Identification and functional analysis of cytochrome P450 complement in Streptomyces virginiae IBL14.

BACKGROUND: As well known, both natural and synthetic steroidal compounds are powerful endocrine disrupting compounds (EDCs) which can cause reproductive toxicity and affect cellular development in mammals and thus are generally regarded as serious contributors to water pollution. Streptomyces virginiae IBL14 is an effective degradative strain for many steroidal compounds and can also catalyze the C25 hydroxylation of diosgenin, the first-ever biotransformation found on the F-ring of diosgenin. RESULTS: To completely elucidate the hydroxylation function of cytochrome P450 genes (CYPs) found during biotransformation of steroids by S. virginiae IBL14, the whole genome sequencing of this strain was carried out via 454 Sequencing Systems. The analytical results of BLASTP showed that the strain IBL14 contains 33 CYPs, 7 ferredoxins and 3 ferredoxin reductases in its 8.0 Mb linear chromosome. CYPs from S. virginiae IBL14 are phylogenetically closed to those of Streptomyces sp. Mg1 and Streptomyces sp. C. One new subfamily was found as per the fact that the CYP Svu001 in S. virginiae IBL14 shares 66% identity only to that (ZP_05001937, protein identifer) from Streptomyces sp. Mg1. Further analysis showed that among all of the 33 CYPs in S. virginiae IBL14, three CYPs are clustered with ferredoxins, one with ferredoxin and ferredoxin reductase and three CYPs with ATP/GTP binding proteins, four CYPs arranged with transcriptional regulatory genes and one CYP located on the upstream of an ATP-binding protein and transcriptional regulators as well as four CYPs associated with other functional genes involved in secondary metabolism and degradation. CONCLUSIONS: These characteristics found in CYPs from S. virginiae IBL14 show that the EXXR motif in the K-helix is not absolutely conserved in CYP157 family and I-helix not absolutely essential for the CYP structure, too. Experimental results showed that both CYP Svh01 and CYP Svu022 are two hydroxylases, capable of bioconverting diosgenone into isonuatigenone and ß-estradiol into estriol, respectively.

Effects of induction starting time and Ca2+ on expression of active penicillin G acylase in Escherichia coli.

Formation of inclusion bodies is an important obstacle to the production of active recombinant protein in Escherichia coli. Thus, soluble expression of penicillin G acylase from Kluyvera citrophila was investigated in BL21(DE3). In this study, the yield of active enzyme was significantly enhanced by the composition of the medium and induction opportunity. When 0.5 mmol/L IPTG was added to complex medium at 15 h after incubation, the volumetric and specific activities of penicillin G acylase both achieved the highest values, respectively. However, aggravation of intracellular proteolysis and decline of enzyme expression were also observed if induction occurred too much later. Ca2+ ion was another critical factor in cell growth and protein expression. When 24 mmol/L Ca2+ ion was adding to the medium at the beginning of fermentation, a greater than 2-fold increase in cell density and a 7-fold increase in volumetric activity of penicillin G acylase were reached. Nevertheless, no significant benefit for recombination protein expression was found when excess Ca2+ was added after induction time. This study demonstrates that the induction starting time and Ca2+ ion are two critical factors for the expression of active penicillin G acylase.

Some characteristics and purification of anti-(human ovarian carcinoma)xanti-(human CD3) single-chain bispecific antibody.

To obtain high-quality AhOC x AhCD3 [anti-(human ovarian carcinoma)xanti-(human CD3)] with high biological activity economically and easily, some characteristics and purification of AhOC x AhCD3, a single chain bispecific antibody, were investigated. During the present study, some important properties of AhOC x AhCD3, such as molecular mass, pI, solubility, stability, hydrophobic ability, molecular status and bioactivity were primarily studied. The molecular mass of AhOC x AhCD3 was determined to be approx. 58.0 kDa by SDS/PAGE (theoretical molecular mass: 56972.5 Da, calculated on the basis of the primary structure) and the pI was approx. 7.5+/-0.4 by IEF (isoelectric focusing; theoretical pI: 8.5, predicted by the ProtParam software tool) respectively. Experiments showed that the solubility of AhOC x AhCD3 increased with the pH increase and that the AhOC x AhCD3 was easily degraded into several fragments during the storage time of samples and the mixtures of both fragment B and fragment C retained normal bioactivity. Particularly, the soluble aggregate/polymer status of AhOC x AhCD3 with bioactivity was verified by non-reducing SDS/PAGE. After optimization of purification process, AhOC x AhCD3 with electrophoretic homogeneity was successfully obtained and the yield was approx. 20%. Some important properties of AhOC x AhCD3 were first observed and a procedure for soluble AhOC x AhCD3 purification in scale-up was first established. Some characteristics of AhOC x AhCD3, especially the stability of AhOC x AhCD3, are critical for the development of this protein pharmaceutical and useful for reference to other proteins.

