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    • 专利摘要:
    Production of steroids 11 α hydroxylated by biotransformation with recombinant bacteria. The present invention provides a method for producing steroid compounds 11 α hydroxylated or derivatives thereof by fermentation of natural sterol or by biotransformation of different steroid synthons with recombinant bacteria, preferably mycobacterium smegmatis or corynebacterium glutamicum. Said recombinant bacteria carry a plasmid comprising a synthetic operon containing the genes cyp509cl2 and rocpr1 of rhizopus oryzae, which code for cytochrome cyp509c12 with activity 11 α -hydroxylase and cytochrome reductase (rocpr1) necessary for cytochrome activity. By means of the method described in the present invention, 11 α can be produced, but without limitation; oh-add or 11 α -oh-ad from natural sterols such as cholesterol or phytosterols, or 11 α -oh-prog, 11 α -oh-doc, 11 α -oh-test, 11 α -oh-dhea, 11 α -oh-ad and/or 11 α -oh-add from their corresponding non-hydroxylated synthons. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2648614A1
    申请号:ES201630701
    申请日:2016-05-30
    公开日:2018-01-04
    发明作者:Carmen FELPETO SANTERO；Beatriz GALÁN SICILIA；José Luis GARCÍA LÓPEZ
    申请人:Consejo Superior de Investigaciones Cientificas CSIC；
    IPC主号:
    专利说明:

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    PRODUCTION OF STEROIDS 11a HYDROXYLATES THROUGH BIOTRANSFORMATION WITH RECOMBINANT BACTERIA
    DESCRIPTION
    The present invention falls within the field of the chemical, pharmaceutical and food industry, particularly within the production processes of hydroxylated steroids 11a and derivatives thereof by microbial biotransformations, both from natural sterols and from non-hydroxylated tuners. .
    STATE OF THE TECHNIQUE
    Steroids are terpenic lipids with a defined structure, which contain a cyclopentaneperhydrophenanthrene or gonane nucleus with four fused rings (A-D); This basic structure is modified by the addition of various functional groups, such as carbonyls and hydroxyls (hydrophilic) or hydrocarbon chains (hydrophobic). The physiological activity of steroids depends on their structure, that is, on the type, number and position of the functional groups attached to the steroid nucleus, as well as on their configurational and structural isomerism, that is, stereoisomería and regioisomería, and on the oxidation state of the rings
    Steroids are widespread in nature and are present in all types of organisms. Thus, hundreds of sterols and related compounds that occur in plants, such as phytosterols, have been identified; in insects, such as ecdysteroids; in vertebrates, such as cholesterol, corticosteroids or steroid hormones; and in lower eukaryotes (yeasts and fungi), such as ergosterol.
    Nowadays, steroid-type pharmaceutical products are of great importance to maintain our quality of life, since many steroids are used as anti-tumor, anti-inflammatory, anti-microbial, anti-viral, anti-fungal, anti- agents. estrogenic, anti-convulsant and antiallergic. In addition, others are used as agents for the prevention and therapy of many other diseases, such as hormone-dependent breast and prostate cancers, certain forms of colon cancer, obesity, diabetes, rheumatoid arthritis, hypertension, asthma
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    eczema, inflammations, metabolic disorders, neurodegenerative diseases in the elderly, or diseases of the central nervous system, among many others. Androgens, anabolic steroids, estrogens, and corticosteroids, among others, are included here.
    Around 300 steroidal drugs have been approved to date for clinical use, and this number tends to grow. Steroid medications are among the most commercialized medical products and represent the second largest category of drugs along with antibiotics. The annual production of steroids is estimated at more than one million tons, with a market of billions of euros.
    Many steroids used as drugs are chemically synthesized, but it has long been known that the microbial transformation of steroids is a powerful tool for the generation of new steroidal drugs, as well as for the efficient production of key intermediates (precursors) for Chemical synthesis of such drugs. The bioconversions allow to modify the steroids in positions of the molecule that are hardly available or accessible to the chemical agents, and the functionalization of the molecule can be carried out regio- and stereo-specifically. Moreover, through bioconversion several reactions can be completed in one step. These and other advantages of biotransformations have meant a wide expansion of microbial technologies in the field of steroids, in such a way that the applications of microorganisms for steroid modification have been reviewed in numerous articles over the past few years. (Donova and Egorova, 2012, Appl Microbiol Biotechnol., 94 (6): 1423-1447).
    The main intermediate products or precursors for the industrial synthesis of steroidal drugs are 4-androsten-3,17-dione (hereinafter, AD) and 1,4-androstadien-3,17-dione (hereinafter, ADD); however, hydroxylated steroids often express greater biological activity compared to their less polar non-hydroxylated analogs. In particular, 11a-hydroxy-progesterone (hereinafter, 11a-OH-PROG), 11a-hydroxy-deoxycorticosterone (hereinafter, 11a-OH-DOC), 11a-hydroxy-testosterone (hereinafter, 11a-OH-TEST) , 11a-hydroxy-4-androsten-3.17-dione (hereinafter 11a-OH-AD) and 11a-hydroxy-1,4-androstadien-3.17-dione (hereinafter 11a-OH-ADD) they are necessary tunes to obtain glucocorticoids that,
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    In addition to being used in replacement therapy, they have pharmacological properties and are used as anti-allergens, anti-inflammatories, immunosuppressants and contraceptives.
    Therefore, the economical and efficient production of these precursors or tuners, preferably hydroxylated ones, is a prevailing necessity in the pharmaceutical industry.
    To obtain the AD and ADD tunings, one of the main raw materials used in the chemical steroid industry are sapogenins, such as diosgenin. Alternatively, some natural sterols are also used as starting materials in the steroid industry, such as 3p-alcohol steroids, which contain a double bond at position 5-6 and an aliphatic side chain at C-17. Among these steroids are cholesterol, which is known as animal sterol, or phytosterols, which are mixtures of plant-derived sterols, mainly of soy origin, such as sitosterol, stigmasterol, campesterol, and brasicasterol. Since the 1980s, the microbial transformation of phytosterol has been a focus of research in the field of steroids.
    Currently, in the industrial production of 11a-hydroxylated steroidal compounds, species belonging to the Aspergillus or Rhizopus genera are used; in particular A. ochraceus, R. oryzae and R. nigricans. These fungi are capable of hydroxylating several steroidal compounds in 11th position, including AD or ADD; but they also modify and hydroxylate to a greater or lesser extent in other positions of the molecule, which drastically lowers production yields.
    To date, these hydroxylated compounds cannot be synthesized from low cost raw materials in a single step; Hydroxylation is done from a syntone (AD or ADD) or from its direct precursors.
    In the specific case of R. oryzae, the enzyme responsible for carrying out 11a-hydroxylations has been identified. It is a cytochrome P450 called Cyp509C12 (European Nucleotide Archive - EBI EIE80372.1) and the cytochrome reductase protein called RoCPR1, which transfers the NADPH necessary for hydroxylase activity (European Nucleotide Archive - EBI EIE89541.1) (Petric et al. , 2010, J Biotechnol., 150 (3): 428-437; WO2011042143A1). Cyp509C12 11a-hydroxylase performs the
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    hydroxylation in position 11a and 6p of various tunings of pharmacological and / or industrial interest, including progesterone (hereinafter, PROG), deoxycorticosterone (hereinafter, DOC), testosterone (hereinafter, TEST), and deoxycortisol (hereinafter, Reichstein's Substance S, RSS).
    The cytochrome 11a-hydroxylase (GenBank DD180525.1) and the cytochrome reductase protein (GenBank AR838156.1) from A. ochraceus have also been identified (US7033807). This 11a-hydroxylase uses as a substrate various tunings of pharmacological and / or industrial interest, including PROG, TEST, AD, ADD, aldone, canrenone, mexrenone and derivatives of the latter.
    The cytochrome CYP509C12 and cytochrome reductase RoCPRI proteins of R. oryzae have been produced heterologously in the Schizosaccharomyces pombe yeast and the hydroxylated products in position 11a and 6p have been identified, as well as other unidentified compounds, from PROG, DOC, TEST and RSS, (Petric et al., 2010, J Biotechnol., 150 (3): 428-437; WO2011042143A1). However, these yeasts are not currently used for industrial production due to the low yields for the hydroxylation of these tuners.
    On the other hand, the cytochrome P450 11a-hydroxylase from A. ochraceus has been expressed in insect cells from a cDNA library. Its enzymatic activity (measured in the microsomal fraction) has been obtained in coexpression with the cytochrome reductase (CPR) of A. ochraceus found by similarity with that already identified of A. niger or in coexpression with a human CPR (US7033807).
    The bacterium Corynebacterium glutamicum is an actinobacterium widely studied for its use as an industrial producer of amino acids, which is why today there are numerous molecular tools for its modification. In fact, since its discovery, the production spectrum of C. glutamicum has been extended in recent decades to different chemicals, materials and fuels through multiple genetic and metabolic engineering strategies. In addition, it should be noted that, despite the great phylogenetic resemblance to the genus Mycobacterium, this bacterium does not use steroids as a source of carbon and energy, since it lacks most of the genes related to its catabolism. Its genome is sequenced and has been extensively studied, becoming a bacterial chassis for industrial biotransformations, with characteristics that the
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    they become an ideal platform for this type of biotransformations such as the multitude of molecular tools for their manipulation, robustness and metabolic vigor.
