US20040259200A1 - Mutated lactococcus strain - Google Patents

Mutated lactococcus strain Download PDF

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US20040259200A1
US20040259200A1 US10/814,674 US81467404A US2004259200A1 US 20040259200 A1 US20040259200 A1 US 20040259200A1 US 81467404 A US81467404 A US 81467404A US 2004259200 A1 US2004259200 A1 US 2004259200A1
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lactate
lactis
glucose
growth
tmb5003
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Ulrika Andersson
Peter Radstrom
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Forskarpatent I SYD AB
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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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/56—Lactic acid

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  • the present invention relates to a new lactate producing strain belonging to the Lactococcus genus, viz. a mutant of Lactococcus lactis spp. lactis 19435 (obtained from ATCC), as well as a use of the mutant for lactate production, and a method for producing lactate.
  • Lactic acid is a naturally occurring organic acid that can be produced by either chemical synthesis or carbohydrate fermentation. Both of these production routes are used commercially [Datta, (1995)]. Chemical synthesis results in racemic lactic acid, while fermentation technologies enable synthesis of a desired stereoisomer of lactic acid. In the literature there are several reports concerning metabolic engineering of lactic acid bacteria in order to assess and enhance their ability in lactic acid production from different carbohydrates. Existing commercial production processes use homolactic organisms such as Lactobacillus delbreckii and Lb. bulgaricus [Datta, (1995)].
  • Lactococcus lactis is one of the most studied organisms used for industrial applications.
  • L. lactis ferments glucose according to a homolactic pathway under non-limiting glucose conditions [Sjöberg, (1995)]. This organism primarily produce the L-isomer of lactic acid that is favoured in applications associated with food since the D-isomer is harmful to humans [Hofvendahl, (1997)].
  • the post-glycolytic pyruvate metabolic pathways play a key role in determining the outcome of a fermentation of L. lactis . Activities of pyruvate converting enzymes changes with different cultivation conditions which lead to the important variations of end product formation [Cocaign-Bousquet, (1996)] (and references therein).
  • Lactic acid is a chemical used in food technology as well as in general chemical industry, including polymer technology. Thus, it can be used to produce polymers or become hydrogenated to produce propylene glycol, and other carbon chemical intermediates. Lactic acid is of interest to produce biocompatible and decomposable polymers, polylactic acid (PLA), used to produce sutures or implants, i.e., to be used within medicinal and veterinary surgery.
  • PLA polylactic acid
  • Lactic acid is an expensive chemical when produced by fermentation of different lactic acid producing micro-organisms, as the fermentation produces lactate, i.e., a salt of lactic acid, which has to be recovered from the fermentation broth also containing proteins and lactic acid producing cells.
  • lactate i.e., a salt of lactic acid
  • the fermentation has to utilise a micro-organism which produces a high concentration of lactate defined as overall concentration, but also defined as specific productivity (Q s ), i.e., grams per gram of substrate, and volumetric productivity (Q v ), i.e., grams per litre of broth per hour.
  • Lactic acid is present in two enantiomeric forms, L-lactic acid, and D-lactic acid.
  • D-lactic acid is toxic to humans, and should not be present in food applications, there is a demand for increased production of L-lactic acid.
  • Lactic acid is produced during fermentation by means of an enzyme, lactate dehydrogenase, LDH.
  • LDH is present in two forms, L-LDH for production of L-lactate, and D-LDH for the production of D-lactate.
  • the lactococci will thus also produce and activate a D-LDH, i.e., a lactate dehydrogenase producing the D-isomer.
  • U.S. Pat. No. 4,885,247 relates to a method for recovery and purification of lactate salts from whole fermentation broth using electrodialysis, whereby the method is stated to efficiently recover the lactate as a concentrated liquid. The lactate recovered is then transformed into lactic acid.
  • Dialog database acc. no. 07450183, General character and taxonomic studies of Lactococcus lactis IO-1 JCM 7638, Jour. of the faculty of agriculture, Kyushu University, 35(1-2):1-8, (1990) by Ishazaki, A., et al, relates to the characterisation of the strain L. lactis IO-1, i.e., the strain discussed above. There is no indication in the paper of which subspecie (spp) L. lactis IO-1 belongs to.
  • STN database acc. no. 0167424, Stimulation of the rate of L-lactate fermentation using Lactococcus lactis IO-1 by periodic electrodialysis L-lactic acid and production, Jour. of Fermentation and Bioengineering, 77(5):508-512, (1994), by Vonktaveesuk, P., et al, discloses electrodialysis of the products obtained by fermentations with strain L. lactis IO-1.
  • the mutant can be used for high yield production of lactate to be used in food technology or as a commodity in chemical industry in general.
  • the present invention is based upon a mutant of Lactococcus lactis spp. lactis wild type strain 19435 (obtained from ATCC), which mutant has been deposited on the 4th of Sep. 2001 at Deutsche Sammlung von Mikroorganismen und Zellkulturen under deposition number DSM 14489 in accordance with the Budapest Treaty.
