Patent Application
20130071888
This disclosure relates to a method for producing 1,5-pentanediamine by the fermentation method.
Polyamide (PA) is a group of important polymers used as raw materials for a series of special plastics to be used in the automobile industry, sports industry and lifestyle industry, and diamines are important raw material monomer components for the polyamides. Diamines are condensed with dicarboxylic acids to form various polymers, and the properties of the polymers vary depending on the chain lengths of the diamines and the dicarboxylic acids.
Conventionally, diamines are chemically produced from materials derived from petroleum via dicarboxylic acids at an intermediate stage, or produced by chemical decarboxylation reaction of amino acids (Suyama and Kane. Yakugaku Zasshi (1965), Vol. 85, pp. 513-533). In consideration of sharp rise in oil prices, the production methods are preferably immediately switched to methods based on biotechnological processes such as fermentation, wherein renewable resources are utilized to synthesize diamines.
In view of this, recent interest has focused on methods for producing 1,5-pentanediamine, which is a diamine having 5 carbon atoms, by fermentation. 1,5-Pentanediamine is also called cadaverine, and is a compound which can be used as raw material monomers for polyamides. 1,5-Pentanediamine is a polyamine which ubiquitously exists in the living body and its biosynthetic system is being elucidated (see Celia White Tabor and a colleague. Microbiological Reviews (1985), Vol. 49, pp. 81-99). In a part of its biosynthetic pathway, L-lysine decarboxylase (hereinafter referred to as LDC), which catalyzes decarboxylation of L-lysine, is known to be involved.
Conventional methods for producing 1,5-pentanediamine by fermentation are based on introduction of an LDC gene to a microorganism, and examples of the methods include a production method by fermentation in a recombinant E. coli (see JP 2002-223770 A), method by further enhancing the lysine production capacity of a coryneform bacterium, which is a lysine-producing microorganism (see JP 2004-222569 A), method by blocking the 1,5-pentanediamine degradation system (see Japanese Translated PCT Patent Application Laid-open No. 2009-531042), and method by supplying lysine decarboxylase by an autonomously replicating vector (see Tateno et al. Appl Microbiol Biotechnol (2009), 81(1), pp. 115-121).
However, there are many problems to be solved in the methods for producing 1,5-pentanediamine by fermentation, and examples of such problems include by-production of lysine in cases of fermentation using a microorganism prepared by introduction of an LDC gene to a coryneform bacterium, which is a lysine-producing microorganism (see Mimitsuka et al. Biosci Biotechnol Biochem (2007), 71(9), pp. 2130-2135). That is, there has been a problem that the culture supernatant contains a large amount of unreacted lysine, which is the immediate precursor of 1,5-pentanediamine in its biosynthesis. Such by-production of lysine prevents improvement of the fermentation yield of 1,5-pentanediamine even with successful production of its precursor, which has been economically problematic. Further, since, in JP '042 and Tateno et al, the LDC gene is supplied by an autonomously replicating vector, the culture needs to be carried out in the presence of an expensive antibiotic or the like. Therefore, use of a microorganism having an LDC gene in its chromosome is demanded for low-cost fermentation production of 1,5-pentanediamine on an industrial scale.
It could therefore be helpful to provide a method for producing 1,5-pentanediamine by fermentation using a microorganism which has an LDC gene in its chromosome and which shows suppressed by-production of L-lysine.
We discovered that by-production of L-lysine can be suppressed by using a coryneform bacterium which has an LDC gene in its chromosome and which maintains a specific activity of lysine decarboxylase of not less than 50 mU/mg protein during culturing for fermentation production of 1,5-pentanediamine. We thus provide:
The lysine decarboxylase (LDC) herein means an enzyme that can convert L-lysine as a substrate to 1,5-pentanediamine by decarboxylation reaction, and may also have another/other enzymatic action(s).
The LDC is not restricted, and is preferably derived from Bacillus halodurans, Bacillus subtilis, Escherichia coli, Selenomonas ruminamtium, Vibrio cholerae, Vibrio parahaemolyticus, Streptomyces coelicolor, Streptomyces pilosus, Eikenella corrodens, Eubacterium acidaminophilum, Salmonella typhimurium, Hafnia alvei, Neisseria meningitidis, Thermoplasma acidophilum or Pyrococcus abyssi. The LDC is more preferably derived from E. coli.
Specific examples of the gene encoding lysine decarboxylase include the genes encoding lysine decarboxylase derived from the above-described organisms. The base sequence of the gene may be redesigned depending on the codon usage of the microorganism used. The base sequences encoding lysine decarboxylase derived from the above-described organisms are registered in a database (GenBank). Among these, the gene derived from E. coli, which has the base sequence shown in SEQ ID NO:1, is preferred.
