CN116179526A - Halohydrin dehalogenase mutant, encoding gene thereof, recombinant plasmid containing encoding gene, genetically engineered bacterium containing recombinant plasmid and application of genetically engineered bacterium - Google Patents

Halohydrin dehalogenase mutant, encoding gene thereof, recombinant plasmid containing encoding gene, genetically engineered bacterium containing recombinant plasmid and application of genetically engineered bacterium Download PDF

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CN116179526A
CN116179526A CN202310271674.4A CN202310271674A CN116179526A CN 116179526 A CN116179526 A CN 116179526A CN 202310271674 A CN202310271674 A CN 202310271674A CN 116179526 A CN116179526 A CN 116179526A
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halohydrin dehalogenase
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黄和
宋金松
蒋可涵
丁继
黄圣博
薛锋
徐晴
张弛
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Abstract

The application discloses a halohydrin dehalogenase mutant, a coding gene thereof, a recombinant plasmid containing the coding gene, a genetic engineering bacterium containing the recombinant plasmid and application thereof, and belongs to the field of genetic engineering. The halohydrin dehalogenase mutant takes the amino acid sequence of the original halohydrin dehalogenase shown in SEQ ID NO.1 as a carrier, and carries out combined mutation on the amino acid sequence, wherein the combined mutation comprises the following steps: mutating asparagine at position 179 to leucine; mutating threonine at position 197 to alanine; mutating leucine at position 198 to tyrosine; mutating serine at position 224 to alanine; and mutating lysine at position 227 to arginine. The combined mutation can change the amino acid sequence directionally, improve the catalytic activity and stereoselectivity of the halohydrin dehalogenase mutant, simplify the number of mutation sites, omit specific transformation operation, save time and labor and have low cost, and are beneficial to industrial production.

Description

Halohydrin dehalogenase mutant, encoding gene thereof, recombinant plasmid containing encoding gene, genetically engineered bacterium containing recombinant plasmid and application of genetically engineered bacterium
Technical Field
The application belongs to the technical field of genetic engineering, and particularly relates to a halohydrin dehalogenase mutant, a coding gene thereof, a recombinant plasmid containing the coding gene, genetic engineering bacteria containing the recombinant plasmid and application thereof.
Background
(S) -1-chloro-3-phenoxy-2-propanol and (S) -1-azido-3-phenoxy-2-propanol are used as common medical intermediates, and can be used in the medical industry and can also be used for preparing dispersing agents and opacifiers with excellent performances. However, when (S) -1-chloro-3-phenoxy-2-propanol and (S) -1-azido-3-phenoxy-2-propanol are synthesized by asymmetric borohydride method, (transfer) hydrogenation method, biocatalytic reduction of R-chloroketone, dynamic kinetic resolution of halohydrin and other methods, production and application are greatly limited because of low optical activity, long time consumption, environmental pollution and other problems.
In order to overcome the defects of the synthesis method, the method can be used for preparing the racemization 1-chloro-3-phenoxy-2-propanol in an azide reagent system by utilizing a halohydrin dehalogenase, and various halohydrin dehalogenase mutants are developed by researchers in order to solve the defects of low stereoselectivity and low yield of wild type halohydrin dehalogenase. For example, patent application No. CN2021112534635 discloses a halohydrin dehalogenase mutant, which has significantly improved stereoselectivity, catalytic activity and substrate yield by mutating the 21 amino acid sequences of wild-type halohydrin dehalogenase and adding 6 amino acids at the N-terminus.
However, the catalytic activity and stereoselectivity of the halohydrin dehalogenase mutant in the prior art still cannot meet the actual demands, and the mutant modification of the original halohydrin dehalogenase has the disadvantages of large workload, complicated operation and high cost, and greatly limits industrial application.
Disclosure of Invention
The purpose of the application is to provide a halohydrin dehalogenase mutant, a coding gene thereof, a recombinant plasmid containing the coding gene, a genetic engineering bacterium containing the recombinant plasmid and application thereof, and aims to solve the problems of low catalytic activity and stereoselectivity of the existing halohydrin dehalogenase mutant; the technical problems of large workload, complicated operation and high cost of mutation transformation.
