Characterization of the versatile monooxygenase CYP109B1 from Bacillus subtilis

The oxidizing activity of CYP109B1 from Bacillus subtilis was reconstituted in vitro with various artificial redox proteins including putidaredoxin reductase and putidaredoxin from Pseudomonas putida, truncated bovine adrenodoxin reductase and adrenodoxin, flavodoxin reductase and flavodoxin from Escherichia coli, and two flavodoxins from B. subtilis (YkuN and YkuP). Binding and oxidation of a broad range of chemically different substrates (fatty acids, n-alkanes, primary n-alcohols, terpenoids like (+)-valencene, α- and β-ionone, and the steroid testosterone) were investigated. CYP109B1was found to oxidize saturated fatty acids (conversion up to 99%) and their methyl and ethyl esters (conversion up to 80%) at subterminal positions with a preference for the carbon atoms C11 and C12 counted from the carboxyl group. For the hydroxylation of primary n-alcohols, the ω_?2 position was preferred. n-Alkanes were not accepted as substrates by CYP109B1. Regioselective hydroxylation of terpenoids α-ionone (～70% conversion) and β-ionone (～ 91% conversion) yielded the allylic alcohols 3-hydroxy-α-ionone and 4-hydroxy-β-ionone, respectively. Furthermore, indole was demonstrated to inhibit fatty acid oxidation.     

CYP109E1 is a novel versatile statin and terpene oxidase from Bacillus megaterium

CYP109E1 is a cytochrome P450 monooxygenase from Bacillus megaterium with a hydroxylation activity for testosterone and vitamin D3. This study reports the screening of a focused library of statins, terpene-derived and steroidal compounds to explore the substrate spectrum of this enzyme. Catalytic activity of CYP109E1 towards the statin drug-precursor compactin and the prodrugs lovastatin and simvastatin as well as biotechnologically relevant terpene compounds including ionones, nootkatone, isolongifolen-9-one, damascones, and β-damascenone was found in vitro. The novel substrates induced a type I spin-shift upon binding to P450 and thus permitted to determine dissociation constants. For the identification of conversion products by NMR spectroscopy, a B. megaterium whole-cell system was applied. NMR analysis revealed for the first time the ability of CYP109E1 to catalyze an industrially highly important reaction, the production of pravastatin from compactin, as well as regioselective oxidations generating drug metabolites (6′β-hydroxy-lovastatin, 3′α-hydroxy-simvastatin, and 4″-hydroxy-simvastatin) and valuable terpene derivatives (3-hydroxy-α-ionone, 4-hydroxy-β-ionone, 11,12-epoxy-nootkatone, 4(R)-hydroxy-isolongifolen-9-one, 3-hydroxy-α-damascone, 4-hydroxy-β-damascone, and 3,4-epoxy-β-damascone). Besides that, a novel compound, 2-hydroxy-β-damascenone, produced by CYP109E1 was identified. Docking calculations using the crystal structure of CYP109E1 rationalized the experimentally observed regioselective hydroxylation and identified important amino acid residues for statin and terpene binding.

     

Transport regulation of recombinant gene expression in E. coli and B. subtilis

Expression kinetics of the lactose (lac) operon in Escherichia coli are reviewed for both wild-type and recombinant cell cultures under chemostatic conditions. A unified model which involves regulation of active inducer (lactose) transport, promoter-operator regulated expression of the lac operon, glucose-mediated inducer exclusion, and catabolite repression is summarized and supporting data is shown to verify its accuracy. The synthesis of alpha-amylase with a recombinant form of Bacillus subtilis is also reviewed to point out generic features in transport regulation, the lac operon model providing a point of departure. While there are many similarities in the influence of transport on both regulating models, there are also important differences. In a chemostat system, the synthesis of alpha-amylase is nongrowth associated, while beta-galactosidase is a growth-associated enzyme. Nevertheless, transport regulation is an important feature in both instances.     

