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WO2025056787A1 - Production de pyruvate par carence en thiamine - Google Patents

Production de pyruvate par carence en thiamine Download PDF

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WO2025056787A1
WO2025056787A1 PCT/EP2024/075691 EP2024075691W WO2025056787A1 WO 2025056787 A1 WO2025056787 A1 WO 2025056787A1 EP 2024075691 W EP2024075691 W EP 2024075691W WO 2025056787 A1 WO2025056787 A1 WO 2025056787A1
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lactis
thiamine
lactic acid
acid bacterium
lactis subsp
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Shuangqing ZHAO
Christian SOLEM
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Danmarks Tekniske Universitet
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    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates

Definitions

  • the present invention relates to lactic acid bacteria deficient in lactate dehydrogenase and/or lactate dehydrogenase activity which become efficient pyruvate producers when starved for thiamine, and to methods of producing pyruvate by incubating or culturing such bacteria in thiamine-deficient media.
  • Pyruvate can be made by classical chemical synthesis or by microbial fermentation, where the latter approach is preferred, especially for sensitive applications such as use in food.
  • Toray Industries in Japan acquired a range of patents (see, e.g., US 4,971,907; JPS6455185A (B2); and JPS6455186A (B2)) relating to fermentative production of pyruvate using the multi-vitamin auxotrophic yeast Candida glabrata (previously called Torulopsus glabrata), and C. glabrata continues to be one of the best pyruvate producers available. This yeast, however, is an opportunistic pathogen and not compatible with food fermentations.
  • Lactococcus lactis is a lactic acid bacterium commonly used in food fermentations. Normally, 95% of the sugar consumed by L. lactis is converted into lactic acid. However, the metabolic flux can be shifted to make these bacteria produce other products.
  • An overview of glucose metabolism in L. lactis is shown in Fig. 1. Recently, L. lactis strain FS1076 was shown to be an efficient pyruvate producer (Suo et al., Front. Bioeng. Biotechnol., 2021).
  • the strain is a substrain of the strain MG1363, which has been genetically engineered to lack the four enzymes lactate dehydrogenase (LDH), phosphate acetyltransferase (PTA), alcohol dehydrogenase (ADH), and acetolactate synthase (ALS).
  • LDH lactate dehydrogenase
  • PTA phosphate acetyltransferase
  • ADH alcohol dehydrogenase
  • ALS acetolactate synthase
  • Lactococcus lactis strain which is deficient in lactate dehydrogenase (LDH) becomes an efficient producer of pyruvate when cultured under conditions where it is starved for thiamine and aerated.
  • LDH lactate dehydrogenase
  • the present invention provides a lactic acid bacterium which efficiently produces pyruvate when grown under certain conditions, and which only requires one genetic modification (causing deficient lactate dehydrogenase and/or lactate dehydrogenase activity).
  • this genetic modification can be easily made using non-GMO methods, such as adaptive evolution and chemical mutagenesis. Therefore, this invention provides a simple and potentially non-GMO method for producing food-grade pyruvate in lactic acid bacteria.
  • the present invention relates to a lactic acid bacterium which is deficient in thiamine and which is deficient in lactate dehydrogenase and/or lactate dehydrogenase activity.
  • the lactic acid bacterium is unable to produce thiamine.
  • the lactic acid bacterium is unable to grow in medium lacking thiamine.
  • the lactic acid bacterium belongs to any one of the following genera: Lactococcus, Pediococcus, Enterococcus, Leukonostoc, Limosilactobacillus, Lactobacillus, and Streptococcus.
  • the lactic acid bacterium belongs to, or is derived from, any one of the following species: Lactococcus lactis, Enterococcus feacium, Enterococcus casseliflavus, Leuconostoc mesenteroides, Lactococcus landensis, Limosilactobacillus reuteri, and Lactobacillus delbrueckii subsp. bulgaricus.
  • the lactic acid bacterium belongs to, or is derived from, any one of the following strains: Lactococcus lactis subsp. biovar diacetylactis RD1M5, Enterococcus feacium L117, Enterococcus casseliflavus L142, Leuconostoc mesenteroides JCFD003, Lactococcus landensis JCFD002, L. landensis JCFD006, L. lactis subsp. cremoris JCFD012, L. lactis subsp. biovar diacetylactis 170B, L. lactis subsp. biovar diacetylactis 17 IB, L.
  • lactis subsp. biovar diacetylactis 170 A L. lactis subsp. biovar diacetylactis SD96, L. lactis subsp. biovar diacetylactis JC0169A, L. lactis subsp. biovar diacetylactis JC0169B, L. lactis subsp. lactis SL195 (JC024), L. lactis subsp. lactis SL69 (JC023), L. lactis subsp. lactis JCFDO25, L. lactis subsp. lactis JCFDO27, L. lactis subsp. lactis JCFD026, L. lactis subsp.
  • lactis 109 L. lactis subsp. lactis 110, L. lactis subsp. lactis 111, L. lactis subsp. lactis 116, L. lactis subsp. lactis 120, L. lactis subsp. lactis 118, L. lactis subsp. lactis 121, L. lactis subsp. lactis 101, L. lactis subsp. lactis KF147, Limosilactobacillus reuteri CRL 1099, L. reuteri CRL 1100, L. reuteri CRL 1101, Lactobacillus delbrueckii subsp. bulgaricus CRL 958, and L. lactis subsp. cremoris CRL 985.
  • the present invention relates to a composition
  • a composition comprising the lactic acid bacterium according to the first aspect, optionally wherein the lactic acid bacterium is in freeze- dried form, and optionally wherein the composition is thiamine-deficient.
  • the present invention relates to a method of producing pyruvate, comprising : culturing the lactic acid bacterium according to the first aspect in medium deficient in thiamine, and under aeration, thereby obtaining pyruvate.
  • the lactic acid bacterium is cultured in the presence of acetate, such as 1.5-150 mM acetate, preferably 15 mM acetate.
  • the lactic acid bacterium is cultured in the presence of a source of oleate, such as in the presence of Tween 80, such as Tween 80 in a concentration of 0.01-1% (w/v), preferably Tween 80 in a concentration of 0.1% (v/v).
  • a source of oleate such as in the presence of Tween 80, such as Tween 80 in a concentration of 0.01-1% (w/v), preferably Tween 80 in a concentration of 0.1% (v/v).
