CN104195158B - The recombination bacillus coli of poly 3-hydroxy butyrate and construction process and purposes are produced in one strain - Google Patents

Abstract

The invention discloses a strain of recombinant Escherichia coli producing poly 3-hydroxybutyrate and its construction method and application. The method is as follows: E.coli? The promoter and ribosome binding site sequences of the sdaA gene of JM109 were replaced with constitutive promoters trc and RBS5; the designed ribosome binding sites were connected with genes pgk, serA, serB, and serC respectively to form fragments, and The transcriptional regulatory sequence Trc-162 constituted the operon PSer, which was integrated into the 58bp site of the serC gene in the genome; then the transcriptional regulatory sequence Trc-162 was inserted 48bp upstream of the operon gene encoding the pyruvate dehydrogenase complex aceEF, and the engineering strain SD06 was constructed. The bacterium constructed by the invention has a clear genetic background, more effectively acquires acetyl coenzyme A, and when glucose is used as a substrate in a basic salt medium, the yield of polytrihydroxybutyrate is increased to 2.66 times that of the wild bacterium.

Description

A recombinant Escherichia coli producing poly-3-hydroxybutyrate and its construction method and use

technical field

The invention belongs to the field of bioengineering technology and application, and in particular relates to a strain of recombinant Escherichia coli producing poly-3-hydroxybutyrate, its construction method and application.

Background technique

Polyhydroxyalkanoate (PHA) is a kind of polymer biopolyester that widely exists in microbial cells. As a carbon source and energy reserve, it has complete biocompatibility and biodegradability, and has the ability to partially replace petrochemical product potential. Poly-3-hydroxybutyrate (PHB) is one of the most commonly studied short-chain monomer polymers, and has broad application prospects in many fields. PHB has the advantages of insolubility in water, moisture resistance, high optical purity, and good oxygen permeability.

In most natural PHB-producing bacteria (including Ralstoniaeutropha, Pseudomonasputida, Aeromonashhydrophila, etc.), the synthesis of PHB consists of three consecutive enzymatic reaction steps, first β-ketothiolase PhaA catalyzes the condensation of two acetyl-CoA to form Acetoacetyl-CoA is further reduced to (R)-3-hydroxybutyryl-CoA under the catalysis of NADPH-dependent acetoacetyl-CoA reductase PhaB, and finally polymerized into PHB by PHA synthase PhaC. Escherichia coli, as the most widely studied microorganism in industrial biotechnology, does not have the synthesis pathway of PHB itself. However, when the heterologous PHB synthesis pathway is introduced, the recombinant Escherichia coli has been widely used as a good PHB production strain due to its rapid growth, availability of a variety of cheap carbon sources and a completely clear genetic manipulation system. Moreover, Escherichia coli does not have PHA degrading enzymes, and the accumulation level of polyester is relatively high, which is easy to break the wall, which is beneficial to the separation and purification of products.

At present, the transformation strategy of PHB-producing Escherichia coli using genetic engineering methods mainly includes increasing the intracellular enrichment of PHB synthesis precursor acetyl-CoA and cofactor NADPH and optimizing the synthesis pathway of PHB. Genetic engineering efforts aimed at improving E. coli PHB productivity by increasing intracellular enrichment of acetyl-CoA have focused on modifying the glycolytic pathway and reducing the expression of acetyl-CoA-derived by-products and competing pathways.

As an important model strain, Escherichia coli has a relatively in-depth understanding of its physiological and biochemical characteristics and genetic background. The relevant molecular biology methods and gene manipulation technologies are relatively mature, which is conducive to the development of metabolic engineering and synthetic biology. Rational design to improve strains.

Contents of the invention

The purpose of the present invention is to overcome the deficiencies of the prior art and provide a recombinant Escherichia coli strain producing poly-3-hydroxybutyrate.

The second object of the present invention is to provide a method for constructing a recombinant Escherichia coli producing poly-3-hydroxybutyrate.

The third object of the present invention is to provide the use of a strain of recombinant Escherichia coli producing poly-3-hydroxybutyrate.

