Microbial hydroxylation of 2-oxatestosterone

An investigation has been undertaken of the microbial biotransformation of 2-oxatestosterone by microorganisms known to hydroxylate conventional steroids, using Aspergillus ochraceus, Bacillus megaterium, Curvularia lunata and Rhizopus arrhizus. A. ochraceus and B. megaterium gave products of 11α- and 15β-hydroxylation, respectively. Biotransformation by C. lunata gave C-11β- and C-14α-hydroxylated products, whereas R. arrhizus produced only the 6β-hydroxy derivative.     

Construction of engineered Arthrobacter simplex with improved performance for cortisone acetate biotransformation

Arthrobacter simplex 156 is a microorganism that is used for steroid drug biotransformation of cortisone acetate (CA) to prednisone acetate (PA). The enzyme 3-ketosteroid-△¹-dehydrogenase encoded by the ksdD gene plays an important role in the bioconversion process. To further improve the biotransformation efficiencies of the industrial strain, a genetic manipulation system for A. simplex 156 was developed. Additional copies of the ksdD gene under the control of the cat promoter (from pXMJ19) were transferred into the strain A. simplex 156 and integrated into the 16S rDNA sites, yielding a series of recombinant strains. One of these recombinant strains, designated A. simplex M158, exhibited superior properties for CA biotransformation. At the substrate concentration of 83.6 g/l, the highest PA production of the recombinant strain reached 66.7 g/l, which is approximately 32.9 % higher than that of wild-type strains, and the incubation time for CA to PA bioconversion was reduced by 20 h. Southern blotting analysis of the recombinant strain indicated two copies of deregulated ksdD genes were integrated into the 16S rDNA sites, which means two of five 16S rRNA operons were insertionally disrupted in the recombinant strain. However, the disruption of the two 16S rRNA operons did not affect the growth rate of the recombinant strain, which survived and thrived under desired conditions. In addition, the new strain was genetically stable for more than 100 generations without the use of antibiotics for selection. These superior characteristics make the new strain more suitable than the wild-type strain for PA production.     

Derivatives of thiocolchicine and its applications to CEM cells treatment using F Magnetic Resonance ex vivo

It was shown, that cultured ex vivo human T-Lymphoblastoid (CEM) cells respond to synthesized thiocolchicine and fluorine thiocolchicine derivatives. The preparation of derivatives with substitution at C-3 and C-7 is described. All compounds were used at concentration from 1 nM to 1000 nM. Inhibitory effects of these compounds were examined in the three-dimensional (3-D) culture and cells morphology during treatment was monitored using 9.4 T MRI system. We performed studies of these compounds in CEM cells ex vivo using ¹H and ¹⁹F Magnetic Resonance Imaging (MRI), ¹⁹F Magnetic Resonance Spectroscopy (MRS), High Performance Liquid Chromatography coupled with Ultra Violet (HPLC-UV) and Electron Impact Mass Spectrometry (EIMS). The results of the multi-technique approach are consistent with the fact that the new derivatives are more efficient than colchicine and thiocolchicine ex vivo.     

High yield production of extracellular recombinant levansucrase by Bacillus megaterium

In this study, a high yield production bioprocess with recombinant Bacillus megaterium for the production of the extracellular enzyme levansucrase (SacB) was developed. For basic optimization of culture parameters and nutrients, a recombinant B. megaterium reporter strain that produced green fluorescent protein under control of a vector-based xylose-inducible promoter was used. It enabled efficient microtiter plate-based screening via fluorescence analysis. A pH value of pH?6, 20 % of dissolved oxygen, 37 °C, and elevated levels of biotin (100 μg?L^?1) were found optimal with regard to high protein yield and reduced overflow metabolism. Among the different compounds tested, fructose and glycerol were identified as the preferred source of carbon. Subsequently, the settings were transferred to a B. megaterium strain recombinantly producing levansucrase SacB based on the plasmid-located xylose-inducible expression system. In shake flask culture under the optimized conditions, the novel strain already secreted the target enzyme in high amounts (14 U?mL^?1 on fructose and 17.2 U?mL^?1 on glycerol). This was further increased in high cell density fed-batch processes up to 55 U?mL^?1, reflecting a levansucrase concentration of 0.52 g?L^?1. This is 100-fold more than previous efforts for this enzyme in B. megaterium and more than 10-fold higher than reported values of other extracellular protein produced in this microorganism so far. The recombinant strain could also handle raw glycerol from biodiesel industry which provided the same amount and quality of the recombinant protein and suggests future implementation into existing biorefinery concepts.     

