CN113481139A - Recombinant bacillus subtilis for producing glycocyamine and construction method thereof - Google Patents
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Abstract
The invention discloses recombinant bacillus subtilis for producing glycocyamine and a construction method thereof, belonging to the technical field of bacillus subtilis metabolic engineering and genetic engineering. The recombinant Bacillus subtilis obtained by the invention is prepared by taking wild Bacillus subtilis as an initial strain and introducing exogenous arginine on a Bacillus subtilis genome: the yield of the recombinant strain with high yield of the glycocyamine obtained by glycine amidine transferase reaches 12.8g/L, and the synthesis of the glycocyamine in bacillus subtilis is realized for the first time. The method lays a foundation for the efficient production of glycocyamine in the bacillus subtilis metabolic engineering.
Description
Technical Field
The invention relates to recombinant bacillus subtilis for producing glycocyamine and a construction method thereof, belonging to the technical field of bacillus subtilis metabolic engineering and genetic engineering.
Background
Guanidinoacetic acid (GAA), also known as guanidinoacetic acid, is a naturally occurring amino acid derivative. Guanidinoacetic acid is currently widely used in animal production as a feed additive to increase muscle production. At present, the guanidine compound is mainly synthesized by chemical synthesis, and most chemical processes have the problems of complex route and environmental unfriendliness, so that the research and development of the guanidine drug synthesis process which is synthesized by a microbial synthesis method and has no pollution to the environment and is economic is of great significance.
Bacillus subtilis has wide industrial application and plays an important role in producing protein and high value-added compounds. At the same time, it is a non-pathogenic microorganism that has been recognized by the U.S. Food and Drug Administration (FDA) as a safe (GRAS) food grade microorganism. In addition, Bacillus subtilis has a number of advantages, including: the growth rate of the cells is high, the fermentation culture time is short, the culture condition requirement is low, the metabolic engineering modification is easy to carry out, the protein secretion capacity is strong, and the like.
The purpose of optimizing metabolic pathways can be achieved by changing the expression strength of the genetic element, and then the high-efficiency synthesis of the target product is realized. The regulation of the expression strength of key genes by promoters is a common and important regulation strategy for controlling the flow of carbon flux to a synthesis pathway of a target product. At present, GAA is produced by expression in Escherichia coli, which belongs to pathogenic strains, can generate endotoxin and is not suitable for serving as a food additive or a feed additive. Bacillus subtilis is more suitable for GAA production than Escherichia coli, but because it does not contain a GAA-producing pathway by itself, it poses a certain difficulty in GAA production using Bacillus subtilis.
Disclosure of Invention
The invention aims to solve the technical problem of providing a recombinant bacillus subtilis strain for producing glycocyamine and a construction method thereof. Promoters with different strengths capable of increasing or reducing protein expression are used for starting the heterologous glycine amidine transferase AGAT, the key gene AGAT in the GAA biosynthetic pathway is inserted into the original nucleotide sequence to realize the up-regulation or down-regulation of the expression quantity of the target gene, and finally, the change of intracellular metabolic flow is realized to improve the yield of the GAA.
The invention provides a recombinant bacillus subtilis which expresses glycine amidine transferase AGAT derived from Amycolatopsis kentuckyensis and knockouts gcvP and/or argI genes in a genome.
In one embodiment, the argI gene is the first gene in the arginolysis pathway and the first gene in the glycine cleavage pathway gcvP.
In one embodiment, the nucleotide sequence of the gene encoding the glycine amidinyltransferase AGAT is as shown in SEQ ID No. 12.
In one embodiment, the glycine amidinase AGAT is expressed using promoters of different strengths; the promoters of different strengths comprise Pxpac、Pycec、PlytR、PsdhB、P43、PodhA、P333、PyvyD、PvegOr P566。
In one embodiment, the promoter Pxpac、Pycec、PlytR、PsdhB、P43、PodhA、P333、PyvyD、Pveg、P566The nucleotide sequences of (A) are respectively shown in SEQ ID NO. 1-10.
In one embodiment, the gcvP gene has a GeneID of 938549; the GeneID of the argI gene was 937760.
In one embodiment, the pHT01 plasmid is used as an expression vector.