The specific activity of LDC is described below. When the amount of LDC that produces 1 μmol of 1,5-pentanediamine per minute is defined as 1 unit (U), the LDC activity is represented by Equation 1 below:
By this, the activity per unit amount of protein, that is, the specific activity, of LDC can be calculated according to Equation 2 below:
The specific activity of LDC means the specific activity per total protein of the coryneform bacterium, and can be calculated by measuring the specific activity using total protein extracted from the coryneform bacterium. The operation of measurement is described in the Example below more specifically.
The method of extraction of protein from the coryneform bacterium is not restricted, and may be a method wherein bacterial cells are homogenized by sonication, method wherein bacterial cells are homogenized using glass beads with stirring by vortexing or the like, or method wherein bacterial cells are homogenized under high pressure; or a combination of these methods. The method is preferably a method using glass beads.
The coryneform bacterium having a gene encoding lysine decarboxylase in its chromosome has an LDC specific activity of not less than 50 mU/mg protein, preferably has an LDC specific activity of not less than 80 mU/mg protein, more preferably has an LDC specific activity of not less than 180 mU/mg protein. Further, as described in the Example below, even in cases where the bacterium has an LDC specific activity of not less than 50 mU/mg protein at a certain time point during the culturing, a decrease in the specific activity after the time point causes accumulation of the precursor L-lysine. Therefore, the coryneform bacterium maintains the above LDC specific activity throughout the culture period.
The term “throughout the culture period” means the period which begins when the coryneform bacterium is inoculated to the fermentation medium and continues while the substrate sugar is being consumed, and the period can be confirmed by measuring the specific activity at an arbitrary time point during the period. The culture period after the beginning of culturing, during which the LDC specific activity is maintained, is preferably not less than 5 hours, more preferably not less than 10 hours, still more preferably not less than 20 hours. The upper limit of the culture period during which the above-described specific activity can be maintained is not restricted.
The method for giving the LDC specific activity as described above is not restricted, and examples of the method include a method wherein the copy number of the gene encoding the lysine decarboxylase to be introduced into the chromosome of the coryneform bacterium is increased, method wherein a strong promoter is used as the promoter of the gene encoding the lysine decarboxylase to be introduced into the chromosome, and method wherein a gene encoding a lysine decarboxylase having an enhanced enzyme activity is employed. Any of these methods may be preferably used, and the method is more preferably the one wherein a strong promoter is used as the promoter of the gene encoding the lysine decarboxylase to be introduced into the chromosome.
The promoter is not restricted, and a promoter that can function in a coryneform bacterium may be generally used. Further, the promoter may be one derived from a different species. Preferred examples of the promoter include:
Examples of the promoter sequence and the base sequence of the gene encoding lysine decarboxylase also include base sequences which are the same as their respective sequences except that one or several amino acids are substituted, deleted, inserted and/or added, as long as their functions are maintained. The term “several” herein means normally about 1 to 40, preferably about 1 to 30, more preferably about 1 to 20, still more preferably about 1 to 9, still more preferably about 1 to 5. Further, examples of the promoter sequence and the base sequence of the gene encoding lysine decarboxylase include base sequences that entirely or partially hybridize with those base sequences or with the complementary strands thereof under stringent conditions, as long as their functions are maintained. The term “polynucleotide that hybridizes under stringent conditions” herein means, for example, a base sequence that hybridizes with a probe(s) having one or more base sequences each having at least 20, preferably 25, more preferably at least 30 continuous sequences arbitrarily selected from the original base sequence, when a known hybridization technique (Current Protocols I Molecular Biology edit. Ausbel et al., (1987) Publish. John Wily & Sons Section 6.3-6.4) or the like is applied. The stringent conditions herein can be achieved, for example, by performing hybridization in the presence of 50% formamide at a temperature of 37° C., at 42° C. for more stringent conditions, or at 65° C. for even more stringent conditions, followed by washing with 0.1× to 2×SSC solution (composition of ×1 SSC solution: 150 mM sodium chloride, 15 mM sodium citrate). The promoter sequence and the base sequence encoding lysine decarboxylase may be sequences having sequence identities of normally not less than 80%, preferably not less than 90%, more preferably not less than 95%, still more preferably not less than 99%. The sequence identity herein means a value calculated by aligning two base sequences such that the number of matched bases is maximum (by insertion of a gap(s), as required) and dividing the number of matched bases by the number of bases of the full-length sequence (in cases where the total number of bases is different between the two sequences, the number of bases of the longer sequence). Such calculation of the homology can be easily carried out using well-known software such as BLAST.
