EP4405471A1 - Genetisch verbesserter mikroorganismusstamm, der grössere mengen flüchtiger organischer verbindungen produzieren kann, und screening-verfahren zur gewinnung solcher stämme - Google Patents

Genetisch verbesserter mikroorganismusstamm, der grössere mengen flüchtiger organischer verbindungen produzieren kann, und screening-verfahren zur gewinnung solcher stämme

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Publication number
EP4405471A1
EP4405471A1 EP22790338.2A EP22790338A EP4405471A1 EP 4405471 A1 EP4405471 A1 EP 4405471A1 EP 22790338 A EP22790338 A EP 22790338A EP 4405471 A1 EP4405471 A1 EP 4405471A1
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Prior art keywords
strains
vocs
strain
coa
activity
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French (fr)
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Mélissa TAN
Jean-Marie Francois
Yanis CARO
Thomas PETIT
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Universite de La Reunion
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Universite de La Reunion
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    • 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
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/62—Carboxylic acid esters
    • 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.)
    • C12N9/001—Oxidoreductases (1.) acting on the CH-CH group of donors (1.3)
    • 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
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • 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
    • C12N9/10—Transferases (2.)
    • C12N9/1096—Transferases (2.) transferring nitrogenous groups (2.6)
    • 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
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • 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
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi

Definitions

  • the invention relates to the field of natural flavorings used in the food industry. More particularly, the invention relates to a strain of genetically modified microorganism capable of producing greater quantities of volatile organic compounds of interest, in particular ⁇ -unsaturated esters. It also relates to a screening method making it possible to select from - natural or modified strains exhibiting these characteristics, and a method for preparing a strain genetically modified to produce satisfactory quantities of volatile organic compounds (VOCs), defined as organic compounds with molecular masses of less than 400 Da.
  • VOCs volatile organic compounds
  • VOCs volatile organic compounds
  • 2-phenylethanol which gives the smell of roses
  • these aromatic compounds are synthesized chemically because of their low cost of production.
  • some of the chemically-synthesized flavors may be altered when integrating human metabolic pathways and be implicated in chronic diseases such as allergies, diarrhea, or cancer.
  • unconventional yeasts show interesting flavor-producing abilities, such as Pichia kluyveri, Torulaspora delbrueckii, Candida stellate, Hanseniaspora delbrueckii, which have been reported to produce high amounts of several flavor compounds like 2- phenylethanol, ethyl octanoate, butanoic acid or phenolic esters.
  • the inventors isolated from Reunion island dragon fruits a strain of filamentous aroma-producing yeast, identified as Saprochaete suaveolens.
  • This species of yeast produces a wide variety of volatile organic compounds (VOCs), including many ⁇ -unsaturated esters (isobutyl tiglate, isoamyl tiglate, butyl tiglate, ethyl tiglate, ethyl 2-methylpropanoate, ethyl 3-methylbut-2-enoate, ethyl but-2-enoate) which is rarely found in the aromatic bouquet of other Saccharomyces and non-Saccharomyces species.
  • the inventors have also demonstrated that the production of these ⁇ -unsaturated esters by 5. suaveolens comes from the catabolism of branched-chain amino acids via the ⁇ -oxidation (BOP) pathway.
  • BOP ⁇ -oxidation
  • FIG. 1 A simplified representation of the metabolic pathways of these branched chain amino acids is shown in Figure 1. Briefly, these branched chain amino acids are first converted to their corresponding branched ⁇ -ketoacid in a transaminase (TA) catalyzed reaction. Then, in Saccharomyces and non-Saccharomyces species, ⁇ -ketoacid enters the Ehrlich pathway (EP) which involves decarboxylation followed by either oxidation to produce carboxylic acids under the action of an aldehyde dehydrogenase (AIDH), or a reduction in higher alcohols under the action of an alcohol dehydrogenase (ADH).
  • TA transaminase
  • EP Ehrlich pathway
  • Esterification of higher alcohols with acetyl-CoA can generate acetates under the catalytic action of ester synthase or alcohol-O-acetyltransferase (AFT).
  • AFT alcohol-O-acetyltransferase
  • a second pathway that has been identified in filamentous fungi but absent in Saccharomyces cerevisiae is the [3-oxidation of branched-chain ⁇ -ketoacid to acetyl-CoA.
  • the ⁇ -keto-acid intermediate is oxidatively decarboxylated to an acyl-CoA, by branched-chain ⁇ -keto acid dehydrogenase (BCKAD) which is the enzyme regulating this pathway.
  • BCKAD branched-chain ⁇ -keto acid dehydrogenase
  • the acyl-CoA produced can be fully degraded to acetyl-CoA (+ propionyl COA in the case of L-leucine and L-isoleucine) via the [3-oxidation pathway.
  • yeasts can produce ethyl esters by condensation of short or medium chain acyl-CoA derived from the degradation of fatty acids with ethanol or higher alcohols (see Figure 1) in a process called “alcoholysis” catalyzed by an acyl-CoA:ethanol-acyltransferase
  • an originality observed in Saprochaete suaveolens is that the enoyl-CoA intermediate derived from the oxidation of amino acids can be esterified with alcohols to give rise to various ct-esters.
  • Agri-food professionals are looking for new sources of natural flavors in order to be able to offer consumers quality products both in terms of taste and food safety.
  • the present invention is based on a common inventive concept, namely the demonstration of the existence of a VOC synthesis pathway based on 6 specific enzymatic activities necessary for the production of VOCs.
