Genetically engineered bacterium for producing polyhydroxyalkanoate by using ethanol and application of genetically engineered bacterium

Abstract

The invention discloses a genetically engineered bacterium for producing polyhydroxyalkanoate by using ethanol and application of the genetically engineered bacterium. On the basis of evolutionary engineering Escherichia coli MEC and WEC capable of assimilating ethanol into acetyl-CoA, a heterologous expression polyester production pathway is constructed, a metabolic pathway and metabolic flux regulation are analyzed, a chassis strain is modified by utilizing a genetic engineering means, and the yield of the acetyl-CoA is improved. The obtained engineering strain can be used for producing glycollic acid monomers, PHB and P (3HB-co-GA) in a culture medium taking ethanol as a carbon source, and a new thought is provided for utilization of ethanol resources and microbial production of PHA.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of bioengineering technology, and particularly relates to a genetically engineered bacterium for producing polyhydroxyalkanoate by using ethanol and application thereof. BACKGROUND

[0002] Polyhydroxyalkanoates (PHAs) are a class of naturally biodegradable polymer materials synthesized by microorganisms, derived from biomass materials, and have excellent biocompatibility and environmental friendliness. In contrast to the negative impact of plastic disposal in landfills, PHA-based bioplastics exhibit biodegradable ability in all anaerobic and aerobic environments, and can be used to manufacture completely compostable, soil and ocean biodegradable commodities. Currently, the use and market prospects of bioplastic material industry are expanding.

[0003] Polyglycolic acid (PGA) based polyhydroxyalkanoates have excellent biocompatibility and degradability. Polyglycolic acid (PGA) will degrade in vivo within 60-90 days, and has better mechanical strength than common degradable plastics such as PLA. PGA is very ideal in applications requiring high strength, controllable degradation time and biocompatibility, especially in medical sutures, orthopedic implants and drug release carriers, tissue engineering scaffolds and the like.

[0004] Poly(3-hydroxybutyric acid-co-glycolic acid) (P(3HB-co-GA)) is also a member of the PHA family, which is polymerized from 3-hydroxybutyryl coenzyme A (3HB-CoA) and hydroxyacetyl coenzyme A (GA-CoA) under the action of PHA synthase. P(3HB-co-GA) will have partial properties of poly(3-hydroxybutyric acid) (PHB) and polyglycolic acid (PGA), and by adjusting the ratio of GA to 3HB, the mechanical properties, hydrolytic degradation rate and biocompatibility of the copolymer can be adjusted, so that it can improve the toughness while maintaining a certain strength, and is suitable for different application scenarios, such as short-term medical implants or long-term packaging materials.

[0005] With the rise of the third generation of biorefinery technology, two-carbon compounds such as ethanol and acetic acid have become one of the main products of carbon fixation. The fermentation raw material has developed from traditional grain-based raw materials to non-grain raw materials, realizing the goal of not competing with the people for grain and not competing with the grain for land. The fuel ethanol industry has made great progress in the past few decades. At present, ethanol can be obtained from renewable resources through various processes, such as hydrolysate of lignocellulose of crop residues, fermentation broth of synthesis gas, hydrolysis and fermentation of starch, and electrochemical fixation of atmospheric CO2, etc. However, there are few studies on the utilization of ethanol by microorganisms. If ethanol can be metabolized by microorganisms to produce high-value chemicals, it will open a new door for the utilization of cheap ethanol resources, and also avoid the high energy consumption and environmental pollution problems of traditional chemical synthesis methods. However, there are few reports on the production of PHA using ethanol as the sole carbon source. SUMMARY

[0006] In view of the deficiencies of the prior art, the present application is dedicated to the modification of the metabolic pathway of Escherichia coli, so as to enable it to produce two types of PHA, PHB and P(3HB-co-GA), using ethanol as the sole carbon source.

[0007] In one aspect, the present application provides a genetically engineered bacterium for producing polyhydroxyalkanoates using ethanol, which is genetically modified from an Escherichia coli starting strain by knocking out glcDEF gene, introducing phaA gene, introducing phaB gene, introducing phaC gene, overexpressing ghrA gene, overexpressing aceA gene and overexpressing aceK gene.

[0008] Preferably, the starting strain is Escherichia coli MEC, and its accession number is CCTCC NO: M 20251599.

[0009] In one or more embodiments, the genetic modification further includes introducing scot gene.

