WO2021059182A1 - Procédé de préparation d'acide ursodésoxycholique - Google Patents

Procédé de préparation d'acide ursodésoxycholique Download PDF

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WO2021059182A1
WO2021059182A1 PCT/IB2020/058932 IB2020058932W WO2021059182A1 WO 2021059182 A1 WO2021059182 A1 WO 2021059182A1 IB 2020058932 W IB2020058932 W IB 2020058932W WO 2021059182 A1 WO2021059182 A1 WO 2021059182A1
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ldh
enzyme
amino acid
dehydrogenase
expression
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PCT/IB2020/058932
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Gianluca Galdi
Silvia RAPACIOLI
Roberto Verga
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Ice S.P.A.
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Priority to CN202080061324.XA priority Critical patent/CN114341350A/zh
Priority to EP20793443.1A priority patent/EP4034643A1/fr
Publication of WO2021059182A1 publication Critical patent/WO2021059182A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01176—12-Alpha-hydroxysteroid dehydrogenase (1.1.1.176)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P33/00—Preparation of steroids
    • C12P33/06—Hydroxylating
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01027—L-Lactate dehydrogenase (1.1.1.27)

Definitions

  • the present invention concerns a chemo-enzymatic process for preparing ursodeoxycholic acid (UDCA) by starting from deoxy cholic acid (DC A), as well as an enzyme having a high 7beta-hydroxylase activity for converting DCA into ursocholic acid (UCA), said conversion being the first step of said process.
  • DC A deoxy cholic acid
  • UCA ursocholic acid
  • Ursodeoxycholic acid is a compound normally present in small quantities in human bile, where it increases the solubilizing capacity of bile in relation to cholesterol.
  • UDCA or 3a,7 -dihydroxy-5 -colanoic acid, has the following formula: UDCA is known for its therapeutic properties, for example in the treatment of hepatic disorders including cholesterol gallstones, primary biliary cholangitis, and sclerosing cholangitis.
  • It is therefore an object of the present invention is to provide a process for the preparation of UDCA that is efficient, economical, and provides high yield and purity of UDCA.
  • the present invention concerns an enzyme having a high 7b- hydroxylase activity, which can be advantageously used in the step of converting deoxycholic acid (DCA) into ursocholic acid (UCA), of said process.
  • DCA deoxycholic acid
  • UCA ursocholic acid
  • the present invention concerns an expression vector comprising the nucleotide encoding sequence for said enzyme.
  • FIG. 1 shows regions of the amino acid sequence of the cytochrome identified for saturation mutagenesis testing, as per the examples. In grey, the mutagenesis target residues;
  • FIG. 5 shows a comparison between the sequence of cytochrome g3484 with those of cytochromes exhibiting 7 -hydroxylase activity.
  • An object of the present invention is therefore an enzyme having a 7-b hydroxylase activity and having SEQ ID N. 1 , wherein independently of one another: the amino acid at position 113 is a hydrophobic amino acid selected from A, E, F, I, L, P, Y, W, and V; the amino acid at position 110 is a hydrogen bond-forming amino acid selected from R, N, Q, S, and T; the amino acid at position 368 is I; the amino acid at position 365 is a hydrogen bond-forming amino acid selected from A, N, Q, S, and T.
  • the amino acid at position 113 is V.
  • the amino acid at position 110 is R or S.
  • the amino acid at position 365 is A or S.
  • the enzyme of the present invention has SEQ ID N. 2.
  • the enzyme of the present invention has SEQ ID N. 3.
  • the amino acid sequence of the enzyme subject of the invention has at least 80% sequence identity matching SEQ ID N. 1 , more preferably the amino acid sequence has a sequence identity at least 85% matching SEQ ID N. 1.
  • a further subject of the present invention is an expression vector comprising the nucleotide encoding sequence for the enzyme having 7-b hydroxylase activity of the present invention.
  • said expression vector comprises the nucleotide sequence SEQ ID N. 11, the nucleotide sequence SEQ ID N. 12, or the nucleotide sequence SEQ ID N. 13 encoding for the enzyme having 7-b hydroxylase activity of the present invention.
  • the enzyme of the present invention is expressed in said expression vector under control of the promoter AOX1, TEF, GUT1, GCW14, GK1, or GAP, preferably the promoter TEF.
  • this expression vector is capable of replicating itself in an eukaryotic cell.