  • the medium is thiamine-deficient milk.
  • the present invention relates to use of the lactic acid bacterium according to the first aspect or the composition according to the second aspect for producing pyruvate.
  • the present invention relates to a kit comprising a) a first lactic acid bacterium according to the first aspect or the composition according to the second aspect and b) a second lactic acid bacterium, or lysate or cell-free extract thereof, capable of producing one or more of o-acetolactate, acetoin, and diacetyl from pyruvate, optionally wherein the first and the second lactic acid bacterium, or lysate or cell-free extract thereof, are in separate vials.
  • the present invention relates to a method of producing one or more of o- acetolactate, acetoin and diacetyl, comprising the steps of: a) culturing the lactic acid bacterium according to the first aspect or the composition according to the second aspect in a culture medium deficient in thiamine, and under aeration, thereby obtaining pyruvate, and b) contacting the pyruvate with a second lactic acid bacterium, or lysate or cell-free extract thereof, capable of producing one or more of o-acetolactate, acetoin, and diacetyl from pyruvate.
  • Fig. 1 Overview of glucose and lactose metabolism in Lactococcus lactis.
  • PDHc pyruvate dehydrogenase complex
  • PFL pyruvate formate lyase
  • ALS o-acetolactate synthase
  • LDH lactate dehydrogenase
  • ADH alcohol dehydrogenase
  • PTA phosphotransacetylase
  • ALD o-acetolactate decarboxylase
  • ACK acetate kinase
  • DR diacetyl reductase
  • Nox NADH oxidase (Adapted from Ferreira and Mendes-Faia, 2020).
  • Fig. 2 Overview of glucose and lactose metabolism in Lactococcus lactis - with indication of principle of the present invention. Crosses indicate pathways that are blocked in L. lactis according to the invention, when starved for thiamine and aerated. Abbreviations: See Fig. 1 legend.
  • Fig. 3 Impact of initial cell density on pyruvate production by L. lactis RD1M5 cultured in SAN medium lacking thiamine. Conical flasks containing SAN medium devoid of thiamine were inoculated with RD1M5 cells to different initial cell densities as indicated and incubated with shaking. Metabolites were measured after 6 h and 16 h. Results are shown as mean ⁇ standard deviation for three replicates.
  • Fig. 4 Effect of incubation temperature on pyruvate production by L. lactis RD1M5 cultured in SAN medium lacking thiamine. Metabolites, cell growth (OD 5 oonm), and pH, were measured at different time points in cultures incubated at the temperatures indicated. RD1M5 was cultured in conical flasks with aeration (shaking) using an initial cell density corresponding to an optical density (OD 5 oonm) of 0.5. Results are shown as mean ⁇ standard deviation for three replicates.
  • Fig. 5 Effect of acetate and Tween 80 on growth and pyruvate production by L. lactis strain RD1M5 in SAN medium lacking thiamine. Metabolites, cell growth (OD 5 oonm), and pH were measured at different time points in cultures of RD1M5 incubated in different media, as indicated.
  • SAN- SAN medium without thiamine
  • SAN- + A SAN medium without thiamine with 15 mM acetate
  • SAN- + A + T SAN medium without thiamine with 15 mM acetate and with 0.1% Tween 80.
  • Fig. 6 Pyruvate production by non-growing L. lactis RD1M5 cells in POM buffer containing 1% glucose. Conical flasks containing different cell densities of RD1M5 cells, as indicated, were incubated at 30°C with shaking. Metabolites were measured after 6 h and 16 h of incubation. Results are shown as mean ⁇ standard deviation for three replicates.
  • Fig. 7 Pyruvate production by L. lactis RD1M5 in milk treated to reduce its thiamine content.
  • Conical flasks containing UHT milk, Florisil treated UHT milk, the latter supplemented with the vitamins present in SAN except thiamine, were supplemented with RD1M5 cells to an initial cell density of OD 5 oonm 0.05 and incubated at 30°C with shaking. Metabolites were measured at different time points, as indicated.
  • Untreated UHT milk contained 139.7 mM lactose and 19.3 mM citrate
  • Florisil-treated UHT milk contained 116.3 mM lactose and 18.6 mM citrate. Results are shown as mean ⁇ standard deviation for three replicates.
  • lactic acid bacterium is meant a bacterium belonging to the Lactobacillales order, which normally produces lactic acid as its main product of carbohydrate fermentation.
  • Lactic acid bacteria are Gram-positive, acid-tolerant bacteria that are often used in food fermentations.
  • Examples of lactic acid bacteria include, but are not limited to, bacteria belonging to the genera Lactococcus, Pediococcus, Enterococcus, Leuconostoc, Limosilactobacillus, Lactobacillus, and Streptococcus.
  • An example of a species of lactic acid bacteria is Lactococcus lactis.
  • a nonlimiting list of species of lactic acid bacteria can be found at the National Center for Biotechnology Information (World-Wide Web (www) address ncbi.
  • Thiamine also known as vitamin Bl, belongs to the group of water-soluble vitamins. As a member of the vitamin B family, thiamine plays a crucial role in supporting various cell functions in living organisms (Tylicki et al., 2018).
  • Thiamine pyrophosphate (TPP) is the active form of intracellular thiamine in L. lactis and serves as a coenzyme in numerous enzymatic reactions (Zhu et al., 2021). TPP forms a coordination bond with a metal ion, which helps anchor the cofactor's pyrophosphate moiety to a protein. This interaction occurs through a specific region of the protein structure (Brown et al., 1994).
  • thiamine as used herein, can mean thiamine in any form, including TPP, depending on the context.
  • thiamine a bacterial cell which contains less than 500 nmol/g dry cell weight, such as 250 nmol/g dry cell weight, such as 100 nmol/g dry cell weight, such as 50 nmol/g dry cell weight, and such as 10 nmol/g dry cell weight, of thiamine. It may also mean a bacterial cell which contains undetectable levels of thiamine, or which does not contain any thiamine (i.e.
  • thiamine-free either due to an inability of the bacterium to produce thiamine, an impaired (such as reduced) ability of the bacterium to take up thiamine, or to it being or having been cultured in a medium which does not contain thiamine or contains very small amounts of thiamine.
