CN113308486A - Method for producing L-phenylglycine by genetic engineering strain biocatalysis - Google Patents
Method for producing L-phenylglycine by genetic engineering strain biocatalysis Download PDFInfo
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- C12Y104/03—Oxidoreductases acting on the CH-NH2 group of donors (1.4) with oxygen as acceptor (1.4.3)
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- C12Y113/11—Oxidoreductases acting on single donors with incorporation of molecular oxygen (oxygenases) (1.13) with incorporation of two atoms of oxygen (1.13.11)
- C12Y113/11046—4-Hydroxymandelate synthase (1.13.11.46)
Abstract
The invention relates to a method for producing L-phenylglycine by genetic engineering strain biocatalysis, which comprises the following steps: connecting a gene for coding L-alpha-amino acid deaminase, a gene for coding hydroxymandelic acid synthetase and a vector by enzyme digestion to obtain a first recombinant plasmid; connecting a gene for coding mandelate dehydrogenase, a gene for coding leucine dehydrogenase and a vector through enzyme digestion to obtain a second recombinant plasmid; and (3) co-transferring the first recombinant plasmid and the second recombinant plasmid into a host cell to obtain the genetically engineered bacterium. According to the embodiment of the invention, the gene engineering bacteria obtained by the method can catalyze L-phenylalanine in a biocatalytic manner, so that the maximum yield of L-phenylglycine can be achieved, and the method has a wide industrial application prospect.
Description
Technical Field
The invention relates to the technical field of bioengineering, in particular to a method for producing L-phenylglycine by gene engineering strain biocatalysis.
Background
L-phenylglycine is a chiral unnatural amino acid, is an important raw material compound and a drug intermediate, can be used for synthesizing beta-lactam antibiotics such as penicillin, victimycin, pristinamycin I and the like, can also be used for synthesizing antitumor drugs taxol, and has wide market prospect in the field of medicine. L-phenylglycine is mainly synthesized by a chemical method, but the chemical synthesis reaction conditions are severe, the process is complex, byproducts are more, the optical purity of the product is lower, and further optical resolution is needed; and a large amount of organic solvents and toxic substances are needed in the synthesis process, so that the industrial pollution is serious.
Compared with chemical synthesis, biosynthesis has the advantages of strong selectivity, mild reaction conditions, environmental friendliness, low equipment requirement and the like, and green and environment-friendly biosynthesis is more concerned and favored with increasingly prominent environmental problems. Therefore, some reports have been made on the biosynthesis of phenylglycine by the phenylhydantoinase conversion method, the D-phenylglycine aminotransferase method, the amino acid dehydrogenase method, and the like. However, they still suffer from certain drawbacks, such as the need for assistance with sulfuric acid, low catalytic activity, or the need for additional coenzyme recycling systems, among others. Therefore, the development of a preparation route of L-phenylglycine, which has high conversion efficiency, good corresponding selectivity, low cost and environmental friendliness, is urgently needed.
Disclosure of Invention
The present invention is directed to solving, at least to some extent, one of the technical problems in the related art. Therefore, the invention aims to provide a method for producing L-phenylglycine by genetic engineering bacteria in a biocatalytic manner. The method can realize the biocatalytic high-efficiency synthesis of the L-phenylglycine by using the L-phenylalanine as a substrate, and has wide industrial application prospect.
To this end, in a first aspect of the present invention, the present invention provides a method for constructing a genetically engineered bacterium producing L-phenylglycine, comprising the steps of:
connecting a gene for coding L-alpha-amino acid deaminase, a gene for coding hydroxymandelic acid synthetase and a vector by enzyme digestion to obtain a first recombinant plasmid;
connecting a gene for coding mandelate dehydrogenase, a gene for coding leucine dehydrogenase and a vector through enzyme digestion to obtain a second recombinant plasmid;
co-transferring said first recombinant plasmid and said second recombinant plasmid into a host cell.
According to the method for constructing the genetically engineered bacteria for producing the L-phenylglycine, disclosed by the embodiment of the invention, the plasmids of co-expression L-alpha-amino acid deaminase (LAAD) and hydroxymandelate synthase (HmaS) are constructed, the plasmids of mandelate dehydrogenase (SMDH) and leucine dehydrogenase (leuDH) are co-expressed, and the two plasmids are simultaneously transferred into host cells to obtain a recombinant strain, wherein the recombinant strain takes the L-phenylalanine as a substrate, and the L-phenylglycine with the yield close to 100% is produced after cascade catalysis.
