Construction and application of Kluyveromyces marxianus strain for producing D-lactic acid

Abstract

The invention discloses construction and application of a Kluyveromyces marxianus strain for producing D-lactic acid, and belongs to the technical field of microorganisms. On the basis of a metabolic engineering modification strategy, lactic dehydrogenase from lactobacillus helveticus is integrated while pyruvate decarboxylase is knocked out, and a D-lactic acid high-yield strain named FMME-LA01 is obtained. The yield of the D-lactic acid can reach 140g / L after fermentation is carried out for 72 hours under the optimized condition of a shake flask, the yield of the D-lactic acid can reach 135g / L after fermentation is carried out for 70 hours in a 7.5 L fermentation tank, the optical purity can reach 99% or above, and the strain has good hereditary stability and is suitable for industrial production.

Description

TECHNICAL FIELD

[0001] The present application relates to the construction and application of a D-lactic acid-producing Kluyveromyces marxianus strain, and belongs to the technical field of microorganisms. BACKGROUND

[0002] Lactic acid, molecular formula C3H6O3, also known as 2-hydroxypropionic acid, is a colorless and clear or slightly yellow viscous liquid. Lactic acid is a very important three-carbon platform compound. Since the molecular structure of lactic acid contains a chiral carbon atom, there are three forms in nature: D-type, L-type and DL-type. As an important chiral intermediate, D-LA is widely used in the fields of pharmaceuticals, cosmetics, etc. such as the synthesis of herbicides, calcium antagonists, and picolinic acid derivatives. The use of chemical resolution method to synthesize D-lactic acid is difficult to be widely used due to the high cost of resolution reagent, difficult separation, and problems such as toxicity and environmental pollution. The biological method for producing D-lactic acid has the advantages of low pollution, low energy consumption, low cost and sustainable development, and has gradually become the mainstream method for synthesizing D-lactic acid, which includes microbial enzyme catalysis and microbial fermentation. Enzyme catalysis has been applied to the industrial production of D-lactic acid, and the level of microbial fermentation method for producing D-lactic acid needs to be further improved. SUMMARY

[0003] The first object of the present application is to provide a D-lactic acid-producing Kluyveromyces marxianus, named Kluyveromyces marxianus FMME-LA01.

[0004] In one embodiment, the Kluyveromyces marxianus knocks out the pdc1 gene and integrates the lactate dehydrogenase gene D-ldh from Lactobacillus helveticus.

[0005] In one embodiment, the nucleotide sequence of the pdc1 gene is shown in SEQ ID NO. 1.

[0006] In one embodiment, the nucleotide sequence of the D-ldh gene is shown in SEQ ID NO. 2.

[0007] In one embodiment, the Kluyveromyces marxianus integrates the D-ldh gene at the position of the pdc1 gene on the genome.

[0008] In one embodiment, the promoter P PGK1 controls the expression of the D-ldh gene.

[0009] In one embodiment, the promoter P PGK1 has a nucleotide sequence shown in SEQ ID NO. 3.

[0010] In one embodiment, Kluyveromyces marxianus NBRC1777 is used as the host.

[0011] The second object of the present application is to provide a microbial preparation containing the Kluyveromyces marxianus.

[0012] In one embodiment, the microbial preparation includes, but is not limited to, a direct-vat starter.

[0013] The third object of the present application is to provide a method for improving the ability of Kluyveromyces marxianus to produce D-lactic acid, which integrates a D-ldh gene at the position of the pdc1 gene on the genome of Kluyveromyces marxianus; optionally, the D-ldh gene is driven by a strong promoter P PGK1 controlling expression.

[0014] The fourth object of the present application is to provide a method for producing D-lactic acid, which adds the Kluyveromyces marxianus to a fermentation system for fermentation.

[0015] In one embodiment, the fermentation system contains ammonium sulfate 5 g / L, potassium dihydrogen phosphate 3 g / L, magnesium sulfate heptahydrate 0.5 g / L, glucose 100 g / L; ethylenediaminetetraacetic acid 15 mg / L, zinc sulfate heptahydrate 4.5 mg / L, cobalt chloride hexahydrate 0.3 mg / L, manganese chloride tetrahydrate 1.0 mg / L, copper sulfate pentahydrate 0.3 mg / L, calcium chloride dihydrate 4.5 mg / L, ferrous sulfate heptahydrate 3.0 mg / L, sodium molybdate dihydrate 0.4 mg / L, boric acid 1.0 mg / L, potassium iodide 0.1 mg / L, filtered and sterilized; biotin 0.05 mg / L, calcium pantothenate 1.0 mg / L, nicotinic acid 1.0 mg / L, myo-inositol 25 mg / L, vitamin B1 1.0 mg / L, pyridoxine 1.0 mg / L, p-aminobenzoic acid 0.2 mg / L, uracil 20 mg / L.

