A CYTOCHROME P450-BASED BIODESULFURIZATION PATHWAY

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

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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.