In one embodiment, Bacillus subtilis168 is used as a starting strain.
The invention provides a method for producing glycocyamine, which takes recombinant bacillus subtilis as a fermentation strain and glycine and arginine as substrates to produce glycocyamine through fermentation.
In one embodiment, the concentration of glycine and arginine in the reaction system is the same and is 15-25 g/L.
In one embodiment, the glycine and arginine are present in the reaction system at a concentration of 20g/L each.
In one embodiment, the OD of the recombinant Bacillus subtilis in the reaction system60060 + -2, and reacting at pH 7.5.
In one embodiment, the reaction system contains 35-45 g/L glucose, 10-15 g/L yeast powder, 5-10 g/L tryptone, 5-10 g/L ammonium sulfate, 10-15 g/L dipotassium hydrogen phosphate trihydrate, 2-5 g/L potassium dihydrogen phosphate, 1-5 g/L magnesium chloride and 5-10 g/L urea.
In one embodiment, the reaction system contains 40g/L glucose, 12g/L yeast powder, 6g/L tryptone, 6g/L ammonium sulfate, 12g/L dipotassium hydrogen phosphate trihydrate, 2.5g/L potassium dihydrogen phosphate, 3g/L magnesium chloride and 6g/L urea.
In one embodiment, the reaction is carried out at 25-40 ℃ for 24-48 h.
In one embodiment, the reaction is carried out at 30 ℃ for 48 h.
The invention provides application of bacillus subtilis in production of glycocyamine.
The invention has the beneficial effects that: the bacillus subtilis belongs to a food safety level microorganism, and the produced GAA meets the food safety requirement and can be directly used as a food additive or a feed additive. According to the invention, the metabolic pathway of GAA production is modified in Bacillus subtilis168, the obtained strain is used for producing GAA through whole-cell transformation, the reaction lasts for 48h, and the final yield can reach 12.8g/L
Detailed Description
Culturing and fermenting recombinant bacillus subtilis seeds:
seed liquid culture medium: 10g/L tryptone, 10g/L sodium chloride and 5g/L yeast powder.
The formula of the fermentation medium is as follows: 40g/L glucose, 12g/L yeast powder, 6g/L tryptone, 6g/L ammonium sulfate, 12g/L dipotassium hydrogen phosphate trihydrate, 2.5g/L potassium dihydrogen phosphate, 3g/L magnesium chloride and 6g/L urea.
Whole cell transformation process: first, single colonies were picked up into 3mL of seed culture medium and cultured at 37 ℃ for 10 hours at 220 rpm. Then, 2.5mL of seed solution was inoculated into 50mL of fermentation medium, and the corresponding antibiotic was added thereto and cultured at 37 ℃ and 220rpm for 20 hours, and a small amount of the bacterial solution was diluted to an appropriate multiple (about 50 times) to measure OD600About 30 ℃ or so, the fermentation broth was centrifuged at 4 ℃ and 4000rpm for 10min to collect cell pellets, which were then washed once with a predetermined volume of 0.01M PBS buffer, pH 7.5. Adding all cells into 25mL of catalytic reaction system containing 20g/L arginine, 20g/L glycine and one-ten-thousandth chloramphenicol, and mixing the bacteria uniformly, wherein the OD in the system is60060. + -.2, the catalytic reaction was carried out in 0.01M PBS buffer, pH 7.5. And adding 5mL of the reaction system into a 50mL centrifuge tube in a reaction container, carrying out whole-cell transformation reaction in a constant-temperature shaking table at 30 ℃ and 220rpm/min, and collecting the concentration change of a sample detection product respectively for 4h, 10h, 24h and 48h of transformation.
The sample detection method comprises the following steps: the detection of guanidinoacetic acid was carried out by means of Agilent high performance liquid chromatography (equipped with UV absorber) on a column of 3.5 μm column (4.6 mm. times.150 mm) from Waters XBridge BEH Amide. The mobile phase was 75% acetonitrile (analytical grade) (ammonia adjusted to pH 10), the detection wavelength was 210nm, and the flow rate was 1 mL/min. Adding 600 mul of 50% acetonitrile (analytically pure) into each 300 mul of sample after whole cell transformation, diluting the sample by 3 times, evenly mixing, centrifuging at 14000rpm/min for 10min, and taking 600 mul of supernatant for detection.
