Recombinant host cells, polypeptides and processes for biosynthesis of hydrocortisone

Abstract

The present disclosure relates to recombinant polypeptides with cytochrome P450 (CYP) activity, engineered recombinant host cells (e.g., Pichia) that heterologously express integrated genes encoding these CYP polypeptides, and processes and compositions comprising these recombinant host cells that can be used in the biosynthesis of hydrocortisone, hydrocortisone precursors, and hydrocortisone derivatives.

Description

RECOMBINANT HOST CELLS, POLYPEPTIDES AND PROCESSES FOR BIOSYNTHESIS OF HYDROCORTISONECROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority of US Provisional Application No. 63 / 579,172, filed August 28, 2023, which is hereby incorporated by reference herein.FIELD

[0002] The present disclosure relates to recombinant polypeptides with cytochrome P450 (CYP) activity, engineered recombinant host cells (e.g., Pichia) that heterologously express integrated genes encoding these CYP polypeptides, and processes and compositions comprising these recombinant host cells that can be used in the biosynthesis of hydrocortisone, hydrocortisone precursors, and hydrocortisone derivatives.REFERENCE TO SEQUENCE LISTING

[0003] The official copy of the Sequence Listing is submitted concurrently with the specification via USPTO Patent Center as an WIPO Standard ST.26 formatted XML file with file name “21896-022WO1.xml”, a creation date of August 23, 2024, and a size of 155,628 bytes. This Sequence Listing filed via USPTO Patent Center is part of the specification and is incorporated in its entirety by reference herein.BACKGROUND

[0004] Hydrocortisone (HC) is a corticosteroid with a number of medicinal uses such antiinflammation and immunosuppression. HC is also a precursor compound for the production of a number of hydrocortisone derivative compounds, such as prednisolone and prednisone, which also have medicinal uses.Hydrocortisone (HC)

[0005] Although widely used, the production of HC still relies largely on chemical synthesis which can be inefficient and expensive. The bioconversion of the HC precursor compound, progesterone to HC requires selective hydroxylations at the 21 , 11 p, and 17a positions on the steroid ring.

[0006] Accordingly, there remains a need for more efficient recombinant polypeptides, recombinant cell systems, and biocatalytic methods for the commercially viable biosynthetic production of HC and HC derivative compounds, such as prednisolone and prednisone.SUMMARY

[0007] The present disclosure relates generally to recombinant host cells comprising heterologous genes that express CYP polypeptides with cytochrome P450 activity capable of selectively hydroxylating hydrocortisone (HC) precursor substrates (e.g., progesterone, or 17a-hydroxyprogesterone) at one or more of the 21 , 11 p, and / or 17a positions on the steroid ring. These recombinant host cells (e.g., the methylotrophic yeast Pichia pastoris) are capable of the biosynthetically producing hydrocortisone (HC) from a number of starting HC precursor substrates (e.g., progesterone, 17a-hydroxyprogesterone, 11 p-deoxycortisol, 21- deoxycortisol). The production of hydrocortisone using recombinant host cell strains in a fermentative bioproduction process in aqueous solution allows for an alternative to standard chemical synthesis of these steroid compounds. Additional engineering of the recombinant genes encoding the CYP polypeptides for heterologous expression in recombinant hosts such as Pichia can allow for further improvement in such fermentative bioproduction of HC and related precursor or derivative compounds. The present disclosure provides a number of recombinant host cell strains with different combinations of two or three different recombinant CYP genes incorporated (e.g., on plasmids or genomically integrated) such that the cell is capable of the bioproduction of hydrocortisone provided with a hydrocortisone precursor substrate compound. The disclosure also provides processes of using these recombinant host cells to produce hydrocortisone and related compounds, and compositions of the recombinant host cells and hydrocortisone, hydrocortisone precursor compounds, and hydrocortisone derivatives. This summary is intended to introduce the subject matter of the present disclosure, but does not cover each and every embodiment, combination, or variation that is contemplated and described within the present disclosure. Further embodiments are contemplated and described by the disclosure of the detailed description, drawings, and claims.

[0008] In at least one embodiment, the present disclosure provides a recombinant host cell comprising a heterologous nucleic acid encoding a first CYP capable of converting 17a- hydroxyprogesterone (compound 2) to 11 -deoxycortisol (compound 5), wherein the first CYP comprises an amino acid sequence having at least 80%, at least 85% at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a sequence of SEQ ID NO: 2, and a heterologous nucleic acid encoding a second CYP capable of converting 11- deoxycortisol (compound 5) to hydrocortisone (compound 8), wherein the second CYPcomprises an amino acid sequence having at least 80%, at least 85% at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a sequence selected from SEQ ID NO: 10, and 16. In at least one embodiment, the host cell further comprises a heterologous nucleic acid encoding a third CYP capable of converting progesterone (compound 1) to 17a-hydroxyprogesterone (compound 2), wherein the third CYP comprises an amino acid sequence having at least 80%, at least 85% at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a sequence selected from SEQ ID NO: 6, 36, 38, 40, and 42.

[0009] In at least one embodiment, the present disclosure provides a recombinant host cell comprising a heterologous nucleic acid encoding a first CYP capable of converting 17a- hydroxyprogesterone (compound 2) to 21 -deoxycortisol (compound 6), wherein the first CYP comprises an amino acid sequence having at least 80%, at least 85% at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a sequence selected from SEQ ID NO: 10 or 16, and a heterologous nucleic acid encoding a second CYP capable of converting 21 -deoxycortisol (compound 6) to hydrocortisone (compound 8), wherein the second CYP comprises an amino acid sequence having at least 80%, at least 85% at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a sequence of SEQ ID NO: 2. In at least one embodiment, the host cell further comprises a heterologous nucleic acid encoding a third CYP capable of converting progesterone (compound 1) to 17a- hydroxyprogesterone (compound 2), wherein the third CYP comprises an amino acid sequence having at least 80%, at least 85% at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a sequence selected from SEQ ID NO: 6, 36, 38, 40, and 42.

[0010] In at least one embodiment, the present disclosure provides a recombinant host cell comprising a heterologous nucleic acid encoding a first CYP capable of converting 11 - hydroxyprogesterone (compound 3) to 21 -deoxycortisol (compound 6), wherein the first CYP comprises an amino acid sequence having at least 80%, at least 85% at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a sequence selected from SEQ ID NO: 6, 36, 38, 40, and 42, and a heterologous nucleic acid encoding a second CYP capable of converting 21 -deoxycortisol (compound 6) to hydrocortisone (compound 8), wherein the second CYP comprises an amino acid sequence having at least 80%, at least85% at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a sequence of SEQ ID NO: 2. In at least one embodiment, the host cell further comprises a heterologous nucleic acid encoding a third CYP capable of converting progesterone (compound 1) to 110-hydroxyprogesterone (compound 3), wherein the third CYP comprises an amino acid sequence having at least 80%, at least 85% at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a sequence selected from SEQ ID NO: 10 or 16.

[0011] In at least one embodiment, the present disclosure provides a recombinant host cell comprising a heterologous nucleic acid encoding a first CYP capable of converting 110- hydroxyprogesterone (compound 3) to corticosterone (compound 7), wherein the first CYP comprises an amino acid sequence having at least 80%, at least 85% at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a sequence of SEQ ID NO: 2, and a heterologous nucleic acid encoding a second CYP capable of converting corticosterone (compound 7) to hydrocortisone (compound 8), wherein the second CYP comprises an amino acid sequence having at least 80%, at least 85% at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a sequence selected from SEQ ID NO: 6, 36, 38, 40, and 42. In at least one embodiment, the host cell further comprises a heterologous nucleic acid encoding a third CYP capable of converting progesterone (compound 1) to 110-hydroxyprogesterone (compound 3), wherein the third CYP comprises an amino acid sequence having at least 80%, at least 85% at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a sequence selected from SEQ ID NO: 10 or 16.

[0012] In at least one embodiment, the present disclosure provides a recombinant host cell comprising a heterologous nucleic acid encoding a first CYP capable of converting 21- hydroxyprogesterone (compound 4) to 11 -deoxycortisol (compound 5), wherein the first CYP comprises an amino acid sequence having at least 80%, at least 85% at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a sequence selected from SEQ ID NO: 6, 36, 38, 40, and 42, and a heterologous nucleic acid encoding a second CYP capable of converting 11 -deoxycortisol (compound 5) to hydrocortisone (compound 8), wherein the second CYP comprises an amino acid sequence having at least 80%, at least 85% at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a sequence selected from SEQ ID NO: 10, and 16. In at least one embodiment, the host cell further comprises a heterologous nucleic acid encoding a third CYP capable of converting progesterone (compound 1) to 21 -hydroxyprogesterone (compound 4), wherein the third CYP comprises an amino acid sequence having at least 80%, at least 85% at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a sequence of SEQ ID NO: 2.

[0013] In at least one embodiment, the present disclosure provides a recombinant host cell comprising a heterologous nucleic acid encoding a first CYP capable of converting 21- hydroxyprogesterone (compound 4) to corticosterone (compound 7), wherein the first CYP comprises an amino acid sequence having at least 80%, at least 85% at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a sequence selected from SEQ ID NO: 10 or 16, and a heterologous nucleic acid encoding a second CYP capable of converting corticosterone (compound 7) to hydrocortisone (compound 8), wherein the second CYP comprises an amino acid sequence having at least 80%, at least 85% at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a sequence selected from SEQ ID NO: 6, 36, 38, 40, and 42. In at least one embodiment, the host cell further comprises a heterologous nucleic acid encoding a third CYP capable of converting progesterone (compound 1) to 21 -hydroxyprogesterone (compound 4), wherein the third CYP comprises an amino acid sequence having at least 80%, at least 85% at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a sequence of SEQ ID NO: 2.

[0014] In any of the embodiments of the recombinant host cells of the present disclosure, the host cell can further comprise a heterologous nucleic acid encoding one or more CPR polypeptides capable of transferring electrons to the first CYP, the second CYP, and / or the third CYP; optionally, wherein the CPR comprises an amino acid sequence having at least 80%, at least 85% at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a sequence selected from SEQ ID NO: 4, 8, 12, 44, 46, and 48.

[0015] In at least one embodiment of the recombinant host cells of the present disclosure, the host cell further comprises a heterologous nucleic acid encoding an enzyme with 3- ketosteroid-A1 -dehydrogenase (“KstD”) activity; optionally, wherein the KstD comprises an amino acid sequence having at least 80%, at least 85% at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a sequence selected from SEQ ID NO: 50, 52, and 54. In at least one embodiment, the host cell further comprises a heterologous nucleic acid encoding an enzyme with 11 p-hydroxysteroid dehydrogenase type 2 (“HSD2”) activity; optionally, wherein the HSD2 comprises an amino acid sequence having at least 80%, at least 85% at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a sequence of SEQ ID NO: 56.

[0016] In any of the embodiments of the recombinant host cells of the present disclosure, the source organism of the host cell can be selected from Pichia pastoris, Saccharomyces cerevisiae, Yarrowia lipolytica, and Escherichia coli.

[0017] In at least one embodiment of a recombinant host of the present disclosure, the heterologous nucleic acid encoding the first, second, and / or third CYP is integrated at one or more sites in the genome of the cell; optionally, integrated at three or more sites. In at least one embodiment, the heterologous nucleic acid encoding the first, second, and / or third CYP is under the control of a promoter system selected from pCAT1 :pFDH1 , pAOX1 and pGal1 / 10. In at least one embodiment, the recombinant host cell is Pichia pastoris and the heterologous nucleic acid is integrated at one or more sites in the genome selected from HIS4, I nt 15, I nt 18, I nt 1 , I nt6 , and AOX1 . In at least one embodiment, the recombinant host cell is Saccharomyces cerevisiae and the heterologous nucleic acid is integrated at one or more sites in the genome selected from X-2, X-4, XI-2, XII-4, NDE1 , XII-5, Gal80, and ROQ1.

[0018] In at least one embodiment, the present disclosure also provides processes using the recombinant host cells for preparing hydrocortisone, a hydrocortisone precursor compound, and / or a hydrocortisone derivative compound, the process comprising: (a) contacting under suitable conditions a recombinant host cell of the present disclosure comprising two or three different heterologous genes encoding two or three different CYP polypeptides, and a substrate compound selected from progesterone (compound 1), 17a-hydroxyprogesterone (compound 2), 1 i p-hydroxyprogesterone (compound 3), 21 -hydroxyprogesterone (compound 4), 1 1 -deoxycortisol (compound 5), 21 -deoxycortisol (compound 6), and corticosterone (compound 7); and (b) recovering the hydrocortisone, hydrocortisone precursor, and / or a hydrocortisone derivative product compound.

[0019] In at least one embodiment of the process, the substrate compound is progesterone (compound 1), and the product compound is hydrocortisone (compound 8). In at least one embodiment of the process, the substrate compound is selected from 17a- hydroxyprogesterone (compound 2), 1 i p-hydroxyprogesterone (compound 3), and 21- hydroxyprogesterone (compound 4); and the product compound is hydrocortisone(compound 8). In at least one embodiment of the process, the substrate compound is selected from 11 -deoxycortisol (compound 5), 21 -deoxycortisol (compound 6), and corticosterone (compound 7); and the product compound is hydrocortisone (compound 8).

[0020] In another embodiment, the present disclosure also provides processes for the preparation of hydrocortisone where the bioconversion process is carried out by contacting a hydrocortisone precursor substrate with two or three different recombinant host cell strains, each incorporating a different heterologous nucleic acid encoding a different CYP polypeptide.

[0021] In at least one embodiment, the present disclosure provides a process for preparing hydrocortisone (compound 8) comprising: (a) contacting 17a-hydroxyprogesterone (compound 2) under suitable conditions with: (i) a recombinant host cell comprising a heterologous nucleic acid encoding a first CYP capable of converting 17a- hydroxyprogesterone (compound 2) to 11 -deoxycortisol (compound 5), wherein the first CYP comprises an amino acid sequence having at least 80%, at least 85% at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a sequence of SEQ ID NO: 2, and (ii) a recombinant host cell comprising a heterologous nucleic acid encoding a second CYP capable of converting 11 -deoxycortisol (compound 5) to hydrocortisone (compound 8), wherein the second CYP comprises an amino acid sequence having at least 80%, at least 85% at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a sequence selected from SEQ ID NO: 10, and 16; and (b) recovering the produced hydrocortisone. In at least one embodiment, the process further comprises contacting progesterone under suitable conditions with a recombinant host cell comprising a heterologous nucleic acid encoding a third CYP capable of converting progesterone (compound 1) to 17a-hydroxyprogesterone (compound 2), wherein the third CYP comprises an amino acid sequence having at least 80%, at least 85% at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a sequence selected from SEQ ID NO: 6, 36, 38, 40, and 42.

[0022] In at least one embodiment, the present disclosure provides a process for preparing hydrocortisone (compound 8) comprising: (a) contacting 17a-hydroxyprogesterone (compound 2) under suitable conditions with: (i) a recombinant host cell comprising a heterologous nucleic acid encoding a first CYP capable of converting 17a- hydroxyprogesterone (compound 2) to 21 -deoxycortisol (compound 6), wherein the first CYP comprises an amino acid sequence having at least 80%, at least 85% at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or at least 99.5% sequence identity to a sequence selected from SEQ ID NO: 10 or 16, and (ii) a recombinant host cell comprising a heterologous nucleic acid encoding a second CYP capable of converting 21 -deoxycortisol (compound 6) to hydrocortisone (compound 8), wherein the second CYP comprises an amino acid sequence having at least 80%, at least 85% at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a sequence of SEQ ID NO: 2; and (b) recovering the produced hydrocortisone. In at least one embodiment, the process further comprises contacting progesterone under suitable conditions with a recombinant host cell comprising a heterologous nucleic acid encoding a third CYP capable of converting progesterone (compound 1) to 17a-hydroxyprogesterone (compound 2), wherein the third CYP comprises an amino acid sequence having at least 80%, at least 85% at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a sequence selected from SEQ ID NO: 6, 36, 38, 40, and 42.

[0023] In at least one embodiment, the present disclosure provides a process for preparing hydrocortisone (compound 8) comprising: (a) contacting 1 ip-hydroxyprogesterone (compound 3) under suitable conditions with: (i) a recombinant host cell comprising a heterologous nucleic acid encoding a first CYP capable of converting 11 p- hydroxyprogesterone (compound 3) to 21 -deoxycortisol (compound 6), wherein the first CYP comprises an amino acid sequence having at least 80%, at least 85% at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a sequence selected from SEQ ID NO: 6, 36, 38, 40, and 42, and (ii) a recombinant host cell comprising a heterologous nucleic acid encoding a second CYP capable of converting 21 -deoxycortisol (compound 6) to hydrocortisone (compound 8), wherein the second CYP comprises an amino acid sequence having at least 80%, at least 85% at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a sequence of SEQ ID NO: 2; and (b) recovering the produced hydrocortisone. In at least one embodiment, the process further comprises contacting progesterone under suitable conditions with a recombinant host cell comprising a heterologous nucleic acid encoding a third CYP capable of converting progesterone (compound 1) to 11 p-hydroxyprogesterone (compound 3), wherein the third CYP comprises an amino acid sequence having at least 80%, at least 85% at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a sequence selected from SEQ ID NO: 10 or 16.

