CN113061642B

CN113061642B - Application of high-throughput screening construction of glycollic acid high-yield bacteria by utilizing biosensor

Info

Publication number: CN113061642B
Application number: CN202110242666.8A
Authority: CN
Inventors: 邓禹; 周胜虎; 徐淑敏; 毛银; 赵磊; 陆雨洁; 肖潇; 王心仪
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2021-03-05
Filing date: 2021-03-05
Publication date: 2024-02-27
Anticipated expiration: 2041-03-05
Also published as: CN113061642A

Abstract

The invention discloses an application of high-throughput screening construction of glycollic acid high-yield bacteria by utilizing a biosensor, and belongs to the technical field of high-throughput screening. The invention combines two biosensors of specificity response glycollic acid to construct a high-efficiency high-flux screening method, and carries out high-flux screening on a polygenic pathway library of metabolite small molecule glycollic acid, thereby realizing optimization of glycollic acid polygenic metabolic pathways in a short time. On the one hand, the screening flux can be greatly enlarged by selecting the high-yield strain based on the resistance of the flat plate, and the high-yield strain can be simply and rapidly enriched; on the other hand, the concentration of the ethanol acid in the fermentation broth is rapidly detected by a method for measuring fluorescence by combining a 96-well plate. The combination of the glycolic acid sensor and the temperature-sensitive replicon can realize repeated elimination of plasmids, and is favorable for iterative high-throughput screening. The screening sensitivity of the target high-yield strain is effectively improved through the improvement, and the enrichment efficiency of the high-yield strain is improved.

Description

Application of high-throughput screening construction of glycollic acid high-yield bacteria by utilizing biosensor

Technical Field

The invention relates to an application of high throughput screening construction of glycollic acid high-yield bacteria by utilizing a biosensor, belonging to the technical field of biochemistry.

Background

Glycolic acid is a type of α -glycolic acid that has been widely used in the industry for various purposes, including cosmetics, chemical cleaning, and degradable materials. In recent years, with the continuous development of metabolic engineering and synthetic biology, the full-biological synthesis method for generating glycolic acid has the advantages of mild reaction, high product purity, readily available raw materials and green and sustainable properties. Earlier studies have enhanced glycolic acid metabolic flux by metabolic engineering means and thus, some expensive inducer-dependent strong promoters (e.g., T7, trc promoters) have been widely used to promote transcription of biochemical synthetic pathways, significantly increasing fermentation costs. In addition, gene expression control is critical to increase recombinant protein production, fine-tune metabolic pathways and reliably express synthetic pathways, and overexpression of the glycolic acid synthetic pathway regulated by a strong promoter typically results in large amounts of protein expression beyond what is actually required, resulting in imbalance in metabolic flux and waste of cellular resources. In recent years, with the development of synthetic biology, powerful natural constitutive promoters provide another approach to increase expression levels of pathway genes to promote accumulation of target products and to implement a plug-and-play architecture for application to hosts with different genetic backgrounds.

The multi-module engineering strategy is one of the most commonly used metabolic pathway optimization strategies, and has been successfully applied to the production of a variety of high value-added chemicals. However, the process of optimizing the polygenic metabolic pathways can result in thousands of different combinations. The lack of high throughput screening methods for glycolic acid producing strains is a major challenge for systems to optimize their synthetic pathways. Aiming at the key problems, the invention establishes a glycollic acid path library and a high-efficiency and feasible high-throughput screening method based on a glycollic acid sensor, thereby rapidly balancing glycollic acid metabolic pathways, improving the growth and production performance of thalli and providing theoretical references for metabolic pathway balance and high-throughput screening of other metabolites.

Disclosure of Invention

Technical problems:

in the prior art, the glycolic acid biosensor screens the glycolic acid production strains through fluorescent signals, and in practical application, the period of the glycolic acid production strains for fermentation production of the glycolic acid is found to be contradictory with fluorescent detection, the difference between the glycolic acid production yields of the strains with short fermentation time is not obvious, and the fluorescent signals with long production time are attenuated; and glycolic acid can be secreted outside cells, so that the fluorescent signal of a single glycolic acid-producing strain in a reaction system is interfered, and the fluorescent signal of the single strain is not real.

The technical scheme is as follows:

it is a first object of the present invention to provide a screening system for glycolic acid-producing strains, comprising a first sensor, a second sensor and a strain deficient in glycolic acid synthesis.

In one embodiment, the first sensor comprises P _glcD glcC, resistance gene and thermo-sensitive replicon Ori101; the genes in the sensor are a glcC gene and a resistance gene in sequence along the direction of gene expression; the P is _glcD Regulating and controlling the expression of the resistance gene; the P is _glcD A binding site for an upper integration GlcC-glyceride allosteric protein; p (P) _glcD Upstream of the cis-element upstream enhancement factor UAS; the promoter P _ffS Upstream of and regulating the expression of glcC.

In one embodiment, the resistance gene includes, but is not limited to, an ampicillin resistance gene, a tetracycline resistance gene, a cycloserine resistance gene.

In one embodiment, the second sensor is constructed according to the method of example 1 of the patent publication No. CN110684792A and is named pGBS-P _ffS -sfgfp。

A second object of the invention is to provide a method of constructing the first sensor.