  • the bacterial cell is deficient in thiamine due to a combination of the above.
  • a bacterium may be deficient in thiamine due to an inability of the bacterium to produce thiamine and it being cultured in a medium which does not contain thiamine or contains very small amounts of thiamine.
  • a bacterium may be deficient in thiamine due to an inability of the bacterium to produce thiamine and a reduced ability to take up thiamine and it being cultured in a medium which only comprises very small amounts of thiamine.
  • a bacterium may also be deficient in thiamine due to a reduced ability of the bacterium to produce thiamine and an inability to take up thiamine.
  • undetectable levels of thiamine is meant an amount of thiamine which is below the detection limit in an assay suitable for measuring thiamine levels, such as an assay described elsewhere herein.
  • thiamine By “deficient in thiamine”, as used herein to describe a cell medium or a composition, is meant a medium or composition which contains less than 200 nM, such as less than 150 nM, such as less than 100 nM, such as less than 75 nM, such as less than 50 nM, such as less than 25 nM, and such as less than 10 nM, thiamine. It may also mean that the cell medium or composition contains undetectable levels of thiamine, or that it does not contain any thiamine (i.e. that it is "thiamine-free”). By “undetectable levels of thiamine” is meant an amount of thiamine which is below the detection limit in an assay suitable for measuring thiamine levels, such as an assay described elsewhere herein.
  • lactate dehydrogenase is meant all variants of the enzyme lactate dehydrogenase (LDH), which in L. lactis, for example, may be the protein encoded by the Idh gene (NCBI locus tag : H0A38_RS02065). The term may also include any of the proteins encoded by the IdhB and IdhX genes in L. lactis, which have more than 30% sequence identity to the W/7-encoded enzyme (Makarova et al., 2006; Liu et al., 2020).
  • lactate dehydrogenase activity is meant the enzymatic catalysis by the lactate dehydrogenase enzyme of the conversion of pyruvate to lactate and of NADH to NAD + .
  • reduced typically means that the amount of LDH protein or functional LDH protein present in the cell is reduced by at least 70%, such as at least 75%, such as at least 85%, such as at least 90%, such as at least 95%, as compared to a control.
  • the control is the unmodified host cell, which means the strain from which the cell deficient in lactate dehydrogenase is derived but which does not comprise the mutation(s) causing the deficiency in lactate dehydrogenase.
  • Methods for measuring mRNA or protein levels such as qPCR and quantitative western blot, are generally known in the art. That the bacterium is "deficient in lactate dehydrogenase", as used herein, may also mean that the bacterium does not contain any LDH enzyme or any functional LDH enzyme, for example due to knockout of the gene encoding LDH or knockdown of its expression.
  • knockdown refers to any of a range of techniques resulting in reduced expression of a gene in a host cell, such as introduction of a mutation in a promoter.
  • knockout refers to any of a range of techniques resulting in abolished expression of a gene in a host cell, such as introduction of a mutation in, or deletion of, the gene.
  • deletion refers to a partial or complete removal of the coding sequence of a gene, which either results in abolished expression of that gene or in the expression of a non-functional gene product.
  • That the bacterium is "deficient in lactate dehydrogenase activity", as used herein, means that the bacterium can produce no more than 30 mM, such as no more than 25 mM, such as no more than 20 mM, such as no more than 15 mM, such as no more than 10 mM, and such as no more than 5 mM, lactate, when cultured in medium with 1% glucose, with a start OD 5 oonm 10, for 16 hours at 30°C with 200 rpm shaking. These conditions were tested in Example 6 herein (results shown in Fig. 6(d)), where less than 25 mM lactate was produced.
  • lactate dehydrogenase activity can also mean that the bacterium produces undetectable levels of lactate, or that it cannot produce any lactate, i.e., the lactate dehydrogenase activity may be reduced or abolished.
  • the production of lactate may be measured using liquid chromatography. Such a method is described under "Analytical methods" in Example 1.
  • acetolactate decarboxylase is meant all variants of the enzyme acetolactate decarboxylase (ALD).
  • ALD is the protein encoded by the gene aldB (NCBI locus tag : llmg_1275).
  • acetolactate decarboxylase activity is meant the enzymatic catalysis by the acetolactate decarboxylase enzyme of the conversion of acetolactate to acetoin and CO 2 .
  • reduced typically means that the amount of ALD protein present in the cell is reduced by at least 50%, such as at least 60%, such as at least 75%, such as at least 90%, such as at least 95%, as compared to a control.
  • the control is the unmodified host cell, which means the strain from which the cell deficient in acetolactate decarboxylase is derived but which does not comprise the mutation(s) causing the deficiency in acetolactate decarboxylase.
  • Methods for measuring mRNA or protein levels such as qPCR and quantitative western blot, are generally known in the art. That the bacterium is "deficient in acetolactate decarboxylase", as used herein, may also mean that the bacterium does not contain any ALD enzyme or any functional ALD enzyme, for example due to knockout of the gene encoding ALD or knockdown of its expression.
  • That a bacterial strain is "deficient in acetolactate decarboxylase activity", as used herein, means that the concentration of pyruvate in the bacterium is at least 10%, preferably at least 50%, higher than the concentration of acetoin in the cell, i.e. that the acetoin: pyruvate ratio in the bacterium is at most 1 : 1.1, such as at most 1 : 1.2, such as at most 1 : 1.3, such as at most 1 : 1.4, and preferably no more than 1 : 1.5.
  • acetoin and pyruvate can be measured using liquid chromatography. Such a method is described under "Analytical methods" in Example 1.
  • culturing is meant that bacteria are incubated in a medium.
  • the bacteria may grow during the incubation, or they may not grow.
  • growth is meant multiplication of cells. Bacterial growth can be assessed by determining if the OD 5 oonm in the bacterial culture increases over time by measuring it at different time points during incubation, as is exemplified in the Examples section herein.
  • Lactococcus lactis strain RD1M5 which is deficient in lactate dehydrogenase (LDH) becomes an efficient producer of pyruvate when cultured under conditions where it is starved for thiamine and aerated.
  • the enzyme pyruvate formate lyase (PFL) is oxygen sensitive and is rapidly inactivated in aerated cultures (Takahashi et al., 1987; Zelcbuch et al., 2016), and the enzymes pyruvate dehydrogenase complex (PDHc) and o-acetolactate synthase (ALS) use thiamine, also known as vitamin Bl, as a co-factor, and depend on it to function (Nam et al., 2004).