[0010] In one or more embodiments, the overexpression of the ghrA gene includes introducing an additional ghrA gene, the overexpression of the aceA gene includes introducing an additional aceA gene, and the overexpression of the aceK gene includes introducing an additional aceK gene.

[0011] In one or more embodiments, the phaA gene, phaB gene, phaC gene, ghrA gene, aceA gene, aceK gene and scot gene are constructed into 1, 2 or more plasmid vectors, and then introduced by plasmid transformation.

[0012] In one or more embodiments, the phaA gene, the phaB gene, the phaC gene, the ghrA gene, the aceA gene and the aceK gene are constructed into a pTrc99a plasmid vector containing a trc promoter, and the scot gene is constructed into a pBAD plasmid vector containing a trc promoter, and then introduced respectively by means of plasmid transformation.

[0013] In one or more embodiments, in the pTrc99a plasmid vector, the ghrA gene is located upstream of the aceA gene and the aceK gene in the order of 5'-3'.

[0014] Preferably, in the pTrc99a plasmid vector, the phaA gene, the phaB gene, the phaC gene, the ghrA gene, the aceA gene and the aceK gene are arranged in sequence in the order of 5'-3'.

[0015] In one or more embodiments, in the pTrc99a plasmid vector, the ghrA gene is located downstream of the aceA gene and the aceK gene in the order of 5'-3'.

[0016] Preferably, in the pTrc99a plasmid vector, the phaA gene, the phaB gene, the phaC gene, the aceA gene, the aceK gene and the ghrA gene are arranged in sequence in the order of 5'-3'.

[0017] In another aspect, the present application provides an application of the genetically engineered bacteria for producing poly(3-hydroxybutyric acid) or poly(3-hydroxybutyric acid-co-glycolic acid) using ethanol as described in any one of the embodiments herein.

[0018] In another aspect, the present application provides a method for producing poly(3-hydroxybutyric acid) or poly(3-hydroxybutyric acid-co-glycolic acid), which comprises genetically modifying an Escherichia coli as a starting strain to obtain genetically engineered bacteria, and culturing the genetically engineered bacteria in an environment containing ethanol; the genetic modification comprises knocking out the glcDEF gene, introducing the phaA gene, introducing the phaB gene, introducing the phaC gene, overexpressing the ghrA gene, overexpressing the aceA gene and overexpressing the aceK gene.

[0019] Preferably, the starting strain is Escherichia coli MEC.

[0020] In one or more embodiments, the genetic modification further comprises introducing the scot gene.

[0021] Compared with the prior art, the present application has the following beneficial effects:

[0022] 1. On the basis of the evolutionarily engineered Escherichia coli MEC and WEC that can assimilate ethanol into acetyl-CoA, the application constructs a heterologous polyester production pathway, analyzes the metabolic pathway and metabolic flow regulation, and modifies the chassis strain by genetic engineering, so that the obtained engineering strain can produce glycolic acid monomer, PHB and P(3HB-co-GA) in a culture medium with ethanol as the carbon source.

[0023] 2. The application also reduces the metabolic consumption of glycolic acid to a certain extent by deleting the key gene glcDEF of the glycolic acid oxidation pathway, thereby enhancing the molar proportion of glycolic acid in P(3HB-co-GA). BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 Metabolic flow chart for genetically engineered bacteria to produce polyhydroxybutyrate (PHB) using ethanol.

[0025] Figure 2 Flow chart for genetically engineered bacteria to produce poly-3-hydroxybutyric acid glycolate (P(3HB-co-GA)) using ethanol. Figure 2 In the formula, phaA is beta-ketothiolase, phaB is acetoacetyl-CoA reductase, scot is 3-ketoacid CoA transferase, and phaCm is a PHA synthase mutant, and the four enzymes are key enzymes in the P(3HB-co-GA) production process.

[0026] Figure 3 GC-MS detection chart of P(3HB-co-GA). 1 H NMR (A), 13 C NMR (B) detection chart.

[0027] Figure 4 GC-MS detection chart of P(3HB-co-GA).

[0028] Deposit Items

[0029] The Escherichia coli in the application is classified and named as Escherichia coli, which has been preserved in the China Center for Type Culture Collection on July 14, 2025, and the preservation number is CCTCC NO: M 20251599. DETAILED DESCRIPTION

[0030] The technology of the application is described in detail below in combination with specific embodiments. Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials, reagents, etc. used in the following examples can be obtained from commercial channels. It should be understood that these examples are only used to illustrate the application and not to limit the scope of the application.

