US7960155B1 | 2011-06-14 | |||
US6100074A | 2000-08-08 | |||
US20110086405A1 | 2011-04-14 | |||
US20090209010A1 | 2009-08-20 |
What is claimed is: 1. An engineered variant of a reductase-fused P450 capable of oxidizing thiophene, benzothiophene, dibenzothiophene and derivatives thereof to the corresponding sulfoxides and sulfone, and wherein the variant favors thiophene oxidation over alkane hydroxylation. 2. The engineered variant of claim 1, wherein the variant has from 2 to 20 mutations with respect to any one of SEQ ID NOS-1-16. 3. The engineered variant of claim 1 or 2, wherein the variant is selected in E. coli grown on medium containing one or more thiophenes as the sole sulfur source. 4. The engineered variant of any one of claims 1 to 3, wherein the P450 is a P450BM3 has a mutation selected from position 87 or position 268. 5. The engineered variant of claim 4, having the substitution F87V. 6. The engineered variant of claim 4, having the substitution T268A. 7. The engineered variant of any one of claims 1 to 6, wherein the variant is expressed in a microbial host cell. 8. The engineered variant of claim 7, wherein the microbial host cell is a species selected from Rhodococcus sp, Sphingomonas sp, Tsukamarella sp, Bacillus sp, Mycobacterium sp, and Nocardia sp. 9. The engineered variant of claim 7 or 8, wherein the microbial host cell comprises at least one component of the 4S oxidative pathway. 10. The engineered variant of claim 9, wherein the one or more components of the 4S oxidative pathway are endogenous. 11. The engineered variant of claim 9, wherein the one or more components of the 4S oxidative pathway are heterologous. 12. The engineered variant of claim 9, wherein the gene encoding the engineered P450 and the gene(s) encoding one or more components of the 4S oxidative pathway are together in an operon, which optionally integrated into the host genome. 13. A microbial cell comprising a biodesulfurizing metabolic pathway: the pathway comprising an engineered P450, a dibenzothiophene sulfone monooxygenase, 2-hydroxybiphenyl-2-sulfmate sulfmolyase, and a flavin reductase, the metabolic pathway converting dibenzothiophene (DBT) compounds to 2- hydroxybiphenyl-2-sulfinate (HBPS), HBPS to 2-hydroxybiphenyl (HBP), and HBP to sulfite. 14. The microbial cell of claim 13, wherein the dibenzothiophene sulfone monooxygenase is DszA or engineered variant thereof. 15. The microbial cell of claim 13 or 14, wherein the 2-hydroxybiphenyl-2- sulfmate sulfmolyase is DszB or engineered variant thereof. 16. The microbial cell of any one of claims 13 to 15, wherein the flavin reductase is DszD or engineered variant thereof. 17. The microbial cell of any one of claims 13 to 16, wherein at least one of the dibenzothiophene sulfone monooxygenase, 2-hydroxybiphenyl-2-sulfmate sulfmolyase, and the flavin reductase is heterologous to the cell. 18. The microbial cell of any one of claims 13 to 17, wherein the pathway enzymes are encoded in an operon, which is optionally integrated into the host genome. 19. The microbial cell of any one of claims 13 to 18, wherein the microbial cell is able to grow on a biphasic water-oil mixture containing aromatic sulfur compounds in the oil layer. 20. The microbial cell of any one of claims 13 to 19, wherein the microbial cell is a species selected from Rhodococcus sp, Sphingomonas sp., Tsukamarella sp., Bacillus sp., Mycobacterium sp., and Nocardia sp. 21. The microbial cell of claim 20, wherein the microbial cell uses said metabolic pathway as a sulfur source but not as a carbon source. 22. The microbial host cell of any one of claims 13 to 21, wherein the cell is an engineered strain of R. opacus, R. erythropolis and B. subtilis. 23. The microbial cell of claim 22, wherein the NADPH metabolism is engineered to increase metabolic efficiency. 24. The microbial host cell of claim 22, wherein the strain is a derivative of R. opacus B-4. 25. The microbial cell of any one of claims 13 to 24, containing the dszABC operon of R. erthropolis IGTS8, with dszC optionally replaced with the engineered P450. 26. The microbial host cell of any one of claims 13 to 25, wherein the P450 is the engineered P450 of any one of claims 1 to 12. 27. A microbial host cell comprising the engineered P450 of any one of claims 1 to 6 encoded in an operon with one or more components of the Dsz pathway. 28. A method for biodesulfurization of petroleum, comprising: culturing the microbial cell of any one of claims 13 to 27 on the oil and water interface of a petroleum product. 29. The method of claim 28, wherein the petroleum has a sulfur level of about 1% or greater. 30. The method of claim 28, performed before or after hydrodesulfurization of the petroleum product. 31. The method of any one of claims 28 to 30, wherein the oil water ratio is less than about 7: 1, less than about 6: 1, less than about 5: 1, less than about 4: 1, or about 3: 1 or less. 32. A method for making a microbial strain for biodesulphurization of petroleum, comprising: culturing an E. coli strain harboring a library of P450BM3 mutants and harboring a Dsz pathway, and selecting mutants capable of growth on medium having thiophenes as the sole sulfur source. 33. The method of claim 32, wherein the library of mutants comprises at least five, or at least ten, or at least 20, or at least 40 mutants at positions 87 and 268, said positions corresponding to any one of SEQ ID NOS: 1-16. |
PRIORITY
This application claims priority to U.S. Provisional Application No. 61/738,863, filed December 18, 2012, which is hereby incorporated by reference in its entirety.
BACKGROUND OF THE INVENTION
Fossil hydrocarbons contain both inorganic and organic forms of sulfur. Pyrite (FeS 2 ) constitutes the most common inorganic form, whereas organic compounds vary from aliphatics, such as thioethers (RSR') and mercaptans (RSH), to aromatic sulfur heterocycles, such as thiophenes. In petroleum, sulfur is found mainly as aliphatic and aromatic organic forms but also as elemental sulfur and hydrogen sulfide. 1 The sulfur content in crude oil varies significantly depending on its type and source, ranging from less than 0.1% to over 5%, and is generally related to its density. Light crudes generally have low sulfur levels (< 1%) and command premium prices. There is, however, an increasingly limited supply of light crudes and production trends are clearly in the direction of heavy oils with higher sulfur content as demonstrated by the increase in the average sulfur content of petroleum across the World from 1.13% in 1990 to 1.27% in 2010 (Table 1).
Of all the biogeochemical cycles (e.g. carbon, nitrogen, phosphorous), it is the sulfur cycle that is most influenced by human activity. Estimates for anthropogenic sulfur emissions into the atmosphere range between two to three times higher than those derived from natural sources. 2 Anthropogenic emissions are mainly caused by burning fossil fuels and mining activities, which release large quantities of sulfur oxides (SO x ). SO x are known to significantly impact the environment (i.e. acid rain and soot) and the climate (i.e. global cooling). 3 There has been a slow decrease in anthropogenic emission from 1970 to 2000, thanks to environmental laws and to improvements in desulfurization technologies. 2 Since 2000, however, sulfur emissions have started to rise at a fast rate due to the increasing consumption of hydrocarbons in emerging economies such as China, India and Brazil. The concentration of sulfur in diesel affects how much soot is produced during the combustion of the fuel. Soot is a fine dispersion of particulate matter that causes various respiratory and cardiovascular diseases. 4 Soot emissions from diesel engines can be drastically reduced by catalytic converters, however, these catalysts are strongly inhibited by sulfur compounds. Sulfate particulate matter also reduces engine life due to corrosion. For these reasons, developed countries now have regulations requiring diesel fuel with ultra- low sulfur levels (< 15 ppm, Table 1).
Hydrodesulfurization (HDS) is the prevailing technology for sulfur removal in an oil refinery. 5 HDS is energy intensive and requires large volumes of hydrogen, which contribute to its high cost. The most common HDS catalysts are based on molybdenum sulfides, promoted by cobalt or nickel with an aluminum oxide support (Co-Mo/Al 2 0 3 , Ni-Mo/Al 2 0 3 ). The process requires high temperatures (290-425 °C) and pressures (1.4 to 20.7 MPa). Current HDS processes are capable of removing aliphatic sulfur compounds with high efficiency, but aromatic compounds are typically recalcitrant to these treatments. Figure 1 shows the qualitative relationship between the molecular structures of sulfur compounds, found in various oil fractions, and their relative reactivity under HDS. Aliphatic sulfur compounds exhibit about 10 times faster HDS rates than the simplest aromatic sulfur compound, thiophene. The HDS reactivity of the aromatics follow the order thiophene > benzothiophene > dibenzo thiophene (DBT) > multi-alkylated dibenzothiophene (Cx-DBT). Removal of recalcitrant DBT and sterically hindered Cx-DBT requires multiple HDS treatments thus consuming large amounts of hydrogen and energy.
Research into novel desulfurization methods has been stimulated by the steady increase in the average sulfur content of globally available petroleum and the ever stricter environmental regulations, including requirements on refineries to reduce C0 2 emissions. Biodesulfurization (BDS) is a promising method that does not require hydrogen, high temperatures or high pressure and generates far less C0 2 than HDS. Importantly, BDS may offer a substrate selectivity that is complementary to HDS, such that the combination of the two methods can deliver ultra-low sulfur fuels.
For at least these reasons, enzymes, microbial cells, and processes for biodesulfurization are needed.
SUMMARY OF THE INVENTION In various aspects, the invention provides engineered cytochrome P450 catalysts for catalyzing aromatic sulfur oxidation to sulfoxides and sulfones. The invention additionally provides engineered microbial catalysts for removing thiophene, benzothiophene, dibenzothiophene and derivatives thereof from petroleum. In some embodiments, the invention provides non-naturally occurring microbial organisms containing a BDS pathway comprising at least one exogenous gene encoding a BDS pathway enzyme and an engineered cytochrome P450 (e.g., a fused heme-reductase P450 as described herein) expressed in a sufficient amount to oxidize DBT related compounds in petroleum. The invention in still other aspects provides methods for making microbial strains for biodesulphurization of petroleum.
For example, in various aspects and embodiments, the invention provides engineered variants of P450 BM3 capable of oxidizing thiophene, benzothiophene, dibenzothiophene and derivatives thereof to the corresponding sulfoxides and sulfone, as well as engineered microbial hosts expressing these engineered variants, optionally with one or more components of the 4S oxidative pathway. In various embodiments, the P450 BM3 variant favors thiophene oxidation over alkane hydroxylation, making it particularly useful for biodesulphurization of petroleum.
The engineered P450 variant may be expressed in a microbial host cell, such as E. coli, and recovered, or alternatively, expressed in a microorganism optionally harboring one or more components of the 4S oxidative pathway (DszA, DszB, DszC, or DszD). For example, the microbial host cell in some embodiments is a species selected from Rhodococcus sp, Sphingomonas sp, Tsukamarella sp, Bacillus sp, Mycobacterium sp, and Nocardia sp. In some embodiments, the microbial host cell comprises at least one component of the 4S oxidative pathway, such as DszA, DszB, and/or DszD, which may be endogenous or heterologous. In some embodiments, the engineered P450 may replace or complement DszC, the first step in the 4S oxidative pathway.
The microbial host is generally selected from hosts that can grow on biphasic water-oil mixtures and are able to utilize thiophene derivatives in the oil layer as their only source of sulfur. These microbes can use sulfur as a nutrient by oxidizing aromatic sulfur compounds to release sulfite ions that can be fixed by the cells.