Alternatively, the first recombinant plasmid is a pET series vector as an expression vector, for example, pETDuet-1; the second recombinant plasmid is pRSF series vector as an expression vector, for example, pRSFDuet-1.
Alternatively, the host cell is an escherichia coli BL21(DE3) competent cell.
In a second aspect of the invention, the invention provides a genetically engineered bacterium for producing L-phenylglycine constructed by the above method. The genetic engineering bacteria take L-phenylalanine as a substrate, and produce L-phenylglycine with the yield close to 100% after cascade catalysis.
In a third aspect of the invention, the invention provides a method for expressing the genetically engineered bacteria, which comprises the steps of inoculating the genetically engineered bacteria in an LB culture medium, culturing overnight, transferring the overnight culture into a TB culture solution according to a ratio of 1:100, culturing for 2-3 h, and adding an inducer for induction culture for 16-24 h.
In the fourth aspect of the invention, the invention provides a method for producing L-phenylglycine, which takes L-phenylalanine as a substrate and takes the genetically engineered bacterium as a biocatalyst. The method takes the L-phenylalanine as a substrate to produce the high-yield L-phenylglycine, and has wide industrial application prospect.
Optionally, the reaction conditions of the method are 30 ℃ and 250rpm for 1-12 h.
In a fifth aspect of the present invention, the present invention provides the use of the above-mentioned process for producing L-phenylglycine for the preparation of L-phenylglycine.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Drawings
FIG. 1 shows the synthesis of L-phenylglycine catalyzed by four enzymes LAAD, HmaS, SMDH and LeuDH according to the present invention;
FIG. 2 is a time chart of the production of L-phenylglycine by Escherichia coli engineering bacteria according to an embodiment of the present invention;
FIG. 3 is a HPLC verification chart of the production of L-phenylglycine by Escherichia coli engineering bacteria according to the embodiment of the present invention;
Detailed Description
The technical solution of the present invention is illustrated by specific examples below. It is to be understood that one or more method steps mentioned in the present invention do not exclude the presence of other method steps before or after the combination step or that other method steps may be inserted between the explicitly mentioned steps; it should also be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Moreover, unless otherwise indicated, the numbering of the various method steps is merely a convenient tool for identifying the various method steps, and is not intended to limit the order in which the method steps are arranged or the scope of the invention in which the invention may be practiced, and changes or modifications in the relative relationship may be made without substantially changing the technical content.
In order to better understand the above technical solutions, exemplary embodiments of the present invention are described in more detail below. While exemplary embodiments of the invention have been shown, it should be understood that the invention may be embodied in various forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.
The test materials adopted by the invention are all common commercial products and can be purchased in the market; the related experiments are all routine experimental methods if not specifically stated.
Sources of materials used: coli BL21(DE3) and TOP10 are commercially available, E.coli BL21(DE3) is used for expression of all genes in the present invention, and TOP10 is used for vector construction. Escherichia coli expression vector pETDuet-1, pRSFDuet-1, from Novagen, Phusion Hi-Fi DNA polymerase, restriction enzyme from Xiamen Lulong biotechnology development company. Plasmid extraction kits, DNA purification kits, gel recovery kits and bacterial genome DNA extraction kits were purchased from Shanghai bioengineering, Inc.
The LB culture medium is: 10 g.L-1Peptone, 5 g. L-1Yeast powder, 5 g.L-1NaCl, the balance double distilled water, and the culture medium sterilized at 121 deg.C under 0.1Mpa for 20 min.
The TB culture medium is: 12 g.L-1Peptone, 24 g. L-1Yeast powder, 2.31 g.L-1KH2PO4、12.54g·L- 1K2HPO40.4% glycerol, and the balance of double distilled water, and sterilizing the culture medium at 121 deg.C under 0.1Mpa for 20 min.
The biosynthesis pathway of L-phenylglycine in the examples of the present invention is shown in FIG. 1:
taking L-phenylalanine as a substrate, synthesizing phenylpyruvic acid under the catalysis of L-alpha-amino acid deaminase LAAD, synthesizing (S) -mandelic acid under the catalysis of hydroxymandelic acid synthase HmaS, synthesizing benzoylformic acid under the catalysis of mandelic acid dehydrogenase SMDH, and finally synthesizing L-phenylglycine under the action of leucine dehydrogenase LeuDH.