[0016] In one embodiment, the inoculation amount is 10-15% (v / v).

[0017] In one embodiment, the inoculation amount is preferably 10%.

[0018] In one embodiment, the fermentation temperature is 35-40°C.

[0019] In one embodiment, the fermentation temperature is preferably 37°C.

[0020] In one embodiment, the fermentation period is 60 h-90 h.

[0021] In one embodiment, the stirring is 300 rpm, and the aeration is 1 vvm.

[0022] In one embodiment, calcium carbonate is added during the fermentation process, and the pH is maintained at 5.0-6.0.

[0023] In one embodiment, glucose is supplemented during the fermentation process.

[0024] In one embodiment, when the glucose content in the medium is reduced to below 10 g / L, glucose is supplemented to maintain the glucose concentration in the fermentation system at 50-60 g / L.

[0025] In one embodiment, the fermentation is carried out by inoculating the activated seed liquid into the fermentation medium; the seed liquid is obtained by culturing the Kluyveromyces marxianus in a seed medium to an OD 600 of 10-15.

[0026] In one embodiment, agar plate culture is carried out before seed culture, which refers to inoculating the strain preserved in a glycerol tube into a YPD solid plate medium, and culturing at 37°C for 1-2 days to obtain single colonies.

[0027] In one embodiment, the fermentation is carried out by inoculating the seed liquid into the fermentation medium at an inoculation amount of 10-15 mL / 100 mL, and culturing at 35-40°C for 60-90 h.

[0028] The present application also protects the use of the Kluyveromyces marxianus, or the microbial inoculant, or the method for producing D-lactic acid, or the method for improving the yield of D-lactic acid in the production of D-lactic acid.

[0029] Advantages:

[0030] The present application uses Kluyveromyces marxianus NBRC1777 as the starting strain, and obtains a Kluyveromyces marxianus with high yield of D-lactic acid through metabolic engineering, which is named FMME-LA01. Under the optimized conditions in a shake flask, the yield of D-lactic acid can reach 140 g / L after 72 h of fermentation, and the yield of D-lactic acid can reach 135 g / L after 70 h of fermentation in a 7.5 L fermenter, the optical purity is greater than 99%, and it has good genetic stability and is suitable for industrial production. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 is the plasmid map of pY26-LhLDH.

[0032] Figure 2 is the plasmid map of pUG6.

[0033] Figure 3 is the fermentation curve of strain FMME-LA01 cultured in a 500 mL shake flask.

[0034] Figure 4 Figure 1 is a fermentation curve chart of strain FMME-LA01 in a 7.5 L fermenter. DETAILED DESCRIPTION

[0035] (1) Culture medium:

[0036] YPD solid medium: peptone 20 g / L, yeast powder 10 g / L, glucose 20 g / L, agar powder 20 g / L.

[0037] Seed medium: containing ammonium sulfate 5 g / L, potassium dihydrogen phosphate 3 g / L, magnesium sulfate heptahydrate 0.5 g / L, glucose 40 g / L; ethylenediaminetetraacetic acid 15 mg / L, zinc sulfate heptahydrate 4.5 mg / L, cobalt chloride hexahydrate 0.3 mg / L, manganese chloride tetrahydrate 1.0 mg / L, copper sulfate pentahydrate 0.3 mg / L, calcium chloride dihydrate 4.5 mg / L, ferrous sulfate heptahydrate 3.0 mg / L, sodium molybdate dihydrate 0.4 mg / L, boric acid 1.0 mg / L, potassium iodide 0.1 mg / L, sterilized by filtration; biotin 0.05 mg / L, calcium pantothenate 1.0 mg / L, nicotinic acid 1.0 mg / L, myo-inositol 25 mg / L, vitamin B1 1.0 mg / L, pyridoxine 1.0 mg / L, p-aminobenzoic acid 0.2 mg / L, uracil 20 mg / L.

