WO1996027678A1

WO1996027678A1 - Process for hydroxylating long-chain alkanes, fatty acids and other alkyl compounds

Info

Publication number: WO1996027678A1
Application number: PCT/DE1996/000410
Authority: WO
Inventors: Thomas Zimmer; Kristina Kaminski; Wolf-Hagen Schunck; Eva KÄRGEL; Ulrich Scheller; Stephan Mauersberger
Original assignee: Max-Delbrück-Centrum für Molekulare Medizin
Priority date: 1995-03-03
Filing date: 1996-03-01
Publication date: 1996-09-12
Also published as: DE19507546A1; DE19507546C2

Abstract

A microbial hydroxylation process is disclosed for selectively oxidising regions of long-chain alkanes, fatty acids and other alkyl compounds. The process should be easy to carry out and produce good yields of oxidation products, in particular hydroxylated fatty acids and long-chain dicarboxylic acids. The object of the invention is to modify yeast by genetic engineering so that when it is cultivated it expresses the required enzymes. The disclosed process is characterised in that the long-chain alkanes, fatty acids and other alkyl compounds are treated with monooxygenase systems that consist of cytochrom P450 and NADPOH cytochrom P450 reductase, and the hydroxylation products are then separated. The monooxygenase systems are produced in the reaction mixture by simultaneous expression of their components in yeast, preferably saccharomyces cerevisiae. The invention relates essentially to a vector for modifying saccharomyces by genetic engineering. On the basis of the structure Yep 51, the vector contains reductase cDNA between the restriction sites Sa1K and BamHI and a second expression cassette bound in the restriction site NruI. The expression cassette consists of the GAL10 promoter, the sequence that codes for cytochrom P450 and the ADH1 terminator.

Description

Process for the hydroxylation of long chain alkanes, fatty acids and other alkyl compounds

description

The invention relates to a process for the hydroxylation of long-chain alkanes and alkyl compounds, in particular fatty acids. It also relates to a vector for implementing this method. The field of application of the invention is biotechnology, in particular the extraction of fatty acid oxidation products such as hydroxy fatty acids and long-chain dicarboxylic acids.

Monooxygenases of the cytochrome P450 type are common in organisms on the entire phylogenetic scale. They catalyze NAD (P) H- and 0_-dependent reactions in various biosynthetic and catabolic pathways. The primary sequences of more than 220 different P450 forms are currently known. These are combined into a superfamily based on characteristic sequence features (review by Nelson et al., DNA Cell Biol. 12 [1993] 1).

In yeasts, such as the alkane-utilizing species Candida maltosa (Vogel et al., Eur. J. Cell Biol. 57 [1992] 285) and in higher eukaryotes, the majority of the P450 forms are localized in the endoplasmic reticulum. The electron transfer required in the reaction cycle takes place via a membrane-based NADPH cytochrome P450 reductase. P450 and reductase components together form the active monooxygenase system. Various strategies have been described in scientific publications and in the patent literature for the functional heterologous expression of such two-component systems (Murakami et al., DNA 5 [1986] 1; Urban et al., Biochem. Soc. Trans. 21 [1993] 1028; Pompon et al., FR-PS-2,679,249) The enormous variety of P450 forms combined with individual chemoselectivity forms great potential for future applications in the synthesis of fine chemicals. The high regio- and stereoselectivity of substrate hydroxylation achieved by some P450 forms is of particular interest, since comparable specificities can hardly be achieved with methods of organic chemistry or only with great effort in synthesis and preparation.

An example of this is the regioselective oxygenation of long-chain alkanes, fatty acids and other alkyl compounds. While chemical oxidation is known to attack tertiary and secondary carbon atoms with great preference and leading to a mixture of oxidation products, some P450 forms are capable , with almost absolute selectivity

Hydroxylation of fatty acids in the ω position, i.e. on the primary carbon atom. P450 forms with this specificity occur primarily in alkane-utilizing yeasts such as C. maltosa and C. tropicaliε and belong to the CYP52 family within the P450 superfamily (review by Müller et al., In Frontier in Biotranεformation 4. K. Ruckpaul, H . Rein, eds., Akademieverlag, Berlin [1991] 87).

To date, the CYP52 family is the most extensive P450 family in microorganisms. For example, 8 different alkane-inducible P450 forms have already been identified for the yeast C. maltosa, which differ significantly in their substrate specificity from various alkyl compounds (Schunck et al., DD-WP 271 339; Schunck et al., DD-WP 292 022) . Recently, the associated reductase component was also cloned from this organism.