  • This pathway stems directly from the establishment of a functional link between, on the one hand, the ability/inability of strains of microorganisms, in particular yeasts, to grow on a medium using only branched amino acids as a source of carbon, and on the other hand their ability to produce VOCs.
  • the inventors first implemented an original screening method based on the ability of strains to grow rapidly on media containing branched-chain amino acids (isoleucine, valine and leucine; so-called “ILV+” strains) as the sole source. of carbon. Then, they studied the ability of ILV+ strains to produce VOCs of interest, in particular ⁇ -unsaturated esters, in order to identify natural strains capable of producing molecules of aromas of interest. Twelve strains producing ⁇ -unsaturated esters were isolated, including two producing ⁇ -unsaturated esters not produced by the reference strain 5. suaveolens, validating the interest of this screening method.
  • the inventors propose to select strains capable of producing VOCs, in particular ⁇ -unsaturated esters, and which are of interest either for the diversity of the aroma molecules produced, or for their level of production. . These strains can then be exploited as is or genetically modified to improve their yield performance.
  • the invention relates to a strain of genetically modified microorganism capable of producing volatile organic compounds characterized in that: it expresses the activities transaminase (TA), branched-chain keto-acid decarboxylase (BCKAD), acyl-CoA dehydrogenase ( ACyD), alcohol acyl transferase (AAT) and acyl CoA hydrolase (ACyH) by expression or overexpression of the genes responsible for these activities; its enoyl-CoA hydratase (ECH) activity is reduced/suppressed, if such activity is present in said microorganism.
  • TA transaminase
  • BCKAD branched-chain keto-acid decarboxylase
  • ACyD acyl-CoA dehydrogenase
  • AAT alcohol acyl transferase
  • ACyH acyl CoA hydrolase
  • the invention also relates to the screening of yeast strains capable of producing VOCs consisting of: select, in natural isolates, the strains capable of growing on a medium containing only branched amino acids (isoleucine, leucine and valine, “ILV+” strains) as carbon source quantify the production of VOCs in the isolated strains select the strains capable of producing the required amount of VOCs.
  • yeast strains capable of producing VOCs consisting of: select, in natural isolates, the strains capable of growing on a medium containing only branched amino acids (isoleucine, leucine and valine, “ILV+” strains) as carbon source quantify the production of VOCs in the isolated strains select the strains capable of producing the required amount of VOCs.
  • It also relates to a method for screening a strain of mutated microorganism exhibiting increased VOC production relative to the non-mutated reference parent strain capable of producing VOCs.
  • the present invention also relates to a process for the preparation of a strain of genetically modified microorganism in which the production of VOCs is increased.
  • the present invention has the advantage of describing a new method for screening strains naturally producing VOCs of interest, in particular ⁇ -unsaturated esters with aromatic notes, based on their ability to grow on a medium containing only amino acids. plugged in as a carbon source. This method is simple and quick.
  • the invention also proposes to screen among strains producing COVs of interest, mutant strains having acquired the capacity to produce COVs in greater quantity.
  • This screening method is simple to analyze and quick to implement. It consists of random mutagenesis followed by selection on a medium containing only branched amino acids as carbon source. Random mutagenesis techniques generate a very large number of strains potentially carrying the desired mutation and therefore require a subsequent screening method that is rapid.
  • the screening of microorganisms for the production of VOCs is today essentially based on chromatographic methods which can prove to be very time-consuming when the number of strains to be analyzed is high.
  • the inventors propose an innovative approach based on physiological screening established by hypothesizing that the ability of strains to grow on a medium containing the branched amino acids isoleucine, leucine or valine (ILV medium) is at the origin of a reduction performance in VOC production. It is therefore a matter of selecting, from a parent strain producing the COV of interest, mutant strains that have lost the ability to grow on ILV medium as the sole carbon source. These mutant strains potentially increased their production of VOCs by promoting the flow of branched-chain amino acids into the flavor-producing pathway. The relevance of this approach is confirmed by the experimental results described below and the identification of a mutant strain meeting these selection criteria.
  • the present invention also describes a genetically modified 5.
  • suaveolens yeast strain capable of producing total VOCs at a level significantly higher than that of currently available strains (experimental data shows an 8-fold increase in the amount of total VOCs) . It makes it possible to envisage production on an industrial scale.
  • the target production thresholds are specific to each farm.
  • the present invention provides a method for preparing a microorganism capable of producing VOCs, in particular ⁇ -unsaturated esters, consisting in: reducing or eliminating the activity of enoyl-CoA hydratase, if this activity is present in the targeted microorganism, and to increase the activity of the acyl-CoA hydrolase if it is present or to provide it if it is not present in the targeted microorganism.
  • This method is applicable to a large number of cells, in particular cells validated for industrial production, such as 5. cerevisae.
  • a first object of the invention relates to a strain of genetically modified microorganism capable of producing volatile organic compounds, characterized in that: it expresses the activities transaminase (TA), branched-chain keto-acid decarboxylase (BCKAD), Acyl-Coa dehydrogenase (ACyD), alcohol acyl transferase (AAT) and acyl CoA hydrolase (ACyH) by expression or overexpression of the genes responsible for these activities; its enoyl-CoA hydratase (ECH) activity is reduced/suppressed, if such activity is present in said microorganism.
  • TA transaminase
  • BCKAD branched-chain keto-acid decarboxylase
  • ACyD Acyl-Coa dehydrogenase
  • AAT alcohol acyl transferase
  • ACyH acyl CoA hydrolase
  • microorganism within the meaning of the invention, is meant within the meaning of the invention preferably a yeast, a bacterium or a fungus. Specific examples are 5. suaveolens, S. cerevisae, E. coli, Aspergillus sp.