[0031] The host bacteria E. coli MEC and WEC used in the following examples are strains of wild-type E. coli MG1655 adapted by culture in ethanol-containing medium, as described in CN117660287A. E. coli MEC and WEC can utilize ethanol and convert it into acetyl-CoA.

[0032] The plasmids used in the following examples, such as pTrc99a, pGRB, pREDCas9, pBAD, etc., are commercial plasmids unless otherwise specified.

[0033] The LB medium, M9 medium, etc. used in the following examples are conventional formulations known in the art unless otherwise specified.

[0034] The plasmids and strains used in the following examples are shown in Table 1 and Table 2, respectively, and the sequence information of the relevant genes is shown in Table 3.

[0035] Table 1: Plasmids used in the examples and their descriptions

[0036]

[0037] Table 2: Strains used in the examples and their descriptions

[0038]

[0039]

[0040] Table 3: Genes and their sequence information

[0041] Genes Description phaA SEQ ID NO: 1 phaB SEQ ID NO: 2 scot SEQ ID NO: 3 phaC (phaCm) SEQ ID NO: 4 aceA SEQ ID NO: 5 aceK SEQ ID NO: 6 ghrA SEQ ID NO: 7

[0042] In the following examples, the trace element stock solution contains FeSO4·7H2O 80 g, AlCl3·6H2O 10 g, ZnSO4·7H2O 2.0 g, CuCl2·2H2O 1.0 g, Na2MoO4·2H2O 2.0 g, MnSO4·H2O 10 g, CoCl2 4.0 g, and H3BO4 0.5 g per liter.

[0043] In the following examples, the pathway for the production of PHB by the E. coli engineering strain using ethanol as a substrate is shown in Figure 1 The pathway for the production of P(3HB-co-GA) using ethanol as a substrate is shown in Figure 2 In the following examples, the pathway for the production of PHB by the E. coli engineering strain using ethanol as a substrate is shown in

[0044] In the following examples, OD 600The determination method of the fermentation liquid sample is as follows: the fermentation liquid sample is diluted with double distilled water to make the OD 600 The absorbance at a wavelength of 600 nm is between 0.2 and 0.8.

[0045] In the following examples, ethanol and glycolic acid in the fermentation liquid sample are detected by liquid chromatography.

[0046] The pretreatment method of the fermentation liquid sample for liquid chromatography detection is as follows: 1 mL of the fermentation liquid sample is centrifuged at 12000 rpm for 10 min as soon as possible, the supernatant is filtered with a 0.22 μm water phase microporous filter, and the filtrate is transferred to a liquid phase vial as a liquid phase sample.

[0047] The liquid phase detection method is as follows: the concentration of the detected substance in each liquid phase sample is detected by high performance liquid chromatography. The detector is a RID differential refractometer detector (purchased from Shimadzu Corporation), the chromatographic column is a 300 mm x 7.8 mm cation column (purchased from Bio-Rad Corporation), the mobile phase is a 5 mM sulfuric acid solution prepared freshly and filtered by a microporous filter, the flow rate of the mobile phase is set to 0.6 mL / min during liquid phase detection, the detection cell temperature is set to 45°C, the column temperature is set to 65°C, and the sample detection time is 23 min.

[0048] The standard curve preparation method is as follows: ethanol, acetic acid and glycolic acid are prepared into standard sample liquids of 10 g / L, 8 g / L, 6 g / L, 4 g / L and 2 g / L in advance, the appearance time of each standard sample peak and the peak area corresponding to different concentrations are obtained, and the standard curves of the concentration-peak area relationship of each are obtained.

[0049] In the following examples, polymers in the fermentation liquid sample are detected by gas chromatography (GC), nuclear magnetic resonance spectroscopy (NMR) and gas chromatography-mass spectrometry (GC-MS).

[0050] In the following examples, the method for preparing the fermentation liquid sample into a freeze-dried sample is as follows:

[0051] (1) Esterification liquid preparation: 1 g of benzoic acid is dissolved in 850 mL of methanol, 150 mL of 98% concentrated sulfuric acid is added in multiple portions, and the esterification liquid is obtained after the liquid is completely cooled after a large amount of heat is released to boiling, and is stored at 4°C for use.