[0024] In at least one embodiment, the present disclosure provides a process for preparing hydrocortisone (compound 8) comprising: (a) contacting 1 ip-hydroxyprogesterone (compound 3) under suitable conditions with: (i) a recombinant host cell comprising a heterologous nucleic acid encoding a first CYP capable of converting 1113- hydroxyprogesterone (compound 3) to corticosterone (compound 7), wherein the first CYP comprises an amino acid sequence having at least 80%, at least 85% at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a sequence of SEQ ID NO: 2, and (ii) a recombinant host cell comprising a heterologous nucleic acid encoding a second CYP capable of converting corticosterone (compound 7) to hydrocortisone (compound 8), wherein the second CYP comprises an amino acid sequence having at least 80%, at least 85% at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a sequence selected from SEQ ID NO: 6, 36, 38, 40, and 42; and (b) recovering the produced hydrocortisone. In at least one embodiment, the process further comprises contacting progesterone under suitable conditions with a recombinant host cell comprising a heterologous nucleic acid encoding a third CYP capable of converting progesterone (compound 1) to 11 p-hydroxyprogesterone (compound 2), wherein the third CYP comprises an amino acid sequence having at least 80%, at least 85% at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a sequence selected from SEQ ID NO: 10 or 16.

[0025] In at least one embodiment, the present disclosure provides a process for preparing hydrocortisone (compound 8) comprising: (a) contacting 21-hydroxyprogesterone (compound 4) under suitable conditions with: (i) a recombinant host cell comprising a heterologous nucleic acid encoding a first CYP capable of converting 21- hydroxyprogesterone (compound 4) to 11 -deoxycortisol (compound 5), wherein the first CYP comprises an amino acid sequence having at least 80%, at least 85% at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a sequence selected from SEQ ID NO: 6, 36, 38, 40, and 42, and (ii) a recombinant host cell comprising a heterologous nucleic acid encoding a second CYP capable of converting 11 -deoxycortisol (compound 5) to hydrocortisone (compound 8), wherein the second CYP comprises an amino acid sequence having at least 80%, at least 85% at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a sequence selected from SEQ ID NO: 10, and 16; and (b) recovering the produced hydrocortisone. In at least one embodiment, theprocess further comprises contacting progesterone under suitable conditions with a recombinant host cell comprising a heterologous nucleic acid encoding a third CYP capable of converting progesterone (compound 1) to 21-hydroxyprogesterone (compound 4), wherein the third CYP comprises an amino acid sequence having at least 80%, at least 85% at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a sequence of SEQ ID NO: 2.

[0026] In at least one embodiment, the present disclosure provides a process for preparing hydrocortisone (compound 8) comprising: (a) contacting 21-hydroxyprogesterone (compound 4) under suitable conditions with: (i) a recombinant host cell comprising a heterologous nucleic acid encoding a first CYP capable of converting 21- hydroxyprogesterone (compound 4) to corticosterone (compound 7), wherein the first CYP comprises an amino acid sequence having at least 80%, at least 85% at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a sequence selected from SEQ ID NO: 10 or 16, and (ii) a recombinant host cell comprising a heterologous nucleic acid encoding a second CYP capable of converting corticosterone (compound 7) to hydrocortisone (compound 8), wherein the second CYP comprises an amino acid sequence having at least 80%, at least 85% at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a sequence selected from SEQ ID NO: 6, 36, 38, 40, and 42; and (b) recovering the produced hydrocortisone. In at least one embodiment, the process further comprises contacting progesterone under suitable conditions with a recombinant host cell comprising a heterologous nucleic acid encoding a third CYP capable of converting progesterone (compound 1) to 21-hydroxyprogesterone (compound 4), wherein the third CYP comprises an amino acid sequence having at least 80%, at least 85% at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a sequence of SEQ ID NO: 2.

[0027] In at least one embodiment of the processes of the present disclosure, the process further comprises: (iii) a recombinant host cell comprising a heterologous nucleic acid encoding an enzyme with 3-ketosteroid-A1 -dehydrogenase (“KstD”) activity capable of converting hydrocortisone (8) to the hydrocortisone derivative prednisolone (9), wherein the KstD comprises an amino acid sequence having at least 80%, at least 85% at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a sequence selected from SEQ ID NO: 50, 52, and 54; and recovering the produced hydrocortisone derivativeprednisolone (9). In at least one embodiment of the processes of the present disclosure, the process further comprises: (iv) a recombinant host cell comprising a heterologous nucleic acid encoding an enzyme with 11 p-hydroxysteroid dehydrogenase type 2 (“HSD2”) activity capable of converting the hydrocortisone derivative prednisolone (9) to prednisone (10), wherein the HSD2 comprises an amino acid sequence having at least 80%, at least 85% at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a sequence of SEQ ID NO: 56; and recovering the produced hydrocortisone derivative prednisone (10).

[0028] In at least one embodiment, the present disclosure also provides a reaction mixture composition comprising a recombinant host cell of the present disclosure and a HC precursor substrate. Accordingly, the present also provides a composition in aqueous solution comprising: (i) a recombinant host cell of the present disclosure comprising one, two, and / or three integrated heterologous nucleic acids encoding a first, second, and / or third CYP; and (ii) a compound selected from: progesterone (compound 1), 17a- hydroxyprogesterone (compound 2), 1 ip-hydroxyprogesterone (compound 3), 21- hydroxyprogesterone (compound 4), 11 -deoxycortisol (compound 5), 21 -deoxycortisol (compound 6), corticosterone (compound 7), hydrocortisone (compound 8), and / or a mixture thereof.BRIEF DESCRIPTION OF THE DRAWINGS

[0029] A better understanding of the novel features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings (also “Figure” and “FIG.” herein), of which:

[0030] FIG. 1A, FIG. 1B, and FIG. 1C depict schemes for twelve reactions (Reaction 1 - Reaction 12) related to the biosynthesis of hydrocortisone (HC) from progesterone via the CYP enzyme catalyzed hydroxylation of the 11 p, 17a, and / or 21 positions of progesterone, and other mono- and di- hydroxylated HC precursor compounds, including other 11 p- hydroxyprogesterone, 17a-hydroxyprogesterone, 21 -hydroxyprogesterone, 11 -deoxycortisol, and 21 -deoxycortisol.

[0031] FIG. 2 is a schematic depiction of the combinatorial assembly of CYP-CPR gene pairs on a plasmid to screen for production of 17a-hydroxyprogesterone as described in Example 3.DETAILED DESCRIPTION

[0032] For the descriptions herein and the appended claims, the singular forms “a”, and “an” include plural referents unless the context clearly indicates otherwise. Thus, for example,reference to “a protein” includes more than one protein, and reference to “a compound” refers to more than one compound. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation. The use of “comprise,” “comprises,” “comprising” “include,” “includes,” and “including” are interchangeable and not intended to be limiting. It is to be further understood that where descriptions of various embodiments use the term “comprising,” those skilled in the art would understand that in some specific instances, an embodiment can be alternatively described using language “consisting essentially of’ or “consisting of.”

[0033] Where a range of values is provided, unless the context clearly dictates otherwise, it is understood that each intervening integer of the value, and each tenth of each intervening integer of the value, unless the context clearly dictates otherwise, between the upper and lower limit of that range, and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of these limits, ranges excluding (i) either or (ii) both of those included limits are also included in the invention. For example, “1 to 50,” includes “2 to 25,” “5 to 20,” “25 to 50,” “1 to 10,” etc.

[0034] Generally, the nomenclature used herein and the techniques and procedures described herein include those that are well understood and commonly employed by those of ordinary skill in the art, such as the common techniques and methodologies described in e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manual (Fourth Edition), Vols. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 2012 (hereinafter “Sambrook”); and Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., originally published in 1987 in book form by Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., and regularly supplemented through 2011 , and now available in journal format online as Current Protocols in Molecular Biology, Vols. 00 - 130, (1987-2020), published by Wiley & Sons, Inc. in the Wiley Online Library (hereinafter “Ausubel”).

[0035] All publications, patents, patent applications, and other documents referenced in this disclosure are hereby incorporated by reference in their entireties for all purposes to the same extent as if each individual publication, patent, patent application or other document were individually indicated to be incorporated by reference herein for all purposes.

[0036] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains. It is to be understood that the terminology used herein is fordescribing particular embodiments only and is not intended to be limiting. For purposes of interpreting this disclosure, the following description of terms will apply and, where appropriate, a term used in the singular form will also include the plural form and vice versa.

[0037] Definitions

[0038] “Hydrocortisone” or “HC” refers to the compound hydrocortisone having the chemical structure of compound 8 (below).

[0039] “HC precursor” as used herein refers to a compound capable of being converted into HC, or a HC derivative, by an enzyme capable producing HC alone, or in combination with another enzyme or a non-enzymatic chemical reaction. HC precursors as referenced in the present disclosure include, but are not limited to, the exemplary compounds summarized in Table 1 (below).

[0040] TABLE 1: Exemplary HC precursor compounds

[0041] “HC derivatives” as referenced in the present disclosure include structural analogs of hydrocortisone (compound 8), including but not limited to the exemplary corticosteroid compounds listed in Table 2 (below).

[0043] “CYP activity” as used herein refers to the catalytic activity of a cytochrome P450 monooxygenase enzyme.

[0044] “CPR activity” as used herein refers to the catalytic activity of a cytochrome P450 reductase enzyme.

[0045] “Conversion” as used herein refers to the enzymatic conversion of a substrate(s) to a corresponding product(s). “Percent conversion” refers to the percent of the substrate that is converted to the product within a period of time under specified conditions. Thus, the “enzymatic activity” or “activity” of an enzymatic conversion can be expressed as “percent conversion” of the substrate to the product.

[0046] “Substrate” as used herein in the context of an enzyme mediated process refers to the compound or molecule acted on by the enzyme.

[0047] “Product” as used herein in the context of an enzyme mediated process refers to the compound or molecule resulting from the activity of the enzyme.

[0048] “Host cell” as used herein refers to a cell capable of being functionally modified with recombinant nucleic acids and functioning to express recombinant products, including polypeptides and compounds produced by activity of the polypeptides.

[0049] “Nucleic acid,” or “polynucleotide” as used herein interchangeably to refer to two or more nucleosides that are covalently linked together. The nucleic acid may be wholly comprised ribonucleosides (e.g., RNA), wholly comprised of 2'-deoxyribonucleotides (e.g., DNA) or mixtures of ribo- and 2'-deoxyribonucleosides. The nucleoside units of the nucleicacid can be linked together via phosphodiester linkages (e.g., as in naturally occurring nucleic acids), or the nucleic acid can include one or more non-natural linkages (e.g., phosphorothioester linkage). Nucleic acid or polynucleotide is intended to include singlestranded or double-stranded molecules, or molecules having both single-stranded regions and double-stranded regions. Nucleic acid or polynucleotide is intended to include molecules composed of the naturally occurring nucleobases (i.e., adenine, guanine, uracil, thymine, and cytosine), or molecules comprising that include one or more modified and / or synthetic nucleobases, such as, for example, inosine, xanthine, hypoxanthine, etc.

[0050] “Protein,” “polypeptide,” and “peptide” are used herein interchangeably to denote a polymer of at least two amino acids covalently linked by an amide bond, regardless of length or post-translational modification (e.g., glycosylation, phosphorylation, lipidation, myristilation, ubiquitination, etc.). As used herein “protein” or “polypeptide” or “peptide” polymer can include D- and L-amino acids, and mixtures of D- and L-amino acids.

[0051] “Naturally-occurring” or “wild-type” as used herein refers to the form as found in nature. For example, a naturally occurring nucleic acid sequence is the sequence present in an organism that can be isolated from a source in nature, and which has not been intentionally modified by human manipulation.

[0052] “Recombinant,” “engineered,” or “non-naturally occurring” when used herein with reference to, e.g., a cell, nucleic acid, or polypeptide, refers to a material, or a material corresponding to the natural or native form of the material, that has been modified in a manner that would not otherwise exist in nature, or is identical thereto but is produced or derived from synthetic materials and / or by manipulation using recombinant techniques. Non-limiting examples include, among others, recombinant cells expressing genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise expressed at a different level.

[0053] “Nucleic acid derived from” as used herein refers to a nucleic acid having a sequence at least substantially identical to a sequence of found in naturally in an organism. For example, cDNA molecules prepared by reverse transcription of mRNA isolated from an organism, or nucleic acid molecules prepared synthetically to have a sequence at least substantially identical to, or which hybridizes to a sequence at least substantially identical to a nucleic sequence found in an organism.

[0054] “Coding sequence” refers to that portion of a nucleic acid (e.g., a gene) that encodes an amino acid sequence of a protein.

[0055] “Heterologous nucleic acid” as used herein refers to any polynucleotide that is introduced into a host cell by laboratory techniques and includes polynucleotides that are removed from a host cell, subjected to laboratory manipulation, and then reintroduced into a host cell.

[0056] “Codon degenerate” describes a nucleotide sequence that has one or more different codons relative to the reference nucleotide sequence, but which encodes a polypeptide that is identical to the polypeptide encoded by a reference nucleotide sequence. The different codons between the nucleotide sequence and the reference nucleotide sequence are called “synonyms” or “synonymous” codons in that they use different triplets of nucleotides to encode the same amino acid in a polypeptide.

[0057] “Codon optimized” refers to changes in the codons of the polynucleotide encoding a protein to those preferentially used in a particular organism such that the encoded protein is efficiently expressed in the organism of interest. Although the genetic code is degenerate in that most amino acids are represented by several different “synonymous” codons, it is well known that codon usage by particular organisms is nonrandom and biased towards particular codon triplets. This codon usage bias may be higher in reference to a given gene, genes of common function or ancestral origin, highly expressed proteins versus low copy number proteins, and the aggregate protein coding regions of an organism's genome. In some embodiments, the polynucleotides encoding the imine reductase enzymes may be codon optimized for optimal production from the host organism selected for expression.

[0058] “Preferred, optimal, high codon usage bias codons” refers to codons that are used at higher frequency in the protein coding regions than other codons that code for the same amino acid. The preferred codons may be determined in relation to codon usage in a single gene, a set of genes of common function or origin, highly expressed genes, the codon frequency in the aggregate protein coding regions of the whole organism, codon frequency in the aggregate protein coding regions of related organisms, or combinations thereof. Codons whose frequency increases with the level of gene expression are typically optimal codons for expression. A variety of methods are known for determining the codon frequency (e.g., codon usage, relative synonymous codon usage) and codon preference in specific organisms, including multivariate analysis, for example, using cluster analysis or correspondence analysis, and the effective number of codons used in a gene (see GCG CodonPreference, Genetics Computer Group Wisconsin Package; CodonW, John Peden, University of Nottingham; McInerney, J. O, 1998, Bioinformatics 14:372-73; Stenico et al., 1994, Nucleic Acids Res. 222437-46; Wright, F., 1990, Gene 87:23-29). Codon usage tables are available for a growing list of organisms (see for example, Wada et al., 1992, Nucleic Acids Res. 20:2111-2118; Nakamura et al., 2000, Nucl. Acids Res. 28:292; Duret, et al., supra; Henaut and Danchin, "Escherichia coli and Salmonella," 1996, Neidhardt, et al. Eds., ASM Press, Washington D.C., p. 2047-2066. The data source for obtaining codon usage may rely on any available nucleotide sequence capable of coding for a protein. These data sets include nucleic acid sequences actually known to encode expressed proteins (e.g., complete protein coding sequences-CDS), expressed sequence tags (ESTS), or predictedcoding regions of genomic sequences (see for example, Mount, D., Bioinformatics: Sequence and Genome Analysis, Chapter 8, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001 ; Uberbacher, E. C., 1996, Methods Enzymol. 266:259-281 ; Tiwari et al., 1997, Comput. Appl. Biosci. 13:263-270).

[0059] “Control sequence’’ as used herein refers to all sequences, which are necessary or advantageous for the expression of a polynucleotide and / or polypeptide as used in the present disclosure. Each control sequence may be native or foreign to the nucleic acid sequence encoding a polypeptide. Such control sequences include, but are not limited to, a leader, a promoter, a polyadenylation sequence, a pro-peptide sequence, a signal peptide sequence, and a transcription terminator. At a minimum, control sequences typically include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the nucleic acid sequence encoding a polypeptide.

[0060] “Operably linked’’ as used herein refers to a configuration in which a control sequence is appropriately placed (e.g., in a functional relationship) at a position relative to a polynucleotide sequence or polypeptide sequence of interest such that the control sequence directs or regulates the expression of the sequence of interest.

[0061] “Promoter sequence” refers to a nucleic acid sequence that is recognized by a host cell for expression of a polynucleotide of interest, such as a coding sequence. The promoter sequence contains transcriptional control sequences, which mediate the expression of a polynucleotide of interest. The promoter may be any nucleic acid sequence which shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.

