FR2772788A1 - Amplification of glucose-6-phosphate dehydrogenase enzyme activity - Google Patents

Abstract

Amplification of glucose-6-phosphate dehydrogenase (G6PDH) enzyme activity in the polyol metabolic pathway of osmotolerant yeast is new. A method to increase the production of a polyol from a stock of osmotolerant yeast by stimulating the carbon flux in the oxidative metabolic path of the pentose phosphate cycle of the yeast, characterized by: (1) amplifying the enzymatic activity of the first enzyme of the metabolic path, that is the G6PDH; (2) culturing the yeast on a fermentation containing a source of carbon that is directly assimilable; and recovering the polyol that is produced. Independent claims are also included for: (1) an osmotolerant yeast characterized in that the enzyme activity of G6PDH is amplified in the yeast; and (2) a DNA sequence corresponding to a portion of the gene encoding the Yamadazyma ohmeri G6PDH, the sequence having a 180 bp sequence (given in the specification).

Description

PROCESS FOR INCREASING THE PRODUCTION OF CERTAIN POLYOLS
BY AN OSMOTOLERATING STRAIN
The present invention relates to a process for increasing the production of certain polyols by an osmotolerant yeast strain grown on a medium containing a directly assimilable carbon source.

 It also relates to an osmotolerant strain whose cultivation on a medium containing a directly assimilable carbon source makes it possible to increase the production of polyols.

 It further relates to a DNA fragment that corresponds to a portion of the gene sequence encoding glucose-6-phosphate dehydrogenase Yamadazyma ohmeri (formerly called Pi chia ohmeri).

 In the context of the present invention, the term "directly assimilable carbon source" is understood to mean a carbon source which, once assimilated by the microorganism, leads to the production of polyols, Examples of such carbon sources are glucose, digalactose, L-sorbose, D-ribose, D-xylose, Darabinose, L-arabinose, D-ribulose, D-xylulose, sucrose, glycerol, D-sorbitol, D-mannitol, galactitol , myo-inositol, ethanol, starch, inulin, D-gluconate, lactate, succinate, citrate, fructose, and mixtures thereof.

Preferably, these carbon sources are chosen from the group comprising glucose, D-galactose,
L-sorbose, sucrose, glycerol, starch and fructose.

Glucose-6-phosphate dehydrogenase (G6PDH) catalyzes the first step of the oxidative pathway of the pentose phosphate cycle. In this reaction, glucose-6-phosphate is oxidized to 6-phospho-glucono lactone with concomitant reduction of coenzyme Nicotinamide-Adenine
Dinucleotide Phosphate (NADP) to NADPH.

Indeed, pyridine nucleotides, Nicotinamide
Adenine-Dinucleotide (NAD) and NADP, are widely used as coenzymes by dehydrogenases. They carry hydride ions (2 electrons and 1 H +). Reduced NADP is the most important reducing agent in biosynthesis.

 G6PDH exists in microorganisms as well as in animals and plants. They can be classified in three groups according to their specificity for coenzymes of the NAD or NADP type.

Thus, the enzymes of the first group react exclusively with NADP. One example is the
G6PDH Escherichia coli. However, some enzymes belonging to the first group react weakly with the
NAD.

 The second group includes the enzymes of the animal species. These enzymes are NADP dependent, but they have a clear, albeit low, activity with NAD.

The enzymes belonging to the third group have no specificity preference for the cofactors, reacting both in the presence of NADP and in the presence of
NAD. By way of examples, mention may be made of the G6PDHs of Leuconostoc inesenteroides and of Pseudomonas aeruginosa.

 Genes coding for G6PDH from Leuconostoc mesenteroides, Leuconostoc dextranicus and Bacillus were isolated and sequenced. This work is covered respectively by US Pat. Nos. 5,244,796, 5,229,286 and 2,401,285.

 The patents cited above describe the isolation and cloning of genes for the purpose of providing a method for preparing G6PDH.