Such a promoter sequence and base sequence encoding lysine decarboxylase can also be obtained from an organism other than coryneform bacteria, and can also be obtained by subjecting a coryneform bacterium-derived base sequence to in vitro mutagenesis or site-directed mutagenesis.
The method of introduction of the promoter sequence and base sequence encoding lysine decarboxylase to the coryneform bacterium is not restricted, and the sequences may be introduced by electroporation (Bio/Technology, (1989), 7, 1067-1070).
Coryneform bacteria are aerobic gram-positive bacilli, and also include bacteria which had previously been classified in the genus Brevibacterium but have now been integrated into the genus Corynebacterium (Int. J. Syst., Bacteriol., (1981) 41, p. 225). Coryneform bacteria also include the bacteria belonging to the genus Brevibacterium, which is very close to the genus Corynebacterium. Examples of such coryneform bacteria include Corynebacterium acetoacidophylum, Corynebacterium acetoglutamicum, Corynebacterium alkanolyticum, Corynebacterium callunae, Corynebacterium glutamicum, Corynebacterium lilium, Corynebacterium mellassecola, Corynebacterium thermoaminogenes, Corynebacterium efficiens, Corynebacterium herculis, Brevibacterium divaricatum, Brevibacterium flavum, Brevibacterium immariophilum, Brevibacterium lactofermentum, Brevibacterium roseurn, Brevibacterium saccharolyticum, Brevibacterium thiogenitalis, Corynebacterium ammoniagenes, Brevibacterium album, Brevibacterium cerinum and Microbacterium ammoniaphilum.
Specific examples of strains of the respective coryneform bacteria include Corynebacterium acetoacidophylum ATCC13870, Corynebacterium acetoglutamicum ATCC15806, Corynebacterium alkanolyticum ATCC21511, Corynebacterium callunae ATCC15991, Corynebacterium glutamicum ATCC13020, ATCC13020 and ATCC13060, Corynebacterium lilium ATCC15990, Corynebacterium mellassecola ATCC17965, Corynebacterium efficiens AJ12340 (accession No. FERM BP-1539), Corynebacterium herculis ATCC13868, Brevibacterium divaricatum ATCC14020, Brevibacterium flavum ATCC13826, ATCC14067 and AJ12418 (accession No. FERM BP-2205), Brevibacterium immariophilum ATCC14068, Brevibacterium lactofermentum ATCC13869, Brevibacterium roseum ATCC13825, Brevibacterium saccharolyticum ATCC 14066, Brevibacterium thiogenitalis ATCC19240, Corynebacterium ammoniagenes ATCC6871 and ATCC6872, Brevibacterium album ATCC15111, Brevibacterium cerinum ATCC15112 and Microbacterium ammoniaphilum ATCC15354.
The above coryneform bacteria are available from, for example, American Type Culture Collection. That is, a corresponding accession number is given to each strain and described in the catalogue of American Type Culture Collection, and each strain can be obtained by reference to this number.
The coryneform bacterium to be used in the method for producing 1,5-pentanediamine, which bacterium has a gene encoding lysine decarboxylase in the chromosome, is preferably a coryneform bacterium having improved productivity of L-lysine, which is a precursor of 1,5-pentanediamine. The method for improving the L-lysine productivity of the coryneform bacterium is not restricted, and a known method may be used. Examples of the method include: a method wherein, as described in JP 2004-222569 A, a strain resistant to S-aminoethylcysteine (AEC) is obtained; and a method wherein, as described in Journal of industrial Microbiol Biotechnol (2006, 33(7) 610-5) and Japanese Translated PCT Patent Application Laid-open No. 2009-531042, the L-lysine productivity is improved by genome breeding. Both of these methods may be preferably employed.
Preferably, the coryneform bacterium has improved L-lysine productivity, the bacterium preferably has aspartate kinase whose feedback inhibition by L-lysine is canceled, and the bacterium more preferably has a mutant aspartate kinase having the same amino acid sequence as described in SEQ ID NO:3 except that the 311th amino acid residue is replaced with an amino acid other than threonine.
Also preferably, the coryneform bacterium has improved L-lysine productivity, the bacterium preferably has decreased homoserine dehydrogenase activity or is deficient for the homoserine dehydrogenase activity. The bacterium is more preferably deficient for the homoserine dehydrogenase activity because of destruction or disruption of the homoserine dehydrogenase gene by insertional mutagenesis.
Examples of the culture method which may be used include batch culture, fed-batch culture and continuous culture. In cases of continuous culture, continuous culture described in JP 2008-104453 A or the like is preferably carried out.