A method for constructing a recombinant escherichia coli producing poly-3-hydroxybutyrate, comprising the steps of:

(1) Design the ribosome binding site code-named RBS5 shown in SEQIDNO.1, and replace the promoter and ribosome binding site sequence of the sdaA gene of EscherichiacoliJM109 with the constitutive promoter trc shown in SEQIDNO.33 and RBS5 to construct engineering strain SD01;

(2) Design the ribosome binding site RBS1 shown in SEQIDNO.2, the ribosome binding site RBS2 shown in SEQIDNO.3, the ribosome binding site RBS3 shown in SEQIDNO.4 and the ribosome binding site RBS3 shown in SEQIDNO.5 The indicated ribosome binding site RBS4;

(3) The gene pgk coding sequence shown in RBS1 and SEQIDNO.36 is connected into fragment 1, the gene serA coding sequence shown in RBS2 and SEQIDNO.37 is connected into fragment 2, and the gene serB shown in RBS3 and SEQIDNO.38 is connected The coding sequence is connected into fragment 3, and the gene serC coding sequence shown in RBS4 and SEQ ID NO.39 is connected into fragment 4;

(4) Fragment 1, Fragment 2, Fragment 3 and Fragment 4 are fused into the operator PSer by overlapping-extension PCR, and integrated into step (1) together with the transcriptional regulatory sequence Trc-162 containing the promoter shown in SEQ ID NO.32 The genomic serC gene-58bp site of the obtained engineering strain SD01 was obtained to obtain the engineering strain SD02;

(5) Insert the sequence shown in SEQ ID NO.32 in the upstream 48bp of the aceEF operon gene encoding the pyruvate dehydrogenase complex complex of the bacterial strain SD02 to construct the engineering strain SD06;

(6) The plasmid pBHR68 containing the PHB synthesis pathway gene was introduced into SD06, and the genotype was E.coliJM109, Ptrc-RBS5-sdaA, Trc-162-RBS1-pgk-RBS2-serA-RBS3-serB-RBS4-serC, Trc -162-aceEF, pBHR68, amp ^r , referred to as recombinant Escherichia coli containing pBHR68E.coliSD06 producing poly-3-hydroxybutyrate.

A recombinant Escherichia coli producing poly-3-hydroxybutyrate constructed by the above method.

Use of the above-mentioned poly-3-hydroxybutyrate-producing recombinant Escherichia coli in the fermentative production of poly-3-hydroxybutyrate.

Advantages of the present invention:

The recombinant Escherichia coli constructed in the present invention produces poly-3-hydroxybutyrate, the genetic background of the strain is cleared, and the entire metabolic pathway is constructed on the genome, the relevant genes are stably expressed during the fermentation process, and the constructed serine The ammonia pathway is a pathway for obtaining pyruvate in parallel with the EMP pathway. After constructing and activating this pathway, it can obtain pyruvate more efficiently. Under the action of the pyruvate dehydrogenase complex, it can efficiently obtain the precursor of PHB, acetyl-CoA, basic salt The fermentation result of the shake flask with medium glucose as the substrate reaches more than 5g/L, which is 2.66 times higher than that of wild bacteria. This laid the foundation for the continuous feeding of subsequent fermenters to increase the output and yield of PHB.

Description of drawings

Figure 1 is the gene manipulation target.

Figure 2 shows the construction of each gene in the serine synthesis pathway.

Figure 3 is the serine synthesis operon PSer.

Figure 4 is an electrophoresis verification map of sdaA gene manipulation.

Figure 5 is the map of pBluescriptSK(-).

Figure 6 is a map of pBHR68.

detailed description

The present invention will be further described below in conjunction with the examples. The following examples are intended to enable those skilled in the art to better understand the present invention, but the present invention is not limited in any way.

The source of the original strain E.coliJM109 used in the present invention is purchased from Transgen ( http://www.transgen.com/ ).

The source of the original strain Corynebacterium glutamicumATCC13032 used in the present invention is American Type Culture Collection (ATCC) ( http://www.atcc.org/ ).

The source of the original strain RalstoniaeutrophaH16 used in the present invention is AmericanTypeCultureCollection (ATCC) ( http://www.atcc.org/ ).

The source of original plasmids pTKRED and pTKS/CS is Addgene (Addgene, https://www.addgene.org/ ).

Other biochemical reagents used were purchased from Sangon Bioengineering (Shanghai) Co., Ltd. ( http://www.sangon.com/ ).

Figure 1 is the gene manipulation target, where PDH represents the pyruvate dehydrogenase complex, and its coding genes include aceEF and lpd.