  • RD1M5 is a thiamine auxotroph, meaning that it cannot produce thiamine itself (see Table 2).
  • the present invention provides a lactic acid bacterium which efficiently produces pyruvate when grown under conditions where it is thiamine-starved and aerated, and which advantageously only requires one genetic modification (causing deficient lactate dehydrogenase and/or lactate dehydrogenase activity).
  • this genetic modification can be easily made using non-GMO methods, such as adaptive evolution and chemical mutagenesis.
  • the present invention provides a simple and potentially non- GMO method for producing food-grade pyruvate in lactic acid bacteria.
  • the present invention relates to a lactic acid bacterium which is deficient in thiamine, and which is deficient in lactate dehydrogenase and/or lactate dehydrogenase activity.
  • the lactic acid bacterium is unable to produce thiamine.
  • the lactic acid bacterium is unable to grow in medium lacking thiamine.
  • the lactic acid bacterium has an impaired uptake of thiamine.
  • the lactic acid bacterium contains less than 500 nmol/g dry cell weight, such as 250 nmol/g dry cell weight, such as 100 nmol/g dry cell weight, such as 50 nmol/g dry cell weight, and such as 10 nmol/g dry cell weight, of thiamine.
  • Quantification of thiamine levels may be performed using an assay such as the assay described in Masuda et al., 2012 (hereby incorporated by reference herein in its entirety), in the section "Thiamine determination" on page 2062.
  • the thiamine concentration is measured in cell culture supernatant and in cells, whereas, in the context of the present disclosure, the thiamine concentration may be measured using this method in cells and any type of cell medium.
  • lactic acid bacteria including L. lactis
  • SAN medium The exact composition of SAN medium can be seen in Table 1, and an experimental procedure that can be used to determine whether a given lactic acid bacterial strain, which can grow in SAN medium, can also grow in SAN medium without thiamine, is described in Examples 1 and 2.
  • Lactic acid bacteria that are able to grow in standard thiamine-containing SAN medium and unable to grow in SAN medium lacking thiamine are considered to be unable to produce thiamine.
  • their ability to grow in thiamine-deficient medium can be tested using other growth media as appropriate.
  • the present inventors have identified several strains of lactic acid bacteria that are unable to produce thiamine.
  • the tested bacterial strains were mainly isolated from sourdough, cheese and beans, and out of 28 strains tested, 27 were found to be thiamine auxotrophs (unable to produce thiamine; these are listed in Table 2). If genetically altered to become deficient in lactate dehydrogenase and/or lactate dehydrogenase activity, all 27 of these strains identified as thiamine auxotrophs are expected to be suitable for use according to the present invention. Furthermore, other strains belonging to any one of the genera of the 27 strains are also expected to have a high likelihood of being thiamine auxotrophs.
  • Lactococcus Pediococcus, Enterococcus, Leuconostoc, Limosilactobacillus, Lactobacillus, and Streptococcus. This can be tested for each strain using the method described in Example 2.
  • the lactic acid bacterium belongs to any one of the following genera: Lactococcus, Pediococcus, Enterococcus, Leuconostoc, Limosilactobacillus, Lactobacillus, and Streptococcus.
  • the lactic acid bacteria strains Limosilactobacillus reuteri CRL 1099, L. reuteri CRL 1100, L. reuteri CRL 1101, Lactobacillus delbrueckii subsp. bulgaricus CRL 958, and L. lactis subsp. cremoris CRL 985 have previously been characterized as thiamine auxotrophic (Teran et al., 2021). These strains are likewise expected to be suitable for use according to the present invention, if genetically altered to become deficient in lactate dehydrogenase and/or lactate dehydrogenase activity.
  • the lactic acid bacterium belongs to, or is derived from, any one of the following species: Lactococcus lactis, Enterococcus feacium, Enterococcus casseliflavus, Leuconostoc mesenteroides, Lactococcus landensis, Limosilactobacillus reuteri, and Lactobacillus delbrueckii subsp. bulgaricus.
  • the lactic acid bacterium belongs to, or is derived from, any one of the following strains: Lactococcus lactis subsp. biovar diacetylactis RD1M5, Enterococcus feacium L117, Enterococcus casseliflavus L142, Leuconostoc mesenteroides JCFD003, Lactococcus landensis JCFD002, L. landensis JCFD006, L. lactis subsp. cremoris JCFD012, L. lactis subsp. biovar diacetylactis 170B, L. lactis subsp. biovar diacetylactis 17 IB, L.
  • lactis subsp. biovar diacetylactis 170 A L. lactis subsp. biovar diacetylactis SD96, L. lactis subsp. biovar diacetylactis JC0169A, L. lactis subsp. biovar diacetylactis JC0169B, L. lactis subsp. lactis SL195 (JC024), L. lactis subsp. lactis SL69 (JC023), L. lactis subsp. lactis JCFD025, L. lactis subsp. lactis JCFD027, L. lactis subsp. lactis JCFD026, L. lactis subsp.
  • lactis 109 L. lactis subsp. lactis 110, L. lactis subsp. lactis 111, L. lactis subsp. lactis 116, L. lactis subsp. lactis 120, L. lactis subsp. lactis 118, L. lactis subsp. lactis 121, L. lactis subsp. lactis 101, L. lactis subsp. lactis KF147, Limosilactobacillus reuteri CRL 1099, L. reuteri CRL 1100, L. reuteri CRL 1101, Lactobacillus delbrueckii subsp. bulgaricus CRL 958, and L.
  • the lactic acid bacterium belongs to any one of the strains L. lactis subsp. biovar diacetylactis JC0169A and L. lactis subsp. biovar diacetylactis JC0169B.
  • the lactic acid bacterium belongs to the strain L. lactis subsp. biovar diacetylactis RD1M5.
  • the strain L. lactis subsp. biovar diacetylactis RD1M5 (herein simply referred to as RD1M5) is a natural (non-GMO) dairy isolate (Liu et al., 2020) which is a natural thiamine auxotroph (see Example 2; Table 2) and lacks lactate dehydrogenase as a result of chemical mutagenesis and selection.
