Effect of bacterial starvation on surfactant biotransformation

Effect of carbon starvation on the rate of dihexylsulfosuccinate (DHSS) biotransformation byComamonas terrigena was determined. The protein content during the starvation was stable in all variants and did not change during the transformation cycle. All starved cultures exhibited a higher biotransformation rate than a non-starved control. Cells ofC. terrigena exposed for 16 h in media with no C source showed the highest specific biotransformation rate (144% of the non-starved culture). Extension of the starvation to 2 d led to a decrease of the rate to close to that found in non-starved cells.     

Engineering neoglycoproteins with multiple O-glycans using repetitive pentapeptide glycosylation units

Controlled protein remodeling with O-linked glycans has been limited by our incomplete understanding of the process of glycosylation. Here we describe a secretable fibroblast growth factor (FGF) with multiple mucin-type O-glycans produced by introducing a minimum pentapeptide glycosylation unit in a decarepeat format at its N- or C-terminus. Expressed in Chinese hamster ovary cells, chemical and biochemical analyses of the resultant proteins (Nm10-FGF and Cm10-FGF, respectively) demonstrated that all O-glycosylation units were glycosylated and the dominant structure was sialylated Gal[1–3]GalNAc. This indicates that minimum O-glycosylation unit in multirepeat format serves as a remarkably efficient acceptor in CHO cells. The Nm10-FGF and Cm10-FGF proteins maintained the mitogenic activity to vascular endothelial cells. In addition, intact Cm10-FGF and its desialylated form interacted with several lectins in the same way as mucin-type glycoproteins. The intact Cm10-FGF with multiple sialylated O-glycans exhibited a longer lifetime in circulating blood, whereas the Cm10-FGF with desialylated O-glycans exhibited a shorter lifetime than the deglycosylated form of Cm10-FGF. Our approach would thus appear to be highly effective for engineering neoglycoproteins, the characteristics of which are determined by their multiple mucin-type O-glycans.     

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.     

Biotransformation of 6,7-epoxygeraniol by fungi

The biotransformation of 6,7-epoxygeraniol by resting cells of selected fungi was investigated. The main product obtained from the transformation in Rhodotorula glutinis and R. marina cultures was 6,7-epoxynerol (5–48% of chloroform extracts), whereas Saccharomyces cerevisiae, Candida parapsilosis and C. kefyr reduced this substrate to 6,7-epoxycitronellol (30–33% of chloroform extracts). Cultures of Yarrowia lipolytica, Botrytis cinerea and S. cerevisiae promoted the cyclisation of 6,7-epoxygeraniol to 2-methyl-2-(2-hydroxyethyl)-5-(2-hydroxyprop-2-yl)tetrahydrofuran (11–99% of chloroform extracts). The biotransformation of 6,7-epoxynerol was also investigated. However, none of the tested micro-organisms converted this compound.     

Characterization of plasmid transformation in Bacillus subtilis: kinetic properties and the effect of DNA conformation.

Summary Transformation of competent cells of Bacillus subtilis with antibiotic resistance plasmid DNA has shown that (a) competence for plasmid and chromosomal DNA develops with similar kinetics; (b) DNA linearized with a variety of restriction endonucleases does not transform; (c) CCC plasmid DNA is inactivated for transformation by a single nick; (d) T4 ligase restores transforming activity to both nicked and linearized DNA; (e) CCC relaxed DNA is fully active in transformation; (f) the DNA concentration-dependence of plasmid transformation is first order; and (g) plasmid transformation proceeds with a low efficiency, requiring the uptake of 10³ to 10⁴ DNA molecules per transformant.Based on this information, a model for the processing of chromosomal, plasmid and transfecting DNA is proposed.Abbreviations Cm Chloramphenicol - Em erythromycin - Km kanamycin - Sm streptomycin - CCC covalently closed circular - TBAB tryptose blood agar base - NCE nicking and closing enzyme In partial fulfillment of the requirements for the doctoral degree in the Department of Microbiology at the New York University School of Medicine, for S.C.     

Transformation of Schizandrin into Gomisin A by Use of Microbial O-Demethylation and Chemical Reactions

The transformation of schizandrin (I) into gomisin A (II) was accomplished by use of a combination of biotransformation and chemical reactions. The biotransformation, microbial O-demethylation of I by Cuntiinghamella echinulata var. elegans (ATCC 9245) produced two novel metabolites [3-norschizandrin (IV) and 2-norschizandrin (VI)] and two known metabolites [gomisin T (III) and 13-norschizandrin (V)]. Among those metabolites, compound III was derived to II by the O-demethylation with a Lewis acid in the presence of an acid scavenger, followed by methylenation.     