    Mycobacterium smegmatis mc2155 is an actinobacterium capable of metabolizing steroids and using them as a source of carbon and energy. In a previous patent, mutants of this strain were developed for the production of tuners such as AD and ADD from phytosterols. In these strains the degradation pathway of cholesterol or phytosterols was blocked, so the degradation of the compound does not follow and ADD (M. smegmatis mc2155 A6039, CECT 8331) and AD (M. smegmatis m22155 A6039A5941, CECT 8332) ( WO2015128534).
    However, to date, no natural or recombinant system capable of producing hydroxylated steroids 11a, derivatives thereof or their hydroxylated precursors (tuners) 11a is known, efficiently, since the producing organisms designed to date (fungi and yeasts) have the disadvantage that they generate many secondary products that complicate the purification of the hydroxylated derivative, which significantly decreases the process performance.
    Therefore, the design of new organisms that allow hydroxylation in position 11a compounds with steroid structure is required to obtain hydroxylated steroids 11a or hydroxylated syntheses 11a efficiently, in an improved production process resulting in higher yields of the final product. In addition, ideally these organisms should allow such production both from non-hydroxylated precursors (tuners) and from natural sterols, thus generating hydroxylated steroidal compounds 11a of high purity.
    DESCRIPTION OF THE INVENTION
    The present invention provides recombinant bacteria capable of efficiently hydroxylating compounds with steroid structure in position 11a. These bacteria therefore allow microbial biotransformation processes to be carried out in which hydroxylated steroidal compounds 11a and hydroxylated precursor intermediates (tunings) 11a are efficiently and economically generated. These precursors are, for example, but not limited to, 11a-hydroxyprogesterone (11a-OH-PROG), 11a-hydroxy-deoxycorticosterone (11a-OH-DOC), 11a-
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    hydroxy-testosterone (11a-OH-TEST), 11a-hydroxy-dehydroepiandrosterone (11a-OH-DHEA), 11a-hydroxy-4-androsten-3.17-dione (11a-OH-AD) and 11a-hydroxy-1 ,4-
    androstadien-3,17-diona (11a-OH-ADD).
    To date, these hydroxylated compounds could not be synthesized directly from low-cost raw materials, such as natural sterols, in a single step, since hydroxylation was performed from an intermediate syntone (AD, ADD, TEST, PROG, etc. .) in a process carried out by fungal CYP / CPR systems such as Rhizopus oryzae or Aspergillus ochraceus. One of the advantages of the present invention is that it allows hydroxylated steroids 11a to be obtained from natural sterols in a single fermentation process.
    In addition, when fungi or yeasts are used for the production of these compounds, many secondary products are generated that complicate the purification of the hydroxylated derivative, which drastically lowers the process performance. However, the present invention represents a solution to this problem, since the developed recombinant bacteria are capable of hydroxylating in a position 11a compounds with steroid structure, both from syntones and natural sterols, generating hydroxylated steroidal compounds 11a of high purity with a high production performance thanks to a drastic reduction of secondary products.
    For the development of the recombinant bacteria of the invention, it has been started from actinobacteria cells, preferably from Corynebacterium glutamicum R31 or Mycobacterium smegmatis mc2 155, where the DNA sequences encoding the enzymes have been heterologously expressed by means of a synthetic operon involved in the 11a-hydroxylase activity in the R. oryzae fungus. Specifically, said enzymes are a cytochrome P450 (CYP509C12) and its corresponding NADPH-dependent cytochrome reductase (RoCPR1). Both enzymes have been expressed in said bacteria through the design and construction of an operon comprising the DNA coding for said cytochrome and said reductase and in which certain modifications / improvements for its expression in bacteria have also been introduced, such as the optimization of codons, restriction sites, consensus sequences, etc.
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    Additionally, the M. smegmatis host strains employed in the present invention as starting cells are preferably mutants that produce AD and ADD tunings from cholesterol and phytosterols. Thus, in the present invention, recombinant bacterial biocatalysts capable of hydroxylating steroids in position 11a have been developed by a one-step biotransformation process, in which both non-hydroxylated and natural sterols can be used as starting material, which does not It can be obtained with fungi or yeasts.
    Particularly, the modified strain of C. glutamicum of the present invention is capable of producing the 11a-hydroxylated tuners: 11a-OH-PROG, 11a-OH-DOC, 11a-OH-TEST, 11a-OH-DHEA, 11a-OH -AD and 11a-OH-ADD substantially pure, from their corresponding non-hydroxylated precursors (tuners) PROG, DOC, TEST, DHEA, AD and ADD, respectively. The modified strain of M. smegmatis CECT 8331 of the present invention is capable of producing substantially pure synthase 11a-hydroxylated 11a-OH-ADD, from natural sterols. The modified strain of M. smegmatis CECT 8332 of the present invention is capable of producing substantially pure synthase 11a-hydroxylated 11a-OH-AD, from natural sterols.
    The advantages derived from the present invention, in particular the use of bacteria against eukaryotic organisms, are therefore:
    - The fact of transferring this 11a hydroxylation capacity of steroids to a bacterium makes it possible to obtain the steroid of industrial interest hydroxylated in 11th position with greater purity and efficiency thanks to a drastic reduction of the secondary reaction products, as however it does not happen directly using fungi or yeasts, which modify steroidal substrates in various positions, contaminating the samples and making purification difficult. The 11a-hydroxylation carried out by the bacteria of the invention facilitates the product recovery processes, by reducing the by-products; and improves production yields by allowing to obtain a product with a higher degree of purity.
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    - They allow to obtain hydroxylated steroidal derivatives 11a, preferably 11a-OH-ADD and 11a-OH-AD, from natural sterols in a single fermentation process, contrary to what happens with fungi and yeasts where the process has been in two steps (from the raw material to the syntone and from this to its hydroxylated derivative 11a).
    Therefore, a first aspect of the present invention relates to a gene or genetic construct comprising a nucleotide sequence encoding the cytochrome CYP509C12 of Rhizopus oryzae and a nucleotide sequence encoding the RoCPR1 reductase of R. oryzae. From now on, this aspect will be referred to as the "gene construct of the invention".
    In a preferred embodiment of this aspect of the invention, the nucleotide sequence encoding the cytochrome CYP509C12 and the nucleotide sequence encoding the RoCPR1 reductase from R. oryzae are in the form of an operon. “Operon” means a functional genetic unit formed by a group or complex of genes capable of exerting a regulation of its own expression by means of the substrates with which the proteins encoded by their genes interact. In the present invention, this operon will also be referred to as "FUN operon".
    The gene construct of the invention may further comprise other regulatory elements of gene expression, such as, for example, but not limited to, promoters, regulators, operators, terminators, inductors, etc. A "promoter" is a control element that is a region of DNA with a sequence that is recognized by RNA polymerase to begin transcription. It is found immediately before the structural genes coding for CYP509C12 and RoCPR1. The promoter referred to in the present invention may be constitutive or inducible, preferably inducible. Examples of prokaryotic promoters include, for example, but not limited to, promoters of the trp, hps, recA, lacZ, lacI, tet, gal, trc, or tacher Escherichia coli genes, or the a-amylase gene promoter of Bacillus subtilis.
    An "operator" is another control element that is a region of DNA with a sequence that is recognized by the regulatory protein. The operator is located between the promoter region and the structural genes. A "regulator" is a DNA sequence that encodes the regulatory protein that recognizes the sequence of the region of the
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    operator. The regulatory gene is close to the structural genes of the operon but is not immediately next. An "inducer" is the substrate or compound whose presence / absence induces the expression of the rest of the genes that make up the operon. It can act by activating the expression, calling itself “activator” or repressing it, calling itself “repressor”.
    The gene construct of the present invention preferably comprises nucleotide sequences in the form of an inducible operon, meaning "inducible operon" which is not normally expressed and is activated in response to an inducing agent that functions as an activator, so that at the moment when the inductor joins the operator, the promoter is activated and transcription of the structural genes begins. The classic model of this type of operon is the lactose operon. Other examples of inducible operons are, but not limited to, those that code for enzymes that participate in the metabolism of substrates such as maltose, arabinose operon, etc. In the present invention the operon is preferably inducible by IPTG.
    In another preferred embodiment, the gene construct of the invention is comprised in an expression vector. The term "expression vector" refers to a DNA molecule in which another DNA fragment can be integrated without losing self-replication. The term "expression vector" refers to a cloning vector suitable for expressing a nucleic acid that has been cloned therein after being introduced into a host cell. Said nucleic acid is generally operatively linked to control sequences. In a more preferred embodiment, the vector is selected from the group consisting of: plasmids, phages, cosmids, phagemids, artificial yeast chromosomes (YAC), artificial bacterial chromosomes (BAC), artificial human chromosomes (HAC), viral vectors, such as adenovirus, retrovirus or any other type of DNA molecule capable of replicating inside a cell, preferably prokaryotic. In an even more preferred embodiment, the gene construct of the invention is comprised in a plasmid. Examples of plasmids into which the gene construct of the invention can be introduced are, but not limited to, pGH, pMV261, pECXK-99E, etc., preferably pMV261 and pECXK-99E.