 Indeed, the applications of G6PDH are in the field of diagnosis, especially for the determination of creatine kinase in case of myocardial infarction. It is therefore essential to have a thermally stable and stable enzyme over time. Such an enzyme has been obtained by genetic engineering in the patents cited above.

The purpose followed in the context of the present invention is quite different. The originality of the present invention lies in the fact of using the stimulation of the expression of the yeast-homologous G6PDH, enzyme of the first group previously described, to amplify the oxidative metabolic pathways leading to the pentose phosphates, thus allowing the production of certain polyols, in this case arabitol, erythritol and ribitol. The increase of the reducing power in
NADPH, concomitant with the stimulation of G6PDH, will be used for the reduction of the precursors of arabitol and erythritol.

 Polyols are sugar alcohols. Most of them are produced industrially because their fields of application are extremely numerous.

 They are used in particular for the stabilization of enzymes, by improving their thermostability and stability in aqueous or organic solvents, for preservation by lyophilization or cryoprotection, and for encapsulation.

 Polyols also find applications in the agro-food sector given their acariogenic properties and their lower caloricity.

 They are also used in cosmetology or pharmacy, particularly with regard to skin care, given their high emollient power and their growth inhibiting properties, properties due to the fact that they are carbon sources difficult to assimilate.

 A number of microorganisms, including osmotolerant yeasts, naturally produce polyols. The latter participate in the osmotic equilibrium, act as carbonaceous reserve compounds, and contribute to the red-ox equilibrium of the microorganisms that produce them.

 Osmotolerant yeasts thus have many industrial potentialities. Under certain conditions they can produce ethanol. Depending on the osmotic pressures imposed on them, and in the presence of salts, they produce glycerol. In the presence of a high density of sugars, such as glucose, they produce erythritol, mannitol, or arabitol.

 Y. ohmeri is an osmotolerant yeast (ATCC 20209 deposit, in The American Type Culture Collection) and is therefore able to grow in an environment with high osmotic pressure.In response to an osmotic shock, Y. ohmeri produces polyols which are osmolytes capable of counterbalancing the osmolarity of the external medium The nature and amount of polyols synthesized depend, among other factors, on the culture conditions, for example, on a medium containing glucose, Y. ohmeri produces D-arabitol This has been demonstrated by the work of Onishi et al (Advances in Food Research, 12, 53).

 The metabolic pathways involved in the production of arabitol in Y. ohmeri are similar to those of Zygosaccharomyces cerevisiae, Z. melli and Z. rouxii.

In 1968, Spencer proposed diagrammatic pathways in Zygosaccharomyces (Prog, Ind., Microbiol., V, 1). The Applicant Company has performed a similar work with regard to the oxidative metabolic pathways of pentose phosphates in Y. ohmeri, the result being shown schematically in FIG.

 For Spencer and also for Onishi, as well as for those skilled in the art considering the pentose phosphate scheme, transketolase, an enzyme that catalyzes a number of rearrangements of the carbon skeleton of metabolites, was considered the key enzyme for the production of polyols by yeast strains.

However, the Applicant Company has succeeded in highlighting that, quite surprisingly and unexpectedly, the amplification of the first enzyme of the oxidative pathway of the pentose phosphate cycle, namely the
G6PDH, led to an increase in polyol yields in osmotolerant yeast.

 Indeed, and contrary to all expectations, the work carried out by the Applicant Company has shown that it is G6PDH (and not transketolase) which is the key enzyme responsible for the biosynthesis of polyols since it is promotes the displacement of the carbon stream towards the cycle of pentose phosphates to the detriment of glycolysis.

 The amplification of this enzyme also promotes an additional supply of NADPH cofactors that can be used for the reduction of ribulose to arabitol and erythritol to erythrose.

 The present invention therefore relates to a process for increasing the production of a polyol by an osmotolerant strain, said polyol being selected from the group consisting of ribitol, arabitol and erythritol, by stimulating the carbon flow in the oxidative metabolic pathway of the pentose phosphate cycle in said strain.