In order to achieve the above purpose, the technical scheme of the application is as follows:
in a first aspect, the present application provides a halohydrin dehalogenase mutant. The halohydrin dehalogenase mutant takes the amino acid sequence of the original halohydrin dehalogenase shown in SEQ ID NO.1 as a carrier, and carries out the following combined mutation on the amino acid sequence:
i: mutating asparagine at position 179 to leucine;
ii: mutating threonine at position 197 to alanine;
iii: mutating leucine at position 198 to tyrosine;
iv: mutating serine at position 224 to alanine;
v: the mutation of lysine at position 227 was made to arginine.
In an alternative implementation of the first aspect, the halohydrin dehalogenase mutant has the amino acid sequence shown in SEQ ID No. 2.
In an alternative implementation of the first aspect, the nucleotide sequence of the halohydrin dehalogenase mutant is shown in SEQ ID No. 3.
In a second aspect the present application provides a gene encoding a halohydrin dehalogenase mutant according to the first aspect. The coding gene is shown as SEQ ID NO. 4.
In a third aspect the present application provides a recombinant plasmid comprising a gene encoding a halohydrin dehalogenase mutant according to the second aspect.
In an alternative implementation of the third aspect, the expression vector of the recombinant plasmid is pET28a (+).
In a fourth aspect, the present application provides a genetically engineered bacterium comprising a gene encoding a halohydrin dehalogenase mutant according to the second aspect.
In an optional implementation manner of the fourth aspect, the expression host of the genetically engineered bacterium is e.coli BL21 (DE 3).
In a fifth aspect, the present application provides an application of the genetically engineered bacterium described in the fourth aspect in catalyzing synthesis of (S) -1-chloro-3-phenoxy-2-propanol from 1-chloro-3-phenoxy-2-propanol.
In an optional implementation manner of the fifth aspect, the application method includes:
s1: suspending genetically engineered bacteria in a suspension containing NaN 3 Is of Tris-SO 4 A buffer solution system to obtain a resolution reaction system;
s2: after adding 1-chloro-3-phenoxy-2-propanol into the resolution reaction system, carrying out oscillation reaction at the temperature of 30 ℃ and the rotating speed of 200r/min, purifying and drying the reaction product, and thus completing the catalytic resolution.
Compared with the prior art, the advantage or beneficial effect of this application includes at least:
according to the halohydrin dehalogenase mutant provided by the first aspect, the amino acid sequence of the original halohydrin dehalogenase shown in SEQ ID NO.1 is subjected to combined mutation, so that the amino acid and the related nucleotide sequence are subjected to directional structure and functional change, the catalytic activity and stereoselectivity of the halohydrin dehalogenase mutant are greatly improved, the number of mutation sites is reduced, the specific transformation operation is omitted, time and labor are saved, the cost is low, and industrial production is facilitated. The test results of the examples show that the specific enzyme activity of the halohydrin dehalogenase mutant reaches 7.5U/g, and the catalytic enantioselectivity reaches 193.09; the ee value of the (S) -1-chloro-3-phenoxy-2-propanol generated by catalysis is more than 99.99%, and the yield reaches 47.49%.
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In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are required to be used in the description of the embodiments will be briefly described below. It is obvious that the drawings in the following description are only some embodiments described in the present application, and that other drawings may be obtained from these drawings without inventive effort for a person of ordinary skill in the art.
FIG. 1 is a high performance liquid chromatogram of the substrate 1-chloro-3-phenoxy-2-propanol provided in the examples herein;
FIG. 2 is a high performance liquid chromatogram of a genetically engineered bacterium catalyzed reaction comprising a halogen alcohol dehalogenase mutant provided in an embodiment of the present application.
Detailed Description
The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. It will be apparent that the described embodiments are some, but not all, of the embodiments of the present application. All other embodiments, which can be made by one of ordinary skill in the art without undue burden from the present disclosure, are within the scope of the present disclosure.
In the following description of the present embodiment, the term "and/or" is used to describe an association relationship of association objects, which means that three relationships may exist, for example, a and/or B may mean: a alone, B alone and both a and B. Wherein A, B may be singular or plural. The character "/" generally indicates that the context-dependent object is an "or" relationship.