[0062] “Percentage of sequence identity,” “percent sequence identity,” “percentage homology,” or “percent homology” are used interchangeably herein to refer to values quantifying comparisons of the sequences of polynucleotides or polypeptides, and are determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (or gaps) as compared to the reference sequence for optimal alignment of the two sequences. The percentage values may be calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Alternatively, the percentage may be calculated by determining the number of positions at which either theidentical nucleic acid base or amino acid residue occurs in both sequences or a nucleic acid base or amino acid residue is aligned with a gap to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Those of skill in the art appreciate that there are many established algorithms available to align two sequences. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman, 1981 , Adv. Appl. Math. 2:482, by the homology alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin Software Package), or by visual inspection (see generally, Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1995 Supplement) (Ausubel)). Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., 1990, J. Mol. Biol. 215: 403-410 and Altschul et al., 1977, Nucleic Acids Res. 3389-3402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information website. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as, the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11 , an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, 1989, Proc Natl Acad Sci USA89:10915). Exemplary determination of sequence alignment and % sequence identity can employ the BESTFIT or GAP programs in the GCG Wisconsin Software package (Accelrys, Madison Wis.), using default parameters provided.

[0063] “Reference sequence” refers to a defined sequence used as a basis for a sequence comparison. A reference sequence may be a subset of a larger sequence, for example, a segment of a full-length nucleic acid or polypeptide sequence. A reference sequence typically is at least 20 nucleotide or amino acid residue units in length but can also be the full length of the nucleic acid or polypeptide. Since two polynucleotides or polypeptides may each (1) comprise a sequence (i.e., a portion of the complete sequence) that is similar between the two sequences, and (2) may further comprise a sequence that is divergent between the two sequences, sequence comparisons between two (or more) polynucleotides or polypeptide are typically performed by comparing sequences of the two polynucleotides or polypeptides over a “comparison window” to identify and compare local regions of sequence similarity. “Comparison window” refers to a conceptual segment of at least about 20 contiguous nucleotide positions or amino acids residues wherein a sequence may be compared to a reference sequence of at least 20 contiguous nucleotides or amino acids and wherein the portion of the sequence in the comparison window may comprise additions or deletions (or gaps) of 20 percent or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.

[0064] “Substantial identity” or “substantially identical” refers to a polynucleotide or polypeptide sequence that has at least 70% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95 % sequence identity, or at least 99% sequence identity, as compared to a reference sequence over a comparison window of at least 20 nucleoside or amino acid residue positions, frequently over a window of at least 30-50 positions, wherein the percentage of sequence identity is calculated by comparing the reference sequence to a sequence that includes deletions or additions which total 20 percent or less of the reference sequence over the window of comparison.

[0065] “Corresponding to,” “reference to,” or “relative to” when used in the context of the numbering of a given amino acid or polynucleotide sequence refers to the numbering of the residues of a specified reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence. In other words, the residue number or residue position of a given polymer is designated with respect to the reference sequence rather than by the actual numerical position of the residue within the given amino acid or polynucleotide sequence. For example, a given amino acid sequence, such as that of an engineered imine reductase, can be aligned to a reference sequence by introducing gaps to optimize residue matches between the two sequences. In these cases, although the gapsare present, the numbering of the residue in the given amino acid or polynucleotide sequence is made with respect to the reference sequence to which it has been aligned.

[0066] “Isolated” as used herein in reference to a molecule means that the molecule (e.g., HC, polynucleotide, polypeptide) is substantially separated from other compounds that naturally accompany it, e.g., protein, lipids, and polynucleotides. The term embraces nucleic acids which have been removed or purified from their naturally occurring environment or expression system (e.g., host cell).

[0067] “Substantially pure” refers to a composition in which a desired molecule is the predominant species present (i.e. , on a molar or weight basis it is more abundant than any other individual macromolecular species in the composition) and is generally a substantially purified composition when the object species comprises at least about 50 percent of the macromolecular species present by mole or % weight.

[0068] “Recovered” as used herein in relation to an enzyme, protein, or compound (e.g., HC), refers to a more or less pure form of the enzyme, protein, or compound.

[0069] Genes Encoding Recombinant Polypeptides with CYP Activity

[0070] The present disclosure provides genes encoding polypeptides with cytochrome P450 (CYP) monooxygenase activity capable of selectively hydroxylating one or more of the hydrocortisone precursor substrate compounds, progesterone (compound 1), 17a- hydroxyprogesterone (compound 2), 1 ip-hydroxyprogesterone (compound 3), 21- hydroxyprogesterone (compound 4), 11 -deoxycortisol (compound 5), 21 -deoxycortisol (compound 6), corticosterone (compound 7), to another hydrocortisone precursor compound, or to hydrocortisone (compound 8), as illustrated by the bioconversion Reactions 1-12 depicted in FIGS. 1A-1C. The bioconversion Reactions 1-12 of FIGS. 1A-1C all involve selective hydroxylation of the 11 p, 17a, or 21 position of one or more of the hydrocortisone precursor compounds 1-7, where the hydroxylation is catalyzed by a CYP polypeptide.Typically, the CYP catalysis occurs in the presence of an electron transfer agent or catalyst, such as cytochrome P450 reductase (CPR) enzyme, as well as the cofactor NADPH, and oxygen.

[0071] As described elsewhere herein, the screening of candidate CYP polypeptides for the desired CYP activity when expressed heterologously from a gene integrated in a recombinant host cell strain (e.g., Pichia pastoris), identified at least 5 exemplary CYP polypeptide that exhibit the unexpected and surprising technical effect of catalyzing one or more of the selective hydroxylations of the 11 p, 17a, or 21 positions involved in the bioconversion Reactions 1-12 of FIGS. 1A-1C when integrated in a recombinant host cell. These exemplary CYP polypeptides, exemplary CPR polypeptides that facilitate electrontransfer to them to facilitate their catalytic hydroxylation, and exemplary genes, with sequences optimized for Pichia or Saccharomyces, that encode them are summarized in Table 3 below (as well as in the following Examples and the accompanying Sequence Listing).

[0072] TABLE 3: CYP and CPR polypeptides with activity useful in bioconversion Reactions 1-12.

[0073] In at least one embodiment, when a host cell (e.g., P. pastoris) is transformed with a heterologous nucleic acid encoding an exemplary homolog CYP polypeptide of the present disclosure (e.g., a CYP polypeptide of Table 3), and the recombinant host cell is fed a hydrocortisone precursor substrate(s), (e.g., 17a-hydroxyprogesterone or 11 -deoxycortisol), the mono- or di-hydroxylated bioconversion product molecule is produced by the host cell. Exemplary bioconversions of Reactions 1-12 using CYP and CPR genes of Table 3 integrated in recombinant host cells are illustrated in the Examples.

[0074] Based on the correlation of recombinant polypeptide functional information provided herein with the sequence information provided in Table 3, the accompanying Sequence Listing, and / or the Examples disclosed herein, one of ordinary skill can recognize that the present disclosure of various CYP polypeptides herein provides a range of recombinant polypeptides having CYP activity capable of one or more of the selective hydroxylations involved in Reactions 1-12 of FIGS. 1A-1C, wherein the polypeptide comprises an amino acid sequence comprising one or more of the amino acid differences or sets of amino acid differences relative to a CYP of any one of SEQ ID NO: 2, 6, 10, or 16, and otherwise have at least 80%, at least 85% at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 2, 6, 10, or 16.

[0075] Additionally, in at least one embodiment, a recombinant polypeptide of the present disclosure having CYP activity capable of one or more of the selective hydroxylations involved in Reactions 1-12 of FIGS. 1A-1C can have an amino acid sequence comprising one or more of the amino acid differences or sets of amino acid differences (relative to SEQ ID NO: 2, 6, 10, or 16), e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11 , 1-12, 1-14, 1- 15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35, 1-40, 1-45, 1-50, 1-55, or 1-60 residue differences. In some embodiments, the number of differences can be 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35, 40, 45, 50, 55, or 60 residue differences. The residue difference can include conservative changes or non-conservative changes. In some embodiments, the residue differences can comprise conservative substitutions and non-conservative substitutions as compared to the reference polypeptides of SEQ ID NO: 2, 6, 10, or 16.

[0076] In some embodiments, the recombinant CYP polypeptides of the present disclosure can be in the form of fusion polypeptides in which the polypeptides are fused to other polypeptides, such as, by way of example and not limitation, antibody tags (e.g., myc epitope), purification sequences (e.g., His tags for binding to metals), and cell localization signals (e.g., secretion signals). Thus, the recombinant polypeptides described herein can be used with or without fusions to other polypeptides. It is also contemplated that the recombinant polypeptides described herein are not restricted to the genetically encoded amino acids. In addition to the genetically encoded amino acids, the polypeptides described herein may be comprised, either in whole or in part, of naturally occurring and / or synthetic non-encoded amino acids.

[0077] In another aspect, the present disclosure provides polynucleotides encoding the recombinant polypeptides having CYP activity capable of one or more of the selective hydroxylations involved in Reactions 1-12 of FIGS. 1A-1C with increased activity and / or yield as described herein (e.g., CYP polypeptides of Table 3). In at least one embodiment, the polynucleotide encoding a recombinant CYP polypeptide comprises an polynucleotide sequence that is at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to a polynucleotide sequence encoding an exemplary polypeptide of SEQ ID NO: 2, 6, 10, or 16. In some embodiments, the polynucleotide encodes a recombinant polypeptide comprising an amino acid sequence that has the percent identity described above and has one or more amino acid residue differences as compared to the polypeptide of SEQ ID NO: 2, 6, 10, or 16.

[0078] In at least one embodiment, the polynucleotide has a sequence encoding a recombinant polypeptide of the present disclosure in which the encoding polynucleotide sequence has one or more neutral codon differences relative to a polynucleotide sequence of SEQ ID NO: 1 , 5, 9, or 15, which codon differences do not encode an amino acid difference but result in increased yield of the desired product compound (e.g., HC) produced by a recombinant host cell in which the polynucleotide sequence is integrated.

[0079] It is also contemplated that the polynucleotides encoding the recombinant polypeptides having CYP activity and increased activity and / or yield as described herein, can include a combination of one or more codon differences relative to SEQ ID NO: 1 , 5, 9, or 15, wherein at least one of the codon differences encodes an amino acid difference as compared to the parent sequence and at least one codon difference is a neutral codon difference that does not encode an amino acid difference as compared to the parent sequence of SEQ ID NO: 2, 6, 10, or 16. Accordingly, in at least one embodiment, the present disclosure provides a polynucleotide sequence encoding a recombinant polypeptide having CYP activity capable of one or more of the selective hydroxylations involved in Reactions 1-12 of FIGS. 1A-1C, wherein the polynucleotide sequence comprises acombination of a codon differences encoding an amino acid difference and a neutral codon difference.

[0080] In at least one embodiment, the polynucleotide comprises a sequence encoding an exemplary recombinant polypeptide having CYP activity as disclosed in Table 3 and the accompanying Sequence Listing. In at least one embodiment, the polynucleotide comprises a sequence of at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to a sequence selected from the group consisting of SEQ ID NO: 1 , 5, 9, or 15. In at least one embodiment, the polynucleotide comprises a codon degenerate sequence of a sequence selected from the group consisting of SEQ ID NO: 1 , 5, 9, or 15.

[0081] The polynucleotide sequences encoding the recombinant polypeptides of the present disclosure may be operatively linked to one or more heterologous regulatory sequences that control gene expression to create a recombinant polynucleotide capable of expressing the polypeptide. Expression constructs containing a heterologous polynucleotide encoding the recombinant polypeptide can be introduced into appropriate host cells to express the corresponding polypeptide. Because of the knowledge of the codons corresponding to the various amino acids, availability of a protein sequence provides a description of all the polynucleotides capable of encoding the subject. The degeneracy of the genetic code, where the same amino acids are encoded by alternative or synonymous codons allows an extremely large number of nucleic acids to be made, all of which encode the improved P450 or CYP enzymes disclosed herein. Thus, having identified a particular amino acid sequence, those skilled in the art could make any number of different nucleic acids by simply modifying the sequence of one or more codons in a way which does not change the amino acid sequence of the protein. In this regard, the present disclosure specifically contemplates each and every possible variation of polynucleotides that could be made by selecting combinations based on the possible codon choices, and all such variations are to be considered specifically disclosed for any polypeptide disclosed herein, including the amino acid sequences presented in Table 3 and the accompanying Sequence Listing.

[0082] The codons can be selected to fit the host cell in which the protein is being produced. For example, preferred codons used in bacteria are used to express the gene in bacteria; preferred codons used in yeast are used for expression in yeast; and preferred codons used in mammals are used for expression in mammalian cells. It is contemplated that all codons need not be replaced to optimize the codon usage of the recombinant polypeptide since the natural sequence will comprise preferred codons and because use of preferred codons may not be required for all amino acid residues. Consequently, codon optimized polynucleotides encoding the recombinant polypeptide may contain preferred codons at about 40%, 50%, 60%, 70%, 80%, or greater than 90% of codon positions of the full-length coding region.

[0083] The present disclosure also provides an expression vector comprising a polynucleotide encoding a recombinant polypeptide having CYP activity capable one or more of the selective hydroxylations involved in Reactions 1-12 of FIGS. 1A-1C, and one or more expression regulating regions such as a promoter, a terminator, a replication origin, or the like, depending on the type of hosts into which they are to be introduced. The various nucleic acid and control sequences described above may be joined together to produce a recombinant expression vector which may include one or more convenient restriction sites to allow for insertion or substitution of the nucleic acid sequence encoding the recombinant polypeptide at such sites. Alternatively, a polynucleotide sequence of the present disclosure may be expressed by inserting the nucleic acid sequence or a nucleic acid construct comprising the sequence into an appropriate vector for expression. In creating the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression. The recombinant expression vector may be any vector (e.g., a plasmid or virus), which can be conveniently subjected to recombinant DNA procedures and can bring about the expression of the polynucleotide sequence. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vectors may be linear or closed circular plasmids.

[0084] The expression vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one which, when introduced into the host cell, is integrated into the genome, and replicated together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids which together contain the total DNA to be introduced into the genome of the host cell, or a transposon may be used. In at least one embodiment, the expression vector further comprises one or more selectable markers, which permit easy selection of transformed cells.

[0085] Use in Recombinant Host Cells

[0086] The recombinant genes of the present disclosure that encode recombinant polypeptides with CYP activity capable of catalyzing one or more of the selective hydroxylations involved in Reactions 1-12 of FIGS. 1A-1C (e.g., exemplary CYP polypeptides of Table 3) can be incorporated in recombinant host cells to enable in vivo biosynthesis of the compound, hydrocortisone (compound 8), hydrocortisone precursors (e.g., compounds 2-7) and / or hydrocortisone derivative compounds. Generally, these recombinant host cells comprise a heterologous nucleic acid that encodes the recombinantpolypeptide with CYP activity, wherein the nucleic acid is operatively linked to one or more control sequences for expression of the polypeptide in the host cell. Host cells for use in expressing recombinant genes encoding the polypeptides with CYP activity of the present disclosure are well known in the art and include but are not limited to, fungal cells, such as Saccharomyces cerevisiae or Pichia pastoris, bacterial cells, such as E. coli, insect cells, such as Drosophila S2 and Spodoptera Sf9, animal cells, such as CHO, COS, BHK, 293, and plant cells. Appropriate mediums and growth conditions for culturing the recombinant host cells so that they express the polypeptide with CYP activity are well known in the art.

[0087] The present disclosure contemplates a variety of recombinant host cells incorporating and capable of expressing one or more different CYP polypeptides thereby allowing the cells to catalyze one or more of the selective 17a, 11 p, and / or 21 position hydroxylations on a hydrocortisone precursor of compounds 1-7 as shown in Reactions 1-12 of FIGS. 1A-1C. For example, the present disclosure provides a recombinant host cell comprising a heterologous nucleic acid encoding a first CYP capable of converting 17a- hydroxyprogesterone (compound 2) to 11 -deoxycortisol (compound 5), and a heterologous nucleic acid encoding a second CYP capable of converting 11 -deoxycortisol (compound 5) to hydrocortisone (compound 8).

[0088] The present disclosure also provides recombinant host cell comprising a heterologous nucleic acid encoding a first CYP capable of converting 17a- hydroxyprogesterone (compound 2) to 21 -deoxycortisol (compound 6), and a heterologous nucleic acid encoding a second CYP capable of converting 21 -deoxycortisol (compound 6) to hydrocortisone (compound 8).

[0089] The present disclosure provides a recombinant host cell comprising a heterologous nucleic acid encoding a first CYP capable of converting 11 p-hydroxyprogesterone (compound 3) to 21 -deoxycortisol (compound 6), and a heterologous nucleic acid encoding a second CYP capable of converting 21 -deoxycortisol (compound 6) to hydrocortisone (compound 8).

[0090] The present disclosure provides a recombinant host cell comprising a heterologous nucleic acid encoding a first CYP capable of converting 1 i p-hydroxyprogesterone (compound 3) to corticosterone (compound 7), and a heterologous nucleic acid encoding a second CYP capable of converting corticosterone (compound 7) to hydrocortisone (compound 8).