In one embodiment, the first sensor is a second sensor pGBS-P _ffS Replacement of the reporter gene sfgfp in sfgfp with a resistance groupBecause of tetA, and named pGBS-P _ffS -tetA。

In one embodiment, the first sensor pGBS-P _ffS tetA is in pGBS-P _ffS Amplification of linearized vector P with primer GBS-F/GBS-R based on sfgfp _ffS -glcC-P _glcD The tetA fragment is amplified by using the plasmid pGLY-2 as a template through the primers tetA-F/tetA-R, and the two fragments are connected.

In one embodiment, the second sensor is constructed according to the method of the patent publication No. CN110684792A and named pGBS-P _ffS -sfgfp。

A third object of the present invention is to provide an application of the above-mentioned glycolic acid screening method in high-throughput screening of microorganisms, comprising the steps of:

1) Transforming the first sensor into cells to be screened, and screening the recombinant cells in a culture medium containing antibiotics;

2) Inducing the supernatant of the cell culture selected in step (1) to a strain defective in glycolic acid synthesis containing a second sensor, and culturing the strain in a high-throughput culture vessel.

The fourth object of the present invention is to provide the application of the above high throughput screening method in screening of glycolic acid producing strains, the specific steps are as follows:

1) The first sensor is put into a glycollic acid producing strain to be screened and coated on a selective agar plate containing antibiotics;

2) Picking single colonies growing on the flat plate in the step 1) to a high-throughput culture container for fermentation culture, centrifuging and collecting a supernatant;

3) Adding the supernatant obtained in the step 2) into an M9 culture medium in a proportion of 8-12% to induce a strain with a second sensor of glycolic acid synthesis defect, measuring a single cell green fluorescence value of the mixed culture after 4-8 hours of induction, and further screening the strain;

4) And (3) culturing the strain screened in the step (3) at the constant temperature of 40-44 ℃ overnight by a shaking table to eliminate the first sensor, and re-screening by shaking fermentation.

In one embodiment, the conditions of the fermentation described in step 2) are 28-32 ℃,250rpm; the fermentation condition in the step 4) is 35-39 ℃ and 250r/min.

In one embodiment, the fermentation medium is M9 medium consisting of M9 salt solution, 8g/L glucose, 2mM MgSO ₄ 、0.1mM CaCl ₂ 50. Mu.g/ml streptomycin and 100. Mu.g/ml ampicillin.

A fifth object of the present invention is to provide a strain for producing glycolic acid, which is obtained by screening by the above-mentioned high throughput screening system.

In one embodiment, the strain is derived from glycolic acid producing strain Mgly6 and comprises the strains consisting of PUTR, respectively _gltA Regulated ycdW gene, by PUTR _gltA 、PUTR _cmk-rpsA 、PUTR _cspA 、PUTR _dnaKJ 、PUTR _grpE Or PUTR _alsRBACE Regulated aceA Gene and expression from PUTR _infC-rplT 、PUTR _infCL 、PUTR _grpE 、PUTR _hupA 、PUTR _rpsU 、PUTR _rpsT 、PUTR _gltA 、PUTR _pheM Or PUTR _cmk-rpsA Regulated gltA gene.

In one embodiment, the strain is derived from glycolic acid producing strain Mgly6 and comprises the strains consisting of PUTR, respectively _gltA Co-regulated ycdW gene and aceA gene and gene derived from PUTR _infC-rplT 、PUTR _infCL Or PUTR _grpE Regulated gltA gene.

In one embodiment, the nucleotide sequence of the ycdW gene is shown as SEQ ID NO.1, the nucleotide sequence of the aceA gene is shown as SEQ ID NO.2, the nucleotide sequence of the gltA gene is shown as SEQ ID NO.3, and the PUTR _gltA The nucleotide sequence of (C) is shown as SEQ ID NO.4, the PUTR _cmk-rpsA The nucleotide sequence of (B) is shown as SEQ ID NO.5, the PUTR _cspA The nucleotide sequence of (C) is shown as SEQ ID NO.6, the PUTR _dnaKJ The nucleotide sequence of (B) is shown as SEQ ID NO.7, the PUTR _grpE The nucleotide sequence of (C) is shown as SEQ ID NO.8, the PUTR _alsRBACE The nucleotide sequence of (B) is shown as SEQ ID NO.9, the PUTR _infC-rplT The nucleotide sequence of (B) is shown as SEQ ID NO.10, the PUTR _infCL The nucleotide sequence of (B) is shown as SEQ ID NO.11, the PUTR _hupA The nucleotide sequence of which is shown as SEQ ID NO.12, the PUTR _rpsU The nucleotide sequence of (C) is shown as SEQ ID NO.13, the PUTR _rpsT The nucleotide sequence of which is shown as SEQ ID NO.14, the PUTR _pheM The nucleotide sequence of (2) is shown as SEQ ID NO. 15.

It is a sixth object of the present invention to provide a method for producing glycolic acid by fermentation using the glycolic acid-producing strain obtained as described above.

In one embodiment, the conditions of the fermentation are: the temperature is 35-39 ℃, the aeration rate is 0.5-1.5vvm, the stirring rotating speed is 350-450rpm, and the PH is regulated to be 7.0 by ammonia water. The initial glucose adding amount is 5-8g/L, and when the glucose in the culture medium to be fermented is consumed to 1-2g/L, the glucose is supplemented, so that the glucose concentration is maintained at 1-4g/L, and the feeding is performed.

A seventh object of the present invention is to provide the use of the high throughput screening method in screening strains which metabolize glycolic acid or upstream and downstream products thereof.