  • the production levels to be achieved depend on the desired VOC.
  • the invention is implemented as soon as the latter is capable of producing VOCs.
  • the invention allows an increase in the total production level of VOCs by a factor of 1.5 or more.
  • the strains according to the invention produce at least 1000 mg/L of total VOCs, and preferably at least 1500 mg/L, even more preferably at least 2000 mg/L of head space after 48 hours of growth .
  • the strains according to the invention produce at least 400 mg/L of alpha-unsaturated esters, and preferably at least 800 mg/L, even more preferably at least 1000 mg/L of headspace after 48 h of growth.
  • VOCs are by nature “volatile” compounds, they are found after production in the gaseous phase located above the culture medium, which is called “head space”; their recovery and quantification can be done from this gaseous phase, or other techniques such as distillation.
  • the modified strains should be considered in comparison with the quantities produced by the same unmodified strains.
  • the M10 strain described in the experimental part produces 1800 mg/L of total VOCs against 231 mg/L for the wild strain.
  • the microorganism is a strain of 5. suaveolens capable of producing VOCs and which has been genetically modified in order to increase this production, in which the activity of acyl CoA hydrolase and reduced/suppressed enoyl-CoA hydratase activity.
  • a strain produces more total VOCs after genetic modification; preferably, it produces 1.5 times, 2 times, 5 times, 10 times more VOC than the natural strain.
  • the VOCs produced or whose production is increased are alpha-unsaturated esters. These compounds are compounds having particularly interesting aromatic properties.
  • ⁇ -unsaturated esters is meant in particular isobutyl tiglate, isoamyl tiglate and ethyl tiglate.
  • a strain according to the invention is a genetically modified strain obtained from an initial strain (before genetic modification) producing a basal level of ⁇ -unsaturated esters and in which the genetic modifications consist of a reduction in the activity of the enzyme enoyl-CoA hydratase and an increase in the activity of acyl-CoA hydrolase compared to the initial strain.
  • Such a strain may be a yeast strain Saprochaete suaveolens.
  • a strain of microorganism according to this embodiment may be a strain in which the activity of the enzyme enoyl-CoA hydratase is reduced and the activity of the enzyme acyl-CoA hydrolase is increased by at least 50% compared to the activity of the initial strain.
  • a strain according to the invention is a genetically modified strain obtained from an initial strain (before genetic modification) not producing ⁇ -unsaturated esters and in which the genetic modifications consist of (i ) an expression or overexpression of transaminase (TA), branched-chain keto-acid decarboxylase (BCKAD), Acyl-CoA dehydrogenase (ACyD), alcohol acyl transferase (AAT) and acyl CoA hydrolase (ACyH) activities (by expression or overexpression genes responsible for these activities) and (ii) a reduction or elimination of the activity of the enzyme enoyl-CoA hydratase (ECH) if it exists.
  • TA transaminase
  • BCKAD branched-chain keto-acid decarboxylase
  • ACyD Acyl-CoA dehydrogenase
  • AAT alcohol acyl transferase
  • ACyH acyl CoA hydrolase
  • EHC enoyl-
  • a second object of the invention relates to a method for screening microorganism strains capable of producing VOCs consisting in: isolating, in natural media, the strains capable of growing on culture media containing at least one branched amino acid (isoleucine, leucine or valine) as sole carbon source quantify the production of ⁇ -unsaturated esters in isolated strains select strains capable of producing the required quantity of ⁇ -unsaturated esters.
  • branched amino acid isoleucine, leucine or valine
  • This method can be used either to directly select strains producing VOCs, in particular ⁇ -unsaturated esters (for example at least 400 mg/L of ⁇ -unsaturated ester headspace), or to select strains producing VOC of interest at a level lower than a target level of interest (for example lower than 400 mg/L of ⁇ -unsaturated esters in headspace) and which can be modified to increase this production.
  • ⁇ -unsaturated esters for example at least 400 mg/L of ⁇ -unsaturated ester headspace
  • a target level of interest for example lower than 400 mg/L of ⁇ -unsaturated esters in headspace
  • this screening method can be integrated as a first step in the methods described below.
  • the natural media in which the strains are sought can be soil samples, plant microbial flora or any other natural medium likely to contain microorganisms.
  • a third subject of the invention relates to a method for screening a mutated microorganism strain exhibiting increased COV production compared to the non-mutated reference parent strain capable of producing COVs, comprising the steps of: a. Random mutagenesis by UV irradiation of isolates of microorganism strains “mother strains” on a medium containing glucose as the sole carbon source b. Transfer of each of the irradiated strains so as to obtain four series of identical strains c.
  • the quantification of the production of VOCs is done by chromatography, in particular gas chromatography.
  • This analysis can be both quantitative and qualitative in terms of the VOCs produced.
  • the selected strains produce at least 400 mg/L of ⁇ -unsaturated esters in headspace.
  • An increase in production can also be evaluated by comparison with the level of production of the strain before mutagenesis, and be applied to VOCs in general or to ⁇ -unsaturated esters, or to a particular ⁇ -unsaturated ester such as tiglate. ethyl.
  • the non-mutated reference parent strains capable of producing ⁇ -unsaturated esters are selected by the strain screening method from natural isolates as described previously.