[0052] (2) Freeze-dried sample preparation: after fermentation, the fermentation liquid sample is centrifuged at 8000 rpm for 10 min, the supernatant is removed, deionized water is added to resuspend the bacterial body, and the centrifugation and resuspension are repeated twice, the last time the bacterial liquid volume is adjusted to 2 mL, and the bacterial liquid is frozen in an ultra-low temperature refrigerator for 6 h, and then immediately transferred to a vacuum freeze-drying machine pre-cooled by starting in advance, and freeze-dried for 60 h.

[0053] In the following examples, the GC or GC-MS detection method for lyophilized samples is as follows:

[0054] (1) Esterification reaction and extraction of polymers: about 15 mg of completely lyophilized sample was weighed and the exact weight was recorded, and the sample was transferred to a glass esterification tube with a cover. 1.5 mL of chloroform and 1.5 mL of esterification solution were added to the sample, respectively. The esterification tube was placed in a 100°C constant temperature metal bath for esterification reaction for 4h. After the esterification was completed, the esterification tube was immediately inserted into an ice box and fully ice-bathed for 20-30 min or more. After adding 750 mL of deionized water, the cover was tightly closed and shaken for 3 min to fully mix the organic and aqueous phases. Then, the aqueous and organic phases were separated by centrifugation at 3000 rpm and 4°C for 3 min.

[0055] (2) Preparation of gas phase sample: A PTFE 0.22 μm water-based needle filter was fitted on a 1 mL syringe without a needle, the plunger was pulled out, and anhydrous sodium sulfate powder was filled in the syringe to the 0.5 mL scale. In a sterile clean environment, the lower organic phase in the esterification tube was taken out with another syringe with a needle and injected into the 1 mL syringe containing the sodium sulfate powder. After a few seconds, the syringe plunger was inserted and pushed, and the liquid was filtered through the microporous membrane and transferred to a sample vial as a gas phase sample.

[0056] (3) Gas phase detection and analysis: GC-2014 gas chromatograph (purchased from Shimadzu Corporation) was used to analyze the monomer composition of the methyl esterified polymers in the gas phase sample and calculate the proportion of the copolymer in the cell dry weight. The chromatographic column was x-5 capillary column with a length of 30 mm and an inner diameter of 0.25 mm. The gas detector was a flame ionization detector. High-purity nitrogen gas cylinder was used to provide nitrogen as the carrier gas, and electrolytic water was used to produce hydrogen as the fuel gas, and air as the combustion-supporting gas. AOC-20S type automatic sampler was used, and anhydrous acetone was used as the cleaning agent. The gas analysis program was set as follows: at the beginning, stay at 54°C for 4 min, then increase the temperature to 125°C at a rate of 5°C / min, and finally increase the temperature to 180°C at a rate of 20°C / min.

[0057] In the following examples, the method for detecting lyophilized samples by NMR is as follows:

[0058] About 20 mg of lyophilized sample was taken in a clean standard nuclear magnetic tube, and a certain amount of deuterated chloroform was added to dissolve it thoroughly, and finally the liquid level in the tube was maintained above 4 cm. Then, the nuclear magnetic tube was placed in a 400 MHz nuclear magnetic resonance spectrometer for detection.

[0059] Example 1: Construction and screening of an engineered strain of Escherichia coli for producing PHB using ethanol

[0060] In this example, an engineered strain of Escherichia coli for producing PHB using ethanol was constructed.

[0061] 1.1 Construction of engineered strains for PHB production using ethanol

[0062] Take 5 μL plasmid pTrc99aABC and transform into 100 μL competent cells of MEC and WEC respectively, plate on plates containing ampicillin (Amp) and incubate at 37°C for 8-12 h. Pick single colonies in 4 mL LB tubes containing Amp and incubate for 12 h. The resulting colonies are the genetically engineered MEC and WEC strains with pTrc99aABC, named JLY-M01 and JLY-W01 respectively. After construction, store as glycerol bacteria for later use.