An eighth object of the present invention is to provide the use of the above strain in glycolic acid biosynthesis.

The beneficial effects are that:

a high-throughput screening method for glycollic acid is established, and the method can be combined to realize the rapid optimization of glycollic acid polygene metabolic pathways. By using the high-throughput screening method established by the invention, the screening of a path library with the capacity of 60 ten thousand is completed within one week, and the optimal strain Mgly6-H1 can produce 45g/L of glycollic acid without using an inducer.

The method is a high-throughput screening method which is established for the first time and aims at producing the glycollic acid by microbial fermentation. The high-throughput screening method adopted by the invention is based on the sensor, and can be used for efficiently screening to obtain the high-yield strain through two rounds of screening. Compared with single-cell fluorescence signal detection of a flow cytometer, the selection strategy based on the resistance of the flat plate can greatly expand the screening flux, avoid the problem of fluorescence quenching accompanied by long production period of glycollic acid, and simultaneously avoid the problem that high-yield cells are difficult to identify due to diffusion of small-molecular compounds secreted to the outside of cells in a solution. In addition, the sensor cells in the logarithmic phase induced by the supernatant of the fermentation broth perfectly balance the contradiction of short half-life period and long production period of fluorescent protein, and further screen low-yield strains.

Drawings

Figure 1 glycolic acid theory of operation and strength of response to glycolic acid at different concentrations. A: glycolic acid sensor pGBS-P _ffS -sfgfp principle of operation; b: glycolic acid sensor pGBS-P _ffS -sfgfp response curve to glycolic acid at different concentrations; c: glycolic acid sensor pGBS-P _ffS The tetA principle of operation; d: glycolic acid sensor pGBS-P _ffS tetA response intensity to different concentrations of glycolic acid.

FIG. 2 high throughput screening method. A: a high throughput screening process; relationship between colony size of B plate and glycolic acid yield; c: fermentation broth induced sensor cell fluorescence value versus glycolate yield.

FIG. 3 pathway library construction process.

FIG. 4 pathway library screening for glycollic acid shake flask fermentation yield. Squares represent OD values, circles represent glycolic acid concentration, triangles represent glucose concentration, and diamonds represent acetic acid concentration.

FIG. 5 fermentation diagram of Mgly6-H1 engineering strain in tank.

Detailed Description

Coli JM109 was used for plasmid construction, MG 1655. Delta. GlcC (published in the patent: publication No. CN 110684792A) was used for characterization of the sensor, and glycolic acid producing strain Mgly6 was stored for the present laboratory (published in the article: systematic analysis of the effects of different nitrogen source and ICDH knockout on glycolate synthesis in Escherichia coli). DNA polymerase was purchased from Takara, and recombinant cloning kit C112 was purchased from Norwegian. The multifunctional enzyme-labeled instrument Cystation 3plate reader (BioTek) is used for detecting the fluorescence intensity of the sample.

The control strain was Mgly625 (Mgly 6 harbors plasmids pJUN-5 and pJUN-YA): this strain is also published in article Systematic analysis of the effects of different nitrogen source and ICDH knockout on glycolate synthesis in Escherichia coli.

Plasmid pGLY-2: published in article Balancing the carbon flux distributions between the TCA cycle and glyoxylate shunt to produce glycolate at high yield and titer in Escherichia coli.

Plasmid pJUN-YA: published in article Systematic analysis of the effects of different nitrogen source and ICDH knockout on glycolate synthesis in Escherichia coli.

Plasmid pJUN-5: published in article Systematic analysis of the effects of different nitrogen source and ICDH knockout on glycolate synthesis in Escherichia coli.

Tetracycline selection agar plates: 6.78 g.L ^-1 Disodium hydrogen phosphate, 3 g.L ^-1 Monopotassium phosphate, 1 g.L ^-1 Ammonium chloride, 0.5 g.L ^-1 Sodium chloride, 10 g.L ^-1 Glucose, 0.24 g.L ^-1 Magnesium sulfate, 0.115 g.L ^-1 Calcium chloride, 8 g.L ^-1 Tryptone, 2 g.L ^-1 Yeast powder, 8g/L glucose, 2g/L agar powder. 50mg/L kanamycin, 50mg/L ampicillin, 50mg/L streptomycin and 30mg/L tetracycline were added prior to plate pouring.

Example 1 design and construction of glycolic acid sensor

As shown in FIG. 1A, pGBS-P _ffS The sfgfp sensor comprises a promoter P _glcD sfGFP protein, promoter P _ffS The glcC gene; the promoter P _glcD Initiation of sfGFP expression, P _glcD A cis-response element enhancement factor UAS is arranged on the reaction chamber; the P is _glcD Contains a binding site for a GlcC-glyceride allosteric protein; downstream of glcC is in turn the kanamycin resistance gene and the thermo-responsive replicon Ori101; the P is _glcD Downstream of replicon Ori101, the glcC is provided with promoter P upstream _ffS . (patent publication No. CN 110684792A).