  • said enzyme is expressed in a cell of P. pastoris, E. coli, Bacillus, S. cerevisiae, K. lactis, or Aspergillus.
  • said enzyme in expressed in a strain of P. pastoris through a fermentation process.
  • Said fermentation process is preferably performed in a culture medium comprising casein peptone, yeast extract, glycerol, and phosphate buffer, at a temperature of 25°C to 35°C, preferably about 30°C, and at a pH of 6.5 to 7.5, preferably about 7.0.
  • said strain of P. pastoris is selected from P. pastoris X-33, P. pastoris KM71H, P. pastoris SMD1168H, P. pastoris M5 (Superman5), and combinations thereof.
  • the strain is P. pastoris X-33.
  • a subject of the present invention is also an enzyme having 7-b hydroxylase activity and SEQ ID N.
  • the amino acid at position 110 is a hydrophobic amino acid selected from A, E, F, I, L, P, Y, W, and V; - the amino acid at position 113 is a hydrogen bond-forming amino acid selected from R, N, Q, S, and T; the amino acid at position 365 is I; the amino acid at position 368 is a hydrogen bond-forming amino acid selected from A, N, Q, S, and T.
  • the amino acid at position 110 is V.
  • the amino acid at position 113 is R or S.
  • the amino acid at position 368 is A or S.
  • a further subject of the present invention is a chemo-enzymatic process for the preparation of ursodeoxycholic acid (UDCA) comprising the steps of: a) converting deoxycholic acid (DCA) into ursocholic acid (UCA), by using the enzyme having a 7 beta-hydroxylase activity of the present invention, b) converting ursocholic acid (UCA) into 12K-UDCA by using a 12a- hydroxysteroid dehydrogenase (12a-HSDH) and lactic-dehydrogenase (LDH), and c) conducting a Wolff-Kishner reduction, thereby obtaining UDCA.
  • DCA deoxycholic acid
  • UUA ursocholic acid
  • LDH lactic-dehydrogenase
  • step a) of the process of the present invention the conversion of DCA to UCA is achieved by using biomass of P. pastoris transformed with the encoding expression vector for the enzyme having 7 beta-hydroxylase activity of the present invention.
  • step a) of conversion cells obtained from fermentation of Pichia pastoris are directly used.
  • the enzyme having 7 beta-hydroxylase activity of the present invention is used free in solution or immobilized onto solid matrices, selected from resins known in the art.
  • Said enzyme is obtained through lysis of the cells transformed with the vector of the present invention by cellular lysis methods known in the art.
  • the collected cells are added to a phosphate buffer 100 mM pH 7.00, containing sorbitol 200 mM, at a final concentration of 20%.
  • the lOx solution of DCA dissolved in organic solvent, preferably DMSO (100 ml in every liter of suspension), is added under stirring.
  • the concentration of DCA is 5-30 g/L, more preferably is about 20 g/L.
  • the entire mixture is maintained under stirring for 48-96 h, more preferably 72 h.
  • the final conversion yield percentage from DCA into UCA is about 60-65%.
  • a dehydrogenase 12oc-HSDH selected from 12a_988, 12a_829, 12a_793, 12a_956, and 12a_698 and a lactic-dehydrogenase LDH selected from LDH_CUPNH, LDH_THECA, LDH_CAEEL, LDA_GEOSE, and LDH_LACPE, are used.
  • step b) a combination of lactic-dehydrogenase LDH_LACPE and dehydrogenase 12a_829, or a combination of lactic-dehydrogenase LDH_LACPE and dehydrogenase 12a_793, is used.
  • the dehydrogenase 12oc-HSDH and LDH enzymes used in step b) of the process of the present invention can be free in the reaction solution or immobilized onto solid matrices, selected from resins known in the art.
  • the concentration of UCA is 20-50 g/L, more preferably about 40 g/L.
  • step b) of the chemo-enzymatic process of the present invention is performed at an incubation temperature of 24-30°C, at a phosphate buffer concentration of 0.1-1 M, and at a pH of 6.5-7.5.
  • the volume of LDH used is 3-6% v/v and the volume of 12a- HSDH used is 3-6% v/v.
  • steps a) and b) are performed in one-pot, i.e. in the same reaction environment, without intermediate separation steps.
  • a variant of the one -pot embodiment is a process wherein, before adding 12a- hydroxysteroid dehydrogenase and lactic-dehydrogenase, unreacted DCA is separated.