[0063] 1.2 Shake flask fermentation screening

[0064] Inoculate JLY-M01 and JLY-W01 in 4 mL liquid LB medium containing Amp and incubate at 37°C, 220 rpm for 10 h as a primary seed liquid. Take 1 mL of the primary seed and inoculate in 50 mL liquid LB shake flask medium containing Amp and incubate at 37°C, 220 rpm for 8 h as a secondary seed liquid. Inoculate the secondary seed liquid in a shake flask fermentation medium (M9 base salt medium with the addition of 10 g / L ethanol and optionally 2 g / L yeast extract) at a 2% inoculation amount (v / v) and incubate at 30°C, 220 rpm. When the OD 600 value reaches 2, add 0.1 mM of inducer IPTG to continue fermentation. Fermentation lasts for 72 h, and take samples every 12 h to detect OD 600 value; after fermentation, take samples to detect OD 600 value, cell dry weight, ethanol concentration and PHB yield, and calculate ethanol consumption. The results are shown in Table 4.

[0065] Composition of M9 base salt medium (per liter): 15.1 g Na2HPO4·12H2O, 3.0 g KH2PO4, 0.5 g NaCl, 1.0 g NH4Cl, 0.5 g MgSO4·7H2O, 0.011 g CaCl2, and 2 mg vitamin B1, 1 mg trace element stock solution.

[0066] Table 4: Results of shake flask fermentation of JLY-M01 and JLY-W01

[0067]

[0068] As can be seen from the results of Table 4, both recombinant bacteria JLY-M01 and JLY-W01 can produce PHB. Under the condition of adding 10 g / L ethanol and 2 g / L yeast extract to the fermentation medium in a shake flask, the PHB production of JLY-M01 is higher than that of JLY-W01; while no yeast extract is added, the difference of PHB production between the two is more obvious, in which the PHB production of JLY-M01 is comparable to that of adding yeast extract, while the PHB production of JLY-W01 is reduced to 2.70 g / L, about 33% lower than that of adding yeast extract. According to the above results, the MEC corresponding to JLY-M01 is selected for subsequent study.

[0069] Example 2: Fermentation test of the engineered strain producing PHB in a 1L fermenter

[0070] The recombinant bacteria JLY-M01 constructed in Example 1 is selected to perform a fermentation test in a 1L fermenter without adding yeast extract to the fermentation medium.

[0071] JLY-M01 is inoculated into 4 mL liquid LB medium containing Amp, and cultured at 37°C, 220 rpm for 10 h as a primary seed liquid. 1 mL of the primary seed is inoculated into 50 mL M9 basic salt medium containing Amp and 10 g / L ethanol, and cultured at 30°C, 220 rpm for 39 h as a secondary seed liquid. The secondary seed liquid is inoculated into a 1L bioreactor containing fermentation medium at an inoculation amount of 10% (v / v) for fermentation culture, and the initial volume of the fermentation medium is 70% of the volume of the reactor; 0.1 mM IPTG is added to induce when the OD reaches about 12. The pH is controlled at 7 and the temperature is controlled at 30°C during the fermentation process; the initial stirring speed of the paddle is 400 rpm and the maximum stirring speed is 700 rpm. Concentrated sulfuric acid with a concentration of 3M and ammonia water with a concentration of 14% (w / v) are used as acid-alkali neutralizing agents during the fermentation process. 10 g / L ethanol is added initially, and the concentration of ethanol is detected by HPLC offline during the fermentation process, and when the concentration is lower than 2 g / L, the ethanol is supplemented to 10 g / L.

[0072] Composition of the fermentation medium in the fermenter (per liter): 15.1 g Na2HPO4·12H2O, 3.0 g KH2PO4, 0.5 g NaCl, 6.0 g NH4Cl, 1 g MgSO4·7H2O, 0.022 g CaCl2, and 4 mg vitamin B1, 2 mg trace element stock solution.

[0073] Table 5: PHB production of JLY-M01 in a 1L fermenter

[0074]

[0075] From the results of Table 5, it can be seen that JLY-M01 successfully achieved the production of PHB in a 1L fermenter. JLY-M01 showed a gradual increasing trend in the synthesis of PHB during fermentation, and the highest dry cell weight (DCW) was 26.59 g / L at the end of fermentation, at which time the OD 600 was 120, the PHB yield was 13.12 g / L, and the intracellular PHB content was 49.33%.

[0076] Since glycolate is mainly metabolized by oxidation to glyoxylate in E. coli cells, this process is catalyzed by glycolate dehydrogenase, and glcDEF is the three main subunits associated with FAD binding of glycolate dehydrogenase. This embodiment knocks out the glcDEF gene by CRISPR-Cas9 technology in order to reduce the metabolic consumption of glycolate converted to glyoxylate.

[0077] The strain knocking out glcDEF based on MEC using CRISPR-Cas9 technology is named MEC-ΔglcDEF, and the specific operation is as follows.