Biosensor pGBS-P _ffS tetA construction, pGBS-P as shown in FIG. 1C _ffS The tetA sensor will pGBS-P _ffS The reporter gene sfGFP protein in sfGFP is replaced by the tetracycline resistance gene tetA, the other structure being unchanged. At the position ofpGBS-P _ffS Amplification of linearized vector P with primer GBS-F/GBS-R based on sfgfp _ffS -glcC-P _glcD The tetA fragment was amplified by the primers tetA-F/tetA-R using plasmid pGLY-2 as template. The plasmid pGBS-P is obtained by assembling two fragments by using a C112 one-step cloning kit _ffS -tetA。

TABLE 1 primer sequence listing

Example 2 response of glycolic acid sensor to glycolic acid

To test the response performance of the glycolic acid sensor, expression of the reporter gene was induced by exogenous addition of glycolic acid at different concentrations.

pGBS-PpffS-sfgfp was introduced into the host cell MG 1655. Delta. GlcC, and single colonies were picked up and inoculated into a kanamycin-resistant liquid LB medium, and cultured overnight at 30℃at 250rpm to prepare a seed solution. The seed liquid is respectively at the initial OD ₆₀₀ An inoculum size of =0.05 was inoculated into M9 medium, wherein the glucose concentration contained was 4g/L. After 7-8h of culture, the OD is left ₆₀₀ Glycolic acid with different concentrations is added to the mixture until the concentration reaches about 0.6, so that the expression of green fluorescent protein is induced. The culture was continued under the same conditions for 6 hours, 50. Mu.L of the sample was sampled and diluted 3 times with PBS buffer solution, and the fluorescence value of single cells was measured by an enzyme-labeled instrument (excitation wavelength 485 nm/emission wavelength 528 nm), and the detection limit of the sensor was 0.1-200mM and the dynamic control range was 79 times as large as that of the sensor (FIG. 1B).

pGBS-P _ffS After introduction of tetA into the host strain MG 1655. Delta. GlcC, single colonies were picked and inoculated into a liquid LB medium having kanamycin resistance, and cultured overnight at 30℃and 250rpm to prepare a seed solution. The seed liquid is respectively at the initial OD ₆₀₀ Inoculum size of =0.05 was transferred to fresh LB medium, while 0, 1, 5, 10, 50, 80mM glycollic acid was added to induce expression of tetracycline resistance gene, incubated at 30deg.C for 2h, 0, 20, 30, 40, 50, 60mg/L tetracycline was added (as shown in FIG. 1D), and after further incubation for 2h, OD in logarithmic phase was measured ₆₀₀ Values were calculated and their specific growth rates calculated. As a result, as shown in FIG. 1D, the specific growth rate of the cells was determinedThe inhibition of tetracycline is obvious, but the specific growth rate of the thalli is gradually increased along with the increase of the concentration of glycollic acid, which shows that the growth rate of the thalli can reflect the concentration of glycollic acid when the tetracycline exists; meanwhile, under the induction of high-concentration glycollic acid, the strain expresses tetracycline resistance genes, is less influenced by the concentration of tetracycline, and can grow normally.

EXAMPLE 3 construction of pathway library

In glycolic acid production, there are three key pathway genes, ycdW, aceA and gltA, respectively, which are controlled by the inducible promoters ptrc and T7 promoter. The Trc and T7 promoters were replaced with a series of promoters with gradient strength to fine tune the metabolic pathway and avoid IPTG. 22 PUTRs (promoter and 5' UTR complex) with gradient strength were amplified and randomly ligated upstream of three pathway genes, respectively, and the constructed plasmid library was co-transformed into the sensor-carrying glycolic acid engineering strain Mgly6 (pGBS-P) _ffS tetA), this strain was named Mgly6-TP6101, generating a final library of theoretical diversity 10648. The specific construction steps are shown in fig. 3:

amplifying the plasmid pJUN-YA containing the gene ycdW by using a primer PycdW-F/PycdW-R to obtain a linearization vector ptrc-ycdWaceA, and amplifying the plasmid pJUN-5 containing the gene gltA by using a primer PgltA-F/PgltA-R to obtain a linearization vector pCDF-gltA; the amplified 22 PUTRs were mixed uniformly at an equimolar ratio (0.06 pmol) and randomly ligated with the linearized vectors ptrc-ycdWaceA and pCDF-gltA to give two plasmid libraries ptrc- α -ycdWaceA and pCDF- γ -gltA.

The plasmid library ptrc-alpha-ycdWaceA containing the gene aceA constructed in the previous step was amplified and linearized using the primer pair PaceA-F/PaceA-R. In order to introduce template diversity as much as possible, the template ptrc-alpha-ycdwaceA was added in excess (200 ng), and after the amplification of the linearized vector was completed, the recovered linearized vector was digested with Dpn I enzyme to remove the excess plasmid template. Then 22 PUTRs were randomly inserted upstream of aceA to obtain the plasmid library ptrc- α -ycdW- β -aceA.

Co-transformation of the constructed plasmid library into the carrying sensor pGBS-P _ffS Glycolic acid of tetAEngineering strain Mgly6, generates a final pathway library with a theoretical diversity of 10648.

