Biocatalytic conversion of avermectin to 4"-oxo-avermectin: heterologous expression of the ema1 cytochrome P450 monooxygenase

The cytochrome P450 monooxygenase Ema1 from Streptomyces tubercidicus R-922 and its homologs from closely related Streptomyces strains are able to catalyze the regioselective oxidation of avermectin into 4"-oxo-avermectin, a key intermediate in the manufacture of the agriculturally important insecticide emamectin benzoate (V. Jungmann, I. Molnár, P. E. Hammer, D. S. Hill, R. Zirkle, T. G. Buckel, D. Buckel, J. M. Ligon, and J. P. Pachlatko, Appl. Environ. Microbiol. 71:6968-6976, 2005). The gene for Ema1 has been expressed in Streptomyces lividans, Streptomyces avermitilis, and solvent-tolerant Pseudomonas putida strains using different promoters and vectors to provide biocatalytically competent cells. Replacing the extremely rare TTA codon with the more frequent CTG codon to encode Leu4 in Ema1 increased the biocatalytic activities of S. lividans strains producing this enzyme. Ferredoxins and ferredoxin reductases were also cloned from Streptomyces coelicolor and biocatalytic Streptomyces strains and tested in ema1 coexpression systems to optimize the electron transport towards Ema1.     

Genome analyses of Streptomyces peucetius ATCC 27952 for the identification and comparison of cytochrome P450 complement with other Streptomyces

We have determined the genome sequence of 8.7 Mb chromosome of Streptomyces peucetius ATCC 27952, which produces clinically important anthracycline chemotherapeutic agents of the polyketide class of antibiotics, daunorubicin and doxorubicin. The cytochrome P450 (CYP) superfamily is represented by 19 sequences in the S. peucetius. Among those, 15 code for functional genes, whereas the remaining four are pseudo genes. CYPs from S. peucetius are phylogenetically close to those of Streptomyces amermitilis. Four CYPs are associated with modular PKS of avermectin and two with doxorubicin biosynthetic gene cluster. CYP252A1 is the new family found in S. peucetius, which shares 38% identity to CYP51 from Streptomyces coelicolor A3 (2). Nine CYPs from S. peucetius are found in the cluster containing various regulatory genes including rar operon, conserved in S. coelicolor A3 (2) and Streptomyces griseus. Although two ferredoxins and four ferredoxin reductases have been identified so far, only one ferredoxin reductase was found in the cluster of CYP147F1 in S. peucetius. To date, 174 CYPs have been described from 45 Streptomyces species in all searchable databases. However, only 18 CYPs are clustered with ferredoxin. The comparative study of cytochrome P450s, ferredoxins, and ferredoxin reductases should be useful for the future development and manipulation of antibiotic biosynthetic pathways.     

Optimization of transformation procedures in avermectin high-producing Streptomyces avermitilis

The optimal pH conditions for efficient transformation of protoplasts and intact cells were established in avermectin high-producing mutants, ATCC31780 and L-9. Among all factors tested, protoplast buffer pH was elucidated as the most important factor influencing transformation efficiency. The optimal pH of the protoplast buffer for the regeneration of ATCC31780 was 6.5, and using this condition, 4.5 × 10⁶ transformants per g of pIJ702 were produced. At pH 6.3, the maximal number of L-9 transformants was 1.6 × 10⁵ per g of the same plasmid. However, the protoplasting process decreased avermectin productivity to half or one-sixth of ATCC31780 or L-9, respectively. To avoid the productivity loss, electroporation of intact cells without lysozyme treatment was developed for these mutant strains even though this method was approximately 100-fold less efficient. In this method, the initial pH of culture medium was elucidated as a critical factor and optimized for both transformation efficiency and avermectin productivity.     

Mutation breeding of high avermectin B<Subscript>1a</Subscript>-producing strain by the combination of high energy carbon heavy ion irradiation and sodium nitrite mutagenesis based on high throughput screening