Penetration and Distribution of Thiocolchicoside through Human Skin: Comparison Between a Commercial Foam (Miotens<Superscript>®</Superscript><Emphasis Type="Bold">) and a Drug Solution</Emphasis>

Penetration and distribution of thiocolchicoside from a commercially available foam (Miotens^® 0.25%, w/v) through human excised full-thickness skin were evaluated using two different in vitro apparatus: a Franz diffusion cell and a Saarbruecken penetration model-based cell. In order to evaluate the intrinsic capability of the drug to penetrate into the skin, a simple drug aqueous solution prepared at the same drug concentration as Miotens^® was also tested. Results showed that both apparatus were suitable to study thiocolchicoside penetration into human skin. Penetrated drug amounts were comparable using the two apparatus, probably because skin acts as “sink” for the drug. Miotens^® was found to significantly promote thiocolchicoside accumulation into full human skin thickness in comparison with the simple drug solution. The mixture of propylene glycol and propylene glycol diperlargonate contained into Miotens^® foam has been proven to be effective to promote penetration of thiocolchicoside into human skin.     

Transformation of ferulic acid to 4-vinyl guaiacol as a major metabolite: a microbial approach

The majority of the flavours and fragrances used worldwide are produced by chemical synthesis at low price. However, consumers prefer natural compounds because of increasing health and nutrition awareness in routine life. Hence, biotransformation is an alternative process to produce natural aroma compounds. Microorganisms have been gradually used more to produce natural aroma compounds with various applications in food, agriculture and pharmaceutical industries. This paper reviews the role of microorganisms in the transformation of ferulic acid to 4-vinyl guaiacol. The microbial processes based on biocatalytic method are discussed in terms of their advantages over chemical synthesis, plant cell cultures and enzyme catalyzed reactions. Thus, the transformation of ferulic acid by microorganisms could have possible use in food, pharmaceutical industry and become an increasingly important platform for the production of natural aroma compounds.     

Studies on the production of shikimic acid using the <Emphasis Type="Italic">aroK</Emphasis> knockout strain of <Emphasis Type="Italic">Bacillus megaterium</Emphasis>

Shikimic acid has various pharmaceutical and industrial applications. It is the sole chemical building block for the antiviral drug oseltamivir (Tamiflu^®) and one of the potent pharmaceutical intermediates with three chiral centres. Here we report a modified strain of Bacillus megaterium with aroK (shikimate kinase) knock out to block the aromatic biosynthetic pathway downstream of shikimic acid. Homologous recombination based gene disruption approach was used for generating aroK knock out mutant of B. megaterium. Shake flask cultivation showed shikimic acid yield of 2.98 g/L which is ~6 times more than the wild type (0.53 g/L). Furthermore, the shikimate kinase activity was assayed and it was 32 % of the wild type. Effect of various carbon sources on the production of shikimic acid was studied and fructose (4 %, w/v) was found to yield maximum shikimic acid (4.94 g/L). The kinetics of growth and shikimic acid production by aroK knockout mutant was studied in 10 L bioreactor and the yield of shikimic acid had increased to 6 g/L which is ~12 fold higher over the wild type. It is evident from the results that aroK gene disruption had an immense effect in enhancing the shikimic acid production.     

Aspergillus niger catalyzes the synthesis of flavonoids from chalcones

Abstract

Flavonoids, which have many biological activities and have been widely used in nature, can be artificially synthesized. However, regioselective cyclization of chalcones is difficult by chemical methods. In this study, we demonstrated that Aspergillus niger is capable of cyclizing chalcones to flavanones, affording a mimic of plant biosynthetic processes. Chalcones 1–6 were biotransformated to the modified chalcones 8–14 and to the flavanones 15–27. The biotransformation showed that enzymatic cyclization and demethylation occurred during the first days of biotransformation; in contrast, hydroxylation is a later process. With a longer culturing time, it is possible to obtain more hydroxylated flavanones with excellent yields.     

