidal cyclic lipopeptide from Bacillus subtilis subsp. subtilis.

BACKGROUND & OBJECTIVES: A cyclic lipopeptide, surfactin produced by a strain of Bacillus subtilis subsp. subtilis (VCRC B471) was found to exhibit activity against both the larval and pupal stages of mosquitoes. The present study was aimed at increasing the production of the mosquitocidal metabolite by modifying the conventional medium. METHODS: Enhancement of mosquitocidal metabolite production was attempted by replacing the existing micronutrients of the conventional NYSM and supplementing the medium with additional amounts of glucose. The LC50 value of culture supernatant (CS) against the larval and pupal stages of Anopheles stephensi was determined. Crude mosquitocidal metabolite (CMM) was separated from the CS, identified by MALDI-TOF analysis and its LC50 dosage requirement for the pupal stage of the above mosquito species determined. RESULTS: The medium containing a new composition of micronutrients and glucose up to 1 per cent resulted in increased metabolite production. The LC50 value of the CS obtained in the improved medium against larvae and pupae of An. stephensi was 5.57 and 0.71 µl/ml, respectively. The yield of CMM was doubled in the improved medium. MALDI-TOF analysis revealed that the CMM was surfactin. INTERPRETATION & CONCLUSIONS: The new improved medium enhanced the production of mosquitocidal metabolite as the dosage required for inciting 50 per cent mortality among the pupal stages of mosquitoes was only half of that required when the metabolite was produced in the conventional medium. The mosquitocidal metabolite was identified as surfactin, a cyclic lipopeptide and biosurfactant.

The biosynthesis of Caryophyllaceae-like cyclic peptides in Saponaria vaccaria L. from DNA-encoded precursors.

Cyclic peptides (CPs) are produced in a very wide range of taxa. Their biosynthesis generally involves either non-ribosomal peptide synthases or ribosome-dependent production of precursor peptides. Plants within the Caryophyllaceae and certain other families produce CPs which generally consist of 5-9 proteinogenic amino acids. The biological roles for these CPs in the plant are not very clear, but many of them have activity in mammalian systems. There is currently very little known about the biosynthesis of CPs in the Caryophyllaceae. A collection of expressed sequence tags from developing seeds of Saponaria vaccaria was investigated for information about CP biosynthesis. This revealed genes that appeared to encode CP precursors which are subsequently cyclized to mature CPs. This was tested and confirmed by the expression of a cDNA encoding a putative precursor of the CP segetalin A in transformed S. vaccaria roots. Similarly, extracts of developing S. vaccaria seeds were shown to catalyze the production of segetalin A from the same putative (synthetic) precursor. Moreover, the presence in S. vaccaria seeds of two segetalins, J [cyclo(FGTHGLPAP)] and K [cyclo(GRVKA)], which was predicted by sequence analysis, was confirmed by liquid chromatography/mass spectrometry. Sequence analysis also predicts the presence of similar CP precursor genes in Dianthus caryophyllus and Citrus spp. The data support the ribosome-dependent biosynthesis of Caryophyllaceae-like CPs in the Caryophyllaceae and Rutaceae.

[Isolation and identification of surfactin producing Bacillus subtilis strain and its effect of surfactin on crude oil].

Strains producing biological surfactants were isolated from formation water of Daqing oil field, Heilongjiang Province, China. From which a lipopeptide producing strain ZW-3 was screened out by PCR of the sfp gene. The morphology, cultural characteristics, physiological, biochemical properties and chemotaxonomy of strain ZW-3 were studied. The strain is rod shaped (0.7-0.8 microm x 2-3 microm), gram-positive, spore-forming and aerobic bacteria. Its (G+C) content was determined to be 42.2 mol%. Phylogenetic analysis based on 16S rRNA gene sequence demonstrated that it was closely related to the genus Bacillus subtilis, and the metabolites of strain ZW-3 was analyzed by thin layer chromatography and high performance liquid chromatography, the result indicated that the biosurfactant strain ZW-3 produced was surfactin. It could reduce surface tension of bacterial fermentation culture medium and water/oil- interfacial tension from 68.82 mN/m to 24.88 mN/m and from 23.53 mN/m to 4.57 mN/m, respectively, and its mixture with 1.8% NaOH could reduce water/oil- interfacial tension to an ultra low level (1.2 x 10(-3) mN/m), Its critical micelle concentration (CMC) was tested to be 33.3 mg/L (3.24 x 10(-5) mol/L)at 25 degrees C, and it had excellent emulsifying (2.89U) and foaming property. All these results showed that this biosurfactant had great potential in pharmaceutics, environmental protection, cosmetic, oil recovery and many other application fields.

Stabilization of hyperactive dihydrofolate reductase by cyanocysteine-mediated backbone cyclization.

Stabilization of an enzyme while maintaining its activity has been a major challenge in protein chemistry. Although it is difficult to simultaneously improve stability and activity of a protein by amino acid substitutions due to the activity-stability trade-off, backbone cyclization by connecting the N and C termini with a linker is promising as a general method of stabilizing a protein without affecting its activity. Recently, we created a hyperactive, methionine- and cysteine-free mutant of dihydrofolate reductase from Escherichia coli, called ANLYF, by introducing seven amino acid substitutions, which, however, destabilized the protein. Here we show that ANLYF is stabilized without a loss of its high activity by a novel backbone cyclization method for unprotected proteins. The method is based on the in vitro cyanocysteine-mediated intramolecular ligation reaction, which can be conducted with relatively high efficiency by a simple procedure and under mild conditions. We also show that the reversibility of thermal denaturation is highly improved by the cyclization. Thus, activity and stability of the protein can be separately improved by amino acid substitutions and backbone cyclization, respectively. We suggest that the cyanocysteine-mediated cyclization method is complementary to the intein-mediated cyclization method in stabilizing a protein without affecting its activity.

Variations in the microcystin production of Planktothrix rubescens (cyanobacteria) assessed from a four-year survey of Lac du Bourget (France) and from laboratory experiments.

Between 1999 and 2002, a routine survey of water quality in the Lac du Bourget was performed to study the dynamics and microcystin (MC) production of Planktothrix rubescens. Using liquid chromatography coupled to diode array detection and mass spectrometry, we found that two main variants ([D-Asp3] and [D-Asp3, Dhb7] microcystin-RR) were produced. The proportion of these two variants was not influenced by the depth or season of sampling. Expressed in microcystin-LR equivalents, high microcystin concentrations were recorded from August to December each year, reaching values of up to 6.7 microg L-1. A significant correlation was found between the microcystin cell content and the cell densities of P. rubescens. Cellular quotas of microcystins ranged from 0.1 to 0.3 pg cell-1. Simultaneously, laboratory experiments were performed on a strain of P. rubescens isolated from the lake to assess the potential impact of various P-PO4 (3-) concentrations on intra- and extracellular microcystin production. Unlike natural populations, this strain only produced [D-Asp3] MC-RR. The intracellular microcystin content was similarly correlated to the cell density, but the cellular quota was slightly higher (0.3-0.7 pg cell-1) than in the natural population. Again, as in the natural population, a linear relationship was found between growth rate and microcystin production rate. These findings support the hypothesis that environmental factors, such as phosphate concentrations, have no direct impact on microcystin production by P. rubescens, but act indirectly by affecting growth rate.