As a culture medium, a normal nutrient medium comprising a carbon source, nitrogen source, inorganic salt and/or the like may be used. Examples of the carbon source which may be used include saccharides such as glucose, fructose, sucrose, maltose and starch hydrolysates; alcohols such as ethanol; and organic acids such as acetic acid, lactic acid and succinic acid. Examples of the nitrogen source which may be used include ammonia; inorganic and organic ammonium salts such as ammonium chloride, ammonium sulfate, ammonium carbonate and ammonium acetate; nitrogen-containing organic compounds such as urea; and nitrogen-containing organic substances such as meat extracts, yeast extracts, corn steep liquor and soybean hydrolysates. Examples of the inorganic salt which may be used include potassium dihydrogen phosphate, dipotassium hydrogen phosphate, ammonium sulfate, sodium chloride, magnesium sulfate and calcium carbonate. Further, as required, micronutrients such as biotin, thiamine, vitamin B6 and the like may be added. Medium additives such as meat extracts, yeast extracts, corn steep liquor and casamino acids may be used as alternatives to these micronutrients.
The culture conditions are not restricted, and the culture is carried out under aerobic conditions, for example, with shaking or by deep aeration stirring culture. The culture temperature is generally 25° C. to 42° C., preferably 28° C. to 38° C. The culture period is normally 1 day to 10 days.
For adjusting the culture pH, ammonia, hydrochloric acid or dicarboxylic acid is preferably used, and dicarboxylic acid is more preferably used. It is preferred to use the neutralizer to control the culture pH to 5 to 8, more preferably 6.5 to 7.5. The state of the neutralizer is not restricted, and the neutralizer may be used as a gas, liquid, solid or an aqueous solution. The neutralizer is especially preferably an aqueous solution.
The dicarboxylic acid to be preferably used as the neutralizer is not restricted, and the dicarboxylic acid is preferably a dicarboxylic acid having substantially no functional group other than the above-described two carboxylic groups. The functional group herein means a reactive group which reacts, during polyamide polymerization reaction (under reaction conditions wherein, for example, the reaction temperature is 250 to 270° C., the pressure is 10 to 20 kg/cm2, and the reaction time is 1 to 5 hour(s)), with an amino group or carboxyl group to cause branching of the polymer or reduction in the degree of crystallinity of the polymer (to a degree of crystallinity of not more than 80%). Examples of the functional group include the amino group and carboxyl group, and other examples of the functional group include acidic groups (e.g., the sulfonic acid group, phosphate group and phenolic hydroxyl group), basic groups (e.g., the hydrazino group), protonic polar groups (e.g., the hydroxyl group), cleavable groups (e.g., the epoxy group and peroxide group) and other highly reactive groups (e.g., isocyanate group). On the other hand, halogen substituents, aromatic substituents, ether groups, ester groups, amide groups and the like are not included in the functional group since their reactivity is low.
The dicarboxylic acid is more preferably a dicarboxylic acid represented by Formula (1), (2) or (3) below:
HOOC—(CH2)m—COOH (1)
(wherein m=0 to 16)
(wherein n, o=0 to 16)
(wherein p, q=0 to 16).
The dicarboxylic acid is still more preferably adipic acid, sebacic acid, 1,12-dodecanedicarboxylic acid, succinic acid, isophthalic acid or terephthalic acid.
1,5-Pentanediamine in the culture liquid exists in the free state or as a salt of 1,5-pentanediamine. In the method for collecting 1,5-pentanediamine from the culture liquid, the microorganism is first removed from the culture liquid. In this step, the bacterial cells may be separated from the culture supernatant after the growth of the microorganism and production of 1,5-pentanediamine as a result of sufficient progress of fermentation (the bacterial cells may be separated by removal by precipitation, centrifugation or membrane filtration), or the bacterial cells may be separated, retained or immobilized on a carrier or the like from the beginning. For collecting 1,5-pentanediamine from the culture liquid from which the microorganism was removed and which contains 1,5-pentanediamine, 1,5-pentanediamine dicarboxylate may be collected by crystallization as described in JP 2009-207495 A. Alternatively, 1,5-pentanediamine may be purified and collected using an NF membrane as described in JP 2009-29872 A. Alternatively, 1,5-pentanediamine may be collected by extraction with a polar organic solvent followed by distillation as described in JP 2009-28045 A.
Our methods will now be described below by way of Examples, but these are merely examples and do not limit the scope of this disclosure.
To prepare Corynebacterium glutamicum capable of synthesizing L-lysine as a precursor of 1,5-pentanediamine, an L-lysine fermentation bacterium was prepared by introduction of an effective mutation to aspartokinase (AK). Referring to the method described in Appl. Microbiol. Biotechnol., (2002), 58, pp. 217-223, the Corynebacterium glutamicum AK-1 strain (hereinafter referred to as the AK-1 strain) was prepared. More specifically, the operation was carried out as follows.