Water-assisted dissemination of conidia of the mycoparasite Coniothyrium minitans in soil

A study was conducted to determine water-assisted dissemination of conidia of Coniothyrium minitans (Cm), a mycoparasite of Sclerotinia sclerotiorum (Ss), in four soils (yellow–brown soil, red-clay soil, fluvo-aquic soil and black soil) and one sand. Conidial suspensions (1×10⁷ conidia mL^?1) of Cm were applied to sieved (2 mm screen) soil or sand in glass tubes to test vertical dissemination (VD) and in aluminum boxes to test horizontal dissemination (HD) of conidia. Results showed that conidia of Cm could be disseminated with water and spread in soil or sand for 16–20 cm vertically and for 5–10 cm horizontally. The conidial concentration of Cm was logarithmically reduced with the increase in depth of VD or the distance of HD. Dissemination of Cm conidia in sand was better than that in four soils. Potting experiments were done to further understand the potential of water-assisted dissemination of Cm conidia in suppression of Ss carpogenic germination. Results showed that more apothecia were produced by Ss sclerotia located at the soil surface than those at 5 and 10 cm in depth. The minimum Cm concentration for suppression of Ss carpogenic germination was 1000 conidia g^?1 soil. Two-season field trials indicated that water-assisted application of Cm was an effective strategy used at the time for transplanting oilseed rape seedlings to suppress Ss carpogenic germination, thereby reducing the primary infection source for sclerotinia diseases of oilseed rape.     

Characterization of sphingosine-1-phosphate lyase activity by electrospray ionization–liquid chromatography/tandem mass spectrometry quantitation of (2E)-hexadecenal

Sphingosine-1-phosphate (S1P) is a sphingolipid signaling molecule crucial for cell survival and proliferation. S1P-mediated signaling is largely controlled through its biosynthesis and degradation, and S1P lyase (S1PL) is the only known enzyme that irreversibly degrades sphingoid base-1-phosphates to phosphoethanolamine and the corresponding fatty aldehydes. S1PL-mediated degradation of S1P results in the formation of (2E)-hexadecenal, whereas hexadecanal is the product of dihydrosphingosine-1-phosphate (DHS1P) degradation. Fatty aldehydes can undergo biotransformation to fatty acids and/or alcohols, making them elusive and rendering the task of fatty aldehyde quantitation challenging. We have developed a simple, highly sensitive, and high-throughput protocol for (2E)-hexadecenal quantitation as a semicarbazone derivative by liquid chromatography–electrospray ionization–tandem mass spectrometry. The approach was applied to determining S1PL activity in vitro with the ability to use as low as 0.25 μg of microsomal protein per assay. The method is also applicable to the use of total tissue homogenate as the source of S1PL. A correction for (2E)-hexadecenal disappearance due to its biotransformation during enzymatic reaction is required, especially at higher protein concentrations. The method was applied to confirm FTY720 as the inhibitor of S1PL with an IC₅₀ value of 52.4 μM.     

Enhanced hydroxylation of imidacloprid by <Emphasis Type="Italic">Stenotrophomonas maltophilia</Emphasis> upon addition of sucrose

Sucrose’s ability to promote the hydroxylation of imidacloprid (IMI) by bacterium Stenotrophomonas maltophilia strain CGMCC 1.1788 was examined. Both growing culture and resting cells could transform IMI into 5-hydroxy IMI. Adding 2% sucrose to the growing culture transformation broth and 5% sucrose to the resting cell transformation broth resulted in biotransformation yields, respectively, 2.5 and 9 times greater than without sucrose. In the growing culture transformation, sucrose increased biomass, which led to enhance hydroxylation of IMI. In the resting cell transformation, sucrose was used not as a carbon source but as an energy source for cofactor regeneration for hydroxylation of IMI. The hydroxylation activity of IMI was promoted eightfold by adding reduced nicotinamide adenine dinucleotide (NADH) to the cell-free extract. The hydroxylation of IMI was significantly inhibited by P450 inhibitor piperonyl butoxide. It seems that the hydroxylation of IMI by S. maltophilia CGMCC 1.1788 might proceed through a system by cooperating with P450 enzyme.     

Transformation of alpha-santonin via two independent pathways by Pseudomonas strain S ATCC 43388

Pseudomonas strain S (ATCC 43388) transforms alpha-santonin in the presence of dicyclohexylcarbodiimide (DCCD) to 1,2-dihydrosantonin (DHS) and two more products, D1 and D2. D2 was identified as 4,5-dihydroxysantonin (DHXS). Cells incubated with semicarbazide (SC) formed DHXS but not DHS. Simultaneous formation of DHS and DHXS is indicative of the presence of two distinct pathways of alpha-santonin biotransformation.     