In the following description of the present embodiments, the term "at least one" means one or more, and "a plurality" means two or more. "at least one of" or the like means any combination of these items, including any combination of single item(s) or plural items(s). For example, "at least one (individual) of a, b, or c," or "at least one (individual) of a, b, and c" may each represent: a, b, c, a-b (i.e., a and b), a-c, b-c, or a-b-c, wherein a, b, c may be single or multiple, respectively.
It should be understood by those skilled in the art that, in the following description of the embodiments of the present application, the sequence number does not mean that the sequence of execution is not sequential, and some or all of the steps may be executed in parallel or sequentially, and the execution sequence of each process should be determined by its functions and inherent logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.
The terminology used in the embodiments of the application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. As used in this application in the examples and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.
In a first aspect, embodiments herein provide a halohydrin dehalogenase mutant. The halohydrin dehalogenase mutant takes the amino acid sequence of the original halohydrin dehalogenase shown in SEQ ID NO.1 as a carrier, and carries out the following combined mutation on the amino acid sequence:
i: mutating asparagine at position 179 to leucine;
ii: mutating threonine at position 197 to alanine;
iii: mutating leucine at position 198 to tyrosine;
iv: mutating serine at position 224 to alanine;
v: the mutation of lysine at position 227 was made to arginine.
It should be noted that the amino acid names in the sequence shown in SEQ ID NO.1 match the single letter abbreviations of the amino acids in Table 1 below.
TABLE 1 names and abbreviations for amino acids
Figure BDA0004134910840000051
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Figure BDA0004134910840000061
Therefore, in the embodiment of the application, the amino acid sequence of the original halohydrin dehalogenase shown in SEQ ID NO.1 is taken as a carrier, and after the combination mutation is carried out on specific sites in the amino acid sequence, the amino acid sequence of the obtained halohydrin dehalogenase mutant is shown in SEQ ID NO. 2. Wherein, the amino acid sequence of the mutant of the embodiment is represented by replacing the corresponding amino acid site in the amino acid sequence of the original halohydrin dehalogenase with a mutant amino acid, and the number of the mutant site corresponds to the amino acid sequence site of the original halohydrin dehalogenase.
Meanwhile, the present examples provide nucleotide sequences based on the amino acid sequences of the original halohydrin dehalogenase described above, the nucleotide sequences of which are shown in SEQ ID NO. 5. Based on the specific nucleotide sequence of each amino acid, the nucleotide sequence of the obtained halohydrin dehalogenase mutant is shown as SEQ ID NO.3 after the combined mutation is carried out in the embodiment of the application. Wherein, the nucleotide sequence of the mutant of the embodiment is represented by replacing the corresponding nucleotide site in the nucleotide sequence of the original halohydrin dehalogenase with the nucleotide of the mutant amino acid, and the number of the mutation site corresponds to the nucleotide sequence site of the original halohydrin dehalogenase.
It will be appreciated by those skilled in the art that the above-described modification methods of the combination mutation are all implemented according to related genetic engineering techniques known in the art, and specific genetic mutation techniques are not particularly limited in the examples herein, so that those skilled in the art can obtain the target mutant by the above-described combination mutation.
In addition, the present examples performed individual mutation on each mutation site to verify the technical effect of the combination mutation described above, and as a result found that individual mutation on each site could not achieve the catalytic synthesis resolution effect of the combination mutation.
The amino acid and the related nucleotide sequence of the original halohydrin dehalogenase shown in SEQ ID NO.1 are subjected to combined mutation, so that the oriented structure and the oriented functionalization of the amino acid and the related nucleotide sequence are changed, the catalytic activity and the stereoselectivity of the halohydrin dehalogenase mutant are greatly improved, the number of mutation sites is obviously reduced, the specific transformation operation is omitted, and the method is time-saving, labor-saving, low in cost and beneficial to industrial production.
In a second aspect, embodiments of the present application also provide a gene encoding the halohydrin dehalogenase mutant of the first aspect. The coding gene is shown as SEQ ID NO. 4.
Wherein, the embodiment of the application forms a coding gene with the halohydrin dehalogenase mutant by coding the halohydrin dehalogenase mutant in the first aspect. In particular to a method for synthesizing coding genes with mutant enzymes by a total synthesis method based on genetic engineering technology. Wherein the coding gene of the halohydrin dehalogenase mutant is shown in SEQ ID NO. 4; meanwhile, for convenience of description, the gene encoding the halohydrin dehalogenase mutant is expressed as HHDH22 in the examples of the present application.