The invention will now be described with reference to specific examples, which are intended to be illustrative only and not to be limiting in any way.
Example 1 construction of recombinant E.coli
1. Construction of pET-LAAD-Hmas plasmid
Taking the sequences shown in SEQ ID NO. 5 and SEQ ID NO. 6 as upstream and downstream primers (table 1), carrying out PCR amplification on an L-alpha-amino acid deaminase gene LAAD (SEQ ID NO. 1), and recovering a target band from the gel; wherein, the PCR amplification conditions are as follows: circulating for 30 times at 98 deg.C for 2min, 98 deg.C for 10s, 56 deg.C for 30s, and 72 deg.C for 1 min; 72 ℃ for 2 min.
PCR amplifying hydroxymandelate synthase gene HmaS (SEQ ID NO:2) by using SEQ ID NO:7 and SEQ ID NO:8 as primers, and recovering target bands from the gel; wherein, the PCR amplification conditions are as follows: circulating for 30 times at 98 deg.C for 2min, 98 deg.C for 10s, 56 deg.C for 30s, and 72 deg.C for 1 min; 72 ℃ for 2 min.
Respectively carrying out BsaI enzyme digestion on an L-alpha-amino acid deaminase gene LAAD and a hydroxymandelate synthase gene Hmas at 37 ℃ for 2 hours, and then carrying out PCR purification to recover enzyme digestion fragments; wherein, the enzyme cutting system is as follows: the 50. mu.L system contained 1. mu.L of BsaI enzyme and 1. mu.g of PCR product.
The purified and recovered fragment is expressed by BamHI and XhoI enzyme digestion, the vector pETDuet-1 is connected, and the connection is carried out for 1 hour at 16 ℃, so as to obtain a connection product, wherein the connection system is as follows: the 20. mu.L system contains 1. mu. L T4 ligase, as well as 1. mu.L pETDuet-1 vector and 2. mu.L of the target gene enzyme digestion product respectively.
The ligation product is transferred into escherichia coli TOP10 competent cells, and then a positive clone colony is obtained through verification of a T7 primer, so that a pET-LAAD-HmaS plasmid is obtained.
2. Construction of pRSF-SMDH-LeuDH plasmid
Taking sequences shown in SEQ ID NO 9 and SEQ ID NO 10 as upstream and downstream primers (table 1), taking a P.Putida KT2440 genome as a template, carrying out PCR amplification on a mandelate dehydrogenase gene SMDH (SEQ ID NO 3), and recovering a target band by using glue; wherein, the PCR amplification conditions are as follows: circulating for 30 times at 98 deg.C for 2min, 98 deg.C for 10s, 56 deg.C for 30s, and 72 deg.C for 1 min; 72 ℃ for 2 min.
Taking the sequences shown in SEQ ID NO. 11 and SEQ ID NO. 12 as upstream and downstream primers (table 1), taking a Bacillus cereus genome as a template, amplifying a leucine dehydrogenase gene LeuDH (SEQ ID NO. 4), and recovering a target band by using glue; wherein, the PCR amplification conditions are as follows: circulating for 30 times at 98 deg.C for 2min, 98 deg.C for 10s, 56 deg.C for 30s, and 72 deg.C for 1 min; 72 ℃ for 2 min.
Carrying out enzyme digestion on a mandelic acid dehydrogenase gene SMDH and a leucine dehydrogenase gene LeuDH for 2 hours at 37 ℃ respectively by BsaI, and then carrying out PCR purification to recover enzyme digestion fragments, wherein the enzyme digestion system is as follows: the 50. mu.L system contained 1. mu.L of BsaI enzyme and 1. mu.g of PCR product.
The purified and recovered fragment was ligated with the expression vector pRSFDuet-1 digested with BamHI and XhoI to obtain pRSF-SMDH-LeuDH plasmid. Wherein, the connector system is: the 20. mu.L system contained 1. mu. L T4 ligase, as well as 1. mu.L pRSFDuet-1 vector and 2. mu.L of the target gene cleavage product, respectively.
3. Obtaining recombinant strains of Escherichia coli
The pET-LAAD-HmaS plasmid and pRSF-SMDH-LeuDH plasmid obtained in the above 1 and 2 were introduced into E.coli BL21(DE3) competent cells by electric shock, and then the strain was inoculated into LB medium (containing two antibiotics of 50. mu.g/mL ampicillin and 50. mu.g/mL kanamycin) to be screened and cultured overnight at 37 ℃ to obtain an E.coli engineered strain.
