RU2752896C1 - Strain of yeast schizosaccharomyces pombe, producing l-lactic acid, containing genes of three different heterologous lactate dehydrogenases in chromosome - Google Patents

Abstract

FIELD: biotechnology.SUBSTANCE: invention relates to biotechnology. A recombinant strain of Schizosaccharomyces pombe VKPM Y-4822, which is a producer of L-lactic acid, is proposed. The strain has a deleted pyruvate decarboxylase PDC1 gene and carries lactate dehydrogenase genes from Lactobacillus pentosus, Lactobacillus acidophilus and Lactobacillus helveticus in the chromosomal DNA. The strain produces L-lactic acid in an amount of 125 g/l for 72 hours of cultivation in a fermenter under aerobic conditions.EFFECT: invention makes it possible to expand the arsenal of recombinant microorganisms that produce L-lactic acid.1 cl, 2 dwg, 3 ex

Description

The invention relates to microbiology and biotechnology and relates to a recombinant strain of the yeast Schizosaccharomyces pombe - a producer of L-lactic acid.

Lactic acid is a platform for the production of various chemical compounds. Over the past decade, interest has increased in the production of a biodegradable polymer of lactic acid polylactide, which occupies a leading position in terms of production among bioplastics. Currently, polylactide is increasingly used, in particular, for the production of environmentally friendly packaging products and biodegradable implants, including using a 3D printer.

In this regard, the development of effective methods for the production of lactic acid is an urgent task.

Traditionally, to obtain lactic acid, microbiological methods based on the cultivation of bacterial strains of lactic acid bacteria are used.

The disadvantage of bacterial strains producing lactic acid is their sensitivity to low pH values and high concentrations of lactic acid in the culture liquid (CL), which leads to a slowdown in culture growth, a decrease in the rate of lactic acid synthesis and, as a consequence, to the premature completion of the fermentation process.

As an alternative to bacterial strains, yeast strains capable of growth and fermentation under conditions of high acidity are widely used.

Yeast strains producing L-lactic acid are obtained by expressing heterologous genes of L-lactate dehydrogenase (L-LDH) in yeast cells, mainly from lactic acid bacteria. In this case, different types of yeast are used as a recipient.

Yeast strains producing L-lactic acid in quantities comparable to the production level were obtained from the yeast Saccharomyces cerevisiae.

The Saccharomyces cerevisiae SP7 strain [Biotechnol. Bioeng. 2015; 112: 751-758], in which the genes PDC1, CYB2 and GPD1 are replaced by the genes of lactate dehydrogenase from Pelodiscus sinensis, and the genes NDE1 and NDE2, which are responsible for extracellular redox balance, are deleted, produces L-lactic acid in the amount of 117 g / L CL in a fermenter on a medium with yeast extract and peptone, while maintaining the pH at 3.5 throughout the entire cultivation process.

Known acid-fast strain Saccharomyces cerevisiae [Metab. Eng. 2016; 35: 38-45], containing heterologous genes of lactate dehydrogenases from Pelodiscus sinensis japonicas and Bos taurus, which produces L-lactic acid in an amount of 142 g / L for 40 hours of cultivation in a fermenter. The disadvantage of this strain - auxotroph for uracil, tryptophan, leucine and histidine, is the need to cultivate it on a medium containing yeast extract, a mixture of amino acids and vitamins, which leads to a significant increase in the cost of the final product.

The yeast Schizosaccharomyces pombe [RU 2268304] is also highly resistant to low pH values and high concentrations of lactic acid.

Known [RU 2650669] strain Schizosaccharomyces pombe VKPM Y-4224, obtained by sequential three-fold introduction of the gene of lactate dehydrogenase from Lactobacillus plantarum into the chromosome of S. pombe yeast with inactivated gene of alcohol dehydrogenase, producing in an amount of 100 g / l of culture in aerobic conditions.

The technical problem to be solved by the claimed invention is the expansion of the arsenal of recombinant microorganisms that produce L-lactic acid.