In other aspects, the invention provides a method for biodesulfurization of petroleum, using the engineered P450 and engineered microbial cells described herein. Additional aspects and embodiments of the invention will be apparent from the following detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. Qualitative relationship between hydrodesulfurization reaction rate and molecular structure for various sulfur compounds found in the distillate fractions equivalent to gasoline, jet fuel and diesel. 5
Figure 2. 4S biodesulfurization pathway. Pathway metabolites: DBT, dibenzothiophene; DBTO, dibenzothiophene sulfoxide; DBT0 2 , dibenzothiophene sulfone; HBPS, 2-hydroxybiphenyl-2-sulfmate; HBP, 2-hydroxybiphenyl. Pathway enzymes: DszC, dibenzothiophene monooxygenase; DszA, dibenzothiophene sulfone monooxygenase; DszB, 2-hydroxybiphenyl-2-sulfmate sulfmolyase; DszD, NADH- dependent flavin reductase. Pathway cofactors: FMN, flavin mononucleotide; FMNH 2 , reduced flavin mononucleotide; NAD, nicotinamide adenine dinucleotide.
Figure 3. Artificial 4S pathway utilizing cytochrome P450 BM3 to oxidize DBT to DBTO and DBT0 2 .
Figure 4. Simplified catalytic cycle for cytochrome P450. Sulfur oxidation can be catalyzed by either compound 0 or compound I whereas alkane hydroxylation can only be catalyzed by compound I.
Figure 5. Calculations on the theoretical product-per-glucose yield (Y PP G)- (1)
DBT oxidation via the 4S pathway requires 3 equivalents of NADH. (2) Theoretical maximum NADH from glucose yield under non-growing aerobic conditions. (3) Overall, the BDS reaction requires 0.25 equivalents of glucose and produces 1.5 equivalents of C0 2 .
DETAILED DESCRIPTION OF THE INVENTION
Two aerobic pathways have been described for sulfur removal. The first is the ring-destructive pathway, which breaks down the aromatic rings as the mechanism for sulfur removal, thus resulting in hydrocarbon loss and a lower calorific value of the fuel. The second is the non-destructive 4S oxidation pathway, which uses DBT as a sulfur source but not as a carbon source and specifically removes sulfur from heterocyclic compounds without disrupting the hydrocarbon skeleton. The 4S pathway has been more intensely investigated, due to its superior commercial potential, as it affords a higher hydrocarbon yield.
Four enzymes (DszA, DszB, DszC, DszD) are involved in catalyzing the four oxidative reactions in the 4S pathway (Figure 2), which is found in diverse microbes including species of Rhodococcus, Bacillus, Gordonia, Paenibacillus, Pseudomonas and Mycobacterium, among others. The first enzyme, dibenzothiophene monooxygenase (DszC), catalyzes two sequential oxidations converting DBT to dibenzothiophene sulfoxide (DBTO) and dibenzothiophene sulfone (DBT0 2 ). The second enzyme, dibenzothiophene sulfone monooxygenase (DszA), converts DBT0 2 to 2-hydroxybiphenyl-2-sulfmate (HBPS), which is the only water-soluble molecule in the pathway. 2-hydroxybiphenyl-2-sulfmate sulfmolyase (DszB) finally converts HPBS to sulfite and 2-hydroxylbiphenyl (HBP). Sulfite is fixed by the microbe and HBP partitions back to the hydrophobic phase. DszC and DszA require a flavin reductase (DszD) to regenerate reduced flavin cofactor (FMNH 2 ).
The main drawback of BDS is its slow reaction rate compared to chemical processes. For example, BDS of crude oil with Mycobacterium goodii X7B removed 59% of total sulfur content (from 3600 to 1478 ppm) over 72 h; 9 BDS of a previously hydrodesulfurized middle distillate fraction with Rhodococcus erythropolis 1-19 removed 67% of total sulfur content (from 1850 to 615 ppm) over 24 h. 10 While it is estimated that a commercial BDS process would need to proceed at 3 mmol Cx-DBT gDCW 1 h "1 , the fastest reported example operates only at 0.28 mmol gDCW 1 h "1 (DCW, dry cell weight of biocatalyst). 7 Therefore, the reaction rate needs to be enhanced by one order of magnitude. To meet this goal, there are two key parameters on which to focus improvements: namely the catalytic rates of the 4S pathway enzymes and the rate of substrate transfer from the oil-water interface to the cells.
The cytochrome P450 from Bacillus megaterium (P450 BM3 or CYP102A1) is the fastest known monooxygenase {k cat = 5,140 min "1 for palmitic acid hydroxylation). 11 Replacing the first enzyme in the 4S pathway, DszC, with a faster monooxygenase (i.e. P450 BM3 ) can lead to faster kinetics for sulfur oxidation. P450 BM3 is a well-studied, soluble, self-sufficient (heme and diflavin reductase domains are fused in a single polypeptide, ~ 120 KDa), long-chain fatty acid monooxygenase. More than a decade of protein engineering attests to the functional plasticity of this biocatalyst. For example, Sulistyaningdyah et al. have reported that the single mutant P450 BM3 -F87V hydroxylates benzothiophene with an initial rate of 230 mm 1 . 13
Other fused P450 enzymes that can be used or engineered according to this disclosure, include those of SEQ ID NOS:2-16, as shown in Table 2 herein. Thus, engineered variants may have from 2 to 20 mutations, for example, with respect to any one of SEQ ID NOS: 1-16.
Thus, in various aspects and embodiments, the invention provides engineered variants of P450 BM3 or variant of other fused P450 capable of oxidizing thiophene, benzothiophene, dibenzothiophene and derivatives thereof to the corresponding sulfoxides and sulfone, as well as engineered microbial hosts expressing these engineered variants, optionally with one or more components of the 4S pathway. In various embodiments, the P450 variant favors thiophene oxidation over alkane hydroxylation, making it particularly useful for biodesulphurization of petroleum.
P450 BM 3 has the amino acid sequence of SEQ ID NO: l . Substitutions, insertions, additions, deletions, and truncations of this sequence are selected to enhance oxidation of thiophene, benzothiophene, and/or dibenzothiophene (including alkylated derivatives thereof), while reducing alkane hydroxylation activity. Such mutations may be made rationally and/or by selection in a suitable screening assay, and/or by directed evolution. P450 BM3 comprises a P450 heme domain of about 55 kDa and a reductase domain of about 65 kDa containing two flavin (FAD and FMN) groups. Numerous crystal structures of P450 BM3 are available to guide mutations. In some embodiments, at least one, at least two, at least three, at least four, or at least five mutations are at positions 47, 82, 94, 142, 205, 226, 290, and 328, which have been shown to exhibit effects on alkane hydroxylation activity. See US Patent 7,524,664, which is hereby incorporated by reference in its entirety for all purposes. In some embodiments, one to twenty deletions, insertions, or substitutions (e.g., one to ten or one to five such alterations) are made in the region of residues 1-32 and 447- 473. In these or other embodiments, residues 69-92, residues 253-271, and residues 434-441 harbor fewer than two, three, or four mutations each. In some embodiments, the derivative has a mutations at one or more (e.g., two, three, four, five, six, seven eight, nine, or ten) of position 25, 26, 42, 47, 51, 52, 58, 72, 74, 75, 78, 81, 82, 86, 87, 88, 94, 100, 106, 107, 135, 142, 158, 162, 168, 175, 177, 181, 184, 188, 197, 205, 225, 226, 228, 236, 239, 252, 255, 260, 261, 263, 264, 265, 267, 268, 274, 281, 290, 324, 328, 329, 330, 340, 353, 354, 363, 366, 393, 401, 434, 435, 437, 438, 440, 442. In these or other embodiments, the variant has a substitution at one or more of position 87 (e.g. F87V) and position 268 (e.g, T268A). The corresponding positions may also be varied in any one of SEQ ID NOS:2-16 in accordance with the above description.
In some embodiments, these and other P450 mutants are screened in E. coli for the desired activity. For example, introduction of a synthetic P450-dsz pathway or operon, where DszC is replaced with the engineered P450 mutants, in E. coli under the control of an inducible promoter and an appropriate selection marker forms the basis of a selection system for engineering P450 variants with activity on DBT and its alkylated derivatives (Cx-DBTs). The library of P450 mutants can be introduced into a suitable E coli strain by transformation or transfection, including via any suitable plasmid or other expression vector. When libraries of transformants are plated on medium (e.g., agar) where Cx-DBT is the only source of sulfur, only the clones bearing a P450 that is active on the target Cx-DBT compound will make the sulfone substrate for DszA that will ultimately make the suflite ions necessary for cell growth. This strategy enables rapid identification of P450 variants with some level of activity on the target Cx-DBT. These variants can be further evolved for efficient catalytic parameters by subsequent chromatography screening (e.g., HPLC or GC) of mutant libraries.
Once P450 variants with high activity on Cx-DBTs are identified, it is important to ensure that they cannot oxidize the long-chain hydrocarbons in middle distillate fractions. P450 oxidation during BDS has to be selective for sulfur compounds so as to not waste valuable NADPH reducing equivalents in futile reactions, and not to oxidize the hydrocarbon fuel and alter its properties. It is noteworthy that P450 BM3 -oxidation of thioethers has been proposed to proceed via the hydroperoxo intermediate (compound 0), 14 whereas the hydroxylation of unfunctionalized C-H bonds is only achieved by the more oxidizing radical ferryl intermediate (compound I, Figure 4). Since the mutation T268A hinders compound I formation, it is anticipated that T268A, and other mutations at position 268 (for example, substitutions that do not contain a side chain having a hydroxyl, including substitutions of G, L, I, V, or other non-polar or hydrophobic amino acid) or mutation of amino acids having a side chain in its 3-dimensional vicinity (e.g., 5 or 10 Angstrom radius), can also be used to favor sulfur oxidation over alkane hydroxylation. Such mutations may be included in mutation libraries and screened in E. coli as described above. For example, the library of mutants may comprise at least five, or at least ten, or at least 20, or at least 40 mutants at positions 87 and 268 (positions corresponding to SEQ ID NO:l, or corresponding position of SEQ ID NOS:2-16 based on an alignment with SEQ ID NO: l).
The engineered P450 variant may be expressed in a microbial host cell, such as E. coli, and recovered, or alternatively, expressed in a microorganism optionally harboring one or more components of the 4S pathway for biodesulphurization of petroleum. For example, the microbial host cell in some embodiments is a species selected from Rhodococcus sp., Sphingomonas sp, Tsukamarella sp, Bacillus sp, and Mycobacterium sp, Nocardia sp. In some embodiments, the microbial host cell comprises at least one component of the 4S oxidative pathway (DszA, DszB, DszC, or DszD. In various embodiments, the engineered P450 replaces DscZ, the first step in the pathway. See Figure 2. Thus, in some embodiments, the microbe expresses a biodesulfurization metabolic pathway: the metabolic pathway comprising P450 or variant thereof (including a variant described herein), a dibenzothiophene sulfone monooxygenase, 2-hydroxybiphenyl-2-sulfmate sulfino lyase, and a flavin reductase. The metabolic pathway may convert dibenzothiophene (DBT) compounds to 2- hydroxybiphenyl-2-sulfinate (HBPS), HBPS to 2-hydroxybiphenyl (HBP), and HBP to sulfite.
