CN117305194A - Amycolatopsis mutant strain and application thereof - Google Patents

Amycolatopsis mutant strain and application thereof Download PDF

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CN117305194A
CN117305194A CN202310234577.8A CN202310234577A CN117305194A CN 117305194 A CN117305194 A CN 117305194A CN 202310234577 A CN202310234577 A CN 202310234577A CN 117305194 A CN117305194 A CN 117305194A
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amycolatopsis
gene
seq
vanillin
vdh
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郑璞
王冠娜
吴丹
陈鹏程
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Jiangnan University
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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12Y102/01067—Vanillin dehydrogenase (1.2.1.67)
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Abstract

The invention discloses a amycolatopsis mutant strain and application thereof, belonging to the technical field of genetic engineering. The invention successfully performs traceless knockout on vanillin dehydrogenase gene (vdh) and 4-hydroxy-3-methoxybenzoyl-beta-hydroxypropionyl coenzyme dehydrogenase gene (phdB) in amycolatopsis CCTCC NO: M2011265 by using a CRISPR/Cas12a gene editing system to obtain amycolatopsis delta vdh delta phdB strain, and the amycolatopsis delta vdh delta phdB strain is fermented in a 3L fermentation tank for 60 hours to produce 20.4g/L of vanillin, and has good industrial application value for substrate ferulic acid molar conversion rate reaching 90.5%.

Description

Amycolatopsis mutant strain and application thereof
Technical Field
The invention relates to a amycolatopsis mutant strain and application thereof, belonging to the technical field of genetic engineering.
Background
Vanillin is one of the most important fragrances in the world, has the reputation of "the king of fragrances", and is widely used in foods, chemical industry, medicine, agriculture and the like. In the aspect of food, the flavoring agent has the effects of assisting fragrance and enhancing flavor; in chemical industry, vanillin can be used for toothpaste, perfumed soap, perfume and various cosmetics, and is a raw material for preparing various toilet products, perfumes and cosmetics; in medicine, vanillin is an important raw material for synthesizing various medicines; vanillin can be used as a yield increasing agent and a ripening agent in agriculture, and can also be used for synthesizing herbicides; in addition, the vanillin can be used as a bactericide, a defoaming agent, an electroplating additive and the like, and the vanillin produced at home and abroad can not meet the current market demands far because of the wide application fields. Vanillin on the market is mainly divided into synthetic vanillin and natural vanillin, wherein the synthetic vanillin takes the dominant position on the market. The production mode of synthesizing vanillin is continuously perfect, and there are nearly 10 chemical synthesis methods, and the market share of the vanillin exceeds 90%. Because the planting of vanilla is affected by climate, the yield is limited, the natural vanillin is extremely expensive and exceeds 100 times of the chemical synthesis method, and the price per kilogram is about 1000 to 4000 dollars. The annual total yield of vanillin reaches 2 ten thousand tons, while the yield of vanillin in China occupies about 70% of the global total yield. The microorganism used in the microbial transformation method has various types, the substrate is mainly renewable resources of ferulic acid, vanillin produced by biological transformation of a natural substrate is called natural vanillin, raw materials containing ferulic acid are used as substrates, and the biological transformation is utilized to produce the vanillin which accords with the edible safety requirements of European Union (EU) and Food and Drug Administration (FDA) on natural essence, and the vanillin has advantages in terms of price and quality compared with chemically synthesized vanillin; the microbial transformation method has short period, high yield and less pollution, and is favorable for industrial production, so the microbial transformation method has become a trend for producing natural vanillin, and is an important way for improving the yield of vanillin produced by microorganisms and realizing industrial production by breeding excellent microbial strains and optimizing fermentation processes.
Amycolatopsis CCTCC NO: M2011265 (described in the Chinese patent publication No. CN 102321563B) is a strain capable of producing vanillin by fermentation using ferulic acid as a precursor. At present, a genome editing method based on specific locus recombination and homologous recombination is already applied to genome modification of amycolatopsis, but the operation method is complex and takes long time, and the construction efficiency of a target strain is reduced. For example, CN 113789292B discloses the knocking out of ROK family transcription regulatory genes ROK-TR1, ROK-TR2 or ROK-TR3 by homologous recombination. After the ROK-TR1 gene is knocked out, the vanillin yield of amycolatopsis is improved by 33% compared with that of an outgoing strain. CN 108138125B patent discloses a amycolatopsis strain derived from amycolatopsis Zyl926 strain with at least one additional copy of FCS and ECH genes encoding feruloyl-CoA synthase and enoyl-CoA hydratase/aldolase integrated at the integration site of phage ψc31. CN 113717914A patent discloses a method for improving homologous recombination efficiency of gene targeting technology in amycolatopsis application by ku1 and/or ku2 gene deletion.