Biotransformation and reduction of estrogenicity of bisphenol A by the biphenyl-degrading Cupriavidus basilensis

The biphenyl-degrading Gram-negative bacterium Cupriavidus basilensis (formerly Ralstonia sp.) SBUG 290 uses various aromatic compounds as carbon and energy sources and has a high capacity to transform bisphenol A (BPA), which is a hormonally active substance structurally related to biphenyl. Biphenyl-grown cells initially hydroxylated BPA and converted it to four additional products by using three different transformation pathways: (a) formation of multiple hydroxylated BPA, (b) ring fission, and (c) transamination followed by acetylation or dimerization. Products of the ring fission pathway were non-toxic and all five products exhibited a significantly reduced estrogenic activity compared to BPA. Cell cultivation with phenol and especially in nutrient broth (NB) resulted in a reduced biotransformation rate and lower product quantities, and NB-grown cells did not produce all five products in detectable amounts. Thus, the question arose whether enzymes of the biphenyl degradation pathway are involved in the transformation of BPA and was addressed by proteomic analyses.

     

Biotransformation of monoterpenes and sesquiterpenes by cell suspension cultures of Achillea millefolium L. ssp. millefolium

The transformation capacity of Achillea millefolium L. ssp. millefolium (yarrow) cell suspension cultures was investigated using geraniol (50mg/l) and borneol, menthol, thymol and farnesols (25mg/l) as substrates. Apart from converting these substrates into several biotransformation products, the cell suspension cultures were also able to glycosylate both the substrates and the biotransformation products. aa]Key Words bb]Achillea millefolium L. ssp. millefolium bb]Yarrow bb]Compositae bb]Biotransformation bb]Glycosylation bb]Geraniol bb]Borneol bb]Menthol bb]Thymol bb]Farnesols     

Stereoselective oxidation of racemic 1-arylethanols by basil cultured cells of <Emphasis Type="Italic">Ocimum </Emphasis><Emphasis Type="Italic">basilicum</Emphasis> cv. <Emphasis Type="Italic">Purpurascens</Emphasis>

The biotransformation of racemic 1-phenylethanol (30 mg) with plant cultured cells of basil (Ocimum basilicum cv. Purpurascens, 5 g wet wt) by shaking 120 rpm at 25°C for 7 days in the dark gave (R)-(+)-1-phenylethanol and acetophenone in 34 and 24% yields, respectively. The biotransformation can be applied to other 1-arylethanols and basil cells oxidized the (S)-alcohols to the corresponding ketones remaining the (R)-alcohols in excellent ee.     

Distinct quantitative trait loci for kidney, cardiac, and aortic mass dissociated from and associated with blood pressure in Dahl congenic rats

Blood pressure (BP) is largely determined by quantitative trait loci (QTLs) in Dahl salt-sensitive (DSS) rats. Little is known about QTLs controlling kidney (K), cardiac (C), and aortic (A) mass (i.e. Km, Cm, and Am, respectively) of DSS rats independent of BP. Their identification can facilitate our understanding of end organ damage. In this work, 36 congenic strains were employed to define QTLs for Km, Cm, and Am either independent of or associated with BP. Five new QTLs, i.e., KmQTLs, that influence Km independent of Cm, Am, and BP were defined. Four new CakmQTLs were defined for Cm, Am, and Km independent of BP. Among them, the CakmC10QTL1 interval contained 13 genes and undefined loci, and none was known to influence the phenotypes in question, paving the way for a novel gene discovery. Among 17 individual QTLs for BP, 14 also affected Cm, Km, and Am, i.e., they are BpcakmQTLs. In contrast, one BpQTL had no effect on Cm, Am, and Kam. Therefore, BP and Cm, Am, and Km have distinct and shared genetic determinants. The discovery of individual Km and Cakm QTLs will likely facilitate the identification of mechanisms underlying renal, cardiac, and/or aortic hypertrophy independent of hypertension. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. Chenda Duong and Sophie Charron contributed equally to this work.     

Assessment of ATR‐NIR and ATR‐MIR spectroscopy as an analytical tool for the quantification of the total polyphenols in Dendrobium huoshanense

In this study, the potentiality of applying attenuated total reflectance near‐infrared (ATR‐NIR) and attenuated total reflectance mid‐infrared (ATR‐MIR) techniques combined with a partial least squares (PLS) regression technology to quantify the total polyphenols (TPs) in Dendrobium huoshanense (DHS) was investigated and compared. The real TP contents in the DHS samples were analysed using methods of reference. The capability of the two IR spectroscopic techniques to quantify the TPs in DHS was assessed by the root‐mean‐square error of calibration (RMSEC) and determination coefficients (R²). The results showed that both NIR and MIR might be used as a fast and simple tool to replace traditional chemical assays for the determination of the TP contents in DHS, and the best NIR model showed slightly better prediction performance [root‐mean‐square error of prediction (RMSEP): 0.307, R²: 0.9122, ratio performance deviation (RPD): 4.43] than the best MIR model (RMSEP: 0.440, R²: 0.9069, RPD: 3.09). Results from this study indicated that both the NIR and MIR models could be used to quantify the TP in DHS, and ATR‐NIR appeared to be the more predominant and more robust technique for the quantification of the TP in DHS.     

