PL226910B1 - New bacterial strain of the Enterobacter kind - Google Patents

Description

Description of the invention

The subject of the invention is a new strain of bacteria of the genus Enterobacter and its use for the production of succinic acid and 2,3-butanediol.

Currently, intensive research is underway around the world to develop effective biotechnological processes that enable the production of industrially important precursors for chemical syntheses using renewable raw materials instead of crude oil. The observed trend is the result of the shrinking global resources of this fossil raw material, its high and unstable prices, as well as the release of huge amounts of carbon dioxide into the atmosphere during the combustion of crude oil and its derivatives. In the case of energy demand, a number of alternative sources of energy acquisition are known, e.g. energy from solar radiation, wind or geothermal sources. When it comes to the production of plastics and other chemicals, the only reasonable way to abandon fossil raw materials seems to be the use of plant biomass and waste materials from its processing, which are ubiquitous on our planet. One of them is glycerol which is a by-product of biodiesel technology. The growing production of glycerol and a nearly 10-fold drop in its price in recent years make it a cheap and easily accessible source of carbon in biotechnological processes. In 2004, the US Department of Energy (DOE) compiled a list of the 12 most desirable chemicals for which an alternative production method should be found (Werpy T, Petersen G. Eds. Top Value Added Chemicals from Biomass. US DoE 2004). Among the compounds presented in the cited report, we can find dicarboxylic acids such as succinic acid, fumaric acid and malic acid.

Microbial production of succinic acid shows the most promising alternative sourcing. At present, only a few microorganisms are known capable of significantly producing succinic acid, in their case either the end product of the fermentation or an intermediate in the Krebs cycle. Among the producers of this acid we can mention the bacteria Actinobacillus succinogenes (Guettler MV, Rumler D, Jain MK. Actinobacillus succinogenes sp. Nov., A novel succinic-acid-producing strain from the bovine rumen. Int J Syst Bacteriol 1999; 49: 207-16 ), Anaerobiospirillium succiniciproducens (Lee PC, Lee WG, Kwon S, Lee SY, Chang HN. Succinic acid production by Anaerobiospirillium succiniciproducens: effects of the H ₂ / CO ₂ supply and glucose concentration. Enzyme Microb Technol 1999; 24: 549-54 ), Mannheimia succiniciproducens (Lee PC, Lee SY, Hong SH, Chang HN. Isolation and characterization of new succinic acid producing bacterium, Mannheimia succiniciproducens MBEL 55E, from bovine rumen. Appl Microbiol Biotechnol 2002; 58: 663-8), Basfia succiniciproducens DD1 (Scholten D, Daegele D. Succinic acid production by a newly isolated bacterium. Biotechnol Lett 2008; 30: 2143-46) and genetically modified Escherichia coli (Vemuri GN, Eiteman MA, Altman E. Succinate production in dual-phase Escherichia coli fermentations depends on the time of transition from aerobic to anaerobic conditions. J Ind Microbiol Biotechnol 2002; 28: 325-32), Corynebacterium glutamicum (Okino S, Noburyu R, Suda M, Jojima T, Inui M, Yukawa H. An efficient succinic acid production process in a metabolically engineered Corynebacterium glutamicum strain. Appl Microbiol Biotechnol 2008; 81: 459-64) and Yarrowia lipolytica yeast (Yuzbashev TV, Yuzbasheva EY, Sobolevskaya TI, Laptev IA, Vybornaya TV, Larina AS, Matsui K, Fukui K, Sineoky SP. Production of succinic acid at low pH by a recombinant strain of the aerobic yeast Yarrowia lipolytica. Biotechnol Bioeng 2010; 107: 673-82) and Saccharomyces cerevisiae (Raab AM, Gebhardt G, Bolotina N, Weuster-Botz D, Lang C. Metabolic engineering of Saccharomyces cerevisiae for the biotechnological production of succinic acid. Metab Eng 2010; 12: 518-25). Often a big problem is the presence of by-products in post-fermentation fluids or the need to use rich, and thus expensive fermentation broths that increase the cost of microbial production of succinic acid and its purification. For the above reasons, it is necessary to search for new microorganisms capable of effective production of succinic acid using cheap, non-competing with food ingredients, sources of carbon and energy, e.g. glycerol. Currently, the natural producers of succinic acid on glycerol are Anaerobiospirilium succiniciproducens (Lee et al., 2001) and Basfia succiniciproducens DD1 (Scholten and Daegele, 2008), in which the presence of succinic acid in the post-culture filtrates was found, but in concentrations too low to be accepted by the technology. succinic acid.