  • a fourth object of the invention relates to a process for the preparation of a strain of genetically modified microorganism in which the production of VOCs is increased, consisting in: a. Have a strain producing a basal level of a-unsaturated esters b. Reduce enoyl-CoA hydratase activity c. Increase acyl-coA hydrolase activity d. Quantify the VOCs produced to select the strains of interest
  • a fifth object of the invention relates to a process for the preparation of a strain of genetically modified microorganism capable of producing VOCs, consisting in: a. Have a strain that does not produce VOCs b. Introduce one or more gene expression cassettes containing the genes responsible for the expression of the enzymes transaminase (TA), branched-chain keto-acid decarboxylase (BCKAD), acyl-CoA dehydrogenase (ACyD), alcohol acyl transferase (AAT) and acyl CoA hydrolase. vs. Reduce or abolish enoyl-CoA hydratase activity by mutagenesis, if such activity is present in said microorganism d. Quantify the VOCs produced to select the strains of interest.
  • TA transaminase
  • BCKAD branched-chain keto-acid decarboxylase
  • ACyD acyl-CoA dehydrogenase
  • AAT alcohol acyl transfera
  • Steps b. etc. can be carried out by mutagenesis of the gene(s) encoding these enzymes.
  • the reduction of the enoyl-CoA hydratase (ECH) activity can be obtained by mutation or deletion of the gene responsible for the ECH activity or of a gene regulating the expression of the ECH gene.
  • transaminase TA
  • BCKAD branched-chain keto acid decarboxylase
  • ACyD acyl-CoA dehydrogenase
  • AAT alcohol acyl transferase
  • ACyH acyl-CoA hydrolase
  • Figure 1 Simplified diagram of branched-chain amino acid catabolism involving the Ehrlich pathway (EP) and the [3-oxidation (BOP) pathway in Saprochaete suaveolens.
  • TA transaminase
  • BCKAD branched chain keto acid dehydrogenase
  • ADH alcohol dehydrogenase
  • DC decarboxylase
  • ACyD FAD-dependent acyl-CoA dehydrogenase
  • ACyH acyl-CoA hydrolase
  • ECH enoyl-CoA hydratase
  • ACyDH -acyl-CoA NAD-dependent dehydrogenase
  • KT -keto acyl-CoA thiolase
  • AAT alcohol acyl transferase
  • ACyH acyl-CoA hydrolase.
  • Figure 2 Representative curve of PC2 versus PCI scores according to the aroma production of the strains and their origins Component analysis was performed using the XLStat Applied Sensory software (2020.1.3).
  • Group 1 included S13, S18, S21, S37, S38, S70, S74, S82, S84, S105 and S106.
  • Group 2 consisted only of S88.
  • Group 3 included S3, S12, S64, S68, S75 and S91.
  • Group 4 included S27, S99 and S100.
  • Figure 3 Cluster analysis of isolated VOC-producing strains. Strains were grouped based on their aroma production and origin using Ward's method and using XL Stat Applied Sensory software (2020.1.3). Automatic truncation (dashed line) identified four consistent groups of strains. Most of the strains were classified in group 1 (11 strains) and group 3 (6 strains). Group 4 included three strains. The S88 strain was found alone in group 2. The dendrogram is more flattened for group 4, suggesting that this group of strains is more homogeneous than the other two groups.
  • Figure 4 Survival rate of 5. suaveolens as a function of UV exposure time. Values are from eight independent experiments, with the standard value shown as a vertical bar.
  • Figure 5 Total VOCs determined by gas chromatography coupled to a mass spectrum detector (HS-SPME-GC/MS) produced by the wild strain of Saprochaete suaveolens and 9 isolated ILV- mutants. Data presented are the mean ⁇ SD of three independent cultures.
  • FIG. 6 Principal component analysis (PCA) of the VOCs produced by the wild strain of wild Saprochaete suaveolens and its 9 ILV- mutants generated by UV irradiation.
  • PCA Principal component analysis
  • Figure 7 Representation by map in color code mode of the COVs determined in the wild type and in 9 ILV- mutants of 5. suaveolens obtained by UV mutagenesis. The data is represented as the ratio of the COV value in the mutant versus the wild type. VOCs have been classified as originating primarily from the Ehrlich pathway (EP), acyl-COA or enoyl-COA intermediates from the oxidation pathway.
  • EP Ehrlich pathway
  • acyl-COA or enoyl-COA intermediates from the oxidation pathway.
  • EXAMPLE 1 Screening of yeast biodiversity from wild animal faeces from South Africa for the production of volatile ⁇ -unsaturated esters
  • Saccharomyces cerevisiae CEN.PK 112-2N van Dijken et al., 2000 was used in this study.
  • a strain of Saprochaete suaveolens previously isolated from Pitaya (Hylecereus polyrhisus) fruits in Reunion Island, France (Grondin et al., 2015a) was also used.
  • Fresh faeces from wild animals were collected aseptically from different locations in zoos, reserves and national parks in South Africa. The collection was carried out under sterile conditions and the samples were then stored at 4°C until their use. In addition, freeze-dried samples of wild animal faeces were obtained from an anonymous donor. A total of 118 samples were studied.
  • swab specimens For swab specimens, the swabs were added directly to 10 mL of sterile Ringer's solution. For tube samples, 1 g of faeces was pre-weighed and dissolved in Ringer's solution. Serial dilutions were then prepared and 100 ⁇ L of each dilution were spread on the surface of Yeast-Peptone-Dextrose agar medium (Biolab diagnostics LTD, South Africa) supplemented with 0.1 gL 1 of chloramphenicol (EMDQ Millipore Corp. Billerica, MA USA) (YPD-chloramphenicol) to promote yeast growth and selection. Petri dishes were then incubated at 30°C for 48 hours until cell colonies were completely formed.