CRISPR/Cas, which naturally occurs in most bacteria and archaea, plays an immune defensive role, and consists of CRISPR arrays and Cas protein-encoding genes, has been widely used in mediating genome editing in various organisms and cell lines. Compared with the gene targeting technology, the CRISPR/Cas gene editing technology has the advantages of high efficiency, rapidness, simplicity and convenience and low cost. CN113061560a discloses that Vdh gene of amycolatopsis cctccc NO: M2011265 is knocked out by using CRISPR/Cas9 editing system (CN 113061560 a). Unlike Cas9, cas12a can not only cleave target DNA, but also cleave RNA precursors transcribed from CRISPR itself, forming mature CrRNA (CRISPRRNA), cas12a protein has a lower molecular weight than Cas9 protein, reducing the difficulty of transferring CRISPR systems into cells, and Cas12a has less toxicity to prokaryotes. Therefore, the CRISPR/Cas12a system mediated gene editing has obvious advantages, greatly expands the selection range of gene editing target sites, and is beneficial to breaking through and overcoming the limitation of low editing efficiency of the CRISPR/Cas9 editing system in amycolatopsis application.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a novel high-yield vanillin mutant amycolatopsis, the molar yield of a fermentation product is higher, and the impurity content such as vanillyl alcohol and vanillic acid content is lower.
The invention provides a amycolatopsis mutant strain, which is obtained by knocking out genes expressing vanillin dehydrogenase and genes expressing 4-hydroxy-3-methoxybenzoyl-beta-hydroxypropionyl CoA dehydrogenase in the genome of amycolatopsis.
In one embodiment of the invention, the mutant strain uses amycolatopsis CCTCC NO: M2011265 as host cell.
In one embodiment of the present invention, the sequence of the gene expressing vanillin dehydrogenase is shown in SEQ ID NO. 1.
In one embodiment of the invention, the gene sequence of the 4-hydroxy-3-methoxybenzoyl-beta-hydroxypropionyl CoA dehydrogenase is shown in SEQ ID NO. 2.
The invention also provides a method for improving the yield of vanillin prepared by amycolatopsis, which comprises the step of knocking out genes expressing vanillin dehydrogenase and genes expressing 4-hydroxy-3-methoxybenzoyl-beta-hydroxypropionyl CoA dehydrogenase in the genome of amycolatopsis.
In one embodiment of the invention, the mutant strain uses amycolatopsis CCTCC NO: M2011265 as host cell.
The invention also provides a method for carrying out gene knockout in amycolatopsis based on the CRISPR/Cas12a editing system, which leads CRISPR/Cas12a editing plasmids WGN01 and WGN02 into amycolatopsis and amycolatopsis delta vdh strains respectively, realizes vdh and phdB gene knockout, simplifies genetic operation, enriches gene editing tools of amycolatopsis, and promotes metabolic engineering modification for improving the performance of amycolatopsis. In addition, it may be suggested for metabolic engineering of other strains lacking genetic manipulation.
The invention also provides a construction method of the amycolatopsis mutant strain, which comprises the following steps:
step one, obtaining a target crRNA sequence: target crRNA is obtained by designing a target sequence of a target amycolatopsis CCTCC NO: M2011265 genome according to the requirement; contains the vdh gene target sequence (SEQ ID NO. 3) and contains the phdB gene target sequence (SEQ ID NO. 4);
step two, constructing a skeleton plasmid pULcrRNA-km-Cas12a containing Cas12a and crRNA;
step three, constructing a traceless knockout vdh gene editing plasmid WGN01;
step four, constructing a traceless knockout phdB gene editing plasmid WGN02;
step five, the editing plasmid WGN01 is electrodeposited into amycolatopsis CCTCC NO: M2011265; obtaining a amycolatopsis delta vdh strain;
step six, the editing plasmid WGN02 is electrically transferred into the amycolatopsis delta vdh strain to obtain the amycolatopsis delta vdh delta phdB strain.