This process is characterized by the fact that the enzymatic activity of the first enzyme of said metabolic pathway, namely glucose-6-phosphate dehydrogenase, is amplified, that said strain is cultivated on a fermentation medium containing a source directly assimilable carbon, and that the polyol thus obtained is recovered.

 In the context of the present invention the "amplified" activity of G6PDH can be obtained by various means.

 According to a preferred embodiment of the invention, the activity of G6PDH is amplified by integrating one or more copies of the gene coding for G6PDH into the strain and / or by replacing the natural promoter of G6PDH with a strong promoter. Examples that may be mentioned include the promoter of the specific NADPH ribulose reductase, the alcohol dehydrogenase (ADCl) promoter, the GAL1 gene promoter, the promoters of the glycolysis genes (in S. cerevisiae or related strains), and the promoter. of the phosphoglycerol kinase gene (PGK). Preferably, the NADPH-specific ribulose reductase promoter is used.

 The osmotolerant strain may be selected from the group consisting of Y. ohmeri, Zygosaccharomyces rouxii, Candida polymorpha and Torulopsis candida strains, or any other strain capable of producing significant amounts of D-arabitol from glucose. Preferably, this strain is Y. ohmeri.

The preferred cultivation conditions are the following
lowing
- temperature between about 25 and about
40 ° C, preferably between about 30 and 380 ° C,
- pH between about 3 and about 7, pre
between about 5 and about 6, and
- carbon source content directly assimilated
in the fermentation medium included in
about 100 and about 600 g / l, preferably
from about 150 to about 400 g / l.

 According to a preferred embodiment of the process according to the invention, a step is added in which the enzymatic activity of the specific NADPH ribulose reductase is substantially reduced or eliminated, thus favoring the production of ribitol.

 Advantageously, this step is carried out by deleting the gene coding for the specific NADPH ribulose reductase.

 According to another preferred embodiment of the process according to the invention, a step is added in which the enzymatic activities of the specific NADPH and NADH ribulose reductases are eliminated or substantially reduced, thus favoring the production of erythritol.

Advantageously, this step is performed by deleting the genes coding for ribulose reductases.
NADPH and NADH specific. Preferably, a step is also added in which a specific NADPH erythrose reductase enzymatic activity is introduced into the strain.

Advantageously, a gene coding for the specific NADPH erythrose reductase enzyme activity is introduced into the strain.

 The invention also relates to an osmotolerant strain in which the activity of the first enzyme of the oxidative pathway of the pentose phosphate ring, that is G6PDH, has been amplified.

The invention further relates to a DNA sequence corresponding to a portion of the gene encoding G6PDH of
Y. ohmeri. This DNA sequence, as well as the deduced amino acid sequence, is given in FIG.

 The invention can be better understood using the non-limiting examples described below.

EXAMPLE 1 Isolation and Cloning of the Gene Coding for the
G6PDH from Yamadazyma ohmeri
Oligonucleotide primers for chain polymerization reactions (PCR) were synthesized from conserved regions of the peptide sequences of several known G6PDHs.

The following primer pairs (degenerate sequences) were used in PCR reactions according to the method described in Current Protocols in Molecular Biology (first edition, 1987, Wiley Interscience), on the plasmid clones of a Y. ohmeri genomic library.
GPD2: 5 'AG [A, G] TCGTTCTGCATCACGTC 3'
GPD4: 5 'GAYCAYTAYYTKGGYAARGA 3'
GPD5: 5 'GAWBACVACVCCVTCCCA 3'
30 cycles were carried out on a device of type
Thermocycler Perkin Elmer 7200, following the temperature profile below
denaturation for 30 seconds at 94 ° C .;
hybridization for 30 seconds at 50 ° C.,
polymerization for one minute at 72 ° C.

 The amplified DNA fragments were then analyzed by sequencing. The method followed was that of the SANGER chain terminations described in the above-mentioned work.