  • Yeast-Peptone-Dextrose agar medium Biolab diagnostics LTD, South Africa
  • chloramphenicol EMDQ Millipore Corp. Billerica, MA USA
  • the colonies were then isolated by repeating the subculture on YPD-chloramphenicol agar medium and stored at 4°C.
  • 1 g of faeces was resuspended in 10 ml of sterile nutrient broth (Merck, South Africa) and vortexed before being incubated at 25°C for 5 days before being inoculated on a YPD-chloramphenicol medium.
  • strains capable of growing on media containing only branched-chain amino acids isoleucine, leucine and valine
  • ILV+ strains The selection of strains capable of growing on media containing only branched-chain amino acids (isoleucine, leucine and valine) as the sole carbon source (ILV+ strains) was carried out as follows. 5 ml of YPD broth was inoculated with each isolated strain and the culture was incubated overnight at 30°C with shaking. After centrifugation of the culture (13,000 rpm, 5 min), the cell pellet was washed twice with 5 mL of sterile physiological water and resuspended in 10 mL of sterile physiological water. Next, a culture of 10 6 cells. mL 1 was prepared in sterile physiological water and cultures of 10 5 and 10 4 cells. mL 1 were prepared by serial dilution.
  • VOCs volatile organic compounds
  • the strains were inoculated into a 20 mL screw cap tube containing 15 mL of slanted YPD-agar medium and incubated at 30°C for 24 hours. Then the vials were sealed and incubated for 24 hours at 30°C.
  • the headspace of the slant cultures was directly subjected to solid-phase microextraction (HS-SPME) using a 2 cm long fiber coated with 50/30 ⁇ m of divinylbenzene /Carboxene on polydimethylsiloxane bonded to a flexible fused silica core (Supelco).
  • octan-1-ol (at 0.5 gL 1 in dichloromethane) were added to the sealed tubes as an internal standard.
  • the fiber was then exposed to the headspace of the strains for 15 min at 30°C and inserted into the injection port at 250°C for 2 min.
  • Metabolites were separated by gas chromatography (GC) using a ZB-5MSI column (30m * 0.32mm * 0.25 ⁇ m film thickness), coupled to a mass spectrometer (Shimadzu GCMS-Q.P2010 Ultra).
  • the carrier gas (H2) was adjusted to a flow rate of 1.4 mL.min -1 .
  • the temperature of the column was maintained at 45° C.
  • Colony PCR was performed using fresh cells as an amplifiable template. The cells were taken directly from a colony of fresh yeast using an lpL loop. Cells were suspended in 100 ⁇ L PCR reaction mixture containing 0.5 ⁇ M ITS1 (5'-TCCGTAGGTGAACCTGCGG 3') or NL1 (5'-GCATATCAATAAGCGGAGGAAAAG) primers, 0.5 ⁇ M ITS4 (5'-TCCTCCGCTTATTGATATGC 3' ) or NL4 (5'-GGTCCGTGTTTCAAGACGG), 200 ⁇ M of each deoxynucleotide, 1x buffer and 2.5 units of DNA polymerase (Qiagen).
  • ITS1 5'-TCCGTAGGTGAACCTGCGG 3'
  • NL1 5'-GCATATCAATAAGCGGAGGAAAAG
  • ITS4 5'-TCCTCCGCTTATTGATATGC 3'
  • NL4 5'-GGTCCGTGTTTCAAGAC
  • the PCR conditions were as follows: initial denaturation at 95°C for 5 min; 35 cycles of denaturation at 94°C for 1 min, 1 min of primer annealing at 55.5°C for 1 min, 1 min of extension at 72°C, and a final extension at 72°C for 10 min.
  • PCR products were analyzed by electrophoresis on 2% agarose gels, with 1 x TAE buffer at 100V for 20 min. Gels were stained with Midori Green (Nippon Genetics Europe) and visualized under UV light. The sizes were estimated by comparison with a 1 kbp DNA ladder (1 kbp ladder, Gene O'ruler, ThermoFischer). Strain identification was performed by sequencing PCR products obtained by Eurofins Genomics (Germany). Sequences were analyzed using BLAST at NCBI (http://www.ncbi.nlm.nih.gov/blast).
  • suaveolens metabolism was used as the basis for the development of a selection method using the ability of yeasts to grow on media containing only branched-chain amino acids (isoleucine, leucine and valine) such as only carbon source (ILV+ strains). Assuming that this method would make it possible to select strains overproducing ⁇ -unsaturated esters, we searched for ILV+ strains among the 119 strains isolated from the faeces of wild animals, while using 5. suaveolens and 5. cerevisiae as controls. positive and negative respectively. Surprisingly, we found that 43 of the 119 strains tested presented the ILV+ criteria.
  • volatilomes of the selected strains i.e. the volatile organic compounds produced by all the ILV+ strains (43 strains isolated from the faeces and the positive control 5. suaveolens S0) were extracted by solid phase microextraction from space top, and analyzed by gas chromatography coupled to a mass spectrometry detector (HS-SPME-GC/MS) after 48 h of growth on a YPD medium at 30°C.
  • PCA principal component analysis
  • the number of acids was not correlated with any of the other parameters, probably because they represent only a small part of the VOCs detected compared to the other categories of VOCs.
  • VOCs come from amino acid catabolism.
  • ethyl 2-methylbutanoate and ethyl 3-methylbutanoate are likely produced in the Ehrlich pathway via the catabolism of isoleucine and leucine respectively (Hazelwood et al., 2008).