Previous attempts, the particular chemoselectivity of P450 forms of the CYP52 family for the production of alkane and fatty acid oxidation products in particular The use of dicarboxylic acids is based on their indirect use in genetically modified (overview by J. Schindler et al., Forum Mikrobiol. 5 [1990] 274) and also genetically modified (Picataggio et al., Bio / Technology 10 [1992] 894) Candi da tribes.

The problem of direct use, namely to establish highly active P450 systems for the regioselective hydroxylation of fatty acids and other alkyl compounds by heterologous expression in a suitable microorganism, is largely unsolved.

The aim of the invention is to provide a microbial hydroxylation process for the regioselective oxidation of long-chain alkanes, fatty acids and other alkyl compounds. It is said to lead to the oxidation products, in particular hydroxy fatty acids and long-chain dicarboxylic acids, in good yields in a simple process. The object of the invention is to genetically modify yeasts so that they express the necessary enzymes when cultivated.

The object of the invention is achieved by monooxygenase systems which consist of cytochrome P450 and NADPH-cytochrome P450 reductase, which are expressed simultaneously in Saccharomyces. The long-chain alkanes, fatty acids or alkyl compounds are brought into cultivation solutions from such genetically modified Saccharomyces, the expressed enzymes causing a regioselective hydroxylation. After the reaction, the hydroxylation products are separated off. A preferred yeast species for the invention is Saccharomyces cerevisiae, a preferred enzyme system are cytochrome P450 forms of the CYP 52 family and Candida maltosa NADPH cytochrome P450 reductase. The long-chain alkyl compounds, especially the fatty acids, are treated according to the invention in cultures of intact cells (cell suspensions). In contrast, for the hydroxylation of alkanes, cell homogenates of the yeast cultures have to be prepared because the cells cannot absorb the alkanes.

The substances to be hydroxylated are added to the cell cultures in organic solvents, preferably in alcoholic solution. In the case of fatty acids, their salts can also be added in aqueous solution.

The process is carried out at low temperature (30-40 ° C), the hydroxylation is usually largely complete after one hour. First of all, regioselective monohydroxylation occurs, predominantly at the terminal or subterminal carbon atom. The point of entry of the OH group is determined by the P450 form used and by the reaction time.

The monohydroxylated product is further hydroxylated during further cultivation. Depending on the procedure, hydroxy compounds, hydroxycarboxylic acids or dicarboxylic acids are obtained. At maximum oxidation, for example, an ω, ω * -dicarboxylic acid is obtained from an alkane. The respective turnover is checked by means of thin layer chromatography, the reaction is preferably terminated with acids, for example with dilute sulfuric acid, as soon as the desired product is formed in sufficient quantity.

The essence of the invention is the vector according to claim 7. It is based on the YEpδl vector and contains reductase cDNA between the restriction sites Sall and BamHI. A second

Expression cassette, consisting of the GALIO promoter, the coding sequence of cytochrome P450 and the ADH1 terminator, is ligated into the restriction site Nrul of this vector. Starting materials and the structure of the vector are shown in Figure 1.

An important feature of the vector for the implementation of the invention is that both ligated genes are under the control of the same promoter. Contrary to expectations, P450 and reductase are formed in the expression in a ratio of approximately 1: 3 ("overexpression"), a ratio which surprisingly leads to the optimal yield in the hydroxylation.

The process according to the invention is distinguished by high hydroxylation rates. For example, lauric acid is hydroxylated about 20 times faster than in the single expression of natural P450 forms, in which only a molar ratio of P450: reductase of approx. 32: 1 is given due to the low expression of the endogenous reductase (see Table 2).

1. DNA sequences used

For the heterologous coexpression of P450 forms and

The following cDNAs are used for reductase:

CYP52A3 - P450Cml (Schunck et al., Biochem. Biophys. Res.

Commun. 161 [1989] 843)

CYP52A4 - P450Cm2 (Schunck et al., Eur. J. Cell Biol. 55

[1991]

336; modified from Zimmer and Schunck, Yeast 11 [1995]

33)

CPR - NADPH cytochrome. P450 reductase from Candida maltosa

(Kärgel et al., 1993 - EMBL accession number X76226)

2. Construction of a suitable vector for heterologous coexpression of cytochrome P450 and reductase

The vectors used and essential steps for the construction of the co-expression vector are shown in Figure 1.