Example 4 evaluation of high throughput screening methods

To verify the feasibility of the high throughput screening method in practical applications, the pathway library constructed in example 3 was plated on tetracycline selection agar plates. As can be seen from example 2: in the presence of tetracycline, the sensor GRT-P is carried _ffS The growth rate of tetA cells reflects glycolic acid concentration; meanwhile, under the induction of high-concentration glycollic acid, the strain expresses tetracycline resistance genes, is less influenced by the concentration of tetracycline, and can grow normally. Under the selective pressure of tetracycline, many colonies grew on the tetracycline selection plate as single colonies of different sizes, indicating a different ability of these colonies to produce glycolic acid. Single colonies with different diameters are picked by a high-throughput bacterial picker (QPix 420, molecular Devices, USA) to prepare seed liquid, colonies with diameters of 0.5-1.0mm are defined as microcolonies, colonies with diameters of 1.0-1.5mm are defined as medium colonies, and colonies with diameters of 1.5-2.5mm are defined as large colonies. Then fermenting with 24-well plate, fermenting for 24 hr, and detecting glycolic acid yield by High Performance Liquid Chromatography (HPLC), primary Bio-Rad berle AminexHPX-87H organic acid column) mobile phase of 5mM H in HPLC detection ₂ SO ₄ The column temperature was 35℃and the UV detector 210nm. As shown in FIG. 2B, the concentration of the glycolic acid produced by the cells positively correlated with the diameter of the colony, and the larger the diameter of the colony, the larger the yield of the glycolic acid produced by the cells.

To verify the feasibility of the 48-well plate screening, fluorescence values were compared to glycolic acid production as measured by high performance liquid chromatography. Firstly, randomly picking a plurality of single colonies on a tetracycline resistance flat plate, fermenting in a 48-pore plate for 24 hours, centrifuging at 4000rpm of the pore plate for 5 minutes to obtain a supernatant, adding the supernatant into an M9 culture medium in a proportion of 10 percent, and inducing the culture to be in a logarithmic phase in advance without producing glycollic acid and containing pGBS-P _ffS Sensor cell MG 1655. Delta. GlcC of sfgfp (pGBS-P) _ffS Sfgfp), after 6h induction 50uL of the sample was diluted appropriately and its green fluorescence value was determined with a microplate reader. (MG 1655. Delta. GlcC (pGBS-P) _ffS Sfgfp) culture method reference patent: publication number CN110684792 a). Along with itAfter that, the remaining fermentation broth was treated with a 0.22 μm filter for HPLC to detect the glycolic acid concentration. As a result, as shown in FIG. 2C, the concentration of glycolic acid is proportional to fluorescence, R ² 0.9038, thereby proving the use of the biosensor pGBS-P _ffS Measurement of glycolic acid production by sfgfp fluorescence is an accurate method.

EXAMPLE 5 high throughput screening of high-yield glycolic acid strains

The biggest challenge faced in the optimization of multiple gene pathways is the need to screen large numbers of libraries, and therefore efficient and viable screening methods are particularly important. As shown in fig. 2, the biosensor constructed according to example 1 established a high throughput screening method, which comprises the following steps:

1) The library of pathways of theoretical diversity 10648 constructed in step example 3 was plated on tetracycline selection agar plates and incubated at 30℃for 12-16h at 250 rpm.

2) Single large colonies (1.5-2.5 mm in diameter) were selected and inoculated in 48-well plates containing 0.5mL M9 medium and cultured at 30℃for 24 hours at 250r/min.

3) Centrifuging the 48-well plate at 4000rpm for 5min in step 2) to obtain supernatant, adding into M9 medium at a ratio of 10%, and inducing sensor cells MG1655 ΔglcC (pGBS-P) cultured in advance to logarithmic phase _ffS Sfgfp), after 6h induction 50uL of the sample was diluted appropriately and its green fluorescence value was determined with a microplate reader. (MG 1655. Delta. GlcC (pGBS-P) _ffS Sfgfp) culture method reference patent: publication number CN110684792 a).

Example 6 constitutive engineering strain shake flask fermentation double Screen

After two rounds of screening, 155 high-yield strains are finally obtained, and the strain is cultured overnight at 42 ℃ in a shaking table to eliminate the sensor pGBS-P _ffS tetA followed by shaking re-screening under fermentation conditions as follows:

fermentation medium: m9 saline+8 g/L glucose+2 mM MgSO ₄ +0.1mM CaCl ₂ +50. Mu.g/ml streptomycin+100. Mu.g/ml ampicillin.

Fermentation conditions: the overnight cultured seed solution was inoculated into 25mL of fermentation medium at 2% by volume, cultured at 37℃for 24 hours at 250r/min, and then centrifuged to obtain a fermentation broth, which was treated with a 0.22 μm filter membrane for HPLC to detect the concentration of glycolic acid. As a result, as shown in FIG. 4, 14% of the strains exceeded the production of Mgly625 strain of the control group, and their production and the PUTR names used for overexpressing the ycdW gene, aceA gene and gltA gene are shown in Table 2, and the corresponding PUTR nucleotide sequences are shown in Table 2. The strain with the highest yield is Mgly6-H1, and the yield is 3.6g/L.

TABLE 2 PUTR for controlling constitutive glycolate producing strain pathway genes

Note that: "-" indicates that the genes ycdW and aceA are jointly controlled by the same promoter.

EXAMPLE 7 production of glycolic acid by fermentation in 5L fermenter

To further increase glycolic acid production, mgly6-H1 was fed to a 5L fermenter for fermentation, the medium was shake flask fermented, and the initial glucose was 5g/L. The fermentation conditions were as follows:

the temperature was 37℃and the aeration rate was 1vvm, the stirring speed was 400rpm, and the pH was maintained at 7.0 by adjusting the pH with ammonia. Glucose is supplemented when the glucose in the culture medium to be fermented is consumed to 1-2g/L, and the glucose concentration is maintained at 1-4 g/L.