[0091] The present disclosure provides a recombinant host cell comprising a heterologous nucleic acid encoding a first CYP capable of converting 21-hydroxyprogesterone (compound 4) to 11 -deoxycortisol (compound 5), and a heterologous nucleic acid encoding a second CYP capable of converting 11 -deoxycortisol (compound 5) to hydrocortisone (compound 8).

[0092] The present disclosure provides a recombinant host cell comprising a heterologous nucleic acid encoding a first CYP capable of converting 21-hydroxyprogesterone (compound 4) to corticosterone (compound 7), and a heterologous nucleic acid encoding a second CYP capable of converting corticosterone (compound 7) to hydrocortisone (compound 8).

[0093] Exemplary genes encoding CYP polypeptides capable of catalyzing Reactions 1-12 of FIGS. 1A-1C are provided in Table 3, the Examples, and the accompanying sequence listing. As described elsewhere herein, it is contemplated that these CYP genes and polypeptides can be further engineered for enhanced traits of bioconversion when incorporated in a recombinant host cell of the present disclosure. Accordingly, it is contemplated that the CYP capable of converting 17a-hydroxyprogesterone (compound 2) to 11 -deoxycortisol (compound 5) can comprise an amino acid sequence having at least 80%, at least 85% at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a sequence of SEQ ID NO: 2, and the CYP capable of converting 11 -deoxycortisol (compound 5) to hydrocortisone (compound 8) can comprise an amino acid sequence having at least 80%, at least 85% at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a sequence selected from SEQ ID NO: 10, and 16.

[0094] The CYP capable of converting 17a-hydroxyprogesterone (compound 2) to 21- deoxycortisol (compound 6) can comprise an amino acid sequence having at least 80%, at least 85% at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a sequence selected from SEQ ID NO: 10 or 16, and the CYP capable of converting 21- deoxycortisol (compound 6) to hydrocortisone (compound 8) can comprise an amino acid sequence having at least 80%, at least 85% at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a sequence of SEQ ID NO: 2.

[0095] The CYP capable of converting 1 i p-hydroxyprogesterone (compound 3) to 21- deoxycortisol (compound 6) can comprise an amino acid sequence having at least 80%, at least 85% at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a sequence selected from SEQ ID NO: 6, 36, 38, 40, and 42, and the CYP capable of converting 21 -deoxycortisol (compound 6) to hydrocortisone (compound 8) can comprise an amino acid sequence having at least 80%, at least 85% at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a sequence of SEQ ID NO: 2.

[0096] The CYP capable of converting 1 i p-hydroxyprogesterone (compound 3) to corticosterone (compound 7) can comprise an amino acid sequence having at least 80%, at least 85% at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a sequence of SEQ ID NO: 2, and the CYP capable of converting corticosterone (compound 7) to hydrocortisone (compound 8) can comprise an amino acid sequence having at least 80%, at least 85% at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a sequence selected from SEQ ID NO: 6, 36, 38, 40, and 42.

[0097] The CYP capable of converting 21-hydroxyprogesterone (compound 4) to 11- deoxycortisol (compound 5) can comprise an amino acid sequence having at least 80%, at least 85% at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a sequence selected from SEQ ID NO: 6, 36, 38, 40, and 42, and the CYP capable of converting 1 -deoxycortisol (compound 5) to hydrocortisone (compound 8) can comprise an amino acid sequence having at least 80%, at least 85% at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a sequence selected from SEQ ID NO: 10 and 16.

[0098] The CYP capable of converting 21-hydroxyprogesterone (compound 4) to corticosterone (compound 7) can comprise an amino acid sequence having at least 80%, at least 85% at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a sequence selected from SEQ ID NO: 10 or 16, and the CYP capable of converting corticosterone (compound 7) to hydrocortisone (compound 8) can comprise an amino acid sequence having at least 80%, at least 85% at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a sequence selected from SEQ ID NO: 6, 36, 38, 40, and 42.

[0099] In any of the above embodiments of a recombinant host cell, the cell can further comprise a heterologous nucleic acid encoding a third CYP capable of converting progesterone (compound 1) to the desired hydrocortisone precursor compound that leads to the next bioconversion reaction for producing hydrocortisone, i.e., 17a-hydroxyprogesterone (compound 2), 1 i p-hydroxyprogesterone (compound 3), or 21-hydroxyprogesterone (compound 4). An exemplary CYP capable of converting progesterone (compound 1) to 17a-hydroxyprogesterone (compound 2) can comprise an amino acid sequence having at least 80%, at least 85% at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%,at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a sequence selected from SEQ ID NO: 6, 36, 38, 40, and 42. An exemplary CYP capable of converting progesterone (compound 1) to 11 p- hydroxyprogesterone (compound 3) can comprise an amino acid sequence having at least 80%, at least 85% at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a sequence selected from SEQ ID NO: 10 or 16. An exemplary CYP capable of converting progesterone (compound 1) to 21-hydroxyprogesterone (compound 2) can comprise an amino acid sequence having at least 80%, at least 85% at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a sequence of SEQ ID NO: 2.

[0100] In any of the above embodiments of a recombinant host cell, the cell can further comprise a heterologous nucleic acid encoding a heterologous nucleic acid encoding an enzyme with 3-ketosteroid-A1 -dehydrogenase (“KstD”) activity capable of catalyzing dehydrogenation of the C-C bond at position 1 of the A ring resulting in the formation of the HC derivative compound, prednisolone (compound (9)). Exemplary KstD enzymes are provided in the Examples herein and can comprise an amino acid sequence having at least 80%, at least 85% at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a sequence selected from SEQ ID NO: 50, 52, and 54.

[0101] In a further embodiment of the recombinant host cell capable of producing prednisolone, it is contemplated that the cell can further comprise a heterologous nucleic acid encoding an enzyme with 11 p-hydroxysteroid dehydrogenase type 2 (“HSD2”) activity capable of catalyzing the oxidation of the hydroxyl group at position 11 of the C ring of prednisolone to a ketone group resulting in the formation of the HC derivative compound, prednisone (compound (10)). Exemplary HSD2 enzymes are provided in the Examples herein and can comprise an amino acid sequence having at least 80%, at least 85% at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a sequence of SEQ ID NO: 56.

[0102] The recombinant host cells can comprise integrated heterologous nucleic acids encoding not only polypeptides with CYP activity capable of catalyzing one or more of the selective hydroxylations involved in Reactions 1-12 of FIGS. 1A-1C, and also other enzymes, such as CPR, and enzymes, such as a KstD and / or a HSD2, capable of producing other compounds, such as precursors for the hydrocortisone precursor substrates, and / or hydrocortisone derivatives, such as prednisolone and / or prednisone. As describedelsewhere herein, nucleic acid sequences encoding pathway enzymes for producing such precursor compounds are known in the art and can readily be used in accordance with the present disclosure. Typically, the nucleic acid sequence encoding the enzymes which form a part of the pathway, further include one or more additional nucleic acid sequences, for example, a nucleic acid sequence controlling expression of the enzymes which form a part of the biosynthetic pathway, and these one or more additional nucleic acid sequences together with the nucleic acid sequence encoding the recombinant polypeptides with CYP activity can be considered a heterologous nucleic acid sequence. A variety of techniques and methodologies are available and well known in the art for introducing heterologous nucleic acid sequences, such as nucleic acid sequences encoding the enzymes (e.g., CYP and CPR), into a host cell so as to attain expression in the host cell. Such techniques are well known to the skilled artisan and can be found in, for example, Sambrook et al., Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press, 2012, Fourth Ed.

[0103] For example, the introduction of the heterologous nucleic acids can include integration of the nucleic acids into specific loci in the genome of a host cell via CRISPR- Cas9 and other techniques, some of which are demonstrated in the Examples herein. Such techniques are well known to the skilled artisan and can, for example, be found in Sambrook and other well-known sources. The number of copies of heterologous genes and their locus of integration in a recombinant host cell’s genome can result in improved biosynthetic production of a desired product, such as HC. For example, a heterologous nucleic acid encoding a polypeptide with CYP activity capable of converting 17a-hydroxyprogesterone to 11 -deoxycortisol can be integrated into a host cell’s genome in 1 , 2, 3, 4, or more copies. Accordingly, it is contemplated that in the recombinant host cells of the present disclosure, the heterologous nucleic acid encoding the recombinant polypeptide having CYP activity can be integrated in the host cell’s genome at one or more loci, including but not limited to the well-known genomic loci in Pichia pastoris of HIS4, Inti 5, Inti 8, Inti , Int6, and AOX1 , or the loci in Saccharomyces cerevisiae of X-2, X-4, XI-2, XII-4, NDE1 , XII-5, Gal80, and ROQ1 .

[0104] One of ordinary skill will recognize that the heterologous nucleic acids encoding the recombinant enzymes with CYP activity capable of catalyzing one or more of the selective hydroxylations involved in Reactions 1-12 of FIGS. 1A-1C, CPR activity, and any other pathway enzymes will further comprise transcriptional promoters capable of controlling expression of the enzymes in the recombinant host cell. Generally, the transcriptional promoters are selected to be compatible with the host cell, so that promoters obtained from bacterial cells are used when a bacterial host cell is selected in accordance herewith, while a fungal promoter is used when a fungal host cell is selected, a plant promoter is used when a plant cell is selected, and so on. Promoters useful in the recombinant host cells of thepresent disclosure may be constitutive or inducible, provided such promoters are operable in the host cells. Promoters that may be used to control expression in fungal host cells, such as Saccharomyces cerevisiae and Pichia pastoris, are well known in the art and include, but are not limited to, inducible promoters, such as a Gall promoter or Gall 0 promoter, a constitutive promoter, such as an alcohol dehydrogenase (ADH) promoter, a glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter, or an S. pombe Nmt, or ADH promoter. Exemplary promoters that may be used to control expression in bacterial cells can include the Escherichia coll promoters, lac, tac, trc, trp or the T7 promoter. Exemplary promoters that may be used to control expression in plant cells include, for example, a Cauliflower Mosaic Virus 35S promoter (Odell et al. (1985) Nature 313:810-812), a ubiquitin promoter (U.S. Pat. No. 5,510,474; Christensen et al. (1989)), or a rice actin promoter (McElroy et al. (1990) Plant Cell 2:163-171). Exemplary promoters that can be used in mammalian cells include, a viral promoter such as an SV40 promoter or a metallothionine promoter. All of these host cell promoters are well known by and readily available to one of ordinary skill in the art. Further nucleic acid control elements useful for controlling expression in a recombinant host cell can include transcriptional terminators, enhancers, and the like, all of which may be used with the heterologous nucleic acids incorporate in the recombinant host cells of the present disclosure.

[0105] A wide variety of techniques are well known in the art for linking transcriptional promoters and other control elements to heterologous nucleic acid sequences encoding pathway genes for biosynthesis of HC, HC precursors, and / or HC derivatives. Such techniques are described in e.g., Sambrook et al., Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press, 2012, Fourth Ed. Accordingly, in at least one embodiment, the heterologous nucleic acid sequences of the present disclosure comprise a promoter capable of controlling expression in a host cell, wherein the promoter is linked to a nucleic acid sequence encoding a recombinant polypeptide of the present disclosure having CYP activity capable of catalyzing one or more of the selective hydroxylations involved in Reactions 1-12 of FIGS. 1A-1C, and as necessary, other enzymes constituting a pathway for production of a precursor, such as HC precursors of compounds 1-7, and / or HC derivative compound precursors. This heterologous nucleic acid sequence can be integrated into a recombinant expression vector which ensures good expression in the desired host cell, wherein the expression vector is suitable for expression in a host cell, meaning that the recombinant expression vector comprises the heterologous nucleic acid sequence linked to any genetic elements required to achieve expression in the host cell. Genetic elements that may be included in the expression vector in this regard include a transcriptional termination region, one or more nucleic acid sequences encoding marker genes, one or more origins of replication, and the like. In some embodiments, the expression vector further comprisesgenetic elements required for the integration of the vector or a portion thereof in the host cell's genome.

[0106] It is also contemplated that in some embodiments an expression vector comprising a heterologous nucleic acid of the present disclosure may further contain a marker gene. Marker genes useful in accordance with the present disclosure include any genes that allow the distinction of transformed cells from non-transformed cells, including all selectable and screenable marker genes. A marker gene may be a resistance marker such as an antibiotic resistance marker against, for example, kanamycin or ampicillin. Screenable markers that may be employed to identify transformants through visual inspection include p-glucuronidase (GUS) (U.S. Pat. Nos. 5,268,463 and 5,599,670) and green fluorescent protein (GFP) (Niedz etal., 1995, Plant Cell Rep., 14: 403).

[0107] In at least one embodiment, the present disclosure also provides a method for producing HC, HC precursors, and / or HC derivatives, wherein a heterologous nucleic acid encoding a recombinant polypeptide having CYP activity of catalyzing one or more of the selective hydroxylations involved in Reactions 1-12 of FIGS. 1A-1C (e.g., an exemplary engineered polypeptide of Table 3) is introduced into a recombinant host cell. The recombinant host cell comprising and capable of expressing the heterologous nucleic acid is then used in a process for the production of the polypeptide, or incorporated into a biocatalytic process that utilizes the CYP activity of the recombinant polypeptide expressed by the host cell for the catalytic hydroxylation of an HC precursor substrate at one or more of the 11 p, 17a, or 21 steroid positions, e.g., the bioconversion of 17a-hydroxyprogesterone to 11 -deoxycortisol. In at least one embodiment, the recombinant host cell can further comprise a pathway of enzymes capable of producing the HC precursor compound (e.g., progesterone, 17a-hydroxyprogesterone) which can act as a substrate for the recombinant polypeptides with CYP activity. In at least one embodiment, the recombinant host cell can further comprise a pathway of enzymes (e.g., KstD, HSD2) capable of using HC as a substrate to produce an HC derivative compound (e.g., prednisolone, prednisone). It is contemplated that a recombinant host cell comprising an integrated heterologous nucleic acid encoding a recombinant polypeptide having CYP activity of the present disclosure can provide improved biosynthesis of a desired product compound ( e.g., HC, HC precursors, and / or HC derivatives) in terms of titer, yield, and production rate, due to the improved characteristics of the expressed CYP activity in the cell engineered with the gene.

[0108] In at least one embodiment, the present disclosure contemplates processes in which a recombinant host expressing one or more genes encoding the desired CYP activity in an aqueous solution is contacted with a substrate (e.g, an HC precursor compound) and the expressed polypeptide catalyzes the bioconversion to the desired HC precursor or HCproduct compound. Thus, in one embodiment, the present disclosure provides a method for producing HC, HC precursors, and / or HC derivatives comprising: (a) culturing a recombinant host cell of the present disclosure in a suitable medium comprising an HC precursor, such as progesterone, or 17a-hydroxyprogesterone; and (b) recovering the desired produced HC, HC precursors, and / or HC derivatives.

[0109] As described and exemplified elsewhere herein, the present disclosure contemplates a number of different biocatalytic routes using one or more recombinant host cells expressing one or more of the CYP polypeptides of Table 3 to carry out the one or more of the selective hydroxylations involved in Reactions 1-12 of FIGS. 1A-1C. The present disclosure contemplates using recombinant host cells engineered to express CYP polypeptides of Table 3 to carry out any of the biosynthetic routes from a hydrocortisone precursor (e.g., progesterone, 17a-hydroxyprogesterone, 11 p-hydroxyprogesterone, 21- hydroxyprogesterone) to produce hydrocortisone as shown in FIGS. 1A-1C.

[0110] For example, it is contemplated that a 17a-hydroxyprogesterone to hydrocortisone bioconversion process exemplified by Reactions 4 and 10 of FIG. 1A can be carried out using a single recombinant host cell strain engineered to express two different CYP genes encoding polypeptides with different hydroxylation selectivity - one CYP that catalyzes the conversion of 17a-hydroxyprogesterone to 11 -deoxycortisol of Reaction 4, and one CYP that catalyzes the conversion of 11 -deoxycortisol to hydrocortisone of Reaction 10. In another example, the biosynthetic production of hydrocortisone can be carried out starting from 11 [3- hydroxyprogesterone via a bioconversion process exemplified by Reactions 7 and 11 of FIG. 1B. Such a process can be carried out using a single recombinant host cell strain engineered to express two different CYP genes encoding polypeptides with different hydroxylation selectivity - one CYP that catalyzes the conversion of 11 p- hydroxyprogesterone to 21 -deoxycortisol of Reaction 7, and one CYP that catalyzes the conversion of 21 -deoxycortisol to hydrocortisone of Reaction 11. In either of these examples, it is also contemplated that the bioconversion process could further start with progesterone and proceed via Reaction 1 to 17a-hydroxyprogesterone (FIG. 1A) or via Reaction 2 to 11 |3-hydroxyprogesterone (FIG. 1B).