[0038] Fermentation medium: containing ammonium sulfate 5 g / L, potassium dihydrogen phosphate 3 g / L, magnesium sulfate heptahydrate 0.5 g / L, glucose 100 g / L; ethylenediaminetetraacetic acid 15 mg / L, zinc sulfate heptahydrate 4.5 mg / L, cobalt chloride hexahydrate 0.3 mg / L, manganese chloride tetrahydrate 1.0 mg / L, copper sulfate pentahydrate 0.3 mg / L, calcium chloride dihydrate 4.5 mg / L, ferrous sulfate heptahydrate 3.0 mg / L, sodium molybdate dihydrate 0.4 mg / L, boric acid 1.0 mg / L, potassium iodide 0.1 mg / L, sterilized by filtration; biotin 0.05 mg / L, calcium pantothenate 1.0 mg / L, nicotinic acid 1.0 mg / L, myo-inositol 25 mg / L, vitamin B1 1.0 mg / L, pyridoxine 1.0 mg / L, p-aminobenzoic acid 0.2 mg / L, uracil 20 mg / L.

[0039] (2) Determination of glucose:

[0040] Pre-treatment of fermentation broth: centrifuge the fermentation broth at 12000 r / min for 10 min to obtain the supernatant. Dilute to an appropriate multiple, and use M-100 biological sensor analyzer to detect the glucose concentration of the fermentation broth.

[0041] (3) Determination of D-lactic acid:

[0042] High performance liquid chromatography: a 10 g / L concentration of D-lactic acid solution was prepared, diluted to 0.2, 0.4, 0.6, 0.8 and 1.0 g / L, and detected by using a high performance liquid chromatograph (HPLC) to obtain the peak time and the peak area corresponding to different concentrations of D-lactic acid. A standard curve was drawn with the concentration of D-lactic acid as the abscissa and the peak area as the ordinate, and a linear regression equation was obtained. The regression coefficient of the linear regression equation should be above 0.99 before it can be used. The instrument was an Agilent high performance liquid chromatograph, the chromatographic column was an Aminex HPX-87H column; the mobile phase was 5 mM dilute sulfuric acid; the flow rate was set to 0.6 mL / min; the detector was a UV detector with a detection wavelength of 210 nm, and the column temperature was 52°C.

[0043] Fermentation broth pretreatment: centrifuge the fermentation broth at 12000 r / min for 10 min to obtain the supernatant. After dilution to an appropriate multiple, the sample was subjected to membrane treatment, and HPLC was used for detection. The obtained peak area was substituted into the linear regression equation, and the obtained result multiplied by the dilution multiple was the concentration of D-lactic acid in the fermentation broth.

[0044] Example 1: Construction of uracil auxotrophic strain

[0045] Kluyveromyces marxianus NBRC1777 was selected as the starting strain for modification. The ura3 gene was first knocked out to construct a strain with uracil auxotrophy. The specific steps were as follows:

[0046] (1) Using the NBRC1777 genome as the template, the primers ura3 up-1 and ura3 up-2 were used to amplify the 500 bp upstream of the ura3 gene, and the primers ura3 down-1 and ura3 down-2 were used to amplify the 500 bp downstream of the ura3 gene; using the plasmid PUG6 as the template, the primers KanMX-1 and KanMX-2 were used to amplify the KanMX gene fragment.

[0047] (2) The three fragments in step (1) were subjected to homologous recombination to construct an expression frame of ura3 upstream homologous arm-KanMX gene-ura3 downstream homologous arm, and the yeast strain NBRC1777 was transformed and plated on YPD plates containing 200 mg / L of G418.

[0048] (3) The positive transformants were selected and sent to the company for sequencing using the primers ura3 up-1 and ura3 down-2 to determine the successful knockout of ura3.

[0049] Example 2: Construction of D-lactic acid-producing recombinant strain

[0050] To reduce the accumulation of ethanol, D-lactate dehydrogenase gene D-ldh (abbreviated as Lh-dldh) from Lactobacillus helveticus was integrated at the site of pyruvate decarboxylase gene pdc1, so as to make the strain produce D-lactic acid while knocking out ethanol. In order to increase the expression of D-lactic acid dehydrogenase, P PGK1 The strong promoter controls Lh-dldh, and the specific steps are as follows:

[0051] (1) Using the genome of NBRC1777 as a template, the primer pdc1 up-1 and pdc1 up-2 were used to amplify the 500bp homologous arm sequence upstream of the pdc1 gene (shown in SEQ ID NO. 1), and the primer pdc1 down-1 and pdc1 down-2 were used to amplify the 500bp homologous arm sequence downstream of the pdc1 gene; the primer PGK-1 and PGK-2 were used to amplify the sequence of the upstream promoter of the pgk gene (shown in SEQ ID NO. 3), and the recombinant plasmid PY26-LhLDH containing the Lh-dldh gene shown in SEQ ID NO. 2 was used as a template, and the primer LDH-1 and LDH-2 were used to amplify the expression frame containing the Lh-dldh gene fragment and ura3.