Table 1: primers for PCR amplification
Example 2 Whole cell bioconversion of recombinant strains
The engineering bacteria of Escherichia coli prepared in example 1 are inoculated into 2mL LB culture solution, and then inoculated into 100mL TB culture solution according to the proportion of 1:100 for amplification culture for 2-3 h, OD600After reaching 0.6, 0.5mM IPTG is added to induce protein expression for 20h at 22 ℃, and the thalli are obtained by centrifugation.
Whole cell transformation system (2 mL): 10g/L dry cell weight, L-phenylalanine (10mM or 40mM), and finally supplemented with phosphate buffer (200mM, pH 8.0). The system reacts for 1-12 h at 30 ℃ and 250 rpm. Then, the sample was centrifuged, and the supernatant was collected and subjected to liquid chromatography to detect the amount of L-phenylglycine produced.
Product quantitative analysis: detecting and analyzing the conversion solution by using a Shimadzu high performance liquid chromatograph, and using a photodiode array detector (with a working wavelength of 210nm) and a Shimadzu C18 chromatographic column (4.6 multiplied by 250mm, 5 mu m), wherein the flow rate is 1ml/min, the column temperature is 40 ℃, and the sample injection amount is 10 mu L; mobile phase: 90% double distilled water, 0.1% trifluoroacetic acid, 10% acetonitrile.
As shown in FIG. 2, the yield of L-phenylglycine was close to 100% by mole after 12 hours of whole-cell catalysis with 10mM or 40mM L-phenylalanine.
As shown in FIG. 3, after 12 hours of whole-cell catalysis with 10mM or 40mM L-phenylalanine, HPLC assay showed complete conversion of L-phenylalanine to L-phenylglycine with no significant accumulation of reaction intermediates.
In summary, according to the embodiments of the present invention, a plasmid co-expressing L- α -amino acid deaminase (LAAD) and hydroxymandelate synthase (HmaS) is constructed, a plasmid co-expressing mandelate dehydrogenase (SMDH) and leucine dehydrogenase (leu) is co-expressed, and the two plasmids are simultaneously transferred into a host cell to obtain a recombinant strain, wherein the recombinant strain uses L-phenylalanine as a substrate and produces L-phenylglycine with a yield close to 100% after cascade catalysis; compared with other published or published inventions, the method does not need a coenzyme circulating system, reduces the production cost, is environment-friendly and has wide industrial application prospect.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above should not be understood to necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples described in this specification can be combined and combined by those skilled in the art.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
SEQUENCE LISTING
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tcaactgtcg tgcaaagtgc gagtggggcg gtaacactaa ccttgataga acccgatcgt 660
aatgccgacc ccgggcaaat tgatgagttc ctaaaagatc accagggtgc gggtgtgcag 720
cacatcgcct ttaattctaa tgacgctgtg cgtgcagtga aagcactgtc agagagaggg 780
gtcgagtttt tgaaaacccc gggggcgtat tacgatcttc taggagagag aataaccctg 840
caaacgcata gtctggatga tttgagagcg accaatgttt tggcagatga ggatcacgga 900
ggtcaacttt ttcaaatctt cacagcgagt acgcacccaa gacacaccat tttttttgaa 960
gtcatcgaaa gacaaggagc gggcactttt ggttccagta atatcaaggc tttatatgag 1020
gcagtggaac tagaacgtac aggtcaaagt gagtttggag ccgcgaggcg ttag 1074
<210> 3
<211> 1182
<212> DNA
<213> Artificial sequence
<400> 3
atgagccgga atctctttaa cgttgaggac tatcgcaagc tcgcgcaaaa gcgcttgcca 60
aagatggtgt acgactatct ggaaggtgga gctgaagacg aatacggggt gaaacacaac 120
cgcgacgtct tccagcaatg gcgattcaaa ccgaagcggc tggtagatgt cagccgccgc 180
agccttcaag cggaagtact tggaaagagg cagtcaatgc ctctcttgat tggcccaact 240
gggctgaatg gtgcgctctg gcctaaaggg gatctcgccc ttgctcaagc ggcaaccaag 300