Microbial mutation breeding has been widely used because it is one of the most efficient and practical breeding strategies in the fermentation industry. However, different mutagenesis methods cause various degrees of DNA damage to individual microorganisms, which lead to diverse characteristics of the mutants. In this study, the effects of four different mutagenesis methods on the mutation breeding of Streptomyces avermitilis for improving avermectin B1a production were investigated with an optimized liquid microtiter plate (MTP) culture system. First, an effective and feasible MTP system for mutant strain screening was evaluated through the optimization of the oxygen transfer rate and rapid titer determination. Then, high energy carbon heavy ion irradiation, diethyl sulfate, ultraviolet- (UV) irradiation combined with lithium chloride, and sodium nitrite were used as the mutagens for mutation breeding, respectively. Results showed that carbon heavy ion irradiation had the advantages of possessing the highest positive mutation rate and mean-production of positive mutant strains in the first generation. Sodium nitrite treatment resulted in mutant strains with better inherited stability than the other three methods. Through the combined treatment of carbon heavy ion irradiation and sodium nitrite treatment, an inheritstable mutant S. avermitilis S-233 with high avermectin B_1a production was successfully obtained. The fermentation verification in a 500-liter (L) bioreactor demonstrated that the avermectin B_1a produced by mutant S. avermitilis S-233 reached 6818 μg/mL, which was 23.8% higher than that of parent strains.     

阿维菌素的生物合成及代谢工程研究进展

阿维菌素(avermectin)是由除虫链霉菌(Streptomycesavermitilis)产生的一种具有杀虫活性的大环内酯类抗生素,在农业和畜牧业中应用广泛。本文综述了有关除虫链霉菌基因组序列分析、阿维菌素的生物合成以及阿维菌素育种和代谢工程的研究进展。     

Synthesis and biological activities of novel 4"-alkylidene avermectin derivatives

Horner-Emmons reaction of 4"-dehydro-5-O-TBDMS-avermectin B(1a) with a variety of phosphorus ylides using LHMDS gave novel 4"-alkylidene avermectin derivatives in high yields. Further modifications led to derivatives bearing diverse functional groups. The new avermectin derivatives showed potent growth inhibitory activity against Artemia salina and Caenorhabditis elegans.     

Alternative production of avermectin components in Streptomyces avermitilis by gene replacement

The avermectins are composed of eight compounds, which exhibit structural differences at three positions. A family of four closely-related major components, A1a, A2a, B1a and B2a, has been identified. Of these components, B1a exhibits the most potent antihelminthic activity. The coexistence of the "1" components and "2" components has been accounted for by the defective dehydratase of aveAI module 2, which appears to be responsible for C22-23 dehydration. Therefore, we have attempted to replace the dehydratase of aveAI module 2 with the functional dehydratase from the erythromycin eryAII module 4, via homologous recombination. Erythromycin polyketide synthetase should contain the sole dehydratase domain, thus generating a saturated chain at the C6-7 of erythromycin. We constructed replacement plasmids with PCR products, by using primers which had been derived from the sequences of avermectin aveAI and the erythromycin eryAII biosynthetic gene cluster. If the original dehydratase of Streptomyces avermitilis were exchanged with the corresponding erythromycin gene located on the replacement plasmid, it would be expected to result in the formation of precursors which contain alkene at C22-23, formed by the dehydratase of erythromycin module 4, and further processed by avermectin polyketide synthase. Consequently, the resulting recombinant strain JW3105, which harbors the dehydratase gene derived from erythromycin, was shown to produce only C22,23-unsaturated avermectin compounds. Our research indicates that the desired compound may be produced via polyketide gene replacement.     

Influence of Transmembrane Helix Mutations on Cytochrome P450-Membrane Interactions and Function

Human cytochrome P450 (CYP) enzymes play an important role in the metabolism of drugs, steroids, fatty acids, and xenobiotics. Microsomal CYPs are anchored in the endoplasmic reticulum membrane by an N-terminal transmembrane (TM) helix that is connected to the globular catalytic domain by a flexible linker sequence. However, the structural and functional importance of the TM-helix is unclear because it has been shown that CYPs can still associate with the membrane and have enzymatic activity in reconstituted systems after truncation or modification of the N-terminal sequence. Here, we investigated the effect of mutations in the N-terminal TM-helix residues of two human steroidogenic enzymes, CYP 17A1 and CYP 19A1, that are major drug targets for cancer therapy. These mutations were originally introduced to increase the expression of the proteins in Escherichia coli. To investigate the effect of the mutations on protein-membrane interactions and function, we carried out coarse-grained and all-atom molecular dynamics simulations of the CYPs in a phospholipid bilayer. We confirmed the orientations of the globular domain in the membrane observed in the simulations by linear dichroism measurements in a Nanodisc. Whereas the behavior of CYP 19A1 was rather insensitive to truncation of the TM-helix, mutations in the TM-helix of CYP 17A1, especially W2A and E3L, led to a gradual drifting of the TM-helix out of the hydrophobic core of the membrane. This instability of the TM-helix could affect interactions with the allosteric redox partner, cytochrome b5, required for CYP 17A1’s lyase activity. Furthermore, the simulations showed that the mutant TM-helix influenced the membrane interactions of the CYP 17A1 globular domain. In some simulations, the mutated TM-helix obstructed the substrate access tunnel from the membrane to the CYP active site, indicating a possible effect on enzyme function.     