[0111] Additionally, the present disclosure contemplates that any of the biosynthetic processes described herein using a recombinant host cell engineered to express two or three different CYP polypeptides, could also be carried out using a mixture of one, two, or three different recombinant host cells each expressing one or more recombinant CYP polypeptides (e.g., CYP of Table 3). Thus, a bioconversion process of the present disclosure can include contacting one or more HC precursors with a mixture of one, two, three, or more recombinant host cells, engineered with one, two, three, or more heterologous genes expressing polypeptides with CYP activity capable of catalyzing one,two, three, or more different selective hydroxylations of HC precursor substrates. Accordingly, a similar 17a-hydroxyprogesterone to hydrocortisone bioconversion process of Reactions 4 and 10 can be carried out using an aqueous solution containing a mixture of two recombinant host cells, each engineered to express one of the two CYP genes encoding a polypeptide with a different hydroxylation selectivity. In such a process, an aqueous solution containing both host cell strains is contacted with the 17a-hydroxyprogesterone and one host cell expresses the CYP polypeptide catalyzing Reaction 4 (the conversion of 17a- hydroxyprogesterone to 11 -deoxycortisol), and the other host cell expresses the CYP polypeptide catalyzing Reaction 10 (the conversion of 11 -deoxycortisol to hydrocortisone).

[0112] In at least one embodiment, the present disclosure provides processes using one, two, or three different recombinant host cells for preparing hydrocortisone (HC), a hydrocortisone precursor compound, and / or a hydrocortisone derivative compound, the process comprising: (a) contacting under suitable conditions the one, two, or three different recombinant host cells of the present disclosure and a substrate compound selected from progesterone (compound 1), 17a-hydroxyprogesterone (compound 2), 11 p- hydroxyprogesterone (compound 3), 21 -hydroxyprogesterone (compound 4), 11- deoxycortisol (compound 5), 21 -deoxycortisol (compound 6), and corticosterone (compound7); and (b) recovering the hydrocortisone, hydrocortisone precursor, and / or a hydrocortisone derivative product compound.

[0113] In at least one embodiment of the process, the substrate compound is progesterone (compound 1), and the product compound is hydrocortisone (compound 8). In at least one embodiment of the process, the substrate compound is selected from 17a- hydroxyprogesterone (compound 2), 1 ip-hydroxyprogesterone (compound 3), and 21- hydroxyprogesterone (compound 4); and the product compound is hydrocortisone (compound 8). In at least one embodiment of the process, the substrate compound is selected from 11 -deoxycortisol (compound 5), 21 -deoxycortisol (compound 6), and corticosterone (compound 7); and the product compound is hydrocortisone (compound 8).

[0114] In at least one embodiment of the processes for preparing hydrocortisone (compound8) of the present disclosure, the process comprises: (a) contacting 17a-hydroxyprogesterone under suitable conditions with a recombinant host cell comprising: (i) an integrated heterologous nucleic acid encoding a first CYP capable of converting 17a- hydroxyprogesterone to 11 -deoxycortisol; and (ii) a heterologous nucleic acid encoding a second CYP capable of converting 11 -deoxycortisol to hydrocortisone; and (b) recovering the produced hydrocortisone. In at least one embodiment of the process, the first CYP comprises an amino acid sequence having at least 80%, at least 85% at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a sequence of SEQ ID NO:2, and the second CYP comprises an amino acid sequence having at least 80%, at least 85% at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a sequence selected from SEQ ID NO: 10, and 16. In at least one embodiment of the process, the cell further comprises an integrated heterologous nucleic acid encoding a CPR capable of transferring electrons to the first CYP, the second CYP, and / or the third CYP; optionally, wherein the CPR comprises an amino acid sequence having at least 80%, at least 85% at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a sequence selected from SEQ ID NO: 4, 8, 12, 44, 46, and 48.

[0115] In another embodiment of the process, the bioconversion of 17a- hydroxyprogesterone to hydrocortisone is carried out using two strains each with a different CYP gene integrated. In at least one embodiment, the process comprises: (a) contacting 17a-hydroxyprogesterone under suitable conditions with: (i) a recombinant host cell comprising an integrated heterologous nucleic acid encoding a first CYP capable of converting 17a-hydroxyprogesterone to 11 -deoxycortisol; and (ii) a recombinant host cell comprising a heterologous nucleic acid encoding a second CYP capable of converting 11- deoxycortisol to hydrocortisone; and (b) recovering the produced hydrocortisone. In at least one embodiment of this process, wherein the first CYP comprises an amino acid sequence having at least 80%, at least 85% at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a sequence of SEQ ID NO: 2, and the second CYP comprises an amino acid sequence having at least 80%, at least 85% at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a sequence selected from SEQ ID NO: 10, and 16. In at least one embodiment of this process, each of the two cells further comprises an integrated heterologous nucleic acid encoding a CPR capable of transferring electrons to the first CYP, the second CYP, and / or the third CYP; optionally, wherein the CPR comprises an amino acid sequence having at least 80%, at least 85% at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a sequence selected from SEQ ID NO: 4, 8, 12, 44, 46, and 48.

[0116] In at least one embodiment, the process is capable of converting progesterone to hydrocortisone using a single strain with three CYP genes integrated. In at least one embodiment, this process comprises: (a) contacting progesterone under suitable conditions with a recombinant host cell comprising: (i) an integrated heterologous nucleic acid encoding a first CYP capable of converting progesterone to 17a-hydroxyprogesterone; (ii) anintegrated heterologous nucleic acid encoding a second CYP capable of converting 17a- hydroxyprogesterone to 11 -deoxycortisol; and (iii) a heterologous nucleic acid encoding a third CYP capable of converting 11 -deoxycortisol to hydrocortisone; and (b) recovering the produced hydrocortisone. In at least one embodiment of this process, the first CYP comprises an amino acid sequence having at least 80%, at least 85% at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a sequence selected from SEQ ID NO: 6, 36, 38, 40, and 42, the second CYP comprises an amino acid sequence having at least 80%, at least 85% at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a sequence of SEQ ID NO: 2, and the third CYP comprises an amino acid sequence having at least 80%, at least 85% at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a sequence selected from SEQ ID NO: 10, and 16. In at least one embodiment of this process, the cell further comprises an integrated heterologous nucleic acid encoding a CPR capable of transferring electrons to the first CYP, the second CYP, and / or the third CYP; optionally, wherein the CPR comprises an amino acid sequence having at least 80%, at least 85% at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a sequence selected from SEQ ID NO: 4, 8, 12, 44, 46, and 48.

[0117] In at least one embodiment, the process is capable of converting progesterone to hydrocortisone using a three strain each with a different CYP genes integrated. In at least one embodiment, this process comprises: (a) contacting progesterone under suitable conditions with: (i) a recombinant host cell comprising an integrated heterologous nucleic acid encoding a first CYP capable of converting progesterone to 17a-hydroxyprogesterone; (ii) a recombinant host cell comprising an integrated heterologous nucleic acid encoding a second CYP capable of converting 17a-hydroxyprogesterone to 11 -deoxycortisol; and (iii) a recombinant host cell comprising a heterologous nucleic acid encoding a third CYP capable of converting 11 -deoxycortisol to hydrocortisone; and (b) recovering the produced hydrocortisone. In at least one embodiment of this process, the first CYP comprises an amino acid sequence having at least 80%, at least 85% at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a sequence selected from SEQ ID NO: 6, 36, 38, 40, and 42, the second CYP comprises an amino acid sequence having at least 80%, at least 85% at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5%sequence identity to a sequence of SEQ ID NO: 2, and the third CYP comprises an amino acid sequence having at least 80%, at least 85% at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a sequence selected from SEQ ID NO: 10, and 16. In at least one embodiment of this process, each of the cells further comprises an integrated heterologous nucleic acid encoding a OPR capable of transferring electrons to the first CYP, the second CYP, and / or the third CYP; optionally, wherein the CPR comprises an amino acid sequence having at least 80%, at least 85% at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a sequence selected from SEQ ID NO: 4, 8, 12, 44, 46, and 48.

[0118] In any of the processes for producing HC of the present disclosure, it is further contemplated that the process can comprise enzymes and / or recombinant host cells capable of further converting the HC to an HC derivative compound. Accordingly, in at least one embodiment, the process can further comprise: (iii) a recombinant host cell comprising a heterologous nucleic acid encoding an enzyme with 3-ketosteroid-A1 -dehydrogenase (“KstD”) activity capable of converting hydrocortisone (8) to the hydrocortisone derivative prednisolone (9). As described elsewhere herein, KstD genes and enzymes useful in the process are provided in the Examples and can comprise an amino acid sequence having at least 80%, at least 85% at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a sequence selected from SEQ ID NO: 50, 52, and 54. In such an embodiment, the process does not require recovering HC, can include a final step of recovering the produced hydrocortisone derivative prednisolone (9).

[0119] In another related embodiment, the processes of the present disclosure capable of producing prednisolone (9) can further comprise: (iv) a recombinant host cell comprising a heterologous nucleic acid encoding an enzyme with 11 p-hydroxysteroid dehydrogenase type 2 (“HSD2”) activity capable of converting the hydrocortisone derivative prednisolone (9) to prednisone (10). As described elsewhere herein, KstD genes and enzymes useful in the process are provided in the Examples and can comprise an amino acid sequence having at least 80%, at least 85% at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a sequence of SEQ ID NO: 56. In such an embodiment, the process does not require recovering HC, or prednisolone, but can include a final step of recovering the produced hydrocortisone derivative prednisone (10).

[0120] Suitable reaction conditions for the biosynthesis of compounds such as HC, HC precursors, and HC derivatives are known in the art and can be used with the recombinantpolypeptides having CYP activity of the present disclosure. Additionally, suitable reaction conditions for the exemplary polypeptides of the present disclosure can be determined using routine techniques known in the art for optimizing biocatalytic reactions. It is contemplated that various ranges of suitable reaction conditions with the recombinant polypeptides of the present disclosure, including but not limited to ranges of pH, temperature, buffer, solvent system, substrate loading, polypeptide loading, co-substrate or co-factor loading, atmosphere, and reaction time. Suitable reaction conditions can be readily determined and optimized for particular reactions by routine experimentation that includes, but is not limited to, contacting the recombinant polypeptide and substrate under experimental reaction conditions of concentration, pH, temperature, solvent conditions, and detecting the production of the desired compound. In at least one embodiment, the suitable reaction conditions comprise a reaction solution of ~pH 7-8, a temperature of 25C to 37C; optionally, the reaction conditions comprise a reaction solution of ~ pH 7 and a temperature of ~30C. In at least one embodiment, the reaction solution is allowed to incubate at a temperature of 25C to 37C for a reaction time of at least 1 , 6, 12, 24, or 48 hours, before the amount of reaction product is determined.

[0121] Associated with the above-described biocatalytic processes for preparing HC, HC derivatives, and HC precursors, the disclosure also provides reaction mixture compositions useful for these processes. For example, in at least one embodiment the disclosure provides a composition comprising the components of a 17a-hydroxyprogesterone to hydrocortisone bioconversion process of the present disclosure that uses a single strain with two different CYP genes integrated. Accordingly, the present also provides a composition in aqueous solution comprising: (i) a recombinant host cell of the present disclosure comprising one, two, and / or three integrated heterologous nucleic acids encoding a first, second, and / or third CYP; and (ii) a compound selected from: progesterone (compound 1), 17a- hydroxyprogesterone (compound 2), 1 ip-hydroxyprogesterone (compound 3), 21- hydroxyprogesterone (compound 4), 11 -deoxycortisol (compound 5), 21 -deoxycortisol (compound 6), corticosterone (compound 7), hydrocortisone (compound 8), and / or a mixture thereof.

[0122] In at least one embodiment, the present disclosure provides a composition comprising: (a) a hydrocortisone precursor selected from any one of compounds 1-7, or a mixture thereof; and (b) one or more recombinant host cells in an aqueous solution, wherein the one or more recombinant host cells comprise: (i) an integrated heterologous nucleic acid encoding a first CYP capable of converting 17a-hydroxyprogesterone (compound 2) to 11- deoxycortisol (compound 5), wherein the first CYP comprises an amino acid sequence having at least 80%, at least 85% at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least99.5% sequence identity to a sequence of SEQ ID NO: 2; and (ii) an integrated heterologous nucleic acid encoding a second CYP capable of converting 11 -deoxycortisol (compound 5) to hydrocortisone (compound 8), wherein the second CYP comprises an amino acid sequence having at least 80%, at least 85% at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a sequence selected from SEQ ID NO: 10, and 16. In at least one embodiment of this composition, the heterologous nucleic acid encoding the first CYP and the heterologous nucleic acid encoding the second CYP are integrated in a single recombinant host cell. In at least one embodiment of this composition, the one or more recombinant host cells further comprise an integrated heterologous nucleic acid encoding a third CYP capable of converting progesterone (compound 1) to 17a-hydroxyprogesterone (compound 2), wherein the third CYP comprises an amino acid sequence having at least 80%, at least 85% at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a sequence selected from SEQ ID NO: 6, 36, 38, 40, and 42.

[0123] In at least one embodiment of this composition, the heterologous nucleic acid encoding the first CYP, the heterologous nucleic acid encoding the second CYP, and the heterologous nucleic acid encoding the third CYP are integrated in a single host cell.

[0124] In at least one embodiment of this composition, the one or more recombinant host cells further comprise an integrated heterologous nucleic acid encoding a CPR capable of transferring electrons to the first CYP, the second CYP, and / or the third CYP; optionally, wherein the CPR comprises an amino acid sequence having at least 80%, at least 85% at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to a sequence selected from SEQ ID NO: 4, 8, 12, 44, 46, and 48.

[0125] In at least one embodiment of the compositions of the present disclosure, the composition comprises the hydrocortisone precursor 17a-hydroxyprogesterone (compound 2). In at least one embodiment, the composition further comprises hydrocortisone (compound 8).EXAMPLES

[0126] Various features and embodiments of the disclosure are illustrated in the following representative examples, which are intended to be illustrative, and not limiting. Those skilled in the art will readily appreciate that the specific examples are only illustrative of the invention as described more fully in the claims which follow thereafter. Every embodiment and feature described in the application should be understood to be interchangeable and combinable with every embodiment contained within.Example 1: Development of Recombinant Strains of Pichia pastoris with CYP Activity for Bioconversion of Hydrocortisone Precursors

[0127] This example illustrates construction of recombinant Pichia pastoris strains with integrated heterologous genes encoding enzymes with CYP and CPR activity for bioconversion reactions for preparing hydrocortisone.

[0128] Material and Methods

[0129] A. Strains designed for 17a hydroxylation of HC precursors

[0130] Two recombinant Pichia strains (HCP001 and HCP002) with one copy of a gene encoding the CYP enzyme, CYP256 (SEQ ID NO: 6) and one copy of the gene encoding the electron transfer CPR enzyme, CPR010 (SEQ ID NO: 8) were created as follows. A single plasmid (wbPlasmid239; SEQ ID NO: 17) containing a zeocin resistance marker, a pUC origin of replication, a HIS4 homologous sequence (SEQ ID NO: 25), and a cassette comprising of tDAS1 :CPR010:pCAT1 :pFDH1 :CYP256:tDAS2 was assembled, using the tDAS1 sequence (SEQ ID NO: 31), pCAT1 :pFDH1 sequence (SEQ ID NO: 29), and tDAS2 sequence (SEQ ID NO: 32). Individual DNA sequences were amplified using PCR, followed by agarose gel purification. Gel-purified PCR amplicons, which contained homologous overlapping ends, were assembled into a plasmid using NEBuilder® HiFi Assembly Mastermix according to standard protocol. Three microliters of the HiFi assembly were used to transform NEB 10-beta E.coli chemical competent cells according to standard protocol and plated on low-salt LB media plates containing 100 pg / mL of zeocin. After 16 h of growth, individual colonies were inoculated in 4 mL of low-salt LB media containing zeocin. Following plasmid recovery and sequence confirmation, a single plasmid was digested with Bbvcl and concentrated using Zymo Research’s DNA Clean and Concentrator Kit (catalog no. D4014). The restriction enzyme cut site, found in the HIS4 homologous sequence, linearized the plasmid, allowing for homologous integration at the HIS4 locus. Approximately 5 pg of linearized plasmid was used to transform a 50 pL aliquot of BG-10 (wild-type) competent cells and a 50 pL aliquot of BG-11 (aox1-KO) competent cells and plated on YPDS media plates containing zeocin. After 3 days of growth, individual colonies were confirmed for integration of the linearized plasmid using PCR and genomic DNA from each individual colony. A single sequence verified clone from the BG-10 transformation was promoted to strain HCP001 , and a single sequence verified clone from the BG-11 transformation was promoted to strain HCP002.

[0131] B. Strains designed for 11 B hydroxylation of HC precursors

[0132] Two recombinant Pichia strains (HCP009 and HCP010) with one copy of a gene encoding CYP258 (SEQ ID NO: 10) and one copy of the gene encoding CPR012 (SEQ ID NO: 12) were created as follows. A single plasmid (wbPlasmid243; SEQ ID NO: 19) containing an ampicillin resistance marker, a nourseothricin resistance marker, a pUC originof replication, an intergenic region 15 homologous sequence (Inti 5; SEQ ID NO: 26), and a cassette comprising tFBA2:CPR012:pCAT1 :pFDH1 :CYP258:tADH2 was assembled as described above. Following plasmid recovery and sequence confirmation, a single plasmid was digested with Hpal and concentrated using Zymo Research’s DNA Clean and Concentrator Kit (catalog no. D4014). The restriction enzyme cut site, found in the Inti 5 homologous sequence, linearized the plasmid, allowing for homologous integration at the Inti 5 locus. Approximately 5 pg of linearized plasmid was used to transform a 50 pL aliquot of BG-10 (wild-type) competent cells and a 50 pL aliquot of BG-11 (aox1-KO) competent cells and plated on YPD media plates containing nourseothricin. After 3 days of growth, individual colonies were confirmed for integration of the linearized plasmid using PCR and genomic DNA from each individual colony. A single sequence verified clone from the BG-10 transformation was promoted to strain HCP009, and a single sequence verified clone from the BG-11 transformation was promoted to strain HCP010.