Embodiment 1SdaA gene upstream +123bp to -636bp sequence replacement

The specific operation is as follows:

Using two pairs of primers sdaApU_F (SEQIDNO.6), sdaApU_R (SEQIDNO.7) and sdaApL_F (SEQIDNO.10), sdaApL_R (SEQIDNO.11), using the genome of E.coliJM109 as a template, using KOD-plus high-fidelity DNA polymerase The upstream and downstream homology arms sdaAp-U and sdaAp-L with sizes of 349bp and 314bp were amplified respectively. Using sdaApT_F (SEQ ID NO.8), T1 (SEQ ID NO.26) and T2 (SEQ ID NO.27), sdaApT_R (SEQ ID NO.9) and plasmid pTKS/CS as a template, use KOD-plus high-fidelity DNA polymerase to amplify the size 875bp and 1113bp upstream and downstream fragments of tetracycline sdaAp-T1 and sdaAp-T2. After gel recovery, use primers sdaApU_F (SEQ ID NO.6), T1 (SEQ ID NO.26) and T2 (SEQ ID NO.27), sdaApL_R (SEQ ID NO.11), and also use KOD-plus high-fidelity DNA polymerase for fusion PCR amplification , and the splicing products sdaAp-UT and sdaAp-TL of the upstream homology arm and tetracycline upstream and tetracycline downstream and downstream homology arms were obtained, with sizes of 1191bp and 1397bp, respectively. After the above two fragments were recovered by gel cutting, primers sdaApU_F (SEQ ID NO.6) and sdaApL_R (SEQ ID NO.11) were used to perform fusion PCR amplification using KOD-plus high-fidelity DNA polymerase to obtain a size of 2010bp (Figure 4). PCR fragment SdaAp-tet of downstream homology arm, tetracycline and constitutive promoter trc (shown in SEQ ID NO.33) and RBS5 sequence (shown in SEQ ID NO.1).

The plasmid pTKRED was transferred into E.coliJM109 strain and made electrotransfer competent; the fragment sdaAp-tet was transferred electrotransfer competently, positive clones were screened with tetracycline, and verified by colony PCR. The tetracycline resistance gene was deleted by inducing the expression of the homing endonuclease I-SceI, and verified by colony PCR, the fragment size was 662bp (Figure 4), thereby obtaining the metabolic engineering strain SD01.

Example 2

Design the ribosome binding site RBS1 shown in SEQIDNO.2, the ribosome binding site RBS2 shown in SEQIDNO.3, the ribosome binding site RBS3 shown in SEQIDNO.4 and the ribosome binding site RBS3 shown in SEQIDNO.5 Ribosome binding site RBS4.

Example 3

The artificially designed ribosome binding site RBS1 and The coding sequence of the pgk gene on the glycolytic pathway (shown in SEQ ID NO.36, NCBI ID: 947414) was amplified to obtain fragment 1 (see FIG. 2 ). (wherein primer pgk-F (SEQ ID NO.16) contains RBS1 sequence)

Using the amplification primers serA-F (SEQIDNO.18)/serA-R (SEQIDNO.19) to use the genome of Corynebacterium glutamicum (Corynebacterium glutamicum) ATCC13032 as a template, and using KOD-plus high-fidelity DNA polymerase, the artificially designed ribose The body binding site RBS2 and the serA gene coding sequence (shown in SEQ ID NO.37, NCBI ID: 3345035) on the serine anabolic pathway were amplified to obtain Fragment 2 (see Figure 2). (wherein the primer serA-F (SEQ ID NO.18) contains the RBS2 sequence)

Using amplification primers serB-F(SEQIDNO.20)/serB-R(SEQIDNO.21) to Escherichia coli JM109 genome as a template, using KOD-plus high-fidelity DNA polymerase, the artificially designed ribosome binding site RBS3 and The coding sequence of the serB gene (shown in SEQ ID NO.38, NCBI ID: 948913) on the serine anabolic pathway was amplified to obtain fragment 3 (see FIG. 2 ). (wherein primer serB-F (SEQ ID NO.20) contains RBS3 sequence)