PL 226 910 B1

The subject of the invention is a new bacterial strain Enterobacter sp. LU1 deposited in the International Collection of Industrial Microbial Cultures of the Institute of Agricultural and Food Biotechnology under the number KKP 2050p.

The essence of the new strain is its use as a production strain in the microbial production of succinic acid on a liquid bacterial medium containing glycerol as a carbon source.

The essence of the new strain is also its use as a production strain in the processes of microbial production of 2,3-butanediol on a liquid bacterial medium containing glucose as a carbon source.

The bacterial strain Enterobacter sp. LU1 No. KKP 2050p has the following nucleotide sequence of the DNA region encoding the 16S rRNA, the sequence of which determines the genus identity of the bacteria:

CGGTAACAGGAAGCAGCTTGCTGCTTCGCTGACGAGTGGCGGACGGGTGAGTAATGTCTG

GGAAACTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTC

GCAAGACCAAAGAGGGGGACCTTCGGGCCTCTTGCCATCGGATGTGCCCAGATGGGATTA

GCTAGTAGGTGGGGTAACGGCTCACCTAGGCGACGATCCCTAGCTGGTCTGAGAGGATGA

CCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATA

TTGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTATGAAGAAGGCCTTCGGGT

TGTAAAGTACTTTCAGCGGGGAGGAAGGCGATAAGGTTAATAACCTTGTCGATTGACGTT

ACCCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCA

AGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTCTGTCAAGTCGGATGTG

AAATCCCCGGGCTCAACCTGGGAACTGCATTCGAAACTGGCAGGCTAGAGTCTTGTAGAG

GGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGC

GAAGGCGGCCCCCTGGACAAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGG

ATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCGACTTGGAGGTTGTGCCCTTGAG

GCGTGGCTTCCGGAGCTAACGCGTTAAGTCGACCGCCTGGGGAGTACGGCCGCAAGGTTA

AAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATG

CAACGCGAAGAACCTTACCTACTCTTGACATCCAGAGAACTTTCCAGAGATGGATTGGTG

CCTTCGGGAACTCTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTT

GGGTTAAGTCCCGCAACGAGCGCAACCCTTATCCTTTGTTGCCAGCGGTTAGGCCGGGAA

CTCAAAGGAGACTGCCAGTGATAAACTGGAGGAAGGTGGGGATGACGTCAAGTCATCATG

GCCCTTACGAGTAGGGCTACACACGTGCTACAATGGCGCATACAAAGAGAAGCGACCTCG

CGAGAGCAAGCGGACCTCATAAAGTGCGTCGTAGTCCGGATTGGAGTCTGCAACTCGACT

CCATGAAGTCGGAATCGCTAGTAATCGTGGATCAGAATGCCACGGTGAATACGTTCCCGG

GCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGTAGCTTA

ACCTTCGGGAGGGCGCTTACCACTTTGTGATTCATGACTGGGG

PL 226 910 B1

The taxonomic affiliation of the new strain to the species of the Enterobacter cloacae complex was determined on the basis of the API 20E biochemical tests. Sequencing of the rpoB gene and API 50 CH tests show the greatest taxonomic similarity to the species Enterobacter hormaechei subsp. Steigerwaltia. The nucleotide sequence of the DNA region encoding the β subunit of bacterial RNA polymerase is as follows:

GTCTCTGGGCGATCTGGATACCCTGATGCCTCAGGATATGATTAACGCGAAGCCGATTTC

TGCGGCAGTGAAAGAGTTCTTCGGTTCCAGCCAGCTGTCTCAGTTCATGGACCAGAACAA

CCCGCTGTCTGAGATCACGCACAAACGTCGTATCTCCGCACTCGGCCCAGGCGGTCTGAC

CCGTGAACGCGCAGGCTTTGAAGTTCGAGACGTACACCCGACTCACTACGGTCGCGTATG

TCCAATCGAAACGCCTGAAGGTCCAAACATCGGTCTGATCAACTCCCTGTCCGTGTACGC

ACAGACGAACGAATACGGTTTCCTCGAGACCCCGTATCGTAAAGTGACCGACGGTGTTGT

TACTGACGAAATTCATTACCTGTCTGCTATTGAAGAAGGCAACTACGTTATCGCTCAGGC

GAACTCCAACCTGGATGACGAAGGCCACTTTGTAGAAGATCTGGTTACCTGCCGTAGCAA

AGGCGAATCCAGCTTGTTCAGCCGCGACCAGGTTGACTACATGGACGTATCCACCCAGCA

GGTGGTATCCGTCGGTGCGTCCCTGATCCCGTTCCTGGAACACGATGACGCCAACCGTGC

ATTGATGGGTGCGAACATGCAACGTCAGGCCGTTCCAACTCTGCGTGCTGATAAGCCGCT

GGTTGGTACCGGTATGGAACGTGCTGTTGCCGTTGACTCTGGTGTTACAGCAGTTGCTAA

GCGTGGCGGTACCGTTCAGTACGTTGACGCATCCCGTATCGTTATCAAAGTTAACGAAGA

CGAGATGTATCCGGGCGAAGCGGGTATCGACATCTACAACCTGACCAAATACACCCGTTC

TAACCAGAACACCTGTATCAACCAGATGCCATGTGTGTCTCTGGGTGAGCCAGTTGAGCG

CGGCGACGTGCTGGCAGACGGTCCGTCCACCGACCTCGGTGAACTGGCGCTCGGTCAGAA

CATGCGCGTAGC

The API 50 CH tests also revealed the fermentation abilities of the new strain against the following saccharides and their derivatives: glycerol, D-arabinose, L-arabinose, D-ribose, D-xylose, D-adonitol, D-galactose, D-glucose, D -fructose, D-mannose, L-rhamnose, D-mannitol, D-sorbitol, Methyl-aD-glucopyronazide, N-acetylglucosamine, arbutin, esculin (weak reaction), salicin, D-cellobiose, D-maltose, D-lactose , D-melibiosis, D-sucrose, D-trehalose, D-rafmose, gentiobiose, D-fucose, L-fucose, D-arabitol, potassium 2-ketogluconate.

The newly isolated bacteria are 1 µm long gram-negative rods and do not produce spores.

The new strain is characterized by the ability to produce succinic acid on a medium where the carbon source is glycerol. Succinic acid, in a concentration of about 2 g / l, is in this case the main product of fermentation. An additional product of metabolic transformations is acetic acid present in a slight amount of about 0.4 g / l. The possible presence of other metabolic products is at a level below their detection limit in the standard method of HPLC analysis using the RI detector in such cases.

The new strain also has the ability to produce 2,3-butanediol on a medium where the carbon source is glucose. In this case, 2,3-butanediol is one of the many fermentation products.

The strain according to the invention was isolated from the content of the goat's rumen using decimal dilutions of the analyzed content in PBS (0.145 M NaCl, 0.01 M Na ₂ HPO ₄ ). The resulting dilutions were plated on isolation media. The medium used was in g / l: glycerol 20; galacturonic acid 10; yeast extract 10; Na2HPO4 x 12H2O 0.78; NaH2HPO4

PL 226 910 B1 x H2O 1.16; MgCl2 x 6H2O 0.2; CaCl2 0.15; NaCl 1; MgCO3 15; Bacteriological agar 15. Single colonies grown on the plates after 48 hours of anaerobic incubation at 37 ° C were reductively screened into fresh media three times to purify the isolated bacteria. Then the purified isolate was cultured for 48 hours in cerebral cardiac broth (BHI medium: glucose 2 g / l; cerebral-cardiac extract 17.5 g / l; peptone 10 g / l; Na2HPO4 2.5 g / l; NaCl 5 g / l; pH 7.2). After completion of the culture, the strain was protected in 20% glycerol and stored at -80 ° C.