Fermentation production of keratinase from Bacillus licheniformisPWD-1 and a recombinant B. subtilis FDB-29

Bacillus licheniformis PWD-1, the parent strain, and B. subtilis FDB-29, a recombinant strain. In both strains, keratinase was induced by proteinaceous media, and repressed by carbohydrates. A seed culture of B. licheniformis PWD-1 at early age, 6–10 h, is crucial to keratinase production during fermentation, but B. subtilis FDB-29 is insensitive to the seed culture age. During the batch fermentation by both strains, the pH changed from 7.0 to 8.5 while the keratinase activity and productivity stayed at high levels. Control of pH, therefore, is not necessary. The temperature for maximum keratinase production is 37°C for both strains, though B. licheniformis is thermophilic and grows best at 50°C. Optimal levels of dissolved oxygen are 10% and 20% for B. licheniformis and B. subtilis respectively. A scale-up procedure using constant temperature at 37°C was adopted for B. subtilis. On the other hand, a temperature-shift procedure by which an 8-h fermentation at 50°C for growth followed by a shift to 37°C for enzyme production was used for B. licheniformis to shorten the fermentation time and increase enzyme productivity. Production of keratinase by B. licheniformis increased by ten-fold following this new procedure. After respective optimization of fermentation conditions, keratinase production by B. licheniformis PWD-1 is approximately 40% higher than that by B. subtilis FDB-29. Received 16 July 1998/ Accepted in revised form 07 March 1999     

Regioselective hydroxylation of norisoprenoids by CYP109D1 from Sorangium cellulosum So ce56

Sesquiterpenes are particularly interesting as flavorings and fragrances or as pharmaceuticals. Regio- or stereoselective functionalizations of terpenes are one of the main goals of synthetic organic chemistry, which are possible through radical reactions but are not selective enough to introduce the desired chiral alcohol function into those compounds. Cytochrome P450 monooxygenases are versatile biocatalysts and are capable of performing selective oxidations of organic molecules. We were able to demonstrate that CYP109D1 from Sorangium cellulosum So ce56 functions as a biocatalyst for the highly regioselective hydroxylation of norisoprenoids, α- and β-ionone, which are important aroma compounds of floral scents. The substrates α- and β-ionone were regioselectively hydroxylated to 3-hydroxy-α-ionone and 4-hydroxy-β-ionone, respectively, which was confirmed by ¹H NMR and ¹³C NMR. The results of docking α- and β-ionone into a homology model of CYP109D1 gave a rational explanation for the regio-selectivity of the hydroxylation. Kinetic studies revealed that α- and β-ionone can be hydroxylated with nearly identical V _max and K _m values. This is the first comprehensive investigation of the regioselective hydroxylation of norisoprenoids by CYP109D1.     

Enhanced production of carboxymethylcellulase of a marine microorganism, Bacillus subtilis subsp. subtilis A-53 in a pilot-scaled bioreactor by a recombinant Escherichia coli JM109/A-53 from rice bran

A gene encoding the carboxymethylcellulase (CMCase) of a marine bacterium, Bacillus subtilis subsp. subtilis A-53, was cloned in Escherichia coli JMB109 and the recombinant strain was named as E. coli JMB109/A-53. The optimal conditions of rice bran, ammonium chloride, and initial pH of the medium for cell growth, extracted by Design Expert Software based on response surface methodology, were 100.0 g/l, 7.5 g/l, and 7.0, respectively, whereas those for production of CMCase were 100.0 g/l, 7.5 g/l, and 8.0. The optimal temperatures for cell growth and the production of CMCase by E. coli JM109/A-53 were found to be and 40 and 35 °C, respectively. The optimal agitation speed and aeration rate of a 7 l bioreactor for cell growth were 400 rpm and 1.5 vvm, whereas those for production of CMCase were 400 rpm and 0.5 vvm. The optimal inner pressure for cell growth was 0.06 MPa, which was the same as that for production of CMCase. The production of CMCase by E. coli JM109/A-53 under optimized conditions was 880.2 U/ml, which was 2.9 times higher than that before optimization. In this study, rice bran and ammonium chloride were developed as carbon and nitrogen source for production of CMCase by a recombinant E. coli JM109/A-53 and the productivity of E. coli JM109/A-53 was 5.9 times higher than that of B. subtilis subp. subtilis A-53.     