 The nucleotide sequence of the GPD42 fragment (from the primer pair GPD2 and GPD4), translated into amino acids and compared to the peptide sequence of S. cerevisiae G6PDH, revealed 70% homology (Fig. 3). This DNA fragment was therefore used as a probe, in order to isolate the complete G6PDH gene from one of the plasmid clones of the Y. ohmeri genomic DNA library.

A plasmid vector carrying a 6700 base pair insert was isolated by the Southern technique
Blot, by applying the protocols described in the aforementioned work.

 The localization of the open reading phase made it possible to reduce this insert to the size of 3500 bp, thanks to the EcoRI restriction sites. The enzymatic digestion conditions were as follows: 37 C, pH 7.5 for 1 hour in 50mM NaCl buffer, 10mM Tris HCl, 10mM MgCl 2, Triton x 100.025%. This restriction fragment was then placed in a Y. ohmeri replicative plasmid vector, which vector contains a homologous origin of replication (Y. ohmeri ARS element) and the Y. ohmeri leu2 gene.

 The resulting plasmid, poGPD4 whose restriction map is shown in FIG. 4, was then reintroduced by electroporation into the auxotrophic host strain for leucine (Yoleu strain, obtained by UV mutagenesis). The transformed strain was named YoGPD.

EXAMPLE 2 Assay of the Activities of G6PDH in the YoGPD Strain
The transformed strain, YoGPD, was cultured on medium containing glucose (150 g / l), yeast extract (3 g / l), KH 2 PO 4 (2 g / l) and MUSO 4 (1 g / l). l).

 The productions were carried out in Erlenmeyer flasks and fermentors of 2 liters, at a temperature of 37 ° C. and at an initial pH of 5%.

 Enzymatic kinetics were performed on the total activity of G6PDH expressed by the transformed strain, in comparison with that expressed by the auxotrophic host strain for leucine (Yoleu-).

 The enzymatic activities were assayed by incubation of the acellular crude extracts in the presence of glucose-6-phosphate and the NADP cofactor. Reduced cofactor (NADPH) formation was followed by noting the change in absorbance at 340 nm, thereby quantifying the level of expression of G6PDH.

The following table illustrates the assay results (expressed in umoles of reduced cofactor formed / min / mg total protein).

<Tb>

<SEP> Time <SEP> YoLeu- <SEP> YoGPD <SEP> Factor
<tb> (hours) <SEP> AS (U / mg) <SEP> AS (U / mg) <SEP> amplification
<tb><SEP> 24 <SEP> 3 <SEP> 2.9 <SEP> 1
<tb><SEP> 48 <SEP> 3 <SEP> 4. <SEP> 1.5
<tb><SEP> 56 <SEP> 3 <SEP> 3.5 <SEP> 1.2
<tb><SEP> 72 <SEP> 2.6 <SEP> 3.2 <SEP> 1.2
<tb><SEP> 80 <SEP> 3 <SEP> 4.2 <SEP> 1.4
<tb><SEP> 96 <SEP> 3 <SEP> 4.5 <SEP> 1.5
<Tb>
This level of expression can be increased by replacing the gene's own promoter with regulatory elements of another yeast promoter known to have better efficacy in this yeast host. One example is the promoter of Ribulose Reductase
NADPH-specific Y. ohmeri.

EXAMPLE 3 Production of arabitol by culturing the YoGPD strain
The culture medium selected for production in Erlenmeyer flasks or fermenters is the same as that described in Example 2.

 In the same way as the host strain, the transformed YoGPD strain produces exclusively arabitol from glucose. However, if the starting strain produces 55 g / l of arabitol (ie a weight yield of 55% on the glucose consumed), the transformed strain produces, under the same conditions, 65 g / l of arabitol, a gain of yield of 10%.

The increase in the specific activity of the
G6PDH between the host strain and the transformed strain is therefore sufficient to deflect metabolic fluxes (carbon flux and additional reducing power) to optimize Y. ohmeri's ability to produce fermentative polyols.