  • ethyl 2-methylbut-2-enoate and ethyl 3-methylbut-2-enoate are probably also produced by the catabolism of isoleucine and leucine, but via the [3 -oxidation (Grondin et al., 2015).
  • the main metabolic pathways involved in the production of the other VOCs detected in this study are the fatty acid [3-oxidation (ethyl hexanoate, ethyl octanoate, etc.), the butanoate pathway (butyl acetate, butyl butanoate, ethyl butanoate, etc.), the propanoate pathway (butyl propanoate) and the pentanoate pathway (ethyl pentanoate ).
  • some esters probably result from the esterification of two units generated by two different metabolic pathways. For example, the formation of 2-methylbutyl butanoate is likely generated by the esterification of 2-methylbutanol (synthesized via the Ehrlich pathway) and butanoic acid (synthesized via the butanoate pathway).
  • Esters represent 37 of the 50 VOCs detected in this study. They are present in the volatilome of all strains except strains S74 and S82 which only produce 2-phenylethanol. S0 (5. suaveolens), S12, and S91 strains exhibited the highest ester diversities in their volatilome. The S0 volatilome exhibited the greatest diversity of esters, with 30 different compounds in this category detected. Of these, 13 esters were not identified in the other strains investigated in this study. However, pentyl butanoate, which gives off an apricot-pineapple aroma frequently used in food and perfumery, was not detected in S0 volatilome, but was detected in S12 and S91 strains. This result is interesting because this compound is mainly present in fruits (apple and cocoa bean) but rarely detected in the volatilome of microorganisms.
  • This strain was incubated in 10 mL of peptone dextrose yeast extract medium (YPD; 20 gL 1 peptone, 20 gL 1 dextrose and 10 gL 1 yeast extract) at 30°C overnight, collected and washed twice with 10 mL of sterile water. An aliquot of 100 pL at 5.10 4 cells. mL 1 was plated on a nitrogen and yeast base medium (YNB) supplemented with 2g. L 1 of glucose (YNB-GIc) before exposure to UV irradiation (254 nm, 30 cm high) at times ranging from 0 and 540 sec. To stop the photoreactions, the plates were kept in the dark and incubated at 30°C for 15 h.
  • YNB nitrogen and yeast base medium
  • UV irradiation 254 nm, 30 cm high
  • the irradiated colonies were replicated on YNB supplemented with 1 gL 1 of isoleucine, leucine or valine or 2 gL 1 of glucose (YNB-lle, YNB-Leu, YNB-Val, YNB -GIc respectively) and incubated for 5 days at 30°C.
  • the colonies developing on YNB-GIc and not on YNB-lle, YNB-Leu or YNB-Val were then subcultured onto YPD agar medium.
  • the confirmation of the selected mutant clones was carried out by an additional growth test on YPD medium followed by YNB-lle.
  • the clones were suspended in 5 ml of YPD and incubated overnight at 30°C. The culture was then centrifuged (13,000 rpm, 5 min) and resuspended in 5 mL of sterile water. Then, 5pL of 10 6 , 10 5 and 10 4 cells. ml 1 prepared in sterile water were deposited in duplicate in a YNB-agar plate containing glucose, leucine, valine or isoleucine as a carbon source. The plates were incubated for 48 hours at 30°C before being read. This procedure was performed two more times to ensure the stability of the mutation. Wild strains of 5. suaveolens and 5. cerevisiae were used as positive and negative controls, respectively.
  • the strains were inoculated into a 20 ml crimp bottle containing 15 ml of YNB agar medium supplemented with 2 gL 1 of glucose and 1 gL 1 of isoleucine (YNB-GIc-lle) and incubated at 30°C for 24 h . Then the flask was sealed and re-incubated for 24 h at 30°C. Before the analysis, 10 ⁇ L of octan-l-ol (at 1 gL 1 in dichloromethane) were added in sealed vials as internal standard.
  • the headspace of the slant cultures was subjected to SPME analysis using a 2 cm long fiber coated with 50/30 ⁇ m divinylbenzene/carboxene on polydimethylsiloxane bonded to a flexible core of fused silica ( Supelco).
  • the fiber was exposed to the headspace for 15 minutes at 30°C and inserted into the injection port at 250°C for 2 minutes.
  • the metabolites were separated by GC, on a ZB-5MSI column (30m * 0.32mm * 0.25pm film thickness), coupled to a mass spectrometer (Shimadzu GCMS-Q.P2010 Ultra).
  • the carrier gas (H2) was adjusted to a flow rate of 2 mL.min -1 .
  • the temperature of the column was maintained at 40° C. for 2 min, brought to 150° C. at 10° C. min ⁇ 1 , then brought to 240° C. at 30° C. min 1 before the end.
  • Volatile organic compounds were identified by comparing their mass spectra and their experimental Kovats index with the NIST database (www.chemdata.nist.gov).
  • Yeast cells were cultured in 250 ml Erlenmeyer flasks containing 50 ml of YNB-GIc or YNB-GIc-lle for 24 h at 30°C. Then, the equivalent of 100 DO units at 600 nm of the culture was collected in 50 ml Falcon tubes by centrifugation (2000 rpm, 4° C., 1 min). Cell pellets were resuspended in 1 ml cold water, transferred to Eppendorf tubes, washed once more with cold water. The pellets obtained after centrifugation (2 min at 10,000 rpm) were stored at -20° C. until their use.
