CN118126919A

CN118126919A - Engineering bacterium for synthesizing ceruloplasmic reticulum and construction method and application thereof

Info

Publication number: CN118126919A
Application number: CN202410284862.5A
Authority: CN
Inventors: 请求不公布姓名
Original assignee: Nanjing Hegu Life Biotechnology Co ltd
Current assignee: Nanjing Hegu Life Biotechnology Co ltd
Priority date: 2024-03-13
Filing date: 2024-03-13
Publication date: 2024-06-04

Abstract

The application relates to engineering bacteria for synthesizing blue, a construction method and application thereof, and belongs to the technical field of natural dye synthesis by a biological method. The engineering bacterium for synthesizing blue viewing, which is disclosed by the application, expresses bpsA, sfp, glnA x and gdhA; and the engineering bacteria are knocked out glsK, proB, aceA and ldh. According to the application, by knocking out proB and aceA genes, site-directed mutagenesis is carried out on the glnA gene, glsK genes are replaced by glnA genes, ldh genes are replaced by gdhA genes, bpsA and sfp genes are integrated, by-product generation can be blocked, flux of pyruvic acid to tricarboxylic acid circulation and alpha-ketoglutarate to glutamic acid is improved, glutamine concentration in organisms is improved, blue synthetase catalytic glutamine synthesis blue viewing is promoted, and thus blue viewing yield is improved.

Description

Engineering bacterium for synthesizing ceruloplasmic reticulum and construction method and application thereof

Technical Field

The application relates to the technical field of natural dye synthesis by a biological method, in particular to engineering bacteria for synthesizing blue-looking, a construction method and application thereof.

Background

Blue-viewing (Indigoidine), which is a natural blue pigment without toxic and side effects, is mainly applied to the dye industry. The synthesis of the blue-viewing in the organism mainly comprises the steps of activating the blue-viewing synthetase by 4' -phosphopantetheinyl transferase, and synthesizing 2 molecules of glutamine in the organism into 1 molecule of blue-viewing.

The pigment can be divided into natural pigment and synthetic pigment, and the synthetic pigment has the advantages of bright color, abundant variety, high production yield, low cost and low price compared with the natural pigment. However, as most of the raw materials used in the synthesis process are toxic compounds, a large amount of the synthetic pigment is easy to poison, cancerogenic and cause allergy, and the synthetic pigment is difficult to degrade, so that the industrial wastewater has high toxicity, difficult degradation and high chromaticity value, and serious environmental pollution is caused. Therefore, the microbial fermentation production of blue pigment has little pollution and low cost, and gradually becomes an alternative scheme for industrial synthesis of pigment, but the current microbial fermentation production of blue pigment in industry has low yield, so that the cost is high and a distance from the large-scale industrial production application is provided. Aiming at the problems in the prior art, the invention discloses a genetic engineering strain for producing blue-looking and a fermentation method, which have the potential of reducing cost, improving efficiency and realizing industrial application.

Disclosure of Invention

The application aims to overcome the defects of the prior art and provide the engineering bacterium which can improve the blue viewing yield by reducing the synthesis of byproducts and improving the accumulation of glutamine in the body.

In order to achieve the above purpose, the technical scheme adopted by the application is as follows:

In a first aspect, the present application provides an engineered bacterium for synthesizing blue viewing, the engineered bacterium expressing blue pigment synthase bpsA, phosphopantetheinyl transferase sfp, glutamine synthase glnA, and glutamate dehydrogenase gdh a;

The engineering bacteria knock out glutaminase glsK, glutamate 5-kinase proB, isocitrate lyase aceA and lactate dehydrogenase ldh;

The engineering bacteria Corynebacterium glutamicum for synthesizing the blue is preserved in China center for type culture collection, chinese Wuhan, and the preservation number is CCTCCNO: m2024243, the preservation date is 2024, 01 and 29.

According to the application, by knocking out proB and aceA genes, site-directed mutagenesis (Y405F, Y405N, Y L) is carried out on the glnA gene, the glsK gene is replaced by the glnA gene, the ldh gene is replaced by the gdhA gene, bpsA and sfp genes are integrated, so that pyruvic acid in corynebacterium glutamicum can be blocked from being converted into lactate, isocitric acid is cracked into succinic acid, glutamic acid is converted into proline and glutamine is hydrolyzed into glutamic acid, flux of pyruvic acid flowing to tricarboxylic acid circulation and alpha-ketoglutarate flowing to glutamic acid is improved, glutamine concentration in organisms is improved, blue synthase catalytic glutamine synthesis is promoted, and blue viewing yield is improved.