[0133] To improve the activity of strains HCP009 and HCP010, each strain was used as a host to integrate a copy of AoCYB5 (SEQ ID NO: 14) at the HIS4 locus. AoCYB5 is a cytochrome b5 protein that improves electron transfer for CYP and CPR activity. These new strains, HCP015 and HCP016, respectively, were created as follows. A single plasmid (wbPlasmid247; SEQ ID NO: 21) containing a zeocin resistance marker, a pUC origin of replication, a HIS4 homologous sequence (SEQ ID NO: 25), and a cassette comprising of pAOX1 :AoCYB5:DAS1 was assembled as described above, using pAOX1 sequence (SEQ ID NO: 30). Following plasmid recovery and sequence confirmation, a single plasmid was digested with Bbvcl and concentrated using Zymo Research’s DNA Clean and Concentrator Kit (catalog no. D4014). The restriction enzyme cut site, found in the HIS4 homologous sequence, linearized the plasmid, allowing for homologous integration at the HIS4 locus. Approximately 5 pg of linearized plasmid was used to transform a 50 pL aliquot of HCP009 competent cells and a 50 pL aliquot of HCP010 competent cells and plated on YPDS media plates containing zeocin. After 3 days of growth, individual colonies were confirmed for integration of the linearized plasmid using PCR and genomic DNA from each individual colony. A single sequence verified clone from the HCP009 transformation was promoted to strain HCP015, and a single sequence verified clone from the HCP010 transformation was promoted to strain HCP016.

[0134] To test another CYP for 1 ip-OH activity, two recombinant Pichia strains (HCP018 and HCP019) with one copy of a gene encoding CYP261 (SEQ ID NO: 16) and one copy of the gene encoding CPR012 (SEQ ID NO: 12) were created as follows. A single plasmid (wbPlasmid309; SEQ ID NO: 23) containing an ampicillin resistance marker, a nourseothricin resistance marker, a pUC origin of replication, an intergenic region 15 homologous sequence (Inti 5; SEQ ID NO: 26), and a cassette comprising oftFBA2:CPR012:pCAT1 :pFDH1 :CYP261 :tADH2 was assembled as described above, using tFAB2 sequence (SEQ ID NO: 33) and tADH2 sequence (SEQ ID NO: 34). Following plasmid recovery and sequence confirmation, a single plasmid was digested with Hpal and concentrated using Zymo Research’s DNA Clean and Concentrator Kit (catalog no. D4014). The restriction enzyme cut site, found in the Inti 5 homologous sequence, linearized the plasmid, allowing for homologous integration at the I nt15 locus. Approximately 5 g of linearized plasmid was used to transform a 50 pL aliquot of BG-10 (wild-type) competent cells and a 50 pL aliquot of BG-11 (aox1-KO) competent cells and plated on YPD media plates containing nourseothricin. After 3 days of growth, individual colonies were confirmed for integration of the linearized plasmid using PCR and genomic DNA from each individual colony. A single sequence verified clone from the BG-10 transformation was promoted to strain HCP018, and a single sequence verified clone from the BG-11 transformation was promoted to strain HCP019.

[0135] C. Strains designed for 21 hydroxylation of HC precursors

[0136] Two recombinant Pichia strains (HCP011 and HCP012) with one copy of a gene encoding CYP259 (SEQ ID NO: 2) and one copy of the gene encoding CPR009 (SEQ ID NO: 4) were created as follows. A single plasmid (wbPlasmid246; SEQ ID NO: 20) containing zeocin resistance marker, a pUC origin of replication, a HIS4 homologous sequence (SEQ ID NO: 25), and a cassette comprising of tDAS1 :CPR009:pCAT1 :pFDH1 :CYP259:tDAS2 was assembled as described above. Following plasmid recovery and sequence confirmation, a single plasmid was digested with Bbvcl and concentrated using Zymo Research’s DNA Clean and Concentrator Kit (catalog no. D4014). The restriction enzyme cut site, found in the HIS4 homologous sequence, linearized the plasmid, allowing for homologous integration at the HIS4 locus. Approximately 5 pg of linearized plasmid was used to transform a 50 pL aliquot of BG-10 (wild-type) competent cells and a 50 pL aliquot of BG-11 (aox1-KO) competent cells and plated on YPDS media plates containing zeocin. After 3 days of growth, individual colonies were confirmed for integration of the linearized plasmid using PCR and genomic DNA from each individual colony. A single sequence verified clone from the BG-10 transformation was promoted to strain HCP011 , and a single sequence verified clone from the BG-11 transformation was promoted to strain HCP012.

[0137] D. Strains designed with two CYPs to perform two hydroxylation reactions

[0138] To test whether two CYPs and their reductase partners can function in the same Pichia strain, two recombinant Pichia strains (HCP017 and HCP020) with one copy of a gene encoding CYP258 (SEQ ID NO: 10), one copy of the gene encoding CPR012 (SEQ ID NO: 12), one copy of the gene encoding AoCYB (SEQ ID NO: 14), one copy of the gene encoding CPR009 (SED ID NO:4), and one copy of CYP259 (SEQ ID NO: 2) was created asfollows. Competent cells for strain HCP016 and HCP015 were prepared; these strains already had CYP258, CPR012, and AoCYB5 integrated and demonstrated 1 i p-OH activity. A single plasmid (wbPlasmid297; SEQ ID NO: 22) containing a hygromycin resistance marker, an ampicillin resistance marker, a pUC origin of replication, an intergenic region 18 homologous sequence (Inti 8; SEQ ID NO: 27), and a cassette comprising of tDAS1 :CPR009:pCAT 1 :pFDH1 :CYP259:tDAS2 was assembled as described above. Following plasmid recovery and sequence confirmation, a single plasmid was digested with Aval and concentrated using Zymo Research’s DNA Clean and Concentrator Kit (catalog no. D4014). The restriction enzyme cut site, found in the Inti 8 homologous sequence, linearized the plasmid, allowing for homologous integration at the I nt18 locus. Approximately 5 pg of linearized plasmid was used to transform a 50 pL aliquot of HCP016 competent cells and a 50 pL aliquot of HCP015 competent cells and plated on YPD media plates containing hygromycin. After 3 days of growth, individual colonies were confirmed for integration of the linearized plasmid using PCR and genomic DNA from each individual colony. A single sequence verified clone from the transformation with HCP016 was promoted to strain HCP017 and a single sequence verified clone from the transformation with HCP015 was promoted to strain HCP020.

[0139] To improve the activity of strains HCP018 and HCP019, each strain was used as a host to integrate a copy of AoCYB5 (SEQ ID NO: 14) at the HIS4 locus using wbPlasmid247 (SEQ ID NO: 21) as described above. Approximately 5 pg of linearized plasmid was used to transform a 50 pL aliquot of HCP018 competent cells and a 50 pL aliquot of HCP019 competent cells and plated on YPDS media plates containing zeocin. After 3 days of growth, individual colonies were confirmed for integration of the linearized plasmid using PCR and genomic DNA from each individual colony. A single sequence verified clone from the HCP018 transformation was promoted to strain HCP022, and a single sequence verified clone from the HCP019 transformation was promoted to strain HCP023.

[0140] E. Strains designed with three CYPs to perform three hydroxylation reactions

[0141] To test whether three CYPs and their reductase partners can function in the same Pichia strain for three hydroxylations, recombinant Pichia strain HCP021 with one copy of a gene encoding CYP258 (SEQ ID NO: 10), one copy of the gene encoding CPR012 (SEQ ID NO: 12), one copy of the gene encoding AoCYB (SEQ ID NO: 14), one copy of the gene encoding CPR009 (SED ID NO: 4), one copy of CYP259 (SEQ ID NO: 2), one copy of the gene encoding CYP256 (SEQ ID NO: 6), and one copy of the gene encoding CPR010 (SEQ ID NO: 8) was created as follows. Competent cells for strain HCP017 were prepared; this strain already had CYP258, CPR012, AoCYB5, CYP259, and CPR009 integrated and demonstrated both 110-OH and 21 -OH activity. A single plasmid (wbPlasmid314; SEQ IDNO: 24) containing a geneticin resistance marker, an ampicillin resistance marker, a pUC origin of replication, an intergenic region 1 homologous sequence (Inti ; SEQ ID NO: 28), and a cassette comprising of tDAS1 :CPR010:pCAT1 :pFDH1 :CYP256:tDAS2 was assembled as described above. Following plasmid recovery and sequence confirmation, a single plasmid was digested with Bgl-ll and concentrated using Zymo Research’s DNA Clean and Concentrator Kit (catalog no. D4014). The restriction enzyme cut site, found in the I nt1 homologous sequence, linearized the plasmid, allowing for homologous integration at the I nt1 locus. Approximately 5 pg of linearized plasmid was used to transform a 50 pL aliquot of HCP017 competent cells plated on YPD media plates containing geneticin. After 3 days of growth, individual colonies were confirmed for integration of the linearized plasmid using PCR and genomic DNA from each individual colony. A single sequence verified clone from the transformation was promoted to strain HCP021 .

[0142] Table 4 (below) summarizes the CYP and CPR genes integrated into recombinantPichia pastoris strains as described in sections A-E above.

[0143] TABLE 4

[0144] Table 5 (below) summarizes the recombinant Pichia pastoris strains built as described above in sections A-E.

[0145] TABLE SExample 2: Screening Recombinant Strains of Pichia pastoris for Production ofHydrocortisone and HC Precursor Intermediates

[0146] This example illustrates screening studies of the recombinant Pichia pastoris strains with integrated heterologous CYP and CPR genes as prepared in Example 1 for activity useful in bioconversion reactions for preparing hydrocortisone.

[0147] Material and Methods

[0148] A. General Screening Method 1

[0149] Individual colonies from any of the strain libraries prepared as described in Example 1 were picked and grown in 3-4 mL of BMGY in round bottom tubes. The seed cultures were placed in shaking incubators for 24 h at 30 C and 250 rpm. Cultures were then sub-cultured by transferring 1 mL of fresh overnight cultures to 100 mL of BMGY in 500 mL baffled flasks. These were grown for 48 h with 2% glycerol spike at 24h. Biomass was harvested via centrifugation (4000 rpm, 10 min), supernatant was removed, and 20 w / v% biomass was suspended in 2.5 mL bioconversion buffer (0.1 M Potassium Phosphate, pH = 7.2, 2% MeOH, 1% DMSO, 1 eq 2-hydroxypropyl-P-cyclodextrin, varying substrates and concentrations as denoted in Tables 7-9) in a 20 mL vial. These were allowed to incubate for (a) 2 h (b) 6 h or (c) 24 h at 30 C, 85% humidity, and 250 rpm prior to whole broth extraction (see LCMS sample preparation).

[0150] B. General Screening Method 2

[0151] Individual colonies from any of the strain libraries prepared as described in Example 1 were picked and grown in 3-4 mL of BMGY in round bottom tubes. The seed cultures were placed in shaking incubators for 24 h at 30 C and 250 rpm. Cultures were then subcultured by transferring 1 mL of fresh overnight cultures to 100 mL of BMGY in 500 mL baffled flasks. These were grown for 48 h with 2% glycerol spike at 24h. The biomass was then induced by harvesting the cells via centrifugation at 4000 rpm for 10 minutes, supernatant was removed, and the cells were suspended in 100 mL of BMMY with 0.5% Methanol. This was incubated for 24-96 h at 30 C, 85% humidity, and 250 rpm adding 0.5% Methanol every 24 h. The induced biomass was harvested via centrifugation (4000 rpm, 10 min), supernatant was removed, and 20 w / v% biomass was suspended in 2.5 mL bioconversion buffer (0.1 M Potassium Phosphate, pH = 7.2, 2% MeOH, 1% DMSO, 1 eq 2- hydroxypropyl-p-cyclodextrin, varying substates and concentrations as denoted in Tables 7- 9) in a 20 mL vial. These were allowed to incubate for (a) 2 h (b) 6 h or (c) 24 h at 30 C, 85% humidity, and 250 rpm prior to whole broth extraction (see LCMS sample preparation).

[0152] C. General Screening Methods 3a, 3b, 3c

[0153] Individual colonies from any of the strain libraries prepared as described in Example 1 were picked and grown in 3-4 mL of BMGY in round bottom tubes. The seed cultures were placed in shaking incubators for 24 h at 30 C and 250 rpm. Cultures were then sub-cultured by transferring 90 pL of fresh overnight cultures to 810 pL of BMGY in Axygen 24 deep well plates ( bottom, 10 mL) and sealed with breathable film. These were incubated in shakingincubators for a subsequent 24h at 30 C, 85% humidity, and 250 rpm. The biomass was then harvested via centrifugation at 4000 rpm for 10 minutes, supernatant was removed, and the remaining biomass was resuspended in 600 pL of bioconversion buffer (0.1 M Potassium Phosphate, pH = 7.2, 2% MeOH, 1% DMSO, 1 eq 2-hydroxypropyl-p- cyclodextrin, varying substates and concentrations as denoted in Tables 7-11) containing substrates of interest (see Table 6 for substrates of interest). These were allowed to incubate at 30 C, 85% humidity, 250 rpm for 2 h (Method 3a), 6 h (Method 3b), or 24 h (Method 3c) prior to whole broth extraction (see LCMS sample preparation).

[0154] D. General Screening Method 4

[0155] Individual colonies from any of the strain libraries prepared as described in Example 1 were picked and grown in 3-4 mL of BMGY in round bottom tubes. The seed cultures were placed in shaking incubators for 24 h at 30 C and 250 rpm. Cultures were then sub-cultured by transferring 90 pL of fresh overnight cultures to 810 pL of BMGY in Axygen 24 deep well plates ( bottom, 10 mL) and sealed with breathable film. These were incubated in shaking incubators for a subsequent 24 h at 30 C, 85% humidity, and 250 rpm. The biomass was harvested via centrifugation at 4000 rpm for 10 minutes, supernatant was removed, and the biomass from two or three separately grown strains (see Examples for strains) were reconstituted together in 600 pL total of bioconversion buffer (0.1 M Potassium Phosphate, pH = 7.2, 2% MeOH, 1% DMSO, 1 eq 2-hydroxypropyl-|3-cyclodextrin, varying substates and concentrations as denoted in Table 11) containing substrates of interest (see Table 6 for substrates of interest). These were allowed to incubate for 24 h at 30 C, 85% humidity, and 250 rpm prior to whole broth extraction (see LCMS sample preparation).

[0156] E. General Screening Method 5

[0157] Individual colonies from any of the strain libraries prepared as described in Example 1 were picked and grown in 3-4 mL of BMGY in round bottom tubes. The seed cultures were placed in shaking incubators for 24 h at 30 C and 250 rpm. Cultures were then sub-cultured by transferring 90 pL of fresh overnight cultures to 810 pL of BMGY in Axygen 24 deep well plates (V bottom, 10 mL) and sealed with breathable film. These were incubated in shaking incubators for a subsequent 24 h at 30 C, 85% humidity, and 250 rpm. The biomass was harvested via centrifugation at 4000 rpm for 10 minutes, supernatant was removed, and the biomass for the 21-OH producing strain (for example HCP011 or HCP012) was reconstituted together in 600 pL total of bioconversion buffer (0.1 M Potassium Phosphate, pH = 7.2, 2% MeOH, 1% DMSO, 1 eq 2-hydroxypropyl-p-cyclodextrin, varying substates and concentrations as denoted in Table 11) containing substrates of interest (see Table 6 for substrates of interest) for 2 h prior to addition of biomass from the 11 p-OH producing strains (for example HCP015 and HCP016) and / or biomass from the 17a-OH producing strains.These were allowed to incubate for a further 24 h at 30 C, 85% humidity, and 250 rpm prior to whole broth extraction (see LCMS sample preparation).

[0158] F. LCMS sample preparation

[0159] The whole culture broth was extracted by the addition of 1 :1 volume of methanol, shaken for 30 min at 30 C, 85% humidity, and 250 rpm, and clarified by centrifugation (4000 rpm for 30 min). Samples were then diluted to an appropriate concentration with methanol and analyzed using an Agilent 1290 UPLC coupled to an Agilent LC / TQ 6470 mass spectrometer. Calibration curves of compounds 1-8 (Table 6) were generated by making 10 serial dilutions starting from a 1 mg / L solution. Calibration curves were generated from collected data using Agilent Quantitative Analysis software which was used to calculate the concentration of each metabolite and intern determine % conversion from starting material to product.