Construction of a colony bank of E. coli containing hybrid plasmids representative of the Bacillus subtilis 168 genome. Expression of functions harbored by the recombinant plasmids in B. subtilis.

Summary A collection of about 2500 clones containing hybrid plasmids representative of nearly the entire genome of B. subtilis 168 was established in E. coli SK1592 by using the poly(dA)·poly(dT) joining method with randomly sheared DNA fragments and plasmid pHV33, a bifunctional vector which can replicate in both E. coli and B. subtilis. Detection of cloned recombinant DNA molecules was based on the insertional inactivation of the Tc gene occurring at the unique BamHI cleavage site present in the vector plasmid.Thirty individual clones of the collection were shown to hybridize specifically with a B. subtilis rRNA probe. CCC-recombinant plasmids extracted from E. coli were pooled in lots of 100 and used to transform auxotrophic mutants of B. subtilis 168. Complementation of these auxotrophic mutations was observed for several markers such as thr, leuA, hisA, glyB and purB. In several cases, markers carried by the recombinant plasmids were lost from the plasmid and integrated into the chromosomal DNA. Loss of genetic markers from the hybrid plasmids did not occur when a rec ^- recipient strain of B. subtilis was used.Abbreviations Ap^R resistance to ampicillin - Tc^R resistance to tetracycline - Cm^R resistance to chloramphenicol - CCC covalently closed circular duplex - Mdal magadalton     

枯草杆菌表达系统的研究进展

枯草杆菌由于具有非致病性、分泌蛋白能力强的特性的良好的发酵基础,是目前原核表达系统中分泌表达外源蛋白较理想的宿主。本阐述枯草杆菌基因表达的一般特点、表达载体、表达类型以及分泌表达存在的问题。     

Biodegradation of dioxins by recombinant Escherichia coli expressing rat CYP1A1 or its mutant

Among polychlorinated dibenzo-p-dioxins (PCDDs), 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TetraCDD) is the most toxic one. Recently, we reported that rat CYP1A1 mutant, F240A, expressed in yeast showed metabolic activity toward 2,3,7,8-TetraCDD. In this study, we successfully expressed N-terminal truncated P450s (Δ1A1 and ΔF240A) in Escherichia coli cells. Kinetic analysis using membrane fractions prepared from the recombinant E. coli cells revealed that ΔF240A has enzymatic properties similar to F240A expressed in yeast. The metabolism of PCDDs by recombinant E. coli cells expressing both ΔF240A and human NADPH-P450 reductase was also examined. When 2,3,7-TriCDD was added to the E. coli cell culture at a final concentration of 10 μM, approximately 90% of the 2,3,7-TriCDD was converted into multiple metabolites within 8 h. These results indicate the possible application of prokaryotic cells expressing ΔF240A to the bioremediation of PCDD-contaminated soil.     

Characterization of the Bacillus subtilis WL-3 mannanase from a recombinant Escherichia coli

A mannanase was purified from a cell-free extract of the recombinant Escherichia coli carrying a Bacillus subtilis WL-3 mannanase gene. The molecular mass of the purified mannanase was 38 kDa as estimated by SDS-PAGE. Optimal conditions for the purified enzyme occurred at pH 6.0 and 60 degrees C. The specific activity of the purified mannanase was 5,900 U/mg on locust bean gum (LBG) galactomannan at pH 6.0 and 50 degrees C. The activity of the enzyme was slightly inhibited by Mg(2+), Ca(2+), EDTA and SDS, and noticeably enhanced by Fe(2+). When the enzyme was incubated at 4 degrees C for one day in the presence of 3 mM Fe(2+), no residual activity of the mannanase was observed. The enzyme showed higher activity on LBG and konjac glucomannan than on guar gum galactomannan. Furthermore, it could hydrolyze xylans such as arabinoxylan, birchwood xylan and oat spelt xylan, while it did not exhibit any activities towards carboxymethylcellulose and para-nitrophenyl-beta-mannopyranoside. The predominant products resulting from the mannanase hydrolysis were mannose, mannobiose and mannotriose for LBG or mannooligosaccharides including mannotriose, mannotetraose, mannopentaose and mannohexaose. The enzyme could hydrolyze mannooligosaccharides larger than mannobiose.     