  • step c) of the process 12K-UDCA is subject to Wolff-Kishner reduction, thereby obtaining UDCA.
  • Wolff-Kishner reduction is a chemical reaction of reductive deoxygenation that, starting from an aldehyde or a ketone, produces an alkane, and has been known in literature for decades. It can therefore also be implemented in the context of the invention, for example following the teaching of Dutcher et al. (“ Studies on the Wolff-Kishner reduction of steroid ketones”, Columbia University, 1939, Journal of the American chemical society, Vol. 61) or the teaching of Szmant (“The Mechanism of the Wolff-Kishner Reduction, Elimination, and Isomerization Reactions” , Angew. Chem. Internat. Edit., 1968, Vol. 7, No. 2).
  • the original method consisted of heating a hydrazone together with sodium ethoxide to about 200°C, but various modifications have been introduced in this method over time.
  • Wolff-Kishner reduction is performed in a single step using aldehyde or ketone, KOH, hydrazine, and ethylene glycol.
  • steps a), b), and c) are performed in one-pot, i.e. in the same reaction environment, without intermediate separation steps.
  • sequences identified, compared to each other according to evolutionary distance, can be divided into 10 subgroups within which the proteins are phylogenetically correlated.
  • the identifying ID's of the 10 selected sequences are set out in Table 1, indicating the percentage of similarity relative to the enzyme of F. sporotrichioides and the microorganism of origin.
  • Table 1 List of the 10 selected sequences for expression and comparison of bioconversion yield relative to the enzyme of F. sporotrichioides.
  • n.d. translation of putative genes, obtained applying sequencing techniques for entire genomes, the true activity of which have never been experimentally demonstrated.
  • CLONING AND TRANSFORMATIONS The sequences were ordered and optimized for expression in P. pastoris.
  • the genes were cloned in vectors for intercellular expression in yeast regulated by a promoter induced by methanol, for example the vector pPICZA.
  • the plasmids obtained transformed the strain of P. pastoris, also capable of expressing the fungal accessory protein, NAD(P)H-p450 reductase, identified as gl 1235, necessary for the operation of the cytochromes, providing the latter the reducing power required to complete the reaction.
  • BIOCONVERSION TESTS The bioconversion tests were developed in two steps: accumulation of biomass that expresses the cytochrome under analysis, and actual bioconversion.
  • the clones were inoculated in enriched medium in the presence of d-aminolevulinic acid, a known precursor of the heme group, and of methanol, the inductor required by the expression system of P. pastoris.
  • the biomass was collected, resuspended in buffer at 20% w/v and utilized for bioconversion reactions in the presence of DCA at 3 g/L.
  • the bile acids were extracted after 3 days utilizing 2 volumes of ethylacetate following acidification of the cellular suspensions.
  • the extracts were analyzed utilizing an INERTSIL ODS-2 column, C18, 250 x 4.6 to 5um, CPS analytical (cat.no. 5020-01128), routinely utilized in analytical laboratories (method EP 8.0).
  • the first three present the highest similarity of sequence in relation to the reference (Table 1) and belong to the same phylogenetic cluster; it is probable that having undergone a similar evolutionary course, they have acquired the same capacity to recognize DCA as a substrate in a more or less specific manner.
  • Protein 10 which exhibits a sequence similarity of 65% relative to the reference, produces a reaction yield around 8% as a consequence of presenting various impurities, absent in the other samples, caused by nonspecific reactions that diminish the efficiency of the cytochrome for the production of UCA from DCA.
  • Cytochrome 3 exhibits a yield of around 30%, without the overoxidized compounds of the reference reaction, but including the appearance of other nonspecific peaks.
  • Protein 2 is interesting in that it offers a conversion yield of 49.7% compared to 40% for the standard. The superior result is because the impurities comprising overoxidized molecules in the standard reaction are present in smaller quantities, even if there is more residual unreacted substrate (approximately 45% compared to the 3% reference).
  • the analysis of the percentage areas of impurity revealed that in the cytochrome g3484 reaction these comprise more than 55% of the bile acids present, compared to a percentage area of 18% in the case of conversion with the identified protein 2.
  • the quantity of relevant product measured with the new cytochrome was 1500 ppm.
  • Protein 2 is thus naturally more selective than cytochrome g3484 in the recognition of DCA as substrate and comprises a better starting point for the identification of the optimum protein for the bioconversion.