  • TM5003 At controlled, monitored fermentations (continuous fermentations) of the new mutant, herein called TM5003, the lactate production has been analysed and calculations made show that TMB5003 has double the volumetric production compared to the wildtype strain 19435, and has a specific productivity which is 1.5 times that of the wildtype strain.
  • the yield of lactate calculated as grams of lactate produced per gram of glucose added at the fermentation, was calculated to be the same for both strains at continuous fermentations.
  • L-LDH, and D-LDH are produced or activated at different growth conditions, and thus the conditions can be chosen to produce optimal amounts of the lactate preferred, in this case the L-lactic acid or L-lactate.
  • Escherichia coli DH5 ⁇ (Life Technologies Inc.) was grown in Luria-Bertani medium at 37° C. and erythromycin was added to a concentration of 250 ⁇ g ml ⁇ 1 when required.
  • L. lactis strains were cultivated at 30° C. in M17 medium (Oxoid) containing 10 g l ⁇ 1 sugar.
  • sugars were autoclaved and added separately to the cultures as well as erythromycin was added to a final concentration of 2 ⁇ g ml ⁇ 1 when required to select for TMB5003.
  • the lactococcal strains were grown in a medium of the following composition (per litre): tryptone (Merck), 5 g; yeast extract (Merck), 5 g; casamino acids (Difco Laboratories), 1 g; K 2 HPO 4 , 2.5 g; KH 2 PO 4 , 2.5 g and MgSO 4 .7 H 2 O, 0.5 g, (pH 6.8).
  • Carbohydrates were added to a final concentration of 10 g l ⁇ 1 respectively.
  • the pH-controlled batch fermentations were performed at 30° C. in fermenters with a working volume of 800 ml. Stirring was set to 250 r.p.m.
  • the controlling device was a laboratory pH meter (Radiometer, Copenhagen, Denmark).
  • the parent cultures were grown overnight in the same media as the respective experimental cultures, in standing batch cultures at 30° C.
  • the inoculum 5% (vol/vol) was centrifuged, washed twice and resuspended in fresh culture medium without sugar, before being added to the experimental cultures.
  • L. lactis spp. lactis ATCC19435 and L. lactis spp. lactis TMB5003 were cultivated in semi-defined medium (SD3) according to van Niel and Hahn-Hägerdahl, (1999). All components except for potassium phosphates and water, were sterile filtered. Glucose was autoclaved and added separately to the medium at a final concentration of 5 g l ⁇ 1 . Continuous cultivations were performed using chemostat conditions in Biostat® A fermenters (B. Braun Biotech International, Germany). The volume in the fermenters was kept at 1 l. The temperature was set at 30° C.
  • Lactococcal cells were withdrawn from the cultivations at appropriate times and harvested by centrifugation at 5 000 ⁇ g, 2° C. for 10 min. The cells were washed twice and resuspended in 20 mM triethanolamine buffer, pH 7.2, containing 0.5 mM EDTA and 0.5 mM dithiotreitol. Disintegration of cells was performed by voltexing (3 ⁇ 5 min) at 8° C. by the use of glass beads (0.5 mm, KEBO). Cell debris was removed by centrifugation at 19,500 ⁇ g, 2° C., for 15 min. Cell extracts were kept at ⁇ 80° C. until used. The protein concentration was determined according to the method of Bradford [Bradford, (1976)]. Bovine serum albumin was used as a standard.
  • the reaction vial was washed twice with 3 ml ice-cold 0.5 M glucose solution and finally the filter equipment was also washed twice with ice-cold 0.5 M glucose solution.
  • the filter was placed in a scintillation vial containing 5 ml scintillation solution (EcoscintTM A, Hinzte AB, Sweden).
  • Assays were run in duplicates and for every cell resuspension and for every glucose concentration a background sample was prepared. These samples were prepared and handled as the other tests but the assay was not started by vortexing, instead 3 ml ice-cold 0.5 M glucose solution was dispensed into the vial and the reaction mixture was filtered immediately.
  • the PCR product was cleaved with restriction enzymes BamHI and RsaI, resulting in a 450 bp product.
  • a minimal integration vector, pFL20 not able to replicate in lactococci, developed by Levander et al., (2001), was digested with suitable restriction enzymes and ligated with the 450 bp malP internal fragment.
  • the resulting construct, denoted pTMB5003 was propagated in E. coli and further transformed into L. lactis spp. lactis 19435: Four transformants were obtained on erythromycin selective plates due to a single cross-over event in the malP of L. lactis . All transformants appeared to have the same growth behaviour in glucose and maltose cultivations, respectively.
  • One transformant, called L. lactis spp. lactis TMB5003 was chosen for further investigations.
  • the new mutant has been tested during glucose fermentation runs, and thereby been compared with the type strain.
  • L. lactis spp. lactis 19435 and L. lactis spp. lactis TMB5003 were grown in pH-controlled batch cultures to assess and compare their growth behaviour on glucose (FIG. 2).
  • the maximum specific growth rate of TMB5003 was twice the one that was determined for wild-type lactococci under the same growth conditions.
  • TMB5003 consumed glucose approximately twice as fast as the wild type.
  • Batch cultivations using lactose or maltose as sole carbon source resulted in no growth for TMB5003 in any of the cultivations while 19435 grew with the same specific growth rate on lactose as in glucose cultivations and with a slightly lower rate on maltose (data not shown).
  • TMB5003 The inability of TMB5003 to ferment lactose was confirmed by investigating the plasmid content of these cells. TMB5003 had lost one plasmid, compared to wild-type lactococci, most likely to be the one harbouring the lac-operon (data not shown) [de Vos, (1990); de Vos, (1989); Maeda, (1986)].
