Fermentative production and direct extraction of (-)-α-bisabolol in metabolically engineered Escherichia coli.

BACKGROUND: (-)-α-Bisabolol, also known as levomenol, is an unsaturated sesquiterpene alcohol that has mainly been used in pharmaceutical and cosmetic products due to its anti-inflammatory and skin-soothing properties. (-)-α-Bisabolol is currently manufactured mainly by steam-distillation of the essential oils extracted from the Brazilian candeia tree that is under threat because its natural habitat is constantly shrinking. Therefore, microbial production of (-)-α-bisabolol plays a key role in the development of its sustainable production from renewable feedstock. RESULTS: Here, we created an Escherichia coli strain producing (-)-α-bisabolol at high titer and developed an in situ extraction method of (-)-α-bisabolol, using natural vegetable oils. We expressed a recently identified (-)-α-bisabolol synthase isolated from German chamomile (Matricaria recutita) (titer: 3 mg/L), converted the acetyl-CoA to mevalonate, using the biosynthetic mevalonate pathway (12.8 mg/L), and overexpressed farnesyl diphosphate synthase to efficiently supply the (-)-α-bisabolol precursor farnesyl diphosphate. Combinatorial expression of the exogenous mevalonate pathway and farnesyl diphosphate synthase enabled a dramatic increase in (-)-α-bisabolol production in the shake flask culture (80 mg/L) and 5 L bioreactor culture (342 mg/L) of engineered E. coli harboring (-)-α-bisabolol synthase. Fed-batch fermentation using a 50 L fermenter was conducted after optimizing culture conditions, resulting in efficient (-)-α-bisabolol production with a titer of 9.1 g/L. Moreover, a green, downstream extraction process using vegetable oils was developed for in situ extraction of (-)-α-bisabolol during fermentation and showed high yield recovery (>98%). CONCLUSIONS: The engineered E. coli strains and economically viable extraction process developed in this study will serve as promising platforms for further development of microbial production of (-)-α-bisabolol at large scale.

Display of native proteins on Bacillus subtilis spores.

In principle, protein display is enabled by fusing target proteins to naturally secreted, surface-anchored protein motifs. In this work, we developed a method of native protein display on the Bacillus spore surface that obviates the need to construct fusion proteins to display a motif. Spore coat proteins are expressed in the mother cell compartment and are subsequently assembled and deposited on the surface of spores. Therefore, target proteins overexpressed in the mother cell compartment during the late sporulation phase were expected to be targeted and displayed on the spore surface. As a proof of principle, we demonstrated the display of carboxymethylcellulase (CMCase) in its native form on the spore surface. The target protein, CMCase, was expressed under the control of the cry1Aa promoter, which is controlled by σ(E) and σ(K) and is expressed in the mother cell compartment. The correct display was confirmed using enzyme activity assays, flow cytometry, and immunogold electron microscopy. In addition, we demonstrated the display of a ß-galactosidase tetramer and confirmed its correct display using enzyme activity assays and protein characterization. This native protein display system, combined with the robust nature of Bacillus spores, will broaden the range of displayable target proteins. Consequently, the applications of display technology will be expanded, including high-throughput screening, vaccines, biosensors, biocatalysis, bioremediation, and other innovative bioprocesses.

Controlled localization of functionally active proteins to inclusion bodies using leucine zippers.

Inclusion bodies (IBs) are typically non-functional particles of aggregated proteins. However, some proteins in fusion with amyloid-like peptides, viral coat proteins, and cellulose binding domains (CBDs) generate IB particles retaining the original functions in cells. Here, we attempted to generate CBD IBs displaying functional leucine zipper proteins (LZs) as bait for localizing cytosolic proteins in E. coli. When a red fluorescent protein was tested as a target protein, microscopic observations showed that the IBs red-fluoresced strongly. When different LZ pairs with KDs of 8-1,000 µM were tested as the bait and prey, the localization of the red fluorescence appeared to change following the affinities between the LZs, as observed by fluorescence imaging and flow cytometry. This result proposed that LZ-tagged CBD IBs can be applied as an in vivo matrix to entrap cytosolic proteins in E. coli while maintaining their original activities. In addition, easy detection of localization to IBs provides a unique platform for the engineering and analyses of protein-protein interactions in E. coli.