As a preferred embodiment of the engineering bacterium, the glutamine synthetase glnA is encoded by a glutamine synthetase glnA containing a mutation site, the encoding sequence of the glutamine synthetase is shown in SEQ No.1, and the mutation site comprises any one of the following 1) -3):

1) Mutation from tyrosine to phenylalanine at position 405;

2) Mutation from tyrosine to asparagine at position 405;

3) Mutation from tyrosine to leucine at position 405.

According to the application, the 405 th site of glnA is subjected to site-directed mutagenesis and then inserted into a glsK knocked-out site, so that the conversion of glutamic acid into glutamine can be promoted, and the concentration of glutamine can be increased.

As a preferred embodiment of the engineering bacterium, the blue pigment synthetase BpsA is derived from Streptomyces lilacinus Streptomyces lavendulae; the coding sequence of the blue pigment synthetase BpsA is shown as SEQ ID NO. 2.

As a preferred embodiment of the engineering bacterium, the phosphopantetheinyl transferase sfp is derived from bacillus subtilis Bacillus subtilis; the coding sequence of the phosphopantetheinyl transferase sfp is shown as SEQ ID NO. 3. Phosphopantetheinyl transferase (Phosphopantetheinyl TRANSFE RASE) is an enzyme that catalyzes phosphopantetheinyl transfer and is capable of activating bpsA to catalyze glutamine synthesis to blue.

As a preferred embodiment of the engineering bacterium of the present application, the glutamate dehydrogenase gdhA is derived from Corynebacterium glutamicum; the coding sequence of the glutamate dehydrogenase gdhA is shown in SEQ ID NO. 4.

In a second aspect, the present application provides a construction method of the above engineering, comprising the steps of:

a1, replacing a glutaminase glsK coding gene on corynebacterium glutamicum with a glutamine synthetase glnA coding gene by using a gene editing technology to obtain engineering bacteria A;

A2, knocking out a glutamic acid 5-kinase proB coding gene on the engineering bacterium A obtained in the step A by utilizing a gene editing technology to obtain an engineering bacterium B;

A3, knocking out an isocitrate lyase aceA coding gene on the engineering bacteria B obtained in the step A2 by utilizing a gene editing technology to obtain engineering bacteria C;

A4, replacing the lactate dehydrogenase ldh coding gene on the engineering bacterium C obtained in the step A3 with a glutamate dehydrogenase gdhA coding gene by utilizing a gene editing technology to obtain engineering bacterium D;

and A5, integrating coding genes of blue pigment synthetase bpsA and phosphopantetheinyl transferase sfp into engineering bacteria D obtained in the step A4 by utilizing a gene editing technology to obtain engineering bacteria E, wherein the engineering bacteria E is engineering bacteria for synthesizing blue.

As a preferred embodiment of the construction method of the present application, the genes encoding the blue pigment synthase BpsA and the phosphopantetheinyl transferase Sfp in step A5 are integrated into the engineering bacterium D after codon optimization.

In a third aspect, the application provides an application of the engineering bacteria in blue viewing synthesis.

In a fourth aspect, the application provides a method for synthesizing blue-viewing, which is obtained by fermenting the seed solution of the engineering bacteria and a fermentation medium in a bioreactor.

The application provides a method for synthesizing blue-viewing, which is prepared into blue-viewing through a series of biochemical reactions by taking glucose as a substrate and inducing engineering bacteria CG 07. Engineering bacteria CG07 adopted in the synthesis process can block pyruvic acid in corynebacterium glutamicum from being converted into lactate, isocitric acid from being cracked into succinic acid, glutamic acid from being converted into proline and glutamine from being hydrolyzed into glutamic acid, can reduce the formation of byproducts, simultaneously improve the level of glutamine, further promote the conversion of alpha-ketoglutarate into glutamic acid and the conversion of glutamic acid into glutamine, activate blue pigment synthase, enable glutamine to be synthesized into blue, and enable the shake flask fermentation yield of blue to be up to 15.48g/L.

As a preferred embodiment of the method of the present application, the fermentation conditions are fermentation at 20℃and 200rpm for 72 to 96 hours;

The bioreactor also contains IPTG solution with the final concentration of 0.8-1.5 mM.

Compared with the prior art, the application has the beneficial effects that:

(1) According to the application, by knocking out proB and aceA genes, site-directed mutagenesis (Y405F, Y405N, Y L) is carried out on the glnA gene, the glsK gene is replaced by the glnA gene, the ldh gene is replaced by the gdhA gene, bpsA and sfp genes are integrated, so that pyruvic acid in corynebacterium glutamicum can be blocked from being converted into lactate, isocitric acid is cracked into succinic acid, glutamic acid is converted into proline and glutamine is hydrolyzed into glutamic acid, flux of pyruvic acid flowing to tricarboxylic acid circulation and alpha-ketoglutarate flowing to glutamic acid is improved, glutamine concentration in organisms is improved, blue synthase catalytic glutamine synthesis is promoted, and blue viewing yield is improved.