PCR was carried out using as an amplification template a genomic DNA solution prepared from the Corynebacterium glutamicum ATCC13032 strain according to a conventional method, and oligonucleotides (SEQ ID NOs:4 and 5) designed by reference to a base sequence of the AK gene (Accession No. BA000036) registered in a database (GenBank) as a primer set, and the obtained product was subjected to electrophoresis in 1% agarose gel, followed by excising the DNA fragment of about 1.3 kb containing the AK gene from the gel and purifying the fragment using Gene Clean Kit. This fragment was digested with BamHI and SphI, and the resulting BamHI-SphI fragment of about 1.3 kb was ligated to the BamHI/SphI gap in pUC19 that had been preliminarily digested with BamHI and SphI. The obtained plasmid was designated pTM47. The obtained AK gene was confirmed to have the same gene sequence as the one registered in the database by sequencing.
To mutate acc (Thr) at positions 931 to 933 in the cloned AK gene to atg (Ile), QuickChange Multi Site-Directed Mutagenesis Kit (manufactured by Stratagene) was used. Details of the experiment were carried out according to the instructions attached to the product. Using pAK1 as an amplification template and the oligonucleotides having the base sequences shown in SEQ ID NOs:6 and 7 as a primer set, the entire sequence of pAK1 was amplified. After treating this PCR product with DpnI, the E. coli JM109 strain was transformed. After extracting the plasmid, the AK gene sequence was analyzed, and it was revealed that the plasmid wherein the mutation of interest was introduced could be obtained. This plasmid was designated pTM49-1. The thus obtained pTM49-1 was digested with SphI and BamHI, and the part containing the AK gene (about 1.3 kb) was obtained by gel extraction, to purify the AK gene to which the mutation was introduced.
PCR was carried out using bacterial cells of the Bacillus subtilis IFO13719 strain as a template and the base sequences shown in SEQ ID NOs:8 and 9 as a primer set, to amplify the sacB gene. The obtained PCR product was digested with SacI, and ligated to pHSG298 (a commercially available pUC-type plasmid having as a selection marker a kanamycin resistance gene, manufactured by Takara Bio Inc.) which had been preliminarily similarly digested with SacI. The obtained plasmid was designated pTM38. Subsequently, the above-described fragment containing the AK gene was ligated to pTM38 digested with SphI and BamHI. The thus prepared plasmid having the sacB gene and the mutant AK gene was designated pTM52.
pTM52 prepared as described above was introduced to the ATCC13032 strain by electroporation [FEMS Microbiology Letters, 65, p. 299 (1989)], and transformants were selected on an agar medium containing LB (tryptone (10 g/l) (manufactured by Bacto), yeast extract (5 g/l) (manufactured by Bacto) and sodium chloride (10 g/l)) supplemented with kanamycin (25 μg/ml). Thereafter, the selected kanamycin-resistant coryneform cells were cultured on a medium supplemented with sucrose, and sucrose-resistant coryneform cells were selected by double crossing-over. Among the thus selected transformants, a strain that can grow on a minimum medium supplemented with 20 mM S-aminoethylcysteine (AEC) was selected, and a genomic DNA solution was prepared therefrom according to a conventional method. PCR was carried out using this genomic DNA as a template and the oligonucleotides (SEQ ID NOs:4 and 5) as a primer set, and the AK gene sequence was analyzed. As a result, it could be confirmed that the sequence at positions 931 to 933 was replaced with atg. Feedback inhibition of aspartokinase by lysine and threonine is canceled in the thus prepared AK-1 strain. Therefore, L-lysine can be synthesized by culturing.
Subsequently, the obtained AK-1 strain was subjected to genetic recombination to prepare a coryneform bacterium having a single copy of E. coli-derived LDC gene in the chromosome and a coryneform bacterium having two copies of E. coli-derived LDC gene in the chromosome by the following methods.
To modify the AK-1 strain into a 1,5-pentanediamine fermentation bacterium, a plasmid pTM45 for introduction of an E. coli-derived LDC gene into the homoserine dehydrogenase locus was prepared. More specifically, this operation was carried out as follows.
PCR was carried out using pHSG298 as a template and the oligonucleotides having the base sequences shown in SEQ ID NOs:10 and 11 as a primer set, to amplify the promoter of the kanamycin resistance gene. Subsequently, this PCR product was digested with BamHI and KpnI, and gel extraction was carried out to purify the promoter of the kanamycin resistance gene (Kmp). Further, a vector pUC19 was digested with BamHI and KpnI, and gel extraction was carried out to purify pUC19. These BamHI/KpnI-digested pUC19 and promoter of the kanamycin resistance gene were ligated to each other. The thus prepared plasmid was designated pKmp.