For example, the microbial host may express a dibenzothiophene sulfone monooxygenase, such as DszA or engineered variant thereof. Examples include those of Rhodococcus erythropolis (AAP80182.1), Rhodococcus sp. DS-3 (ABE26644.1), Rhodococcus sp. EU-32 (AGT97385.1), Nocardia globerula (AAU14820.1), Gordonia alkanivorans (AAU14817.1), Sorangium cellulosum (AAY32964.1), Mycobacterium sp. (e.g., BAC41357.1) or Bur kholderia sp. (e.g., YP 005043164.1). For example, the engineered variant may have from one to twenty, or from one to ten, or from one to five amino acid changes with respect to one or more of the above sequences, including substitutions, deletions, and insertions with respect to the DszA. DszA of Rhodococcus erythropolis is shown herein as SEQ ID NO: 17. In some embodiments, the DszA enzyme comprises an amino acid sequence that is at least 70%, at least 80%, at least 90%, or at least 95% identical to SEQ ID NO: 17. Substitutions or other alterations in amino acid sequence can be guided by any known crystal structure or homology model.
In these or other embodiments, the microbial cell expresses a 2- hydroxybiphenyl-2-sulfinate sulfinolyase, e.g., DszB, or engineered variant thereof. For example, the engineered variant may have from one to twenty, or from one to ten, or from one to five amino acid changes, including substitutions, deletions, and insertions with respect to DszB. Examples include those of Rhodococcus erythropolis (AAP80183.1, AAU 14821.1), Rhodococcus sp. (AAA99483.1), Rhodococcus sp. DS- 3 (ABE26645.1), Nocardia globerula (AAU14824.1), Gordonia alkanivorans (AAP49448.1), Sorangium cellulosum (AAY32965), Mycobacterium sp. (e.g., BAC41358) or Burkholderia sp. (e.g., WP 006399409.1). For example, the engineered variant may have from one to twenty, or from one to ten, or from one to five amino acid changes with respect to one or more of the above sequences, including substitutions, deletions, and insertions with respect to the DszB. DszB of Rhodococcus erythropolis is shown herein as SEQ ID NO: 18. In some embodiments, the DszB enzyme comprises an amino acid sequence that is at least 70%, at least 80%, at least 90%, or at least 95% identical to SEQ ID NO: 18. Substitutions or other alterations in amino acid sequence can be guided by any known crystal structure or homology model.
In these or other embodiments, the microbial cell expresses a flavin reductase
(e.g., DszD) or engineered variant thereof. For example, the engineered variant may have from one to twenty, or from one to ten, or from one to five amino acid changes, including substitutions, deletions, and insertions with respect to DszD. Examples include those of Rhodococcus erythropolis (ABV44406.1, BAB 18470.1), Rhodococcus sp. DS-3 (ABE26647.1), Rhodococcus sp. SDUZAWQ (AAV49164.1), Rhodococcus opacus B4 (BAH52549.1), Rhodococcus sp. IGTS8 (AAC38226.1), Nocardia asteroides NBRC 15531 (GAD84696.1), Gordonia sp. (GAB24076.1), Sorangium cellulosum (AAY32968.1). For example, the engineered variant may have from one to twenty, or from one to ten, or from one to five amino acid changes with respect to one or more of the above sequences, including substitutions, deletions, and insertions with respect to the DszD. DszD of Rhodococcus erythropolis is shown herein as SEQ ID NO: 19. In some embodiments, the DszD enzyme comprises an amino acid sequence that is at least 70%>, at least 80%>, at least 90%>, or at least 95%> identical to SEQ ID NO: 19. Substitutions or other alterations in amino acid sequence can be guided by any known crystal structure or homology model.
The engineered P450, as well as any other desired heterologous enzyme, can be plasmid encoded or can be integrated into the host genome through known methods. In some embodiments, the engineered P450 and the components of the 4S pathway (e.g., DszA, DszB, and DszD) are constructed as a monocistronic operon that is genomically integrated. In some embodiments, the cells contain on average from two to twenty, or from two to ten, copies of the operon, to thereby increase expression levels of the key enzymes.
The microbial host is generally selected from hosts (e.g., various yeasts and bacteria) that can grow on biphasic water-oil mixtures and are able to utilize thiophene derivatives in the oil layer as their only source of sulfur. These microbes can use sulfur as a nutrient by oxidizing aromatic sulfur compounds to release sulfite ions that can be fixed by the cells. Bacteria of the type Sphingomonas, Tsukamarella, Bacillus, Mycobacterium, Pseudomonas, Nocardia, and Rhodococcus have all exhibited this biodesulfurization property.
In some embodiments, the host cell is a Rhodococcus sp., such as R. erythropolis and Rhodococcus opacus. In Rhodococcus erythropolis strain IGTS8, the constitutively expressed Dsz enzymes (from the dszABC operon) are soluble and presumably found in the cytoplasm. DBT and its metabolites have not been described as being actively transported into (or out of) Rhodococcus cells.
The limiting steps for BDS are thought to be diffusion of DBT from the oil- water interface into the cells, the supply of reducing equivalents and enzyme turnover rates for specific substrates. For hydrophilic bacteria, such as Pseudomonas, mass transfer of DBT from oil to cells can be rate limiting, whereas for hydrophobic bacteria, such as Rhodococcus, enzyme rates can be rate limiting. Thus, in some embodiments, the invention employs Rhodococcus sp. as the host cell, with the engineered P450, and components of the 4S pathway, which may also be engineered to increase turnover rate.
In some embodiments, the host cell contains an endogenous 4S oxidative pathway. In some embodiments, an active DszC gene is replaced with (or complemented by) the engineered P450 (that is, DszC is deleted or inactivated), or in other embodiments, the engineered P450 works alongside the endogenous DszC to increase productivity of this monooxygenase step. In some embodiments, a heterologous P450-DszA-DszB-DszD pathway or operon (in any order) is added to cells (including by plasmid or by genomic integration as described) that also contain an active endogenous 4S pathway. In some embodiments, one or more of the endogenous genes are replaced with engineered variants thereof, having modified substrate selectivity or reaction rate. In still other embodiments, the metabolic pathway is constructed completely of heterologous (e.g., non-endogenous) genes. For example, one or more of the dibenzothiophene sulfone monooxygenase, 2- hydroxybiphenyl-2-sulfmate sulfinolyase, and the flavin reductase is heterologous to the cell.
In various embodiments, the microbial cell has one or more properties that are useful for biodesulphurization of petroleum. In one embodiment, a synthetic P450- Dsz pathway or operon (as described) is introduced into the hydrophobic bacterium Rhodococcus opacus. R. opacus B-4, originally isolated from gasoline-contaminated soil (with high aromatics content), is metabolically active in anhydrous solvents and preferentially exists at the oil-water interface in biphasic reaction media. 18 Kawaguchi et al. have constructed a recombinant R. opacus that expresses the dszABC operon of R. erythropolis IGTS8, and reported resting cells BDS activity at WOR as low as 0.5 and even at anhydrous organic solvents. 16 Interestingly, R. opacus was found to accumulate at the emulsion layer where DBT transfer can occur to the cells. The proposed recombinant R. opacus P450-Dsz strain can be constructed by using the published protocols for R. opacus genetic transformation. 19 It is noteworthy that Rosloniec et al. have recently reported the expression of P450 BM3 variants in the closely related Rhodococcus erythropolis, which is also a relevant BDS host. 20
Thus, in some embodiments, the host cell is able to grow on a biphasic water- oil mixture containing thiophene compounds in the oil layer, and use thiophenes as a source of sulfur, but not as a source of carbon. For example, the microbial cell may be a species selected from Sphingomonas sp., Tsukamarella sp., Bacillus sp., Mycobacterium sp., Nocardia sp, and Rhodococcus sp. In some embodiments, the cell is an engineered strain of R. opacus, R. erythropolis and B. subtilis, and said strain removes DBT derivatives from petroleum; and makes DBT0 2 , HBPS and HBP with efficient product per glucose yields.
B. subtilis is also an attractive candidate host for biodesulfurization since it endogenously carries the 4S pathway as well as two self-sufficient cytochrome P450s (CYP102A2 and CYP102A3), and is a well developed genetic engineering host. Additionally, the moderately thermophilic nature of B. subtilis (50 °C) can reduce cooling costs, since the hydrocarbon feed in a refinery will come from a much hotter upstream process. Higher temperatures also lower the viscosity of crude oil facilitating inter-phase mass transfer. Finally, the DszC catalyzed step can be rate determining for B. subtilis, such that replacing DszC by the faster P450 is likely to increase the overall BDS rate.
In some embodiments, the efficiency of the strain is enhanced by NADPH metabolism engineering. BDS has intensive cofactor requirements. Conversion of DBT to HBP requires 3 equivalents of NADH (Figure 5), which must be regenerated in vivo by the oxidation of a suitable carbon source (i.e. glucose). Figure 5 shows that the theoretical maximum product-per-glucose yield (Yppo) is 4 under non-growing aerobic conditions. Additionally, the process should produce at least 1.5 equivalents of carbon dioxide. The NAD(P)H metabolism of the host can be engineered towards these theoretical limits by employing similar strategies as described by Fasan et al. (e.g. partial inactivation of the endogenous respiratory chain and removal of competing fermentation pathways). 24
In some embodiments, the strain is a derivative of R. opacus B-4. In some embodiments, the microorganism contains the dszABC operon of R. erthropolis IGTS8.
In some embodiments, the components of the 4S oxidative pathway are DszA and DszD only. For example, engineered P450, DszA and DszD are heterologously introduced to R. opacus so that HBPS is the final pathway product. HBPS is water soluble and can be used as a building block for surfactants, 22 thus serving as a platform for the production of high-value chemicals. Thus, in these embodiments, HBPS is recovered as a substrate for synthesis of industrial chemicals or pharmaceuticals, (including surfactants).
In another embodiment, only the engineered P450BM3 is heterologously introduced to the host cell (e.g., R. opacus) so that the sulfone becomes the final oxidation product. This alleviates the metabolic burden on the cells and simplifies the kinetics for the overall process, as the truncated pathway is not limited by the potentially slower downstream enzymes (DszA and DszB). Thus, in these embodiments, the endogenous 4S oxidative pathway may be deleted or inactivated. Dibenzothiophene sulfone can be selectively removed by extraction as it is more polar than dibenzothiophene and other hydrocarbons in petroleum.
In other aspects, the invention provides a method for biodesulfurization of petroleum. In these aspects, the engineered P450 catalyst is introduced into an appropriate host for BDS applications as described. The choice of host needs to take at least three factors into account: i) tolerance to petroleum and its aromatic compounds; ii) hydrophobicity of cells so that the overall kinetics is not limited by mass transfer; and iii) ability to express the engineered P450 (e.g., engineered Since the organic solvents present in petroleum are frequently toxic to common industrial bacteria such as E. coli, BDS processes are typically conducted under biphasic reaction conditions with high water-to-oil ratios (WOR). That is, the microbial cells are separately grown to the desired cell density in purely aqueous media and are then transferred to the BDS reactor where they are mixed with a given volume of oil. WOR of 3: 1 up to 9: 1 has been attempted. In idealized process conditions, BDS should be performed in the absence or with a minimum proportion of water. This is because high WOR leads to reduced BDS activity due to low interface mass transfer and contact efficiency between oil and water. Additionally, the 4S pathway is inhibited by its final product 2-HBP (which is also a potent phenolic biocide), and HBP accumulation is reduced with decreasing WOR, as higher concentrations of solvent facilitate 2-HBP diffusion from cells. Finally, a lower WOR facilitates biocatalyst separation and recovery, and reduces water treatment costs. In various embodiments, the oil water ratio is less than about 7: 1, less than about 6:1, less than about 5: 1, less than about 4: 1 , or is about 3 : 1 or less.