(2) The application provides a method for synthesizing blue-viewing, which is prepared into blue-viewing through a series of biochemical reactions by taking glucose as a substrate and inducing engineering bacteria CG 07. Engineering bacteria CG07 adopted in the synthesis process can block pyruvic acid in corynebacterium glutamicum from being converted into lactate, isocitric acid from being cracked into succinic acid, glutamic acid from being converted into proline and glutamine from being hydrolyzed into glutamic acid, can reduce the formation of byproducts, simultaneously improve the level of glutamine, further promote the conversion of alpha-ketoglutarate into glutamic acid and the conversion of glutamic acid into glutamine, activate blue pigment synthase, enable glutamine to be synthesized into blue, and enable the shake flask fermentation yield of blue to be up to 15.48g/L.

(3) Compared with the prior art, the engineering bacterium has the advantages of reducing cost, improving efficiency, realizing industrial production of blue viewing, along with environmental protection, safety, stability and no chemical pollution.

Drawings

FIG. 1 shows the whole process of blue-looking biosynthesis of the present application.

Detailed Description

For a better description of the objects, technical solutions and advantages of the present application, the present application will be further described with reference to the following specific examples.

Materials, reagents and the like used in the examples, comparative examples and experimental examples were commercially available unless otherwise specified.

Coli DH 5. Alpha. (hereinafter DH 5. Alpha.) was used for vector construction, and was commercially available.

Corynebacterium glutamicum (Corynebacterium glutamicum) ATCC 13032 (referred to as ATCC 13032 hereinafter) was purchased from North Biotech Co.Ltd.

Plasmids pTargetF, 95S-rhtA, pJYS 3-Delta crtYf, pTRCmob were purchased from biological wind.

High-fidelity DNA polymerase, restriction endonuclease, in-Snap Assembly Master Mix available from TAKARA.

Plasmid extraction kits, DNA purification kits, gel recovery kits, bacterial genomic DNA extraction kits were purchased from OMEGA.

LB medium: 5g/L yeast powder, 10g/L peptone and 10g/L NaCl, adding water to make up to 1L total volume, and sterilizing at 121deg.C for 20min; if the solid culture medium is prepared, 15-20g/L agar powder is added.

BHISG medium: 37g/L brain heart infusion, 10g/L glucose, adding water to make up to 1L total volume, and sterilizing at 121deg.C for 20min; if the solid culture medium is prepared, adding 15-20g/L agar powder; brain heart infusion was purchased from south kyuno laboratories, inc.

GAP medium: 70g/L glucose, 20g/L ammonium sulfate, 1g/L potassium dihydrogen phosphate, 0.01g/L ferric sulfate, 0.01g/L manganese sulfate, 4 mu g/L biotin, 20 mu g/L thiamine hydrochloride, 15mL/L commercial soy sauce and 50g/L calcium carbonate, adding water to make up to 1L total volume, sterilizing at 121 ℃ for 20min, wherein the commercial soy sauce can be any commercially available soy sauce.

In the following examples and comparative examples, unless otherwise specified, the plasmid transfer into E.coli DH 5. Alpha. Was a chemical transformation comprising the steps of: adding the transformation system into DH5 alpha transformation competence melted on ice, carrying out heat shock at 42 ℃ for 1min after ice bath for 30min, carrying out ice bath for 2min again, adding 900uL of precooled LB, and incubating at 37 ℃ and 220rpm for 60min; 100 mu L of the incubated bacterial liquid is added into LB culture medium containing streptomycin resistance, and the bacterial liquid is cultured at 30 ℃/37 ℃ overnight.

The electric shock conversion comprises the following steps: adding 200-500 mug of fragment to be converted into the prepared electrotransformation competent cells, gently mixing uniformly, adding into an electrotransformation cup, and carrying out ice bath for 5-10min; after 1.8KV electric shock is carried out for 2-3 times, a BHISG culture medium preheated at 46 ℃ is rapidly added, and heat shock is carried out in a water bath kettle at 46 ℃ for 6 minutes; then placing the conversion solution after heat shock in a shaking table at 30 ℃ for incubation for 2 hours; after the incubation, 100. Mu.L of the transformation solution was spread on BHISG plates and incubated in a 30℃incubator for 48 hours.

The Gibson assembly technique (hereinafter Gibson) operates with reference to the following references:

Gibson,D.G.,et al.(2009).Enzymatic assembly of DNA molecules up to s everal hundred kilobases.Nature Methods,6(5),343-345.