In one embodiment of the present invention, the crRNA sequence containing the target sequence of the vdh gene in the first step is shown in SEQ ID NO.3, and the crRNA sequence containing the target sequence of the phdB gene is shown in SEQ ID NO. 4.
In one embodiment of the invention, the crRNA method for designing a target sequence containing the vdh gene is: the spacer sequence of the vdh gene was designed using the CRISPR RGEN Tools (rgenome. Net) website; out-of-frame Score greater than 66 and RGEN Target sequence with GC content of 40-60% were selected from 23nt results of the obtained series of sgRNAs, and the sequences are shown in SEQ ID NO.3, respectively.
In one embodiment of the invention, crRNA is designed that contains the phdB gene target sequence: the spacer sequence of phdB gene was obtained using a CRISPR RGEN Tools (rgenome. Net) website design; out-of-frame Score greater than 66 and RGEN Target sequence with GC content of 40-60% were selected from 23nt results of the obtained series of sgRNAs, and the sequences are shown in SEQ ID NO.4, respectively.
In one embodiment of the present invention, in the second step, the construction method of the backbone plasmid pULcrRNA-km-Cas12a containing Cas12a and crRNA comprises the following steps: the Km (SEQ ID NO. 32) promoter fragment from the Cas12a fragment (SEQ ID NO. 27) from Francisella tularensis subsp.Novicida, the pLYZYP01 plasmid (described in the patent application text with publication No. CN 113061560A) and the linearized pULcrRNA (disclosed in Crispr-Cas12a-assisted GenomeEditing in Amycolatopsis mediterranei) plasmids were joined by a single cloning procedure to give the plasmid pULcrRNA-Km-Cas12a.
In one embodiment of the invention, step three, the pULcrRNA-km-Cas12a plasmid is taken as a template, a CRISPR RGEN Tools (rgenome.net) website is used for designing a spacer sequence of the vdh gene, primers are designed, the primer sequences shown in SEQ ID No.7 and SEQ ID No.8 are subjected to PCR amplification, and the PCR products are transferred into escherichia coli for cyclization after purification, and the pULcrRNA delta vdh plasmid is obtained;
then the genome of amycolatopsis CCTCC No. M2011265 is used as a template, and primer sequences shown as SEQ ID No.9 and SEQ ID No.10 are used for amplifying homologous arm fragments (SEQ ID No.28 and SEQ ID No. 29) at the upstream and downstream of the vdh gene by using primer sequences shown as SEQ ID No.11 and SEQ ID No.12 respectively; then, recovering the amplified upstream and downstream products by using a DNA recovery kit, carrying out overlay recombination by using primer sequences shown as SEQ ID NO.9 and SEQ ID NO.12 to obtain a vdh gene homologous arm fragment, and connecting the vdh gene homologous arm fragment with a pULcrRNA delta vdh plasmid by using a one-step cloning method to obtain a knockout plasmid WGN01;
in one embodiment of the present invention, the WGN01 is a spacer sequence of the vdh gene and a homology arm upstream and downstream of the vdh gene added on the pULcrRNA-km-Cas12a plasmid.
In one embodiment of the invention, step four, a pULcrRNA-km-Cas12a plasmid is taken as a template, a CRISPR RGEN Tools (rgenome.net) website is used for designing a spacer sequence of a phdB gene, a primer is designed, a primer sequence shown in SEQ ID NO.13 and SEQ ID NO.14 is subjected to PCR amplification, and after the PCR product is purified, the PCR product is transferred into escherichia coli for cyclization, so that pULcrRNA delta phdB plasmid is obtained;
then, using pUC-GW-kana plasmid as a template, respectively carrying out PCR amplification by using primer sequences shown in SEQ ID NO.15 and SEQ ID NO.16, SEQ ID NO.17 and SEQ ID NO.18 to obtain a kasO p promoter fragment, a gapdh promoter fragment, and replacing a km promoter and an hsp60 promoter on pULcrRNA ΔphdB grains by a cloning method to obtain a plasmid pULcrRNA ΔphdB1;
then the genome of amycolatopsis CCTCC No. M2011265 is used as a template, the primer sequences shown as SEQ ID No.19 and SEQ ID No.20 are used for amplifying homologous arm fragments (SEQ ID No.30 and SEQ ID No. 31) at the upstream and downstream of the phdB gene by using the primer sequences shown as SEQ ID No.21 and SEQ ID No.22 respectively; and then, recovering the amplified upstream and downstream products by using a DNA recovery kit, carrying out overlay recombination by using primer sequences shown as SEQ ID NO.19 and SEQ ID NO.22, and connecting the obtained phdB gene homology arm fragments with a linearized pULcrRNA delta phdB1 plasmid by a one-step cloning method to obtain a knockout plasmid WGN02.