Subsequently, PCR was carried out using cells of the E. coli JM109 strain as a template and the oligonucleotides having the base sequences shown in SEQ ID NOs:12 and 13 as a primer set, to amplify the LDC gene. The amplified LDC gene was digested with NcoI and SacI, and gel extraction was carried out to purify the LDC gene. Further, the plasmid pKmp was digested with NcoI and SacI, and gel extraction was carried out to purify pKmp. These NcoI/SacI-digested pKmp and LDC gene were ligated to each other. The thus prepared plasmid was designated pTM24.
Subsequently, PCR was carried out using cells of the ATCC13032 strain as a template and the oligonucleotides having the base sequences shown in SEQ ID NOs:14 and 15 as a primer set, to amplify the horn gene. The amplified hom gene was digested with SphI and BamHI, and gel extraction was carried out to purify the hom gene. Further, the above-described plasmid pTM38 was digested with SphI and BamHI, and gel extraction was carried out to purify pTM38. These SphI/BamHI-digested pTM38 and hom gene were ligated to each other. The thus prepared plasmid was designated pTM44.
Subsequently, PCR was carried out using the plasmid pTM24 as a template and the oligonucleotides having the base sequences shown in SEQ ID NOs:16 and 17 as a primer set, to amplify a Kmp-LDC gene fragment. The amplified Kmp-LDC gene fragment was digested with a restriction enzyme Aor51HI, and gel extraction was carried out to purify the Kmp-LDC gene fragment. pTM44 prepared as described above was digested with Aor51HI, and gel extraction was carried out to purify pTM44. These Aor51HI-digested pTM44 and Kmp-LDC gene fragment were ligated to each other. The thus prepared plasmid was designated pTM45.
Subsequently, the plasmid pTM45 was introduced to the AK-1 strain by electroporation [FEMS Microbiology Letters, 65, p. 299 (1989)], and kanamycin-resistant strains were selected, followed by culturing the kanamycin-resistant strains on a medium supplemented with sucrose and selecting sucrose-resistant coryneform cells lacking the sacB gene by the double crossing-over method which is described in Biosci. Biotechnol. Biochem (2007), 71(9), 2130-5 as a method for preparing the TM45 strain. From the thus selected transformant, a genomic DNA solution was prepared according to a conventional method. Using this genomic DNA as a template and the oligonucleotides (SEQ ID NOs:18 and 19) as a primer set, PCR was carried out to obtain a product, which was then subjected to electrophoresis in 1.0% agarose gel. As a re-suit, a single band of 3.5 kb was observed. By this, the selected transformant could be confirmed to have the LDC gene inserted at the horn locus. This transformant was designated the Corynebacterium glutamicum TM4552 strain (hereinafter referred to as the TM4552 strain for short).
Thereafter, a plasmid for introduction of the LDC gene into the lactate dehydrogenase (LDH) locus was prepared.
As a promoter for expression of the LDC gene, the promoter (SEQ ID NO:2, hereinafter referred to as Pdiv for short) of the divIVA gene (SEQ ID NO:20) of the ATCC13032 strain was used.
First, PCR was carried out using as a template the genomic DNA of the ATCC13032 strain prepared according to a conventional method and the oligonucleotides having the base sequences shown in SEQ ID NOs:21 and 22 as a primer set, to clone Pdiv. For the PCR amplification reaction, KOD-plus polymerase (manufactured by Toyobo Co., Ltd.), and the reaction buffer, dNTP mix and the like attached to the polymerase were used. The reaction system was prepared such that 20 μmol/sample of the primers and 1 U/sample of KOD-Plus polymerase were contained in 50 μl of the reaction solution. Using a PCR amplification device iCycler (manufactured by BIO-RAD), the reaction solution was subjected to heat denaturation at 94° C. for 5 minutes, followed by 30 cycles of 94° C. (heat denaturation) for 30 seconds, 60° C. (annealing of the primers) for 30 seconds and 68° C. (extension of the complementary strand) for 30 seconds, after which the reaction solution was cooled to a temperature of 4° C. The primers for amplification of the gene (SEQ ID NOs:21 and 22) were prepared such that a BamHI recognition site and an NcoI recognition site were attached to the 5′-end and the 3′-end, respectively.