gccggcatcc cgttcgtgct gtcgaccgcc tccaacatgt ccattgaaga cctcgctcgt 360
cagtgtgatg gcgatctatg gttccagctc tatgtgatcc accgagagat cgcgcagggg 420
atggtgctca aagccctgca ctccggttac accacgctgg tactcacaac agacgtcgcg 480
gttaatggct atcgcgagcg ggacctgcac aaccgattca agatgccgat gagctacacc 540
ccaaaggtga tgctggacgg atgcctgcat ccacgctggt cgctcgatct ggtgcgccac 600
ggcatgccgc aactggccaa cttcgtcagc agtcaaacgt ccagcttaga gatgcaggca 660
gcattgatga gccgccaaat ggatgccagt ttcaactggg aggcattgag atggctgcgt 720
gacctctggc cgcacaaact cctcgtaaag gggttgctca gtgctgagga cgccgatcgg 780
tgcatcgctg aaggtgccga cggcgtgatc ctgtcaaacc acggcggtcg ccaactcgat 840
tgcgcggtat cgccaatgga agttttggct caatcggtag cgaaaactgg aaaaccagtg 900
cttatcgata gcggcttccg acggggttcg gacatcgtta aagcgcttgc gctaggtgct 960
gaggctgtac tcctgggacg tgcaactttg tatggccttg cagcacgagg tgaaacgggt 1020
gttggcgagg tgctaaccct cctcaaagcg gatatcgacc gcactctggc ccagattgga 1080
tgccctgaca tcacctccct ttctcctgat tacctccaaa gcgagggagt gactaacacc 1140
gctccagtcg atcacctcat tggtaaagga acacacgcat ga 1182
<210> 4
<211> 1101
<212> DNA
<213> Artificial sequence
<400> 4
atgacattag aaatcttcga atacttagaa aaatatgatt atgagcaagt agtattttgt 60
caagataaag aatctggttt aaaagcaatt attgcaattc atgatacaac acttggaccg 120
gctcttggtg gaacaagaat gtggacatat gattctgaag aagcggcgat tgaagatgca 180
ttgcgtcttg caaaagggat gacatacaaa aacgcagcag ctggtttaaa cttaggtggt 240
gcgaaaacag taattatcgg tgatcctcgt aaagataaga gcgaagcaat gttccgtgca 300
ctaggacgtt atatccaagg actaaacgga cgttacatta cagctgaaga tgttggtaca 360
acagtagatg atatggatat tatccatgaa gaaactgact ttgtaacagg tatctcacca 420
tcattcggtt cttctggtaa cccatctccg gtaactgcat acggtgttta ccgtggtatg 480
aaagcagctg caaaagaagc tttcggtact gacaatttag aaggaaaagt aattgctgtt 540
caaggcgttg gtaacgtagc atatcaccta tgcaaacatt tacacgctga aggagcaaaa 600
ttaattgtta cagatattaa taaagaagct gtacaacgtg ctgtagaaga attcggtgca 660
tcagcagttg aaccaaatga aatttacggt gttgaatgcg atatttacgc accatgtgca 720
ctaggcgcaa cagttaatga tgaaactatt ccacaactta aagcaaaagt aatcgcaggt 780
tctgcgaata accaattaaa agaagatcgt catggtgaca tcattcatga aatgggtatt 840
gtatacgcac cagattatgt aattaatgca ggtggcgtaa ttaacgtagc agacgaatta 900
tatggataca atagagaacg tgcactaaaa cgtgttgagt ctatttatga cacgattgca 960
aaagtaatcg aaatttcaaa acgcgatggc atagcaactt atgtagcggc agatcgtcta 1020
gctgaagagc gcattgcaag cttgaagaat tctcgtagca cttacttacg caacggtcac 1080
gatattatta gccgtcgcta a 1101
<210> 5
<211> 37
<212> DNA
<213> Artificial sequence
<400> 5
ttggtctcgg atccgatgaa catttcacgt cgcaagc 37
<210> 6
<211> 36
<212> DNA
<213> Artificial sequence
<400> 6
ttggtctcat cctttacttc ttaaaacgat ccaaac 36
<210> 7
<211> 39
<212> DNA
<213> Artificial sequence
<400> 7
ttggtctcaa ggagatatat tatgcagaat ttcgaaatc 39
<210> 8
<211> 32
<212> DNA
<213> Artificial sequence
<400> 8
ttggtctcct cgagctaacg cctcgcggct cc 32
<210> 9
<211> 36
<212> DNA
<213> Artificial sequence
<400> 9
ttggtctcgg atccgatgag ccggaatctc tttaac 36
<210> 10
<211> 33
<212> DNA
<213> Artificial sequence
<400> 10
ttggtctcat ccttcatgcg tgtgttcctt tac 33
<210> 11
<211> 41
<212> DNA
<213> Artificial sequence
<400> 11
ttggtctcaa ggagataact ttatgacatt agaaatcttc g 41
<210> 12
<211> 36
<212> DNA
<213> Artificial sequence
<400> 12
ttggtctcct cgagttagcg acggctaata atatcg 36
Claims (8)
1. A method for constructing a genetic engineering bacterium for producing L-phenylglycine is characterized by comprising the following steps:
connecting a gene for coding L-alpha-amino acid deaminase, a gene for coding hydroxymandelic acid synthetase and a vector by enzyme digestion to obtain a first recombinant plasmid;
connecting a gene for coding mandelate dehydrogenase, a gene for coding leucine dehydrogenase and a vector through enzyme digestion to obtain a second recombinant plasmid;
co-transferring said first recombinant plasmid and said second recombinant plasmid into a host cell.