[0078] 3.1 Construction of pGRB-sgRNA and Donor fragment

[0079] The sgRNA sequence of glcDEF is designed, and the DNA sequence of sgRNA is cloned into the pGRB vector. The primer pair glcDEFsgRNA-F / glcDEFsgRNA-R is used for PCR amplification to obtain a linearized pGRB fragment with the glcDEF-sgRNA sequence, which is recovered after purification and transformed into DH5α calcium competent cells for self-circularization to construct the plasmid pGRB-sgRNA glcDEF , and sequencing verification is performed. The primer pairs glcDEFup-F / glcDEFup-R and glcDEFdown-F / glcDEFdown-R are used to obtain the glcDEF upstream and downstream homologous arm fragments by PCR amplification of the host strain MEC; then the upstream and downstream homologous arm fragments are fused by overlap PCR to obtain the Donor fragment.

[0080] Table 6: Primer and sgRNA sequences

[0081] 3.2 glcDEF gene knockout

[0082] The plasmid pREDCas9 (containing kanamycin resistance), the plasmid pGRB-sgRNA glcDEFThe positive clones were selected by plating on LB plates containing both ampicillin and spectinomycin, and incubating at 30°C for about 12 hours.

[0083] 3.3 Plasmid resistance elimination

[0084] pGRB-sgRNA glcDEF Plasmid elimination: the positive clones with successful glcDEF knockout were inoculated in LB medium containing 100 μg / mL spectinomycin and 0.2% w / v arabinose, and incubated at 30°C for about 10 hours. Then, 1 μL of the bacterial solution was inoculated in LB medium containing 100 μg / mL spectinomycin and 0.2% w / v arabinose, and incubated at 30°C for about 10 hours. Then, the bacteria were streaked on LB plates containing 100 μg / mL spectinomycin and 0.2% w / v arabinose, and incubated for about 10 hours. Then, the bacteria were streaked on ampicillin-resistant plates. The strain that grew on the LB plates containing spectinomycin and arabinose but did not grow on the ampicillin-resistant plates was the strain in which the pGRB-sgRNA plasmid was eliminated. glcDEF Plasmid-free strain.

[0085] pREDCas9 plasmid elimination: the single colony in which the pGRB-sgRNA plasmid was eliminated was inoculated in LB medium, and incubated at 42°C for about 10 hours. Then, 1 μL of the bacterial solution was inoculated in LB medium, and incubated at 42°C for about 10 hours. Then, the bacteria were streaked on LB plates without antibiotics, and incubated for about 10 hours. Then, the bacteria were streaked on spectinomycin-resistant plates. The strain that grew on the LB plates without antibiotics but did not grow on the spectinomycin-resistant plates was the strain in which the pREDCas9 plasmid was eliminated.

[0086] Example 4: Construction of an engineered strain for producing P(3HB-co-GA) using ethanol and glycolic acid

[0087] Example 1 verified that the plasmid pTrc99aABC can enable E. coli to use ethanol to synthesize PHB. In this example, the MEC-ΔglcDEF obtained in Example 3 was used as the starting strain, and the plasmid pTrc99aABC and the plasmid pBAD-Ptrc-scot were transformed into the strain to obtain the recombinant strain JLY-M02. The recombinant strain JLY-M02 was used to further verify whether the expression of the CoA transferase scot can convert exogenous glycolic acid into hydroxyacetyl-CoA, and whether the hydroxyacetyl-CoA can copolymerize with the precursor 3-hydroxybutyryl-CoA of PHB to form P(3HB-co-GA).

[0088] The method of Example 1 was used for shake flask fermentation. The fermentation medium was M9 basic salt medium supplemented with 10 g / L ethanol, 2 g / L yeast extract, and 1 g / L, 2 g / L, or 3 g / L sodium glycolate. After the fermentation was completed, samples of the fermentation broth were taken for detection of OD 600The values ​​of ethanol and glycolic acid concentrations, as well as the polymer content (wt%) and GA content (mol%) in the cells, were measured, and the consumption of ethanol and glycolic acid was calculated. The results are shown in Table 7.