  • cytochrome 2 derived from Fusarium graminearum, is the most promising candidate for further increasing the reaction yield. This protein does not promote oxidation reaction towards undesired products, which constitute the contaminants responsible for the relatively lower yield when utilizing cytochrome g3484, and immediately enables a conversion yield DCA -> UCA greater than 49%.
  • DCA substrate in question
  • the cytochrome 2 was used for computer modeling in silicon of a three-dimensional proteic structure, exploiting similar proteins as scaffolds, the known structures of which are available in the RCSB-PDB database (Research Center for Structural Biology- Protein Data Bank).
  • the highlighted positions 3 and 12 of the protein are the carbons bonded to an OH group, while position 7 is the target position for the hydroxylation in question.
  • the first docking analyses identified the residues of the protein that interact with the substrate, enabling the accommodation of the bond pocket with the correct orientation to cause the reaction of the carbon at position 7 with the oxygen activated by the iron atom of the heme group, the characteristic catalytic center of cytochromes.
  • Table 2 summarizes the results of the analyses, showing the amino acid residues of the protein, which according to the model could be in contact with the DC A and condition the outcome of hydroxylation in beta of the carbon 7.
  • Table 2 List of amino acid residues of cytochrome 2 that interact in the bond pocket with specific substrate atoms (carbon atoms of the rings, C, or substituent hydroxyl groups, OH). Val301 could be correlated with the position of the heme group, conditioning its reactivity. Particularly significant are the amino acids close to the hydroxyl group at position 12, since the docking studies observed that the hydrogen bonds between the hydroxyl group at 12 and the biocatalyst promote oxidation at b of the carbon 7 when the substrate in the bond pocket has its side b facing the heme.
  • a commonly used method for directed evolution tests as an alternative to rational mutagenesis is random mutagenesis, which exploits techniques of molecular biology to introduce random mutations in known sequences and create enzymatic variants that are not definable a priori.
  • Error-prone PCR enables the introduction of random mutations in a selected DNA sequence, exploiting the PCR polymerase chain reaction ) technique of molecular biology, which normally extends DNA sequences with high precision.
  • Altering the reaction conditions for example by introducing manganese salts or modifying the concentrations of the dNTPs, induces the DNA polymerase under reaction to utilize incorrect nucleotides during the polymerization of the DNA, resulting in the introduction of mutations into the extended DNA sequence.
  • Ep-PCR is a commonly used technique in directed evolution tests to obtain variants of a reference nucleotide sequence that code for proteins with different amino acids from wild- type, in order to confer the initial protein new and potentially more interesting characteristics according to the intended use.
  • This technique enabled the creation of a pool of sequences that code for proteins with random mutations, cloned in the commercial vector pPICZA for intracellular expression in yeast regulated by a promoter induced by methanol.
  • the plasmids obtained were utilized to transform the previously selected strain, also capable of expressing the fungal accessory protein, NAD(P)H-p450 reductase, known as gl 1235, necessary for the functioning of cytochromes in that it provides reducing power for the completion of the reaction.
  • the clones were used in expression and bioconversion tests.
  • the cytochrome 2 sequence was used as a PCR reaction template in which the sequence was amplified in the presence of primer with degenerations associated to each of the 4 residues, thus introducing random mutations only in the four selected positions.
  • the primers utilized to introduce the maximum possible number of mutations, reducing the probability of obtaining stop codons to a minimum, are as follows:
  • a pool of sequences was obtained, cloned in the commercial vector pPICZA through intracellular expression in yeast regulated by a promoter induced by methanol.
  • the plasmids obtained were used to transform the previously selected strain of P. pastoris, also capable of expressing the fungal accessory protein, NAD(P)Fl-p450 reductase.
  • EXPRESSION AND BIOCONVERSION TESTS The clones obtained from the transformations of P. pastoris using the sequences obtained from random mutagenesis were utilized for HTS testing for expression in P.
  • the growth step included inoculating one clone per microplate well in buffered medium in the presence of the inductor (methanol), at a concentration of 2% (v/v). After three days of growth the cellular paste was separated from the exhausted medium by centrifugation and used for preparation of bioconversion tests, following resuspension at 20% wwcp/V in reaction buffer and with addition of DCA substrate to a final concentration of 3 g/L.
  • Clone A achieved a conversion yield of 60.6%, obtaining 1820 ppm of UCA and 850 ppm residues of unconverted substrate (29% residue).