EXAMPLE 1 construction of plasmid pHT01-agat
Synthesizing a nucleotide sequence (shown as SEQ ID NO. 12) of an encoding gene AGAT according to glycine amidino transferase AGAT (shown as an amino acid sequence in SEQ ID NO. 11) from Amycolatopsis kentuckyensis, designing a primer, respectively connecting the sequences with the nucleotide sequences shown as SEQ ID NO. 1-10 and the SEQ ID NO.12 to obtain recombinant fragments, respectively connecting the recombinant fragments to pHT01 plasmids in a Gibson assembly mode, and constructing to obtain recombinant plasmids containing different promoter expression AGATs.
With the promoter P333Primers fx-PS7333-F and fx-PS7333-R were designed for the examples,
fx-PS7333-F：
5’-
tcctacaattcttgatataatattctcatagtttgaaaaaggaggtgataaaaATGAGAACAGATACAAGAATTGTTAATT CATGGAATG-3’，
fx-PS7333-R：
5’-aatattatatcaagaattgtaggattaagcaactgaaatttttaagtcaaaattctatgattcctcaagagctcgaattcactggccgtc-3’；
with the promoter P566Primers fx-p10-F and fx-p10-R were designed as examples,
fx-p10-F：
5’-
tcttttgagaatatgttatattatcagaaaggaggtgataaaaATGAGAACAGATACAAGAATTGTTAATTCATGGAATG-3’，
fx-p10-R：
5’-gataatataacatattctcaaaagagtgtcaaccctctatttcgagaggccgttttttgagctcgaattcactggccgtc-3’。
TABLE 1 respective promoter sequences
Example 2 construction of recombinant Bacillus subtilis containing pHT01 plasmids with different AGAT expression intensities
The plasmids constructed in example 1 were transformed into Bacillus subtilis168, respectively, and plated on plates at 37 ℃ until single colonies grew.
And selecting a single colony, verifying by using primers yz-PHT01-F and yz-PHT01-R to generate a 1.7kb band, and verifying that the recombinant bacillus subtilis is successfully constructed.
yz-PHT01-F：5’-gataataagggtaactattgccgatcgtccattc-3’，
yz-PHT01-R：5’-gtacagggactattcctaataagccgatattagcc-3’。
Example 3 ligation of reporter Gene GFP after AGAT to characterize the amount of enzyme expressed
In order to measure the expression level of AGAT, a reporter gene GFP was ligated behind the AGAT gene to construct a fusion protein AGAT-GFP, which was ligated to pHT01 plasmid, and the promoters were the ten promoters with different strengths as described above. The sequence with the nucleotide sequence shown as SEQ ID NO. 1-10 is simultaneously connected with SEQ ID NO.12 and SEQ ID NO.14 respectively to obtain a recombinant fragment, and the obtained recombinant fragment is connected to pHT01 plasmid in a Gibson assembly mode to construct recombinant plasmid containing different promoter expression fusion proteins AGAT-GFP. The constructed plasmids are respectively transformed into Bacillus subtilis168, and the Bacillus subtilis is coated on a plate at 37 ℃ until a single colony grows out. Single colonies were picked and cultured in 96-well plates and fluorescence was detected. The relative expression intensity of AGAT is expressed by the relative expression level of fluorescent protein, and the fluorescence values (fluorescence/OD) of the ten promoters are 300, 453, 497, 550, 580, 670, 742, 899, 1030, and 1245. With the promoter PxpacFor control, the relative expression intensities of AGAT were 1, 1.51, 1.65, 1.83, 1.93, 2.23, 2.47, 2.99, 3.43, 4.15 (table 2).
Example 4 Effect of promoters of different strengths on the production of GAA in engineered bacteria
A single colony of the recombinant strain of example 2 was first picked up and cultured in 3mL of seed culture medium at 37 ℃ and 220rpm for 10 hours. Then, 2.5mL of seed solution was inoculated into 50mL of fermentation medium, and the corresponding antibiotic was added to the medium and cultured at 37 ℃ and 220rpm for 15-20 hours, and after cell pellet was collected by centrifugation at 4000rpm for 10min, the pellet was washed with PBS buffer and resuspended in 3mL, and simultaneously substrate was added to perform whole cell transformation.