In one embodiment of the invention, the WGN02 is to replace hsp60 and Km promoters with gapdh and kasO p promoters in the pULcrRNA-Km-Cas12a plasmid, while adding a spacer sequence of the phdB gene and a homology arm upstream and downstream of the phdB gene; the sequences of gapdh and kasO p promoters are shown as SEQ ID No.5 and SEQ ID No.6, respectively.
In one embodiment of the invention, step five and step six are respectively performing electrotransformation on the editing plasmids WGN01 and WGN02 into amycolatopsis and amycolatopsis delta vdh strains to perform vdh and phdB gene knockout, and respectively obtaining amycolatopsis delta vdh strains; obtaining the amycolatopsis delta vdh delta phdB strain.
In one embodiment of the invention, the editing plasmid is electrotransferred into amycolatopsis, characterized in that plasmids WGN01 and WGN02 are first edited for demethylation; after obtaining the demethylated plasmid and heating to unwind, 0.5-2 μg of plasmid is added into 50-200 μl of competent cells, the mixture is placed on ice for 10min, and then transferred into an electric rotating cup after ice bath under the electric rotating conditions of 1800-2000V and 5ms. After about 4 hours of incubation after electric shock, the bacteria were resuspended and plated on the present medium containing apramycin and incubated at 30℃for 5-7 days.
The invention also provides a method for preparing vanillin, which comprises the steps of adding the mutant strain into a reaction system containing ferulic acid, and converting to obtain vanillin.
In one embodiment of the present invention, the reaction conditions in the reaction system are: and (3) a growth stage: accumulation of the cells, inoculating the seed solution into 1.5L fermentation medium at an inoculum size of 3-10% (v/v)The pH is controlled to be 6.8-7.2, the temperature is 26-35 ℃, and the stirring speed is 500 r.min -1 The aeration rate is 2vvm, and the culture is carried out for about 16-24 hours, thus obtaining the culture solution.
Conversion stage: and (3) intermittently adding substrate ferulic acid with a final concentration of 8-12g/L into the culture solution, simultaneously raising the temperature to 35-40 ℃, converting for 60-72h, and stopping fermentation after the ferulic acid is basically consumed.
The invention also provides application of the mutant strain in preparation of vanillin or vanillin-containing products.
Advantageous effects
The CRISPR/Cas12a gene editing system is constructed in the amycolatopsis CCTCC NO: M2011265 for the first time, has the advantages of high integration efficiency and relatively simple genetic operation, and can be used for carrying out other genetic operations on the amycolatopsis CCTCC NO: M2011265. The novel mutant strain obtained by the method disclosed by the invention has the advantages that the novel mutant strain amycolatopsis delta vdh delta phdB knocked out vdh and phdB genes, and the novel mutant strain is fermented for 60 hours to produce 20.4g/L of vanillin under the condition of adding 30g/L substrate ferulic acid in a 3L fermentation tank, so that the novel mutant strain has good industrial application value for the substrate ferulic acid molar conversion rate reaching 90.5%.
Drawings
Fig. 1: PCR verification and sequencing graphs of Amycolatopsis Deltavdh strain and Amycolatopsis Deltavdh DeltaphdB strain.
Detailed Description
The present invention will be described in further detail with reference to specific examples. It should be understood that the following examples are illustrative of the present invention and are not intended to limit the scope of the present invention.
The following examples relate to the following media:
YEME medium: 180-220g/L of sucrose, 10-20g/L of glucose, 5-10g/L of peptone, 3-5g/L of malt extract powder, 3-5g/L g of yeast powder and pH 7.2.