  • Clone B instead achieved a conversion of 65%, obtaining 1950 ppm of product and 700 ppm of unreacted DCA (23.3%).
  • the two mutations obtained were subjected to sequencing to identify which nucleotide substitutions and therefore which mutations in the amino acid sequence had been introduced.
  • the two mutated residues were localized in target regions for the saturation mutagenesis described in the previous section.
  • the first docking analysis results identified these sites as regions of interaction between substrate and protein. It is possible that the change in charge or polarity and the variation in steric bulk of the lateral amino acid chain modified the bonding site with the protein substrate, permitting improved accommodation of the substrate in the bond pocket and consequently optimizing the reaction.
  • the proteic sequence coded by the nucleotide sequence is shown in Figure 2.
  • the new nucleotide sequence was cloned in the commercial vector pPICZA for intercellular expression in yeast regulated by a promoter induced by methanol.
  • the plasmid obtained was utilized to transform the previously selected strain of P. pastoris, also capable of expressing the fungal accessory protein, NAD(P)H-p450 reductase.
  • Seql 1 was the starting point for the second cycle of mutagenesis. Attention was directed not only to eliminating unwanted collateral reactions, for example during the first step of modification of the enzyme, but also to reduce possible inhibition of the substrate, such as to increase the concentration of DCA in reaction in order to achieve useful conversions for the step of industrialization of the process.
  • the second cycle of mutagenesis was focused on certain positions identified in bioinformatics analysis as important not only for increasing affinity between substrate and protein, but also to eliminate residues that could create a second bond pocket for the DCA.
  • the bioinformatics substrate-cytochrome interaction models identified the potential presence of a secondary region where the DCA can position itself thereby hindering entry to the active site.
  • Table 3 lists the main residues that, from the comparison of at least three docking models, are potentially involved in the formation of a second bonding site with the DCA.
  • Table 3 List of residues involved in the formation of a secondary site for interaction between protein and DCA in a plurality of docking models.
  • the residues were selected as targets for the saturation mutagenesis tests, which involve substituting each of the 6 residues listed in Table 3 with all of the other 20 L-amino acids, obtaining 206 possible different sequence combinations for testing.
  • Seql 1 was utilized as a reaction template for PCR in which the sequence was amplified in the presence of primer with degenerations associated to each of the 6 residues, such as to introduce random mutations only in the 6 selected positions.
  • the primers utilized to introduce the maximum possible number of mutations, reducing to a minimum the probability of obtaining stop codons, are described below.
  • the pool of sequences obtained was cloned in the commercial vector pPICZA for intercellular expression in yeast regulated by a promoter induced by methanol.
  • the plasmids obtained were utilized to transform the previously selected strain of P. pastoris, also capable of expressing the fungal accessory protein, NAD(P)H-p450 reductase.
  • EXPRESSION AND BIOCONVERSION TESTS
  • the clones obtained from the transformations of P. pastoris were used for HTS tests for expression in P. pastoris and bioconversion, making use of a system of miniaturization in 24 well microplates (2 ml of culture/well) for cellular growth, and 96 deep well microplates for the bioconversion step, such as to reduce the working volumes and enable wide spectrum screening of the various clones simultaneously, up to 192 clones per test.
  • the growing step included inoculation of one clone per microplate well in buffered medium in presence of inductor (methanol), at a concentration of 2% (v/v). After three days of growth, the cellular paste was separated from the exhausted medium by centrifugation and used to prepare bioconversion tests, following resuspension at 20% wwcp/V in reaction buffer. Compared to the initial screenings, the concentration of substrate provided was increased from 3 to 5 g/L, such as to satisfy two requirements: select enzymes capable of converting significant quantities of DCA from a perspective of industrialization of the process, and not to render screening of small volumes difficult due to the poor solubility of the bile acid in question.
  • clone 73 capable of producing 4500 ppm of UCA relative to 350 ppm of unreacted substrate, compared to 1950 ppm of product from the best clone of the first cycle of mutagenesis.
  • the mutated enzyme enables a yield of 90%, compared to the 65% obtained during the previous processing step.
  • the increased yield compared to the best clone of the first mutagenesis is a consequence of both greater consumption of DCA and increased selectivity in relation to the substrate, resulting in diminished overoxidized impurities. Measurements revealed both a reduction in unreacted substrate, and a reduction of the area of the peak corresponding to a compound presumably more polar than the required product.
  • Clone 73 was subject to sequencing to identify the nucleotide substitutions introduced during the mutagenesis cycle.