Under the condition of whole cell transformation for 48h and other conditions being the same, the GAA yield in the final reaction solution of the sample of the promoter SEQ ID NO.3 is about 1.89 g/L; while the higher strength promoter SEQ ID NO.10 sample yielded about 9.53g/L of GAA in the final reaction solution, which was 5.04 times that of the promoter SEQ ID NO.3 sample.
TABLE 2 Effect of promoters with different expression intensities inserted before the coding sequence of key genes on GAA production
Example 5 construction of argI and gcvP knockout strains
P in the selected example 4566The recombinant bacillus subtilis as a promoter realizes the knockout of argI (GeneID: 937760) and gcvP (GeneID: 938549) on a bacillus subtilis genome in a fusion PCR mode, the knockout methods of the two genes are the same, and different resistance genes are required to be selected as screening markers. The fusion PCR knockout frame comprises three parts, namely upstream of a knockout site of a bacillus subtilis genome, a resistance gene and downstream of the knockout site, and after target fragments are obtained by PCR amplification of all fragments respectively, the target fragments are connected through one-round and two-round fusion PCR. The integration frame can be directly transformed into a bacillus subtilis competence, the target gene is knocked out through a homologous recombination system, the resistance gene is used as a screening label, a transformant is coated on a resistance plate, a single colony is selected for colony PCR verification, and the recombinant bacillus subtilis with gcvP knocked out on a genome respectively and the recombinant bacillus subtilis with gcvP knocked out and argI knocked out simultaneously are constructed.
Example 6 Effect of Bacillus subtilis genome knockout of argI and gcvP on the production of guanidinoacetic acid by recombinant strains
The recombinant strain with the genomic deletion of gcvP obtained in example 5 was transformed into whole cells, and the concentration of the product was measured after fermentation for 48 hours, which indicated that the yield of guanidinoacetic acid was 10.2 g/L. The recombinant strain obtained in example 4, from which gcvP and argI were simultaneously knocked out on the genome, was transformed into whole cells, and the concentration of the product was measured after fermentation for 48 hours, showing that the final yield of guanidinoacetic acid was 12.8 g/L. 109.4mM GAA was obtained from 266.7mM glycine and 114.8mM arginine, with GAA conversions of arginine and glycine of 95.3 mol% and 41 mol%, respectively.
Substrate conversion rate is the number of moles of substrate actually participating in the reaction/total number of moles of substrate × 100%.
Comparative example 1
Replacing SEQ ID NO.12 in example 1 with a sequence shown in SEQ ID NO.13, constructing a recombinant plasmid in the manner of example 1, transferring the recombinant plasmid into Bacillus subtilis168 to construct a recombinant bacterium, performing whole-cell transformation in the manner of example 4, and determining GAA in the reaction solution, wherein the result shows that GAA is not detected.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
SEQUENCE LISTING