Analysis of results: samples are taken every 2 hours in the fermentation process, the fermentation liquid is separated from thalli by centrifugation at 12000rpm for 2min, and the fermentation liquid is treated by a 0.22 mu m filter membrane and is used for detecting the concentration of glycollic acid, glucose and byproduct acetic acid by HPLC. As shown in FIG. 5, the OD value of the cells increases rapidly from 0 to 28h, and after 28h, the cells tend to be stable and reach the highest OD at 108h ₆₀₀ With a value of 18.8, the glycolic acid yield was continuously accumulated throughout the fermentation period, reaching a maximum at 108h and a yield of 45g/L. After fermentation to 23h, the glycolic acid yield reached 12.4g/L, reaching the highest yield, i.e. 0.77g/g glucose, 91.4% of the theoretical yield, the average yield over the fermentation period was 0.54g/g glucose, 64.0% of the theoretical yield.

Comparative example:

use of the biosensor pGBS-P _ffS Sfgfp pair examples3, screening the obtained plasmid library:

sensor pGBS-P _ffS The plasmid library obtained in example 3 was co-transformed into strain Mgly6 (pGBS-P) _ffS Sfgfp), cultured at 30 ℃ for 24 hours, followed by flow screening. As a result, the detected signal values were all found to be low and not referenced. Probably due to the longer incubation time of the strain, fluorescence quenching was caused; in addition, since glycolic acid is a small molecular substance secreted to the outside of cells, intracellular fluorescent signals are detected in the screening process of a flow cytometer, and a strain with a real high yield of glycolic acid cannot be screened in the screening process.

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.

SEQUENCE LISTING

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tcgaaaaaag gctacatcaa cagcctcggc gcactgactg gcggtcaggc gctgcaacag 240