For example, the method comprises culturing the microbial cell as described herein on the oil and water interface of a petroleum product in a BDS reactor. In some embodiments, the petroleum has a sulfur level of about 1% or greater, or about 2% or greater, or about 3% or greater. The biodesulfurization may be performed before or after hydrodesulfurization of the petroleum product, to remove aromatic sulfur compounds, while removing aliphatic sulfur compounds by HDS. References
1. Hyne, J. B., Desulfurization of fossil fuels. ACS Symposium Series 1979, 90
(Chem. Energy), 45-65.
2. Smith, S. J.; Pitcher, H.; Wigley, T. M. L., Global and regional anthropogenic sulfur dioxide emissions. Global and Planetary Change 2001, 29 (1-2), 99-119.
3. Smith, S. J.; van Aardenne, J.; Klimont, Z.; Andres, R. J.; Volke, A.; Arias, S.
D., Anthropogenic sulfur dioxide emissions: 1850-2005. Atmospheric Chemistry and
Physics 2011, 77 (3), 1101-1116.
4. (a) Miller, K. A.; Siscovick, D. S.; Sheppard, L.; Shepherd, K.; Sullivan, J. H.;
Anderson, G. L.; Kaufman, J. D., Long-Term Exposure to Air Pollution and Incidence of Cardiovascular Events in Women. New England Journal of Medicine 2007, 356 (5),
447-458; (b) Brook, R. D.; Franklin, B.; Cascio, W.; Hong, Y. L.; Howard, G.; Lipsett,
M.; Luepker, R.; Mittleman, M.; Samet, J.; Smith, S. C; Tager, I., Air pollution and cardiovascular disease - A statement for healthcare professionals from the expert panel on population and prevention science of the American Heart Association.
Circulation 2004, 109 (21), 2655-2671.
5. Song, C, An overview of new approaches to deep desulfurization for ultra- clean gasoline, diesel fuel and jet fuel. Catalysis Today 2003, 86 (1-4), 211-263.
6. Mohebali, G.; Ball, A. S., Biocatalytic desulfurization (BDS) of petrodiesel fuels. Microbiology (Reading, United Kingdom) 2008, 154 (Copyright (C) 2011 American Chemical Society (ACS). All Rights Reserved.), 2169-2183.
7. Kilbane, J. J., Microbial biocatalyst developments to upgrade fossil fuels.
Current Opinion in Biotechnology 2006, 17 (3), 305-314.
8. Monticello, D. J., Biodesulfurization and the upgrading of petroleum
distillates. Current Opinion in Biotechnology 2000, 11 (6), 540-546.
9. Li, F.; Zhang, Z.; Feng, J.; Cai, X.; Xu, P., Biodesulfurization of DBT in tetradecane and crude oil by a facultative thermophilic bacterium Mycobacterium goodii X7B. Journal of Biotechnology 2007, 127 (2), 222-228.
10. Folsom, B. R.; Schieche, D. R.; DiGrazia, P. M.; Werner, J.; Palmer, S.,
Microbial desulfurization of alkylated dibenzothiophenes from a hydrodesulfurized middle distillate by Rhodococcus erythropolis 1-19. Applied and Environmental Microbiology 1999, 65 (11), 4967-4972. 11. Noble, M. A.; Miles, C. S.; Chapman, S. K.; Lysek, D. A.; MacKay, A. C; Reid, G. A.; Hanzlik, R. P.; Munro, A. W., Roles of key active-site residues in flavocytochrome P450 BM3. The Biochemical journal 1999, 339 ( Pt 2), 371-9.
12. Whitehouse, C. J. C; Bell, S. G.; Wong, L.-L., P 450BM3 (CYP102A1): connecting the dots. Chemical Society Reviews 2012, 41 (3), 1218-1260.
13. Sulistyaningdyah, W. T.; Ogawa, J.; Li, Q.-S.; Maeda, C; Yano, Y.; Schmid, R. D.; Shimizu, S., Hydroxylation activity of P450 BM-3 mutant F87V towards aromatic compounds and its application to the synthesis of hydroquinone derivatives from phenolic compounds. Applied Microbiology and Biotechnology 2005, 67 (4), 556-562.
14. (a) Cryle, M. J.; De Voss, J. J., Is the ferric hydroperoxy species responsible for sulfur oxidation in cytochrome P450s? Angewandte Chemie, International Edition 2006, 45 (48), 8221-8223; (b) Li, C; Zhang, L.; Zhang, C; Hirao, H.; Wu, W.; Shaik, S., Which oxidant is really responsible for sulfur oxidation by cytochrome P450? Angewandte Chemie, International Edition 2007, 46 (43), 8168-8170.
15. Clark, J. P.; Miles, C. S.; Mowat, C. G.; Walkinshaw, M. D.; Reid, G. A.; Daff, S. N.; Chapman, S. K., The role of Thr268 and Phe393 in cytochrome P 450 BM3. Journal of Inorganic Biochemistry 2006, 100 (5-6), 1075-1090.
16. Kawaguchi, H.; Kobayashi, H.; Sato, K., Metabolic engineering of
hydrophobic Rhodococcus opacus for biodesulfurization in oil-water biphasic reaction mixtures. Journal of Bioscience and Bio engineering 2012, 113 (3), 360-366.
17. Na, K.-S.; Kuroda, A.; Takiguchi, N.; Ikeda, T.; Ohtake, H.; Kato, J., Isolation and characterization of benzene -tolerant Rhodococcus opacus strains. Journal of Bioscience and Bio engineering 2005, 99 (4), 378-382.
18. (a) Yamashita, S.; Satoi, M.; Iwasa, Y.; Honda, K.; Sameshima, Y.; Omasa, T.; Kato, J.; Ohtake, H., Utilization of hydrophobic bacterium Rhodococcus opacus B-4 as whole-cell catalyst in anhydrous organic solvents. Applied Microbiology and Biotechnology 2007, 74 (4), 761-767; (b) Honda, K.; Yamashita, S.; Nakagawa, H.; Sameshima, Y.; Omasa, T.; Kato, J.; Ohtake, H., Stabilization of water-in-oil emulsion by Rhodococcus opacus B-4 and its application to biotransformation.
Applied Microbiology and Biotechnology 2008, 78 (5), 767-773.
19. Na, K.-S.; Nagayasu, K.; Kuroda, A.; Takiguchi, N.; Ikeda, T.; Ohtake, H.; Kato, J., Development of a genetic transformation system for benzene -tolerant Rhodococcus opacus strains. Journal of Bioscience and Bioengineering 2005, 99 (4), 408-414.
20. Rosloniec, K. Z. Steroid trasnformation by Rhodococcus strains and bacterial cytochrome P450 enzymes. University of Groningen, 2011.
21. Chandra Srivastava, V., An evaluation of desulfurization technologies for sulfur removal from liquid fuels. RSC Advances 2012, 2 (3), 759-783.
22. Pacheco, M. A.; Lange, E. A.; Lin, Q., Recent advances in biodesulfurization (BDS) of diesel fuel. World Refining 1999, (Sulfur 2000), 37-40.
23. Ohshiro, T.; Ishii, Y.; Matsubara, T.; Ueda, K.; Izumi, Y.; Kino, K.; Kirimura, K., Dibenzothiophene desulfurizing enzymes from moderately thermophilic bacterium Bacillus subtilis WU-S2B: Purification, characterization and
overexpression. Journal of Bioscience and Bioengineering 2005, 100 (3), 266-273.
24. Fasan, R.; Crook, N. C; Peters, M. W.; Meinhold, P.; Buelter, T.; Landwehr, M.; Cirino, P. C; Arnold, F. H., Improved product-per-glucose yields in P450- dependent propane biotransformations using engineered Escherichia coli. Biotechnol. Bioeng. 2011, 108 (3), 500-510.
