Genetically engineered strains producing polylactic acid and methods for producing polylactic acid

Abstract

Provided is a genetically engineered strain for producing polylactic acid and a method for producing polylactic acid, which comprises the steps of synthesizing D-lactate dehydrogenase gene, propionyl coenzyme A transferase gene and polyhydroxy fatty acid ester synthase gene into a target gene, double enzymatically cleaving with a vector, purifying, transforming a receptor cell to obtain a recombinant plasmid, extracting the DNA of the recombinant plasmid, introducing it into an engineered strain, extracting and verifying it, and obtaining a genetically engineered cell strain, and then cultivating it in a medium, introducing carbon dioxide into the medium, centrifuging under light conditions, drying and recovering it.The present invention greatly improves the biomass of cyanobacteria by modifying natural cyanobacteria and adopting a high-density fermentation strategy, and the present invention solves the problem that only sugar-derived raw materials are used to produce polylactic acid in the conventional technology.

Description

[Technical Field]

[0001] The present invention relates to the field of bioengineering technology, and more specifically to a genetically engineered bacterial strain that produces polylactic acid (PLA) and a method for producing PLA. [Background technology]

[0002] PLA is a highly promising biodegradable material with broad application prospects in fields such as disposable packaging, agriculture, pharmaceuticals, and 3D printing. Currently, PLA is mainly produced by generating lactic acid esters through microbial fermentation, and then synthesizing PLA by converting the lactic acid esters to lactide, which then undergoes cyclization of the lactide ring. However, such chemical polymerization reactions are demanding in terms of equipment, and the removal of necessary solvents and chain coupling agents is difficult, which significantly limits the progress of commercial PLA production. Therefore, developing and preparing methods for high-performance PLA is extremely important. Currently, bio-based PLA production relies mainly on sugar-derived raw materials, which also creates competition with industrial production and food supplies, posing a potential risk. At the same time, with the progress of industrialization, greenhouse gas CO2 emissions are becoming increasingly severe, and the problem of global warming is becoming serious. [Overview of the Initiative] [Problems that the invention aims to solve]

[0003] To address the shortcomings of conventional technologies, the present invention aims to provide genetically engineered bacterial strains that produce PLA and methods for producing PLA. [Means for solving the problem]

[0004] To achieve the above objective, the solution of the present invention is as follows.

[0005] (1) Synechococcus elongatusA genetically engineered strain, wherein by integrating the coding sequence of an exogenous D-lactic acid dehydrogenase gene, the coding sequence of an exogenous propionyl-CoA transferase gene, and the coding sequence of an exogenous polyhydroxy fatty acid ester synthase gene into the genome of the genetically engineered strain, the genetically engineered strain can express an exogenous D-lactic acid dehydrogenase, an exogenous propionyl-CoA transferase, and an exogenous polyhydroxy fatty acid ester synthase.

[0006] (2) According to the genetically engineered strain described in (1), the coding sequence of the D-lactic acid dehydrogenase gene is under the control of the P trc promoter, the P psba promoter or the P cpc560 promoter.

[0007] (3) According to the genetically engineered strain described in (1), the coding sequence of the propionyl-CoA transferase gene is under the control of the P trc promoter, the P psba promoter or the P cpc560 promoter.

[0008] (4) According to the genetically engineered strain described in (1), the coding sequence of the polyhydroxy fatty acid ester synthase gene is under the control of the P trc promoter, the P psba promoter or the P cpc560 promoter.

[0009] (5) According to the genetically engineered strain described in (1), the coding sequence of the exogenous D-lactic acid dehydrogenase gene is derived from Lactobacillus bulgaricus , the coding sequence of the exogenous propionyl-CoA transferase gene is derived from Clostridium propionicum , and the coding sequence of the exogenous polyhydroxy fatty acid ester synthase gene is derived from Pseudomonas.

[0010] (6) According to the genetically engineered bacterial strain described in (1), by integrating the coding sequence of an exogenous acetyl coenzyme A synthase gene into the genome of the genetically engineered bacterial strain, the genetically engineered bacterial strain can express an exogenous acetyl coenzyme A synthase gene.