Characterization of diverse natural variants of CYP102A1 found within a species of Bacillus megaterium.

An extreme diversity of substrates and catalytic reactions of cytochrome P450 (P450) enzymes is considered to be the consequence of evolutionary adaptation driven by different metabolic or environmental demands. Here we report the presence of numerous natural variants of P450 BM3 (CYP102A1) within a species of Bacillus megaterium. Extensive amino acid substitutions (up to 5% of the total 1049 amino acid residues) were identified from the variants. Phylogenetic analyses suggest that this P450 gene evolve more rapidly than the rRNA gene locus. It was found that key catalytic residues in the substrate channel and active site are retained. Although there were no apparent variations in hydroxylation activity towards myristic acid (C14) and palmitic acid (C16), the hydroxylation rates of lauric acid (C12) by the variants varied in the range of >25-fold. Interestingly, catalytic activities of the variants are promiscuous towards non-natural substrates including human P450 substrates. It can be suggested that CYP102A1 variants can acquire new catalytic activities through site-specific mutations distal to the active site.

Generation of human chiral metabolites of simvastatin and lovastatin by bacterial CYP102A1 mutants.

Recently, the wild-type and mutant forms of cytochrome P450 BM3 (CYP102A1) from Bacillus megaterium were found to oxidize various xenobiotic substrates, including pharmaceuticals, of human P450 enzymes. Simvastatin and lovastatin, which are used to treat hyperlipidemia and hypercholesterolemia, are oxidized by human CYP3A4/5 to produce several metabolites, including 6'ß-hydroxy (OH), 3″-OH, and exomethylene products. In this report, we show that the oxidation of simvastatin and lovastatin was catalyzed by wild-type CYP102A1 and a set of its mutants, which were generated by site-directed and random mutagenesis. One major hydroxylated product (6'ß-OH) and one minor product (6'-exomethylene), but not other products, were produced by CYP102A1 mutants. Formation of the metabolites was confirmed by high-performance liquid chromatography, liquid chromatography-mass spectroscopy, and NMR. Chemical methods to synthesize the metabolites of simvastatin and lovastatin have not been reported. These results demonstrate that CYP102A1 mutants can be used to produce human metabolites, especially chiral metabolites, of simvastatin and lovastatin. Our computational findings suggest that a conformational change in the cavity of the mutant active sites is related to the activity change. The modeling results also suggest that the activity change results from the movement of several specific residues in the active sites of the mutants. Furthermore, our computational findings suggest a correlation between the stabilization of the binding site and the catalytic efficiency of CYP102A1 mutants toward simvastatin and lovastatin.

Surface display of heme- and diflavin-containing cytochrome P450 BM3 in Escherichia coli: a whole cell biocatalyst for oxidation.

Cytochrome P450 enzymes (P450s) are involved in the synthesis of a wide variety of valuable products and in the degradation of numerous toxic compounds. The P450 BM3 (CYP102A1) from Bacillus megaterium was the first P450 discovered to be fused to its redox partner, a mammalian-like diflavin reductase. Here, we report the development of a whole cell biocatalyst using ice-nucleation protein (Inp) from Pseudomonas syringae to display a heme- and diflavin-containing oxidoreductase, P450 BM3 (a single, 119-kDa polypeptide with domains of both an oxygenase and a reductase) on the surface of Escherichia coli. Surface localization and functionality of the fusion protein containing P450 BM3 were verified by flow cytometry and measurement of enzymatic activities. The results of this study comprise the first report of microbial cell-surface display of heme- and diflavin-containing enzyme. This system should allow us to select and develop oxidoreductases containing heme and/or flavins, into practically useful whole-cell biocatalysts for extensive biotechnological applications including selective synthesis of new chemicals and pharmaceuticals, bioconversion, bioremediation, live vaccine development, and bio-chip development.