In a third aspect, embodiments of the present application also provide a recombinant plasmid comprising a gene encoding the halohydrin dehalogenase mutant of the second aspect.
Among them, the present embodiment provides a method for preparing the recombinant plasmid described above, preferably comprising:
carrying out double enzyme digestion on the encoding gene of the halohydrin dehalogenase mutant and an expression vector pET28a of escherichia coli by using restriction endonucleases Nco I and Xho I respectively for 3-6 hours, recovering enzyme digestion products, and connecting for 16 hours at the temperature of 16 ℃ by using T4DNA ligase to obtain recombinant plasmids containing the encoding gene of the halohydrin dehalogenase mutant; meanwhile, for convenience of description, the recombinant plasmid is expressed as pET28a-HHDH22 in the examples of the present application.
The specific sources of T4DNA ligase, restriction enzymes Nco I and Xho I in the examples of the present application are not particularly limited, and may be obtained, for example, from Ferretens company in a commercially available manner or synthesized according to a genetic engineering method known in the related art.
In a fourth aspect, embodiments of the present application also provide a genetically engineered bacterium comprising a gene encoding the halohydrin dehalogenase mutant of the second aspect.
In the embodiment of the application, escherichia coli is preferably used as a host, and the recombinant plasmid is expressed to synthesize the genetically engineered bacterium, which specifically comprises the following steps:
s41: transforming recombinant plasmid pET28a-HHDH22 into E.coli BL21 (DE 3) receptor bacteria, coating on LB agar plates containing kanamycin (with the mass concentration of 50 mg/L), and culturing for 12 hours at 37 ℃ to enable single colonies to grow on the plates; after randomly picking single colony and carrying out single clone, inoculating the single colony into LB liquid culture medium for culturing for 12 hours, extracting plasmids for sequencing, and screening out genetic engineering bacteria according to a sequencing result, wherein the genetic engineering bacteria are expressed as positive clone E.coli BL21 (DE 3)/pET 28a-HHDH22.
S42: inoculating the genetically engineered bacteria into 4mL of LB culture medium containing 50mg/L kanamycin, and initially culturing for 12h at 37 ℃ and a rotating speed of 200 r/min; the initially cultured genetically engineered bacteria were inoculated at an inoculum size of 1vt.% into a new 30mL LB medium containing kanamycin at a mass concentration of 50mg/L, and cultured at a temperature of 37℃and a rotational speed of 200r/min to an optical density (OD 600 ) 0.6-0.8; then adding isopropyl-beta-D-thiopyran galactoside (IPTG) inducer to a final concentration of 0.1mM, and carrying out induction culture for 16h under the conditions of 25 ℃ and 200r/min of rotating speed; finally, the cells were centrifuged at 5000 Xg for 5min and collected, and the cells were washed with Tris-SO having pH=7.0 4 The buffer solution was subjected to resuspension washing, and centrifuged at 13000 Xg for 1min, and the wet cells were collected and stored at-20℃for further use.
The composition of the LB agar plate includes: 10.0g/L tryptone, 5.0g/L, naCl and 10.0g/L yeast powder; the composition of the LB solid medium comprises: 10.0g/L tryptone, 5.0g/L, naCl 10.0.0 g/L yeast powder and 15.0g/L agar. Wherein, the LB agar plate and the LB solid medium are autoclaved at a temperature of 121 ℃ for 20min.
The embodiment of the application utilizes the coding gene to code the halohydrin dehalogenase mutant, and expresses the recombination force carrying the coding gene, so that the genetically engineered bacterium can be obtained. Researches show that the purity of (S) -1-chloro-3-phenoxy-2-propanol and (S) -1-azido-3-phenoxy-2-propanol are higher than 99.99% and the yield is over 45% through the catalytic synthesis of the genetically engineered bacteria. Compared with the catalytic reaction of the original halohydrin dehalogenase, the enantioselectivity is improved by 11.9 times.
In a specific embodiment, the expression host of the genetically engineered bacterium is preferably E.coli BL21 (DE 3).