  • RD1M5 the Idh gene, encoding lactate dehydrogenase in L. lactis, has an eight- nucleotide insertion which causes a frameshift mutation. The result is decreased lactate formation by this strain (Liu et al., 2020).
  • the lactic acid bacterium belongs to the strain L. lactis subsp. biovar diacetylactis RD1M5 or has at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, and such as at least 99%, sequence identity to this strain.
  • lactic acid bacteria that are not thiamine auxotrophs can be made thiamine auxotrophic using the non-GMO method described below, i.e. by random mutagenesis and selection.
  • a given lactic acid bacteria strain can be made thiamine auxotrophic, or impaired in its ability to take up thiamine, or deficient in lactate dehydrogenase and/or lactate dehydrogenase activity, or any combination of these, using genetic modifications such as targeted genome modifications or, preferably, using non-GMO methods such as random mutagenesis and selection, or any combination thereof.
  • Genetic alterations resulting in reduced or abolished activity of lactate dehydrogenase can include a mutation or deletion in the coding sequence of that protein which results in the expression of non-functional or less functional protein.
  • genetic alterations resulting in reduced or abolished expression of the lactate dehydrogenase gene and/or reduced or abolished activity of lactate dehydrogenase may be indirect, meaning that they are not genetic alterations in the gene itself.
  • Such genetic alterations may for example include the introduction of a nucleic acid sequence that reduces the expression of the lactate dehydrogenase gene, e.g., a repressor that inhibits expression of the gene.
  • the amount of LDH protein or functional LDH protein present in the cell is reduced by at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, as compared to a control cell.
  • L. lactis for example, the enzyme lactate dehydrogenase (LDH), the activity of which is reduced or abolished, may be the protein encoded by the Idh gene (NCBI locus tag: H0A38_RS02065). However, it may also be the protein encoded by the IdhB or IdhX genes.
  • LDH lactate dehydrogenase
  • the control cell to which the LDH amount or activity is to be compared is usually the unmodified host cell, which means the strain from which the cell deficient in lactate dehydrogenase and/or lactate dehydrogenase is derived but which does not comprise the mutation(s) causing the deficiency in lactate dehydrogenase (activity).
  • control cell may be the strain SD96 from which it is derived (Liu et al., 2020).
  • Standard recombinant DNA and molecular cloning techniques useful for carrying out embodiments of the present invention are well known in the art and are described by, e.g., Sambrook, J., Fritsch, E. F., and Maniatis, T. (2012). Molecular cloning : A laboratory manual, 4th ed. Cold Spring Harbor Laboratory: Cold Spring Harbor, New York, and by Silhavy, T. J., Bennan, M. L., and Enquist, L. W. (1984). Experiments with gene fusions. Cold Spring Harbor Laboratory: Cold Spring Harbor, New York.
  • CRISPR Clustered regularly interspaced short palindromic repeats
  • a particularly preferred method of the present invention e.g. for reducing or abolishing the activity of lactate dehydrogenase, is random mutagenesis and selection.
  • This method can also be used for creating thiamine auxotrophic mutants from a strain that produces thiamine or for creating mutants that have an impaired uptake of thiamine.
  • An example of such a method is mutagenesis followed by a penicillin enrichment step and screening (or in short "penicillin enrichment”).
  • penicillin enrichment or in short "penicillin enrichment”
  • the library of mutated bacteria can be transferred to a defined medium without thiamine which contains penicillin, or another antibiotic that only kills actively growing cells (0- lactam antibiotics, cycloserine). Mutants unable to synthesize thiamine will not grow, and hence will survive this treatment. The survivors are plated on solid defined medium containing thiamine, which will allow auxotrophic mutants to grow, and then replica-plated onto solid medium without thiamine. Colonies only appearing on the first plate with thiamine, but not on the plate without thiamine, will be the thiamine auxotrophic mutants.
  • Penicillin enrichment can also be used to generate mutants that have an impaired ability to take up thiamine from the culture medium, for example using the following protocol:
  • thiamine transport genes potentially can lead to loss of function/reduced ability to take up thiamine from the cell's surroundings.
  • the library of mutated bacteria can be transferred to a defined medium containing "normal" levels of thiamine which also contains penicillin, or another antibiotic that only kills actively growing cells (0-lactam antibiotics, cycloserine). Mutants with an impaired ability to take up thiamine will not grow, and hence will survive this treatment.
  • a composition comprising a thiamine-deficient lactic acid bacterium
  • the present invention relates to a composition
  • a composition comprising the lactic acid bacterium according to embodiments of the first aspect of the invention, optionally wherein the lactic acid bacterium is in freeze-dried form, and optionally wherein the composition is thiamine-deficient.
  • the composition comprises a cell medium or buffer.
  • the cell medium or buffer is thiamine-deficient.
  • the composition comprises a lyophilization (freeze-drying) medium or components of a lyophilization medium.
  • composition comprising the lactic acid bacterium according to the invention
  • freeze-dried form it has the advantage that it can be stored for long periods of time and transported long distances.
  • the lactic acid bacterium deficient in lactate dehydrogenase and/or lactate dehydrogenase activity belongs to any one of the following genera: Lactococcus, Pediococcus, Enterococcus, Leuconostoc, Limosilactobacillus, Lactobacillus, and Streptococcus.
  • the lactic acid bacterium belongs to, or is derived from, any one of the following species: Lactococcus lactis, Enterococcus feacium, Enterococcus casseliflavus, Leuconostoc mesenteroides, Lactococcus landensis, Limosilactobacillus reuteri, and Lactobacillus delbrueckii subsp. bulgaricus.
  • the lactic acid bacterium deficient in lactate dehydrogenase and/or lactate dehydrogenase activity belongs to, or is derived from, any one of the following strains: Lactococcus lactis subsp. biovar diacetylactis RD1M5, Enterococcus feacium L117, Enterococcus casseliflavus L142, Leuconostoc mesenteroides JCFD003, Lactococcus landensis JCFD002, L. landensis JCFD006, L. lactis subsp. cremoris JCFD012, L. lactis subsp. biovar diacetylactis 170B, L.