25. http ://www.dieselnet. com/standards/br/ fuel .php .
Table 1. Sulfur levels (weight %) in the global supplies of crude oil and emission standards (ppm sulfur) for selected regions. 21 ' 25
Sequences Table 2
Sequence Listing
SEQ ID NO:l
CYP102A1
Cytochrome P450 (BM3)
Bacillus megaterium
GenBank Accession No. AAA87602
>gi I 142798 I gb I AAA87602.1 I cytochrome P-450 :NADPH-P-450 reductase precursor [Bacillus megaterium]
TIKEMPQPK TFGELKNLPL LNTDKPVQAL MKIADELGEI FKFEAPGRVT RYLSSQRLIK EACDESRFDK NLSQALKFVR DFAGDGLFTS WTHEKNWKKA HNILLPSFSQ QAMKGYHAMM VDIAVQLVQK WERLNADEHI EVPEDMTRLT LDTIGLCGFN YRFNSFYRDQ PHPFITSMVR ALDEAMNKLQ RANPDDPAYD ENKRQFQEDI KVMNDLVDKI IADRKASGEQ SDDLLTHMLN GKDPETGEPL DDENIRYQI I TFLIAGHETT SGLLSFALYF LVKNPHVLQK AAEEAARVLV DPVPSYKQVK QLKYVGMVLN EALRLWPTAP AFSLYAKEDT VLGGEYPLEK GDELMVLIPQ LHRDKTIWGD DVEEFRPERF ENPSAIPQHA FKPFGNGQRA CIGQQFALHE ATLVLGMMLK HFDFEDHTNY ELDIKETLTL KPEGFWKAK SKKIPLGGIP SPSTEQSAKK VRKKAENAHN TPLLVLYGSN MGTAEGTARD LADIAMSKGF APQVATLDSH AGNLPREGAV LIVTASYNGH PPDNAKQFVD WLDQASADEV KGVRYSVFGC GDKNWATTYQ KVPAFIDETL AAKGAENIAD RGEADASDDF EGTYEEWREH MWSDVAAYFN LDIENSEDNK STLSLQFVDS AADMPLAKMH GAFSTNWAS KELQQPGSAR STRHLEIELP KEASYQEGDH LGVIPRNYEG IVNRVTARFG LDASQQIRLE AEEEKLAHLP LAKTVSVEEL LQYVELQDPV TRTQLRAMAA KTVCPPHKVE LEALLEKQAY KEQVLAKRLT MLELLEKYPA CEMKFSEFIA LLPSIRPRYY SISSSPRVDE KQASI VSW SGEAWSGYGE YKGIASNYLA ELQEGDTITC FISTPQSEFT LPKDPETPLI MVGPGTGVAP FRGFVQARKQ LKEQGQSLGE AHLYFGCRSP HEDYLYQEEL ENAQSEGI IT LHTAFSRMPN QPKTYVQHVM EQDGKKLIEL LDQGAHFYIC GDGSQMAPAV EATLMKSYAD VHQVSEADAR LWLQQLEEKG RYAKDVWAG
SEQ ID NO: 2 CYP102A1
B. megaterium DSM 32
Uniprot Accession No. P14779
>sp I P14779 I CPXB_BACME Bifunctional P-450/NADPH-P450 reductase
OS=Bacillus megaterium GN=cypl02Al PE=1 SV=2
1 MTIKEMPQPK TFGELKNLPL LNTDKPVQAL MKIADELGEI FKFEAPGRVT
RYLSSQRLIK
61 EACDESRFDK NLSQALKFVR DFAGDGLFTS WTHEKNWKKA HNILLPSFSQ
QAMKGYHAMM
121 VDIAVQLVQK WERLNADEHI EVPEDMTRLT LDTIGLCGFN YRFNSFYRDQ
PHPFITSMVR
181 ALDEAMNKLQ RANPDDPAYD ENKRQFQEDI KVMNDLVDKI IADRKASGEQ
SDDLLTHMLN
241 GKDPETGEPL DDENIRYQI I TFLIAGHETT SGLLSFALYF LVKNPHVLQK
AAEEAARVLV
301 DPVPSYKQVK QLKYVGMVLN EALRLWPTAP AFSLYAKEDT VLGGEYPLEK
GDELMVLIPQ
361 LHRDKTIWGD DVEEFRPERF ENPSAIPQHA FKPFGNGQRA CIGQQFALHE
ATLVLGMMLK
421 HFDFEDHTNY ELDIKETLTL KPEGFWKAK SKKIPLGGIP SPSTEQSAKK
VRKKAENAHN
481 TPLLVLYGSN MGTAEGTARD LADIAMSKGF APQVATLDSH AGNLPREGAV
LIVTASYNGH
541 PPDNAKQFVD WLDQASADEV KGVRYSVFGC GDKNWATTYQ KVPAFIDETL
AAKGAENIAD
601 RGEADASDDF EGTYEEWREH MWSDVAAYFN LDIENSEDNK STLSLQFVDS
AADMPLAKMH
661 GAFSTNWAS KELQQPGSAR STRHLEIELP KEASYQEGDH LGVIPRNYEG
IVNRVTARFG
721 LDASQQIRLE AEEEKLAHLP LAKTVSVEEL LQYVELQDPV TRTQLRAMAA
KTVCPPHKVE
781 LEALLEKQAY KEQVLAKRLT MLELLEKYPA CEMKFSEFIA LLPSIRPRYY
SISSSPRVDE
841 KQASITVSW SGEAWSGYGE YKGIASNYLA ELQEGDTITC FISTPQSEFT
LPKDPETPLI
901 MVGPGTGVAP FRGFVQARKQ LKEQGQSLGE AHLYFGCRSP HEDYLYQEEL
ENAQSEGI IT
961 LHTAFSRMPN QPKTYVQHVM EQDGKKLIEL LDQGAHFYIC GDGSQMAPAV
EATLMKSYAD
1021 VHQVSEADAR LWLQQLEEKG RYAKDVWAG
SEQ ID NO: 3
CYP102A5
B . cereus ATCC14579
GenBank Accession No. AAP10153
>gi I 29896875 I gb I AAP10153.1 I NADPH-cytochrome P450 reductase [Bacillus cereus ATCC 14579]
1 MEKKVSAIPQ PKTYGPLGNL PLIDKDKPTL SFIKIAEEYG PIFQIQTLSD
TIIWSGHEL
61 VAEVCDETRF DKSIEGALAK VRAFAGDGLF TSETHEPNWK KAHNILMPTF
SQRAMKDYHA
121 MMVDIAVQLV QKWARLNPNE NVDVPEDMTR LTLDTIGLCG FNYRFNSFYR
ETPHPFITSM
181 TRALDEAMHQ LQRLDIEDKL MWRTKRQFQH DIQSMFSLVD NIIAERKSSG
DQEENDLLSR
241 MLNVPDPETG EKLDDENIRF QIITFLIAGH ETTSGLLSFA IYFLLKNPDK
LKKAYEEVDR
301 VLTDPTPTYQ QVMKLKYMRM ILNESLRLWP TAPAFSLYAK EDTVIGGKYP
IKKGEDRISV 361 LIPQLHRDKD AWGDNVEEFQ PERFEELDKV PHHAYKPFGN GQRACIGMQF ALHEA LVMG
421 MLLQHFELID YQNYQLDVKQ TLTLKPGDFK IRILPRKQTI SHPTVLAPTE DKLKNDEIKQ
481 HVQKTPSIIG ADNLSLLVLY GSDTGVAEGI ARELADTASL EGVQTEWAL NDRIGSLPKE
541 GAVLIVTSSY NGKPPSNAGQ FVQWLEELKP DELKGVQYAV FGCGDHNWAS TYQRIPRYID
601 EQMAQKGATR FSKRGEADAS GDFEEQLEQW KQNMWSDAMK AFGLELNKNM EKERSTLSLQ
661 FVSRLGGSPL ARTYEAVYAS ILENRELQSS SSDRSTRHIE VSLPEGATYK EGDHLGVLPV
721 NSEKNINRIL KRFGLNGKDQ VILSASGRSI NHIPLDSPVS LLALLSYSVE VQEAATRAQI
781 REMVTFTACP PHKKELEALL EEGVYHEQIL KKRISMLDLL EKYEACEIRF ERFLELLPAL
841 KPRYYSISSS PLVAHNRLSI TVGWNAPAW SGEGTYEGVA SNYLAQRHNK DEIICFIRTP
901 QSNFELPKDP ETPIIMVGPG TGIAPFRGFL QARRVQKQKG MNLGQAHLYF GCRHPEKDYL
961 YRTELENDER DGLISLHTAF SRLEGHPKTY VQHLIKQDRI NLISLLDNGA HLYICGDGSK
1021 MAPDVEDTLC QAYQEIHEVS EQEARNWLDR VQDEGRYGKD VWAGI
SEQ ID NO: 4
CYP102A7
B . licheniformis ATTC1458
GenBank Accession No. YP 079990
>gi I 52081199 I ref I YP_079990.1 I cytochrome P450 / NADPH- ferrihemoprotein reductase [Bacillus licheniformis DSM 13 =
14580]
1 MNKLDGIPIP KTYGPLGNLP LLDKNRVSQS LWKIADEMGP IFQFKFADAI GVFVSSHELV
61 KEVSEESRFD KNMGKGLLKV REFSGDGLFT SWTEEPNWRK AHNILLPSFS QKAMKGYHPM
121 MQDIAVQLIQ KWSRLNQDES IDVPDDMTRL TLDTIGLCGF NYRFNSFYRE GQHPFIESMV
181 RGLSEAMRQT KRFPLQDKLM IQTKRRFNSD VESMFSLVDR I IADRKQAES ESGNDLLSLM
241 LHAKDPETGE KLDDENIRYQ I ITFLIAGHE TTSGLLSFAI YLLLKHPDKL KKAYEEADRV
301 LTDPVPSYKQ VQQLKYIRMI LNESIRLWPT APAFSLYAKE ETVIGGKYLI PKGQSVTVLI
361 PKLHRDQSVW GEDAEAFRPE RFEQMDSIPA HAYKPFGNGQ RACIGMQFAL HEATLVLGMI
421 LQYFDLEDHA NYQLKIKESL TLKPDGFTIR VRPRKKEAMT AMPGAQPEEN GRQEERPSAP
481 AAENTHGTPL LVLYGSNLGT AEEIAKELAE EAREQGFHSR TAELDQYAGA IPAEGAVIIV
541 TASYNGNPPD CAKEFVNWLE HDQTDDLRGV KYAVFGCGNR SWASTYQRIP RLIDSVLEKK
601 GAQRLHKLGE GDAGDDFEGQ FESWKYDLWP LLRTEFSLAE PEPNQTETDR QALSVEFVNA
661 PAASPLAKAY QVFTAKISAN RELQCEKSGR STRHIEISLP EGAAYQEGDH LGVLPQNSEV
721 LIGRVFQRFG LNGNEQILIS GRNQASHLPL ERPVHVKDLF QHCVELQEPA TRAQIRELAA
781 HTVCPPHQRE LEDLLKDDVY KDQVLNKRLT MLDLLEQYPA CELPFARFLA LLPPLKPRYY 841 SISSSPQLNP RQTSITVSW SGPALSGRGH YKGVASNYLA GLEPGDAISC
FIREPQSGFR
901 LPEDPETPVI MVGPGTGIAP YRGFLQARRI QRDAGVKLGE AHLYFGCRRP
NEDFLYRDEL
961 EQAEKDGIVH LHTAFSRLEG RPKTYVQDLL REDAALLIHL LNEGGRLYVC
GDGSRMAPAV
1021 EQALCEAYRI VQGASREESQ SWLSALLEEG RYAKDVWDGG VSQHNVKADC IART
SEQ ID NO: 5
CYPX
B. thuringiensis serovar konkukian
str .97-27
GenBank Accession No. YP 037304
>gi I 49480099 I ref I YP_037304.1 I NADPH-cytochrome P450 reductase
[Bacillus thuringiensis serovar konkukian str. 97-27]
1 MDKKVSAIPQ PKTYGPLGNL PLIDKDKPTL SFIKLAEEYG PIFQIQTLSD
TIIWSGHEL
61 VAEVCDETRF DKSIEGALAK VRAFAGDGLF TSETDEPNWK KAHNILMPTF
SQRAMKDYHA
121 MMVDIAVQLV QKWARLNPNE NVDVPEDMTR LTLDTIGLCG FNYRFNSFYR