Gibson,D.G.,et al.(2010).Creation of a bacteria1 cell controlled by a che mically synthesized genome.Science,329(5987),52-56.

Example 1

The embodiment provides engineering bacteria for synthesizing blue-looking and a construction method thereof, wherein the construction method comprises the following steps:

1. Construction of engineering bacterium CG01

1.1, Taking plasmid pTargetF as a template, and carrying out PCR (polymerase chain reaction) amplification by utilizing PT-N24R and PT-N24F primers to obtain a pTarget-N24 plasmid skeleton;

Carrying out PCR (polymerase chain reaction) amplification by using pJYS-delta crtYf as a template and 23119-crRNA-F and 23119-crRNA-R primers to obtain a crRNA-crtYf fragment;

assembling the pTarget-N24 plasmid backbone and the crRNA-crtYf fragment by Gibson to obtain a pTarget-N24-crtYf fragment;

Taking pTarget-N24-crtYf as a template, utilizing pT-N24-glsK-F and pT-N24-glsK-R primers for PCR amplification, adopting Dpn I enzyme digestion, and transferring into DH5 alpha to obtain pTarget-N24-glsK plasmid;

The PT-N24R, PT-N24F, 23119-crRNA-R, pT-N24-glsK-F and pT-N24-glsK-R primer sequences are shown in Table 1.

1.2 PCR amplification with ATCC 13032 as template and Ptuf-F and Ptuf-R primers to give the ptuf promoter;

Taking the 95S-rhtA plasmid as a template, and carrying out PCR amplification by using 95S-F and 95S-Ptuf-R primers to obtain a ptuf-S plasmid skeleton;

Using ATCC 13032 as a template, carrying out PCR amplification by using glnA-Ptuf-F and glnA ^Y405F -R primers to obtain a glnA ^Y405F upstream fragment, and carrying out PCR amplification by using glnA ^Y405F -F and glnA-Ptuf-R primers to obtain a glnA ^Y405F downstream fragment; the glnA sequence is shown in SEQ ID NO.1;

The ptuf-S plasmid skeleton, ptuf promoter and glnA ^Y405F upstream and downstream fragment are assembled through Gibson, and transferred into DH5 alpha to obtain ptuf-S-glnA ^Y405F plasmid;

Ptuf-F, ptuf-R, 95S-F, 95S-Ptuf-R, glnA. Times. Ptuf-F, glnA ^Y405F-R、glnA^Y405F -F and glnA. Times. Ptuf-R primer sequences are shown in Table 1.

1.3 Using ATCC 13032 as a template, glsK-up1200F and glsK: : glnA-up 500R primer PCR amplification gives Δ glsK: : a glnA up fragment;

Using ATCC 13032 as a template, using glsK: : glnA. Times. -Down500F-T1 and glsK-Down1200R primers PCR amplified to give Δ glsK: : a glnA down fragment;

Carrying out PCR amplification by using ptuf-S-glnA ^Y405F obtained in the step 1.2 as a template and using Ptuf-F and rrnB-R1-T1 primers to obtain ptuf-glnA ^Y405F fragments;

At delta glsK: : glnA up, ptuf-glnA ^Y405F and Δ glsK: : glnA down was used as a template and PCR was performed using overlapping extension of S3-glsK-up1000F and S3-glsK-down1000R primers to give Δ glsK: : a glnA ^Y405F donor fusion fragment;

glsK-up1200F, glsK: : glnA up500R, glsK: : the glnA-Down 500F-T1, glsK-Down1200R, rrnB-R1-T1, S3-glsK-up1000F and S3-glsK-Down1000R primer sequences are shown in Table 1.

1.4 PCR amplification of 23119-crRNA-F and 23119-crRNA-R primers using pTarget-N24-glsK obtained in step 1.1 as a template to obtain crRNA-glsK fragment;

Carrying out PCR amplification by using pJYS-delta crtYf as a template and using pJYS3-vector-F and pJYS3-vector-R primers to obtain a pJYS plasmid skeleton;

crRNA-glsK, Δ glsK was assembled by Gibson: : the glnA ^Y405F donor and pJYS plasmid backbone was transformed into DH 5. Alpha. To give pJYS 3-. DELTA. glsK: : glnA ^Y405F plasmid.

1.5 Using shock transformation method pJYS 3-. DELTA. glsK from step 1.4: : the glnA ^Y405F plasmid was transferred into ATCC 13032 and screened by colony PCR to give Δ glsK: : the target strain of glnA ^Y405F is used for obtaining engineering bacterium CG01.