Continuous testing system for Baeyer-Villiger biooxidation using recombinant Escherichia coli expressing cyclohexanone monooxygenase encapsulated in polyelectrolyte complex capsules

An original strategy for universal laboratory testing of Baeyer-Villiger monooxygenases based on continuous packed-bed minireactor connected with flow calorimeter and integrated with bubble-free oxygenation is reported. Model enantioselective Baeyer-Villiger biooxidations of rac-bicyclo[3.2.0]hept-2-en-6-one to corresponding lactones (1R,5S)-3-oxabicyclo-[3.3.0]oct-6-en-3-one and (1S,5R)-2-oxabicyclo-[3.3.0]oct-6-en-3-one as important chiral synthons for the synthesis of bioactive compounds were performed in the minireactor equipped with a column packed with encapsulated recombinant cells Escherichia coli overexpressing cyclohexanone monooxygenase. The cells were encapsulated in polyelectrolyte complex capsules formed by reaction of oppositely charged polymers utilizing highly reproducible and controlled encapsulation process. Encapsulated cells tested in minireactor exhibited high operational stability with 4 complete substrate conversions to products and 6 conversions above 80% within 14 repeated consecutive biooxidation tests. Moreover, encapsulated cells showed high enzyme stability during 91 days of storage with substrate conversions above 80% up to 60 days of storage. Furthermore, usable thermometric signal of Baeyer-Villiger biooxidation obtained by flow calorimetry using encapsulated cells was utilized for preparatory kinetic study in order to guarantee sub-inhibitory initial substrate concentration for biooxidation tests.     

枯芽孢杆菌α—淀粉酶基在在大肠杆菌中的表达及其产物的分泌

The E. coli which carrying the alpha-amylase gene fragment cloned from B. subtilis secreted the gene products into the medium. The reason is the exogenous gene fragment act on the cell wall of E. coli by some way, gives rise to the change of its structure. It leads up to the alpha-amylase and some periplasm proteins passing through the cell wall into the medium. It also causes the change of host colonial morphology. The secrete process are non-specific.     

Overexpression of a recombinant wild-type and His-tagged Bacillus subtilis glycine oxidase in Escherichia coli.

We have cloned the gene coding for the Bacillus subtilis glycine oxidase (GO), a new flavoprotein that oxidizes glycine and sarcosine to the corresponding alpha-keto acid, ammonia and hydrogen peroxide. By inserting the DNA encoding for GO into the multiple cloning site of the expression vector pT7.7 we produced a recombinant plasmid (pT7-GO). The pT7-GO encodes a fully active fusion protein with six additional residues at the N-terminus of GO (MARIRA). In BL21(DE3)pLysS Escherichia coli cells, and under optimal isopropyl thio-beta-D-galactoside induction conditions, soluble and active chimeric GO was expressed up to 1.14 U g(-1) of cell (and a fermentation yield of 3.82 U x L(-1) of fermentation broth). An N-terminal His-tagged protein (HisGO) was also successfully expressed in E. coli as a soluble protein and a fully active holoenzyme. HisGO represents approximately 3.9% of the total soluble protein content of the cell. The His-tagged GO was purified in a single step by nickel-chelate chromatography to a specific activity of 1.06 U x mg(-1) protein at 25 degrees C and with a yield of 98%. The characterization of the purified enzyme showed that GO is a homotetramer of approximately 180 kDa with the spectral properties typical of flavoproteins. GO exhibits good thermal stability, with a Tm of 46 degrees C after 30 min incubation; its stability is maximal in the 7.0-8.5 pH range. A comparison of amino-acid sequence and substrate specificity indicates that GO has similarities to other flavoenzymes acting on primary amines and on D-amino acids.     