Seed culture medium: glucose 5-10g/L; yeast extract 10-15g/L; na (Na) 2 HPO 4 4-5g/L；KH 2 PO 4 1-2g/L；NaCl 0.2-.0.5g/L；MgSO 4 ·7H 2 O 0.2-0.5g/L；CaCl 2 ·2H 2 O0.05-0.1 g/L; pH7.2, kill at 115 DEG CBacteria for 20min.
Fermentation medium: 80-120g/L sucrose; 5-10g/L yeast extract; na (Na) 2 HPO 4 4-8g/L；KH 2 PO 4 1-2g/L；NaCl 0.2-0.5g/L；MgSO 4 ·7H2O 0.2-0.5g/L；CaCl 2 ·2H 2 O0.05-0.1 g/L; pH7.2, and sterilizing at 115 deg.C for 20min.
Reagents and plasmid sources used in the following examples:
1. pULcrRNA plasmid, construction method is disclosed in Crispr-cas12a-assisted Genome Editing in Amycolatopsis mediterranei.
2. Escherichia coli JM109, escherichia coli JM are commercially available.
The amplification system, amplification procedure, cleavage reaction system and one-step cloning reaction system involved in the following examples were as follows:
using high fidelity enzymesHS DNA Polymerase with GC Buffer the whole plasmid fragment was amplified, 50. Mu.L of the amplification system, the amplification system and the amplification procedure were as follows:
(1) Amplification system
PCR system: volume 50 μl, high fidelity enzyme HS:0.5 μl, dNTPs:4 μl, GC Buffer: 25. Mu.L, primer p1 (10. Mu.M): 2. Mu.L, primer p2 (10. Mu.M): 2. Mu.L, template (pULcrRNA): 1ng, ddH2O, to 50. Mu.L.
(2) Amplification procedure
Pre-denaturation at 98 ℃,3min, denaturation at 98 ℃ for 10sec, annealing at 60 ℃,5sec, ×30, extension at 72 ℃,1min/kb, total extension at 72 ℃,10 min, incubation at 4 ℃.
(3) The enzyme digestion reaction system is as follows:
the total volume of the digestion system was 50. Mu.L, restriction enzyme 1. Mu.L, PCR product 44. Mu.L, 10X QuickCut Buffer
5 μL, incubated at 37℃for 30min.
(4) Cloning a reaction system in one step: linearization vector is less than or equal to 200ng, insertion fragment is less than or equal to 200ng,5 XCE MultiS Buffer
4. Mu.L, exnase MultiS 2. Mu.L, ddH2O to 20. Mu.L, incubated at 37℃for 30min.
Example 1: acquisition of target crRNA sequences
Obtaining a target crRNA sequence: target crRNA is obtained by target sequence design of target amycolatopsis CCTCC NO: M2011265 genome according to the requirement, and the specific steps are as follows:
(1) The crRNA method for designing the target sequence containing the vdh gene comprises the following steps: the spacer sequence of the vdh gene was designed using the CRISPR RGEN Tools (rgenome. Net) website; selecting RGEN Target sequences with Out-of-frame Score greater than 66 and GC content of 40-60% in 23nt results of the obtained series of sgRNAs; the sequence of the polypeptide is shown as SEQ ID NO. 3.
(2) Designing crRNA containing phdB gene target sequence: the spacer sequence of phdB gene was obtained using a CRISPR RGEN Tools (rgenome. Net) website design; out-of-frame Score greater than 66 and RGEN Target sequence with GC content of 40-60% was selected from 23nt results of the obtained series of sgRNAs, and the sequence is shown in SEQ ID NO. 4.