Evaluation of chitosan hydrochloride-alginate as enteric micro-probiotic-carrier with dual protective barriers.

In this study, the cells-free and cells-loaded chitosan hydrochloride-alginate (CHC-Alg) microcapsules were firstly fabricated with polyelectrolyte complexes via an orifice-polymerization method. Scanning electron microscope images showed that the CHC-Alg microcapsules had a typical shell-core structure and the model probiotic cells (Bacillus licheniformis) were embedded in the core in cells-loaded microcapsules. The microcapsules prepared had good thermal stability and moisture property (3.89%). Cells survival and release studies showed that the number of probiotic cells released from the cells-loaded microcapsules (approx. 6.36logCFUml-1) was 6.19logCFUml-1 when they were performed in the simulated gastric fluid (SGF, pH 2.0) for 1h and subsequently in the simulated intestinal fluid (SIF, 0.3%) for 4h. The CHC-Alg microcapsules with favorable swelling performances were helpful to permeate the harsh acid to protect the cells in the SGF (pH 2.0). The CHC-Alg microcapsules effectively protected the model probiotic cells, which was attributed to the "dual protective barriers" of the shell-core structure, that is, the primary barrier of the Alg hydrogel layer formed with a compact polymer matrix and the secondary barrier of the PEC film formed on the surface. The microcapsules prepared could be used as an enteric micro-probiotic-carrier for designing potential probiotic delivery systems.

Effect of the cross-linking agent on performances of NaCS-CS/WSC microcapsules.

Based on the properties of oppositely charged natural polysaccharides, the polyelectrolyte complexes (PECs) prepared with chitosan-related polycationic polyelectrolytes and cellulose-related polyanionic polyelectrolytes have been widely concerned for their potential applications as micro-drug-carriers for colon. However, the poor mechanical property of the PECs becomes the obstacle encountered in practical applications. This study investigated the effect of the cross-linking agent (sodium polyphosphate, PPS) on the performances of sodium cellulose sulfate -chitosan/water soluble chitosan (NaCS-CS/WSC) microcapsules. The results revealed that PPS could penetrate through the PEC film and form tighter interior structures compared with the microcapsules without the addition of cross-linking agent. The NaCS-CS microcapsules and NaCS-WSC microcapsules with or without PPS had distinct microstructures, which could be ascribed to the different physicochemical properties of CS and WSC. During the formation process, CS can be dissolved in water under acidic conditions, while WSC can be directly dissolved and protonated in acid-free aqueous providing NH3(+) groups quickly, which resulted in the microstructure's difference. Further analysis showed the NaCS-CS-PPS microcapsules and NaCS-WSC-PPS microcapsules had lower swelling ratios due to their tighter interior microstructures that formed. The cross-linking agent had important effect on the total mass of PECs that produced; moreover, the decline of zeta potential of NaCS-CS-PPS microcapsules was lower than that of NaCS-CS microcapsules, similar trend was found in the NaCS-WSC-PPS microcapsules compared with NaCS-WSC microcapsules, indicating the PPS participated in the interactions and played a role in the microcapsules' formation process.

Extracellular production of human parathyroid hormone as a thioredoxin fusion form in Escherichia coli by chemical permeabilization combined with heat treatment.