Heterologous expression of nonribosomal peptide synthetases in B. subtilis: construction of a bi-functional B subtilis/E coli shuttle vector system

A major obstacle in investigating the biosynthesis of pharmacologically important peptide antibiotics is the heterologous expression of the giant biosynthetic genes. Recently, the genetically engineered strain Bacillus subtilis KE30 has been reported as an excellent surrogate host for the heterologous expression of an entire nonribosomal peptide synthetase (NRPS) gene cluster. In this study, we expand the applicability of this strain, by the development of four Escherichia coli/B. subtilis shuttle expression vectors. Comparative overproduction of hybrid NRPS proteins derived from both organisms revealed a significant beneficial effect of overproducing proteins in B. subtilis KE30 as underlined by the production of stable nondegradative proteins, as well as the formation of active phosphopantetheinylated holo-proteins.     

Partial suppression of rec B rec C mutations in E. coli by plasmid pBR 322 containing a Bacillus subtilis chromosome insertion

ATP-dependent DNAase genes of Bac. subtilis were originally cloned in E. coli plasmid pBR322. These genes are expressed in rec BC mutant E. coli cells leading to a complete recovery of the enzyme activity. Bac. subtilis enzyme suppresses reparative properties of the rec BC mutant to a considerable extent but is unable to replace functionally the E. coli mutant enzyme in recombination process.     

Transformation of Bacillus subtilis and Escherichia coli by a hybrid plasmid pCD1.

The gene thyP3 from Bacillus subtilis bacteriophage phi 3T was cloned in the plasmid pMB9. The resulting chimeric plasmid, pCD1, is effective in transforming both Escherichia coli and Bacillus subtilis to thymine prototrophy. The activity of the thyP3 gene product, thymidylate synthetase, was assayed and found to be 9 times greater in a transformed strain of Escherichia coli than in a phi 3T lysogen of Bacillus subtilis. The physical location of restriction sites has been determined for two related plasmids pCD1 and pCD2. Hybridization studies clearly indicate that the plasmid gene responsible for Thy+ transformation is the gene from the bacteriophage phi 3T. The lack of restriction in this transformation process is consistent with our previous studies using bacterial DNA in heterospecific exchanges indicating that the nucleotide sequence surrounding the gene is the dominant factor in determining interspecific transformation.     

Molecular cloning of a tetracycline-resistance determinant from Bacillus subtilis chromosomal DNA and its expression in Escherichia coli and B. subtilis

Bacillus subtilis GSY908 DNA fragments (5.1 and 4.4 kilobase pairs (kb)) containing a tetracycline-resistance determinant were cloned in Escherichia coli using a shuttle plasmid vector pLS353. Restriction endonucelase analysis showed that the 4.4 kb fragment is a spontaneous deletion derivative of the 5.1 kb fragment. E. coli tetracycline-resistance transformants carrying pLS353 with the 5.1 kb fragment (named pTBS1) and that with 4.4 kb fragment (pTBS1.1) could grow at tetracycline concentrations up to 80 and 50 micrograms per ml, respectively. B. subtilis MI112 and RM125 were transformed by pTBS1, resulting in isolation of transformants of MI112 maintaining pTBS1 and RM125 maintaining either pTBS1 or pTBS1.1. Maximum tetracycline concentrations permitting growth of plasmidless MI112 and MI112 with pTBS1 were 4 and 10 micrograms per ml, respectively, while those of plasmidless RM125, RM125 with pTBS1 and RM125 with pTBS1.1 were 7, 50 and 80 micrograms per ml, respectively. It was interesting to note that the tetracycline-resistance level in E. coli conferred by the 5.1 kb fragment is higher than that conferred by the 4.4 kb fragment, but in B. subtilis the 4.4 kb fragment, in contrast, confers a higher level of tetracycline resistance. The level of tetracycline resistance in B. subtilis conferred by the cloned determinant clearly depends on the host strain. The tetracycline resistance conferred by the cloned determinant was associated with decreased accumulation of the drug into the cells. However, it was constitutive in E. coli, but inducible in B. subtilis. The cloned tetracycline-resistance determinant was detected specifically on the chromosome of B. subtilis Marburg 168 derivatives.