  • the inability of the mutant strain to ferment maltose was due to the fact that the maltose operon was disrupted by insertion of pTMB5003 in the maltose phosphorylase encoding gene, malP (Nilsson and R ⁇ dström, (2001)). It is believed that the alteration in the maltose operon does not promote the effect on glucose metabolism in TMB5003 and therefore this is not further discussed in the current presentation.
  • TMB5003 did also have an improved specific growth rate when cultivated on mannose, but not on trehalose, also suggested to be transported by PTS (Nilsson and R ⁇ dström, (2001)), further speculations occur concerning the mannose specific components of the mannose/glucose uptake system to be affected in the mutant strain.
  • L. lactis phosphofructokinase PFK
  • PK pyruvate kinase
  • LDH lactate dehydrogenase
  • the genes encoding these enzymes are located together in an operon, denoted as the las-operon, on the chromosome.
  • Recent results have been obtained concerning the role of PFK on glycolytic flux in L. lactis [Andersen, (2001)]. The conclusions were that glycolytic and lactate fluxes were decreased proportionally by a twofold reduction of PFK activity.
  • the concentrations of glucose were varied between 0 to 80 mM, and the specific uptake rate varied between 10 to 150 nmol/min/mg cells added, with regard to the wildtype strain, and between 5 to 55 nmol/min/mg cells added with regard to the new mutant.
  • FIG. 2 shows both growth on glucose (optical density graph, OD-curve), and glucose consumption of the wildtype strain 19435, and TMB5003, respectively.
  • the L-lactate produced and recovered can be used to adjust pH in food, as a taste enhancer in food, as well as a preservative of food, whereby the preservative effect is due to a lowering of pH, as well as due to the weak acid itself, which prevents growth of a number of micro-organisms in food and feedstuff. Accumulation of anions intracellularly or uncoupling of ATP-syntase is thereby the most probable mechanisms of growth inhibition.
  • the lactate can be used for the treatment of paper and metallic surfaces.
  • the lactate can be used for a polymerisation into poly-lactic acid, PLA, which is a biodegradable polymer. Further, the lactate can be used in the production of other compounds, such as propylene glycol, propylene oxide, acetaldehyde, ethanol, acrylates, and acrylic esters.
  • PLA as such can be applied in medical applications in the form of implants and sutures, production of items used for controlled release of drugs, and pesticides.
  • the polylactate can be used in the manufacture of package materials, as well as biodegradable disposable items.
  • the present strain can be grown in different media, such as complex media based on tryptone, yeast extracts, and casamino acids. Glucose can be added as an external carbon source. However, complex media are not preferred, as the amount of sugar (glucose) which results in lactate formation shall be controlled. In tryptone and yeast extracts there will be unknown components present which may make this difficult.
  • a semi-defined media such as SD3 and an addition of glucose up to 5 g/l can be used.
  • the semi-defined medium (E. W. J van Niel and B. Hahn-Hägerdal (1999), “Nutritient requirements of lactococci in defined growth media”, Applied Microbial Biotechnology 52:617-627) used for cultivating the present strain TMB5003 in the investigations made is composed of SD3 medium (per liter) Casamino acids 10 g K 2 HPO 4 2.5 g KH 2 PO 4 2.5 g MgSO 4 .7H 2 O 0.5 g Yeast nitrogen base 5 g (except casamino acids (Difco)) Asparagine 0.4 g Reduced glutathione 10 mg Uracil 60 mg Adenine 30 mg Guanine 30 mg Vitamin solution 10 ml Trace element solution 1 ml Glucose 5 g Vitamin solution (per liter) D-biotin 10 mg Pyridoxal-HCl 206 mg Folic acid 100 mg Riboflavin 100 mg Niacinamide 100 mg Thiamine-HCl 100 mg Ca-D-panthotenate
  • the present mutant TMB5003 is grown at high dilution rate, i.e., with a complete addition of glucose, and absolutely no restricted addition thereof to obtain and maintain a maximal lactate production.
  • a dilution rate of at least 0.5 h ⁇ 1 , preferably at least 0.7 h ⁇ 1 , and most preferably at least 0.8 h ⁇ 1 is used.
  • the lactate production tends to drop to the benefit of formation of by-products such as other acids such as acetate (acetic acid) and formate (formic acid).
  • the limiting factor of the wildtype strain to produce lactate is the ability of the strain to transport glucose.
  • the flux by means of the glucolysis is probably not limiting.
  • the final pyruvate metabolism may be limiting.
  • LDH is the enzyme which transforms pyruvate into lactate and this enzyme has turned out to have different temperature optima depending on which strain of lactococci that is used at the fermentation of glucose. pH, as well, may have an effect upon the activity of LDH.
  • the redox balance influences the efficiency of the LDH.
  • the presence of the co-factors NAD + /NADH is the controlling factor.
  • the conditions for enhancing L-LDH activity are fermentation in a medium having a pH above 6, and a temperature of up to 30° C., at which conditions L-lactate is substantially the only isomer produced, while D-LDH is activated at pH 4-5, and 33.5 to 40° C.
  • the fermentation of the present mutant should be carried out at such conditions that enhance activity of L-LDH to enhance formation of L-lactate.
  • the pH and temperature conditions for production of L-lactate are pH 6 to 7, preferably 6.0 to 6.5, and using a temperature of 25 to 30° C., preferably 27.5 to 30° C.
  • FIG. 1A shows the specific glucose uptake rate Lactococcus lactis spp. lactis 19435 cultivated at a dilution rate of 0.4 h ⁇ 1 .
  • FIG. 1B shows the specific glucose uptake rate by Lactococcus lactis spp. lactis TMB5003 cultivated at a dilution rate of 0.8 h ⁇ 1 .
  • FIG. 2 shows the glucose consumption versus time, and the change of optical density (OD) versus time.