[0052] (2) The four fragments in step (1) were subjected to homologous recombination to construct the expression frame of pdc1 upstream homologous arm-PGK promoter-Lh-dldh gene-CYC1 terminator-ura3 expression frame-pdc1 downstream homologous arm, and the yeast strain after knocking out ura3 was transformed

[0053] NBRC1777 was coated on YNB plate.

[0054] (3) The positive transformants were selected and sent to the company for sequencing using primers pdc1 up-1 and pdc1 down-2 to determine the successful knockout of pdc1.

[0055] The correct recombinant yeast strain was named FMME-LA01.

[0056] Table 1 Primers used for PCR amplification

[0057]

[0058] Example 3: Shake flask production of D-lactic acid by strain FMME-LA01

[0059] The strain FMME-LA01 was inoculated on a solid YPD plate and cultured at 32-37°C for 20-30h, and after a single colony grew, a single colony was inoculated in 50ml YPD liquid medium, and after 18h of culture, the seed culture medium was inoculated in 50ml YPD liquid medium at an initial OD 600=1 was inoculated into the fermentation medium (500 mL flask containing 100 mL fermentation medium) and fermented at 32-37 °C, 200 rpm for 120 h. The fermentation broth was centrifuged and the supernatant was collected to determine the D-lactic acid content in the fermentation broth by HPLC. The results showed that the lactic acid yield of strain FMME-LA01 reached 140 g / L after 72 h fermentation, the yield was 0.64 g / g glucose, and the production intensity was 1.94 g / L / h.

[0060] Example 4: Passage stability of Kluyveromyces marxianus FMME-LA01

[0061] The Kluyveromyces marxianus FMME-LA01 constructed in Example 2 was prepared into seed liquid according to the method of Example 3, and was subjected to continuous passage culture for 12 generations. The strain after passage culture was inoculated into the fermentation medium and subjected to shake flask fermentation according to the method of Example 3. The fermentation conditions were the same as those of Example 3, and the D-lactic acid yield was determined after fermentation. The D-lactic acid yield after passage culture is shown in Table 2.

[0062] Table 2 D-lactic acid yield of Kluyveromyces marxianus FMME-LA01 after passage culture

[0063]

[0064] Example 5: Fermentation of Kluyveromyces marxianus FMME-LA01 in a 7.5 L fermenter

[0065] (1) Seed activation and culture

[0066] Plate activation: one loop of strain was inoculated from the preservation tube to solid YPD medium, and incubated at 37 °C for 24 h;

[0067] Primary seed culture: a well-grown single colony was picked from the plate and inoculated into a 250 mL flask containing 50 mL YPD medium. The reciprocating shaker was set at 200 rpm and incubated at 37 °C for about 12 h, until the OD600 value of the primary seed liquid was between 5 and 6. 600

[0068] Secondary seed culture: the OD600 value of the primary seed liquid was between 5 and 6. The secondary seed culture was inoculated into a 500 mL flask containing 100 mL seed medium, and the reciprocating shaker was set at 200 rpm and incubated at 37 °C for about 12 h, until the OD600 value of the secondary seed liquid was between 10 and 15. 600

[0069] (2) Fermentation culture

[0070] The recombinant strain was subjected to fermentation culture in a 7.5 L fermenter, and the specific fermentation conditions were as follows:

[0071] ​​Fermentation medium: containing ammonium sulfate 5 g / L, potassium dihydrogen phosphate 3 g / L, magnesium sulfate heptahydrate 0.5 g / L, glucose 100 g / L; ethylenediaminetetraacetic acid 15 mg / L, zinc sulfate heptahydrate 4.5 mg / L, cobalt chloride hexahydrate 0.3 mg / L, manganese chloride tetrahydrate 1.0 mg / L, copper sulfate pentahydrate 0.3 mg / L, calcium chloride dihydrate 4.5 mg / L, ferrous sulfate heptahydrate 3.0 mg / L, sodium molybdate dihydrate 0.4 mg / L, boric acid 1.0 mg / L, potassium iodide 0.1 mg / L, filtered and sterilized; biotin 0.05 mg / L, calcium pantothenate 1.0 mg / L, nicotinic acid 1.0 mg / L, myo-inositol 25 mg / L, vitamin B1 1.0 mg / L, pyridoxine 1.0 mg / L, p-aminobenzoic acid 0.2 mg / L, uracil 20 mg / L.