Example 4 Construction of the strain RR-GPD
The Applicant Company has a strain of Y.

 Ribitol-producing osteri, a strain obtained by chromosomal deletion of the NADPH-specific ribulose reductase gene (as disclosed in French Patent Application No. 97 04506) This strain is also auxotrophic for uracil.

 The gene coding for the G6PDH enzyme activity was taken from the vector poGPD4 by means of the SalI-NotI sites and reintroduced at the SalI-NotI sites of the vector plig3 (restriction map Fig. 5) containing a homologous origin of replication (ARS element). Y. Ohmeri) and the Y. ohmeri URA 3 gene.

 The resulting plasmid poGPD5 (restriction map Fig. 6) was then reintroduced by electroporation into the strain RR-URA- (strain obtained by chromosomal deletion of the homologous URA 3 gene as specified in the aforementioned patent application). The transformed strain was named RR-GPD.

Example 5: Production of ribitol by culturing strain RR-GPD.

 The culture medium selected for production in 2 liter Erlenmeyer flasks and fermenters contains glucose at 200 g / l, corn steep liquor at 60 g / l and MUSO4 at 2 g / l.

 In the same way as the host strain, the transformed strain produces ribitol from glucose, with a yield of 30% by weight based on the amount of glucose consumed. However, if the starting strain produces about 4% ribulose in addition to ribitol, the strain transformed into 10% product, a yield gain of 6%.

 At the end of fermentation, the must was filtered, decolorized on black, demineralized, concentrated to 40% dry matter and then hydrogenated in the presence of 5% Raney nickel, at a pressure of 50 bar and at a temperature of 100 ° C. for 3 hours The hydrogenated syrup obtained contained 89% ribitol and 10% arabitol.

 Consequently, the final yield of ribitol increased by 5% relative to the yield obtained with the untransformed strain, under the same operating conditions.

Claims (10)

 A process for increasing the production of a polyol by an osmotolerant yeast strain, said polyol being selected from the group consisting of ribitol, arabitol and erythritol, by stimulating the carbon flux in the oxidative metabolic pathway of the cycle pentose phosphates of said strain, characterized by the fact that the enzymatic activity of the first  enzyme of said metabolic pathway, namely glucose  6-phosphate dehydrogenase, - that the strain obtained is cultured on an iron medium  containing a carbon source directly as  similar and - that we recover the polyol thus produced.  2. Process according to claim 1, characterized in that the enzymatic activity of the specific NADPH ribulose reductase is eliminated or substantially reduced, thereby favoring the production of ribitol.  3. Process according to either of Claims 1 and 2, characterized in that the enzymatic activities of NADPH and NADH-specific ribulose reductases are eliminated or substantially reduced, thus favoring the production of erythritol.  4. Method according to claim 3, characterized in that one introduces an enzymatic activity of the specific erythrose reductase NADPH type in the strain.  5. osmotolerant strain, characterized in that the enzymatic activity of glucose-6-phosphate dehydrogenase is amplified in said strain.  6. osmotolerant strain according to claim 5, characterized in that it is selected from the group comprising Yamadazyma ohmeri, Zygosaccharomyces rouxii, Candida polymorpha, and Torulopsis candida, and preferably from Yamadazyma ohmeri strains.  7. Osmotolerant strain according to either of Claims 5 and 6, characterized in that the enzymatic activity of the specific NADPH ribulose reductase is eliminated or substantially reduced.  8. osmotolerant strain according to either of claims 5 and 6, characterized in that the enzymatic activities of ribulose reductases NADPH and Specific NADH are eliminated or substantially reduced.  9. osmotolerant strain according to claim 8, characterized in that an enzymatic activity of the erythrose reductase type has been introduced into the strain.  10. DNA sequence, corresponding to a portion of the gene encoding Yamadazyma ohmeri glucose-6-phosphate dehydrogenase, as depicted in Figure 2.