<110> university of south of the Yangtze river
<120> recombinant bacillus subtilis for producing glycocyamine and construction method thereof
<130> BAA210928A
<160> 14
<170> PatentIn version 3.3
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<212> DNA
<213> Artificial sequence
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gaatcgtcga cctgtcatag acctgaaaag gtcttttttt gtactcttaa taataaaaag 60
aagatgaaac ttgtttaagg attgaacgta gtagataata atattaaaac tgagaaagga 120
ggtgataaaa 130
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<212> DNA
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gaatcgtcga cctgattatt cttaactttt acgaaacttt gatataataa caaacgtata 60
tattagtaat ttacggctta ttttccttgt gagcgtaaaa ataaatgtga ctataaagga 120
ggtgataaaa 130
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<212> DNA
<213> Artificial sequence
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gaatcgtcga cctgaaacaa tgaaactttt ttttataaaa aacgactatt ttaggatttc 60
attcttgtat taaatagagt tgtatttatt ggaaatttaa ctcataatga aagtaattta 120
aaggaggtga taaaa 135
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<212> DNA
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gaatcgtcga cctgttttct tgacgccctt ttgagggagg agtaaaatga aattgtcaat 60
aaatcttaat aaagtgctta caattgaaag aagtggggga agagattaaa ggaggtgata 120
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gaatcgtcga ctgataggtg gtatgttttc gcttgaactt ttaaatacag ccattgaaca 60
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cgccggggct gtttgcgttc ttgccgtgat ttcgtgtacc attggtttac ttattttttt 180
gccaaggctg taatggctga aaattcttac atttatttta catttttaga aatgggcgtg 240
aaaaaaagcg cgcgattatg taaaatataa agtgatagcg gtaccattat aggtaagaga 300
ggaatgtaca c 311
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ggtgataaaa 130
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Met Arg Thr Asp Thr Arg Ile Val Asn Ser Trp Asn Glu Trp Asp Thr
1 5 10 15
Leu Gln Glu Val Val Val Gly Thr Ala Glu Asn Ala Cys Phe Glu Pro
20 25 30
Thr Glu Pro Gly His Arg Pro Gln Glu Arg Gly Leu Pro Glu Pro Arg
35 40 45
Pro Phe Pro Thr Gly Pro Lys Pro Arg Glu Leu Asn Glu Lys Ala Glu
50 55 60
Glu Glu Leu Ala Gly Leu Val Ser Leu Leu Glu Thr His Gly Val Thr
65 70 75 80
Val Arg Arg Pro Ser Pro Arg Asp Tyr Ser Ile Pro Leu Lys Thr Pro
85 90 95
Thr Phe Glu Val Glu Asn Gln Tyr Cys Ala Val Cys Pro Arg Asp Val
100 105 110
Met Ile Thr Leu Gly His Glu Ile Leu Glu Ala Thr Met Ser Arg Arg
115 120 125
Ser Arg Tyr Phe Glu Tyr Glu Ala Tyr Arg Ser Leu Val Tyr Glu Tyr
130 135 140
Trp Asp Gln Asp Pro Gln Met Thr Trp Ser Val Ala Pro Lys Pro Ser
145 150 155 160
Met Ala Asp Glu Met Tyr Arg Gln Asp Phe Trp Thr Trp Pro Leu Ser
165 170 175
Lys Arg His Glu Glu Met His Asn Phe Glu Phe Cys Val Thr Gln Asp
180 185 190
Glu Val Val Phe Asp Ala Ala Asp Met Ala Arg Met Gly Lys Asp Ile
195 200 205
Phe Val Gln Glu Ser Met Thr Thr Asn Arg Ala Gly Ile Arg Trp Leu
210 215 220
Thr Arg His Leu Glu Pro Lys Gly Phe Arg Val His Pro Val His Phe
225 230 235 240
Pro Leu Asp Tyr Phe Pro Ser His Ile Asp Ala Thr Phe Ile Pro Leu
245 250 255
Arg Ala Gly Leu Val Leu Thr Asn Pro Glu Arg Pro Ile Ser Ser Gly
260 265 270
Glu Glu Lys Leu Phe Leu Ala Asn Asp Trp Glu Phe Val Thr Ala Pro
275 280 285
Gln Pro Leu Thr Gly Asn Asp Glu Met Pro Arg Tyr Cys Gln Ser Ser
290 295 300