The PCR amplification fragment was purified and the fragment was phosphorylated at its ends with T4 polynucleotide Kinase (manufactured by Takara Bio Inc.), followed by being ligated into pUC118 vector (which had been prepared by digestion with a restriction enzyme HincIII and dephosphorylation of the cleavage site). The ligation was carried out using DNA Ligation Kit Ver.2 (manufactured by Takara Bio Inc.). The ligation solution was used to transform competent cells of E. coli DH5α (manufactured by Takara Bio Inc.), and the trans-formed cells were plated on an LB plate supplemented with 50 μg/mL ampicillin. The cells were cultured overnight. From grown colonies, plasmid DNA was recovered by miniprep, and the DNA was digested with restriction enzymes BamHI and NcoI, followed by selecting plasmids to which the Pdiv fragment was inserted. All the series of operations were carried out according to the protocols attached to the products. The thus prepared plasmid was designated pCG5.
Subsequently, PCR was carried out using cells of the E. coli JM109 strain as a template, to clone the LDC gene. The PCR reaction was carried out in the same manner as described above, and the primers for amplification of the gene (SEQ ID NOs:23 and 24) were prepared such that an NcoI recognition site and a SacI recognition site were attached to the 5′-end and the 3′-end, respectively. The obtained PCR amplification fragment comprising the LDC gene was purified and cloned into pUC118 in the same manner as described above. The prepared plasmid was designated pCG11.
Subsequently, pCG11 was digested with restriction enzymes NcoI and XbaI, and the fragment of about 2.1 kb containing the LDC gene was excised and purified, followed by ligating the fragment to pCG5 that had been preliminarily digested with restriction enzymes NcoI and XbaI. The obtained plasmid, which has a fragment in which the LDC gene is linked downstream of Pdiv, was designated pCG13.
(II) Preparation of Plasmid to be Introduced into LDH Locus
As the homologous region to be introduced into the LDH locus, the 5′-end and 3′-end regions in the LDH gene each having a length of 500 bp were cloned. Each of the regions was subjected to PCR using the genome of the ATCC13032 strain as a template and SEQ ID NOs:25 and 26 (5′-end region) or SEQ ID NOs:27 and 28 (3′-end region) as a primer set in the same manner as described above. The primers for amplification of the 5′-end region (SEQ ID NOs:25 and 26) were prepared such that a BglII recognition sequence was added to each of the 5′-end-side and the 3′-end-side, and the primers for amplification of the 3′-end region (SEQ ID NOs:27 and 28) were prepared such that an SalI recognition sequence was loaded on the 5′-end-side and an SphI recognition sequence was loaded on the 3′-end-side. The obtained PCR amplification fragments each having a length of about 500 bp and comprising the 5′-end or 3′-end region of the LDH gene were purified, and each of the purified fragments was cloned into pUC118 in the same manner as described above. The prepared plasmids were designated pCG28 (5′-end region) and pCG29 (3′-end region).
Subsequently, pCG13 was digested with restriction enzymes BamHI and XbaI to excise the fragment of about 2.4 kb containing the Pdiv-LDC gene and the fragment was purified, followed by ligating the fragment to the above-described pTM38 that had been preliminarily digested with restriction enzymes BamHI and XbaI. The obtained plasmid was designated pCG33.
Subsequently, pCG29 was digested with restriction enzymes SalI and SphI, and the fragment containing the 3′-end region of the LDH gene was excised and purified, followed by ligating the fragment to pCG33 that had been preliminarily digested with SalI and SphI. The obtained plasmid was designated pCG37.
Finally, pCG28 was digested with restriction enzymes BglII and BamHI, and the fragment containing the 5′-end region of the LDH gene was excised and purified, followed by ligating the fragment to pCG37 that had been preliminarily digested with BglII and BamHI. The obtained plasmid was designated pCG41.
(iii) Introduction of pCG41 into Chromosome
To the above-prepared TM4552 strain, pCG41 prepared as described above was introduced, and transformants were selected on an agar medium containing LB (tryptone (10 g/l) (manufactured by Bacto), yeast extract (5 g/l) (manufactured by Bacto) and sodium chloride (10 g/l)) supplemented with kanamycin (25 μg/ml). Thereafter, the selected kanamycin-resistant coryneform cells were cultured on a medium supplemented with sucrose, and sucrose-resistant coryneform cells were selected by double crossing-over in the same manner as in Example 1. From the thus selected transformant, a genomic DNA solution was prepared. Using this genomic DNA as a template and the oligonucleotides (SEQ ID NOs:25 and 28) as a primer set, PCR was carried out, and the obtained product was subjected to electrophoresis in 1.0% agarose gel. As a result, a single band of about 3.3 kb was observed. It should be noted that a fragment of about 2 kb is expected in the case of the strain in which pCG41 is not introduced. Thus, it could be confirmed that the selected transformant has the Pdiv-LDC gene fragment inserted at the LDH locus. This transformant was designated the Corynebacterium glutamicum CG4541 strain (hereinafter referred to as the CG4541 strain for short).