  • lactis subsp. biovar diacetylactis 17 IB L. lactis subsp. biovar diacetylactis 170 A, L. lactis subsp. biovar diacetylactis SD96, L. lactis subsp. biovar diacetylactis JC0169A, L. lactis subsp. biovar diacetylactis JC0169B, L. lactis subsp. lactis SL195 (JC024), L. lactis subsp. lactis SL69 (JC023), L. lactis subsp. lactis JCFD025, L. lactis subsp. lactis JCFD027, L.
  • the lactic acid bacterium deficient in lactate dehydrogenase and/or lactate dehydrogenase activity belongs to any one of the strains L. lactis subsp. biovar diacetylactis JC0169A and L. lactis subsp. biovar diacetylactis JC0169B.
  • the lactic acid bacterium deficient in lactate dehydrogenase and/or lactate dehydrogenase activity belongs to the strain L. lactis subsp. biovar diacetylactis RD1M5.
  • the lactic acid bacterium deficient in lactate dehydrogenase and/or lactate dehydrogenase activity belongs to the strain L. lactis subsp. biovar diacetylactis RD1M5 or has at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, and such as at least 99%, sequence identity to this strain.
  • the lactic acid bacterium deficient in lactate dehydrogenase and/or lactate dehydrogenase activity is deficient in lactate dehydrogenase and/or lactate dehydrogenase activity as a result of some type of targeted genetic modification, whereas in other embodiments it is due to non-GMO methods, such as random mutagenesis and selection. Therefore, in some embodiments, the method is preceded by a step of generating and selecting a lactic acid bacterium deficient in lactate dehydrogenase and/or lactate dehydrogenase activity, optionally by a method of performing targeted genomic modifications and/or by a non- GMO method, such as random mutagenesis and selection.
  • the lactic acid bacterium deficient in lactate dehydrogenase and/or lactate dehydrogenase activity is unable to produce thiamine (i.e., is thiamine auxotrophic) and/or has an impaired uptake of thiamine.
  • the inability to produce thiamine and/or the impaired thiamine uptake is a result of some type of targeted genetic modification, whereas in other embodiments it is due to non-GMO methods, such as random mutagenesis and selection.
  • the method is preceded by a step of generating and selecting a lactic acid bacterium, which is:
  • the characteristics under A), the characteristics under B), or the characteristics under both A) and B have been generated and selected using targeted genomic modifications, whereas in other embodiments, the characteristics under A), the characteristics under B), or the characteristics under both A) and B), have been generated and selected using a non-GMO method, such as random mutagenesis and selection.
  • the medium contains less than 200 nM, such as less than 150 nM, such as less than 100 nM, such as less than 75 nM, such as less than 50 nM, such as less than 25 nM, and such as less than 10 nM, thiamine.
  • Quantification of thiamine levels may be performed using an assay such as the assay described in Masuda et al., 2012 in the section "Thiamine determination" on page 2062.
  • the thiamine concentration is measured in cell culture supernatant and in cells, whereas, in the context of the present disclosure, the thiamine concentration may be measured using this method in cells and any type of cell medium.
  • thiamine-deficient medium to choose for a given strain of lactic acid bacteria.
  • An example of such a medium is SAN thiamine+acetate medium.
  • the medium is thiamine-deficient milk.
  • the present invention relates to a method of producing pyruvate, comprising : culturing the lactic acid bacterium according to embodiments of the first aspect of the invention in medium deficient in thiamine, and under aeration, thereby obtaining pyruvate.
  • the lactic acid bacterium deficient in lactate dehydrogenase and/or lactate dehydrogenase activity is deficient in lactate dehydrogenase and/or lactate dehydrogenase activity as a result of some type of targeted genetic modification, whereas in other embodiments it is due to non-GMO methods, such as random mutagenesis and selection. Therefore, in some embodiments, the method is preceded by a step of generating and selecting a lactic acid bacterium deficient in lactate dehydrogenase and/or lactate dehydrogenase activity, optionally by a method of performing targeted genomic modifications and/or by a non-GMO method, such as random mutagenesis and selection.
  • the lactic acid bacterium deficient in lactate dehydrogenase and/or lactate dehydrogenase activity is unable to produce thiamine (i.e., is thiamine auxotrophic) and/or has an impaired uptake of thiamine.
  • the inability to produce thiamine and/or the impaired thiamine uptake is a result of some type of targeted genetic modification, whereas in other embodiments it is due to non-GMO methods, such as random mutagenesis and selection.
  • the method is preceded by a step of generating and selecting a lactic acid bacterium, which is:
  • the characteristics under A), the characteristics under B), or the characteristics under both A) and B have been generated and selected using targeted genomic modifications, whereas in other embodiments, the characteristics under A), the characteristics under B), or the characteristics under both A) and B), have been generated and selected using a non-GMO method, such as random mutagenesis and selection.
  • the lactic acid bacterium is cultured in the presence of acetate, such as 1.5-150 mM acetate, preferably 15 mM acetate.
  • the lactic acid bacterium grows during the cultivation, whereas in other embodiments the lactic acid bacterium is not growing during the cultivation.
  • the lactic acid bacterium is cultured in the presence of a source of oleate, such as in the presence of Tween 80, such as Tween 80 in a concentration of 0.01-1% (v/v), preferably Tween 80 in a concentration of 0.1% (v/v).
  • a source of oleate such as in the presence of Tween 80, such as Tween 80 in a concentration of 0.01-1% (v/v), preferably Tween 80 in a concentration of 0.1% (v/v).
  • the lactic acid bacterium is cultivated at 25-35°C, such as at 25°C, such as at 26°C, such as at 27°C, such as at 28°C, such as at 29°C, such as at 30°C, such as at 31°C, such as at 32°C, such as at 33°C, such as at 34°C, and such as at 35°C, preferably at 30°C.
  • the lactic acid bacterium is cultivated between 6 and 16 hours, such as for 6 hours, such as for 7 hours, such as for 8 hours, such as for 9 hours, such as for 10 hours, such as for 11 hours, such as for 12 hours, such as for 13 hours, such as for 14 hours, such as for 15 hours, and such as for 16 hours, preferably for 6 hours.
  • thiamine-deficient medium examples include SAN -thiamine medium, and POM buffer with 1% glucose.
  • the medium is thiamine-deficient milk.
  • the present invention relates to use of the lactic acid bacterium according to the first aspect of the invention or the composition according to the second aspect of the invention for producing pyruvate.