TABLE 1 primers used in construction process of engineering bacterium CG01

2. Construction of engineering bacterium CG04

2.1 PCR amplification with pTarget-N24-crtYf obtained in step 1.1 as template and pT-N24-proB-F and pT-N24-proB-R primers, digestion of the template with Dpn I enzyme, and transfer into DH 5. Alpha. To obtain pTarget-N24-proB plasmid. The pT-N24-proB-F and pT-N24-proB-R primer sequences are shown in Table 2.

2.2 PCR amplification with ATCC 13032 as template, using proB-up1200F and proB-up500R primers to obtain a ΔproBup fragment;

Using ATCC 13032 as a template, and utilizing proB-down500F and proB-down1200R primers to carry out PCR amplification to obtain a delta proB down fragment;

Delta proB up and delta proB down are used as templates, and S3-proB-up1000F and S3-proB-down1000R primers are used for overlapping extension PCR amplification to obtain delta proB down fusion fragments;

the primer sequences of proB-up1200F, proB-up500R, proB-down500F, proB-down1200R, S-proB-up 1000F and S3-proB-down1000R are shown in Table 2.

2.3 PCR amplification of 23119-crRNA-F and 23119-crRNA-R primers to obtain crRNA-proB fragments by using the pTarget-N24-proB obtained in the step 2.1 as a template;

Assembling crRNA-proB, delta proB donor and pJYS plasmid skeletons through Gibson, and transferring into DH5 alpha to obtain pJYS-delta proB plasmid;

The 23119-crRNA-F, 23119-crRNA-R, pJYS-vector-F and pJYS-vector-R primer sequences are shown in Table 2.

2.4 Transferring pJYS-delta proB plasmid obtained in the step 1.3 into engineering bacterium CG01 obtained in the step 1.5 by utilizing an electric shock transformation method, and screening a target strain with delta proB by colony PCR to obtain engineering bacterium CG04.

TABLE 2 primers used in construction process of engineering bacterium CG04

3. Construction of engineering bacterium CG05

3.1 Taking the pTarget-N24-crtYf obtained in the step 1.1 as a template, utilizing pT-N24-aceA-F and pT-N24-aceA-R primers for PCR amplification, digesting the template by Dpn I enzyme, and transferring into DH5 alpha to obtain pTarget-N24-proB plasmid; the pT-N24-aceA-F and pT-N24-aceA-R primer sequences are shown in Table 3.

3.2 PCR amplification with ATCC 13032 as template and aceA-up1200F and aceA-up500R primers to obtain ΔaceA up fragment;

Using ATCC 13032 as a template, and carrying out PCR amplification by using aceA-Down500F and aceA-Down1200R primers to obtain a DeltaaceA down fragment;

Delta aceA up and delta aceA down are used as templates, and S3-aceA-up1000F and S3-aceA-down1000R primers are used for overlapping extension PCR amplification to obtain delta aceA donor fusion fragments;

The primer sequences of aceA-up1200F, aceA-up500R, aceA-down500F, aceA-down1200R, S-aceA-up 1000F and S3-aceA-down1000R are shown in Table 3.

3.3 PCR amplification with pTarget-N24-aceA as template and 23119-crRNA-F and 23119-crRNA-R primers to obtain crRNA-aceA fragment;

assembling crRNA-aceA, ΔaceA donor and pJYS plasmid skeletons through Gibson, and transferring into DH5 alpha to obtain pJYS- ΔaceA plasmid;

The 23119-crRNA-F, 23119-crRNA-R, pJYS-vector-F and pJYS-vector-R primer sequences are shown in Table 1.

3.4 Transferring pJYS-delta aceA plasmid obtained in the step 3.3 into engineering bacterium CG04 obtained in the step 2.4 by utilizing an electric shock transformation method, and screening a target strain of delta aceA by colony PCR to obtain engineering bacterium CG05.

TABLE 3 primers used in construction process of engineering bacterium CG05

4. Construction of engineering bacterium CG06

4.1 Using pTarget-N24-crtYf obtained in step 1.1 as template, using pT-N24-ldh-F and pT-N24-ldh-R primer to PCR amplify' Dpn I enzyme digestion template, transferring into DH5a to obtain pTarget-N24-ldh plasmid. The pT-N24-1dh-F and pT-N24-ldh-R primer sequences are shown in Table 4.

4.2 PCR amplification with ATCC 13032 as template and pTuf-Nco I-gdhA-F and pTuf-EcoR I-gdhA-R primers to obtain gdhA fragment, the gdhA coding sequence being shown in SEQ ID NO. 4;

Carrying out PCR amplification by using ptuf-S-glnA as a template and using Ptuf-Vector-F and Ptuf-Vector-R primers to obtain a ptuf-S plasmid skeleton;

Assembling ptuf-S plasmid skeleton and gdhA by Gibson, and transferring into DH5a to obtain ptuf-S-gdhA plasmid;

the pT-N24-ldh-F, pT-N24-ldh-R, pTuf-Nco I-gdhA-F, pTuf-EcoR I-gdhA-R, ptuf-Ve ctor-F and Ptuf-Vector-R primer sequences are shown in Table 4.