ETPHPFITSM
181 TRALDEAMHQ LQRLDIEDKL MWRTKRQFQH DIQSMFSLVD NIIAERKSSE
NQEENDLLSR
241 MLNVQDPETG EKLDDENIRF QIITFLIAGH ETTSGLLSFA IYFLLKNPDK
LKKAYEEVDR
301 VLTDSTPTYQ QVMKLKYIRM ILNESLRLWP TAPAFSLYAK EDTVIGGKYP
IKKGEDRISV
361 LIPQLHRDKD AWGDDVEEFQ PERFEELDKV PHHAYKPFGN GQRACIGMQF
ALHEATLVMG
421 MLLQHFEFID YEDYQLDVKQ TLTLKPGDFK IRIVPRNQTI SHTTVLAPTE
EKLKKHEIKK
481 QVQKTPSIIG ADNLSLLVLY GSDTGVAEGI ARELADTASL EGVQTEWAL
NDRIGSLPKE
541 GAVLIVTSSY NGKPPSNAGQ FVQWLEELKP DELKGVQYAV FGCGDHNWAS
TYQRIPRYID
601 EQMAQKGATR FSTRGEADAS GDFEEQLEQW KQSMWSDAMK AFGLELNKNM
EKERSTLSLQ
661 FVSRLGGSPL ARTYEAVYAS ILENRELQSS SSERSTRHIE ISLPEGATYK
EGDHLGVLPI
721 NNEKNVNRIL KRFGLNGKDQ VILSASGRSV NHIPLDSPVR LYDLLSYSVE
VQEAATRAQI
781 REMVTFTACP PHKKELESLL EDGVYQEQIL KKRISMLDLL EKYEACEIRF
ERFLELLPAL
841 KPRYYSISSS PLVAQDRLSI TVGWNAPAW SGEGTYEGVA SNYLAQRHNK
DEIICFIRTP
901 QSNFQLPENP ETPIIMVGPG TGIAPFRGFL QARRVQKQKG MKVGEAHLYF
GCRHPEKDYL
961 YRTELENDER DGLISLHTAF SRLEGHPKTY VQHVIKEDRI HLISLLDNGA
HLYICGDGSK
1021 MAPDVEDTLC QAYQEIHEVS EQEARNWLDR LQEEGRYGKD VWAGI
SEQ ID NO: 6
CYP102E1
R. metallidurans CH34
GenBank Accession No. YP 585608
>gi I 94312398 I ref I YP_585608.1 I putative bifunctional P-450 :NADPH-P450 reductase 2 [Cupriavidus metallidurans CH34]
1 MSTATPAAAL EPIPRDPGWP IFGNLFQITP GEVGQHLLAR SRHHDGIFEL
DFAGKRVPFV 61 SSVALASELC DATRFRKIIG PPLSYLRDMA GDGLFTAHSD EPNWGCAHRI LMPAFSQRAM
121 KAYFDVMLRV ANRLVDKWDR QGPDADIAVA DDMTRLTLDT IALAGFGYDF ASFASDELDP
181 FVMAMVGALG EAMQKLTRLP IQDRFMGRAH RQAAEDIAYM RNLVDDVIRQ RRVSPTSGMD
241 LLNLMLEARD PETDRRLDDA NIRNQVITFL IAGHETTSGL LTFALYELLR NPGVLAQAYA
301 EVDTVLPGDA LPVYADLARM PVLDRVLKET LRLWPTAPAF AVAPFDDWL GGRYRLRKDR
361 RISWLTALH RDPKVWANPE RFDIDRFLPE NEAKLPAHAY MPFGQGERAC IGRQFALTEA
421 KLALALMLRN FAFQDPHDYQ FRLKETLTIK PDQFVLRVRR RRPHERFVTR QASQAVADAA
481 QTDVRGHGQA MTVLCASSLG TARELAEQIH AGAIAAGFDA KLADLDDAVG VLPTSGLVW
541 VAATYNGRAP DSARKFEAML DADDASGYRA NGMRLALLGC GNSQWATYQA FPRRVFDFFI
601 TAGAVPLLPR GEADGNGDFD QAAERWLAQL WQALQADGAG TGGLGVDVQV RSMAAIRAET
661 LPAGTQAFTV LSNDELVGDP SGLWDFSIEA PRTSTRDIRL QLPPGITYRT GDHIAVWPQN
721 DAQLVSELCE RLDLDPDAQA TISAPHGMGR GLPIDQALPV RQLLTHFIEL QDWSRQTLR
781 ALAQATRCPF TKQSIEQLAS DDAEHGYATK WARRLGILD VLVEHPAIAL TLQELLACTV
841 PMRPRLYSIA SSPLVSPDVA TLLVGTVCAP ALSGRGQFRG VASTWLQHLP PGARVSASIR
901 TPNPPFAPDP DPAAPMLLIG PGTGIAPFRG FLEERALRKM AGNAVTPAQL YFGCRHPQHD
961 WLYREDIERW AGQGWEVHP AYSWPDAPR YVQDLLWQRR EQVWAQVRDG ATIYVCGDGR
1021 RMAPAVRQTL IEIGMAQGGM TDKAASDWFG GLVAQGRYRQ DVFN
SEQ ID NO: 7
CYP505X
A. fumigatus Af293
GenBank Accession No. EAL92660
>gi I 66852335 I gb I EAL92660.1 I P450 family fatty acid hydroxyla putative [Aspergillus fumigatus Af293]
1 MSESKTVPIP GPRGVPLLGN IYDIEQEVPL RSINLMADQY GPIYRLTTFG WSRVFVSTHE
61 LVDEVCDEER FTKWTAGLN QIRNGVHDGL FTANFPGEEN WAIAHRVLVP AFGPLSIRGM
121 FDEMYDIATQ LVMKWARHGP TVPIMVTDDF TRLTLDTIAL CAMGTRFNSF YHEEMHPFVE
181 AMVGLLQGSG DRARRPALLN NLPTSENSKY WDDIAFLRNL AQELVEARRK NPEDKKDLLN
241 ALILGRDPKT GKGLTDESII DNMITFLIAG HETTSGLLSF LFYYLLKTPN AYKKAQEEVD
301 SWGRRKITV EDMSRLPYLN AVMRETLRLR STAPLIAVHA HPEKNKEDPV TLGGGKYVLN
361 KDEPIVIILD KLHRDPQVYG PDAEEFKPER MLDENFEKLP KNAWKPFGNG MRACIGRPFA
421 WQEALLWAI LLQNFNFQMD DPSYNLHIKQ TLTIKPKDFH MRATLRHGLD ATKLGIALSG
481 SADRAPPESS GAASRVRKQA TPPAGQLKPM HIFFGSNTGT CETFARRLAD DAVGYGFAAD
541 VQSLDSAMQN VPKDEPWFI TASYEGQPPD NAAHFFEWLS ALKENELEGV NYAVFGCGHH 601 DWQATFHRIP KAVNQLVAEH GGNRLCDLGL ADAANSDMFT DFDSWGESTF WPAITSKFGG
661 GKSDEPKPSS SLQVEVSTGM RASTLGLQLQ EGLVIDNQLL SAPDVPAKRM IRFKLPSDMS
721 YRCGDYLAVL PVNPTSWRR AIRRFDLPWD AMLTIRKPSQ APKGSTSIPL DTPISAFELL
781 STYVELSQPA SKRDLTALAD AAITDADAQA ELRYLASSPT RFTEEIVKKR MSPLDLLIRY
841 PSIKLPVGDF LAMLPPMRVR QYSISSSPLA DPSECSITFS VLNAPALAAA SLPPAERAEA
901 EQYMGVASTY LSELKPGERA HIAVRPSHSG FKPPMDLKAP MIMACAGSGL APFRGFIMDR
961 AEKIRGRRSS VGADGQLPEV EQPAKAILYV GCRTKGKDDI HATELAEWAQ LGAVDVRWAY
1021 SRPEDGSKGR HVQDLMLEDR EELVSLFDQG ARIYVCGSTG VGNGVRQACK DIYLERRRQL
1081 RQAARERGEE VPAEEDEDAA AEQFLDNLRT KERYATDVFT
SEQ ID NO: 8
CYP505A8
A. nidulans FGSC A4
GenBank Accession No. EAA58234
>gi I 40739044 I gb I EAA58234.1 I hypothetical protein AN6835.2 [Aspergillus nidulans FGSC A4 ]
1 MAEIPEPKGL PLIGNIGTID QEFPLGSMVA LAEEHGEIYR LRFPGRTVW VSTHALVNET
61 CDEKRFRKSV NSALAHVREG VHDGLFTAKM GEVNWEIAHR VLMPAFGPLS IRGMFDEMHD
121 IASQLALKWA RYGPDCPIMV TDDFTRLTLD TLALCSMGYR FNSYYSPVLH PFIEAMGDFL
181 TEAGEKPRRP PLPAVFFRNR DQKFQDDIAV LRDTAQGVLQ ARKEGKSDRN DLLSAMLRGV
241 DSQTGQKMTD ESIMDNLITF LIAGHETTSG LLSFVFYQLL KHPETYRTAQ QEVDNWGQG
301 VIEVSHLSKL PYINSVLRET LRLNATIPLF TVEAFEDTLL AGKYPVKAGE TIVNLLAKSH
361 LDPEVYGEDA LEFKPERMSD ELFNARLKQF PSAWKPFGNG MRACIGRPFA WQEALLVMAM
421 LLQNFDFSLA DPNYDLKFKQ TLTIKPKDMF MKARLRHGLT PTTLERRLAG LAVESATQDK
481 IVTNPADNSV TGTRLTILYG SNSGTCETLA RRIAADAPSK GFHVMRFDGL DSGRSALPTD
541 HPWIVTSSY EGQPPENAKQ FVSWLEELEQ QNESLQLKGV DFAVFGCFKE WAQTFHRIPK
601 LVDSLLEKLG GSRLTDLGLA DVSTDELFST FETWADDVLW PRLVAQYGAD GKTQAHGSSA
661 GHEAASNAAV EVTVSNSRTQ ALRQDVGQAM WETRLLTAE SEKERRKKHL EIRLPDGVSY
721 TAGDYLAVLP INPPETVRRA MRQFKLSWDA QITIAPSGPT TALPTDGPIA ANDIFSTYVE
781 LSQPATRKDL RIMADATTDP DVQKILRTYA NETYTAEILT KSISVLDILE QHPAIDLPLG
841 TFLLMLPSMR MRQYSISSSP LLTPTTATIT ISVLDAPSRS RSNGSRHLGV ATSYLDSLSV
901 GDHLQVTVRK NPSSGFRLPS EPETTPMICI AAGSGIAPFR AFLQERAVMM EQDKDRKLAP
961 ALLFFGCRAP GIDDLYREQL EEWQARGWD ARWAFSRQSD DTKGCRHVDD RILADREDW
1021 KLWRDGARVY VCGSGALAQS VRSAMVTVLR DEMETTGDGS DNGKAEKWFD EQRNVRYVMD 1081 VFD
SEQ ID NO: 9
CYP505A3
A. oryzae ATCC42149
Uniprot Accession No. Q2U4F1
>gi I 121928062 I sp I Q2U4F1 I Q2U4F1_ASP0R Cytochrome P450
1 MRQNDNEKQI CPIPGPQGLP FLGNILDIDL DNGTMSTLKI AKTYYPIFKF TFAGETSIVI
61 NSVALLSELC DETRFHKHVS FGLELLRSGT HDGLFTAYDH EKNWELAHRL LVPAFGPLRI
121 REMFPQMHDI AQQLCLKWQR YGPRRPLNLV DDFTRTTLDT IALCAMGYRF NSFYSEGDFH
181 PFIKSMVRFL KEAETQATLP SFISNLRVRA KRRTQLDIDL MRTVCREIVT ERRQTNLDHK
241 NDLLDTMLTS RDSLSGDALS DESIIDNILT FLVAGHETTS GLLSFAVYYL L PDAMAKA
301 AHEVDDWGD QELTIEHLSM LKYLNAILRE TLRLMPTAPG FSVTPYKPEI IGGKYEVKPG
361 DSLDVFLAAV HRDPAVYGSD ADEFRPERMS DEHFQKLPAN SWKPFGNGKR SCIGRAFAWQ
421 EALMILALIL QSFSLNLVDR GYTLKLKESL TIKPDNLWAY ATPRPGRNVL HTRLALQTNS
481 THPEGLMSLK HETVESQPAT ILYGSNSGTC EALAHRLAIE MSSKGRFVCK VQPMDAIEHR
541 RLPRGQPVII ITGSYDGRPP ENARHFVKWL QSLKGNDLEG IQYAVFGCGL PGHHDWSTTF
601 YKIPTLIDTI MAEHGGARLA PRGSADTAED DPFAELESWS ERSVWPGLEA AFDLVRHNSS