Purification of CYP2B-like protein from feral leaping mullet (Liza saliens) liver microsomes and its biocatalytic, molecular, and immunological characterization

In this study, CYP2B-immunoreactive protein was purified to electrophoretic homogeneity from the liver microsomes of leaping mullet. The purified cytochrome P450 (CYP) gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis having a M(r) of 49,300 Da. Absolute absorption spectrum of the purified CYP showed a maximum at 417 nm and CO-difference spectrum of dithionite-reduced cytochrome P450 gave a peak at 450 nm. The purified CYP was found to be active in N-demethylation of benzphetamine, erythromycin, and ethylmorphine, and O-dealkylation of pentoxyresorufin in the reconstituted system. However, it was unable to catalyze O-dealkylation of ethoxyresorufin, methoxyresorufin, benzyloxyresorufin, and hydroxylation of lauric acid and aniline. The purified CYP showed strong cross-reactivity with anti-sheep lung CYP2B, a homologue of CYP2B4. N-terminal amino acid sequence of the mullet P450 had the highest degree of homology with CYP2Bs among the known CYPs. Spectral, electrophoretic, immunochemical, N-terminal amino acid sequence, and biocatalytic properties of the purified CYP are most similar to those of mammalian cytochrome P4502B. All these data indicate that the purified CYP is certainly 2B-like. In this study, we not only purified biocatalytically active CYP2B-like protein from fish, but also demonstrated detailed functional properties of CYP2B-like protein for the first time.     

Cytochrome p450 complement (CYPome) of the avermectin-producer Streptomyces avermitilis and comparison to that of Streptomyces coelicolor A3(2)

The genus Streptomyces produces about two-thirds of naturally occurring antibiotics and a wide array of other secondary metabolites, including antihelminthic agents, antitumor agents, antifungal agents, and herbicides. The newly completed genome sequence of the avermectin-producing bacterium Streptomyces avermitilis contains 33 cytochromes p450 (CYPs), many more than the 18 observed in Streptomyces coelicolor A3(2). Some of the likely metabolic functions are reported together with their genomic location and bioinformatic analysis. Seven entirely new CYP families were found together with close homologues of some forms observed in S. coelicolor A3(2). The presence of unusual CYP forms associated with conservons is revealed and of these, CYP157 forms in both S. avermitilis and S. coelicolor A3(2) deviate from the previously accepted rule for an EXXR motif within the K-helix of CYPs. Amongst this range of CYPs are forms associated with avermectin, filipin, geosmin, and pentalenolactone biosynthesis as well as unknown pathways of secondary metabolism.     

Targeting cytochrome P450 enzymes: a new approach in anti-cancer drug development

Cytochrome P450s (CYPs) represent a large class of heme-containing enzymes that catalyze the metabolism of multitudes of substrates both endogenous and exogenous. Until recently, however, CYPs have been largely overlooked in cancer drug development, acknowledged only for their role in phase I metabolism of chemotherapeutics. The first successful strategy targeting CYP enzymes in cancer therapy was the development of potent inhibitors of CYP19 (aromatase) for the treatment of breast cancer. Aromatase inhibitors ushered in a new era in hormone ablation therapy for estrogen dependent cancers, and have paved the way for similar strategies (i.e., inhibition of CYP17) that combat androgen dependent prostate cancer. Identification of CYPs involved in the inactivation of anti-cancer metabolites of vitamin D(3) and vitamin A has triggered development of agents that target these enzymes as well. The discovery of the over-expression of exogenous metabolizing CYPs, such as CYP1B1, in cancer cells has roused interest in the development of inhibitors for chemoprevention and of prodrugs designed to be activated by CYPs only in cancer cells. Finally, the expression of CYPs within tumors has been utilized in the development of bioreductive molecules that are activated by CYPs only under hypoxic conditions. This review offers the first comprehensive analysis of strategies in drug development that either inhibit or exploit CYP enzymes for the treatment of cancer.     