4.3 Using ATCC 13032 as a template, ldh-up1200F and ldh: : the gdhA-up500R primer was amplified by PCR to give Δldh: : a gdhA up fragment;

using ATCC 13032 as a template, using ldh: : the gdhA-down500F and ldh-down1200R primers were PCR amplified to give Δldh: : a gdhA down fragment;

carrying out PCR amplification by using ptuf-S-gdhA as a template and utilizing Ptuf-F and rrnB-R1-T1 primers to obtain a ptuf-gdhA fragment;

At Δldh: : gdhA up, ptuf-gdhA, and Δldh: : gdhA down is used as a template, and S3-ldh-up1000F and S3-ldh-down1000R primers are used for overlapping extension PCR amplification to obtain delta ldh: : a gdhA donor fusion fragment;

ldh-up1200F, ldh: : gdhA-up500R, ldh: : the gdhA-Down500F, ldh-Down1200R, S-1 dh-up1000F and S3-ldh-Down1000R primer sequences are shown in Table 4, and the Ptuf-F and rrnB-R1-T1 primer sequences are shown in Table 1.

4.4 PCR amplification of 23119-crRNA-F and 23119-crRNA-R primers to obtain crRNA-ldh fragments by using the pTarget-N24-ldh obtained in the step 4.1 as a template;

crRNA-ldh, Δldh, was assembled by Gibson: : the gdhA donor and pJYS plasmid backbone was transformed into DH 5. Alpha. To give pJYS. Delta. Ldh: : a gdhA plasmid;

4.5 Using shock transformation, pJYS 3-. DELTA.ldh from step 4.4: : transferring the gdhA plasmid into engineering bacterium CG05 obtained in the step 3.4, and obtaining delta ldh through colony PCR screening: : the target strain of gdhA is obtained to obtain engineering bacterium CG06.

TABLE 4 primer used in construction process of engineering bacterium CG06

5. Construction of engineering bacterium CG07

5.1 Codon optimization is carried out on a blue pigment synthetase BpsA gene sequence from Streptomyces lilacinus Streptomyces lavendulae, a bpsA gene fragment is synthesized, and a bpsA sequence after codon optimization is shown as SEQ ID NO. 2.

5.2 Codon optimization is carried out on a phosphopantetheinyl transferase Sfp gene sequence from bacillus subtilis Bacillus subtilis, so that a Sfp gene fragment is synthesized, and the Sfp sequence after codon optimization is shown as SEQ ID NO. 3.

5.3 PCR amplification is carried out by taking bpsA gene fragment obtained in the step 5.1 as a template and utilizing bpsA-TRC-F and bpsA-TRC-R primers to obtain a fragment bpsA with a homology arm;

Using the sfp gene fragment obtained in the step 5.2 as a template, and carrying out PCR amplification by using sfp-TRC-F and sfp-TRC-R primers to obtain a sfp fragment with a homology arm;

Carrying out PCR (polymerase chain reaction) amplification by using pTRCmob plasmid as a template and using pTRCmob-vectorF and pTRCmob-vectorR primers to obtain a pTRCmob plasmid skeleton;

assembling bpsA, sfp and pTRCmob plasmid skeletons through Gibson, and transferring into DH5 alpha to obtain pTRCmob-bpsA-sfp plasmid;

The bpsA-TRC-F, bpsA-TRC-R, sfp-TRC-F, sfp-TRC-R, pTRCmob-vectorF and pTRCmob-vectorR primer sequences are shown in Table 5.

5.4 Transferring pTRCmob-bpsA-sfp plasmid obtained in the step 5.3 into engineering bacterium CG06 obtained in the step 4.5 by utilizing an electric shock conversion method, screening a target strain of bpsA-sfp by colony PCR to obtain engineering bacterium CG07, wherein the engineering bacterium CG07 is engineering bacterium for synthesizing cerulous blue, and the engineering bacterium for synthesizing cerulous blue is preserved in China center for type culture collection with a preservation number of CCTCC NO: m2024243, the preservation date is 2024, 01 and 29.

TABLE 5 primers used in construction of engineering bacterium CG07

Example 2

The present example provides an engineering bacterium for synthesizing blue viewing and a construction method thereof, and the construction method is similar to that of example 1, except that the glnA ^Y405F -R primer in step 1.2 is replaced by a glnA ^Y405N -R primer, the glnA ^Y405F -F primer is replaced by a glnA ^Y405N -F primer, the engineering bacterium obtained in step 1.5 is named CG02, the other steps and parameter conditions are unchanged, and the glnA ^Y405N -R and glnA ^Y405N -F primers adopted are shown in Table 6.