A pET system encoding the fusion protein gene of thioredoxin (Trx) and human parathyroid hormone (hPTH) was introduced into Escherichia coli BL21 (DE3). Recombinant Trx-hPTH fusion protein was expressed in soluble form in the cytoplasm of the E. coli transformant. To recover Trx-hPTH from the E. coli culture efficiently, a novel tactic was developed by adding Triton X-100 into the fermentation culture at the exponential growth phase of E. coli and by heat treatment of the culture at the end of the fermentation. A concentration of 1% (v/v) Triton X-100 was added into the culture at the same time as IPTG addition after optimization. Under these conditions, addition of Triton X-100 had little effect on the cell growth, but more than 75% of the total recombinant Trx-hPTH was released into the fermentation broth. Also, a much higher volumetric yield of recombinant Trx-hPTH could be obtained with protein release compared to yield without protein release. Simultaneously, owing to the highly thermal stability of Trx-hPTH fusion protein, heat treatment of the fermentation broth at 80 degrees C for 15 min at the end of fermentation was employed for primary purification. Results demonstrated that heat treatment not only boosted further release of the recombinant Trx-hPTH fusion protein into the fermentation broth but also precipitated/denatured most of the nontarget proteins released in the broth. The tactics described herein integrated the fermentation process with subsequent recovery steps and thus provided a valuable and economical method for the production of Trx-hPTH and maybe some other Trx fusions in E. coli.

Studies on the function and catalytic mechanism of O-methyltransferases SviOMT02, SviOMT03 and SviOMT06 from Streptomyces virginiae IBL14.

To identify the fuctions of the nine putative O-methyltransferase genes in Streptomyces virginiae IBL14, the evolutionary and functional relationship of these genes in its 8.0 Mb linear chromosome was set up via sequence comparison with those of other Streptomyces species. Further, the functions and catalytic mechanism of the three genes sviOMT02, sviOMT03 and sviOMT06 from this strain were studied through experimental and computational approaches. As a result, the nine putative O-methyltransferases belong to methyltransf_2 superfamily, amdomet-MTases superfamily, and leucine carboxyl methyltransferase superfamily, and are phylogenetically close to those of Streptomyces sp. C. The products of genes sviOMT03 and sviOMT06 could catalyze O-methylation of caffeic acid to form ferulic acid. Computational analysis indicated that the O-methylation mechanism of SviOMT03 and SviOMT06 proceeds from a direct transfer of the SAM-methyl group to caffeic acid with inversion of symmetry aided by a divalent metal ion in a SN2-like mechanism. Particularly, the conservative polar amino acid residues in SviOMT03 and SviOMT06, including Lys143 that reacts with caffeic acid, Ser74, Asp140 and Tyr149 that react with S-adenosyl methionine, and His142 (SviOMT03) or His171 (SviOMT06) that transfers the 3-hydroxyl proton of substrate caffeic acid, probably be essential in their O-methylation.

Construction of leaky strains and extracellular production of exogenous proteins in recombinant Escherichia coli.

In this study, a strategy of the construction of leaky strains for the extracellular production of target proteins was exploited, in which the genes mrcA, mrcB, pal and lpp (as a control) from Escherichia coli were knocked out by using single- and/or double-gene deletion methods. Then the recombinant strains for the expression of exogenous target proteins including Trx-hPTH (human parathyroid hormone 1-84 coupled with thioredoxin as a fusion partner) and reteplase were reconstructed to test the secretory efficiency of the leaky strains. Finally, the fermentation experiments of the target proteins from these recombinant leaky strains were carried out in basic media (Modified R media) and complex media (Terrific Broth media) in flasks or fermenters. The results demonstrated that the resultant leaky strains were genetically stable and had a similar growth profile in the complex media as compared with the original strain, and the secretory levels of target proteins into Modified R media from the strains with double-gene deletion (up to 88.9%/mrcA lpp-pth) are higher than the excretory levels from the strains with single-gene deletion (up to 71.1%/lpp-pth) and the host E. coli JM109 (DE3) (near zero). The highest level of extracellular production of Trx-hPTH in fermenters is up to 680 mg l(-1).

Microbial biotransformation: recent developments on steroid drugs.