    Due to the degeneracy of the genetic code, in which several nucleotide triplets give rise to the same amino acid, there are several sequences of
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    nucleotides that give rise to the same amino acid sequence. The terms "nucleotide sequence", "nucleotide sequence", "nucleic acid", "oligonucleotide" and "polynucleotide" are used interchangeably herein and refer to a polymeric form of nucleotides of any length that may or may not be chemical or biochemically modified, thus referring to any polyiribonucleotide or polydeoxyribonucleotide, both single-stranded and double-stranded.
    The nucleotide sequences comprised in the gene construct of the invention may comprise, in addition to the coding sequence, other elements, such as, but not limited to, non-coding sequences at the 5 'or 3' ends, ribosome binding sites, or sequences stabilizers These polynucleotides can additionally also include coding sequences for additional amino acids that may be useful, for example, but not limited to increasing the stability of the peptide generated from it or allowing a better purification thereof.
    In another preferred embodiment of the gene construct of the invention, the cytochrome Cyp509C12 is that described in the European Nucleotide Archive - EBI EIE80372.1 and the cytochrome reductase protein RoCPRI is that described in the European Nucleotide Archive - EBI EIE89541.1.
    In a more preferred embodiment, the nucleotide sequences encoding the cytochrome CYP509C12 and the RoCPRI reductase of Rhizopus oryzae are optimized for expression in bacteria, that is, the codons of said nucleotide sequences are optimized. In an even more preferred embodiment, the nucleotide sequence encoding the cytochrome CYP509C12 is SEQ ID NO: 1. In another preferred embodiment, the nucleotide sequence encoding the RoCPRI reductase is SEQ ID NO: 2.
    In another preferred embodiment, the gene construct of the invention further comprises a consensus sequence for a ribosome binding site or upstream Shine Dalgarno sequence of each of the start codons (ATG) of each nucleotide sequence, to achieve optimal translation of mRNA in bacteria In a more preferred embodiment, this consensus sequence is located 6 bp (6 nucleotides) upstream of each of the start codons of each sequence
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    nucleotide In an even more preferred embodiment, this consensus sequence is SEQ ID NO: 3.
    In another preferred embodiment, the gene construct of the invention further
    It comprises at least two restriction sites to facilitate different cloning options. Examples of restriction sites that can be introduced into the gene construct of the invention are, but not limited to, BamHI, PstI, SacI, NcoI, PvuII, AvrII, MfeI, NdeI, EcoRI and / or XbaI.
    In another preferred embodiment, the gene construct of the invention further
    it comprises a codon that gives rise to an alanine located in the second position of each of the two translated proteins. In this way, the translation of recombinant proteins in bacterial ribosomes is increased. Codons that give rise to alanine are GCU, GCC, GCA or GCG.
    An example of a structural arrangement of the gene construct of the invention is shown in Fig. 1.
    The gene construct of the invention can be introduced into a bacterial cell such that said construct is maintained as a chromosomal integrant or as a self-replicating extrachromosomal vector, for example, a plasmid, a minichromosome, or an artificial chromosome. The gene construct of the invention may comprise any means or element to ensure its self-replication. Alternatively, the gene construct of the invention may be one that, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome (s) in which it has been integrated .
    Therefore, another aspect of the invention relates to a bacterial cell that comprises and expresses the gene construct of the invention, hereafter referred to as "bacterial cell of the invention" or "cell of the invention".
    The bacterial cell of the invention expresses cytochrome CYP509C12 and RoCPRI reductase from Rhizopus oryzae because it comprises the gene construct of the invention, in a self-replicating form or integrated in its chromosome. The term "expression" includes any stage involved in the production of these enzymes that
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    includes, but is not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.
    The bacterial cell must be transformed into a competent cell, preferably electrocompetent, for the subsequent introduction of the gene construct of the invention. Methods for producing electrocompetent cells are well known to those skilled in the art. Preferably, the method employed in the present invention is RbCl and thermal shock or that described in Parish and Stoker (1998, Mycobacteria Protocols. Totowa, N.J., Humana Press).
    Various methods for the transformation of bacterial cells with gene constructs are well known in the art. The method preferably used in the present invention for the introduction of the gene construct of the invention into a bacterial cell is electroporation.
    In another preferred embodiment, the bacterial cell of the invention is an actinobacteria cell. In a more preferred embodiment, said cell is a cell of the Corynebacterium glutamicum species, even more preferably C. glutamicum R31. In another preferred embodiment, said cell is a cell of the Mycobacterium smegmatis species, even more preferably M. smegmatis mc2 155.
    In a more preferred embodiment, the bacterial cell of the invention of the species Mycobacterium smegmatis further comprises functionally inactivated or totally or partially deleted at least one endogenous nucleotide sequence encoding the reductase component of the enzyme 3-ketosteroid-9a-hydroxylase. In an even more preferred embodiment, this cell comprising functionally inactivated or totally or partially deleted at least one endogenous nucleotide sequence encoding the reductase component of the 3- ketosteroid 9a-hydroxylase enzyme is M. smegmatis CECT 8331 cell.
    In another preferred embodiment, this cell of the invention of the Mycobacterium smegmatis species which further comprises functionally inactivated or totally or partially deleted at least one endogenous nucleotide sequence encoding the reductase component of the enzyme 3-ketosteroid-9a-hydroxylase, further comprises functionally inactivated or totally or partially deleted at least one endogenous nucleotide sequence encoding the 3-ketosteroid-A1- enzyme
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    dehydrogenase In an even more preferred embodiment, this cell comprising functionally inactivated or totally or partially deleted at least one endogenous nucleotide sequence encoding the reductase component of the 3- ketosteroid 9a-hydroxylase enzyme and which further comprises functionally inactivated or totally or partially deleted At least one endogenous nucleotide sequence encoding the 3-ketosteroid-A1-dehydrogenase enzyme is the M. smegmatis CECT 8332 cell.
    These strains CECT 8331 and CECT 8332, described in WO2015128534, have blocked the path of degradation of cholesterol or phytosterols, so that the degradation of the compound does not follow and ADD accumulates (M. smegmatis mc2155 A6039, CECT 8331) and AD (M smegmatis mc2155 A6039A5941, CECT 8332). These strains are preferably used as starting cells in this invention for the development of the bacterial cell of the invention comprising the gene construct of the invention.
    The wild bacterium M. smegmatis mc2 155 cannot use substances AD and ADD as a carbon source when these substances are added to a culture medium, despite being intermediates of bacterial cholesterol catabolism. In this way, the M. smegmatis mc2155 bacterium is an ideal bacterium for the production of the intermediate AD and ADD necessary for the production of their corresponding hydroxylated derivatives, since once secreted to the medium they cannot be catabolized by the metabolism of the bacteria.
    The term "deletion of a gene" as used in the present invention refers to the total or partial elimination of a gene by total or partial elimination of the DNA sequence that characterizes that gene in the genome of a bacterium.
    The term "functionally inactivated nucleotide sequence" as used in the present invention refers to a nucleotide sequence that is not capable of exerting its functionality, that is, it is not capable of providing a functional enzyme.
    Another aspect of the invention relates to the use of the bacterial cell of the invention for the production of hydroxylated steroids 11a, hydroxylated steroidal compounds 11a or derivatives thereof, or hydroxylated 11a syntheses.
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    In a preferred embodiment of this aspect of the invention, hydroxylated steroids 11a or hydroxylated syntheses 11a are produced from non-hydroxylated syntones. In a more preferred embodiment, the hydroxylated 11a syntheses produced are 11a-OH-PROG, 11a-OH-DOC, 11a-OH-TEST, 11a-OH-DHEA, 11a-OH-AD and / or 11a-OH-ADD and the non-hydroxylated tunings from which the former are produced are PROG, DOC, TEST, DHEA, AD and / or ADD, respectively. In an even more preferred embodiment, the producer cell of these indicated hydroxylated 11a syntheses, preferably 11a-OH-PROG from PROG, is the cell of the invention of the species C. glutamicum.
    In another preferred embodiment, the hydroxylated syntone 11a produced is 11a-OH-ADD and is produced from natural sterols. In a more preferred embodiment, the bacterial cell producing this indicated hydroxylated syntone 11a is the cell of the invention of the species M. smegmatis which further comprises functionally inactivated or totally or partially deleted at least one endogenous nucleotide sequence encoding the reductase component of the enzyme 3- ketosteroid-9a-hydroxylase, preferably strain CECT 8331.
    In another preferred embodiment, the hydroxylated syntone 11a produced is 11a-OH-AD and is produced from natural sterols. In a more preferred embodiment, the bacterial cell producing this indicated hydroxylated syntone 11a is the cell of the invention of the species M. smegmatis which further comprises functionally inactivated or totally or partially deleted at least one endogenous nucleotide sequence encoding the reductase component of the enzyme 3- ketosteroid-9a-hydroxylase and at least one endogenous nucleotide sequence encoding the enzyme 3-ketosteroid-A1-dehydrogenase, preferably strain CECT 8332.