  • the reaction mixture containing 50 mM potassium phosphate buffer pH 7.4, 2 mM EDTA, 0.2 mM DTT, 0.5 mM TPP, 5 mM MgSO4, 0.5 mM CoA-SH and 1.5 mM NAD+ was initiated by the addition of 0.2 mM 2-oxo-4-methylpentanoic acid.
  • Enoyl-CoA hydratase (ECH) activity was determined according to [24] using 0.225 mM crotonyl-CoA as substrate. Conversion of this substrate to 3-hydroxy-butanoyl-CoA was measured at 263 nm at pH 8.0 in 45 mM Tris-HCl pH 8.0.
  • DC activity was determined using 2-oxo-4-methylpentanoic acid as a substrate in a reaction mixture containing 50 mM Na+citrate pH 6.2, 100 mM KCl, 0.2 mM of DTT, 0.5 mM TPP, 5 mM MgSO, 0.2 mM NADH and 5 U/mL yeast alcohol dehydrogenase.
  • the reaction was started by the addition of 0.2 mM 2-oxo-4-methylpentanoic acid and the oxidation of NAD+ was monitored at 340 nm.
  • Alcohol dehydrogenase (ADH) was tested with 2 mM 2-methylpropanal or 100 mM ethanol.
  • the reduction of 0.2 mM NADH to NAD+ was monitored at 340 nm in a mixture containing 50 mM Na+citrate buffer pH 6.2, 100 mM KCl, 0.2 mM DTT, 0.5 mM TPP and 5 mM MgSO4.
  • the reaction was carried out in 50 mM potassium phosphate pH 7.4, 100 mM KCl, 0.2 mM DTT, 0.5 mM TPP, 5 mM MgSO4 and 0.2 mM NADH. The reaction was initiated with 100 mM ethanol.
  • aldehyde dehydrogenase (AIDH) activity the reaction was carried out in the presence of 50 mM Na+-citrate buffer pH 6.2, 100 mM KCl, 0.2 mM DTT, 0.5 mM TPP, 5 mM MgSO4 and 1.5 mM NAD+.
  • the reaction was initiated by the addition of 2 mM 2-methylpropanal and followed at 340 nm by the reduction of NAD+ to NADH.
  • Acyl-CoA dehydrogenase (ACyD) activity was determined after reduction of DCIP to DCIPH2 at 655 nm.
  • the reaction was carried out in 50 mM potassium phosphate buffer pH 7.4, 2 mM EDTA, 100 mM KCI, 0.1 mM FAD+, 0.5 mM DCIP and 0.2 mM DTT and was initiated by the addition of 0.2 mM 2-methylbutanoyl-CoA.
  • Alcohol acyltransferase (AAT) was tested in the hydrolytic (esterase) sense, which was achieved according to [25] using p-nitrophenyl butyrate which is hydrolyzed to butyrate and p-nitrophenol, which absorbs at 415 nm .
  • the reaction was carried out in a mixture containing 50 mM potassium phosphate pH 7.4, 2 mM EDTA and 100 mM KCl and started with the addition of 2 mM p-nitrophenyl butyrate.
  • the assay of the acyl-CoA hydrolase (ACyH) activity was carried out in a 10 mM potassium phosphate buffer (pH 6.5), 50 mM MgCl2 and 0.1 mM DTNB.
  • the reaction was started by the addition of 0.1 mM 2-methylbutanoyl-CoA and the release of CoASH was monitored at 415 nm, which corresponds to the production of reduced DTNBH.
  • the protein concentration was determined at 550 nm by the Bradford method [26], with bovine serum albumin (BSA, Sigma-Aldrich) as standard. All the tests were carried out in triplicate from independent cultures and for each culture, the tests were carried out in duplicate.
  • mutants M2 to M10 To screen for mutants that lost the ability to grow on media containing isoleucine, leucine, or valine as the sole carbon source, these 15,000 irradiated clones were replicated on YNB-lle medium plates, YNB-Leu and YNB-Val geloses. Only 10 failed to grow out of the three branched chain amino acids. These 10 clones were then cultured again on YNB-GIc, and tested again for growth/absence of growth on non-permeable YNB-Leu/Ile/Val medium. With this procedure, only one of the 10 UV-irradiated clones was lost, thus leaving 9 potentially stable ILV- mutants, designated mutants M2 to M10.
  • VOCs Volatile organic compounds produced by the wild type of 5.
  • suaveolens and the 9 ILV- mutants were extracted by solid phase microextraction from space and then analyzed by gas chromatography coupled with a mass spectral detector (HS-SPME-GC/MS) after 48 h of growth on YNB agar tubes containing 20 gL-1 of glucose and 1 gL-1 of isoleucine (YNB-GIc-lle) at 30°C. Assuming similar growth of mutants and wild-type 5. suaveolens on agar plates, we found that mutants M9 and MIO had total COV production levels 1.8 and 8 times higher respectively than wild type 5.
  • PCA principal component analysis
  • the second axis (PC2) which represents about 10% of the total variance, separated the M9 mutant from the wild-type (WT) strain and the M5 mutant, which were roughly grouped in the same group, indicating that this mutant exhibited a similar COV profile to the wild type.
  • the COV profiles of the M9 and M10 mutants were the most different from each other and from the wild type, which is also consistent with the higher amount of COVs produced by these mutants.
  • M2, M3, M4, M6, M7 and M8 mutants were clustered and not far apart from M5 and WT, these data also suggest that these mutants have a very similar COV profile to each other and not significantly different from the wild type.
  • this PCR analysis showed that UV mutagenesis succeeded in generating at least two interesting mutants with a very different COV profile from the wild type.