Lys Trp Val Ser Ile Asn Val Leu Ser Ile Ser Pro Ser Lys Ile Ile
305 310 315 320
Val Glu Glu Gln Glu Lys Pro Leu Gln Asp Leu Leu Cys Ser Leu Gly
325 330 335
Phe Glu Val Leu Pro Leu Pro Phe Arg His Val Tyr Glu Tyr Gly Gly
340 345 350
Ser Leu His Cys Ala Thr Trp Asp Val Arg Arg Asp Gly Gly Cys Glu
355 360 365
Asp Tyr Phe Pro Asn Gln Asn Val
370 375
<210> 12
<211> 1131
<212> DNA
<213> Artificial sequence
<400> 12
atgagaacag atacaagaat tgttaattca tggaatgaat gggatacact gcaagaggtt 60
gttgtgggca cagcagaaaa tgcatgcttt gaaccgacag aaccgggcca tagaccgcaa 120
gaaagaggcc tgccggaacc gagaccgttt ccgacgggcc cgaaaccgag agaactgaat 180
gaaaaagcag aagaggaact ggcgggcctg gtttcactgc tggaaacaca tggcgttaca 240
gttagaagac cgtcaccgag agattattca attccgctga aaacaccgac atttgaagtt 300
gaaaatcaat attgcgcagt ttgcccgaga gatgttatga ttacactggg ccatgaaatt 360
ctggaagcaa caatgtcaag aagatcaaga tattttgaat atgaagcata tagatcactg 420
gtttatgaat attgggatca agatccgcaa atgacatggt cagttgcacc gaaaccgtca 480
atggcagatg aaatgtatag acaagatttt tggacatggc cgctgtcaaa aagacatgaa 540
gaaatgcata attttgaatt ttgcgttaca caagatgaag ttgtttttga tgcagcagat 600
atggcaagaa tgggcaaaga tatttttgtt caagaatcaa tgacaacaaa tagagcgggc 660
attagatggc tgacaagaca tctggaaccg aaaggcttta gagttcatcc ggttcatttt 720
ccgctggatt atttcccgtc acatattgat gcaacattta ttccgctgag agcgggcctg 780
gttctgacaa atccggaaag accgatttca agcggcgaag aaaaactgtt tctggcaaat 840
gattgggaat ttgttacagc accgcaaccg ctgacgggca atgatgaaat gccgagatat 900
tgccaatcat caaaatgggt ttcaattaat gttctgtcaa tttcaccgtc aaaaattatt 960
gttgaggagc aagaaaaacc gctgcaagat ctgctgtgct cactgggctt tgaagttctg 1020
ccgctgccgt ttagacatgt ttatgaatac ggcggctcac tgcattgcgc aacatgggat 1080
gttagaagag atggcggctg cgaagattat tttccgaatc aaaatgttta a 1131
<210> 13
<211> 318
<212> DNA
<213> Artificial sequence
<400> 13
atgattctgt atgatacata tccgctgtca gaagaaacat ggcatacaca tcaatttaca 60
tttattaaag atcatgcatt tagactgctg caaccgggcg gcgttctgac atattgcaat 120
ctgacatcat ggggcgaact gctgaaaaca aaatcagata ttgaaaaaat gtttgaagaa 180
acacaaattg gccatctggt tgaagcgggc tttaaaaaag aaaatattag aacaacagtt 240
atggatctgg ttccgccgca agattgcaga tattattcat ttcataaaat gattacaccg 300
acaattatta aacattaa 318
<210> 14
<211> 711
<212> DNA
<213> Artificial sequence
<400> 14
agtaaaggag aagaactttt cactggagtt gtcccaattc ttgttgaatt agatggtgat 60
gttaatgggc acaaattttc tgtcagtgga gagggtgaag gtgatgcaac atacggaaaa 120
cttaccctta aatttatttg cactactgga aagcttcctg ttccttggcc aacacttgtc 180
actactctta cttatggtgt tcaatgcttt tcaagatacc cagatcatat gaagcggcac 240
gacttcttca agagcgccat gcctgaggga tacgtgcagg agaggaccat cttcttcaag 300
gacgacggga actacaagac acgtgctgaa gtcaagtttg agggagacac cctcgtcaac 360
agaatcgagc ttaagggaat cgatttcaag gaggacggaa acatcctcgg ccacaagttg 420
gaatacaact acaactccca caacgtatac atcatggcag acaaacaaaa gaatggaatc 480
aaagttaact tcaaaattag acacaacatt gaagatggaa gcgttcaact agcagaccat 540
tatcaacaaa atactccaat tggcgatggc cctgtccttt taccagacaa ccattacctg 600
tccacacaat ctgccctttc gaaagatccc aacgaaaaga gagaccacat ggtccttctt 660
gagtttgtaa cagctgctgg gattacacat ggcatggatg aactatacaa a 711
Claims (10)
1. Recombinant Bacillus subtilis which expresses Amycolatopsis kentuckyensis-derived glycine amidinotransferase AGAT and has a genome in which a gcvP gene and/or an argI gene is knocked out.