A new cytochrome P450 system from Bacillus megaterium DSM319 for the hydroxylation of 11-keto-β-boswellic acid (KBA)

In the genome of Bacillus megaterium DSM319, a strain who has recently been sequenced to fully exploit its potential for biotechnological purposes, we identified a gene encoding the cytochrome P450 CYP106A1 as well as genes encoding potential redox partners of CYP106A1. We cloned, expressed, and purified CYP106A1 and five potential autologous redox partners, one flavodoxin and four ferredoxins. The flavodoxin and three ferredoxins were able to support the activity of CYP106A1 displaying the first cloned natural redox partners of a cytochrome P450 from B. megaterium. The CYP106A1 system was able to convert the pentacyclic triterpene 11-keto-β-boswellic acid (KBA) belonging to the main bioactive constituents of Boswellia serrata gum resin extracts, which are used to treat inflammatory disorders and arthritic diseases. In order to provide sufficient amounts of the KBA products to characterize them structurally by NMR spectroscopy, recombinant whole-cell biocatalysts were constructed based on B. megaterium MS941. The main product has been identified as 7β-hydroxy-KBA, while the side product (～20 %) was shown to be a mixture of 7β,15α-dihydroxy-KBA and 15α-hydroxy-KBA. Without further optimization 560.7 mg l^?1 day^?1 of the main product, 7β-hydroxy-KBA, could be obtained thus providing a suitable starting point for the efficient production of modified KBA by chemical tailoring to produce novel KBA derivatives with increased bioavailability and this way more efficient drugs.     

Isolation of Bacillus megaterium Mutants That Produce High Levels of Heterologous Protein,and Their Use to Construct a Highly Mosquitocidal Strain

A xylose-regulated plasmid expression system for producing high levels of recombinant proteins in Bacillus megaterium has recently been described [Appl Microbiol Biotechnol 35:594, 1991]. Using an antibiotic resistance protein as the expressed protein, we have been able to select mutant plasmids that produce increased levels of heterologous protein. The mutant plasmids show increased segregational stability and have lost the ability to be transformed into Escherichia coli. The same selection protocol has been used to isolate a mutant strain producing high levels of the Bacillus sphaericus mosquitocidal binary toxin. This strain shows toxicity to Culex quinquefasciatus larvae that is comparable to B. sphaericus 2362 and higher than a B. megaterium strain with the original expression plasmid. This approach may be generally useful for high-level regulated protein expression in B. megaterium. Received: 6 December 1996 / Accepted: 28 January 1997     

Biotransformation of dl-lactate to pyruvate by a newly isolated Serratia marcescens ZJB-07166

The production of pyruvate using biotransformation from dl-lactate has been recently drawn more and more attentions due to the wide applications of pyruvate in chemicals, drugs, and agrochemicals industries. In the current study, a strain ZJB-07166, which was capable of converting dl-lactate to pyruvate, was newly isolated and characterized and later identified as Serratia marcescens based on the morphology, physiological tests, ATB system and its 16S rDNA sequence. The strain S. marcescens ZJB-07166 was applied in biotransformation of dl-lactate to pyruvate and the detailed time courses for cultivation and biotransformation were investigated. The optimum nitrogen source and carbon source in the microorganism culture for production of lactate dehydrogenase were NH₄Cl and dl-lactate, respectively. The optimum substrate concentration for biotransformation was around 40 mM and EDTA had an obvious stabilizing effect on pyruvate in biotransformation process. The pyruvate production concentration of 210 mM was achieved under the optimum conditions. These results demonstrated that the newly isolated S. marcescens ZJB-07166 was a promising strain for pyruvate production in industrial scale.     

Megacinogenic plasmid from Bacillus megaterium 216

Summary Ability to produce megacin A, a bacteriocin of b. megaterium, was transferred from the strain B. megaterium 216 into auxotrophic derivatives of the strain B. megaterium KM via protoplast fusion and polyethylene-glycol-induced protoplast transformation by plasmid DNA, respectively. A 30.9 megadalton plasmid was detected in cells with MegA phenotype, and the loss of this phenotype was accompanied in each case with the elimination of that plasmid. The megacinogenic plasmid pBM309 has a single site for both BamIII and XhoI. It is cleaved by the endonucleases SalI, BglII, PstI, PvuII, and EcoRI into 3, 3, 4, 4, and 9 fragments, respectively. The physical map of this plasmid is presented.     

High production of poly(3‐hydroxybutyrate) from a wild Bacillus megaterium Bolivian strain

Aim

Taking into account that a novel strain of Bacillus megaterium was isolated from Uyuni salt lake (Bolivia) in a previous work, the objectives of this new study were to determine the maximal Poly‐3‐hydroxybutyrate production potential of B. megaterium strain uyuni S29 in an industrial conventional media, the possibility that the strain accumulates different types of polyhydroxyalkanoates, the cellular morphology during the biosynthesis process and the characterization of the produced biopolymers.