  • suaveolens are essentially ester type compounds. Two types of esters have been detected, namely acetate esters which are formed by condensation of a higher alcohol generally produced in the Ehrlich pathway with acetyl-CoA and ethyl esters which are formed by condensation of an acyl-CoA with ethanol or another alcohol such as methanol, propanol or butanol [19]. While these two types of esters have also been found in the VOCs of mutants and wild-type 5. suaveolens, other types of esters have been identified.
  • esters can be produced from an acid such as 2-methylbutanoate acid which must be activated as a CoA-intermediate to condense with ethanol to give ethyl 2-methylbutanoate.
  • These esters have been classified in the category of compounds deriving from the Ehrlich pathway (EP) due to the higher alcohols and acids that come from this pathway.
  • Another category are esters which are formed by the condensation of an acyl-COA with ethanol or a higher alcohol such as octanol to give octyl acetate, octyl propanoate and butanoate of octyl.
  • BOP [3-oxidation
  • VOCs whose acid precursor is an enoyl-COA which can be esterified with ethanol (i.e. ethyl tiglate, ethyl but-2-enoate) or with a higher alcohol from the Ehrlich pathway such as 2-methylbutanol and 2-methylpropanol, to give 3-methylbutyl-2-methylbut-2Z-enoate (angelate d isoamyl) and 2-methylpropyl-2-methylbut-2E-enoate (isobutyl tiglate).
  • ethanol i.e. ethyl tiglate, ethyl but-2-enoate
  • 2-methylpropanol 2-methylpropanol
  • esters obtained by condensation of acyl-CoA with ethanol and of enoyl-CoA with ethanol or alcohols derived from EP i.e. ethyl tiglate, butyl tiglate, isobutyl tiglate, etc.
  • EP i.e. ethyl tiglate, butyl tiglate, isobutyl tiglate, etc.
  • the loss of Enoyl-CoA hydratase activity in the oxidation pathway may explain the higher COV production in the M10 mutant.
  • the Ehrlich pathway involves a decarboxylase (DC) and an alcohol dehydrogenase (ADH) for the reductive pathway or an aldehyde dehydrogenase for the oxidative pathway.
  • DC decarboxylase
  • ADH alcohol dehydrogenase
  • aldehyde dehydrogenase for the oxidative pathway.
  • BCKAD branched-chain ⁇ -keto-dehydrogenase
  • This acyl-COA intermediate can lose the CoA by an acyl-CoA hydrolase (AcyH) to give the corresponding acid, to be reduced to enoyl-CoA by an FAD+ dependent acyl-CoA dehydrogenase (ACyD) or even esterified to ethyl esters by a medium-chain fatty acid ester synthase [28].
  • acyl-CoA hydrolase AcyH
  • ACAD FAD+ dependent acyl-CoA dehydrogenase
  • enoyl-COA can be esterified into esters by an alcohol acetyltransferase, the nature of which remains to be identified.
  • suaveolens The enzymes of these two metabolic pathways were therefore determined in the crude extracts of the wild type and the M10 mutant of 5. suaveolens. Overall, the enzyme activities measured in the crude extracts of wild-type and mutant strains of 5. suaveolens were highly variable between cultures, which could be explained by the unexpected difficulty in breaking cells and obtain cell disruption reproducible. However, statistical analysis of the data allows us to be confident about enzymatic differences, when they occurred, between the mutant type and the wild type. Thus, the DC activity of the wild type of 5. suaveolens and of the M10 mutant was approximately comparable, whether the yeasts were cultured in YNB glucose supplemented or not with 1 gL 1 of isoleucine.
  • BCKAD activity which catalyzes the initial step of branched-chain amino acid oxidation, was also measured. It was found that while this enzyme showed similar activity between the mutant type and the wild type, its activity was 4 to 10 times lower than that of the enzymes downstream of the pathway. These data appear to be consistent with previous reports that argue that this enzyme may be rate-limiting in the catabolism of branched-chain amino acids through the P-oxidation pathway as has been reported in mammalian cells. In addition, this enzyme shows an activity at least 4 times lower than Ehrlich pathway enzymes, which could indicate that branched-chain amino acids would be preferentially catabolized by the Ehrlich pathway (EP), at least when yeast is grown with glucose as the carbon source.
  • EP Ehrlich pathway
  • suaveolens As mentioned previously, the overall activity of this esterase was similar in the two strains but was 2 times higher in a synthetic medium containing glucose and isoleucine, suggesting a positive effect of branched amino acids on the expression of genes encoding this esterase.
  • these enzymatic analyzes support the idea that the increased COV production in the M10 mutant of 5.
  • suaveolens can be explained by the almost complete absence of enoyl-CoA hydratase (ECH) activity.This loss of ECH activity in turn leads to an increased availability of the acyl-CoA and enoyl-CoA precursors for ester synthesis, and may explain the inability of this mutant to grow on branched amino acids.
  • ECH enoyl-CoA hydratase
  • UV mutagenesis has been employed to generate S. suaveolens mutants that harbor higher VOC production from branched-chain amino acids. Our selection was based on the inability to grow with these amino acids as the sole carbon source. It made it possible to isolate 9 mutants, one of which (the M10 mutant) proved to present 8 times more COV than the wild strain of 5. suaveolens. By determining the specific activities of key enzymes involved in isoleucine catabolism, we provided evidence that the higher production of VOCs observed in this mutant of 5. suaveolens is mainly due to the loss of activity of the isoleucine. enoyl-CoA hydratase and an increase in acyl-CoA hydrolase activity.

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