Biotransformation of the high‐molecular weight polycyclic aromatic hydrocarbon (PAH) benzo[k]fluoranthene by Sphingobium sp. strain KK22 and identification of new products of non‐alternant PAH biodegradation by liquid chromatography electrospray ionization tandem mass spectrometry

A pathway for the biotransformation of the environmental pollutant and high‐molecular weight polycyclic aromatic hydrocarbon (PAH) benzo[k]fluoranthene by a soil bacterium was constructed through analyses of results from liquid chromatography negative electrospray ionization tandem mass spectrometry (LC/ESI(–)‐MS/MS). Exposure of Sphingobium sp. strain KK22 to benzo[k]fluoranthene resulted in transformation to four‐, three‐ and two‐aromatic ring products. The structurally similar four‐ and three‐ring non‐alternant PAHs fluoranthene and acenaphthylene were also biotransformed by strain KK22, and LC/ESI(–)‐MS/MS analyses of these products confirmed the lower biotransformation pathway proposed for benzo[k]fluoranthene. In all, seven products from benzo[k]fluoranthene and seven products from fluoranthene were revealed and included previously unreported products from both PAHs. Benzo[k]fluoranthene biotransformation proceeded through ortho‐cleavage of 8,9‐dihydroxy‐benzo[k]fluoranthene to 8‐carboxyfluoranthenyl‐9‐propenic acid and 9‐hydroxy‐fluoranthene‐8‐carboxylic acid, and was followed by meta‐cleavage to produce 3‐(2‐formylacenaphthylen‐1‐yl)‐2‐hydroxy‐prop‐2‐enoic acid. The fluoranthene pathway converged with the benzo[k]fluoranthene pathway through detection of the three‐ring product, 2‐formylacenaphthylene‐1‐carboxylic acid. Production of key downstream metabolites, 1,8‐naphthalic anhydride and 1‐naphthoic acid from benzo[k]fluoranthene, fluoranthene and acenaphthylene biotransformations provided evidence for a common pathway by strain KK22 for all three PAHs through acenaphthoquinone. Quantitative analysis of benzo[k]fluoranthene biotransformation by strain KK22 confirmed biodegradation. This is the first pathway proposed for the biotransformation of benzo[k]fluoranthene by a bacterium.     

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.     

Rapid and Unambiguous Detection of DNase I Hypersensitive Site in Rare Population of Cells

DNase I hypersensitive (DHS) sites are important for understanding cis regulation of gene expression. However, existing methods for detecting DHS sites in small numbers of cells can lead to ambiguous results. Here we describe a simple new method, in which DNA fragments with ends generated by DNase I digestion are isolated and used as templates for two PCR reactions. In the first PCR, primers are derived from sequences up- and down-stream of the DHS site. If the DHS site exists in the cells, the first PCR will not produce PCR products due to the cuts of the templates by DNase I between the primer sequences. In the second PCR, one primer is derived from sequence outside the DHS site and the other from the adaptor. This will produce a smear of PCR products of different sizes due to cuts by DNase I at different positions at the DHS site. With this design, we detected a DHS site at the CD4 gene in two CD4 T cell populations using as few as 2×10⁴ cells. We further validated this method by detecting a DHS site of the IL-4 gene that is specifically present in type 2 but not type 1 T helper cells. Overall, this method overcomes the interference by genomic DNA not cut by DNase I at the DHS site, thereby offering unambiguous detection of DHS sites in the cells.     

Cloning of two chloramphenicol acetyltransferase genes from Clostridium butyricum and their expression in Escherichia coli and Bacillus subtilis

Summary Two non-homologous chloramphenicol (Cm) acetyltransferase (CAT) genes, designated catA and catB, were cloned from Clostridium butyricum type strains and characterized by restriction mapping. Both genes are efficiently expressed in Escherichia coli and Bacillus subtilis. In contrast to analogous genes from staphylococci and bacilli, gene expression is not dependent on induction by Cm. The genes are considered as chromosomal, since no association with endogenous plasmids was detectable. Southern hybridization revealed a homology between catA and the staphylococcal Cm resistance plasmid, pC194. The subunit size of the clostridial CAT enzymes expressed in E. coli was determined as 22.5 kDa (catA) and 24 kDa (catB), respectively. The C. butyricum cat genes provide potentially useful selection markers for the construction of cloning vectors from cryptic clostridial plasmids.