Methods and Results

The micro‐organism was first tested in a 3‐L bioreactor obtaining a high specific growth rate of 1·64 h^?1. A second fed‐batch experiment was carried out in shaking flasks, reaching up to 70% PHB of cell dry mass. The biosynthesized polymers were extracted by two different extraction procedures and characterized. The results showed that all of them were PHB with thermal properties different to the conventional PHB. The micrographs taken by TEM show the different cell morphology during the fermentation process.

Conclusions

In this previous study, the strain not only grew properly in the industrial conditions proposed without spore formation, but also produced and accumulated a large content of PHB, never reached before for its genus. Therefore, if the culture conditions can be optimized, the biopolymer production could be increased.

Significance and Impact of the Study

The impact of the study has related to the area of the biomaterials and their production. The study provides new data related to the high production of PHB from the wild novel strain B. megaterium uyuni S29, the highest polymer accumulation for the genus Bacillus without spores formation.     

Macrokinetic model for Gluconobacter oxydans in 2-keto-L-gulonic acid mixed culture

A set of kinetic models have been developed for the production of 2-keto-L-gulonic acid from L-sorbose by a mixed culture of Gluconobacter oxydans and Bacillus megaterium. A metabolic pathway is proposed for Gluconobacter oxydans, and a macrokinetic model has been developed for Gluconobacter oxydans, where the balances of some key metabolites, ATP and NADH are taken into account. An unstructured model is proposed for concomitant bacterium Bacillus megaterium. In the macrokinetic model and unstructured model, the mechanism of interaction between Gluconobacter oxydans and Bacillus megaterium is investigated and modeled. The specific substrate uptake rate and the specific growth rate obtained from the macrokinetic model are then coupled into a bioreactor model such that the relationship between the substrate feeding rate and the main state variables, such as the medium volume, the biomass concentrations, the substrate, and the is set up. A closed loop regulator model is introduced to approximate the induction of enzyme pool during lag phase after inoculation. Experimental results demonstrate that the model is able to describe 2-keto-L-gulonic acid fermentation process with reasonable accuracy.     

Overexpression of a type II 3-dehydroquinate dehydratase enhances the biotransformation of quinate to 3-dehydroshikimate in Gluconobacter oxydans

Shikimate and 3-dehydroshikimate are useful chemical intermediates for the synthesis of various compounds, including the antiviral drug oseltamivir. Here, we show an almost stoichiometric biotransformation of quinate to 3-dehydroshikimate by an engineered Gluconobacter oxydans strain. Even under pH control, 3-dehydroshikimate was barely detected during the growth of the wild-type G. oxydans strain NBRC3244 on the medium containing quinate, suggesting that the activity of 3-dehydroquinate dehydratase (DHQase) is the rate-limiting step. To identify the gene encoding G. oxydans DHQase, we overexpressed the gox0437 gene from the G. oxydans strain ATCC621H, which is homologous to the aroQ gene for type II DHQase, in Escherichia coli and detected high DHQase activity in cell-free extracts. We identified the aroQ gene in a draft genome sequence of G. oxydans NBRC3244 and constructed G. oxydans NBRC3244 strains harboring plasmids containing aroQ and different types of promoters. All recombinant G. oxydans strains produced a significant amount of 3-dehydroshikimate from quinate, and differences between promoters affected 3-dehydroshikimate production levels with little statistical significance. By using the recombinant NBRC3244 strain harboring aroQ driven by the lac promoter, a sequential pH adjustment for each step of the biotransformation was determined to be crucial because 3-dehydroshikimate production was enhanced. Under optimal conditions with a shift in pH, the strain could efficiently produce a nearly equimolar amount of 3-dehydroshikimate from quinate. In the present study, one of the important steps to convert quinate to shikimate by fermenting G. oxydans cells was investigated.     