[0160] G. LCMS analytical methods, instrumentation, and parameters

[0161] Analytical Method 1: Analysis of the compounds 1, 2, 3, and 4, (see Table 6 below) were performed on an Agilent 1290 Infinity II UPLC coupled to an Agilent 6490 LC / TQ-MS system equipped with an ESI-MS probe operating in positive, selective ionization mode (SIM) mode. The compounds were monitored using the m / z values listed in Table 6 and identified based on retention time of corresponding standards. Chromatographic separation was performed using an Agilent Eclipse Plus C18 RRHD 1 .8 pM (3.0 x 50 mm) column. Elution of compounds was accomplished utilizing a gradient (solvent A: 100% Water + 0.1% Formic Acid and solvent B: 100% Acetonitrile + 0.1% Formic Acid) starting with a 0.2 min isocratic step consisting of 45% B followed by a 2.0 min linear gradient to 60% B and an isocratic plateau for 0.3 min at 60% B with a flow rate of 1 .0 mL / min.

[0162] Analytical Method 2: Analysis of compounds 1, 2, 3, 4, 5, 6, 7, and 8 (see Table 6: Compounds screened) were performed on an Agilent 1290 Infinity II UPLC coupled to an Agilent 6490 LC / TQ-MS system equipped with an ESI-MS probe operating in positive, SIM mode. Compounds of interest were monitored for using m / z values listed in Table 6 and identified based on retention time of corresponding standards. Chromatographic separation was performed using an Agilent Eclipse Plus C18 RRHD 1 .8 pM (3.0 x 50 mm) column. Elution of compounds was accomplished utilizing a gradient (solvent A: 100% Water + 0.1% Formic Acid and solvent B: 100% Acetonitrile + 0.1% Formic Acid) starting with a 0.2 min isocratic step consisting of 28% B followed by a 3.8 min linear gradient to 45% B and an isocratic plateau at 60% for 0.4 min with a flow rate of 1.0 mL / min.

[0163] TABLE 6: Compounds and summary LCMS analytical parameters

[0164] Results

[0165] The bioconversion of progesterone (compound (1)) to hydrocortisone (compound (8)) requires three discrete hydroxylation events at the steroid ring positions 17a, 21 , and 11 p. Using the above-described screening and analytical methods, the Pichia pastoris strains expressing either three different P450 enzymes or combinations of two or three P450 enzymes (HCP001 - HCP020) were shown to differentially hydroxylate the HC precursor compounds (1) - (7) to provide the product hydrocortisone (compound (8)) or a hydrocortisone precursor intermediate (compounds (2) - (7)) via one of more of the bioconversion reactions catalyzed by one or more CYP enzymes as shown in FIGS. 1A-1C (i.e., Reactions 1-12).

[0166] Table 7 (below) summarizes exemplary results using General Screening Methods 1c, 2c, or 3c (and Analytical Method 1), to show that Pichia strains expressing CYP256 cancarry out Reaction 1 by selectively hydroxylating the 17a position of progesterone (compound (1)) to produce mono-hydroxylated 17a-hydroxyprogesterone (compound (2)).

[0167] TABLE 7

[0168] Table 8 (below) summarizes exemplary results using General Screening Methods 1a, 1b, 2b, 2c, or 3a (and Analytical Method 1), to show that Pichia strains expressing CYP258 or CYP261 can carry out Reaction 2 by selectively hydroxylating the 11 p position of progesterone (compound (1)) to produce mono-hydroxylated 11 p-hydroxyprogesterone (compound (3)).

[0169] TABLE S

[0170] Table 9 (below) summarizes exemplary results using General Screening Methods 1c, 2c, or 3c (and Analytical Method 1), to show that Pichia strains expressing CYP259 can carry out Reaction 3 by selectively hydroxylating the 21 position of progesterone (compound (1)) to produce mono-hydroxylated 21 -hydroxyprogesterone (compound (4)).

[0171] TABLE 9

[0172] Table 10 (below) summarizes exemplary results using General Screening Method 3c (and Analytical Method 2) to show that a Pichia strain (HCP011) expressing CYP259 can carry out Reaction 4 by selectively hydroxylating the 21 position of 17a-hydroxyprogesterone (2) to produce di-hydroxylated 11 -deoxycortisol (compound (5) and (using General Screening Method 3b and Analytical Method 2) can carry out Reaction 5 by selectivelyhydroxylating the 21 position of 11|3-hydroxyprogesterone (3) to produce di-hydroxylated corticosterone (compound (7)).

[0173] Table 10 also summarizes exemplary results using General Screening Method 3a (and Analytical Method 2) to show that Pichia strains (HCP015 and HCP016) expressing CYP258 can carry out Reaction 6 by selectively hydroxylating the 11 p position of 17a- hydroxyprogesterone (2) to produce di-hydroxylated 21 -deoxycortisol (compound (6) and (using General Screening Method 3a and Analytical Method 2) can carry out Reaction 7 by selectively hydroxylating the 11 position of 21 -hydroxyprogesterone (2) to produce di- hydroxylated corticosterone (compound (7)).

[0174] Table 10 also summarizes exemplary results using General Screening Method 3a (and Analytical Method 2) to show that Pichia strains (HCP018 and HCP019) expressing CYP261 can carry out Reaction 7 by selectively hydroxylating the 11 p position of 21- hydroxyprogesterone (2) to produce di-hydroxylated corticosterone (compound (7)).

[0175] TABLE 10

[0176] Table 11 (below) shows exemplary results obtained using the General Screening Methods 3a, 3b, 3c, 4, or 5 (and Analytical Method 2) to show that Pichia strains expressing CYP258, CYP259, CYP256, and / or CYP261 , or mixtures of the Pichia strains can carry out Reactions 8, 9, and / or 10, to produce Hydrocortisone (8).

[0177] TABLE 11

[0178] As shown in Table 11 , the Pichia strains HCP017 and HCP020, that express both CYP258 and CYP259, can carry out Reaction 10 that includes the hydroxylation of both the 11 p and 21 positions of the precursor 17a-hydroxyprogesterone (2) to produce the product Hydrocortisone (8). Additionally, mixtures of two Pichia strains, where one expresses CYP258 and the other expresses CYP259 (e.g., HCP011 and HCP015, or HCP012 and HCP016) can carry out the bioconversion Reaction 10 to produce the product Hydrocortisone (8) from the precursor 17a-hydroxyprogesterone (2).Example 3: Screening CYPs in Recombinant Strains of Pichia pastoris for Production of 17a-Hydroxylated HC Precursor Intermediates

[0179] This example illustrates screening studies of various CYPs in recombinant Pichia pastoris strains for activity useful in 17a-hydroxylation reactions useful for preparing hydrocortisone.

[0180] Material and Methods

[0181] A. Plasmid Screening:

[0182] Pichia pastoris strains expressing different CYP and CPR enzyme pair combinations were generated as plasmid constructs as illustrated in the schematic of FIG. 2. The generated plasmid constructs with CYP and CPR pairs were screened on the plasmid for the ability to hydroxylate progesterone to generate 17a-hydroxyprogesterone (Reaction 1 of FIG. 1A). The specific CYP and CPR enzymes screened are shown in Table 12 below.

[0183] TABLE 12

[0184] Screening assays were performed by growing transfected cells for 24-48 h in BMGY media after which the biomass was harvested and the bioconversion was performed in a buffer containing 0.1 M phosphate pH 7, 2% methanol and 200 mg / L progesterone. Product concentrations were determined by mass spectrometry.

[0185] Results - Plasmid Screening

[0186] Four new CYP / CPR pairs exhibiting activity in 17a-hydroxylation of progesterone to 17a-hydroxyprogesterone were identified. The amount of substrate converted to product (expressed as percent conversion) is shown in Table 13 below. Amino acid and polynucleotide sequences of the CYP and CPRs are provided in Table 1 above and the accompanying Sequence Listing.

[0187] TABLE 13

[0188] B. Pichia Integrated Screening

[0189] Based on the promising plasmid screening results shown in Table 13, Pichia pastoris strains were prepared with the following CYP / CPR pairs genomically integrated: (1) CYP265 / CPR009; and (2) CYP267 / CPR010. The Pichia strains with integrated pairs were then screened to verify 17a-hydroxylation of progesterone to generate 17a- hydroxyprogesterone, as illustrated in Reaction 1 of FIG. 1A. The Pichia strain assays were performed by growing the cells for 24-48 h in BMGY media after which the biomass was harvested and the bioconversion was performed in a buffer containing 0.1 M phosphate pH 7 , 2% methanol and 200 mg / L progesterone. Product concentrations were determined by mass spectrometry.

[0190] Results - Pichia Screening

[0191] As shown by the results summarized in Table 14 below, the CYP265 / CPR009 and CYP267 / CPR010 pairs integrated in Pichia exhibited activity in 17a-hydroxylation of progesterone to 17a-OH-progesterone.

[0192] TABLE 14Example 4: Biosynthesis of HC Derivative Compound, Prednisolone

[0193] This example demonstrates how the addition of recombinant 3-ketosteroid-A1- dehydrogenase (“KstD”) enzyme to a biosynthetic process for the production of hydrocortisone and / or a hydrocortisone precursor compound, such as 17a- hydroxyprogesterone, can result in the production of the HC derivative compound, prednisolone. Prednisolone is a corticosteroid with several medicinal applications such as for arthritis, immune system disorders, cancer, and severe allergies. Although widely used, its production still relies largely on chemical synthesis which can be inefficient and expensive.

[0194] In the simplest biosynthetic embodiment, prednisolone can be produced by contacting HC with the enzyme, 3-ketosteroid-A1 -dehydrogenase (“KstD”) which catalyzes dehydrogenation of the C-C bond at position 1 of the A ring resulting in the formation of prednisolone, as shown in Scheme 1 below.Scheme 1hydrocortisone pre ixoSone

[0195] Alternatively, prednisolone can be produced via a multienzyme process in which an HC precursor compound such as progesterone or 17a-progesterone is used as the starting compound. The recombinant CYP and CPR enzymes as described in Examples 1-3 are used to convert the precursor to HC and the additional KstD enzyme catalyzes the final dehydrogenation at position 1 to produce prednisolone. For example, the hydrocortisone (HC) precursor 17a-progesterone can be used as the starting compound as shown in Scheme 2 below.

[0196] As shown in Scheme 2, the HC precursor 17a-hydroxyprogesterone is converted to HC using two CYPs. One that hydroxylates the 21 -position, such as CYP259, and one that hydroxylates the 11 p-position, such as CYP261. The 17a-hydroxyprogesterone can be contacted with the two CYPs in a single pot reaction. The HC compound produced by 17a- hydroxyprogesterone in the two CYP reaction is then contacted with recombinant KstD resulting in the formation of prednisolone.

[0197] Material and Methods

[0198] A. Pichia strains with integrated KstD enzyme

[0199] Three KstD enzymes (KstD1 , KstD2, and KstD3) were identified from the literature and their genes were synthesized for introduction into Pichia. The KstD genes and source information is summarized in Table 15 below.

[0200] Table 15: Candidate KstD enzymes

[0201] Pichia strains each engineered with one of the three specific 3-ketosteroid-D1- dehydrogenases, KTD1 , KTD2, or KTD3 were prepared. The Pichia strain names and genotypes are summarized in Table 16 below.

[0202] Table 16. KstD Pichia strain genotypes

[0203] B. Screening of Pichia strains carrying one of 3 different 3-ketosteroid- A1- dehydrogenase genes (KstD1, KstD2 or KstD3)

[0204] Materials and methods

[0205] The three ketosteroid-dehydrogenases KstD1 (SEQ ID NO: 50), KstD2 (SEQ ID NO: 52) or KstD3 (SEQ ID NO: 54) (see Table 15) were codon optimized for expression in Pichia pastoris, synthesized, and integrated into the genome of P. pastoris at the AOX1 locus and under the control of the AOX1 promoter. These strains were tested in three separate experiments (JT-56, JT-57, and JT-59) for their ability to convert hydrocortisone into prednisolone under specific growth and bioconversion conditions.

[0206] Experiment JT-56: Pichia strains PLP002, PLP004, and PLP006, harboring single copies of codon-optimized genes encoding KstD1 (SEQ ID NO: 49), KstD2 (SEQ ID NO: 51 , or KstD3 (SEQ ID NO: 53, respectively, at the AOX1 locus, were grown in shake flasks for 48 h, pre-induced or not pre-induced with MeOH for 24 h, after which the biomass from each culture was harvested and resuspended in bioconversion buffer containing 200 mg / L of hydrocortisone, 0.1 M phosphate buffer, and 2% MeOH at pH 7. After 24 h, the samples were extracted for analysis.

[0207] Experiment JT-57: assay conditions described above (JT-56 experiment) were followed for this experiment where only the no pre-induction process was repeated with the same strains.

[0208] Experiment JT-59: assay conditions described above (JT-56 experiment) were followed for this experiment and higher substrate loading (1 g / L hydrocortisone) was tested on PLP002 with and without 24 h preinduction.

[0209] Results

[0210] Experiment JT-56: As shown by the results summarized in Table 17, PLP002 (harboring KTD1) was the best performing strain and converted almost 100% of the substrate to prednisolone. PLP004 produced prednisolone in lesser amounts, while PLP006 showed no activity. Similar results were obtained between the pre-induction and no induction, i.e. the MeOH contained in the bioconversion buffer was sufficient to activate the AOX1 promoter.

[0211] Experiment JT-57: As shown by the results summarized in Table 17, PLP002 was the best performing strain with 100% of hydrocortisone converted to prednisolone.

[0212] Experiment JT-59: As shown by the results summarized in Table 17, 24 h preinduction leads to higher conversion of hydrocortisone to prednisolone.

[0213] TABLE 17: Results of experiments JT-56, JT-57, and JT-59

[0214] C. Screening of Pichia strain carrying 4 genes: CYP281 (11 p-OH), CYP259 (21- OH), CPR009 (reductase) and KstT1 (3-ketosteroid- A1 -dehydrogenase)

[0215] Materials and Methods

[0216] Experiment PD-82: The conversion of 17-a-hydroxyprogesterone (1 g / L) to prednisolone was tested on strain PLP025 with and without methyl-p-cyclodextrin. As shown by the genotype in Table 16, PLP025 includes two CYPs, 21 -position hydroxylating CYP, CYP259, and the 11 p-position hydroxylating CYP, CYP261 , and the CPR, CPR009, along with KstD1 , integrated in a Pichia strain. PLP025 single colonies were inoculated in 4 mL BMGY in screw-cap tubes and incubated for 18 hours at 30°C in a shaking incubator. 1 ml of the overnight culture was inoculated into 100 ml of fresh BMGY in a 500 ml baffled flask and incubated for 48 hours at 250 RPM and 85% humidity. 500 mg of wet biomass was added to a 20 ml scintillated glass vial with 2.5 ml of 0.1 M phosphate buffer pH 7, 2% MeOH and 1 g / L 17a-hydroxyprogesterone with and without 1 eq methyl-p-cyclodextrin. Bioconversion reactions were carried out at 30°C, 250 RPM for 24 hours. 1 :1 MeOH extraction (v / v) followed by 1000-fold dilution was performed before loading the samples for analysis on a LC-MS-QQQ.

[0217] Results

[0218] As shown by the results of experiment PD-82 summarized in Table 18 below, both conditions resulted in bioconversion of 17-a-hydroxyprogesterone (1 g / L) to prednisolone. Higher yields were observed with methyl-p-cyclodextrin present in the reaction matrix.

[0219] TABLE 18: Results of experiment PD-82Example 5: Biosynthesis of HC Derivative Compound, Prednisone

[0220] This example expands on Example 4 by demonstrating the addition of recombinant enzyme with 1 i p-hydroxysteroid dehydrogenase type 2 (“HSD2”) activity to a biosynthetic process for the production prednisolone from hydrocortisone and / or a hydrocortisone precursor compound, such as 17a-hydroxyprogesterone, can result in the production of the further HC derivative compound, prednisone. Although widely used as a medicine, the production of prednisone still relies largely on chemical synthesis which can be inefficient and expensive.

[0221] In the simplest biosynthetic embodiment, prednisone can be produced by contacting prednisolone with an HSD2 enzyme, such as the human HSD2 of SEQ ID NO: 56, which catalyzes oxidation of the hydroxyl group at position 11 of the C ring of prednisolone to a ketone group, thereby resulting in the formation of prednisone, as shown in Scheme 3 below.Scheme 3

[0222] Alternatively, prednisone can be produced using precursor prednisolone produced via a multienzyme process from HC, or an HC precursor compound, such as progesterone or 17a-hydroxyprogesterone, comprising a series of recombinant CYP and CPR enzymes as described in Examples 1-3, and an additional enzyme KstD enzyme to produce prednisolone as described in Example 4 (see e.g., Scheme 2).

[0223] Materials and Methods

[0224] The production of prednisone was achieved by engineering strains of Pichia pastoris with a codon-optimized version of the HSD2 gene of SEQ ID NO: 55 (see e.g., Albiston, et al., “Cloning and tissue distribution of the human 11|3-hydroxysteroid dehydrogenase type 2 enzyme,” Molecular and cellular endocrinology, 105(2), R11-R17; (1994)). The resulting engineered strain names and genotypes are listed in Table 19 below.