P R Z Y K Ł A D 1

The Enterobacter sp. LU1 strain was grown on a liquid medium with the following composition in g / l: glycerol 20; yeast extract 10; Na2HPO4 x 12H2O 0.78; NaH2HPO4 x H2O 1.16; MgCl2 x 6 H2O 0.2; CaCl2 0.15; NaCl 1; MgCO3 15; at 37 ° C, shaking 160 rpm, in sealed 20 ml injection bottles. When inoculating the microorganism, 2 μΐ of a vitamin solution was also added to the nutrient medium (vitamin B ₁₂ 0.01 mg / ml, biotin 0.2 mg / ml, thiamine 0.5 mg / ml, riboflavin 0.5 mg / ml, niacin 0.5 mg / ml, calcium pantothenate 0.5 mg / ml, p-aminobenzoate 0.5 mg / ml, vitamin B6 1 mg / ml, lipoic acid 0.5 mg / ml). After 96 hours, the cultured cells were separated from the medium by centrifugation (5 min. 7800 xg, 4 ° C), the supernatant obtained in this way was diluted twice, and then deproteinized with Carrez salt (salt I - 15% rr K4 [Fe (CN) ] ₆ x 3H ₂ O; salt II - 30% yr ZnSO ₄ x 7H ₂ O). The sample prepared in this way was analyzed for the presence of fermentation products by means of HPLC, using an Aminex HPX-87H organic acid column and a refractive index detector (Knauer). Elution was carried out with 30 mM sulfuric acid at 42 ° C, with an eluent flow rate of 0.3 ml / min, and 2.19 g / L of succinic acid was present. The exact results of the study are presented in the Table.

P R Z Y K Ł A D II

The Enterobacter sp. LU1 strain was grown in the liquid medium with the following composition in g / l: glucose; yeast extract 10; Na2HPO4 x 12H2O 0.78; NaH2HPO4 x H2O 1.16; MgCl2 x 6 H2O 0.2; CaCl2 0.15; NaCl 1; MgCO3 15; at 37 ° C in 20 ml sealed injection bottles. When inoculating the microorganism, 2 μl of vitamin solution (vitamin B12 0.01 mg / ml, biotin 0.2 mg / ml, thiamine 0.5 mg / ml, riboflavin 0.5 mg / ml, niacin 0.5 mg) was also added to the nutrient medium. / ml, calcium pantothenate 0.5 mg / ml, p-aminobenzoate 0.5 mg / ml, vitamin B6 1 mg / ml, lipoic acid 0.5 mg / ml). After 96 hours, the cultured cells were separated from the medium by centrifugation, and the supernatant obtained in this way was diluted two times, and then deproteinized with Carrez salt (salt I - 15% rr K4 [Fe (CN)] 6 x 3 H2O; salt II - 30 % yr ZnSO4 x 7 H2O). The sample prepared in this way was analyzed for the presence of fermentation products, determining the presence of 2,3-butanediol in the amount of 4.06 g / l. The exact results of the study are presented in the Table. The chromatographic separation of the deproteinized digestate was carried out on an Aminex HPX-87H column at 42 ° C and a flow of 0.3 ml / min. The eluent used was 30 mM sulfuric acid.

T a b e l a. Products of metabolism of the Enterobacter sp. LU1 strain

Example (source coal) Production Succinic acid 2.3- -butanediol Acid form Acid acetic Acid lactic acid Ethanol [g / l] [g / l] [g / l] [g / l] [g / l] [g / l] AND (Glycerol) 2.19 - - 0.46 - - II (Glucose) 0.69 4.06 1.5 1.34 1.21 1.76

Claims (3)

Patent claims 1. A new bacterial strain Enterobacter sp. LU1 deposited in the International Collection of Cultures of Industrial Microorganisms of the Institute of Agricultural and Food Biotechnology under the number KKP 2050p. 2. Use of the bacterial strain Enterobacter sp. LU1 No. KKP 2050p in the microbial production of succinic acid on a liquid bacterial medium containing glycerol as a carbon source. 3. Application of the bacterial strain Enterobacter sp. LU1 No. KKP 2050p in the microbial production of succinic acid and 2,3-butanediol on a liquid bacterial medium containing glucose as a carbon source.