  • the amino acid sequence resulting from these substitutions is reported in Table 4, in comparison with the template (Seqll) and the wild-type sequence (protein 2).
  • Two mutations were introduced during the processing step described: the glutamic acid residue at position 110 mutated with a serine residue, and the leucine residue at 365 mutated into a serine residue.
  • the change in polarity, charge, and steric bulk of the residues modified 10 the three-dimensional structure of the enzyme, facilitating entry of the DCA into the active site and increasing the affinity of the protein for the substrate in question.
  • the substitution of the glutamic acid at position 110 with a serine may have eliminated the saline bridge between the glutamic acid and the arginine residue at position 220, at least partially modifying the secondary bond pocket between DCA and protein, 15 which was responsible for the effect of substrate inhibition identified previously.
  • the sum of mutations from the sequential cycles of mutagenesis resulted not only in achievement of a minimum reaction yield of 70%, the final target defined for step 4, but also exceeded this value by achieving a conversion percentage of 90%.
  • the mutagenesis activity also partially eliminated the effect of inhibition by the substrate, making it possible to exceed 2 g/L of hydroxylated substrate, which constituted the maximum conversion limit at the end of the first cycle of mutagenesis.
  • the first critical step for scaling up the fermentation was the elimination of methanol, which was used as an indicator for expression in the commercial Pichia pastoris system utilized for the expression of the cytochrome in question, in order to avoid problems of flammability and toxicity.
  • a transition was made from the 3 ml of culture utilized during the FIT screening step to 100 ml of culture medium in a conical flask.
  • a biomass production was achieved of 120 g/L in 96 h of total fermentation.
  • the second scale-up step involved a transition from culture in conical flask to a 2 L fermenter. This was an essential step and already indicative of the foreseeable yield of fermenters on the pilot scale, due to the possibility of controlling fundamental parameters for the growth of the yeast in culture, including pFl, temperature, and oxygenation. It was also possible to assess alternative industrial raw materials in a buffered saline medium, in the presence of a carbon source and protein extracts and/or hydrolysate to supply the yeast with nutrition, vitamins, and cofactors required not only for growth but also for the heterologous expression of the cytochrome.
  • the applied experimental design enabled identification of the best combination of components as alternatives to the enriched medium in the conical flask, in particular eliminating the addition of pure cofactors essential for the synthesis of complex proteins like cytochromes, which is difficult to sustain economically in industrial fermentation.
  • the resulting medium not only maintained the yield (125 gwcp/L) achieved with enriched medium even though using industrial components, but the times were also halved: maximum production was achieved in 48 h of fermentation instead of 96 h.
  • INCREASED DCA LOADING The biomass produced was utilized in reaction at 20% w/v at scalar concentrations of DCA: 5 g/L, 10 g/L, 20 g/L, 30 g/L.
  • reaction kinetics were assessed by preparing three different concentrations of wet cell paste (wcp), specifically 10%, 20%, 30% w/v, utilizing an initial 20 g/L of DCA.
  • wcp wet cell paste
  • the bile acids were extracted at successive intervals to assess the development of the reaction. As emerges in Ligure 3, maximum conversion was achieved at 20% w/v of biomass in 72 h. Longer time scales at higher concentrations of biocatalyst did not improve the yield, while lower concentrations of enzyme did not allow the required performance to be achieved.
  • Biocatalyst (biomass) stability tests were conducted assessing parameters including: absence or presence of stabilizers (glycerol or sorbitol), preservation temperature (specifically -80°C, -20°C, 4°C, and 25°C), and preservation times (7, 14, 30 days). Stability was assessed and expressed as bioconversion yield, in presence of 10 g/L of DCA, utilizing biomass preserved under the different conditions
  • Table 6 List of 12a-HSDH selected for expression in E. coli. Similarity is expressed as a percentage of similarity of amino acid sequence compared to 12a-HSDH of Bacillus sp (ID Databank: A3IBN0 described as “predicted short chain dehydrogenase”).
  • LDH_GEOSE and LDH_THECA exhibit mutations relative to the sequences recorded in the database since these modifications confer non-dependence on fructose 1.6-bisphosphate.
  • LDH_GEOSE exhibits the mutations R104C, Q189L, N293S, while the sequence LDH_THECA exhibits six mutations, specifically
  • the genes that code for the selected enzymes were chemically synthesized after being subjected to optimization for expression in the micro-organism selected as host.