[0225] TABLE 19. Genotypes of strains engineered with HsHSD2

[0226] Experiment PD-64: strains PLP026 and PLP027 (and BG-10 control strain) were tested for their ability to convert prednisolone to prednisone under specific growth and bioconversion conditions. The strains were grown in shake flasks for 48 h, after which the biomass from each culture was harvested and resuspended in bioconversion buffer containing 100 mg / L of prednisolone from a 100X DMSO stock, 0.1 M phosphate buffer, 2% MeOH at pH 7, with or without 10 mg NAD+. After 18 h, the samples were extracted for analysis.

[0227] Results

[0228] Both strains PLP026 and PLP027 were able to convert the majority of supplied prednisolone to prednisone, with total yields ranging from 41 mg / L to 51 mg / L. There was no significant difference in prednisone production between the two strains, indicating the presence or absence of AOX1 is not a factor in bioconversion efficiency at this level of substrate. The control strain BG-10 (wild-type) and a blank buffer were also used as negative controls and yielded no prednisone. The quantitation for both the control and blank indicate that the precursor compound, prednisolone is stable as a substrate and not converted to other compounds.

[0229] While the foregoing disclosure of the present invention has been described in some detail by way of example and illustration for purposes of clarity and understanding, this disclosure including the examples, descriptions, and embodiments described herein are for illustrative purposes, are intended to be exemplary, and should not be construed as limiting the present disclosure. It will be clear to one skilled in the art that various modifications or changes to the examples, descriptions, and embodiments described herein can be made and are to be included within the spirit and purview of this disclosure and the appended claims. Further, one of skill in the art will recognize a number of equivalent methods and procedure to those described herein. All such equivalents are to be understood to be within the scope of the present disclosure and are covered by the appended claims.

[0230] Additional embodiments of the invention are set forth in the following claims.

[0231] The disclosures of all publications, patent applications, patents, or other documents mentioned herein are expressly incorporated by reference in their entirety for all purposes to the same extent as if each such individual publication, patent, patent application or otherdocument were individually specifically indicated to be incorporated by reference herein in its entirety for all purposes and were set forth in its entirety herein. In case of conflict, the present specification, including specified terms, will control.

Claims

CLAIMSWhat is claimed is:

1. A recombinant host cell comprising: a heterologous nucleic acid encoding a first CYP capable of converting 17a- hydroxyprogesterone (compound 2) to 11 -deoxycortisol (compound 5), wherein the first CYP comprises an amino acid sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 2, and a heterologous nucleic acid encoding a second CYP capable of converting 11 -deoxycortisol (compound 5) to hydrocortisone (compound 8), wherein the second CYP comprises an amino acid sequence having at least 80% sequence identity to a sequence selected from SEQ ID NO: 16, and 10, and 16; or a heterologous nucleic acid encoding a first CYP capable of converting 17a- hydroxyprogesterone (compound 2) to 21 -deoxycortisol (compound 6), wherein the first CYP comprises an amino acid sequence having at least 80% sequence identity to a sequence selected from SEQ ID NO: 16, and 10, and a heterologous nucleic acid encoding a second CYP capable of converting 21 -deoxycortisol (compound 6) to hydrocortisone (compound 8), wherein the second CYP comprises an amino acid sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 2.

2. The cell of claim 1 , wherein the host cell further comprises a heterologous nucleic acid encoding a third CYP capable of converting progesterone (compound 1) to 17a- hydroxyprogesterone (compound 2), wherein the third CYP comprises an amino acid sequence having at least 80% sequence identity to a sequence selected from SEQ ID NO: 36, 6, 38, 40, and 42.

3. A recombinant host cell comprising: a heterologous nucleic acid encoding a first CYP capable of converting 11 p- hydroxyprogesterone (compound 3) to 21 -deoxycortisol (compound 6), wherein the first CYP comprises an amino acid sequence having at least 80% sequence identity to a sequence selected from SEQ ID NO: 36, 6, 38, 40, and 42, and a heterologous nucleic acid encoding a second CYP capable of converting 21 -deoxycortisol (compound 6) to hydrocortisone (compound 8), wherein the second CYP comprises an amino acid sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 2; or a heterologous nucleic acid encoding a first CYP capable of converting 11 p- hydroxyprogesterone (compound 3) to corticosterone (compound 7), wherein the first CYP comprises an amino acid sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 2, and a heterologous nucleic acid encoding a second CYP capable of converting corticosterone (compound 7) to hydrocortisone (compound 8),wherein the second CYP comprises an amino acid sequence having at least 80% sequence identity to a sequence selected from SEQ ID NO: 36, 6, 38, 40, and 42.

4. The cell of claim 3, wherein the host cell further comprises a heterologous nucleic acid encoding a third CYP capable of converting progesterone (compound 1) to 11 p- hydroxyprogesterone (compound 3), wherein the third CYP comprises an amino acid sequence having at least 80% sequence identity to a sequence selected from SEQ ID NO: 16, and 10.

5. A recombinant host cell comprising: a heterologous nucleic acid encoding a first CYP capable of converting 21- hydroxyprogesterone (compound 4) to 11 -deoxycortisol (compound 5), wherein the first CYP comprises an amino acid sequence having at least 80% sequence identity to a sequence selected from SEQ ID NO: 36, 6, 38, 40, and 42 , and a heterologous nucleic acid encoding a second CYP capable of converting 11 -deoxycortisol (compound 5) to hydrocortisone (compound 8), wherein the second CYP comprises an amino acid sequence having at least 80% sequence identity to a sequence selected from SEQ ID NO: 16, and 10; or a heterologous nucleic acid encoding a first CYP capable of converting 21- hydroxyprogesterone (compound 4) to corticosterone (compound 7), wherein the first CYP comprises an amino acid sequence having at least 80% sequence identity to a sequence selected from SEQ ID NO: 16, and 10, and a heterologous nucleic acid encoding a second CYP capable of converting corticosterone (compound 7) to hydrocortisone (compound 8), wherein the second CYP comprises an amino acid sequence having at least 80% sequence identity to a sequence selected from SEQ ID NO: 36, 6, 38, 40, and 42.

6. The cell of claim 5, wherein the host cell further comprises a heterologous nucleic acid encoding a third CYP capable of converting progesterone (compound 1) to 21- hydroxyprogesterone (compound 4), wherein the third CYP comprises an amino acid sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 2.

7. The cell of any one of claims 1-6, wherein the host cell further comprises: a heterologous nucleic acid encoding a CPR capable of transferring electrons to the first CYP, the second CYP, and / or the third CYP; optionally, wherein the CPR comprises an amino acid sequence having at least 80% sequence identity to a sequence selected from SEQ ID NO: 4, 8, 12, 44, 46, and 48.

8. The cell of any one of claims 1-7, wherein the host cell further comprises: a heterologous nucleic acid encoding an enzyme with 3-ketosteroid-A1- dehydrogenase (“KstD”) activity; optionally, wherein the KstD comprises an amino acid sequence having at least 80% sequence identity to a sequence selected from SEQ ID NO: 50, 52, and 54.

9. The cell of claim 8, wherein the host cell further comprises: a heterologous nucleic acid encoding an enzyme with 11 p-hydroxysteroid dehydrogenase type 2 (“HSD2”) activity; optionally, wherein the HSD2 comprises an amino acid sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 56.

10. The cell of any one of claims 1-9, wherein the source organism of the cell is selected from Pichia pastoris, Saccharomyces cerevisiae, Yarrowia lipolytica, and Escherichia coli.11 . The cell of any one of claims 1-10, wherein the heterologous nucleic acid encoding the first, second, and / or third CYP is integrated at one or more sites in the genome of the cell; optionally, integrated at three or more sites.

12. The cell of any one of claims 1-11 , wherein the heterologous nucleic acid encoding the first and / or second CYP is under the control of a promoter system selected from pCAT1 :pFDH1, pAOX1 and pGal1 / 10.

13. The cell of any one of claims 1-12, wherein the recombinant host cell is Pichia pastoris and the heterologous nucleic acid is integrated at one or more sites in the genome selected from HIS4, Inti 5, Int18, Inti , Int6, and AOX1.

14. The cell of any one of claims 1-13, wherein the recombinant host cell is Saccharomyces cerevisiae and the heterologous nucleic acid is integrated at one or more sites in the genome selected from X-2, X-4, XI-2, XII-4, NDE1 , XII-5, Gal80, and ROQ1.

15. A process for preparing hydrocortisone, a hydrocortisone precursor, and / or a hydrocortisone derivative comprising:(a) contacting under suitable conditions a recombinant host cell of any one of claims 1- 14 and a substrate compound selected from progesterone (compound 1), 17a- hydroxyprogesterone (compound 2), 11 p-hydroxyprogesterone (compound 3), 21- hydroxyprogesterone (compound 4), 11 -deoxycortisol (compound 5), 21- deoxycortisol (compound 6), corticosterone (compound 7), and hydrocortisone (compound 8); and(b) recovering the hydrocortisone, hydrocortisone precursor, and / or a hydrocortisone derivative product compound.

16. The process of claim 15, wherein the substrate compound is progesterone (compound 1), and the product compound is hydrocortisone (compound 8).

17. The process of claim 15, wherein the substrate compound is selected from 17a- hydroxyprogesterone (compound 2), 11 p-hydroxyprogesterone (compound 3), and 21- hydroxyprogesterone (compound 4); and the product compound is hydrocortisone (compound 8).

18. The process of claim 15, wherein the substrate compound is selected from 11- deoxycortisol (compound 5), 21 -deoxycortisol (compound 6), and corticosterone (compound 7); and the product compound is hydrocortisone (compound 8).

19. The process of claim 15, wherein the substrate compound is selected from progesterone (compound 1) or 17a-hydroxyprogesterone (compound 2), and the product compound is prednisolone (compound 9), or prednisone (compound 10).

20. A composition in aqueous solution comprising:(i) a cell of any one of claims 1-14; and(ii) a compound selected from: progesterone (compound 1), 17a- hydroxyprogesterone (compound 2), 11 (3-hydroxyprogesterone (compound 3), 21- hydroxyprogesterone (compound 4), 11 -deoxycortisol (compound 5), 21 -deoxycortisol (compound 6), corticosterone (compound 7), hydrocortisone (compound 8), prednisolone (compound 9), and a mixture thereof.21 . A process for preparing hydrocortisone or a hydrocortisone derivative comprising:(a) contacting 17a-hydroxyprogesterone under suitable conditions with:(i) a recombinant host cell comprising a heterologous nucleic acid encoding afirst CYP capable of converting 17a-hydroxyprogesterone (compound 2) to 11- deoxycortisol (compound 5), wherein the first CYP comprises an amino acid sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 2; and(ii) a recombinant host cell comprising a heterologous nucleic acid encoding a second CYP capable of converting 11 -deoxycortisol (compound 5) to hydrocortisone (compound 8), wherein the second CYP comprises an amino acid sequence having at least 80% sequence identity to a sequence selected from SEQ ID NO: 16, and 10; and(b) recovering the produced hydrocortisone or hydrocortisone derivative.

22. A process for preparing hydrocortisone or a hydrocortisone derivative comprising:(a) contacting 17a-hydroxyprogesterone under suitable conditions with:(i) a recombinant host cell comprising a heterologous nucleic acid encoding a first CYP capable of converting 17a-hydroxyprogesterone (compound 2) to 21- deoxycortisol (compound 6), wherein the first CYP comprises an amino acid sequence having at least 80% sequence identity to a sequence selected from SEQ ID NO: 10 or 16;and(ii) a recombinant host cell comprising a heterologous nucleic acid encoding a second CYP capable of converting 21 -deoxycortisol (compound 6) to hydrocortisone (compound 8), wherein the second CYP comprises an amino acid sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 2 ; and(b) recovering the produced hydrocortisone or hydrocortisone derivative.

23. The process of any one of claims 21-22, wherein the process further comprises contacting progesterone (compound 1) under suitable conditions with a recombinant host cell comprising a heterologous nucleic acid encoding a third CYP capable of converting progesterone (compound 1) to 17a-hydroxyprogesterone (compound 2), wherein the third CYP comprises an amino acid sequence having at least 80% sequence identity to a sequence selected from SEQ ID NO: 36, 6, 38, 40, and 42.

24. A process for preparing hydrocortisone or a hydrocortisone derivative comprising:(a) contacting 1 ip-hydroxyprogesterone under suitable conditions with:(i) a recombinant host cell comprising a heterologous nucleic acid encoding a first CYP capable of converting 11 p-hydroxyprogesterone (compound 3) to 21- deoxycortisol (compound 6), wherein the first CYP comprises an amino acidsequence having at least 80% sequence identity to a sequence selected from SEQ ID NO: 36, 6, 38, 40, and 42 , and(ii) a recombinant host cell comprising a heterologous nucleic acid encoding a second CYP capable of converting 21 -deoxycortisol (compound 6) to hydrocortisone (compound 8), wherein the second CYP comprises an amino acid sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 2; and(b) recovering the produced hydrocortisone or hydrocortisone derivative.

25. A process for preparing hydrocortisone or a hydrocortisone derivative comprising:(a) contacting 1 ip-hydroxyprogesterone under suitable conditions with:(i) a recombinant host cell comprising a heterologous nucleic acid encoding a first CYP capable of converting 11 p-hydroxyprogesterone (compound 3) to corticosterone (compound 7), wherein the first CYP comprises an amino acid sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 2 , and(ii) a recombinant host cell comprising a heterologous nucleic acid encoding a second CYP capable of converting corticosterone (compound 7) to hydrocortisone (compound 8), wherein the second CYP comprises an amino acid sequence having at least 80% sequence identity to a sequence selected from SEQ ID NO: 36, 6, 38, 40, and 42; and(b) recovering the produced hydrocortisone or hydrocortisone derivative.

26. The process of any one of claims 24-25, wherein the process further comprises contacting progesterone (compound 1) under suitable conditions with a recombinant host cell comprising a heterologous nucleic acid encoding a third CYP capable of converting progesterone (compound 1) to 11 p-hydroxyprogesterone (compound 3), wherein the third CYP comprises an amino acid sequence having at least 80% sequence identity to a sequence selected from SEQ ID NO: 16, and 10.

27. A process for preparing hydrocortisone or a hydrocortisone derivative comprising:(a) contacting 21 -hydroxyprogesterone under suitable conditions with:(i) a recombinant host cell comprising a heterologous nucleic acid encoding a first CYP capable of converting 21-hydroxyprogesterone (compound 4) to 11- deoxycortisol (compound 5), wherein the first CYP comprises an amino acid sequence having at least 80% sequence identity to a sequence selected from SEQ ID NO: 36, 6, 38, 40, and 42 , and(ii) a recombinant host cell comprising a heterologous nucleic acid encoding a second CYP capable of converting 11 -deoxycortisol (compound 5) to hydrocortisone (compound 8), wherein the second CYP comprises an amino acid sequence having at least 80% sequence identity to a sequence selected from SEQ ID NO: 10, and 16; and(b) recovering the produced hydrocortisone or hydrocortisone derivative.

28. A process for preparing hydrocortisone or a hydrocortisone derivative comprising:(a) contacting 21-hydroxyprogesterone under suitable conditions with:(i) a recombinant host cell comprising a heterologous nucleic acid encoding a first CYP capable of converting 21-hydroxyprogesterone (compound 4) to corticosterone (compound 7), wherein the first CYP comprises an amino acid sequence having at least 80% sequence identity to a sequence selected from SEQ ID NO: 16, or 10; and(ii) a recombinant host cell comprising a heterologous nucleic acid encoding a second CYP capable of converting corticosterone (compound 7) to hydrocortisone (compound 8), wherein the second CYP comprises an amino acid sequence having at least 80% sequence identity to a sequence selected from SEQ ID NO: 36, 6, 38, 40, and 42.(b) recovering the produced hydrocortisone or hydrocortisone derivative.

29. The process of any one of claims 27-28, wherein the process further comprises contacting progesterone (compound 1) under suitable conditions with a recombinant host cell comprising a heterologous nucleic acid encoding a third CYP capable of converting progesterone (compound 1) to 21-hydroxyprogesterone (compound 4), wherein the third CYP comprises an amino acid sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 2.

30. The process of any one of claims 21-29, wherein the process further comprises:(iii) a recombinant host cell comprising a heterologous nucleic acid encoding an enzyme with 3-ketosteroid-A1 -dehydrogenase (“KstD”) activity capable of converting hydrocortisone (8) to the hydrocortisone derivative prednisolone (9), wherein the KstD comprises an amino acid sequence having at least 80% sequence identity to a sequence selected from SEQ ID NO: 50, 52, and 54; and recovering the produced hydrocortisone derivative prednisolone (9).31 . The process of claim 30, wherein the process further comprises:(iv) a recombinant host cell comprising a heterologous nucleic acid encoding an enzyme with 1 i p-hydroxysteroid dehydrogenase type 2 (“HSD2”) activity capable of converting the hydrocortisone derivative prednisolone (9) to prednisone (10), wherein the HSD2 comprises an amino acid sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 56; and recovering the produced hydrocortisone derivative prednisone (10).

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