In a first step, the starting vector YEp51 (Broach et al., In Experimental Manipulation of Gene Expression. M. Inoye, ed., Academic Press, NY, [1983], pp. 83-117), in which the cDNA of NADPH cytochrome P450 reductase is integrated using the restriction sites Sal I and BamR I, a linker consisting of the two oligonucleotides

5'-CGAGGCGCGCCTCGAGCGGCCGCTCG-3 'and

3'-GCTCCGCGCGGAGCTCGCCGGCGAGC-5 'ligated into the Nru I site. This modification is necessary for the installation of two unique restriction sites to integrate a second expression cassette (Asc I and Not I). To construct this second expression unit consisting of the GALIO promoter, the P450 sequence and the ADHL terminator, PCR reactions are first carried out in order to obtain the GALIO promoter and the ADH1 terminator. The GALIO promoter is made from the vector YEpδl with the two primers 5'- GGGGTACCGGCGCGCCTTACGACGTAGGATC-3 'and 5'-

CGGGATCCAAGGGAGAGCGTCGAC-3 'amplified, digested by means of restrictionases Kpn I and BamH I and ligated into the restriction sites Kpn I and BamH I of the vector pUCBM21 (Boehringer Mannheim). The resulting vector is used to amplify the ADH1 terminator fragment which is primed from the vector pAAH5 (Ammerer, Meth. Enzym. in the BamH I / EcöR I location. In the newly created vector, the Sal I site must finally be destroyed by digestion with EcöR V, treatment with T4 DNA polymerase and religation. Now, using the restriction sites Sal I and BamH I, the cDNAs of P450Cml and P450Cm2 are integrated between the GALIO promoter and the ADH1 terminator.

As the last step, the respective P450 expression unit is recloned into the vector YEp51R using the restriction sites Asc I and Not I.

3. transformation of Saccharomyces cerevisiae,

Cultivation and expression

With the plasmids constructed for heterologous coexpression, the strain Saccharomyces cerevisiae GRF18

(α, his 3-11, his 3-15, leu 2-3, leu 2-112, can ^r ) by the method of Keszenman-Pereyra and Hieda (Curr. Genet. 13 [1988] 21). The transformants obtained are in 500 ml shaking flasks in yeast minimal medium (1.34% YNB- yeast nitrogen base) with additions of 1 mg / 1 FeCl3, 100 mg / l histidine and 2% raffinose in a

Shaking frequency of 240 rpm and a temperature of 28-30 "C. After reaching a cell count of 0.5 to 1.0 x 10 ⁸ cells per ml, the addition of 2% galactose induces the GAL10 promoter and thus the simultaneous expression of P450 and reductase Lowering the shaking frequency and thus reducing the oxygen content of the culture has a positive effect on both the P450 and the reductase content. Under optimized cultivation conditions (semi-anaerobic growth at a shaking frequency of approx. 60 to 80 rpm), the expression rates shown in Table 1 can be achieved for both components of the P450 monooxygenase system (values in brackets). The indicated cultivation regime can also be modified in such a way that glucose is used as the cheaper carbon source (see Scheller et al., J. Biol. Chem. 269 [1994] 12779). However, induction with galactose is only effective after complete glucose consumption.

4. Determination of the hydroxylase activity of the microsomal membrane fractions

For the enrichment of P450 and NADPH cytochrome P450 reductase in the microsomal membrane fraction, the cell biomass obtained is first centrifuged off and the microsomal membrane fraction is obtained therefrom by mechanical cell disruption in the homogenizer (Dyno-Mühle), differential centrifugation and subsequent calcium chloride precipitation. The specific microsomal P450 or reductase content for the various S. cerevisiae transformants is 260-760 pmol / mg protein and is shown in Table 2. The catalytic activity in the hydroxylation of radioactively labeled lauric acid (C12: 0) and n-hexadecane by icrosomal P450Cml and P450Cm2 is determined in a potassium phosphate buffer system (200 mM, pH 7.4), each of which has 1 μmol of the corresponding substrate, 0.2 - 0.5 nmol microsomal P450 per ml reaction mixture and the components of a NADPH-regenerating system (MgCl2, KC1, glucose-6-phosphate and glucose-6-phosphate dehydrogenase). The enzyme reaction is through Addition of NADPH started and, depending on the desired product quantity / product profile, stopped after 5-30 minutes of incubation at 30 ° C. by adding dilute sulfuric acid. The detection of the products obtained by chloroform / methanol extraction and separated by subsequent thin-layer chromatography can be carried out by computer-assisted evaluation of the thin-layer radiograms in a TLC linear analyzer system. Compared to the microsomal membrane fractions from transformants in which P450Cml and P450Cm2 are heterologously expressed without the redox partner reductase, the microsomes obtained after P450 and reductase co-expression can increase the lauric acid hydroxylase activity by up to 20 times and increase the n- Hexadecane hydroxylase activity can be measured. As can be seen from Table 2, the increase in the reaction rate can be attributed to an optimized molar P450: reductase ratio, which is 1: 3 for coexpression, but only 32: 1 (YEp51Cml) or 14: 1 (YEp51Cm2) for single expression.