The technical result of the claimed invention is a recombinant strain of the yeast Schizosaccharomyces pombe Sp-MK6 VKPM Y-4822 with a deleted pyruvate decarboxylase gene PDC1, which carries heterologous genes of lactate dehydrogenases from Lactobacillus pentosus, Lactobacillus acidophilus Lactobacillus acid Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus Lactobacillus

The Schizosaccharomyces pombe Sp-MK6 VKPM Y-4822 strain was obtained by introducing the following modifications into the parental S. pombe MM-11 strain containing the L-LDH gene from Lactobacillus pentosus and the KanMX gene providing resistance to geneticin (G418) of the following modifications:

- sequential deletion of the KanMX, URA4 and PDC1 genes from the chromosomal DNA of the S. pombe MM-11 strain;

- integration into the chromosomal DNA of a cassette containing the URA4 gene and the lactate dehydrogenase gene from Lactobacillus acidophilus;

- integration into the chromosomal DNA of a cassette containing the KanMX gene and the lactate dehydrogenase gene from Lactobacillus helveticus;

- deletion of the KanMX gene from chromosomal DNA.

The invention is illustrated by the following graphical figures:

FIG. 1. Transformation cassette containing the URA4 gene and the lactate dehydrogenase gene from Lactobacillus acidophilus.

FIG. 2. A transformation cassette containing the KanMX gene and the lactate dehydrogenase gene from Lactobacillus helveticus.

Example 1. Construction of the inventive strain

1.1. Deletion of the selective marker KanMX at lox sites from the chromosomal DNA of the S. pombe MM-11 strain

Deletion of the KanMX selectable marker from the chromosomal DNA of the S. pombe MM-11 strain is carried out by recombination at lox sites using Cre recombinase. [Biosci Biotechnol Biochem 2004; 68 (3): 545-550; Yeast 2006; 23 (11): 813-823].

Strain MM-11 is transformed with a plasmid containing the cre-recombinase gene under the control of the nmt1 promoter with a selective marker Hph, which provides resistance to hygromycin. Transformants are selected on YES medium of composition (wt.%): Yeast extract - 0.5; glucose - 3.0; the rest is water, with hygromycin (50 μg / ml). A single colony is inoculated into a tube with thiamine-free PMG mineral medium [Pombe Glutamate medium, https://domsife.usc.edu/pombenet/media/]. The tube is incubated on a shaker (250 rpm) at a temperature of 30 ° for 24 hours. The culture is inoculated to separate colonies on a plate with YES medium. Individual colonies are transferred using a replicator onto media YES, YES with geneticin (100 μg / ml) and YES with hygromycin (50 μg / ml). As a result, a colony sensitive to geneticin and hygromycin is selected and named Sp-MK1.

1.2. Deletion of the URA4 gene from the chromosome of the S. pombe Sp-MK1 strain

Prepare competent cells of the S. pombe Sp-MK1 strain, which are transformed by electroporation with a linear plasmid containing the yeast selectable marker KanMX and the Up- and Dn- sequences combined by PCR upstream and downstream of the URA4 gene with regulatory elements.

The selection of transformants is carried out on agar medium YES with geneticin (100 μg / ml) for 5 days at a temperature of 30 ° C.

The T1 transformant is selected with the specified insertion site of the linear plasmid in the Up arm of the URA4 gene.

Deletion of the URA4 and KanMX ws genes of the chromosomal DNA of the T1 transformant was carried out on the PMG mineral medium with uracil (200 μg / ml) with the addition of 5-fluoroorotic acid (1 wt%) according to the method [https://domsife.usc.edu/pombenet/ drugs /]. Deletion of the URA4 gene and the KanMX selectable marker occurs by direct repeats resulting from the insertion of the plasmid, by the mechanism of homologous recombination. Chromosomal DNA is isolated from individual colonies and analyzed for the presence of a deletion of the URA4 and KanMX genes by PCR. Selected strain S. pombe Sp-MK2, which contains the construct pCMV-LDHpent Δura4.

1.3. Deletion of the PDC1 gene from the chromosomal DNA of the S. pombe Sp-MK2 strain

Competent cells of the S. pombe Sp-MK2 strain are transformed by electroporation with a linear plasmid containing the yeast selection marker URA4 and the Up- and Dn- sequences combined by PCR, located above and below the structural part of the PDC1 gene.

The selection of transformants is carried out on agar medium PMG for 5 days at a temperature of 30 ° C.

Selected transformant T2 with a predetermined insertion site of the linear plasmid in the Up-arm of the PDC1 gene.

Next, the URA4 and PDC1 genes are deleted from the chromosomal DNA of the T2 transformant as described in clause 1.2. Chromosomal DNA is isolated from individual colonies and analyzed for the presence of a deletion of the URA4 and PDC1 genes by PCR. Selected strain S. pombe Sp-MK3, which contains the construct pCMV-LDHpent Δura4 Δpdc1.