gcgaaagcgg gtattgaagc agtctatctg tcgggatggc aggtagcggc ggacgctaac 300

ctggcggcca gcatgtatcc ggatcagtcg ctctatccgg caaactcggt gccagctgtg 360

gtggagcgga tcaacaacac cttccgtcgt gccgatcaga tccaatggtc cgcgggcatt 420

gagccgggcg atccgcgcta tgtcgattac ttcctgccga tcgttgccga tgcggaagcc 480

ggttttggcg gtgtcctgaa tgcctttgaa ctgatgaaag cgatgattga agccggtgca 540

gcggcagttc acttcgaaga tcagctggcg tcagtgaaga aatgcggtca catgggcggc 600

aaagttttag tgccaactca ggaagctatt cagaaactgg tcgcggcgcg tctggcagct 660

gacgtgacgg gcgttccaac cctgctggtt gcccgtaccg atgctgatgc ggcggatctg 720

atcacctccg attgcgaccc gtatgacagc gaatttatta ccggcgagcg taccagtgaa 780

ggcttcttcc gtactcatgc gggcattgag caagcgatca gccgtggcct ggcgtatgcg 840

ccatatgctg acctggtctg gtgtgaaacc tccacgccgg atctggaact ggcgcgtcgc 900

tttgcacaag ctatccacgc gaaatatccg ggcaaactgc tggcttataa ctgctcgccg 960

tcgttcaact ggcagaaaaa cctcgacgac aaaactattg ccagcttcca gcagcagctg 1020

tcggatatgg gctacaagtt ccagttcatc accctggcag gtatccacag catgtggttc 1080

aacatgtttg acctggcaaa cgcctatgcc cagggcgagg gtatgaagca ctacgttgag 1140

aaagtgcagc agccggaatt tgccgccgcg aaagatggct ataccttcgt atctcaccag 1200

caggaagtgg gtacaggtta cttcgataaa gtgacgacta ttattcaggg cggcacgtct 1260

tcagtcaccg cgctgaccgg ctccactgaa gaatcgcagt tctaa 1305

<210> 3

<211> 1284

<212> DNA

<213> artificial sequence

<400> 3

atggctgata caaaagcaaa actcaccctc aacggggata cagctgttga actggatgtg 60

ctgaaaggca cgctgggtca agatgttatt gatatccgta ctctcggttc aaaaggtgtg 120

ttcacctttg acccaggctt cacttcaacc gcatcctgcg aatctaaaat tacttttatt 180

gatggtgatg aaggtatttt gctgcaccgc ggtttcccga tcgatcagct ggcgaccgat 240

tctaactacc tggaagtttg ttacatcctg ctgaatggtg aaaaaccgac tcaggaacag 300

tatgacgaat ttaaaactac ggtgacccgt cataccatga tccacgagca gattacccgt 360

ctgttccatg ctttccgtcg cgactcgcat ccaatggcag tcatgtgtgg tattaccggc 420

gcgctggcgg cgttctatca cgactcgctg gatgttaaca atcctcgtca ccgtgaaatt 480

gccgcgttcc gcctgctgtc gaaaatgccg accatggccg cgatgtgtta caagtattcc 540

attggtcagc catttgttta cccgcgcaac gatctctcct acgccggtaa cttcctgaat 600

atgatgttct ccacgccgtg cgaaccgtat gaagttaatc cgattctgga acgtgctatg 660

gaccgtattc tgatcctgca cgctgaccat gaacagaacg cctctacctc caccgtgcgt 720

accgctggct cttcgggtgc gaacccgttt gcctgtatcg cagcaggtat tgcttcactg 780

tggggacctg cgcacggcgg tgctaacgaa gcggcgctga aaatgctgga agaaatcagc 840

tccgttaaac acattccgga atttgttcgt cgtgcgaaag acaaaaatga ttctttccgc 900

ctgatgggct tcggtcaccg cgtgtacaaa aattacgacc cgcgcgccac cgtaatgcgt 960

gaaacctgcc atgaagtgct gaaagagctg ggcacgaagg atgacctgct ggaagtggct 1020

atggagctgg aaaacatcgc gctgaacgac ccgtacttta tcgagaagaa actgtacccg 1080

aacgtcgatt tctactctgg tatcatcctg aaagcgatgg gtattccgtc ttccatgttc 1140

accgtcattt tcgcaatggc acgtaccgtt ggctggatcg cccactggag cgaaatgcac 1200

agtgacggta tgaagattgc ccgtccgcgt cagctgtata caggatatga aaaacgcgac 1260

tttaaaagcg atatcaagcg ttaa 1284

<210> 4

<211> 359

<212> DNA

<213> artificial sequence

<400> 4

aatcattcaa caaagttgtt acaaacatta ccaggaaaag catataatgc gtaaaagtta 60

tgaagtcggt atttcaccta agattaactt atgtaacagt gtggaagtat tgaccaattc 120

attcgggaca gttattagtg gtagacaagt ttaataattc ggattgctaa gtacttgatt 180

cgccatttat tcgtcatcaa tggatccttt acctgcaagc gcccagagct ctgtacccag 240

gttttcccct ctttcacaga gcggcgagcc aaataaaaaa cgggtaaagc caggttgatg 300

tgcgaaggca aatttaagtt ccggcagtct tacgcaataa ggcgctaagg agaccttaa 359

<210> 5

<211> 95

<212> DNA

<213> artificial sequence

<400> 5

aaactggttt ttgcacacaa cgttaacgat ttgtggcgtc ggcgcgtata atgcgcgcgg 60

ttatgttaac ggtacgcctg ttttaaggag ataaa 95

<210> 6

<211> 220

<212> DNA

<213> artificial sequence

<400> 6

aatcataaat atgaaaaata attgttgcat cacccgccaa tgcgtggctt aatgcacatc 60

aacggtttga cgtacagacc attaaagcag tgtagtaagg caagtccctt caagagttat 120

cgttgatacc cctcgtagtg cacattcctt taacgcttca aaatctgtaa agcacgccat 180

atcgccgaaa ggcacactta attattaaag gtaatacact 220

<210> 7

<211> 175

<212> DNA

<213> artificial sequence

<400> 7

gcacaaaaaa tttttgcatc tcccccttga tgacgtggtt tacgacccca tttagtagtc 60

aaccgcagtg agtgagtctg caaaaaaatg aaattgggca gttgaaacca gacgtttcgc 120

ccctattaca gactcacaac cacatgatga ccgaatatat agtggagacg tttag 175

<210> 8

<211> 99

<212> DNA

<213> artificial sequence

<400> 8

gattgatgac aatgtgagtg cttcccttga aaccctgaaa ctgatcccca taataagcga 60

agttagcgag atgaatgcga aaaaaacgcg gagaaattc 99

<210> 9

<211> 205

<212> DNA

<213> artificial sequence

<400> 9

agcaacatct atcatctaaa aaaccagaaa aacaaataac atcatgtttt taaactaatt 60

aaatgaaata aaattttaag ccactcgcca ttgttcacaa taaaataaac tttataaatt 120

ttattttttt gtgaagtcgc cagcatcttt tctgttcttg ctgtggtgat atagtggcgt 180

cttcaattca aggacaagag aacgt 205

<210> 10

<211> 240

<212> DNA

<213> artificial sequence

<400> 10

gcgggcattc gtgttaaagc agacttgaga aatgagaaga ttggctttaa aatccgcgag 60

cacactttgc gtcgcgtccc atatatgctg gtctgtggtg ataaagaggt ggaatcaggc 120

aaagttgccg ttcgcacccg ccgtggtaaa gacctgggaa gcatggacgt aaatgaagtg 180

atcgagaagc tgcaacaaga gattcgcagc cgcagtctta aacaattgga ggaataaggt 240

<210> 11

<211> 768

<212> DNA

<213> artificial sequence

<400> 11

actgaagaac aaattcgcga tgaagttaac ggatgtatcc gtttagtcta tgatatgtac 60

agcacttttg gcttcgagaa gatcgtcgtc aaactctcca ctcgtcctga aaaacgtatt 120