Using the TM4552 strain (Comparative Example 1) and CG4541 strain (Example 1) prepared as described above, a 1,5-pentanediamine fermentation test was carried out.
Into 5 mL of the medium shown in Table 1 which was sterilized, one platinum loop of each of the CG4541 strain (Example 3) and TM4552 strain (Comparative Example 1) was inoculated, and pre-preculture was carried out at 30° C. for 24 hours with shaking. The entire volume of the obtained pre-preculture was inoculated into 45 ml of the same medium as in the pre-preculture, and preculture was carried out at 30° C. at 120 rpm for 24 hours. Thereafter, the entire volume of the obtained preculture was inoculated into 1000 ml of the medium which is the same as the one shown in Table 1 except that the glucose concentration is 150 g/L, and culturing was carried out under aeration with sterilized air at 0.07 vvm at 30° C. at a stirring blade rotation speed of 800 rpm at a controlled pH of 6.7. As neutralizers, an aqueous sulfuric acid solution (3 M) and aqueous ammonia (3 M) were used.
The result of culturing of the CG4541 strain is shown in
The LDH specific activities of the CG4541 strain (Example 1) and TM4552 strain (Comparative Example 1) cultured as described above were measured over time.
A 10-ml aliquot of the culture liquid of each strain cultured as described above was sampled over time, and the cells were collected by centrifugation at 4000 rpm for 5 minutes, followed by suspending the cells in 2 ml of a buffer (50 mM Tris-HCl, pH 8). In each of 2-mL screw-cap tubes (manufactured by WATSON), 0.4 g of glass beads (0.1 mm diameter, manufactured by As One Corporation) were placed, and 1 ml of the bacterial suspension was added thereto. Subsequently, homogenization at 4000 rpm for 1 minute was repeated 5 times, and the supernatant was collected by centrifugation at 12000 rpm for 5 minutes. The obtained supernatant was used as a raw enzyme liquid in the subsequent measurement of the specific activity of lysine decarboxylase. For measurement of the protein concentration, BCA Protein Assay Kit (manufactured by PIERCE) was used. The protein concentration in the raw enzyme liquid of the CG4541 strain is shown in Table 2, and the protein concentration in the raw enzyme liquid of the TM4552 strain is shown in Table 3.
Using the thus obtained raw enzyme liquid, the lysine decarboxylase activity was measured using L-lysine as a substrate. The composition of the reaction solution is shown in Table 4. The reaction was performed at 37° C. for 30 minutes, and this was followed by boiling for 10 minutes for termination of the reaction. Using the supernatant obtained after centrifugation, the concentrations of L-lysine and 1,5-pentanediamine produced were measured by HPLC as described above, and the LDC specific activity per unit amount of protein was calculated according to Equation 1. Changes in the LDC specific activity over time are shown in
As shown by the results shown in
Table 5 shows the LDC specific activity and the concentrations of 1,5-pentanediamine and L-lysine in the culture liquid observed for the CG4541 strain, and Table 6 shows the LDC specific activity and the concentrations of 1,5-pentanediamine and L-lysine in the culture liquid observed for the TM4552 strain.
As shown by the results shown in Table 6, in the case of the TM4552 strain, the LDC specific activity was 83 mU/mg protein and no accumulation of L-lysine in the culture liquid was observed at Hour 25 of the culturing, but the LDC specific activity decreased to 39 mU/mg protein and accumulation of L-lysine in the culture liquid was observed at Hour 45 of the culturing. After this, the specific activity of LDC continued to decrease and accumulated L-lysine continued to increase. On the other hand, based on the results shown in table 5, in the case of the CG4541 strain, the LDC specific activity during the culturing was maintained at not less than 180 mU/mg protein, and no accumulation of L-lysine occurred at all in the culture liquid.
As promoters for expression of the LDC gene, the CG4541 strain uses the promoters of the kanamycin resistance gene and the divIVA gene. Since the LDC specific activity of the TM4552 strain, which uses only the promoter of the kanamycin resistance gene, was about 80 mU/mg protein even when the specific activity was highest, it is thought that the maintenance of high LDC specific activity in the CG4541 strain was due to the effect of the promoter of the divIVA gene.
Our method produces 1,5-pentanediamine which can be used as a raw material for polyamides.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2010-091602 | Apr 2010 | JP | national |
This is a §371 of International Application No. PCT/JP2011/058987, with an international filing date of Apr. 11, 2011 (WO 2011/129293 A1, published Oct. 20, 2011), which is based on Japanese Patent Application No. 2010-091602, filed Apr. 12, 2010, the subject matter of which is incorporated by reference.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP2011/058987 | 4/11/2011 | WO | 00 | 11/13/2012 |