Example 2: editing plasmid WGN01 construction
The method comprises the following specific steps:
(1) The Km promoter fragment (SEQ ID NO. 28) on the pLYZYP01 plasmid, which is derived from Francisella tularensis subsp.Novicida Cas12a fragment (SEQ ID NO. 27) and the linearized pULcrRNA plasmid are connected by a cloning method to obtain a plasmid pULcrRNA-Km-Cas12a;
(2) Then taking pULcrRNA-km-Cas12a plasmid as a template, using CRISPR RGEN Tools (rgenome.net) website to design a spacer sequence (SEQ ID NO. 3) of the vdh gene, designing a primer, performing PCR amplification by adopting the primer sequences shown in SEQ ID NO.7 and SEQ ID NO.8, purifying a PCR product, digesting by DpnI enzyme, transferring into escherichia coli, and cyclizing to obtain pULcrRNA delta vdh plasmid;
(3) Then the genome of amycolatopsis CCTCC No. M2011265 is used as a template, and primer sequences shown as SEQ ID No.9 and SEQ ID No.10 are used for amplifying homologous arm fragments (SEQ ID No.28 and SEQ ID No. 29) at the upstream and downstream of the vdh gene by using primer sequences shown as SEQ ID No.11 and SEQ ID No.12 respectively; and (2) recovering the amplified upstream and downstream products by using a DNA recovery kit, performing overlay recombination by using primer sequences shown as SEQ ID NO.9 and SEQ ID NO.12, and connecting the obtained upstream and downstream homology arm fragments of the vdh gene with the pULcrRNA delta vdh plasmid obtained in the step (2) by a one-step cloning method to obtain a knockout plasmid WGN01.
Example 3: editing plasmid WGN02 construction
The method comprises the following specific steps:
(1) Using the pULcrRNA-km-Cas12a plasmid obtained in example 2 as a template, using CRISPR RGEN Tools (rgenome. Net) website to design a spacer sequence (SEQ ID NO. 4) of phdB gene, designing primers, and carrying out PCR amplification on primer sequences shown in SEQ ID NO.13 and SEQ ID NO.14 to obtain a PCR product, purifying the PCR product, digesting the PCR product by DpnI enzyme, transferring the purified PCR product into escherichia coli, and cyclizing the purified PCR product to obtain pULcrRNA delta phdB plasmid;
(2) The synthesized kasO p promoter fragment (SEQ ID NO. 6) and gapdh promoter fragment (SEQ ID NO. 5) were used to replace the km promoter and hsp60 promoter on the pULcrRNA ΔphdB plasmid obtained in step (1) by a cloning method to obtain the plasmid pULcrRNA ΔphdB1-kasO p-gapdh, respectively.
(3) Then the genome of amycolatopsis CCTCC No. M2011265 is used as a template, the primer sequences shown as SEQ ID No.19 and SEQ ID No.20 are used for amplifying homologous arm fragments (SEQ ID No.31 and SEQ ID No. 32) at the upstream and downstream of the phdB gene by using the primer sequences shown as SEQ ID No.21 and SEQ ID No.22 respectively; and (2) recovering the amplified upstream and downstream products by using a DNA recovery kit, performing overlay recombination by using primer sequences shown as SEQ ID NO.19 and SEQ ID NO.22, and connecting the obtained phdB gene homology arm fragments with the linearized pULcrRNA delta phdB1-kasO p-gapdh plasmid obtained in the step (2) by a one-step cloning method to obtain a knockout plasmid WGN02.
Example 4: amycolatopsis mutant strain
The method comprises the following specific steps:
(1) Competent preparation:
the amycolatopsis CCTCC NO: M2011265 is inoculated into fresh YEME culture medium from an inclined plane at 28-30 ℃ and 180-220 r.min -1 After 36-48h of cultivation, the medium was transferred to fresh YEME medium at 28-30℃and 180-220 r.mi with an inoculum size of 5% (v/v)n -1 The culture is carried out overnight for about 12 hours, and the OD reaches between about 0.7 and 0.8.
Placing the thalli on ice for 30min;4 ℃,5000 r.min -1 Centrifuging for 5min to collect thalli; the supernatant was decanted, washed 2-3 times with ice-bath shock buffer (0.5M sucrose, 15% glycerol) and then the bacteria was resuspended with 1-2mL shock buffer. The bacterial solutions were filled into 1.5mL centrifuge tubes at 100. Mu.L portions.
(2) Electric conversion:
the editing plasmids WGN01 and WGN02 prepared in examples 2 and 3 were transformed into E.coli JM110 for demethylation; the plasmids WGN01 and WGN02 obtained;
and (3) respectively transferring:
transferring WGN01 into amycolatopsis strain to obtain amycolatopsis CCTCC NO: M2011265 Deltavdh; the WGN02 is introduced into a amycolatopsis CCTCC NO: M2011265 delta vdh strain to obtain amycolatopsis CCTCC NO: M2011265 delta vdh delta phdB;
immediately placing on ice at 90-98deg.C for 10min for cooling, mixing 1 μg plasmid and 100 μl of competent cells in step (1), transferring into an electric rotating cup after ice bath, placing in an electric rotating instrument for 1800V electric shock for 5ms, adding Ben's culture medium at 28deg.C for 220 r.min -1 Incubate for about 4 hours and then spread on the medium containing apramycin and incubate at 30℃for 5-7d.