  • the optimization algorithm exploits the degeneration of the genetic code to conceive nucleotide sequences that, although coded for the protein identical to the one required, enable transcription and optimal translation in the selected expression system, stabilizing the messenger RNA, facilitating the ribosome bond, and eliminating regions that induce premature interruptions in translation.
  • pET21 12a_988 for cytoplasmic expression, which exhibits a promoter inducible using lactose or synthetic analogues, for example IPTG (Isopropyl b-d-l- thiogalactopyranoside), and confers resistance to ampicillin.
  • the constructed vectors were used to transform strains of E. coli hyperproducers, commonly used for heterologous expression. Also transformed was the strain prepared previously by deletion of the hdhA gene, which codes for the endogenous enzyme 7a- HSDH, responsible for unwanted collateral reactions (strain AhdhA). A transforming clone for each strain was propagated in elective liquid medium and then cryopreserved in different quantities. These samples represented the starting point for all subsequent expression tests.
  • the preliminary tests using the glycerinates obtained were conducted in minimal medium with induction from lactose.
  • Qualitative and semi-quantitative analysis of expression was conducted using SDS-PAGE (electrophoresis on polyacrylamide gel in presence of sodium dodecyl sulfate), when the expected molecular weight of the proteins is between 32.8 kDa (LDH_THECA) and 36.8 kDa (LDH_CUPNH).
  • LDH_THECA 32.8 kDa
  • LH_CUPNH 36.8 kDa
  • Inductor concentrations lactose: 2 g/L or 10 g/L;
  • the lactic-dehydrogenase enzymes were purified applying a process of mechanical lysis that involves treatment of the cells using ultrasound in the presence of detergents to facilitate the solubilization of the membrane.
  • the soluble fraction suitably separated from the insoluble fraction, was subject to a first qualitative analysis using SDS-PAGE.
  • the band corresponding to the enzymes LDH_LACPE, LDH_CAEEL, and LDH_CUPNH is visible on polyacrylamide gel, including a more intense band for the first enzyme, which is thus the most soluble among the five proteins of interest.
  • the enzymes LDH_GEOSE and LDH_THECA are instead not visible.
  • the enzymes were titrated applying the method “UV Bict LDH” developed previously.
  • the standard test conditions include incubation of the enzyme in phosphate buffer 100 mM, pH 7 at ambient temperature. The activity levels measured are presented in Table 10.
  • LDH_LACPE The enzyme resulting most soluble in the qualitative analyses, LDH_LACPE, exhibited greater activity under standard conditions. Lower solubility also corresponds with lower activity, as observed for the enzymes LDH_GEOSE and LDH_THECA.
  • Titration is an activity value established in vitro under standard conditions. It is thus necessary to assess the performance of the LDH under the target reaction conditions since these could influence the activity either positively or negatively for each specific protein.
  • TRANSFORMATION AND EXPRESSION TESTING OF 12a-HSDH As in the case of the LDH, the constructed vectors were used to transform strains of E. coli hyperproducers, commonly used for heterologous expression, including the strain AhdhA. A clone transformed for each strain was propagated in selective liquid medium and then cryopreserved. These frozen samples represent the starting point for all the subsequent expression tests.
  • the enzymes were titrated using the method “Uv Bict 12a-HSDH” previously defined.
  • the standard test conditions include incubation of the enzyme in phosphate buffer 100 mM, pH 8.5 at ambient temperature.
  • Table 11 shows the values obtained, including the activity of the enzyme Oxlll (lot 12A010113/1), which is used in standard reactions.
  • Table 14 shows the activity values (U/ml) recorded at specific times following sampling (1 h, 4 h, 20 h) from the reaction mixture.
  • Table 14 Titer measured using the “Method Uv-Bict LDH” following incubation at subsequent times in reaction mixture. The values in the table are expressed as units/ml (U/ml).
  • Table 14 shows that the eukaryotic proteins (LDH_LDH.A and LDH_CAEEL) lost more than 60% of their activity after an hour and the enzymatic titer remained constant over the following hours.
  • the LDH_CUPNH, LDH_GEOSE, and LDH_THECA are stable, but the activity remained too low to achieve the target yields.
  • LDH_LACPE though exhibiting an initial titer much higher than the other proteins, dropped sharply in activity after an hour, proving to be extremely unstable in the bioconversion of interest.