Table 6 primer used in construction process of engineering bacterium CG02 and CG03

Example 3

The present example provides an engineering bacterium for synthesizing cerulous blue and a construction method thereof, and the construction method is similar to that of example 1, except that the glnA ^Y405F -R primer in step 1.2 is replaced with the glnA ^Y405L -R primer, the glnA ^Y405F -F primer is replaced with the glnA ^Y405L -F primer, the engineering bacterium obtained in step 1.5 is named CG03, the other steps and parameter conditions are unchanged, and the glnA ^Y405L -R and glnA ^Y405L -F primers are shown in Table 6.

Example 4

The embodiment provides a method for synthesizing blue viewing, which comprises the following steps:

(1) Culturing engineering bacterium CG07 of example 1 in BHISG solid culture medium at 30deg.C for 48h, selecting single colony, inoculating into 5mL BHISG culture medium, culturing at 30deg.C at 220rpm for 12-16h to obtain CG07 first-order seed solution;

(2) Inoculating 350 mu L of the CG07 first-stage seed liquid obtained in the step (1) into a 35mL BHISG culture medium, and culturing at 30 ℃ and 220rpm for 16 hours to obtain a CG07 second-stage seed liquid;

(3) Centrifuging the CG07 secondary seed solution obtained in the step (2) at 4 ℃ and 4500rpm for 10min, reserving thalli, transferring into 25mL of GAP culture medium, culturing for 3h at 30 ℃ and 200rpm, adding IPTG solution to make the final concentration of the mixture 1mM, culturing for 72h at 20 ℃ and 200rpm, and obtaining blue viewing.

The synthetic route of engineering bacterium CG07 of the application for observing blue is shown in figure 1.

As shown in figure 1, the engineering bacterium CG07 of the application takes glucose as a substrate, pyruvic acid is generated through a glycolysis pathway, acetyl coenzyme A is generated under the action of pyruvic acid dehydrogenase, acetyl coenzyme A enters tricarboxylic acid to circulate, citric acid is generated under the action of citric acid synthase, citric acid generates isocitric acid under the catalysis of aconitase, isocitric acid generates alpha-ketoglutarate under the catalysis of isocitric acid dehydrogenase, alpha-ketoglutarate generates glutamic acid under the catalysis of glutamate dehydrogenase, glutamic acid generates glutamine under the catalysis of glutamine synthase, phosphopantetheinyl transferase can activate blue pigment synthase, and glutamine generates blue pigment synthase under the catalysis of activated blue pigment synthase.

Comparative example 1

The comparative example provides engineering bacteria for synthesizing blue-looking bacteria and a construction method thereof, and the engineering bacteria comprise the following steps:

The pTRCmob-bpsA-sfp plasmid was constructed according to steps 5.1-5.4 of example 1, and pTRCmob-bpsA-sfp was transferred into ATCC 13032 by electric shock transformation to obtain engineering bacterium CG08.

Comparative example 2

The comparative example provides an engineering bacterium for synthesizing cerulous blue and a construction method thereof, and the construction method is similar to that of comparative example 1, except that ATCC 13032 is replaced by engineering bacterium CG01 obtained in step 1.5 in example 1, and the prepared engineering bacterium is named CG09.

Comparative example 3

The comparative example provides an engineering bacterium for synthesizing cerulous blue and a construction method thereof, and the construction method is similar to that of comparative example 1, except that ATCC 13032 is replaced by engineering bacterium CG02 obtained in step 1.5 in example 2, and the prepared engineering bacterium is named CG10.

Comparative example 4

The comparative example provides an engineering bacterium for synthesizing cerulous blue and a construction method thereof, and the construction method is similar to that of comparative example 1, except that ATCC 13032 is replaced by engineering bacterium CG03 obtained in step 1.5 in example 3, and the prepared engineering bacterium is named CG11.

Comparative example 5

The comparative example provides an engineering bacterium for synthesizing cerulous blue, which has the construction method similar to that of comparative example 1, except that ATCC 13032 is replaced by engineering bacterium CG04 obtained in step 2.4 in example 1, and the prepared engineering bacterium is named CG12.

Comparative example 6

The comparative example provides an engineering bacterium for synthesizing cerulous blue, which has the construction method similar to that of comparative example 1, except that ATCC 13032 is replaced by engineering bacterium CG05 obtained in step 3.4 in example 1, and the prepared engineering bacterium is named CG13.