In a fifth aspect, the embodiment of the application also provides an application of the genetically engineered bacterium in the fourth aspect in catalyzing synthesis of (S) -1-chloro-3-phenoxy-2-propanol from 1-chloro-3-phenoxy-2-propanol.
The specific method for catalytically splitting the substrate 1-chloro-3-phenoxy-2-propanol by using the genetically engineered bacterium comprises the following steps:
s51: 50mg of genetically engineered bacteria were weighed and suspended in 1mL of 30mM NaN-containing solution 3 Is of Tris-SO 4 In a buffer system (ph=7.0, 50 mM), a resolution reaction system was obtained.
S51: after adding 1-chloro-3-phenoxy-2-propanol with the concentration of 20mM into the resolution reaction system, carrying out oscillation reaction for 40-80min under the conditions of the temperature of 30 ℃ and the rotating speed of 200 r/min. Wherein, in the oscillation reaction, 0.4mL is sampled at regular time, 900 mu L of ethyl acetate is added into the sample, the oscillation reaction is carried out for 20min under the conditions of the temperature of 30 ℃ and the rotating speed of 200r/min, 13000 Xg is used for centrifugation for 1min, 500 mu L of ethyl acetate is taken out, after air drying overnight, 1mL of mixed reagent (n-hexane: isopropanol=1:1) is adopted for re-dissolution, and 0.22 mu m of organic film is used for filtration, thus the catalytic resolution of the substrate 1-chloro-3-phenoxy-2-propanol can be completed.
The test results of the examples show that the genetically engineered bacteria preferentially hydrolyze (R) -1-chloro-3-phenoxy-2-propanol. Wherein the specific enzyme activity of the halohydrin dehalogenase mutant is 7.5U/g, the enantioselectivity catalyzed by the halohydrin dehalogenase mutant reaches 193.09, the ee value of the (S) -1-chloro-3-phenoxy-2-propanol catalyzed by the halohydrin dehalogenase mutant is more than 99.99%, and the yield is 47.49%.
In order to further illustrate the technical effects of the genetically engineered bacterium containing the halohydrin dehalogenase mutant, the embodiment of the application provides an application test of the genetically engineered bacterium containing the original halohydrin dehalogenase in catalyzing and splitting a substrate 1-chloro-3-phenoxy-2-propanol. The test method is the same as the specific operation and parameter setting of the catalytic splitting method, except that the genetically engineered bacteria containing the halogen alcohol dehalogenase mutant are replaced by genetically engineered bacteria containing the original halogen alcohol dehalogenase.
The test results show that the genetically engineered bacteria containing the original halohydrin dehalogenase also preferentially hydrolyze (R) -1-chloro-3-phenoxy-2-propanol. Wherein, the specific enzyme activity of the original halohydrin dehalogenase is 3.4U/g, and the catalytic enantiomer selectivity is 16.2. Therefore, the enantiomer selectivity catalyzed by the genetically engineered bacterium comprising the halohydrin dehalogenase mutant is 11.9 times that of the genetically engineered bacterium comprising the original halohydrin dehalogenase, and the enzyme activity of the halohydrin dehalogenase mutant is 2.2 times that of the original halohydrin dehalogenase.
The high performance liquid phase analysis of the substrate 1-chloro-3-phenoxy-2-propanol in the examples of the application comprises the following specific steps:
using the Agilent-1220 system, column type: chiralcel OD-H column (Daicel Co., japan;4.6X250mmL,5 μm); chromatographic conditions: the column temperature is 35 ℃;
the chromatographic parameters are: the mobile phase is n-hexane: isopropanol=88:12 (v/v); the flow rate is 0.8mL/min; the UV wavelength was 220nm and the result was shown in FIG. 1. Among them, FIG. 1 shows a high performance liquid chromatogram of the substrate 1-chloro-3-phenoxy-2-propanol.
As can be seen from FIG. 1, the retention time of (S) -1-chloro-3-phenoxy-2-propanol in the standard 1-chloro-3-phenoxy-2-propanol was about 11.3min, and the retention time of (R) -1-chloro-3-phenoxy-2-propanol was about 18.6min.