661 DGTGKSTRIT IRSPYTLRAA HETAWHQVR VLTSAETTKK VHVELALPDT INYRPGDHLA
721 ILPLNSRQSV QRVLSLFQIG SDTILYMTSS SATSLPTDTP ISAHDLLSGY VELNQVATPT
781 SLRSLAAKAT DEKTAEYLEA LATDRYTTEV RGNHLSLLDI LESYSVPSIE IQHYIQMLPL
841 LRPRQYTISS SPRLNRGQAS LTVSVMERAD VGGPRNCAGV ASNYLASCTP GSILRVSLRQ
901 ANPDFRLPDE SCSHPIIMVA AGSGIAPFRA FVQERSVRQK EGI ILPPAFL FFGCRRADLD
961 DLYREELDAF EEQGWTLFR AFSRAQSESH GCKYVQDLLW MERVRVKTLW GQDAKVFVCG
1021 SVRMNEGVKA IISKIVSPTP TEELARRYIA ETFI
SEQ ID NO: 10
CYPX
A. oryzae ATCC42149
Uniprot Accession No. Q2UNA2
>gi I 121938553 I sp I Q2UNA2 I Q2UNA2_ASPOR Cytochrome P450
1 MSTPKAEPVP IPGPRGVPLM GNILDIESEI PLRSLEMMAD TYGPIYRLTT FGFSRCMISS
61 HELAAEVFDE ERFTKKIMAG LSELRHGIHD GLFTAHMGEE NWEIAHRVLM PAFGPLNIQN
121 MFDEMHDIAT QLVMKWARQG PKQKIMVTDD FTRLTLDTIA LCAMGTRFNS FYSEEMHPFV
181 DAMVGMLKTA GDRSRRPGLV NNLPTTENNK YWEDIDYLRN LCKELVDTRK KNPTDKKDLL
241 NALINGRDPK TGKGMSYDSI IDNMITFLIA GHETTSGSLS FAFYNMLKNP QAYQKAQEEV 301 DRVIGRRRIT VEDLQKLPYI TAVMRETLRL TPTAPAIAVG PHPTKNHEDP
VTLGNGKYVL
361 GKDEPCALLL GKIQRDPKVY GPDAEEFKPE RMLDEHFNKL PKHAWKPFGN
GMRACIGRPF
421 AWQEALLVIA MLLQNFNFQM DDPSYNIQLK QTLTIKPNHF YMRAALREGL
DAVHLGSALS
481 ASSSEHADHA AGHGKAGAAK KGADLKPMHV YYGSNTGTCE AFARRLADDA
TSYGYSAEVE
541 SLDSAKDSIP KNGPWFITA SYEGQPPDNA AHFFEWLSAL KGDKPLDGVN
YAVFGCGHHD
601 WQTTFYRIPK EVNRLVGENG ANRLCEIGLA DTANADIVTD FDTWGETSFW
PAVAAKFGSN
661 TQGSQKSSTF RVEVSSGHRA TTLGLQLQEG LWENTLLTQ AGVPAKRTIR
FKLPTDTQYK
721 CGDYLAILPV NPSTWRKVM SRFDLPWDAV LRIEKASPSS SKHISIPMDT
QVSAYDLFAT
781 YVELSQPASK RDLAVLADAA AVDPETQAEL QAIASDPARF AEISQKRISV
LDLLLQYPSI
841 NLAIGDFVAM LPPMRVRQYS ISSSPLVDPT ECSITFSVLK APSLAALTKE
DEYLGVASTY
901 LSELRSGERV QLSVRPSHTG FKPPTELSTP MIMACAGSGL APFRGFVMDR
AEKIRGRRSS
961 GSMPEQPAKA ILYAGCRTQG KDDIHADELA EWEKIGAVEV RRAYSRPSDG
SKGTHVQDLM
1021 MEDKKELIDL FESGARIYVC GTPGVGNAVR DSIKSMFLER REEIRRIAKE
KGEPVSDDDE
1081 ETAFEKFLDD MKTKERYTTD IFA
SEQ ID NO: 11
CYP505A1
F. oxysporum
Uniprot Accession No. Q9Y8G7
>gi I 22653677 I sp I Q9Y8G7.1 I C505_FUSOX RecName : Full=Bifunctional P- 450 :NADPH-P450 reductase; AltName: Full=Cytochrome P450foxy; AltName: Full=Fatty acid omega-hydroxylase; Includes: RecName: Full=Cytochrome P450 505; Includes: RecName: Full=NADPH--cytochrome P450 reductase
1 maesvpipep pgyplignlg eftsnplsdl nrladtygpi frlrlgakap
ifvssnslin
61 evcdekrfkk tlksvlsqvr egvhdglfta fedepnwgka hrilvpafgp
lsirgmfpem
121 hdiatqlcmk farhgprtpi dtsdnftrla Idtlalcamd frfysyykee
lhpfieamgd
181 fltesgnrnr rppfapnfly raanekfygd ialmksvade vvaarkasps
drkdllaaml
241 ngvdpqtgek lsdenitnql itfliaghet tsgtlsfamy qllknpeays
kvqkevdevv
301 grgpvlvehl tklpyisavl retlrlnspi tafgleaidd tflggkylvk
kgeivtalls
361 rghvdpvvyg ndadkfiper mlddefarln keypncwkpf gngkracigr
pfawqeslla
421 mvvlfqnfnf tmtdpnyale ikqtltikpd hfyinatlrh gmtptelehv
lagngatsss
481 thnikaaanl dakagsgkpm aifygsnsgt cealanrlas dapshgfsat
tvgpldqakq
541 nlpedrpvvi vtasyegqpp snaahfikwm edldgndmek vsyavfacgh
hdwvetfhri
601 pklvdstlek rggtrlvpmg sadaatsdmf sdfeawediv Iwpglkekyk
isdeesggqk
661 gllvevstpr ktslrqdvee alvvaektlt ksgpakkhie iqlpsamtyk
agdylailpl 721 npkstvarvf rrfslawdsf lkiqsegptt lptnvaisaf dvfsayvels qpatkrnila
781 laeatedkdt iqelerlagd ayqaeispkr vsvldllekf pavalpissy lamlppmrvr
841 qysissspfa dpskltltys lldapslsgq grhvgvatnf Ishltagdkl hvsvrassea
901 fhlpsdaekt piicvaagtg laplrgfiqe raamlaagrt lapallffgc rnpeiddlya
961 eeferwekmg avdvrraysr atdksegcky vqdrvyhdra dvfkvwdqga kvficgsrei
1021 gkavedvcvr laiekaqqng rdvteemara wfersrnerf atdvfd
SEQ ID NO: 12
CYPX
G. moniliformis
GenBank Accession No. AAG27132
>gi I 11035011 I gb I AAG27132.1 I Fum6p [Fusarium verticillioides]
1 MSATALFTRR SVSTSNPELR PIPGPKPLPL LGNLFDFDFD NLTKSLGELG KIHGPIYSIT
61 FGASTEIMVT SREIAQELCD ETRFCKLPGG ALDVMKAWG DGLFTAETSN PKWAIAHRII
121 TPLFGAMRIR GMFDDMKDIC EQMCLRWARF GPDEPLNVCD NMTKLTLDTI ALC IDYRFN
181 SFYRENGAAH PFAEAWDVM TESFDQSNLP DFVNNYVRFR AMAKFKRQAA ELRRQTEELI
241 AARRQNPVDR DDLLNAMLSA KDPKTGEGLS PESIVDNLLT FLIAGHETTS SLLSFCFYYL
301 LENPHVLRRV QQEVDTWGS D I VDHLSS MPYLEAVLRE TLRLRDPGPG FYVKPLKDEV
361 VAGKYAVNKD QPLFIVFDSV HRDQSTYGAD ADEFRPERML KDGFDKLPPC AWKPFGNGVR
421 ACVGRPFAMQ QAILAVAMVL HKFDLVKDES YTLKYHVTMT VRPVGFTMKV RLRQGQRATD
481 LAMGLHRGHS QEASAAASPS RASLKRLSSD VNGDDTDHKS QIAVLYASNS GSCEALAYRL
541 AAEATERGFG IRAVDWNNA IDRIPVGSPV ILITASYNGE PADDAQEFVP WLKSLESGRL
601 NGVKFAVFGN GHRDWANTLF AVPRLIDSEL ARCGAERVSL MGVSDTCDSS DPFSDFERWI
661 DEKLFPELET PHGPGGVKNG DRAVPRQELQ VSLGQPPRIT MRKGYVRAIV TEARSLSSPG
721 VPEKRHLELL LPKDFNYKAG DHVYILPRNS PRDWRALSY FGLGEDTLIT IRNTARKLSL
781 GLPLDTPITA TDLLGAYVEL GRTASLKNLW TLVDAAGHGS RAALLSLTEP ERFRAEVQDR
841 HVSILDLLER FPDIDLSLSC FLPMLAQIRP RAYSFSSAPD WKPGHATLTY TWDFA PAT
901 QGINGSSKSK AVGDGTAWQ RQGLASSYLS SLGPGTSLYV SLHRASPYFC LQKSTSLPVI
961 MVGAGTGLAP FRAFLQERRM AAEGAKQRFG PALLFFGCRG PRLDSLYSVE LEAYETIGLV
1021 QVRRAYSRDP SAQDAQGCKY VTDRLGKCRD EVARLWMDGA QVLVCGGKKM ANDVLEVLGP
1081 MLLEIDQKRG ETTAKTWEW RARLDKSRYV EEVYV
SEQ ID NO: 13
CYP505A7
G. zeae PHI
GenBank Accession No. EAA67736 >gi I 42544893 I gb I ΕΑΆ67736.1 I C505_FUSOX Bifunctional P-450 :NADPH-P450 reductase (Fatty acid omega-hydroxylase) (P450foxy) [Gibberella zeae PH-1]
1 MAESVPIPEP PGYPLIGNLG EFKTNPLNDL NRLADTYGPI FRLHLGSKTP
TFVSSNAFIN
61 EVCDEKRFKK TLKSVLSWR EGVHDGLFTA FEDEPNWGKA HRILIPAFGP
LSIRNMFPEM
121 HEIANQLCMK LARHGPHTPV DASDNFTRLA LDTLALCAMD FRFNSYYKEE
LHPFIEAMGD
181 FLLESGNRNR RPAFAPNFLY RAANDKFYAD IALMKSVADE WATRKQNPT
DRKDLLAAML
241 EGVDPQTGEK LSDDNITNQL ITFLIAGHET TSGTLSFAMY HLLKNPEAYN
KLQKEIDEVI
301 GRDPVTVEHL TKLPYLSAVL RETLRISSPI TGFGVEAIED TFLGGKYLIK
KGETVLSVLS
361 RGHVDPWYG PDAEKFVPER MLDDEFARLN KEFPNCWKPF GNGKRACIGR
PFAWQESLLA
421 MALLFQNFNF TQTDPNYELQ IKQNLTIKPD NFFFNCTLRH GMTPTDLEGQ
LAGKGATTSI
481 ASHIKAPAAS KGAKASNGKP MAIYYGSNSG TCEALANRLA SDAAGHGFSA
SVIGTLDQAK
541 QNLPEDRPW IVTASYEGQP PSNAAHFIKW MEDLAGNEME KVSYAVFGCG
HHDWVDTFLR
601 IPKLVDTTLE QRGGTRLVPM GSADAATSDM FSDFEAWEDT VLWPSLKEKY
NVTDDEASGQ
661 RGLLVEVTTP RKTTLRQDVE EALWSEKTL TKTGPAKKHI EIQLPSGMTY
KAGDYLAILP
721 LNPRKTVSRV FRRFSLAWDS FLKIQSDGPT TLPINIAISA FDVFSAYVEL
SQPATKRNIL
781 ALSEATEDKA TIQELEKLAG DAYQEDVSAK KVSVLDLLEK YPAVALPISS
YLAMLPPMRV
841 RQYSISSSPF ADPSKLTLTY SLLDAPSLSG QGRHVGVATN FLSQLIAGDK
LHISVRASSA
901 AFHLPSDPET TPIICVAAGT GLAPFRGFIQ ERAAMLAAGR KLAPALLFFG
CRDPENDDLY
961 AEELARWEQM GAVDVRRAYS RATDKSEGCK YVQDRIYHDR ADVFKVWDQG