Using amplification primers serC-F(SEQIDNO.22)/serC-R(SEQIDNO.23) to Escherichia coli JM109 genome as a template, using KOD-plus high-fidelity DNA polymerase, the artificially designed ribosome binding site RBS4 and The coding sequence of the serC gene (shown in SEQ ID NO.39, NCBI ID: 945527) on the serine anabolic pathway was amplified to obtain fragment 4 (see Figure 2). (wherein primer serC-F (SEQ ID NO.22) contains RBS4 sequence)

Example 4

Utilize the amplification primer pgk-F (SEQIDNO.16)/serC-R (SEQIDNO.23), with the fragment 1, fragment 2, fragment 3 and fragment 4 obtained in Example 3 as templates, by overlapping-extending the polymerase chain The four genes were spliced together in the order of RBS1-pgk-RBS2-serA ^Δ197 -RBS3-serB-RBS4-serC and the operon was named PSer (see FIG. 3 ).

Example 5: Genomic serC upstream 58bp site inserted into the PSer operon

Using serU_F (SEQIDNO.14) and serU_R (SEQIDNO.15) as primers, using the E.coliJM109 genome as a template, using KOD-plus high-fidelity DNA polymerase to amplify the upstream homology arm ser-U with a size of 574bp, manipulate The sub-PSer acts as a downstream homology arm. Use serT_F (SEQ ID NO.34), T1 (SEQ ID NO.26) and T2 (SEQ ID NO.27), serT_R (SEQ ID NO.35) to use plasmid pTKS/CS as a template, and use KOD-plus high-fidelity DNA polymerase to amplify to obtain the size They are 895bp and 1159bp tetracycline upstream and downstream fragments ser-T1 and ser-T2. After recovery by cutting the gel, use primers serU_F (SEQ ID NO.14), T1 (SEQ ID NO.26) and T2 (SEQ ID NO.27), serC_R (SEQ ID NO.23), and also use KOD-plus high-fidelity DNA polymerase for fusion PCR amplification , the splicing products ser-UT and ser-TL of the upstream homology arm and tetracycline upstream and tetracycline downstream and downstream homology arms were obtained, with sizes of 1430bp and 5569bp, respectively. After gel cutting and recovery, use primers serU_F and serC_R, and also use KOD-plus high-fidelity DNA polymerase to carry out fusion PCR amplification, and obtain the transcriptional regulatory sequence of 6421bp upstream and downstream homology arms, tetracycline, Trc-162 (SEQ ID NO.32) And the PCR fragment ser-tet of the PSer operon.

The plasmid pTKRED was transferred into the E.coliSD01 strain and made electrotransfer competent; the fragment ser-tet was transferred into the electrotransfer competent obtained in this step, positive clones were screened with tetracycline, and verified by colony PCR to obtain tetracycline-bearing strain E. coliSD02tet. The tetracycline resistance gene was deleted by inducing the expression of homing endonuclease I-SceI, and verified by colony PCR to obtain E.coliSD02 strain.

Example 6: Inserting the Trc-162 transcriptional regulatory sequence into the upstream 48bp site of the pyruvate dehydrogenase complex coding gene aceEF gene

Using two pairs of primers, PaceEFU_F (SEQIDNO.28), PaceEFU_R (SEQIDNO.29) and PaceEFL_F (SEQIDNO.30), PaceEFL_R (SEQIDNO.31), using the genome of E.coliJM109 as a template, using KOD-plus high-fidelity DNA polymerase The upstream and downstream homology arms PaceEF-U and PaceEF-L with the size of 415bp and 272bp were amplified respectively. Using TetF (SEQ ID NO.12) and TetR (SEQ ID NO.13) as primers and the genome of strain E.coliSD02tet as a template, the tetracycline fragment tet with a size of 1551 bp was amplified using KOD-plus high-fidelity DNA polymerase. The upper and lower homology arms and tet fragments were recovered by gel cutting, and the primers PaceEFU_F (SEQ ID NO.28) and PaceEFL_R (SEQ ID NO.31) were used to perform fusion PCR amplification using KOD-plus high-fidelity DNA polymerase to obtain a size of 2167bp. Downstream homology arm, tetracycline PCR fragment and PaceEF-tet of the transcriptional regulatory sequence Trc-162.