(3) Identification of positive clones and elimination of plasmids:
identifying transformants by colony PCR, extracting genome from correct transformants, performing PCR amplification, purifying and recovering amplified correct bands, and finally performing sequencing verification, and performing knock-out verification by using primers vdh shown as SEQ ID NO.23 and SEQ ID NO. 24; the phdB gene knockout using the primers shown in SEQ ID No.25 and SEQ ID No.26 was verified (FIG. 1).
The results showed that the efficiency of the vdh gene knockout was 21.4%; the phdB gene knockout efficiency is 58.0%.
Transferring the correct mutant bacteria to a non-resistant Benshi liquid culture medium for 2 generations, and eliminating plasmids to obtain two amycolatopsis mutant strains Deltavdh (CCTCC NO: M2011265 Deltavdh strain) and amycolatopsis mutant strain Deltavdh DeltaphdB (CCTCC NO: M2011265 Deltavdh DeltaphdB).
Example 5: vanillin production by fermentation of mutant amycolatopsis delta vdh and amycolatopsis delta vdh delta phdB
The method comprises the following specific steps:
(1) Preparing seed liquid:
inoculating 2 loops of fresh bacterial colony original strain amycolatopsis CCTCC NO: M2011265, amycolatopsis mutant strain delta vdh and amycolatopsis mutant strain delta vdh delta phdB to 150mL/1000mL seed culture medium; culturing at 30 ℃ and 220rpm for about 24 hours; OD is between 6 and 8; seed solutions are prepared respectively.
(2) The fermentation method comprises the following steps: the two-stage method is a growth stage and a transformation stage respectively.
And (3) a growth stage: the bacterial cells accumulate, the seed solution is inoculated into 1.5L fermentation medium according to the inoculation amount of 10% (v/v), the pH is controlled to 7.2, the temperature is 30 ℃, and the stirring speed is 500 r.min -1 The aeration rate was 2vvm, and the culture was carried out for about 16 hours to obtain a culture solution.
Conversion stage: and (3) intermittently adding substrate ferulic acid with a final concentration of 8-12g/L (fed-batch addition, 200mL each time and a final concentration of 8-12 g/L) into the culture solution, raising the temperature to 35 ℃, converting for 60h, and stopping fermentation after the ferulic acid is basically consumed.
The results show that the vanillin yields of the original strain amycolatopsis CCTCC NO: M2011265, the amycolatopsis mutant strain Deltavdh strain and the amycolatopsis mutant strain Deltavdh DeltaphdB strain are respectively 10.60g/L, 14.60g/L and 20.44g/L; the concentration of the vanillic acid is respectively 2.45g/L, 0.45g/L and 0.15g/L;
the yield of vanillin of the amycolatopsis mutant strain delta vdh delta phdB of the double-knock strain is improved by about 92.4 percent compared with that of the wild amycolatopsis CCTCC NO: M2011265;
compared with the wild type amycolatopsis CCTCC NO: M2011265, the amycolatopsis delta vdh of the single-knockout strain is improved by about 37.7 percent, the amycolatopsis delta vdh delta phdB vanillin degradation rate of the double-knockout strain is reduced by about 95 percent, and the molar conversion rate of the double-knockout strain is up to 90.5 percent.
In conclusion, the amycolatopsis mutant strain Deltavdh DeltaphdB realizes the remarkable improvement of the vanillin yield by traceless knockout of vdh and phdB genes, remarkably reduces the yield of the byproduct vanillic acid, and shows good industrial application value
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and 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.

Claims (10)

1. A amycolatopsis mutant strain, characterized in that the mutant strain is a gene for knocking out a gene expressing vanillin dehydrogenase and a gene expressing 4-hydroxy-3-methoxybenzoyl-beta-hydroxypropionyl CoA dehydrogenase in the genome of amycolatopsis.