    The "natural sterols" referred to in the present invention are preferably cholesterol or phytosterols, although other steroids 3-p alcohols could also be used as starting material. Examples of phytosterols are, but not limited to, sitosterol, stigmasterol, campesterol or brasicasterol The natural sterols used in the present invention may be only cholesterol or a phytosterol, or a mixture of phytosterols.
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    The "hydroxylated steroids 11a" obtainable according to the present invention are those compounds with hydroxylated steroid structure in position 11a, preferably those that are part of the families of Estrogens, Androgens, Progestogens and Glucocorticoids; for example, estrone, estradiol, testosterone or fluoxymestrone, among others.
    Another aspect of the invention relates to a method of microbial (bacterial) biotransformation of steroidal substrates in their corresponding hydroxylated derivatives 11a or process for the production of hydroxylated steroids 11a or hydroxylated syntheses 11a comprising the steps of:
    to. contacting a culture of the bacterial cell of the invention with a steroidal substrate,
    b. incubate the mixture from step (a) under fermentation conditions, and
    C. separate from the culture medium the hydroxylated steroids 11a or the hydroxylated 11a syntheses produced after the incubation of step (b).
    From now on this procedure will be referred to as "method or method of the invention".
    The process of the invention can be carried out both at the laboratory and at the industrial level, although preferably it is carried out at the industrial level in a bioreactor.
    In a preferred embodiment of the method of the invention, the culture medium of step (a) comprises an additional carbon source, more preferably glycerol.
    In another preferred embodiment, the culture medium of step (a) comprises a surfactant. Said surfactant is selected, but not limited to, from polysorbate 80 (Tween 80 ™) or polysorbate 20 (Tween 20 ™). The culture medium of step (a) may also comprise phosphate buffer.
    In another preferred embodiment, the culture medium of step (a) comprises at least one antibiotic. Said antibiotic is selected, but not limited to, from among kanamycin, gentamicin, or any combination thereof.
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    In general, the cultivation of step (a) can be carried out, for example, but not limited to, in liquid medium or solid medium. Culture media and conditions for bacterial cell growth are widely known to those skilled in the art. The culture medium comprises all the elements and nutrients necessary for the growth and survival of the bacterial cell of the invention and to favor its fermentation activity. Thus, said culture medium may comprise, but not limited to, carbon sources (for example, glucose, sucrose, glycerol or mannitol), sources of vitamins, amino acids, inorganic salts, etc.
    In order for the bacterial cell of the invention to grow properly in the culture medium, it must meet a series of conditions such as temperature, agitation, humidity, and adequate oxygen pressure, as well as a correct degree of acidity or alkalinity (pH ). Likewise, the culture medium must be free of all contaminating microorganisms.
    As for the fermentation conditions suitable for application in step (b) of the process of the invention, preferably said conditions include stirring and incubation at a temperature between 30 and 37 ° C. More preferably, Ta is 30 ° C when the bacterial cell of the invention is of the species C. glutamicum and 37 ° C when the bacterial cell of the invention is of the species M. smegmatis. The incubation is preferably carried out for a time between 25 and 35 h, more preferably for 30 h when the bacterial cell of the invention is of the species C. glutamicum. Cultures of the bacterial cell of the invention must be induced by the corresponding activator compound if the promoter employed in the gene construct of the invention is inducible. Thus, preferably the bacterial culture in the method of the invention is induced with IPTG.
    In another preferred embodiment, the steroidal substrate of step (a) is selected from the list consisting of: phytosterols, cholesterol, PROG, DOC, TEST, DHEA, AD or ADD, or any combination thereof.
    In a more preferred embodiment of the method of the invention, the starting steroidal substrate of step (a) is phytosterols and / or cholesterol, the hydroxylated syntone 11a produced is 11a-OH-AD and the bacterial cell used for production is the cell of the invention of the species M. smegmatis which also comprises inactivated
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    functionally or partially or totally deleted at least one endogenous nucleotide sequence encoding the reductase component of the 3- ketosteroid 9a-hydroxylase enzyme and at least one endogenous nucleotide sequence encoding the 3-ketosteroid-A1-dehydrogenase enzyme, preferably strain CECT 8332.
    In another preferred embodiment of the method of the invention, the starting steroidal substrate of step (a) is phytosterols and / or cholesterol, the 11a-hydroxylated syntone produced is 11a-OH-ADD and the bacterial cell used for production is the cell of the invention of the species M. smegmatis which further comprises functionally inactivated or totally or partially deleted at least one endogenous nucleotide sequence encoding the reductase component of the 3- ketosteroid 9a-hydroxylase enzyme, preferably strain CECT 8331.
    In another preferred embodiment of the method of the invention, the starting steroidal substrate of step (a) is AD, ADD, PROG, DOC, DHEA and / or TEST, preferably PROG, the hydroxylated syntone 11a produced is 11a-OH-PROG, 11a-OH-DOC, 11a-OH-TEST, 11a-OH-DHEA, 11a-OH-AD and / or 11a-OH-ADD, preferably 11a-OH-PROG, and the bacterial producing cell is the bacterial cell of the invention, more preferably the cell of the invention of the species C. glutamicum.
    The process of the invention may comprise other additional steps or steps, such as a previous step of preparing and sterilizing a mixture of tuners or natural sterols or both, preferably in a polyalcohol or vegetable oil, with or without the addition of salts. minerals and / or a step of preparing and growing a culture of the bacterial cell of the invention prior to its exposure to the steroidal substrate.
    The hydroxylated steroids 11a or hydroxylated syntheses 11a produced by the process of the invention are secreted, together with other metabolites or compounds, in the culture medium, and these can be recovered directly from the medium. Thus, after the incubation of step (b) of the method of the invention, the cell pellet can be separated to obtain a solution comprising the hydroxylated steroid (s) 11 or the hydroxylated syntone 11 s produced. These products are obtained with a high purity and with a higher production yield than when fungi or yeasts are used in similar biotransformation processes.
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    Following the incubation of step (b) of the process of the invention an extraction step takes place in which the sediments are discarded and the supernatants are selected. Said supernatants are subsequently used to obtain hydroxylated steroids 11a or hydroxylated 11a syntones present therein.
    These compounds secreted to the culture medium by the bacterial cell of the invention can be recovered from the medium using methods known in the art. For example, by conventional procedures including, but not limited to, centrifugation, filtration, extraction, evaporation and / or precipitation.
    These compounds produced by the bacterial cell of the invention in culture can be purified by a variety of methods known in the art including, but not limited to, chromatography (eg, HPLC, HPLC-DAD, TLC, LC-MS, ion exchange, affinity , hydrophobic, chromato-focus, and molecular exclusion), electrophoretic procedures (for example, preparatory isoelectric focusing), differential solubility (for example, precipitation with ammonium sulfate), SDS-PAGE, precipitation or extraction, in order to obtain hydroxylated steroids 11a or substantially pure hydroxylated 11a tunes.
    These compounds produced by the bacterial cell of the invention in culture can be detected using methods known in the art. These detection procedures may include, for example, but not limited to, chromatography, NMR, mass spectrometry, or the like.
    The present invention thus relates to genetically modified strains of M. smegmatis mc2 155 containing a synthetic operon (FUN) encoding the cytochrome CYP509C12 and the cytochrome reductase RoCPR1 of R. oryzae, as well as the processes for producing 11a -OH-AD and 11a-OH-ADD from natural sterols, such as cholesterol or phytosterols, using said modified strains.
    The present invention also relates to genetically modified strains of C. glutamicum R31 containing a synthetic operon (FUN) encoding cytochrome CYP509C12 and cytochrome reductase RoCPR1 from R. oryzae, as well as processes for producing 11a-OH-PROG , 11a-OH-DOC, 11a-OH-DHEA and / or 11a-OH-TEST, among others, in a substantially pure manner from the corresponding tunings.
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    The invention relates to the use of the recombinant strain M. smegmatis CECT 8331, which is a mutant strain derived from M. smegmatis mc2 155 comprising at least one nucleotide sequence encoding the reductase component of the 3-ketosteroid enzyme 9a- Functionally inactivated or partially or totally deleted hydroxylase, which carries a plasmid comprising the FUN operon, which is capable of producing 11a-OH-ADD in a substantially pure manner. The invention relates to the use of said bacterial strain for the production of 11a-OH-ADD in substantially pure form by fermentation of natural sterols.
    The invention relates to the use of the recombinant strain M. smegmatis CECT 8332, which is a mutant strain derived from M. smegmatis mc2 155 comprising at least one nucleotide sequence encoding the reductase component of the 3-ketosteroid-9a enzyme - functionally inactivated or partially or totally deleted hydroxylase, as well as at least one nucleotide sequence encoding the functionally inactivated or totally or partially deleted 3-ketosteroid-M-dehydrogenase enzyme, which carries a plasmid comprising the FUN operon, which is capable of producing 11a-OH-AD in a substantially pure manner. The invention relates to the use of said bacterial strain for the production of 11a-OH-AD in substantially pure form by fermentation of natural sterols.