The plasmid pTKRED was transferred into the E.coliSD02 strain and made electrotransfer competent; the fragment PaceEF-tet obtained in the above step was transferred into the electrotransfer competent obtained in this step, positive clones were screened with tetracycline, and verified by colony PCR. The tetracycline resistance gene was deleted by inducing the expression of homing endonuclease I-SceI, and verified by colony PCR to obtain the metabolic engineering strain SD06.

Embodiment 7: Import PHB synthesis pathway and carry out PHB batch shake flask fermentation

Utilizing primers PHB-F (SEQ ID NO.24), PHB-R (SEQ ID NO.25), using the genome of Ralstoniaeutropha H16 as a template, and using KOD-plus high-fidelity DNA polymerase to amplify the PHB-containing synthetic manipulation The 5271bp fragment of the phaCAB operon was cloned into the plasmid pBluescriptSK(-) at the SmaI/EcoRI site (see Figure 5, purchased from Addgene). After the plasmid was digested, enzyme-ligated, transformed, and verified, the plasmid pBHR68( See Figure 6), and the plasmid was transferred into the SD06 Escherichia coli constructed above and the wild strain JM109 respectively to obtain PHB-producing recombinant Escherichia coli named SD06s and JM109s respectively.

The inoculation method is as follows: firstly, activate the strains of the strain SD06s and the control strain JM109s on the LB solid plate respectively, after culturing overnight at 37°C, pick a single colony and inoculate a test tube containing 5mL of LB medium, cultivate for 12 hours, and inoculate 1% to 50mL containing The MS medium of 10g/L glucose and 1g/L yeast extract is used as a seed bottle, cultured for 12 hours, inoculated into 100mL MS medium containing 20g/L glucose (or xylose), 1g/L yeast extract, The initial inoculum OD was 0.04.

The fermentation conditions were as follows: the strain SD06s and the control strain JM109s were respectively cultured in MS medium containing 1 g/L yeast extract (100 mL in a 500 mL Erlenmeyer flask, shaker speed 220 rpm) at 37°C for 48 hours. When 20g/L glucose was added to the medium, SD06s bacteria could accumulate 4.63g/LPHB, accounting for 69.12% of dry cell weight, and the control strain JM109s detected 2.08g/LPHB, accounting for 53.96% of dry cell weight. The strain SD06s and the control strain JM109s were cultured in MS medium containing 1 g/L yeast extract and 20 g/L glucose (100 mL in a 500 mL Erlenmeyer flask, shaker speed 220 rpm) at 37°C for 48 hours. When 20g/L xylose was added to the medium, SD06s bacteria could accumulate 3.90g/LPHB, accounting for 69.11% of dry cell weight, and the control strain JM109s detected 1.79g/LPHB, accounting for 47.5% of dry cell weight.

The formula of LB liquid medium is: 10g/L peptone, 5g/L yeast extract, 10g/L NaCl, adjust the pH to 7.0, and sterilize under 0.1Mpa pressure for 20min.

The formula of LB solid medium is: 10g/L peptone, 5g/L yeast extract, 10g/L NaCl, adjust the pH to 7.0, add 2% agar, and sterilize under 0.1Mpa pressure for 20min.

MS medium formula: 2.0g/L(NH ₄ ) ₂ SO ₄ , 0.4g/LMgSO ₄ ·7H ₂ O, 9.65g/LNa ₂ HPO ₄ ·12H ₂ O, 1.5g/LKH ₂ PO ₄ , 0.05g/ LFe(III)–NH ₄ –citrate, 0.02g/LCaCl ₂ , and 1mL/L trace element solution

Trace element solution formula: 100mg/LZnSO ₄ 7H ₂ O, 30mg/LMnCl ₂ 4H ₂ O, 300mg/LH ₃ BO ₃ , 200mg/LCoCl ₂ 6H ₂ O, 6.4mg/LCuSO ₄ , 20mg/LNiCl ₂ 6H ₂ O, 30 mg/L NaMoO ₄ ·2H ₂ O and 0.5 mol/L HCl.