1.4. Introduction of the L-LDH gene of lactate dehydrogenase from Lactobacillus acidophilus into the chromosomal DNA of S. pombe Sp-MK3 strain

The introduction of the L-LDH gene of lactate dehydrogenase from Lactobacillus acidophilus into the chromosomal DNA of the Sp-MK3 strain is carried out by transformation with a cassette (Fig. 1) containing the L-LDH gene from Lactobacillus acidophilus, the transcription of which is under the control of the pHsp9 promoter and the yeast transcription terminator tbGH the selective marker is the uracil gene URA4, flanked by direct dr repeats and providing prototrophy for uracil.

The selection of transformants is carried out on agar medium PMG for 5 days at a temperature of 30 ° C.

Selected transformants with the insertion of a cassette containing the L-LDH gene from Lactobacillus acidophilus into the chromosomal DNA of S. pombe.

The transformants are cultured in a liquid nutrient medium to determine the production of L-lactic acid.

The inoculum culture is grown in test tubes (50 ml) with 3 ml of YPD liquid nutrient medium of the composition (wt%): yeast extract - 1.0; peptone - 1.5; glucose - 2.0; the rest is water, at 30 ° C for 24 hours on a rocking chair (250 rpm). The inoculum is introduced into the fermentation medium in a ratio of 1/10 vol. The cultivation is carried out at 30 ° C on a shaker (250 rpm) in a fermentation medium of PS with the composition (wt%): corn extract - 2, potassium phosphate buffer with pH 6.0 - 5, the rest is water, with the addition of glucose (20 wt%). .%) in test tubes (50 ml) with a working volume of 5 ml. Fermentation is continued for 72 hours. The concentration of L-lactic acid in the CL is determined according to the HPLC method [Acta Biotechnol. 1990; 10 (5): 459-468]. Based on the results of fermentation, the most productive strain S. pombe Sp-MK4 is selected, which contains the pCMV-LDHpent Δpdc1 pHsp9-LDHaci URA4 construct.

1.5. Introduction of the L-LDH lactate dehydrogenase gene from Lactobacillus helveticus into the chromosomal DNA of S. pombe Sp-MK4 strain

Introduction of the L-LDH gene of lactate dehydrogenase from Lactobacillus helveticus into the chromosomal DNA of S. pombe Sp-MK4 strain is carried out by transformation with a cassette (Fig. 2) containing the L-LDH gene from Lactobacillus helveticus, the transcription of which is under the control of the pHsp9 promoter tbGH transcription ... As a selectable marker, the gene for resistance to geneticin KanMX flanked by sites lox66 and lox71 is used, the transcription of which is under the control of the pTEF promoter and the tTEF transcription terminator.

The selection of transformants is carried out on agar medium YES with geneticin (G418) in an amount of 100 μg / ml for 5 days at a temperature of 30 ° C.

Selected transformants with the insertion of a cassette containing the L-LDH gene from Lactobacillus helveticus into the chromosomal DNA of S. pombe.

The transformants are cultivated in a liquid nutrient medium to determine the production of L-lactic acid, as described in paragraph 1.4, and the most productive transformant Sp-MK5 is selected, which contains the construct pCMV-LDHpent Δpdc1 pHsp9-LDHaci URA4 pHsp9-LDHhelv KanMX.

1.6. Obtaining the claimed strain by deleting the selective marker KanMX at lox sites from the chromosomal DNA of the S. pombe Sp-MK5 strain

Deletion of the selectable marker KanMX at lox sites from the chromosomal DNA of the S. pombe Sp-MK5 strain is carried out as described in clause 1.1.

A colony sensitive to genicin and hygromycin is selected. The strain was named S. pombe Sp-MK6 and contains the construct pCMV-LDHpent Δpdc1 pHsp9-LDHaci URA4 pHsp9-LDHhelv.

As a result, a strain is obtained in which the PDC1 pyruvate decarboxylase gene is deleted, and the genes of lactate dehydrogenases L-LDH from Lactobacillus pentosus, Lactobacillus acidophilus and Lactobacillus helveticus are integrated into the chromosomal DNA. The strain is capable of producing L-lactic acid.

The strain was deposited at the Bioresource Center All-Russian Collection of Industrial Microorganisms (BRC VKPM) NRC "Kurchatov Institute" - GosNIIgenetics (117545 Moscow, 1st Dorozhniy proezd, 1) as Schizosaccharomyces pombe VKPM Y-4822.

The strain is characterized by the following features.

Cultural and morphological characteristics

When cultured at a temperature of 30 ° C for 72 h on agar medium PMG, the culture forms colonies of a white or slightly creamy shade, rounded, convex, with a smooth edge.