    The invention relates to the use of the recombinant strain C. glutamicum R31 which carries a plasmid comprising the FUN operon, which is capable of producing hydroxylated steroids in 11th position (11a-OH-PROG, 11a-OH-DOC, 11a- OH-TEST, 11a-OH-DHEA, 11a-OH-AD and 11a-OH-ADD) from their corresponding tuners (PROG, DOC, TEST, DHEA, AD and ADD, respectively) in a substantially pure manner. The invention relates to the use of said bacterial strain for the production of 11a-OH-PROG, 11a-OH-DOC, 11a-OH-TEST, 11a-OH-DHEA, 11a-OH-AD and 11a-OH-ADD in substantially pure form from their corresponding tunings: PROG, DOC, TEST, DHEA, AD and ADD, respectively.
    Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following examples and figures are provided by way of illustration, and are not intended to be limiting of the present invention.
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    DESCRIPTION OF THE FIGURES FIG. 1. Scheme of the FUN operon.
    FIG. 2. Biotransformation of PROG in 11a-OH-PROG using strain C. glutamicum R31 (pXKFUN). The consumption of PROG (gray dotted line) and the appearance of 11a-OH-PROG (continuous black line) are represented.
    FIG. 3. Chromatograms of LC-MS showing the production of 11aOH-progesterone from progesterone in strain C. glutamicum R31 (pECXK-99E) used as a negative control (A) and strain C. glutamicum R31 (pXKFUN) (B) . PROG, 11a-OH-PROG and TEST, which has been used as an internal standard (ISTD). Mass spectra obtained from the m / z ions between 150-400 (full sean) present in the sample (above line). Mass spectrum of the characteristic 315 ion of the PROG compound (middle line). Mass spectrum of ion 331.3 characteristic of compound 11a-OH-PROG (bottom line).
    FIG. 4. Biotransformation of cholesterol (CHO) in 11a-OH-ADD using strain M. smegmatis CECT 8331 (pMVFUN). CHO consumption and the appearance of 11a-OH-ADD and other by-products are shown: ADD and 11a-OH-AD.
    FIG. 5. Biotransformation of phytosterols (FITO) in 11a-OH-ADD using strain M. smegmatis CECT 8331 (pMVFUN). It shows the consumption of phytosterols and the appearance of 11a-OH-ADD and other by-products: ADD.
    FIG. 6. Biotransformation of cholesterol (CHO) in 11a-OH-AD using strain M. smegmatis CECT 8332 (pMVFUN). CHO consumption and the appearance of 11a-OH-AD and other by-products are shown: AD, 11a-OH-ADD and 4-HBC (22OH-23,24-bisnorchol-4-en-3-one).
    FIG. 7. Biotransformation of phytosterols (FITO) in 11a-OH-AD using strain M. smegmatis CECT 8332 (pMVFUN). The consumption of FITO and the appearance of 11a-OH-AD and other by-products are shown: AD and 4-HBC (22OH-23,24-bisnorchol-4-en-3-a).
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    EXAMPLES
    Next, the invention will be illustrated by tests carried out by the inventors that demonstrate the effectiveness of recombinant bacterial cells designed in the production of hydroxylated steroids 11a or their derivatives, both from syntones and natural sterols.
    EXAMPLE 1. Bacteria used and culture methods.
    The bacterial strains used in this invention are:
    1. - Mycobacterium smegmatis mc1 2 155 (ept-1, mc2 mutant efficient for electroporation).
    2. - Mycobacterium smegmatis mc2 155 A6039 (M. smegmatis mc2 155 with the deleted MSMEG_6039 gene) (strain CECT8331).
    3. - Mycobacterium smegmatis mc2 155 A6039A5941 (M. smegmatis mc2 155 with the deleted MSMEG_6039 and MSMEG_5941 genes) (CECT8332 strain).
    4. - Corynebacterium glutamicum R31 (MeLisR, AecR efficient for electroporation).
    5. - Escherichia coli DH10B (F-, mcrA, A (mrr-hsdRMS-mcrBC), f80AlacZDM15AlacX74, deoR, recAI, endAI, araD139, A (ara, leu) 7697, galU, galK, rpsL, nupG, A) ( Invitrogen).
    The plasmids used in this invention for cloning, mutation and gene expression, together with their most relevant characteristics are shown below:
    1. - Plasmid pMV261 (Expression vector in mycobacteria, under the control of the PhsPso promoter, KmR).
    2. - Plasmid pECXK-99E (Bifunctional vector E. coli / C. Glutamicum, which contains the lacIq gene, KmR).
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    All solutions and culture media used in this invention were sterilized by moist heat in an autoclave at 121 ° C and 1 atm pressure, or by filtration using sterile Millipore filters of 0.2 ^ m in diameter.
    The rich medium used to grow E. coli cells was the Lysogenic Broth (LB) medium. Cultures in solid medium were performed with LB medium to which Bacto Agar (Pronadisa) was added at 1.5% (w / v). If necessary antibiotics were added at the following final concentrations: ampicillin (100 ^ g ml-1 ), Kanamycin (50 ^ g ml-1), chloramphenicol (20 ^ g ml-1). E. coli cells were grown at 37 ° C. The cultures in liquid medium were carried out in an orbital shaker at 250 rpm. Growth in liquid medium was followed by 600 nm turbidimetry (DO600) using a UVMini-1240 spectrophotometer (Shimadzu).
    For the cultivation of mycobacteria, the rich medium Bacto Middlebrook 7H10 Agar (7H10) (Difco) was used as a solid medium, and Middlebrook 7H9 Broth (7H9) (Difco) as a liquid medium. Media 7H9 and 7H10 were supplied with 0.2% (v / v) of glycerol and 10% (v / v) of Middlebrook ADC Enrichment (ADC) (Difco) (7H9 / Gli / ADC and 7H10 / Gli / ADC) . In all cultures in liquid medium 0.05% (v / v) of Tween80 (Sigma) previously autoclaved was added to avoid aggregation of the cells (7H9 / Gli / ADC / Tween). If necessary, antibiotics were added at the following concentrations: kanamycin (20 ^ g ml-1) and gentamicin (5 ^ g ml-1). M. smegmatis cells were grown at 37 ° C. The cultures in liquid medium were carried out in an orbital shaker at 250 rpm. Growth in liquid medium was followed by 600 nm turbidimetry (DO600) using a UV-1240 UV spectrophotometer (Shimadzu).
    The rich medium Tryptic Soy Agar (TSA) (Difco) was used as solid medium and Tryptic Soy Broth (TSB) (Difco) was used as a liquid medium for the culture of corinebacteria. If necessary, kanamycin (25 ^ g ml-1) was added. C. glutamicum cells were grown at 30 ° C. The cultures in liquid medium were carried out in an orbital shaker at 250 rpm. Growth in liquid medium was followed by 600 nm turbidimetry (DO600) using a UVMini-1240 spectrophotometer (Shimadzu).
    EXAMPLE 2. Methods of genetic transformation for the construction of recombinant strains.
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    A. - TRANSFORMATION OF E. coli CELLS
    E. coli cells were genetically modified by transformation after making them competent by the method of RbCl and thermal shock.
    B. - TRANSFORMATION OF M. smegmatis CELLS
    1. - Preparation of electrocompetent cells of M. smegmatis.
    The preparation of electrocompetent cells of M. smegmatis was carried out according to a previously described protocol (Parish and Stoker, 1998, Mycobacteria Protocols. Totowa, N.J., Humana Press). It starts from a stationary phase pre-circle (approximately 30 h) (10 ml in 100 ml flask) in 7H9 / Gli / ADC / Tween medium. Fresh medium is inoculated with DO600 0.01 (200 ml 7H9 / Gli / ADC / Tween in 1L flask) and incubated with stirring (200 rpm) at 30 ° C to an OD600 of 0.8-1 (approximately 18 h of culture). The culture is then maintained 1.5 h on ice and centrifuged in 4 50 ml conical tubes at 3000 x g at 4 ° C for 10 min. Once the supernatant is decanted, the cells are resuspended by gently turning in 200 ml of a 10% (v / v) solution of glycerol and 0.05% (v / v) of Tween-80 (Gli / Tween) cooled in ice. The cell suspension is then centrifuged, the supernatant is removed and resuspended again in 100 ml of the same Gli / Tween solution. After a third centrifugation step, 25 ml of Gli / Tween are added. The cells are resuspended by turning and allowed to stand to settle the aggregates. Subsequently, the suspension is taken without the aggregates with a sterile pipette. The suspension is taken to a clean ice-cold conical tube and centrifuged. Finally, the supernatant is removed, the cells are resuspended in 1.5 ml of cold Gli / Tween and distributed in 200 µl aliquots that are stored at -80 ° C until use.
    2. - Transformation of M. smegmatis cells by electroporation.
    To an aliquot of 200 µl of electrocompetent cells is added 1 ^ g of the DNA with which they will be transformed. This mixture is deposited in an electroporation cuvette (Cell Projects, 50 x 2 mm) and incubated for 10 min on ice. It is then electroporated in a Gene Pulser electroporator (Biorad) at 2.5 kV, 25 ^ F and 1000 Q and the cuvette is incubated on ice for another 10 min. Subsequently, 1 ml is added
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    of 7H9 / Gli / ADC / Tween medium to the cuvette and the liquid is passed to a 15 ml conical tube. The cells are incubated 4 h at 37 ° C with shaking (250 rpm) before sowing them in solid medium 7H10 / Gli / ADC with the necessary antibiotics.