[0072] The fermentation medium was sterilized at 115°C for 15 min. After sterilization, it was installed to the control console, and the temperature control was turned on. After the temperature cooled to 37°C, inoculation was prepared.

[0073] Inoculation amount: when the seed liquid OD reached 10, the prepared seed liquid was inoculated into the fermentation tank at an inoculation amount of 10%, and the inoculation OD was controlled to be 1;

[0074] Fermentation temperature: during the fermentation process, the temperature control was turned on, and the fermentation temperature was maintained at 37°C;

[0075] Fermentation pH: the pH was adjusted to between 5 and 6 using calcium carbonate;

[0076] Dissolved oxygen conditions: aeration rate 1 vvm, rotation speed 300 rpm;

[0077] During the fermentation process, when the initial glucose in the fermentation medium was reduced to 10 g / L, a glucose solution with a concentration of 800 g / L was added at one time to control the glucose concentration in the reaction system to be 50-60 g / L.

[0078] The fermentation conditions were monitored regularly during the fermentation process, and the results showed that after 70 h of fermentation, the D-lactic acid yield was basically stable, the D-lactic acid yield could reach 136 g / L, the yield was 0.67 g / L glucose, the production intensity was 1.94 g / L / h, and the optical purity of the product was ≥99%.

[0079] Comparative example:

[0080] The specific embodiment is the same as that of Example 2, except that the D-ldh gene from Lactobacillus helveticus is replaced by the D-ldh gene from Leuconostoc mesenteroides, Escherichia coli and Lactobacillus plantarum (the nucleotide sequences are shown in SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6, respectively), respectively, and the fermentation is carried out according to the method of Example 3. The results show that the lactic acid production of the modified strains is 102 g / L, 123 g / L and 97 g / L, respectively, after 72 h, which is lower than that of the strain expressing the ldh gene from Lactobacillus helveticus, indicating that the ldh gene from Lactobacillus helveticus has the highest catalytic efficiency in vivo in the NBRC1777 strain.

[0081] Although the present application has been disclosed with the preferred embodiments as above, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, and therefore the protection scope of the present application should be defined by the claims.

Claims

1. A D-lactic acid producing strain of Kluyveromyces marxianus, characterized in that, The expression of a pdc1 gene is reduced, and a D-ldh gene from Lactobacillus helveticus is expressed; the nucleotide sequence of the pdc1 gene is shown as SEQ ID NO. 1; and the nucleotide sequence of the D-ldh gene is shown as SEQ ID NO.

2.

2. The K. marxianus of claim 1, characterized in that, The D-ldh gene is integrated at the position of the pdc1 gene on the genome.

3. The Kluyveromyces marxianus of claim 1 or 2, characterized in that, The promoter P PGK1 controls the expression of the D-Idh gene.

4. The Kluyveromyces marxianus of any one of claims 1 to 3, characterized in that, The starting strain is Kluyveromyces marxianus NBRC1777.

5. A microbial preparation containing the Kluyveromyces marxianus according to any one of claims 1 to 4.

6. A method for improving the ability of K. marxianus to produce D-lactic acid, characterized in that, The D-ldh gene is integrated at the position of the pdc1 gene on the genome of the Kluyveromyces marxianus.

7. A method for the fermentative production of D-lactic acid, characterized in that The Kluyveromyces marxianus according to any one of claims 1 to 4 is cultured in a medium at 35 to 40°C for 60 to 90 hours.

8. The method of claim 7, wherein, The fermentation is inoculating the activated seed liquid into the fermentation medium; the seed liquid is culturing the K. marxianus in the seed medium to OD 600 10-15.

9. The method according to claim 7 or 8, characterized in that, Glucose is supplemented during the fermentation.

10. Use of the Kluyveromyces marxianus according to any one of claims 1 to 4, the microbial preparation according to claim 5, or the method according to any one of claims 6 to 9 in the production of D-lactic acid.

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