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SE0103294-4 2001-10-03
SE0103294A SE0103294D0 (sv) 2001-10-04 2001-10-04 Lactate producing lactococcus strain
PCT/SE2002/001805 WO2003029440A1 (en) 2001-10-03 2002-10-03 Mutated lactococcus strain

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GB2419600A (en) * 2004-10-12 2006-05-03 Danisco Streptococcus with increased rate of glucose metabolism/ lactate synthesis

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MX341691B (es) * 2009-12-11 2016-07-05 Inst Tecnologico Estudios Superiores Monterrey Proceso para la produccion de l-lactato mediante fermentacion de lactosuero con lactococcus lactis subespecie lactis.
US9501647B2 (en) * 2014-12-13 2016-11-22 Security Scorecard, Inc. Calculating and benchmarking an entity's cybersecurity risk score

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US5798237A (en) * 1995-10-10 1998-08-25 Midwest Research Institute Recombinant lactobacillus for fermentation of xylose to lactic acid and lactate

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GB2419600A (en) * 2004-10-12 2006-05-03 Danisco Streptococcus with increased rate of glucose metabolism/ lactate synthesis

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SE0103294D0 (sv) 2001-10-04
ATE349512T1 (de) 2007-01-15
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DE60217142T2 (de) 2007-10-04
US20060270011A1 (en) 2006-11-30
CA2462489A1 (en) 2003-04-10
US7537925B2 (en) 2009-05-26
EP1432789A1 (en) 2004-06-30
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