    C.- TRANSFORMATION OF C. glutamicum CELLS
    1. - Preparation of electrocompetent cells of C. glutamicum.
    For the preparation of the cells, one starts from a stationary phase pre-circle (approximately 30 h) (10 ml of medium in a 100 ml flask) in TSB medium. The fresh medium is inoculated with a DO600 0.01 (200 ml TSB in 1L flask) and incubated under stirring (200 rpm) at 30 ° C to an OD600 of 1.2-1.5 (approximately 6 h of culture). The culture is then maintained 30 min on ice and centrifuged in 4 50 ml conical tubes at 3000 x g at 4 ° C for 10 min. Once the supernatant is decanted, the cells are resuspended by gently turning in 200 ml of a 10% (v / v) solution of ice-cold glycerol. The cell suspension is then centrifuged, the supernatant is removed and resuspended again in 100 ml of the same solution. After a third centrifugation step, 25 ml of 10% (v / v) glycerol are added. The cells are resuspended by turning and allowed to settle to settle the aggregates. Subsequently, the suspension is taken without the aggregates with a sterile pipette. The suspension is taken to a clean ice-cold conical tube and centrifuged. Finally, the supernatant is removed, the cells are resuspended in 2.0 ml of 10% (v / v) glycerol and distributed in 50 µl aliquots that are stored at -80 ° C until use.
    2. - Transformation of C. glutamicum cells by electroporation.
    To a 50 µl aliquot of electrocompetent cells, 1 ^ g of the DNA with which it is to be transformed is added. This mixture is passed to an electroporation cuvette (Cell Projects, 50 x 2 mm) and incubated for 10 min on ice. It is then electroporated in a Gene Pulser electroporator (Biorad) at 2.5 kV, 25 ^ F and 200 Q. Subsequently, 1 ml of TSB medium is added to the cuvette and passed to a 15 ml conical tube. The cells are incubated 1 h at 30 ° C with shaking (200 rpm) before sowing them in solid TSA medium with the necessary antibiotics.
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    EXAMPLE 3. Design and construction of the FUN operon and the recombinant plasmids that carry it.
    To carry out the heterologous production of the 11a-hydroxylase activity in bacteria, the synthetic operon called FUN was designed, which contains the genes cyp509C12 and roCPRI of R. oryzae, which encode the cytochrome CYP509C12 with 11a-hydroxylase activity and the cytochrome protein RoCPRI reductase necessary for its activity, respectively. The use of codons was optimized manually for the Mycobacterium and Rhodococcus bacteria, while maintaining a 82.8% nucleotide identity percentage for cytochrome and 83.24% for CPR. To achieve optimal translation of the mRNA, a Shine Dalgarno / RBS sequence (SEQ ID NO. 3: AAAGGGAG) 6 nucleotides was added upstream from the respective start codons of each gene. Various restriction sites were also added to facilitate different cloning options. In addition, alanine was added as the second amino acid to increase protein translation. The designed operon was synthesized by chemical synthesis and was initially cloned into plasmid pGH generating the recombinant plasmid pGH-FUN that was used to transform competent E. coli DH10B cells. Plasmid pGH-FUN was sequenced to verify that the cloned operon sequence was correct. The scheme of the FUN operon can be seen in Figure 1.
    Plasmid pGH-FUN was digested with BamHI and EcoRI to release the fragment containing the FUN operon and this was subcloned into plasmid pMV261, with double origin of replication for E. coli / Mycobacterium; resulting in the vector pMVFUN that will express the genes under control of the constitutive promoter Phps. Plasmid pMVFUN was initially cloned into E. coli DH10B where it was sequenced to verify that the construction was correct. Plasmids pMV261 and pMVFUN were subsequently isolated from carrier E. coli strains and transformed into M. smegmatis by electroporation.
    On the other hand, plasmid pGH-FUN was digested with SacI and Xbal to release the fragment containing the FUN operon and this was subcloned into plasmid pECXK-99E, with double origin of replication for E. coli / C. glutamicum resulting in the pXKFUN vector that will express the genes under control of the Ptrc promoter, inducible by IPTG. Plasmid pXKFUN was initially cloned in E. coli DH10B where it was sequenced to verify that the construction was correct. Plasmids pECXK-99E and pXKFUN
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    were subsequently isolated from carrier E. coli strains and transformed into C. glutamicum by electroporation.
    EXAMPLE 4. Steroid analysis techniques.
    1. - Extraction methods
    A. - Steroid extraction in reaction systems with resting cells.
    The extraction of the steroids to be analyzed from reaction systems with resting cells was carried out by collecting aliquots (1 ml) of the reaction to which the corresponding internal standards (testosterone) were added to the concentration of interest (250 ^ M) before beginning the extraction process. The samples with the internal standard are extracted in 10 ml of chloroform and homogenized using a Bullet Blender 50-DX-CE homogenizer at speed 12, for 3 min. After rupture, the cells are centrifuged 10 min at 4000 x g at 4 ° C and subsequently the tubes are frozen at -80 ° C for 10-15 min to properly separate the two phases. The organic phase is collected, passed into 50 ml conical tubes and evaporated completely at 70 ° C.
    B. - Steroid extraction in reaction systems with cells in culture.
    The steroid extraction to be analyzed by the different chromatographic techniques in reaction systems with cells in culture was carried out by collecting aliquots (0.2 ml) of the different cultures to which the corresponding internal standards were added to the concentration of interest , indicated in each case, before starting the extraction process. Samples with the internal standard are extracted in 0.5 ml of chloroform and homogenized using a vortex for 30 s. The samples are frozen at -80 ° C for 10-15 min to properly separate the two phases. The organic phase is collected, passed into 2 ml eppendorf tubes and evaporated completely at 70 ° C.
    2. - Chromatographic methods
    A.- FINE LAYER CHROMATOGRAPH (TLC).
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    The TLC plates used (Silica gel 60 F254 20 x 20 cm, Merck) are trimmed leaving 1 cm of margin on each side, 8 cm of stroke and 1 cm of separation between sample and sample. The different samples extracted are resuspended in acetonitrile in a volume of between 10-20 ^ l depending on the concentration expected in each case. To perform the TLC, 10 ^ 1 of each sample is loaded onto the TLC plate and a mixture of chloroform: ethanol (95: 5) (Merck) is used as the mobile phase. The different steroids of biotransformations are revealed with a solution of 20% (v / v) H2SO4.
    B. - HIGH EFFICIENCY LIQUID CHROMATOGRAPH COUPLED TO SERIAL PHOTODY DETECTOR (HPLC-DAD) AND LIQUID CHROMATOGRAPH COUPLED TO MASS SPECTROMETRY (LC-MS).
    The detection and quantification of the products generated in the steroid biotransformation reactions was performed by HPLC-DAD-MS using a liquid chromatography equipment (Surveyor Plus LC) equipped with an automatic injector coupled in series to a DAD detector that allowed to monitor elution at DO245 and with an ion trap (LXQ) equipped with a source of atmospheric pressure chemical ionization (APCI) and a source of isoelectronebulization (IES), all supplied by Thermo Electron (San Jose, CA, USA). The data was processed with the Xcalibur software (Thermo Fisher Scientific, San Jose, CA, USA). The separation is carried out by means of a C18 reverse phase column and at a specific gradient in each case.
    EXAMPLE 5. Biotransformation of PROG in 11a-OH-PROG using strain C. glutamicum R31 (pXKFUN).
    For progesterone biotransformation (PROG) assays with resting cells of C. glutamicum R31 (pXKFUN) 50 mM phosphate buffer (pH 7.4) was used, to which the steroids were added at a final concentration of 0.5 mM, from a steroid solution prepared at 5 mM in 10% (v / v) of Tyloxapol, the final concentration thereof being therefore 1% (v / v). Samples were taken to analyze steroid biotransformation for 72 h. To obtain the biomass used in biotransformation, a total volume of 200 ml of cells was cultured
    C. glutamicum grown in TSB medium for 30 h at 30 ° C and 250 rpm in 1 flask
    l from a DO600 of 0.1, using a 48 h pre-circle grown in TSB medium, to
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    30 ° C and stirred at 250 rpm. If necessary, kanamycin (25 g ml-1) and 0.5 mM 5-aminolevulinic acid were added. The cultures were induced with 1 mM IPTG when they reached an OD600 of 1.5. These cells were collected by centrifugation (10 min, 4000 x g) and washed twice with 50 mM phosphate buffer (pH 7.4).
    - Identification and quantification of PROG and 11a-OH-PROG.
    Chromatographic separation was performed on a Mediterranean Sea C18 column (150 mm x 4.6 mm internal diameter, 5 ^ m particle size) (Teknokroma, Barcelona). The mobile phases used contained water and 0.1% formic acid (A), acetonitrile and 0.1% formic acid (B). The flow used was 1 ml min-1 and the linear gradient used is shown in table 1.
     Time (min)  % A% B
     0  50 50
     5  50 50
     35  20 80
     40  20 80
     Four. Five  50 50
     60  50 50
    Table 1.