About 300 of steroid drugs have been identified since the research and development to steroid drugs was triggered in 1950's. Although much progress in microbial biotransformation on steroid drugs is well-achieved, many efforts are ongoing in order to improve the efficiency of steroidal drug production as well as to degrade their derivatives. In this work an overview of recent and important findings related to patents and various microbial biotransformations of steroidal compounds including dehydrogenation/reduction, hydroxylation, esterification, methylation, halogenation and methoxylation is presented. Both metabolic pathway and genetic manipulation and screening for new strains capable of metabolizing/transforming steroid drugs, as the powerful endocrine disruptors, are introduced since they are serious environmental contaminants.

New microbiological transformations of steroids by Streptomyces virginiae IBL-14.

A bacterium Streptomyces virginiae IBL-14 capable of effective degradation of diosgenin was isolated from activated sludge for treatment of waste from a steroidal drug factory. From the culture broth of diosgenin degradation, 11 compounds were purified and then identified, eight of which were previously unidentified compounds including 1-dehydroisonuatigenone [VI], nuatigenone [VIII], 1-dehydronuatigenone [X], 26-acetyl-nuatigenone [XII], 6-methoxy-6-dehydrodiosgenone [XIII], 6-methoxy-6-dehydroisonuatigenone [XIV], 6-methoxy-6-dehydronuatigenone [XV], and 6-dimethoxy-7alpha-hydroxyldiosgenone [XVI]. Additionally, two important microbial transformations of diosgenin (6-methoxylation and C25-tertiary carbon hydroxylation) were found. Two valuable chemical reactions of the steroids (structural rearrangement and esterification)were also confirmed. As a result, a new metabolic pathway of diosgenin metabolism was postulated.

Identification of Escherichia coli host cell for high plasmid stability and improved production of antihuman ovarian carcinoma x antihuman CD3 single-chain bispecific antibody.

To improve the plasmid stability during the production of antihuman ovarian carcinoma x antihuman CD3 single-chain bispecific antibody (AhOCxAhCD3), the Escherichia coli BL21(DE3) host cell was optimized serially. Firstly, an isogenic recombination-deficient (recA (-)) derivative of BL21(DE3), namely BLR(DE3), was used as host instead of BL21(DE3). Although the segregational plasmid stability was greatly improved, AhOCxAhCD3 yield was not improved due to the severe growth inhibition of plasmid-bearing BLR(DE3) cells and the competitive plasmid instability after induction. Secondly, a mutant BLR(DE3), namely BLRM(DE3), was screened by using LB agar plates plus ampicillin and isopropyl-beta-D: -thiogalactopyranoside. Using this new host, growth inhibition of recombinant cells after induction was eliminated, and plasmids could be stably maintained even after long-time induction in a nonselective medium. At last, about 1.2 g/l AhOCxAhCD3, which was about thrice as much as those of recombinant BL21(DE3) and BLR(DE3) strains, was yielded.

Partial purification of human parathyroid hormone 1-84 as a thioredoxin fusion form in recombinant Escherichia coli by thermoosmotic shock.

A modified purification method, thermoosmotic shock (osmotic shock coupled with heat-treatment) for heat-stable proteins, was devised in the purification of Trx-hPTH (1-84) (human parathyroid hormone coupled with thioredoxin as a fusion partner) from E. coli. Thermoosmotic shock can integrate the functions of extraction and crude separation of fusion protein Trx-hPTH (1-84). To improve the purification efficiency, thermoosmotic shock conditions were optimized and achieved as follows: the optimized high osmotic solution containing 20mM Tris-HCl buffer (pH 8.0), 1mM EDTA, and 25% sucrose; the low osmotic solution containing 20mM Tris-HCl buffer (pH 8.0), 1mM EDTA, and the heat-treatment temperature of 100 degrees C for 10 min. Using this method, the purity of Trx-hPTH (1-84) was up to 73% and the yield was up to 72%, respectively. In addition, the two separation methods of both thermoosmotic shock and affinity chromatography have been compared, indicating that thermoosmotic shock is an economical and feasible way for the fusion protein separation. Besides, the thermoosmotic shock method may be used for the purification of some proteins of thermal stability without N-terminal His-tag.