When cultured in a liquid YES medium for 2 days, the cells are rod-shaped and 3-4 µm in diameter and 7-14 µm in length. The precipitate is forming.

Vegetative reproduction - cell division in half.

Physiological and biochemical signs

The culture is capable of fermenting glucose, sucrose, maltose and raffinose. Not capable of fermenting galactose, lactose and melibiose. Assimilates sucrose, maltose, raffinose as the only carbon source. Does not assimilate galactose, cellobiose, trehalose, lactose, xylose, arabinose, ribose, rhamnose, erythritol, ribitol, mannitol, starch, succinic, citric acids, inositol. The culture does not assimilate nitrates, is unable to grow on a medium without vitamins.

Optimal conditions for maintaining the strain

Temperature 30 ° С, pH 6, complete yeast medium YES.

The strain is stored in lyophilized form or in a liquid YES medium with glycerol (2 wt.%) At -70 ° C.

The inventive strain Schizosaccharomyces pombe Sp-MK6 VKPM Y-4822 retains the ability to form L-lactic acid after 10 successive passages on a complete medium.

Example 2. The level of production of L-lactic acid during the cultivation of the claimed strain in a test tube

The Schizosaccharomyces pombe Sp-MK6 strain is cultured in a liquid nutrient medium to determine the level of L-lactic acid production. The original strain of Schizosaccharomyces pombe MM-11 was used as a control.

The inoculum culture is grown in test tubes (50 ml) with 3 ml of YPD liquid nutrient medium for 24 h on a shaker (250 rpm) at 30 ° C. The inoculum is introduced into the fermentation medium in a ratio of 1/10 vol. The cultivation is carried out in a FS medium at 30 ° C on a shaker (250 rpm) in 50 ml test tubes with a working volume of 5 ml for 72 hours. The concentration of L-lactic acid in the CL is determined according to the HPLC method.

The production of L-lactic acid in the Schizosaccharomyces pombe Sp-MK6 strain is 102 g / L, in the control Schizosaccharomyces pombe MM-11 strain - 60 g / L.

Example 3. The level of production of L-lactic acid during the cultivation of the inventive strain in a fermenter

The cell culture of strain Sp-MK6 is grown on plates with agar YPD medium for 48 hours at 30 ° C. The resulting biomass is resuspended in sterile distilled water. YPD liquid inoculum is used to prepare inoculum in shake flasks. Cell suspension is added to two shaker flasks with a volume of 750 ml with a working volume of 50 ml of liquid inoculum medium YPD until an optical density (OD ₆₀₀ ) of 0.1 is obtained. The flasks are incubated on a shaker at 220 rpm and a temperature of 30 ° C until the culture reaches an optical density of 8.0.

To carry out the main process of biosynthesis in the fermenter KF-103 (LLC-firm "Prointech") use the medium of the following composition (wt.%): K ₂ NRO ₄ - 0.57; KH ₂ PO ₄ - 0.34; corn extract - 2.0; glucose - 19.0; antifoam agent Breox - 0.1; water is the rest.

The culture grown in shaking flasks is introduced into the fermenter until the starting optical density is 2.0. The cultivation is carried out in a fermenter with a volume of Zle with an initial working volume of 1 l at 29 ° C; initial stirring 300 rpm and aeration - 1.0 l / min. The pO ₂ value is maintained at 20.0% (of the saturation of the medium with air) using cascade control of the stirrer rotation speed. pH = 5.0 is maintained by titration with 25% aqueous ammonia solution during the first 24 hours of cultivation, then the titration is turned off.

After 24 h of cultivation, glucose is sterilely added to the CL up to the initial concentration, and cultivation is continued up to 72 h.

The Schizosaccharomyces pombe Sp-MK6 VKPM Y-4822 strain produces L-lactic acid in an amount of 125 g / L for 72 hours of cultivation in a fermenter under aerobic conditions. Conversion 56%, synthesis rate 1.7 g / L / h, final pH = 2.7.

Thus, a highly productive prototrophic strain Schizosaccharomyces pombe Sp-MK6 VKPM Y-4822 was constructed, capable of producing L-lactic acid at acidic pH values in a poor medium with corn extract (waste of starch-syrup and glucose production). The resulting strain does not possess antibiotic resistance.

Claims (1)

Recombinant strain of yeast Schizosaccharomyces pombe VKPM Y-4822 with a deleted pyruvate decarboxylase gene PDC1, carrying genes for heterologous lactate dehydrogenases from Lactobacillus pentosus, Lactobacillus acidophilus.

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