    During this gradient, the eluent was analyzed by DAD and by the mass spectrometer from minute 1 to 50. The working conditions of the equipment were: IES ionization source, capillary temperature (350 ° C), fogging coverage ( 60 ° C), capillary voltage (9 V), amplifier (400 Vp), source current (100 ^ A). High purity nitrogen was used as auxiliary gas and nebulizer. The quantitative analysis was performed by the internal standard method, from dilutions of the stock solutions of PROG and 11a-OH-PROG at concentrations between
    500,000 ^ M and 15,625 ^ M, to which 250 ^ M of testosterone was added as internal standard. The extraction of analytes was carried out in the same manner as in the samples to be analyzed (see example 4, section 1). Once the extraction is done,
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    the residues were resuspended in 500 µl of acetonitrile (LC-MaScan, LAB-SCAN) of which 25 ^ l were injected for chromatographic analysis.
    The results of a biotransformation experiment of PROG in 11a-OH-PROG can be seen in Figures 2 and 3.
    Strain C. glutamicum R31 (pXKFUN) is also capable of hydroxylating 11a other steroids other than PROG and converting them into their corresponding hydroxylated derivatives 11a. For example, when the strain C. glutamicum R31 (pXKFUN) is supplied as a substrate, instead of PROG, substances such as DOC, TEST, AD or dehydroepiandrosterone (hereinafter, DHEA), at a concentration of 0.5 mM, The bacterial strain is capable of synthesizing 11a-OH-DOC, 11a-OH-TEST, 11a-OH-AD or 11a-OH-DHEA, respectively, with yields of 41.5 ± 3.6%, 49.3 ± 0 , 5%, 38.0 ± 7.6% and 22.3 ± 8.7%, respectively.
    EXAMPLE 6. Biotransformation of CHO in 11a-OH-ADD using strain M. smegmatis CECT 8331 (pMVFUN).
    For the biotransformation tests of cholesterol with growing cells of M. smegmatis CECT 8331 (pMVFUN), 7H9 medium without supplements (without glycerol and without ADC) was used to which 18 mM glycerol and cholesterol were added to the final concentration of 1 mM, contained in 3.6% (v / v) of Tyloxapol. If necessary, kanamycin (20 ^ g ml "1) and 0.5 mM 5-aminolevulinic acid were added from the moment of inoculation. A total volume of 20 ml of M. smegmatis cell culture was grown at 37 ° C and 250 rpm in a 100 ml flask from a DO600 of 0.1 using a 48 h pre-circle grown in 7H9 / Gli / ADC / Tween medium, at 37 ° C and stirred at 250 rpm. Samples were taken to analyze the steroid biotransformation and growth was monitored for 96 h.
    - Identification and quantification of ADD and 11a-OH-ADD using CHO as substrate
    Chromatographic separation was performed on a Tracer Excel 120 ODSB C18 column (150 mm x 4.6 mm internal diameter, 5 ^ m particle size) (Teknokroma, Barcelona). The mobile phases used contained water and 0.1% formic acid (A),
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    acetonitrile and 0.1% formic acid (B), isopropanol and 0.1% formic acid (C). The flow used was 1 ml min "1 and the linear gradient used is shown in table 2.
     Time (min)  % A% B% C
     0  50 50  0
     5  50 50 0
     fifteen  20 71 9
     twenty  4 87 9
     40  0 85 15
     41  0 85 15
     42  50 50 0
     52  50 50 0
    Table 2.
    During this gradient, the eluent was analyzed by DAD and by the mass spectrometer from minute 1 to 52. The working conditions of the equipment were: APCI ionization source, capillary temperature 275 ° C, vaporization temperature 425 ° C , capillary voltage 39 V, corona discharge voltage 6.00 kV, source current 6.00 ^ A and 15 eV for collision dissociation energy. High purity nitrogen was used as auxiliary gas and nebulizer.
    The quantitative analysis was performed by the internal standard method, from dilutions of the CHO stock solution, in concentrations between
    1000,000 ^ M and 15,625 ^ M, to which 500 ^ M of testosterone was added as internal standard. The extraction of analytes was carried out in the same manner as in the samples to be analyzed (see example 4, section 1). After extraction, the residues were resuspended in 1500 µl of acetonitrile (LC-MaScan, LAB-SCAN) of which 25 ^ l were injected for chromatographic analysis.
    The quantifications of 11a-OH-ADD were performed by calculating the reaction performance using the corrected areas, as there was no pure product. The yield with respect to the substrate (cholesterol) is calculated as:
    5
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    25
    ^ 11a-OH-ADD / CHO -
    / liaOH-ADD and ISTD)
    U ^ WADD '
    AD [n lSTDj
    V ISTD.
    „/ LiaOH — AD + A (-
    M
    liaOH-ADD
    and 74-HB ^ 71,4-HB ^ / CHOV
    ) + A (ISTD) + A (ISTD) + A (/ STO,)]
    The result of an experiment can be seen in Figure 4.
    EXAMPLE 7. Biotransformation of FITO in 11a-OH-ADD using strain M. smegmatis CECT 8331 (pMVFUN).
    For the biotransformation tests of phytosterols with growing cells of M. smegmatis CECT 8331 (pMVFUN) 7H9 medium without supplements (without glycerol and without ADC) was used to which 18 mM glycerol and phytosterols were added to the final concentration of 1 mM, contained in 3.6% (v / v) of Tyloxapol. If necessary, kanamycin (20 g ml-1) and 0.5 mM 5-aminolevulinic acid were added from the moment of inoculation. A total volume of 20 ml of M. smegmatis cell culture was grown at 37 ° C and 250 rpm in a 100 ml flask from a DO600 of 0.1 using a 48 h pre-circle grown in 7H9 / Gli / ADC / medium Tween, at 37 ° C and stirred at 250 rpm. Samples were taken to analyze steroid biotransformation and growth was monitored for 96 h.
    - Identification and quantification of ADD and 11a-OH-ADD using as FITO substrate
    Chromatographic separation was performed on a Tracer Excel 120 ODSB C18 column (150 mm x 4.6 mm internal diameter, 5 ^ m particle size) (Teknokroma, Barcelona). The mobile phases used contained water and 0.1% formic acid (A), acetonitrile and 0.1% formic acid (B), isopropanol and 0.1% formic acid (C). The flow used was 1ml min-1 and the linear gradient used is shown in the following table 3:
     Time (min)  % A% B% C
     0  50 50  0
     5  50 50 0
     fifteen  20 71 9
     twenty  0 91 9
    5
    10
    fifteen
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    25
    30
    35
     40  0 70 30
     41  0 85 15
     42  50 50 0
     52  50 50 0
    Table 3.
    During this gradient, the eluent was analyzed by DAD and by the mass spectrometer from minute 1 to 52. The working conditions of the equipment were: APCI ionization source, capillary temperature 275 ° C, vaporization temperature 425 ° C , capillary voltage 39 V, corona discharge voltage 6.00 kV, source current 6.00 ^ A and 15 eV for collision dissociation energy. High purity nitrogen was used as auxiliary gas and nebulizer.
    The quantitative analysis was performed by the internal standard method, from dilutions of the FITO stock solution, in concentrations between
    1000,000 ^ M and 15,625 ^ M, to which 500 ^ M of testosterone was added as internal standard. The extraction of analytes was carried out in the same manner as in the samples to be analyzed. After extraction, the residues were resuspended in 1500 µl of acetonitrile (LC-MaScan, LAB-SCAN) of which 25 ^ l were injected for chromatographic analysis.
    For the preparation of the culture medium, as for the calibration line, phytosterols solutions were prepared at a concentration of 1 mM using the molecular weight of p-sitosterol, the most abundant of the phytosterols present in the mixture, for the calculation. However, for the quantification a correction was made based on the relative abundance of its components: p-sitosterol (83.61%), campesterol (7.59%) and stigmasterol (8.79%) and taking into account molecular weight the concentration of them was calculated from each of them, so that in the real concentration in the samples prepared at 1 mM concentration (initial culture medium) it was as follows: 0.84 mM p-sitosterol, 0.08 mM of campesterol and 0.09 mM of stigmasterol. In the quantification, both for the calculation of consumption and for the calculation of yields, the concentration of phytosterols is expressed as the sum of the concentrations of its components, e.g. for the initial time the concentration of phytosterols would be 0.84 + 0.08 + 0.08 = 1 mM). As can be seen, although this correction was made, it is negligible.
    5
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    25
    30
    The quantifications of ADD and 11aOH-ADD were performed by calculating the reaction performance using the corrected areas, as there was no pure product. The yield with respect to the substrate (phytosterols) is calculated as:
    ^ 11a-OH-ADD / FITO -
    / liaOH-ADD and ISTD)

    ADD and „/ liaOH-AD + ^ (- ^ -
    M
    liaOH-ADD
    and, / 4-HBCy, / 1,4-HBCy, / FITOy-,) + ^ (ISTD) + ^ (ISTD) + ^ (/ STO,)]
    The result of an experiment can be seen in Figure 5.
    EXAMPLE 8. Biotransformation of CHO in 11a-OH-AD using strain M. smegmatis CECT 8332 (pMVFUN).