Engineering bacterial cytochrome P450 (P450) BM3 into a prototype with human P450 enzyme activity using indigo formation.

Human cytochrome P450 (P450) enzymes metabolize a variety of endogenous and xenobiotic compounds, including steroids, drugs, and environmental chemicals. In this study, we examine the possibility that bacterial P450 BM3 (CYP102A1) mutants with indole oxidation activity have the catalytic activities of human P450 enzymes. Error-prone polymerase chain reaction was carried out on the heme domain-coding region of the wild-type gene to generate a CYP102A1 DNA library. The library was transformed into Escherichia coli for expression of the P450 mutants. A colorimetric colony-based method was adopted for primary screening of the mutants. When the P450 activities were measured at the whole-cell level, some of the blue colonies, but not the white colonies, possessed apparent oxidation activity toward coumarin and 7-ethoxycoumarin, which are typical human P450 substrates that produce fluorescent products. Coumarin is oxidized by the CYP102A1 mutants to produce two metabolites, 7-hydroxycoumarin and 3-hydroxycoumarin. In addition, 7-ethoxycoumarin is simultaneously oxidized to 7-hydroxycoumarin by O-deethylation reaction and to 3-hydroxy,7-ethoxycoumarin by 3-hydroxylation reactions. Highly active mutants are also able to metabolize several other human P450 substrates, including phenacetin, ethoxyresorufin, and chlorzoxazone. These results indicate that indigo formation provides a simple assay for identifying CYP102A1 mutants with a greater potential for human P450 activity. Furthermore, our computational findings suggest a correlation between the stabilization of the binding site and the catalytic efficiency of CYP102A1 mutants toward coumarin: the more stable the structure in the binding site, the lower the energy barrier and the higher the catalytic efficiency.

Spore display using Bacillus thuringiensis exosporium protein InhA.

A new spore display method is presented that enables recombinant proteins to be displayed on the surface of Bacillus spores via fusion with InhA, an exosporium component of Bacillus thuringiensis. The green fluorescent protein and beta-galactosidase as model proteins were fused to the C-terminal region of InhA, respectively. The surface expression of the proteins on the spores was confirmed by flow cytometry, confocal laser scanning microscopy, measurement of the enzyme activity, and an immunogold electron microscopy analysis. InhA-mediated anchoring of foreign proteins in the exosporium of Bacillus spores can provide a new method of microbial display, thereby broadening the potential for novel applications of microbial display.

Generation of the human metabolite piceatannol from the anticancer-preventive agent resveratrol by bacterial cytochrome P450 BM3.

In recent studies, the wild-type and mutant forms of cytochrome P450 (P450) BM3 (CYP102A1) from Bacillus megaterium were found to metabolize various drugs through reactions similar to those catalyzed by human P450 enzymes. Therefore, it was suggested that CYP102A1 can be used to produce large quantities of the metabolites of human P450-catalyzed reactions. trans-Resveratrol (3,4',5-trihydroxystilbene), an anticancer-preventive agent, is oxidized by human P450 1A2 to produce two major metabolites, piceatannol (3,5,3',4'-tetrahydroxystilbene) and another hydroxylated product. In this report, we show that the oxidation of trans-resveratrol, a human P450 1A2 substrate, is catalyzed by wild-type and a set of CYP102A1 mutants. One major hydroxylated product, piceatannol, was produced as a result of the hydroxylation reaction. Other hydroxylated products were not produced. Piceatannol formation was confirmed by high-performance liquid chromatography and gas chromatograph-mass spectrometry by comparing the metabolite with the authentic piceatannol compound. These results demonstrate that CYP102A1 mutants can be used to produce piceatannol, a human metabolite of resveratrol.