AKVFICGSRE
1021 IGKAVEDICV RLAMERSEAT QEGKGATEEK AREWFERSRN ERFATDVFD
SEQ ID NO: 14
CYP505C2
G. zeae PHIa
GenBank Accession No. EAA77183
>gi I 42554340 I gb I EAA77183.1 I hypothetical protein FG07596.1
[Gibberella zeae PH-1]
1 MAIKDGGKKS GQIPGPKGLP VLGNLFDLDL SDSLTSLINI GQKYAPIFSL
ELGGHREVMI
61 CSRDLLDELC DETRFHKIVT GGVDKLRPLA GDGLFTAQHG NHDWGIAHRI
LMPLFGPLKI
121 REMFDDMQDV SEQLCLKWAR LGPSATIDVA NDFTRLTLDT IALCTMGYRF
NSFYSNDKMH
181 PFVDSMVAAL IDADKQSMFP DFIGACRVKA LSAFRKHAAI MKGTCNELIQ
ERRKNPIEGT
241 DLLTAMMEGK DPKTGEGMSD DLIVQNLITF LIAGHETTSG LLSFAFYYLL
ENPHTLEKAR
301 AEVDEWGDQ ALNVDHLTKM PYVNMILRET LRLMPTAPGF FVTPHKDEI I
GGKYAVPANE
361 SLFCFLHLIH RDPKVWGADA EEFRPERMAD EFFEALPKNA WKPFGNGMRG
CIGREFAWQE 421 AKLITVMILQ NFELSKADPS YKLKIKQSLT IKPDGFNMHA KLRNDRKVSG LFKAPSLSSQ
481 QPSLSSRQSI NAINAKDLKP ISIFYGSNTG TCEALAQKLS ADCVASGFMP SKPLPLDMAT
541 KNLSKDGPNI LLAASYDGRP SDNAEEFTKW AESLKPGELE GVQFAVFGCG HKDWVSTYFK
601 IPKILDKCLA DAGAERLVEI GLTDASTGRL YSDFDDWENQ KLFTELSKRQ GVTPTDDSHL
661 ELNVTVIQPQ NNDMGGNFKR AEWENTLLT YPGVSRKHSL LLKLPKDMEY TPGDHVLVLP
721 KNPPQLVEQA MSCFGVDSDT ALTISSKRPT FLPTDTPILI SSLLSSLVEL SQTVSRTSLK
781 RLADFADDDD TKACVERIAG DDYTVEVEEQ RMSLLDILRK YPGINMPLST FLSMLPQMRP
841 RTYSFASAPE WKQGHGMLLF SWEAEEGTV SRPGGLATNY MAQLRQGDSI LVEPRPCRPE
901 LRTTMMLPEP KVPIIMIAVG AGLAPFLGYL QKRFLQAQSQ RTALPPCTLL FGCRGAKMDD
961 ICRAQLDEYS RAGWSVHRA YSRDPDSQCK YVQGLVTKHS ETLAKQWAQG AIVMVCSGKK
1021 VSDGVMNVLS PILFAEEKRS GMTGADSVDV WRQNVPKERM ILEVFG
SEQ ID NO: 15
CYP505A5
M. grisea 70-15 syn
GenBank Accession No. XP 365223
>gi I 145601517 I ref I XP_365223.2 I hypothetical protein MGG_01925 [Magnaporthe oryzae 70-15]
1 MFFLSSSLAY MAATQSRDWA SFGVSLPSTA LGRHLQAAMP FLSEENHKSQ GTVLIPDAQG
61 PIPFLGSVPL VDPELPSQSL QRLARQYGEI YRFVIPGRQS PILVSTHALV NELCDEKRFK
121 KKVAAALLGL REAIHDGLFT AHNDEPNWGI AHRILMPAFG PMAIKGMFDE MHDVASQMIL
181 KWARHGSTTP IMVSDDFTRL TLDTIALCSM GYRFNSFYHD SMHEFIEAMT CWMKESGNKT
241 RRLLPDVFYR TTDKKWHDDA EILRRTADEV LKARKENPSG RKDLLTAMIE GVDPKTGGKL
301 SDSSIIDNLI TFLIAGHETT SGMLSFAFYL LLKNPTAYRK AQQEIDDLCG REPITVEHLS
361 KMPYITAVLR ETLRLYSTIP AFWEAIEDT WGGKYAIPK NHPIFLMIAE SHRDPKVYGD
421 DAQEFEPERM LDGQFERRNR EFPNSWKPFG NGMRGCIGRA FAWQEALLIT AMLLQNFNFV
481 MHDPAYQLSI KENLTLKPDN FYMRAILRHG MSPTELERSI SGVAPTGNKT PPRNATRTSS
541 PDPEDGGIPM SIYYGSNSGT CESLAHKLAV DASAQGFKAE TVDVLDAANQ KLPAGNRGPV
601 VLITASYEGL PPDNAKHFVE WLENLKGGDE LVDTSYAVFG CGHQDWTKTF HRIPKLVDEK
661 LAEHGAVRLA PLGLSNAAHG DMFVDFETWE FETLWPALAD RYKTGAGRQD AAATDLTAAL
721 SQLSVEVSHP RAADLRQDVG EAVWAARDL TAPGAPPKRH MEIRLPKTGG RVHYSAGDYL
781 AVLPVNPKST VERAMRRFGL AWDAHVTIRS GGRTTLPTGA PVSAREVLSS YVELTQPATK
841 RGIAVLAGAV TGGPAAEQEQ AKAALLDLAG DSYALEVSAK RVGVLDLLER FPACAVPFGT 901 FLALLPPMRV RQYSISSSPL WNDEHATLTY SVLSAPSLAD PARTHVGVAS
SYLAGLGEGD
961 HLHVALRPSH VAFRLPSPET PWCVCAGSG MAPFRAFAQE RAALVGAGRK
VAPLLLFFGC
1021 REPGVDDLYR EELEGWEAKG VLSVRRAYSR RTEQSEGCRY VQDRLLKNRA
EVKSLWSQDA
1081 KVFVCGSREV AEGVKEAMFK WAGKEGSSE EVQAWYEEVR NVRYASDIFD
SEQ ID NO: 16
CYP505A2
N. crassa OR74 A
GenBank Accession No. XP 961848
>gi I 85104987 I ref I XP_961848.1 I bifunctional P-450 :NADPH-P450 reductase [Neurospora crassa OR74A]
1 MSSDETPQTI PIPGPPGLPL VGNSFDIDTE FPLGSMLNFA DQYGEIFRLN
FPGRNTVFVT
61 SQALVHELCD EKRFQKTVNS ALHEIRHGIH DGLFTARNDE PNWGIAHRIL
MPAFGPMAIQ
121 NMFPEMHEIA SQLALKWARH GPNQSIKVTD DFTRLTLDTI ALCSMDYRFN
SYYHDDMHPF
181 IDAMASFLVE SGNRSRRPAL PAFMYSKVDR KFYDDIRVLR ETAEGVLKSR
KEHPSERKDL
241 LTAMLDGVDP KTGGKLSDDS I IDNLITFLI AGHETTSGLL SFAFVQLLKN
PETYRKAQKE
301 VDDVCGKGPI KLEHMNKLHY IAAVLRETLR LCPTIPVIGV ESKEDTVIGG
KYEVSKGQPF
361 ALLFAKSHVD PAVYGDTAND FDPERMLDEN FERLNKEFPD CWKPFGNGMR
ACIGRPFAWQ
421 EALLVMAVCL QNFNFMPEDP NYTLQYKQTL TTKPKGFYMR AMLRDGMSAL
DLERRLKGEL
481 VAPKPTAQGP VSGQPKKSGE GKPISIYYGS NTGTCETFAQ RLASDAEAHG
FTATI IDSLD
541 AANQNLPKDR PWFITASYE GQPPDNAALF VGWLESLTGN ELEGVQYAVF
GCGHHDWAQT
601 FHRIPKLVDN TVSERGGDRI CSLGLADAGK GEMFTEFEQW EDEVFWPAME
EKYEVSRKED
661 DNEALLQSGL TVNFSKPRSS TLRQDVQEAV WDAKTITAP GAPPKRHIEV
QLSSDSGAYR
721 SGDYLAVLPI NPKETVNRVM RRFQLAWDTN ITIEASRQTT ILPTGVPMPV
HDVLGAYVEL
781 SQPATKKNIL ALAEAADNAE TKATLRQLAG PEYTEKITSR RVSILDLLEQ
FPSIPLPFSS
841 FLSLLPPMRV RQYSISSSPL WNPSHVTLTY SLLESPSLSN PDKKHVGVAT
SYLASLEAGD
901 KLNVSIRPSH KAFHLPVDAD KTPLIMIAAG SGLAPFRGFV QERAAQIAAG
RSLAPAMLFY
961 GCRHPEQDDL YRDEFDKWES IGAVSVRRAF SRCPESQETK GCKYVGDRLW
EDREEVTGLW
1021 DRGAKVYVCG SREVGESVKK VWRIALERQ KMIVEAREKG ELDSLPEGIV
EGLKLKGLTV
1081 EDVEVSEERA LKWFEGIRNE RYATDVFD
SEQ ID NO: 17
DszA
Rhodococcus erythropolis
GenBank Accession No. AAP80182.1
mtqqrqmhla gffsagnvth ahgawrhtda sndflsgkyy qhiartlerg kfdllflpdg lavedsygdn Idtgvglggq gavalepasv vatmaavteh Iglgatisat yyppyhvarv fatldqlsgg rvswn vtsl ndaearnfgi nqhlehdary dradefleav kklwnswded alvldkaagv fadpakvhyv dhhgewlnvr gplqvprspq gepvilqagl sprgrrfagk waeavfslap nlevmqatyq gikaevdaag rdpdqtkift avmpvlgesq avaqerleyl nslvhpevgl stlsshtgin laaypldtpi kdilrdlqdr nvptqlhmfa aathseeltl aemgrrygtn vgfvpqwagt geqiadelir hfeggaadgf iispaflpgs ydefvdqvvp vlqdrgyfrt eyqgntlrdh lglrvpqlqg qps
SEQ ID NO: 18
DszB
Rhodococcus erythropolis
GenBank Accession No. AAP80183.1
mtsrvdpanp gseldsaird tltysncpvp nalltasesg fldaagield vlsgqqgtvh ftydqpaytr fggeipplls eglrapgrtr llgitpllgr qgffvrddsp itaaadlagr rigvsasair ilrgqlgdyl eldpwrqtlv algswearal lhtlehgelg vddvelvpis spgvdvpaeq leesatvkga dlfpdvargq aavlasgdvd alyswlpwag elqatgarpv vdlglderna yasvwtvssg Ivrqrpglvq rlvdaavdag Iwardhsdav tslhaanlgv stgavgqgfg adfqqrlvpr ldhdalalle rtqqflltnn llqepvaldq waapefInns lnrhr
SEQ ID NO: 20
DszD
Rhodococcus erythropolis
GenBank Accession No. ABV44406.1 msdkpnavss httpdvpeva atpelstgic agdyraalrr hpagvtvvtl dsgtgpvgft atsfssvsle pplvsfniae tsssinalka aeslvihllg ehqqhlaqrf arsadqrfad eslwavldtg epvlhgtpsw mrvkvdqlip vgdhtlvigl vtrvhaeedd esaaapllyh egkyyrptpl gq
References
1. Ruettinger, R.T., L.P. Wen, and A.J. Fulco, Coding nucleotide, 5' regulatory, and deduced amino acid sequences of P-450BM-3, a single peptide cytochrome P-450:NADPH-P-450 reductase from Bacillus megaterium. J Biol Chem, 1989. 264(19): p. 10987-95.
2. Weis, R., et al., A Diversified Library of Bacterial and Fungal Bifunctional Cytochrome P450 Enzymes for Drug Metabolite Synthesis. Advanced
Synthesis & Catalysis, 2009. 351(13): p. 2140-2146.
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