    For the biotransformation tests of cholesterol with growing cells of M. smegmatis CECT 8332 (pMVFUN) the 7H9 medium without supplements (without glycerol and without ADC) was used to which 18 mM glycerol and cholesterol were added to the final concentration of 1 mM, contained in 3.6% (v / v) of Tyloxapol. If necessary, kanamycin (20 g ml-1) and 0.5 mM 5-aminolevulinic acid were added from the moment of inoculation. A total volume of 20 ml of M. smegmatis cell culture was grown at 37 ° C and 250 rpm in a 100 ml flask from a DO600 of 0.1 using a 48 h pre-circle grown in 7H9 / Gli / ADC / medium Tween, at 37 ° C and stirred at 250 rpm. Samples were taken to analyze steroid biotransformation and growth was monitored for 96 h.
    The identification and quantification of AD and 11a-OH-AD was carried out using the methodology contained in example 6. The quantification 11a-OH-AD was also carried out through the calculation of reaction performance using the corrected areas, as it did not provide of pure product. The yield with respect to the CHO substrate is calculated as:
    ^ 11a-OH-AD / CHO =
    / íQQH-ADy ISTD)
    KtÍtMtÜ)
    ADD and „/ liaOH-AD
    + 4 — FT
    M
    liaOH-ADD
    and, / 4-HBCy, / 1,4-HBCy, / CHOV) + ^ (ISTD) + ^ (ISTD) + ^ (/ STO,)]
    The result of an experiment can be seen in Figure 6.
    EXAMPLE 9. Biotransformation of FITO in 11a-OH-AD using strain M. smegmatis CECT 8332 (pMVFUN).
    For the biotransformation tests of phytosterols with growing cells of M. smegmatis CECT 8332 (pMVFUN), 7H9 medium was used without supplements (without glycerol and without ADC) to which 18 mM glycerol and phytosterols were added to the final concentration of 1 mM, contained in 3.6% (v / v) of Tyloxapol. If necessary, kanamycin (20 g ml-1) and 0.5 mM 5-aminolevulinic acid were added from the moment of inoculation. A total volume of 20 ml of M. smegmatis cell culture was grown at 37 ° C and 250 rpm in a 100 ml flask from a DO600 of 0.1 using a 48 h pre-circle grown in 7H9 / Gli / ADC / medium Tween, at 37 ° C and stirred at 250 rpm. Samples were taken to analyze the biotransformation of 10 steroids and growth was monitored for 96 h.
    fifteen
    The identification and quantification of AD and 11a-OH-AD was carried out using the methodology contained in Example 7. The quantification 11a-OH-AD was also performed by calculating the reaction performance using the corrected areas, as it did not provide of pure product. The yield with respect to the substrate (FITO) is calculated as:
    ^ 1
    1 a-OH-AD / FITO -
    / liaOH-DNA ISTD)

    ADD „/ liaOH-AD
    + K — F5Í
    M
    liaOH-ADD
    and, / 4 — HBCy, / 1,4 — HBCy, / FiTOy-,) + ^ (ISTD) + ^ (ISTD) + ^ (/ Sto)]
    The result of an experiment can be seen in Figure 7.
    twenty
    权利要求:
    Claims (29)
    [1]
    5
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    35
    1. Gene construct comprising a nucleotide sequence encoding the cytochrome CYP509C12 of Rhizopus oryzae and a nucleotide sequence encoding the RoCPR1 reductase of Rhizopus oryzae.
    [2]
    2. Gene construct according to claim 1, wherein the nucleotide sequence encoding the cytochrome CYP509C12 is SEQ ID NO: 1.
    [3]
    3. Gene construct according to any one of claims 1 or 2, wherein the nucleotide sequence encoding the RoCPR1 reductase is SEQ ID NO: 2.
    [4]
    4. Gene construct according to any one of claims 1 to 3, further comprising a Shine Dalgarno consensus sequence upstream of each of the start codons of each nucleotide sequence.
    [5]
    5. Gene construct according to claim 4, wherein the consensus sequence is located 6 bp upstream of each of the start codons of each nucleotide sequence.
    [6]
    6. Gene construct according to any one of claims 1 to 5, further comprising at least two restriction sites.
    [7]
    7. Gene construct according to any one of claims 1 to 6, further comprising a codon that results in an alanine located in the second position of each of the two translated proteins.
    [8]
    8. Bacterial cell comprising the gene construct according to any one of claims 1 to 7.
    [9]
    9. Bacterial cell according to claim 8, which is an actinobacteria cell.
    [10]
    10. Bacterial cell according to any of claims 8 or 9, which is a cell of the species Corynebacterium glutamicum.
    5
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    [11]
    11. Bacterial cell according to any of claims 8 or 9, which is a cell of the species Mycobacterium smegmatis.
    [12]
    12. Bacterial cell according to claim 11, further comprising
    functionally inactivated or totally or partially deleted at least one endogenous nucleotide sequence encoding the reductase component of the 3-ketosteroid-9a-hydroxylase enzyme.
    [13]
    13. Bacterial cell according to claim 12, further comprising
    functionally inactivated or totally or partially deleted at least one endogenous nucleotide sequence encoding the 3-ketosteroid-A1-dehydrogenase enzyme.
    [14]
    14. Bacterial cell according to claim 12, which is a cell of M. smegmatis CECT 8331.
    [15]
    15. Bacterial cell according to claim 13, which is a cell of M. smegmatis CECT 8332.
    [16]
    16. Use of the bacterial cell according to any of claims 8 to 15 for the production of hydroxylated 11a steroids or hydroxylated 11a syntheses.
    [17]
    17. Use of the bacterial cell according to claim 16, wherein the hydroxylated steroids 11a or the hydroxylated tunings 11a are produced from non-hydroxylated tuners.
    [18]
    18. Use of the bacterial cell according to claim 17, wherein the hydroxylated 11a tuners are 11a-OH-PROG, 11a-OH-DOC, 11a-OH-TEST, 11a-OH-DHEA, 11a-OH-AD and / or 11a-OH-ADD and non-hydroxylated syntones are PROG, DOC, TEST, DHEA, AD and / or ADD.
    [19]
    19. Use of the bacterial cell according to claim 18, wherein the cell is the cell according to claim 10.
    5
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    [20]
    20. Use of the bacterial cell according to claim 16, wherein the hydroxylated syntone 11a is 11a-OH-ADD produced from natural sterols and the bacterial cell is the cell according to any of claims 12 or 14.
    [21]
    21. Use of the bacterial cell according to claim 16, wherein the hydroxylated syntone 11a is 11a-OH-AD produced from natural sterols and the bacterial cell is the cell according to any of claims 13 or 15.
    [22]
    22. Use of the bacterial cell according to any of claims 20 or 21, wherein the natural sterols are cholesterol or phytosterols.
    [23]
    23. Process for the production of hydroxylated steroids 11a or hydroxylated 11a syntones comprising the steps of:
    to. contacting a bacterial cell culture according to any of claims 8 to 15 with a steroidal substrate,
    b. incubate the mixture from step (a) under fermentation conditions, and
    C. separate from the culture medium the hydroxylated steroids 11a or the hydroxylated 11a syntheses produced after the incubation of step (b).
    [24]
    24. Method according to claim 23, wherein the culture medium comprises glycerol.
    [25]
    25. Method according to any of claims 23 or 24, wherein the steroidal substrate is selected from the list consisting of: phytosterols, cholesterol, PROG, DOC, TEST, DHEA, AD or ADD, or any combination thereof.
    [26]
    26. A method according to any one of claims 23 to 25, wherein the steroidal substrate is phytosterols or cholesterol, the hydroxylated syntone 11a is 11a-OH-AD and the bacterial cell is the cell according to any of claims 13 or 15.
    [27]
    27. A method according to any of claims 23 to 25, wherein the steroidal substrate is phytosterols or cholesterol, the hydroxylated syntone 11a is
    10
    11a-OH-ADD and the bacterial cell is the cell according to any of claims 12 or 14.
    [28]
    28. Method according to any of claims 23 to 25, wherein the steroidal substrate is AD, ADD, PROG, DOC, DHEA and / or TEST, the hydroxylated syntone 11a is 11a-OH-PROG, 11a-OH-DOC, 11a- OH-TEST, 11a-OH-DHEA, 11a-OH-AD and / or 11a-OH-ADD and the bacterial cell is the cell according to any of claims 8 to 15.
    [29]
    29. The method according to claim 28, wherein the bacterial cell is the cell according to claim 10.
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    同族专利:
    公开号 | 公开日
    WO2017207844A1|2017-12-07|
    ES2648614B1|2018-10-15|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    GB9615032D0|1996-07-17|1996-09-04|Univ Dundee|Enzyme system|
    EP1352054A2|2000-10-30|2003-10-15|Pharmacia Corporation|Aspergillus ochraceus 11 alpha hydrolase and oxidoreductase|
    AT545706T|2009-10-05|2012-03-15|Univ Ljubljani|CYTOCHROM P450 FROM RHIZOPUS ORYZAE AND USES THEREOF|CN108752408B|2018-07-10|2020-01-14|武汉工程大学|Method for recovering and refining androstenedione from solid leftovers of androstenedione prepared by microbial method|
    CN109536562B|2018-11-09|2022-02-22|天津科技大学|Method for preparing steroid drug intermediate by fermenting and converting phytosterol through mixed bacteria|
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