[0089] Table 7: Test results of shake-flask fermentation broth of JLY-M02

[0090]

[0091] like Figure 3 As shown, the chemical shifts of the sample's 1H NMR and 13C NMR are in good agreement with those of P(3HB-co-GA). Figure 4 The presence of methyl glycolate was also detected in the GC-MS image, confirming the successful incorporation of glycolic acid into the polymer. This indicates that the recombinant strain JLY-M02 can generate P(3HB-co-GA) in a glycolic acid-containing medium. However, as shown in Table 7, the addition of 1, 2, or 3 g / L of exogenous sodium glycolate had little effect on glycolic acid consumption, the GA component in the polymer, or the total polymer content.

[0092] Example 5: Construction of an engineered strain for endogenous glycolic acid synthesis

[0093] Since Escherichia coli is basically unable to synthesize glycolic acid endogenously, in order to provide sufficient glycolic acid monomers for polymer production, this embodiment overexpresses the relevant genes based on MEC to open up the synthesis pathway from ethanol to glycolic acid, thereby constructing an engineered strain capable of endogenously synthesizing glycolic acid.

[0094] The pathway for E. coli to produce glycolic acid from isocitrate involves three key enzymes: isocitrate lyase, isocitrate dehydrogenase kinase, and glyoxylate reductase, controlled by three key genes, aceA, aceK, and ghrA, respectively. In E. coli, these enzymes are mostly inactive. Isocitrate lyase (aceA) catalyzes the synthesis of glyoxylate from isocitrate; isocitrate dehydrogenase kinase / phosphorylase (aceK) phosphorylates isocitrate lyase, partially blocking the conversion of isocitrate to α-ketoglutarate; and glyoxylate reductase (ghrA) catalyzes the reduction of glyoxylate to glycolic acid.

[0095] The trc promoter sequence, aceA, aceK, and ghrA genes were sequentially inserted into the high-copy pTrc99a plasmid backbone, or the trc promoter sequence, ghrA, aceA, and aceK genes were sequentially inserted to construct two glycolic acid overexpression plasmids, pAKG (pTrc99a-Ptrc-aceAK-ghrA) and pGAK (pTrc99a-Ptrc-ghrA-aceAK), respectively. These plasmids were then introduced into MEC to obtain the engineered strains JLY-M03 and JLY-M04.

[0096] Shake flask fermentation was carried out according to the method of Example 1, and the fermentation medium was 10 g / L ethanol and 2 g / L yeast extract added to M9 base salt medium. After the fermentation was completed, samples of the fermentation broth were taken for detection of OD 600 values, ethanol and glycolic acid concentrations, and ethanol consumption was calculated, and the detection results are shown in Table 8.

[0097] Table 8: Glycolic acid production of genetically engineered bacteria JLY-M03, JLY-M04

[0098]

[0099] From the results in Table 8, it can be seen that both JLY-M03 and JLY-M04 can produce glycolic acid from ethanol, with production of 1.04 g / L and 3.96 g / L, respectively, and no other by-products are formed. The pGAK plasmid carried by JLY-M04 and the pAKG plasmid carried by JLY-M03 differ in the expression order of the three genes on the plasmid, and the glycolic acid production of JLY-M04 is significantly higher than that of JLY-M03, indicating that for the synthesis of glycolic acid, the preferential expression of glyoxylic acid reductase (ghrA) directly catalyzing the reduction of glyoxylic acid to glycolic acid is more important.

[0100] Example 6: Construction of an engineered strain for producing P(3HB-co-GA) from ethanol

[0101] Example 4 constructed an engineered strain for producing P(3HB-co-GA) from ethanol and glycolic acid, Example 5 constructed an engineered strain for accumulating glycolic acid from ethanol, and this example aims to combine the features of the above two to construct an engineered strain for producing P(3HB-co-GA) from ethanol as the sole carbon source.

[0102] In the two plasmids pAKG (pTrc99a-Ptrc-aceAK-ghrA) and pGAK (pTrc99a-Ptrc-ghrA-aceAK) constructed in Example 5, the trc promoter sequence and the phaA gene, phaB gene and phaC gene were inserted in order to obtain plasmids p99aAKG (pTrc99aABC-Ptrc-aceAK-ghrA) and p99aGAK (pTrc99aABC-Ptrc-ghrA-aceAK), and were introduced into MEC, while the plasmid pBAD-Ptrc-scot was also introduced to obtain engineered strains JLY-M05 and JLY-M06; the plasmids p99aAKG and p99aGAK were introduced into MEC-ΔglcDEF, while the plasmid pBAD-Ptrc-scot was also introduced to obtain engineered strains JLY-M07 and JLY-M08.