2. The method of claim 1, wherein the first recombinant plasmid is a pET-series vector as an expression vector; the second recombinant plasmid is pRSF series vector as an expression vector.
3. The method of claim 1, wherein the host cell is an escherichia coli BL21(DE3) competent cell.
4. A genetically engineered bacterium which is constructed by the method according to any one of claims 1 to 3 and produces L-phenylglycine.
5. A method for expressing the genetically engineered bacterium of claim 4,
the genetic engineering bacteria are firstly inoculated in an LB culture medium for overnight culture, then the overnight culture is transferred into a TB culture solution for culture for 2-3 h according to the proportion of 1:100, and then an inducer is added for induction culture for 16-24 h.
6. A method for producing L-phenylglycine, characterized in that L-phenylalanine is used as a substrate, and the genetically engineered bacterium of claim 4 is used as a biocatalyst.
7. The method of claim 6, wherein the reaction is carried out at 30 ℃ and 250rpm for 1-12 h.
8. Use of the process according to claims 6 to 7 for the preparation of L-phenylglycine.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102086462A (en) * | 2009-12-02 | 2011-06-08 | 中国科学院上海生命科学研究院 | Method for preparing chiral monomer mandelic acid |
| WO2017122747A1 (en) * | 2016-01-12 | 2017-07-20 | Ajinomoto Co., Inc. | Method for producing benzaldehyde |
| CN108103038A (en) * | 2017-12-15 | 2018-06-01 | 江南大学 | A kind of unicellular factory for efficiently synthesizing L- phenylglycines and its structure and application |
| CN110452920A (en) * | 2019-07-11 | 2019-11-15 | 南阳师范学院 | A kind of genetic engineering bacterium and with D, L- mandelic acid is the method that substrate prepares L- phenylglycine |
| CN111647616A (en) * | 2020-06-23 | 2020-09-11 | 厦门大学 | Construction method and application of escherichia coli engineering bacteria for producing protocatechuic acid |
-
2021
- 2021-04-13 CN CN202110394270.5A patent/CN113308486A/en active Pending
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102086462A (en) * | 2009-12-02 | 2011-06-08 | 中国科学院上海生命科学研究院 | Method for preparing chiral monomer mandelic acid |
| WO2017122747A1 (en) * | 2016-01-12 | 2017-07-20 | Ajinomoto Co., Inc. | Method for producing benzaldehyde |
| CN108103038A (en) * | 2017-12-15 | 2018-06-01 | 江南大学 | A kind of unicellular factory for efficiently synthesizing L- phenylglycines and its structure and application |
| CN110452920A (en) * | 2019-07-11 | 2019-11-15 | 南阳师范学院 | A kind of genetic engineering bacterium and with D, L- mandelic acid is the method that substrate prepares L- phenylglycine |
| CN111647616A (en) * | 2020-06-23 | 2020-09-11 | 厦门大学 | Construction method and application of escherichia coli engineering bacteria for producing protocatechuic acid |
Non-Patent Citations (2)
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| SHUANG PING LIU: "Heterologous pathway for the production of l-phenylglycine fromglucose by E. coli", JOURNAL OF BIOTECHNOLOGY, pages 91 - 97 * |
| YULING ZHU等: "One-Pot Synthesis of Aromatic Amines from Renewable Feedstocks via Whole-Cell Biocatalysis", CHEMISTRY EUROPE, pages 14292 - 14295 * |
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