Effect example

Engineering bacteria CG07 of example 1 and engineering bacteria CG08-CG13 of comparative examples 1-6 are synthesized to form blue according to the method of example 4.

The OD value is measured at 612nm wavelength by using 1-18 mug/mL of blue-viewing standard substance solution, and a standard curve is drawn by taking blue-viewing concentration as an abscissa and OD612 as an ordinate, and the obtained standard curve is Y=0.0694X+0.0087 and R ² =0.999.

The OD values of the different samples at 612nm were measured using an ultraviolet spectrophotometer, and the blue-viewing yield was calculated from the standard curve, the results are shown in table 7, and the synthetic route is shown in fig. 1.

TABLE 7 shake flask fermentation yield (g/L) for blue viewing

Group of	Corresponding engineering bacteria	Blue viewing yield (g/L)
			Example 1	CG07	15.48
Comparative example 1	CG08	4.85
			Comparative example 2	CG09	7.90
Comparative example 3	CG10	5.93
			Comparative example 4	CG11	6.32
Comparative example 5	CGl2	11.61
			Comparative example 6	CG13	13.87

As shown in Table 7, engineering bacterium CG07 of the application can successfully synthesize blue-viewing under IPTG induction by shake flask fermentation, and the yield of blue-viewing is 15.48g/L.

A number of byproducts occur during the biosynthesis of blue viewing: pyruvic acid generates lactate under the action of lactate dehydrogenase, isocitrate generates succinic acid under the action of isocitrate lyase, glutamic acid generates proline under the action of glutamate 5-kinase, and glutamine generates glutamic acid under the action of glutaminase. As shown in Table 7, the blue viewing yields of the engineering bacteria obtained in examples 1-3 are higher than those of the engineering bacteria obtained in comparative examples 1-6, and the blue viewing yields are improved by knocking out the encoding genes of lactate dehydrogenase, isocitrate lyase, glutamate 5-kinase and glutaminase, reducing the generation of byproducts and improving the flux of the paths related to the generation of blue viewing.

Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present application and not for limiting the scope of the present application, and although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present application may be modified or substituted equally without departing from the spirit and scope of the technical solution of the present application.

Claims

1. An engineering bacterium for synthesizing blue viewing, wherein the engineering bacterium expresses blue pigment synthetase bpsA, phosphopantetheinyl transferase sfp, glutamine synthetase glnA and glutamate dehydrogenase gdhA;

the engineering bacteria for synthesizing the cerulous blue are preserved in China Center for Type Culture Collection (CCTCC) NO: m2024243, the preservation date is 2024, 01 and 29.

2. The engineering bacterium according to claim 1, wherein the glutamine synthetase glnA is encoded by a glutamine synthetase glnA containing a mutation site, and the coding sequence of the glutamine synthetase is shown in SEQ No.1, and the mutation site comprises any one of the following 1) -3):

1) Mutation from tyrosine to phenylalanine at position 405;

2) Mutation from tyrosine to asparagine at position 405;

3) Mutation from tyrosine to leucine at position 405.

3. The engineered bacterium of claim 1, wherein said blue pigment synthase bpsA is derived from streptomyces lavendulae Streptomyces lavendulae; the coding sequence of the blue pigment synthetase bpsA is shown as SEQ ID NO. 2.

4. The engineered bacterium of claim 1, wherein said phosphopantetheinyl transferase sfp is derived from bacillus subtilis Bacillus subtilis; the coding sequence of the phosphopantetheinyl transferase sfp is shown as SEQ ID NO. 3.

5. The engineered bacterium of claim 1, wherein said glutamate dehydrogenase gdhA is derived from corynebacterium glutamicum; the coding sequence of the glutamate dehydrogenase gdhA is shown in SEQ ID NO. 4.

6. The method for constructing engineering bacteria according to any one of claims 1 to 5, comprising the steps of:

a5, transferring the plasmid with the coding genes of the blue pigment synthetase bpsA and the phosphopantetheinyl transferase sfp into the engineering bacteria D obtained in the step A4 to obtain engineering bacteria E, wherein the engineering bacteria E is engineering bacteria for synthesizing blue.

7. The method according to claim 6, wherein in step A5, the genes encoding blue dye synthase bpsA and phosphopantetheinyl transferase sfp are codon optimized and integrated into engineering bacterium D.

8. The use of an engineered bacterium of any one of claims 1-5 in the synthesis of cerulosa.

9. A method for synthesizing blue-viewing, characterized in that the seed solution of the engineering bacterium according to any one of claims 1-5 and a fermentation medium are fermented in a bioreactor.

10. The method of claim 9, wherein the fermentation conditions are fermentation at 20 ℃ at 200rpm for 72-96 hours;