  • a kit comprising a thiamine-deficient lactic acid bacterium and a second lactic acid bacterium
  • the present invention relates to a kit comprising a) a first lactic acid bacterium according to the first aspect of the invention or the composition according to the second aspect of the invention and b) a second lactic acid bacterium, or lysate or cell-free extract thereof, capable of producing one or more of o-acetolactate, acetoin, and diacetyl from pyruvate, optionally wherein the first and the second lactic acid bacterium, or lysate or cell-free extract thereof, are in separate vials.
  • the second lactic acid bacterium, or lysate or cell-free extract thereof is deficient in acetolactate decarboxylase and/or acetolactate decarboxylase activity.
  • the amount of acetolactate decarboxylase (ALD) protein in the second lactic acid bacterium, or lysate or cell-free extract thereof is reduced by at least 50%, such as at least 60%, such as at least 75%, such as at least 90%, such as at least 95%, as compared to a control.
  • ALD acetolactate decarboxylase
  • the control is the unmodified host cell, which means the strain from which the cell deficient in acetolactate decarboxylase is derived but which does not comprise the mutation(s) causing the deficiency in acetolactate decarboxylase.
  • Lactic acid bacteria suitable for use as the second lactic acid bacterium according to the sixth aspect of the invention include Streptococcus spp. and Lactococcus spp. bacteria, such as bacteria selected from any one of the species: Streptococcus agalactiae, S. bovis, S. casei, S. cremoris, S. durans, S. diacetylactis, S. faecalis, S. faecium, S. lactis, S. thermophilus, Lactococcus lactis ssp. lactis (such as L. lactis ssp. lactis biovar. diacetylactis DI), and L. lactis ssp. cremoris (such as L. lactis subsp. cremoris MG1363).
  • Streptococcus spp. and Lactococcus spp. bacteria such as bacteria selected from any one of the species: Streptococcus a
  • lactis subsp. cremoris MG1363 (MG1363 AldhB AldhX Apta AadhE AbutBA AaldB) is deficient in both acetolactate decarboxylase and acetolactate decarboxylase activity (Dorau et al., Microb. Cell Fact., 2019).
  • the kit according to the sixth aspect of the invention enables a method for producing one or more of o-acetolactate, acetoin and diacetyl, as described below.
  • the present invention relates to a method of producing one or more of o- acetolactate, acetoin and diacetyl, comprising the steps of: a) culturing the lactic acid bacterium according to the first aspect of the invention or the composition according to the second aspect of the invention in a culture medium deficient in thiamine, and under aeration, thereby obtaining pyruvate, and b) contacting the pyruvate with a second lactic acid bacterium, or lysate or cell-free extract thereof, capable of producing one or more of o-acetolactate, acetoin, and diacetyl from pyruvate.
  • the second lactic acid bacterium, or lysate or cell-free extract thereof is deficient in acetolactate decarboxylase activity
  • the acetoin :o-acetolactate ratio in the cell medium or composition is at least 1 : 1.1, such as at least 1 : 1.2, such as at least 1 : 1.3, such as at least 1 : 1.4, and preferably more than 1 : 1.5.
  • TTC (2,3,5- triphenyltetrazolium chloride), Florisil, L-isoleucine, peptone, and agar were purchased from Merck Millipore (Darmstadt, Germany). Cytidine was purchased from TCI Chemicals (Japan).
  • the POM buffer contained 1 mM MgCI 2 , 50 mM K 2 HPO 4 , and 50 mM KH 2 PO 4 to adjust the pH to 7.4.
  • 0.9% (w/v) NaCI was used for washing cells.
  • POM buffer and 0.9% NaCI solutions were sterilized by autoclavation for 15 min at 121°C.
  • Ultra-heat-treated (UHT) milk (15 g/L fat) was purchased from the local supermarket (Milsani, Albertslund, Denmark).
  • TTC plates were prepared as described previously, however, with some modifications (Monnet et al., 2000). Briefly, 15 g casein peptone, 5 g yeast extract, 0.5 g MgSO 4 , 15 agar, and 0.5 g ascorbic acid were dissolved in 890 mL water. The pH was adjusted to 7.0 and autoclaved for 15 min at 121°C. Lactose with 10% (w/v) was also autoclaved in the same way. After cooling to 50°C, 10 mL filtered sterile TTC solution (10 g/L) and 100 mL lactose (100 g/L) were added. The agar medium was swirled gently and poured into Petri dishes.
  • M17 was bought from oxoid (Thermo Fisher Scientific, USA) and supplemented with 1% lactose (LM17). Briefly, 37.25 g M17 powder was dissolved in 900 mL of distilled water and sterilized by autoclaving at 121°C for 15 minutes. After cooling to 50°C, 100 mL sterile lactose solution (10% w/v) was aseptically added to the autoclaved M17. In the slant medium, 15 g/L agar was added. SAN medium (does not contain acetate) is a chemically defined minimal growth medium for cultivating L. lactis (Jensen and Hammer, 1993).
  • SAN -thiamine+acetate medium is based on SAN medium without thiamine and with 15 mM acetate added.
  • SAN -thiamine+acetate+Tween 80 is SAN- thiamine+acetate mec
  • Table 1 Composition of SAN medium (adapted from Jensen and Hammer, 1993J
  • L. lactis subsp. lactis biovar diacetylactis RD1M5 (RD1M5) is a strain of technological interest.
  • This strain is an LDH-deficient strain obtained via adaptive evolution and chemical mutagenesis from L. lactis subsp. lactis biovar diacetylactis SD96 (Liu et al., 2020).
  • TTC is a water-soluble compound, which can be reduced to the red compound triphenyl formazan (TPF) if the pH is not too low. Lactate dehydrogenase deficient strains form red colonies on TTC plates as they are unable to form lactic acid and thus the pH in the vicinity of the colony is high enough for
  • the frozen culture was spread on LM17 (0.5% lactose) agar plates for 24 h at 30°C, and then a single colony was inoculated into 50 mL LM17 (0.5% lactose) and incubated at 30°C without shaking for 10 h to prepare a preculture.