Purification and identification of dTDP-oleandrose, the precursor of the oleandrose units of the avermectins

The nucleotide sugar precursor of the oleandrose units of the avermectins has been purified from a mutant of Streptomyces avermitilis, which does not synthesize any avermectins but which converts avermectin aglycones to their respective disaccharides. This precursor has been identified as dTDP-oleandrose. The purification was achieved by anion exchange and reverse phase high performance liquid chromatography. The purified nucleotide sugar had an absorption spectra characteristic of thymidine, released dTMP when treated with phosphodiesterase, and possessed an NMR spectrum in which three resonances characteristic of oleandrose were seen in addition to the thymidine signals. The enzyme, avermectin aglycone dTDP-oleandrose glycosyltransferase, which catalyzes the stepwise addition of oleandrose to the avermectin aglycones, has been demonstrated in cell-free extracts and (NH4)2SO4 fractions of cell-free extracts of S. avermitilis. The enzyme is specific for dTDP-oleandrose as the glycosyl donor but utilizes all avermectin aglycones as glycosyl acceptors. The stoichiometry between dTDP-oleandrose consumed in the reaction and oleandrose units transferred to the avermectin mono- and disaccharide was found to be 1:1.     

Improved preparation and detection of cytochrome P450 isoforms using MS methods

Cytochromes P450 (CYPs) are a superfamily of mixed function oxidases, which in the liver have great significance to the pharmaceutical industry because their expression will determine the fate of most clinical agents. CYPs are also targets for inhibitors of hormone‐dependent diseases and conversion of prodrugs to active agents in normal and cancer tissues. We have applied simple modifications to established methods of isolating CYPs, using 8 M urea to solubilise microsomal proteins and specific molecular weight gel bands for in‐gel digestion in combination with nanoHPLC MALDI MS to acquire peptide MS/MS spectra for database searching. As a consequence of the changes we significantly improved the yield of proteomic data, identifying 26 mouse CYPs (CYP1a2, 2a4, 2a5, 2a12, 2b9, 2c29, 2c37, 2c39, 2c40, 2c50, 2c54, 2c70, 2d9, 2d10, 2d26, 2e1, 2f2, 2j5, 3a11, 3a13, 3a25, 3a41, 4a14, 4f14, 8b1 and 27a1) with an average sequence coverage of 30.1%, including some previously undetected highly homologous isoforms. In addition, other important enzymes in drug metabolism are also identified. There is a divergence of opinion over the expression of CYP1a1 in liver and we could not detect the presence of this isoform. In order to provide definitive evidence of the ability to detect CYP1a1, we analysed CHO cells transfected with human CYP1A1 and identified unique peptides that differentiated this isoform from human CYP1A2.     

Association of functionally important polymorphisms in cytochrome P450s with squamous cell carcinoma of head and neck

Head and neck squamous cell carcinoma (HNSCC), a common malignancy that possibly involves a combination of exposure to the carcinogens and inherited genetic differences in the enzymes catalyzing their metabolism. Alcohol and tobacco consumption are the primary environmental risk factors, while polymorphism in various biotransformation enzymes such as cytochrome P450s (CYPs) and glutathione S-transferases, (GSTs) has been implicated as the major genetic risk factors for the development of HNSCC. The functionally important polymorphisms in these CYPs (1AI*2A, 1A1*2C, 1B1*2, 2E1*5B, 2E1*6, 2C19*2, 2D6*4, 2D6*10) and GSTs (GSTM1-null or GSTT1-null) were found to be significantly associated with HNSCC risk. Significant differences in the distribution of certain haplotypes of CYPs have also been reported and prevalence of certain genotype combinations of CYPs and GSTS in cases has indicated the importance of gene-gene interactions in HNSCC risk. Alcohol or tobacco use (smoking and chewing) were also found to interact synergistically with variant genotypes of these CYPs and GSTS in significantly enhancing HNSCC risk. This increase in risk associated with the variant genotypes with tobacco or alcohol use have further demonstrated the importance of gene-environment interactions in determining the susceptibility to HNSCC.     

A chemiluminescent assay for measuring avermectin binding sites

Avermectins are potent, broad spectrum anthelmintics. We describe the synthesis of a biologically active chemiluminescent compound, 22,23-dihydroavermectin B1a, 4"-[4-[[4-[ethyl(1,2,3,4-tetrahydro-1,4-dioxo-6- phthalazinyl)amino]butyl]amino]-4-oxobutanoate] (ivermectin-luminol). This compound retains its anthelmintic properties and provides an extremely sensitive nonradioactive probe to study avermectin binding sites.     