Functional expression in Bacillus subtilis of mammalian NADPH-cytochrome P450 oxidoreductase and its spore-display.

The technology for over-expressing NADPH-cytochrome P450 reductase (CPR), a diflavin-containing enzyme, offers the opportunity to develop enzymatic systems for environmental detoxication and bioconversions of drugs, pesticides and fine chemicals. In this study, Bacillus subtilis was chosen to express rat CPR (rCPR) because of its capacities for high protein production and spore formation. rCPR was expressed in B. subtilis DB104 under the transcriptional control of an IPTG-inducible fusion promoter of P(groE) and P(tac). The expressed rCPR was released into the culture medium after sporulation by autolysis of the host cell. It was associated with and displayed on the spore surfaces; this was confirmed by measuring rCPR activity in purified spores and analyzing its accessibility to anti-rCPR antibodies using flow cytometry. The spore-displayed rCPR was able to reduce cytochrome c and ferricyanide, and also assisted in the O-deethylation of 7-ethoxyresorufin and 7-ethoxy-4-trifluoromethylcoumarin (EFC) by human cytochrome P450 1A2, indicating that it was functionally active. Spore surface display of rCPR in B. subtilis appears to be useful for preparing cytochrome P450-related enzymes, and spore biocatalysts of rCPR are likely to have wide biotechnological applications.

Generation of human metabolites of 7-ethoxycoumarin by bacterial cytochrome P450 BM3.

Recently, wild-type and mutant forms of bacterial cytochrome P450 BM3 (CYP102A1) have been found to metabolize various drugs through reactions similar to those catalyzed by human cytochromes P450 (P450s). Therefore, it has been suggested that CYP102A1 may be used to produce large quantities of the metabolites of human P450-catalyzed reactions. In this report, we show that the oxidation of 7-ethoxycoumarin, a typical human P450 substrate, is catalyzed by both wild-type and mutant forms of CYP102A1. Two major products were produced as a result of O-deethylation and 3-hydroxylation reactions. These results demonstrate that CYP102A1 mutants catalyze the same reactions as human P450s. High noncompetitive intermolecular kinetic deuterium isotope effects were observed for 7-ethoxycoumarin O-deethylation in the CYP102A1 system. These results suggest that there is a common mechanism for the oxidation reactions catalyzed by both the bacterial CYP102A1 and human P450 enzymes.

Cohnella laeviribosi sp. nov., isolated from a volcanic pond.

A novel thermophilic and endospore-forming Gram-positive bacterium capable of assimilating and isomerizing l-ribose was isolated from a volcanic area in Likupang, Indonesia. The isolate, RI-39(T), was able to grow at high temperatures (37-60 degrees C); optimum growth was observed at pH 6.5 and 45 degrees C. The strain contained MK-7 (87 %) as the main respiratory quinone and had a DNA G+C content of 51 mol%. The major cellular fatty acids of the isolate were iso-C(16 : 0) and anteiso-C(15 : 0) and the predominant polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and lysyl-phosphatidylglycerol. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the isolate represents an evolutionary lineage that is distinct from those of other Cohnella species. Based on morphological, physiological and chemotaxonomic characteristics and 16S rRNA gene sequence comparisons, it is proposed that strain RI-39(T) represents a novel species, Cohnella laeviribosi sp. nov. The type strain is RI-39(T) (=KCTC 3987(T) =KCCM 10653P(T) =CCUG 52217(T)).

Bacterial surface display of GFP(uv) on bacillus subtilis spores.