2. The amycolatopsis mutant strain of claim 1, wherein the mutant strain is a vanillin-producing amycolatopsis host cell.
3. The amycolatopsis mutant strain according to claim 2, wherein the sequence of the gene expressing vanillin dehydrogenase is shown in SEQ ID No. 1; the sequence of the gene of the 4-hydroxy-3-methoxybenzoyl-beta-hydroxypropionyl CoA dehydrogenase is shown as SEQ ID NO. 2.
4. A method for constructing a amycolatopsis mutant strain according to any one of claims 1-3, characterized in that the method comprises the steps of:
step one, obtaining a target crRNA sequence: target crRNA is obtained by target sequence design of target amycolatopsis CCTCC NO: M2011265 genome according to the requirement: contains a vdh gene target sequence and a phdB gene target sequence;
constructing a skeleton plasmid containing Cas12a and crRNA;
step three, constructing a traceless knockout vdh gene editing plasmid WGN01: adding a spacer sequence of the vdh gene and homologous arms on the upstream and downstream of the vdh gene on the skeleton plasmid of the second step;
step four, constructing a traceless knockout phdB gene editing plasmid WGN02: replacing hsp60 and Km promoters on the skeleton plasmid of the second step with gapdh and kasO p promoters respectively, and simultaneously adding a spacer sequence of the phdB gene and upstream and downstream homology arms of the phdB gene; the sequences of gapdh and kasO p promoters are shown in SEQ ID No.5 and SEQ ID No.6 respectively;
step five, the editing plasmid WGN01 is electrically transferred into amycolatopsis; obtaining a amycolatopsis delta vdh strain;
step six, the editing plasmid WGN02 is electrically transferred into the amycolatopsis delta vdh strain to obtain the amycolatopsis delta vdh delta phdB strain.
5. The method according to claim 4, wherein the crRNA containing the target sequence of the vdh gene is shown in SEQ ID NO. 3.
6. The construction method according to claim 5, wherein the crRNA containing the phdB gene target sequence is shown in SEQ ID NO. 4.
7. A method for improving the yield of vanillin prepared by amycolatopsis is characterized by knocking out the gene of the amycolatopsis CCTCC NO: M2011265 expressed vanillin dehydrogenase and the gene of 4-hydroxy-3-methoxybenzoyl-beta-hydroxypropionyl CoA dehydrogenase.
8. The method according to claim 7, wherein the sequence of the gene expressing vanillin dehydrogenase is shown in SEQ ID No. 1; the sequence of the gene of the 4-hydroxy-3-methoxybenzoyl-beta-hydroxypropionyl CoA dehydrogenase is shown as SEQ ID NO. 2.
9. A method for preparing vanillin, which is characterized in that the mutant strain of any one of claims 1-3 is inoculated into a fermentation medium containing ferulic acid and fermented to obtain vanillin.
10. Use of a mutant according to any one of claims 1 to 3 for the preparation of vanillin or a product containing vanillin.
CN202310234577.8A 2023-03-10 2023-03-10 Amycolatopsis mutant strain and application thereof Pending CN117305194A (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113061560A (en) * 2021-03-11 2021-07-02 江南大学 Genetically engineered bacterium of amycolatopsis as well as construction method and application thereof
WO2022133254A1 (en) * 2020-12-18 2022-06-23 Basf Se Amycolatopsis strains for vanillin production with suppressed vanillic acid formation

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022133254A1 (en) * 2020-12-18 2022-06-23 Basf Se Amycolatopsis strains for vanillin production with suppressed vanillic acid formation
CN113061560A (en) * 2021-03-11 2021-07-02 江南大学 Genetically engineered bacterium of amycolatopsis as well as construction method and application thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
FLORIAN MEYER: "A proteomic analysis of ferulic acid metabolism in Amycolatopsis sp. ATCC 39116", 《APPLIED MICROBIOLOGY AND BIOTECHNOLOGY》, vol. 102, 16 May 2018 (2018-05-16), pages 6128 *
HUI QIAN: "Strategy for Producing the High-Quality Glycopeptide Antibiotic A82846B in Amycolatopsis orientalis Based on the CRISPR-Cas12a System", 《ACS SYNTH. BIOL》, vol. 10, no. 11, 19 November 2021 (2021-11-19), pages 3 *

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