  • the other LDH instead exhibited a deterioration in performance, shifting into acidic conditions.
  • Reactions were thus implemented varying parameters comprising: incubation temperature (30°C / 24°C), volume of LDH (6% v/v or 3% v/v), volume of 12a-HSDH (3% v/v or 6% v/v), and concentration of phosphate buffer pH7 (1M or 0.1 M). The yields were assessed applying the “Method Ox from CA to 12K-CDCA”.
  • the discriminating element is the molar concentration of the phosphate buffer in which the reaction occurs, considering that the target was only achieved using phosphate buffer at a concentration of 100 mM. It can be noted that the concentration of LDH can be halved compared to the standard, since probably in these conditions, the enzyme is very active and greatly in excess.
  • the enzymes LDH_LACPE, 12A_829 and 12A_793 were expressed in a fermentation process on laboratory scale (conical flask of 500 ml) utilizing the strains and optimal conditions described herein above.
  • the proteins were purified according to the methods described herein above.
  • the enzymes, in the form of cell lysates, were then resuspended at 25% v/v in glycerol in order to achieve improved protein and titer stability.
  • a structural bioinformatics analysis of the enzyme revealed a number of other optional mutations in order to potentially increase performance, possibly in order to further reduce substrate inhibition (acting on the so-called “secondary pockets” in the three-dimensional structure of the enzyme).
  • the residues were selected as the target for saturation mutagenesis tests, which aim to substitute all of the 6 residues with all of the other 19 L- amino acids, obtaining 19 6 possible different sequence combinations for testing.
  • Seqll was used as a PCR reaction template in which the sequence was amplified in presence of a primer with degenerations affecting each of the 8 residues, so as to introduce random mutations in only the 8 selected positions. 5 different primers were used to introduce the maximum possible number of mutations, reducing the probability of obtaining stop codons to a minimum.
  • the pool of sequences obtained was cloned in the vector for intercellular expression in yeast regulated by a constituting promoter, described herein above, thus eliminating the use of methanol as inductor foreseen in commercial vectors, but difficult to apply in fermentations on an industrial scale (risks of flammability and toxicity).
  • the plasmids obtained were utilized to transform the strain of P. pastoris selected previously, also capable of expressing the fungal accessory protein, NAD(P)H-p450 reductase. EXPRESSION AND BIOCONVERSION TESTING
  • the clones derived from the transformations of P. pastoris using the sequences obtained from random mutagenesis were utilized for HTS expression and bioconversion tests, utilizing a 24 well microplate miniaturization system (2 ml of culture/well) for cellular growth, and 96 deep well microplates for the bioconversion step, such as to reduce the operating volumes and enable wide spectrum screening on different clones simultaneously, up to 192 clones per test.
  • the growth step involved inoculating one clone per microplate well in buffered medium. After three days of growth, the cellular paste was separated from the exhausted medium by centrifugation and used to prepare bioconversion tests, following resuspension at 20% wwcp/V in reaction buffer and addition of DCA substrate to a final concentration of 20 g/L.
  • the biocatalyst is comprised in the biomass itself, it is advantageous to have a yeast clone that grows as quickly as possible, for equal expression of the cytochrome of interest, such as to limit the fermentation times and consequently the costs.
  • the cellular paste was separated from the exhausted medium by way of centrifugation and used to prepare bioconversion tests, following suspension at 20% wwcp/V in reaction buffer with addition of DCA substrate at a final concentration of 20 g/L.
  • the three clones did not exhibit higher growth rates compared to the yeast clone that expresses Seq73.
  • SEQ ID N. 11 (nucleotide sequence Protein2)
  • TTGGGT GTT A ATC AT GTTTT GGGT ACTTCT ACTGAGTGGC ACCC A ATT A ACC CTGGTGAAGATATTATGAGAATCGTTTCTAGAATGTCTTCTAGAATTTTTAT

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Abstract

L'invention concerne un procédé chimio-enzymatique pour la préparation d'acide ursodésoxycholique (UDCA) à partir de l'acide désoxycholique (DCA), comprenant la préparation d'une enzyme ayant une activité de 7 bêta-hydroxylase élevée pour la conversion de l'acide désoxycholique (DCA) en acide ursocholique (UCA), ladite conversion étant la première étape dans ledit procédé.
PCT/IB2020/058932 2019-09-27 2020-09-24 Procédé de préparation d'acide ursodésoxycholique WO2021059182A1 (fr)

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