2. The recombinant Bacillus subtilis of claim 1, wherein the nucleotide sequence of the gene encoding the glycinamidine transferase AGAT is set forth in SEQ ID No. 12.
3. The recombinant bacillus subtilis of claim 1 wherein the glycine amidinyltransferase AGAT is expressed using promoters of different strengths; the promoters of different strengths comprise Pxpac、Pycec、PlytR、PsdhB、P43、PodhA、P333、PyvyD、PvegOr P566。
4. The recombinant Bacillus subtilis of any one of claims 1 to 3 wherein the gcvP gene has a GeneID of 938549; the GeneID of the argI gene was 937760.
5. The recombinant Bacillus subtilis of claim 4 wherein the pHT01 plasmid is used as an expression vector.
6. A method for producing glycocyamine, characterized in that, the recombinant Bacillus subtilis of any one of claims 1 to 5 is used as a fermentation strain, glycine and arginine are used as substrates, and glycocyamine is produced by fermentation.
7. The method according to claim 6, wherein the concentration of the glycine and the arginine in the reaction system is the same and is 15-25 g/L; OD of the recombinant bacillus subtilis in a reaction system600Is 55 to 65.
8. The method of claim 6, wherein the reaction system comprises 35-45 g/L glucose, 10-15 g/L yeast powder, 5-10 g/L tryptone, 5-10 g/L ammonium sulfate, 10-15 g/L dipotassium hydrogen phosphate trihydrate, 2-5 g/L potassium dihydrogen phosphate, 1-5 g/L magnesium chloride, and 5-10 g/L urea.
9. The method according to any one of claims 6 to 8, wherein the reaction is carried out at 25 to 40 ℃ for 24 to 48 hours.
10. Use of the Bacillus subtilis of any one of claims 1 to 5 for the production of glycocyamine.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2023232583A1 (en) | 2022-06-03 | 2023-12-07 | Evonik Operations Gmbh | Improved biotechnological method for producing guanidino acetic acid (gaa) by using nadh-dependent dehydrogenases |
| WO2024094483A1 (en) * | 2022-11-03 | 2024-05-10 | Evonik Operations Gmbh | Improved biotechnological process to produce guanidinoacetic acid (gaa) by targeted introduction or by increasing the activity of a transmembrane transport protein belonging to the amino acid-polyamine-organocation superfamily |
| WO2024094481A1 (en) * | 2022-11-03 | 2024-05-10 | Evonik Operations Gmbh | Improved biotechnological process to produce guanidinoacetic acid (gaa) by targeted introduction or by increasing the activity of a transmembrane exporter protein |
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| CN111748506A (en) * | 2019-03-29 | 2020-10-09 | 中国科学院微生物研究所 | Engineering bacterium for producing glycocyamine and construction method and application thereof |
| WO2021122400A1 (en) * | 2019-12-19 | 2021-06-24 | Evonik Operations Gmbh | Method for the fermentative production of guanidinoacetic acid |
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| CN111748506A (en) * | 2019-03-29 | 2020-10-09 | 中国科学院微生物研究所 | Engineering bacterium for producing glycocyamine and construction method and application thereof |
| WO2021122400A1 (en) * | 2019-12-19 | 2021-06-24 | Evonik Operations Gmbh | Method for the fermentative production of guanidinoacetic acid |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023232583A1 (en) | 2022-06-03 | 2023-12-07 | Evonik Operations Gmbh | Improved biotechnological method for producing guanidino acetic acid (gaa) by using nadh-dependent dehydrogenases |
| WO2024094483A1 (en) * | 2022-11-03 | 2024-05-10 | Evonik Operations Gmbh | Improved biotechnological process to produce guanidinoacetic acid (gaa) by targeted introduction or by increasing the activity of a transmembrane transport protein belonging to the amino acid-polyamine-organocation superfamily |
| WO2024094481A1 (en) * | 2022-11-03 | 2024-05-10 | Evonik Operations Gmbh | Improved biotechnological process to produce guanidinoacetic acid (gaa) by targeted introduction or by increasing the activity of a transmembrane exporter protein |
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