5. Biotransformation of fatty acids using intact yeast cells

To a ml of an induced cell culture (S. cerevisiae transformed with Plas id YEp51Cm2-R), its P450 content

0.4 nmol, 200 nmol be added [l- ^14C] lauric acid (16.7 BMQ / mmol). After an incubation of 20 min or 1.5 h at 30 ° C. (with shaking), the cell pellet and the aqueous supernatant are separated by centrifugation (3000 × g for 5 min). The pellet and supernatant are then extracted as described in Sanglard and Loper (Gene 76 [1989] 121) and the analysis of the products formed by means of thin layer chromatography (see Figure 2). The total amount of radioactivity used after 20 min is distributed as follows: 35% hydroxy lauric acid, 15% dodecanedioic acid and 50% lauric acid. 80% of the products formed can be detected in the supernatant and 83% of the remaining starting substrate in the cell pellet.

After a reaction time of 1.5 h, a complete

Sales of lauric acid reached. As the main one

Reaction product occurs the dodecanedioic acid.

No reaction conversions can be observed when using a control strain (Sacharomyces cerevisiae GRF18 transformed with starting plasmid YEp51).

Legends

Fig. 1. Construction of plasmids for the simultaneous expression of cytochrome P450 forms and the NADPH cytochrome P450 reductase. Each of the complete P450 expression cassettes can be cleaved from the vector pUCBM21-P450 using the restriction sites Asc I and Not I and ligated into the vector YEpδlR. The specified restriction sites are: A - Asc I, B - BamH I, N - Not I, S- Sal I.

Fig.2. Thin-layer chro atographic analysis of the product pattern in the biotransformation of lauric acid using intact yeast cells. The plasmids used to transform Saccharomyces cerevisiae and the reaction times are: YEp51 - 20 min (lanes 1 and 2), YEp51Cm2-R - 20 min (lanes 3 and 4), YEp51Cm2-R - 1.5 h (lanes 5 and 6 ). The product analysis can be carried out separately according to cell pellet (lanes 1, 3 and 5) and supernatant (lanes 2, 4 and 6). A - [1- ¹⁴ C] lauric acid; B - [1-

¹ C] dodecanedioic acid; C - [l- ¹⁴ C] hydroxy-lauric acid.

SPARE BLADE (RULE 26)

Claims

claims

1. A process for the hydroxylation of long-chain alkanes, fatty acids and other alkyl compounds, characterized in that the long-chain alkanes, fatty acids or other alkyl compounds are treated with monooxygenase systems consisting of cytochrome P450 and NADPH-cytochrome P450 reductase, and the hydroxylation products are separated off.

2. The method according to claim 1, characterized in that cytochrome P450 forms of the CYP52 family and Candida maltosa NADPH cytochrome P450 reductase are used as the monooxygenase system.

3. The method according to claim 1 and 2, characterized in that the monooxygenase systems in the reaction mixture by simultaneous expression of their components in yeasts, preferably in Saccharomyces cerevisiae, are prepared.

4. The method according to claim 1-3, characterized in that the long-chain alkanes are implemented in cell homogenates of Saccharomyces.

5. The method according to claim 1-3, characterized in that the long-chain alkyl compounds are implemented in cell suspensions of Saccharomyces.

6. The method according to claim 1-5, characterized in that the long-chain alkanes, fatty acids or other alkyl compounds are added to the cell homogenates or suspensions in organic, preferably alcoholic solution.

7. Vector for the genetic modification of Saccharomyces, based on the basic structure Yep 51 and characterized by

- Reductase cDNA between the restriction sites Sall and BamHI

- And a 2nd expression cassette, ligated into the restriction site Nrul and consisting of the GALIO promoter, the coding sequence of cytochrome P450 and the ADH1 terminator.