ggcagcgacg aaatgtggga tcgtgctgag gcggacctgg cggttgcgct ggaagaaaac 180

aacatcccgt ttgaatatca actgggtgaa ggcgctttct acggtccgaa aattgaattt 240

accctgtatg actgcctcga tcgtgcatgg cagtgcggta cagtacagct ggacttctct 300

ttgccgtctc gtctgagcgc ttcttatgta ggcgaagaca atgaacgtaa agtaccggta 360

atgattcacc gcgcaattct ggggtcgatg gaacgtttca tcggtatcct gaccgaagag 420

ttcgctggtt tcttcccgac ctggcttgcg ccggttcagg ttgttatcat gaatattacc 480

gattcacagt ctgaatacgt taacgaattg acgcaaaaac tatcaaatgc gggcattcgt 540

gttaaagcag acttgagaaa tgagaagatt ggctttaaaa tccgcgagca cactttgcgt 600

cgcgtcccat atatgctggt ctgtggtgat aaagaggtgg aatcaggcaa agttgccgtt 660

cgcacccgcc gtggtaaaga cctgggaagc atggacgtaa atgaagtgat cgagaagctg 720

caacaagaga ttcgcagccg cagtcttaaa caattggagg aataaggt 768

<210> 12

<211> 189

<212> DNA

<213> artificial sequence

<400> 12

taagaccaga aaacgtgatt taacgcctga tttgtcgtac ctggagtctt ccctttcgcc 60

ccccgtctgg tctacatttg gggggcgaaa aaaagtggct atcggtgcgt gtatgcagga 120

gagtgctatt ctggcatttc cgtcgcactc gatgcttagc aagcgataaa cacattgtaa 180

ggataactt 189

<210> 13

<211> 195

<212> DNA

<213> artificial sequence

<400> 13

atgcgggttg atgtaaaact ttgttcgccc ctggagaaag cctcgtgtat actcctcacc 60

cttataaaag tccctttcaa aaaaggccgc ggtgctttac aaagcagcag caattgcagt 120

aaaattccgc accattttga aataagctgg cgttgatgcc agcggcaaac cgaattaatc 180

aaaggtgaga ggcac 195

<210> 14

<211> 192

<212> DNA

<213> artificial sequence

<400> 14

tgctgcaatt tttatcgcgg aaaagctgta ttcacacccc gcaagctggt agaatcctgc 60

gccatcacta cgtaacgagt gccggcacat taacggcgct tatttgcaca aatccattga 120

caaaagaagg ctaaaagggc atattcctcg gcctttgaat tgtccatata gaacacattt 180

gggagttgga cc 192

<210> 15

<211> 91

<212> DNA

<213> artificial sequence

<400> 15

tttcgtttca acgccatcaa aacattgact tttatcgccg tagccttttc aataaaggtc 60

ttttgaagag taccaaaagg taacgcaagc a 91

Claims

1. A glycolic acid-producing strain comprising, starting from glycolic acid-producing strain Mgly6, PUTR _gltA Regulated and controlledycdWGene, by PUTR _gltA Regulated and controlledaceAGene and the gene derived from PUTR _hupA 、PUTR _rpsT Or PUTR _gltA Regulated and controlledgltAA gene;

or comprises the components respectively made of PUTR _gltA Regulated and controlledycdWGene, by PUTR _cmk-rpsA Regulation and controlA kind of electronic deviceaceAGene and the gene derived from PUTR _rpsU Regulated and controlledgltAA gene;

or comprises the components respectively made of PUTR _gltA Regulated and controlledycdWGene, by PUTR _cspA Regulated and controlledaceAGene and the gene derived from PUTR _hupA Regulated and controlledgltAA gene;

or comprises the components respectively made of PUTR _gltA Regulated and controlledycdWGene, by PUTR _dnaKJ Regulated and controlledaceAGene and the gene derived from PUTR _pheM Regulated and controlledgltAA gene;

or comprises the components respectively made of PUTR _gltA Regulated and controlledycdWGene, by PUTR _grpE Regulated and controlledaceAGene and the gene derived from PUTR _rpsT Regulated and controlledgltAA gene;

or comprises the components respectively made of PUTR _gltA Regulated and controlledycdWGene, by PUTR _alsRBACE Regulated and controlledaceAGene and the gene derived from PUTR _cmK-rpsA Regulated and controlledgltAA gene;

or comprises the components respectively made of PUTR _gltA Co-regulatedycdWGene and geneaceAGene and the gene derived from PUTR _infC-rplT 、PUTR _infCL Or PUTR _grpE Regulated and controlledgltAA gene;

wherein the saidycdWThe nucleotide sequence of the gene is shown as SEQ ID NO.1, and the gene is shown in the specificationaceAThe nucleotide sequence of the gene is shown as SEQ ID NO.2, and thegltAThe nucleotide sequence of the gene is shown as SEQ ID NO.3, and the PUTR _gltA The nucleotide sequence of (C) is shown as SEQ ID NO.4, the PUTR _cmk-rpsA The nucleotide sequence of (B) is shown as SEQ ID NO.5, the PUTR _cspA The nucleotide sequence of (C) is shown as SEQ ID NO.6, the PUTR _dnaKJ The nucleotide sequence of (B) is shown as SEQ ID NO.7, the PUTR _grpE The nucleotide sequence of (C) is shown as SEQ ID NO.8, the PUTR _alsRBACE The nucleotide sequence of (B) is shown as SEQ ID NO.9, the PUTR _infC-rplT The nucleotide sequence of (B) is shown as SEQ ID NO.10, the PUTR _infCL The nucleotide sequence of (B) is shown as SEQ ID NO.11, the PUTR _hupA The nucleotide sequence of which is shown as SEQ ID NO.12, the PUTR _rpsU The nucleotide sequence of (C) is shown as SEQ ID NO.13, the PUTR _rpsT The nucleotide sequence of which is shown as SEQ ID NO.14, the PUTR _pheM The nucleotide sequence of (2) is shown as SEQ ID NO. 15.

2. A method for producing glycolic acid, characterized in that fermentation is carried out using the glycolic acid-producing strain of claim 1.

3. The method according to claim 2, wherein the fermentation conditions are: the temperature is 35-39 ℃, the ventilation is 0.5-1.5vvm, the stirring rotating speed is 350-450rpm, and the PH is regulated to be 7.0 by ammonia water; the initial glucose addition amount is 5-8g/L, and glucose is added when glucose is consumed to 1-2g/L, so as to maintain glucose concentration at 1-4 g/L.

4. Use of the glycolic acid-producing strain of claim 1 in glycolic acid biosynthesis.