To analyze a cotG-based Bacillus subtilis spore display system directly, GFP(uv) was expressed on the surface of Bacillus subtilis spores. When GFP(uv) was fused to the C-terminal of the cotG structural gene and expressed, the existence of a CotG-GFP(uv) fusion protein on the B. subtilis spore was confirmed by flow cytometry confocal microscopic analysis. When the cotG anchoring motif was deleted, no fluorescence emission was observed under flow cytometry and confocal microscopic analysis from the purified spore, confirming the essential role of CotG as an anchoring motif. This GFP(uv) displaying spore might be used for another signaling application triggered by intracellular or extracellular stimuli.

Identification and expression of GH-8 family chitosanases from several Bacillus thuringiensis subspecies.

The Gram-positive spore-forming bacterium, Bacillus thuringiensis, a member of the Bacillus cereus group, produces chitosanases that catalyze the hydrolysis of chitosan to chitosan-oligosaccharides (COS). Although fungal and bacterial chitosanases belonging to other glycoside hydrolase (GH) families have been characterized in a variety of microorganisms, knowledge on the genetics and phylogeny of the GH-8 chitosanases remains limited. Nine genes encoding chitosanases were cloned from 29 different serovar strains of B. thuringiensis and they were expressed in Escherichia coli. The ORFs of the chitosanases contained 1,359 nucleotides and the protein products had high levels of sequence identity (>96%) to other Bacillus species GH-8 chitosanases. Thin-layer chromatography and HPLC analyses demonstrated that these enzymes hydrolyzed chitosan to a chitosan-trimer and a chitosan-tetramer as major products, and this could be useful in the production of COS. In addition, a simple plate assay was developed, involving a soluble chitosan, for high-throughput screening of chitosanases. This system allowed screening for mutant enzymes with higher enzyme activity generated by error-prone PCR, indicating that it can be used for directed chitosanase evolution.

The bacterial P450 BM3: a prototype for a biocatalyst with human P450 activities.

The use of cytochrome P450 (P450 or CYP) enzymes as biocatalysts for the production of fine chemicals, including pharmaceuticals, has been of increasing interest, primarily owing to their catalytic diversity and broad substrate range. CYP102A1 (P450 BM3) from Bacillus megaterium integrates an entire monooxygenase system into one polypeptide and represents an appropriate prokaryotic model for industrial applications of mammalian P450 activities. CYP102A1 not only exhibits the highest catalytic activity ever detected in a P450 monooxygenase but also provides a potentially versatile biocatalyst for the production of human P450 metabolites. CYP102A1 can be further engineered to be a drug-metabolizing enzyme, making it a promising candidate to use as a biocatalyst in drug discovery and synthesis.

Characterization of a novel D-lyxose isomerase from Cohnella laevoribosii RI-39 sp. nov.

A newly isolated bacterium, Cohnella laevoribosii RI-39, could grow in a defined medium with L-ribose as the sole carbon source. A 21-kDa protein isomerizing L-ribose to L-ribulose, as well as D-lyxose to D-xylulose, was purified to homogeneity from this bacterium. Based on the N-terminal and internal amino acid sequences of the purified enzyme obtained by N-terminal sequencing and quantitative time of flight mass spectrometry-mass spectrometry analyses, a 549-bp gene (lyxA) encoding D-lyxose (L-ribose) isomerase was cloned and expressed in Escherichia coli. The purified endogenous enzyme and the recombinant enzyme formed homodimers that were activated by Mn(2+). C. laevoribosii D-lyxose (L-ribose) isomerase (CLLI) exhibits maximal activity at pH 6.5 and 70 degrees C in the presence of Mn(2+) for D-lyxose and L-ribose, and its isoelectric point (pI) is 4.2 (calculated pI, 4.9). The enzyme is specific for D-lyxose, L-ribose, and D-mannose, with apparent K(m) values of 22.4 +/- 1.5 mM, 121.7 +/- 10.8 mM, and 34.0 +/- 1.1 mM, respectively. The catalytic efficiencies (k(cat)/K(m)) of CLLI were 84.9 +/- 5.8 mM(-1) s(-1) for D-lyxose (V(max), 5,434.8 U mg(-1)), 0.2 mM(-1) s(-1) for L-ribose (V(max), 75.5 +/- 6.0 U mg(-1)), and 1.4 +/- 0.1 mM(-1) s(-1) for D-mannose (V(max), 131.8 +/- 7.4 U mg(-1)). The ability of lyxA to permit E. coli cells to grow on D-lyxose and L-ribose and homology searches of other sugar-related enzymes, as well as previously described sugar isomerases, suggest that CLLI is a novel type of rare sugar isomerase.