  • pyruvate, glucose, acetate, lactate, citrate, and acetoin concentrations were determined utilizing an advanced high-performance liquid chromatography system incorporating an Aminex HPX-87H column (Bio-Rad, Hercules, USA) and a Shodex RI-101 refractive index detector (Showa Denko K.K., Tokyo, Japan). Moreover, pyruvate was quantified utilizing a UV detector set at a wavelength of 210 nm. The mobile phase, consisting of 5 mM H 2 SO 4 , was used at a flow rate of 0.5 mL/min, and the column oven temperature was consistently maintained at 60°C.
  • a single colony from an LM17 agar plate was inoculated into liquid LM17 medium in a conical flask which subsequently was incubated at 30°C for 16 h.
  • Cells were harvested by centrifugation at 10,000 rpm for 5 min, and the pellet washed three times with 0.9% NaCI solution, followed by resuspension in the same solution to attain the original volume.
  • Each cell suspension was inoculated into 5 mL of SAN thiamine and SAN medium to an initial optical density (ODeoonm) of 0.05 and incubated at 30°C for 24 h. After this, each culture was subcultured four times in the same medium for 24 h under the same conditions using a 1% inoculum during each transfer.
  • This dense cell suspension was diluted in SAN -thiamine medium to obtain 50 ml suspensions of cells with cell density corresponding to OD 5 oonm-values of 0.05, 0.25, 0.5, 1, and 2, respectively.
  • the samples were incubated at 30°C with 200 rpm shaking, and after 6 h and 16 h samples were acquired for HPLC analysis, and pH was determined concurrently.
  • the lactate concentration starts to decrease when the start cell densities increase beyond a certain point; again, this may be explained by higher levels of thiamine/TPP in the medium due to more cells, whereby PDHc and ALS can keep some of their function and to some extent keep the NAD + /NADH balance.
  • Example 3 it was shown that the RD1M5 strain of L. lactis can produce pyruvate when it is starved for thiamine and aerated.
  • the purpose of this experiment was to test the influence of incubation temperature on this process.
  • Suspensions 50 ml of RD1M5 in SAN -thiamine medium adjusted to a cell density of 0.5 were prepared as described in Example 3. These were incubated at different temperatures: 25, 30, 35, 39, and 45°C with 200 rpm shaking, and samples were taken at various times for HPLC analysis. The pH and optical density (OD 5 oonm) were measured concurrently.
  • Acetyl-CoA is essential for fatty acid synthesis, which supports L. lactis growth.
  • the acetyl-CoA can be synthesized from acetate by acetate kinase and phosphotransacetylase, which has been shown to increase the pool of intracellular acetyl-CoA (Nordkvist et al., 2003). Lactic acid bacteria are dependent on biotin for their growth. However, it has been documented that oleate or its derivative, such as Tween 80, can effectively substitute for biotin.
  • Tween 80 is recognized for its ability to supply exogenous oleic acid, which can integrate into the membranes of lactic acid bacteria and stimulate their proliferation (Potter & Elvehjem, 1947; Station & Rouge, 1945). The purpose of this experiment was to test if it is possible to produce thiamine-deficient cells that can produce pyruvate by culturing RD1M5 cells in SAN thiamine medium with addition of acetate and Tween 80.
  • SAN’ thiamine medium could not support the growth of RD1M5.
  • RD1M5 grew in both SAN’ thiamine+acetate and SAN’ thiamine+acetate+Tween 80 medium, where a cell density corresponding to an OD 5 oonm of about 1.0 was obtained after 16 h.
  • RD1M5 grew faster in the medium containing Tween 80 than in the SAN -thiamine+acetate medium.
  • the purpose of this experiment was to test if thiamine-starved RD1M5 cells can produce pyruvate in a different medium than SAN medium, i.e. POM buffer with 1% glucose.
  • RD1M5 cells were incubated in LM17 medium, growing at 30°C for 16 h. Cells were harvested by centrifugation, and washed three times with the same volume of 0.9% NaCI solution. To obtain thiamine-starved cells, the washed cells were added to SAN -thiamine+acetate medium adjusted to a start cell density corresponding to an OD 5 oonm of 0.05 and grown for 16 h at 30°C with 200 rpm shaking, after which the cells were harvested by centrifugation and washed three times with 0.9% NaCI solution. The thiamine-starved cells were re-suspended in 0.9% NaCI solution.
  • pyruvate can also be produced in non-growing thiamine-starved cells in POM buffer (1% glucose).
  • the production of the by-product acetoin also increased with increasing start cell density. This may be due to a limited extent of thiamine/TPP-dependent functioning of ALS. However, the acetoin production was still much lower than for the thiamine-enriched cells, as is shown in Fig. 3(c).
  • the Florisil particles are settled at the bottom of the beaker.
  • the acetic acid solution is poured off, and the Florisil beads are washed 3 times using 300 mL water to remove extra acetic acid and Florisil dust.
  • Filter paper was placed in a Buchner funnel, and the washed Florisil particles were placed in it and allowed to drain.
  • the first 50 mL of milk was added to a Buchner funnel with the rate of filtration, and the filtered milk (still some water mixed with milk) was discarded. Then, 250 mL milk was continuously added to a Buchner funnel, and the filtered milk was collected as thiamine-deficient milk. After that, the treated milk was heated at 100°C for 30 min to sterilize the treated milk.
  • RD1M5 was cultured in LM17 medium, growing at 30°C for 10 h. The culture was centrifuged, and the cells were washed three times with the same volume of 0.9% NaCI solution. Then the washed cells were put in 10 mL treated UHT milk or UHT milk in a 250 mL shaking flask with a start OD 5 oonm of 0.05 and incubated at 30°C for 24 h. Samples were taken after 6 h, 12 h, and 24 h, to determine metabolites by HPLC. Results
  • TTC 2,3,5-Triphenyltetrazolium chloride
  • EP0247646 Bl (UNILEVER NV)
  • EP0483888 Bl (UNILEVER NV)

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Abstract

L'invention concerne des bactéries lactiques déficientes en thiamine et déficientes en lactate déshydrogénase et/ou dont l'activité de la lactate déshydrogénase est déficiente, des procédés de production de telles bactéries, et des procédés de production de pyruvate par culture de telles bactéries en milieu déficient en thiamine et dans des conditions d'aération.
PCT/EP2024/075691 2023-09-14 2024-09-13 Production de pyruvate par carence en thiamine Pending WO2025056787A1 (fr)

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