Synthetic biology of avermectin for production improvement and structure diversification

Natural products are still key sources of current clinical drugs and innovative therapeutic agents. Since wild‐type microorganisms only produce natural products in very small quantities, yields of production strains need to be improved by breaking down the precise genetic and biochemical circuitry. Herein, we use avermectins as an example of production improvement and chemical structure diversification by synthetic biology. Avermectins are macrocyclic lactones produced by Streptomyces avermitilis and are well known and widely used for antiparasitic therapy. Given the importance of this molecule and its derivatives, many efforts and strategies were employed to improve avermectin production and generate new active analogues. This review describes the current status of synthetic strategies successfully applied for developing natural‐product‐producing strains and discusses future prospects for the application of enhanced avermectin production.     

Comparison of the 1.85 A structure of CYP154A1 from Streptomyces coelicolor A3(2) with the closely related CYP154C1 and CYPs from antibiotic biosynthetic pathways

The genus Streptomyces produces two-thirds of microbially derived antibiotics. Polyketides form the largest and most diverse group of these natural products. Antibiotic diversity of polyketides is generated during their biosynthesis by several means, including postpolyketide modification performed by oxidoreductases, a broad group of enzymes including cytochrome P450 monooxygenases (CYPs). CYPs catalyze site-specific oxidation of macrolide antibiotic precursors significantly affecting antibiotic activity. Efficient manipulation of Streptomyces CYPs in generating new antibiotics will require identification and/or engineering of monooxygenases with activities toward a diverse array of chemical substrates. To begin to link structure to function of CYPs involved in secondary metabolic pathways of industrially important species, we determined the X-ray structure of Streptomyces coelicolor A3(2) CYP154A1 at 1.85 A and analyzed it in the context of the closely related CYP154C1 and more distant CYPs from polyketide synthase (EryF) and nonribosomal peptide synthetase (OxyB) biosynthetic pathways. In contrast to CYP154C1, CYP154A1 reveals an active site inaccessible from the molecular surface, and an absence of catalytic activities observed for CYP154C1. Systematic variations in the amino acid patterns and length of the surface HI loop correlate with degree of rotation of the F and G helices relative to the active site in CYP154A1-related CYPs, presumably regulating the degree of active site accessibility and its dimensions. Heme in CYP154A1 is in a 180 degrees flipped orientation compared with most other structurally determined CYPs.     

The ER protein Ema19 facilitates the degradation of nonimported mitochondrial precursor proteins

For the biogenesis of mitochondria, hundreds of proteins need to be targeted from the cytosol into the various compartments of this organelle. The intramitochondrial targeting routes these proteins take to reach their respective location in the organelle are well understood. However, the early targeting processes, from cytosolic ribosomes to the membrane of the organelle, are still largely unknown. In this study, we present evidence that an integral membrane protein of the endoplasmic reticulum (ER), Ema19, plays a role in this process. Mutants lacking Ema19 show an increased stability of mitochondrial precursor proteins, indicating that Ema19 promotes the proteolytic degradation of nonproductive precursors. The deletion of Ema19 improves the growth of respiration-deficient cells, suggesting that Ema19-mediated degradation can compete with productive protein import into mitochondria. Ema19 is the yeast representative of a conserved protein family. The human Ema19 homologue is known as sigma 2 receptor or TMEM97. Though its molecular function is not known, previous studies suggested a role of the sigma 2 receptor as a quality control factor in the ER, compatible with our observations about Ema19. More globally, our data provide an additional demonstration of the important role of the ER in mitochondrial protein targeting.     

Identification of universal selectivity‐determining positions in cytochrome P450 monooxygenases by systematic sequence‐based literature mining

Cytochrome P450 monooxygenases (CYPs) are a large, highly diverse protein family with a common fold. The sequences, structures, and functions of CYPs have been extensively studied resulting in more than 53,000 scientific articles. A sequence‐based literature mining algorithm was designed to systematically analyze this wealth of information on SNPs, designed mutations, structural interactions, or functional roles of individual residues. Structurally corresponding positions in different CYPs were compared and universal selectivity‐determining positions were identified. Based on the Cytochrome P450 Engineering Database ( www.CYPED.BioCatNet.de ) and a standard numbering scheme for all CYPs, 4000 residues in 168 CYPs mentioned in 2400 articles could be assigned to 440 structurally corresponding standard positions of the CYP fold, covering 96% of all standard positions. Seventeen individual standard positions were mentioned in the context of more than 32 different CYPs. The majority of these most frequently mentioned positions are located on the six substrate recognition sites and are involved in control of selectivity, such as the well‐studied position 87 in CYP102A1 (P450_BM‐3) which was mentioned in the articles on 63 different CYPs. The recurrent citation of the 17 frequently mentioned positions for different CYPs suggests their universal functional relevance. Proteins 2015; 83:1593–1603. © 2015 Wiley Periodicals, Inc.