Overexpression of cytochrome p450 125 in <Emphasis Type="Italic">Mycobacterium</Emphasis>: a rational strategy in the promotion of phytosterol biotransformation

Androst-4-ene-3, 17-dione (AD) and androst-1, 4-diene-3, 17-dione (ADD) are generally produced by the biotransformation of phytosterols in Mycobacterium. The AD (D) production increases when the strain has high NAD⁺/NADH ratio. To enhance the AD (D) production in Mycobacterium neoaurum TCCC 11978 (MNR M3), a rational strategy was developed through overexpression of a gene involved in the phytosterol degradation pathway; NAD⁺ was generated as well. Proteomic analysis of MNR cultured with and without phytosterols showed that the steroid C27-monooxygenase (Cyp125-3), which performs sequential oxidations of the sterol side chain at the C27 position and has the oxidative cofactor of NAD⁺ generated, played an important role in the phytosterol biotransformation process of MNR M3. To improve the productivity of AD (D), the cyp125-3 gene was overexpressed in MNR M3. The specific activity of Cyp125-3 in the recombinant strain MNR M3C3 was improved by 22% than that in MNR M3. The NAD⁺/NADH ratio in MNR M3C3 was 131% higher than that in the parent strain. During phytosterol biotransformation, the conversion of sterols increased from 84 to 96%, and the yield of AD (D) by MNR M3C3 was increased by approximately 18% for 96 h fermentation. This rational strain modification strategy may also be applied to develop strains with important application values for efficient production of cofactor-dependent metabolites.     

Simple and efficient preparation of ginsenoside (S)-Rg2 from ginsenoside Re by biotransformation with Cellulosimicrobium sp. 21

A novel ginsenoside-hydrolyzing strain was isolated from ginseng-cultivation soil in Changbai Mountain (China). The strain was identified as Cellulosimicrobium sp. 21 by 16S rDNA sequencing. Using the β-glucosidases secreted from Cellulosimicrobium sp. 21, protopanaxatriol-type ginsenoside Re was converted to the highly active neuroprotective molecule (S)-Rg2 by removal of the C-20-glucopyranosyl residue. The α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranose at the C-6 position of Rg2 was not further attacked by Cellulosimicrobium sp. 21, so the transformation shows high specificity. To simplify the transformation and product-preparation process, a simple and efficient transformation system was developed in a phosphate buffer system instead of organic media. The optimum conditions for transforming ginsenoside Re into Rg2 by Cellulosimicrobium sp. 21 were determined through single-factor experiments and response surface methodology. Under the optimized conditions: transformation buffer, 50 mM phosphate buffer, at pH: 7.00; temperature: 27.6°C; substrate concentration: 0.50 mg/ml; biotransformation period: 12 h; the biotransformation efficiency reached 89.8% (molar ratio) in 2-L reaction system. This simple biotransformation with high specificity and efficiency has potential for use in Rg2 preparation in the pharmaceutical industry.     

Production of a pharmaceutical intermediate via biohydroxylation using whole cells of Rhodococcus rubropertinctus N82

Rhodococcus rubropertinctus N82 possesses unique regiospecific hydroxylation activity in biotransformation of compounds. In this study, the ability of whole cells of the strain R. rubropertinctus N82 in biotransformation was studied. The hydroxylation activity resulted in transforming 6,7-dihydro-4H-thieno[3,2-c]-pyridine-5-carboxylic acid tert-butyl ester (LS1) into 2-hydroxy-6,7-dihydro-4H-thieno[3,2-c]-pyridine-5-carboxylic acid tert-butyl ester (LP1), a pharmaceutical intermediate. By optimizing conditions for the hydroxylating biotransformation using whole cells of R. rubropertinctus N82 as biocatalyst, 3.3?mM LP1 was successfully produced from 4?mM LS1 with a molar yield of 83%. Thus, effective method was newly developed to produce LP1, which is a synthetic intermediate of a platelet inhibitor active pharmaceutical ingredient drug, prasugrel.     

Bacillus megaterium mediated mineralization of calcium carbonate as biogenic surface treatment of green building materials

Microbially induced calcium carbonate precipitation is a biomineralization process that has various applications in remediation and restoration of range of building materials. In the present study, calcifying bacteria, Bacillus megaterium SS3 isolated from calcareous soil was applied as biosealant to enhance the durability of low energy, green building materials (soil–cement blocks). This bacterial isolate produced high amounts of urease, carbonic anhydrase, extra polymeric substances and biofilm. The calcium carbonate polymorphs produced by B. megaterium SS3 were analyzed by scanning electron microscopy, confocal laser scanning microscopy, X-ray diffraction and Fourier transmission infra red spectroscopy. These results suggested that calcite is the most predominant carbonate formed by this bacteria followed by vaterite. Application of B. megaterium SS3 as biogenic surface treatment led to 40 % decrease in water absorption, 31 % decrease in porosity and 18 % increase in compressive strength of low energy building materials. From the present investigation, it is clear that surface treatment of building materials by B. megaterium SS3 is very effective and eco friendly way of biodeposition of coherent carbonates that enhances the durability of building materials.