[0103] The shake flask fermentation was carried out according to the method of Example 1, and the fermentation medium was 10 g / L ethanol and 2 g / L yeast extract added on the basis of M9 basic salt medium. After the fermentation was completed, the fermentation liquid sample was taken for detection of OD 600 value, ethanol concentration, and polymer content (wt%) and GA content (mol%) in the polymer in the cells, and the ethanol consumption was calculated, and the detection results are shown in Table 9.

[0104] Table 9: Detection results of shake flask fermentation liquid of JLY-M05 to JLY-M08

[0105]

[0106] Methyl glycolate and methyl 3-hydroxybutyrate were also detected in the samples of JLY-M05 and JLY-M06 by NMR and GC-MS, indicating that JLY-M05 and JLY-M06 can generate P(3HB-co-GA) using ethanol as the sole carbon source. From the results in Table 9, it can be seen that under the condition of 10 g / L ethanol, using JLY-M05 for fermentation, the polymer content can account for 31.05% of the dry weight of the cells, and the GA component content in the polymer is 4.21 mol%; using JLY-M06 for fermentation, the polymer content accounts for a slight decrease, but the GA content in the polymer is significantly increased to 10.43 mol%. Compared with JLY-M05, JLY-M07 constructed by knocking out the glcDEF gene has a slight increase in polymer content, and the GA component also has a significant increase from 4.21 mol% to 8.90 mol%. Compared with JLY-M06, JLY-M08 has a decrease in polymer content and GA component, which may be because the knockout of the glcDEF gene and the superposition of the p99aGAK plasmid make the metabolic flow be excessively directed to glycolic acid production, affecting the metabolic balance of the cells.

[0107] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A genetically engineered bacterium producing polyhydroxyalkanoate using ethanol, characterized by comprising a gene encoding a polyhydroxyalkanoate synthase and a gene encoding an alcohol dehydrogenase. The genetically engineered bacteria are obtained by knocking out glcDEF gene, introducing phaA gene, introducing phaB gene, introducing phaC gene, overexpressing ghrA gene, overexpressing aceA gene and overexpressing aceK gene based on Escherichia coli as a starting strain.

2. The genetically engineered bacteria of claim 1, wherein, The genetic modification further includes introducing scot gene.

3. The genetically engineered bacteria of claim 2, wherein, The overexpression of the ghrA gene includes introducing additional ghrA gene, the overexpression of the aceA gene includes introducing additional aceA gene, and the overexpression of the aceK gene includes introducing additional aceK gene.

4. The genetically engineered bacteria of claim 3, wherein, The phaA gene, phaB gene, phaC gene, ghrA gene, aceA gene, aceK gene and scot gene are constructed into 1, 2 or more plasmid vectors and then introduced by plasmid transformation.

5. The genetically engineered bacteria as described in claim 4, characterized in that, The phaA gene, phaB gene, phaC gene, ghrA gene, aceA gene and aceK gene are constructed into 1 pTrc99a plasmid vector containing trc promoter, and the scot gene is constructed into 1 pBAD plasmid vector containing trc promoter, and then introduced by plasmid transformation respectively.

6. The genetically engineered bacteria as described in claim 5, characterized in that, In the pTrc99a plasmid vector, the ghrA gene is located upstream of the aceA gene and the aceK gene in the order of 5'-3'.

7. The genetically engineered bacteria as described in claim 5, characterized in that, In the pTrc99a plasmid vector, the ghrA gene is located downstream of the aceA gene and the aceK gene in the order of 5'-3'.

8. Use of the genetically engineered bacteria for producing poly(3-hydroxybutyric acid) or poly(3-hydroxybutyric acid-co-glycolic acid) by utilizing ethanol to produce polyhydroxyalkanoate according to any one of claims 1-7.

9. A process for the production of poly(3-hydroxybutyric acid) or poly(3-hydroxybutyric acid-co-glycolic acid) characterized in that, The method includes obtaining genetically engineered bacteria by genetic modification based on Escherichia coli as a starting strain and culturing in an environment containing ethanol; and the genetic modification includes knocking out glcDEF gene, introducing phaA gene, introducing phaB gene, introducing phaC gene, overexpressing ghrA gene, overexpressing aceA gene and overexpressing aceK gene.

10. The method of claim 8, wherein the genetic modification further includes introducing scot gene.

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