MS medium preparation method: first prepare mother liquor, weigh 10g (NH ₄ ) ₂ SO ₄ and 2gMgSO ₄ , add water to make up to 200mL, store in a blue cap bottle, sterilize by high-pressure steam at 121°C, and become component I; Take 38.27gNa ₂ HPO ₄ and 15gKH ₂ PO ₄ , add water to make up to 200mL, store in a blue cap bottle, and sterilize with high pressure steam at 121°C to become component II; weigh 5g ferric ammonium citrate and 2gCaCl ₂ 2H ₂ O, measure 41.7mL of concentrated hydrochloric acid (12mol/L) and add water to make up to 1000mL, store in a blue cap bottle to become component III; take 100mL of component III, 90mL of distilled water, after autoclaving at 121°C, add 10mL Trace element solution, and adjust the pH to 4.5-5.5 with NaOH. When inoculating, each 100 mL of MS medium includes 2 mL of component I, 2 mL of component II, 2 mL of component IV, 4 mL of 500 g/L glucose aqueous solution, and 90 mL of sterilized distilled water (with yeast extract at a final concentration of 1 g/L).

Cell esterification and poly-3-hydroxybutyrate gas chromatographic detection method: take 40-60 mg of freeze-dried cells in an esterification tube, add 2 ml of esterification solution to the esterification tube (measure 485 ml of anhydrous methanol, 15ml of 98% concentrated sulfuric acid, weighed 0.5g of benzoic acid) and 2ml of chloroform, sealed with a rubber stopper and a screw cap, and processed in an oven at 100 degrees Celsius for 4 hours. After the esterification is complete, cool the esterified solution to room temperature, add 1ml of deionized water, mix and let stand for 1 hour, absorb the lower clear chloroform phase for gas phase detection.

Gas chromatography heating program: use HP-5 gas-phase capillary column, keep at 80°C for 2 minutes; raise the temperature to 140°C at a rate of 30°C per minute; then increase the temperature to 250°C at a rate of 40°C per minute.

As can be seen from the shake flask fermentation results, the PHB-producing Escherichia coli strain constructed by the present invention can achieve higher PHB accumulation and has a good application prospect, and the resulting strain can be used for other products that use acetyl-CoA as a precursor production of products.

Claims (3)

1. the construction method of a strain producing the recombinant escherichia coli of poly-3-hydroxybutyrate is characterized in that comprising the steps: (1) Design the ribosome binding site code-named RBS5 as shown in SEQIDNO.1, and replace the promoter and ribosome binding site sequence of the sdaA gene of Escherichiacoli JM109 with a constitutive promoter as shown in SEQIDNO.33 trc and RBS5, construct engineering strain SD01; (2) Design the ribosome binding site RBS1 as shown in SEQIDNO.2, the ribosome binding site RBS2 as shown in SEQIDNO.3, the ribosome binding site RBS3 as shown in SEQIDNO.4 and the ribosome binding site RBS3 as shown in SEQIDNO.5 The indicated ribosome binding site RBS4; (3) The gene pgk coding sequence shown in RBS1 and SEQIDNO.36 is connected into fragment 1, the gene serA coding sequence shown in RBS2 and SEQIDNO.37 is connected into fragment 2, and the gene serB shown in RBS3 and SEQIDNO.38 is connected The coding sequence is connected into fragment 3, and the gene serC coding sequence shown in RBS4 and SEQ ID NO.39 is connected into fragment 4; (4) Fragment 1, Fragment 2, Fragment 3 and Fragment 4 are fused into the operator PSer by overlapping-extension PCR, and integrated into step (1) together with the transcriptional regulatory sequence Trc-162 containing the promoter shown in SEQ ID NO.32 The 58bp site upstream of the genome serC gene of the obtained engineering strain SD01 was obtained to obtain the engineering strain SD02; (5) Insert the sequence shown in SEQ ID NO.32 at the upstream 48bp site of the aceEF operon gene encoding the pyruvate dehydrogenase complex complex of the bacterial strain SD02 to construct the engineering strain SD06; (6) The plasmid pBHR68 containing the PHB synthesis pathway gene was introduced into SD06, and the genotype was E.coli JM109, Ptrc-RBS5- sdaA , Trc-162-RBS1- pgk -RBS2- serA -RBS3- serB -RBS4- serC , Trc- 162- aceEF , pBHR68,amp ^r , referred to as recombinant Escherichia coli containing pBHR68 E.coli SD06 producing poly-3-hydroxybutyrate. 2. a recombinant escherichia coli producing poly-3-hydroxybutyrate constructed by the method of claim 1. 3. the purposes of a recombinant escherichia coli producing poly-3-hydroxybutyrate according to claim 2 in fermentative production of poly-3-hydroxybutyrate.