Transgalactosylation in a water-solvent biphasic reaction system with beta-galactosidase displayed on the surfaces of Bacillus subtilis spores.

The ever-increasing industrial demand for biocatalysis necessitates innovations in the preparation and stabilization of biocatalysts. In this study, we demonstrated that beta-galactosidase (beta-Gal) displayed on Bacillus spores by fusion to the spore coat proteins (CotG) may be used as a whole-cell immobilized biocatalyst for transgalactosylation in water-solvent biphasic reaction systems. The resulting spores had a specific hydrolytic activity of 5 x 10(3) U/g (dry weight) of spores. The beta-Gal was tightly attached to the spore surface and was more stable in the presence of various organic solvents than its native form was. The thermostability of the spore-displayed enzyme was also increased, and the enzyme was further stabilized by chemically cross-linking it with glutaraldehyde. With spore-displayed beta-Gal, octyl-beta-D-galactopyranoside was synthesized at concentrations up to 27.7 mM (8.1 g/liter) with a conversion yield of 27.7% (wt/wt) after 24 h from 100 mM lactose and 100 mM octanol dissolved in phosphate buffer and ethyl ether, respectively. Interestingly, the spores were found to partition mainly at the interface between the water and solvent phases, and they were more available to catalysis between the two phases, as determined by light microscopy and confocal fluorescence microscopy. We propose that spore display not only offers a new and facile way to construct robust biocatalysts but also provides a novel basis for phase transfer biocatalytic processes.

Molecular cloning and characterization of NAD(+)-dependent xylitol dehydrogenase from Candida tropicalis ATCC 20913.

Induction of xylitol dehydrogenase of Candida tropicalis ATCC 20913 by various carbon sources was investigated. The enzyme activity was induced when the yeast was grown on l-arabinose and d-xylose. A novel gene encoding the enzyme was cloned and characterized. The 1,095-bp coding sequence of the gene encodes a polypeptide of 364 amino acids, with a molecular mass of 39.4 kDa. Sequence analysis of the putative protein showed it to be a member of the zinc-containing alcohol dehydrogenase family and to have homology to xylitol dehydrogenase genes from other yeasts and fungi. The recombinant xylitol dehydrogenase expressed in Escherichia coli oxidized polyols such as xylitol and d-sorbitol and reduced ketoses such as d-xylulose and d-fructose. It required exclusively NAD or NADH as a cofactor.

Functional expression of mammalian NADPH-cytochrome P450 oxidoreductase on the cell surface of Escherichia coli.

To develop a whole-cell oxidoreductase system without the practical limitation of substrate/product transport, easy preparation, stability of enzymes, and low expression levels, we here report the development of a whole cell biocatalyst displaying rat NADPH-cytochrome P450 oxidoreductase (CPR, 77-kDa) on the surface of Escherichia coli by using ice-nucleation protein from Pseudomonas syringae. Surface localization and functionality of the CPR were verified by flow cytometry, electron microscopy, and measurements of enzyme activities. The results of this study comprise the first report of microbial cell-surface display of diflavin-containing mammalian enzymes. This system will allow us to select and develop oxidoreductases, containing bulky and complex prosthetic groups of FAD and FMN, into practically useful whole-cell biocatalysts for broad biological and biotechnological applications including the selective synthesis of new chemicals and pharmaceuticals, bioconversion, bioremediation, and bio-chip development.