Substrate ee S ＝[(S-R)/(S+R)]×100％，
Alternatively, the substrate ee S ＝[(R-S)/(R+S)]×100％；
E＝ln[(1-c)×(1-ee S )]/ln[(1-c)×(1+ee S )]。
Wherein R and S are the peak areas of the (R) -and (S) -substrates and c is the rac-substrate conversion.
Meanwhile, the results of the present example using genetically engineered bacteria comprising halogen alcohol dehalogenase mutants to catalyze the resolution of the substrate 1-chloro-3-phenoxy-2-propanol are shown in FIG. 2. Wherein, FIG. 2 shows a high performance liquid chromatogram of a genetically engineered bacterium catalyzed reaction comprising a halohydrin dehalogenase mutant.
As can be seen from FIG. 2, (S) -1-chloro-3-phenoxy-2-propanol showed a peak at 11.3min, and (R) -1-chloro-3-phenoxy-2-propanol was completely converted without a peak, with a higher yield of 47.4%.
In summary, the halohydrin dehalogenase mutant contained based on the genetically engineered bacterium has excellent performances of high specific activity and high stereoselectivity. Therefore, after the genetically engineered bacterium is used for catalyzing 1-chloro-3-phenoxy-2-propanol to synthesize (S) -1-chloro-3-phenoxy-2-propanol, the enantioselectivity reaches 193.09, and the ee value of the (S) -1-chloro-3-phenoxy-2-propanol generated by the catalysis is more than 99.99%, and the yield reaches 47.49%.
Various embodiments in this specification are described in an incremental manner, and identical or similar parts of the various embodiments are referred to each other, with each embodiment focusing on differences from the other embodiments.
The above embodiments are only for illustrating the technical solution of the present application, and not for limiting the present application; although the present application has been described in detail with reference to the foregoing embodiments, it should be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced with equivalents; such modifications and substitutions do not depart from the spirit of the corresponding technical solutions.

Claims (10)

1. A halohydrin dehalogenase mutant, which is characterized in that the amino acid sequence of the original halohydrin dehalogenase shown in SEQ ID NO.1 is taken as a carrier, and the amino acid sequence is subjected to the following combined mutation:
i: mutating asparagine at position 179 to leucine;
ii: mutating threonine at position 197 to alanine;
iii: mutating leucine at position 198 to tyrosine;
iv: mutating serine at position 224 to alanine;
v: the mutation of lysine at position 227 was made to arginine.
2. The halohydrin dehalogenase mutant according to claim 1, wherein the halohydrin dehalogenase mutant has the amino acid sequence shown in SEQ ID No. 2.
3. The halohydrin dehalogenase mutant according to claim 2, wherein the nucleotide sequence of the halohydrin dehalogenase mutant is shown in SEQ ID No. 3.
4. A gene encoding a halohydrin dehalogenase mutant according to any one of claims 1 to 3, wherein the encoding gene is shown in SEQ ID No. 4.
5. A recombinant plasmid comprising a gene encoding the halohydrin dehalogenase mutant according to claim 4.
6. The recombinant plasmid according to claim 5, wherein the expression vector of the recombinant plasmid is pET28a (+).
7. A genetically engineered bacterium comprising a gene encoding the halohydrin dehalogenase mutant according to claim 4.
8. The genetically engineered bacterium of claim 7, wherein the genetically engineered bacterium has an expression host of e.collbl21 (DE 3).
9. Use of the genetically engineered bacterium of claim 7 or 8 for catalyzing synthesis of (S) -1-chloro-3-phenoxy-2-propanol from 1-chloro-3-phenoxy-2-propanol.
10. The use according to claim 9, wherein the application method comprises:
s1: suspending genetically engineered bacteria in a suspension containing NaN 3 Is of Tris-SO 4 A buffer solution system to obtain a resolution reaction system;
s2: after adding 1-chloro-3-phenoxy-2-propanol into the resolution reaction system, carrying out oscillation reaction at the temperature of 30 ℃ and the rotating speed of 200r/min, purifying and drying the reaction product, and thus completing the catalytic resolution.
CN202310271674.4A 2023-03-20 2023-03-20 Halohydrin dehalogenase mutant, encoding gene thereof, recombinant plasmid containing encoding gene, genetically engineered bacterium containing recombinant plasmid and application of genetically engineered bacterium Pending CN116179526A (en)

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