[0011] (7) According to the genetically engineered bacterial strain described in (1), the expression of the acetate kinase gene in the genetically engineered bacterial strain is knocked down.

[0012] (8) According to the genetically engineered bacterial strains described in (7), the genome of the genetically engineered bacterial strain has an exogenous sRNA coding sequence that suppresses the expression of the acetate kinase gene. The aforementioned exogenous sRNA is a small regulatory RNA. ru.

[0013] (9) According to the genetically engineered bacterial strain described in (1), the acetyl coenzyme A carboxylase gene of the genetically engineered bacterial strain, β-Ketoacyl-ACP Synthetic enzymes II The expression of the gene and / or the β-ketoacyl-ACP synthase III gene is knocked down.

[0014] (10) According to the genetically engineered bacterial strains described in (9), the genome of the genetically engineered bacterial strain contains the acetyl coenzyme A carboxylase gene, β-Ketoacyl-ACP Synthetic enzymes II The coding sequences of exogenous sRNAs that can suppress the expression of the gene and / or the β-ketoacyl-ACP synthase III gene are integrated. The aforementioned exogenous sRNA is a small regulatory RNA. ru.

[0015] (11) According to the genetically engineered bacterial strain described in any of (1)-(10), the genetically engineered bacterial strain is Synechococcus elongatus This is the PCC 7942 strain.

[0016] (12) A method for producing polylactic acid using carbon dioxide, 1) Any of the items listed in (1)-(11) Synechococcus elongatus The steps include providing a genetically engineered bacterial strain, 2) Introducing carbon dioxide into light ShootingThe steps include culturing the genetically engineered bacterial strain under certain conditions, 3) Growth of the genetically engineered bacterial strain optical density The process includes the step of collecting and drying the genetically engineered bacterial strain when it reaches its maximum value, and recovering the polylactic acid within the strain.

[0017] (13) According to the method described in (12), in step 2), the amount of carbon dioxide to be permeated is 1.5 vvm, and the volume ratio of introduced carbon dioxide to air is 1:(1-10).

[0018] (14) According to the method of (12), in step 2) the light illumination Shooting The intensity is 125 μmol photon quantum m -2 s -1 From 1000 μmol of photons m -2 s -1 Less than, preferably, the light Shooting The intensity is 125-500 μmol photons m -2 s -1 That is the case.

[0019] (15) According to the method of (12), in step 2), the genetically engineered bacterial strain is cultured in such a medium, the medium comprising sodium carbonate, sodium nitrate, dipotassium hydrogen phosphate, magnesium sulfate, calcium chloride, citric acid, ferric ammonium citrate, ethylenediaminetetraacetic acid, boric acid, manganese chloride, zinc sulfate, sodium molybdate, copper sulfate, and cobalt nitrate.

[0020] (16) According to the method described in (15), in addition to the composition of the culture medium, Na2CO3, MgSO4·7H2O, NaNO3, KH2PO4 and trace elements compounds Add, preferably the trace elements compounds The selected element is chosen from H3BO3, MnCl2·7H2O, ZnSO4·7H2O, Na2MoO4·2H2O, CuSO4·5H2O, and Co(NO3)2·6H2O.

[0021] (17) According to the method of (15) or (16), the final concentration of sodium carbonate in the culture medium is 0.25-1.5 g / L.

[0022] (18) According to the method of (12), in step 2), the genetically engineered bacterial strain is cultured in the presence of an inducer, preferably one or more inducers selected from IPTG and theophylline.

[0023] (19) According to the method of (12), in step 3) to collect the genetically engineered strain, mix the culture medium containing the polymer electrolyte flocculant and the genetically engineered strain for 5 minutes, let it stand for 5 minutes, and after standing, discard the supernatant and allow the precipitate to settle. collection This includes, preferably, that the polymer electrolyte flocculant is a polyacrylamide flocculant.

[0024] (20) According to the method described in (12), in step 3), the polylactic acid in the genetically engineered strain is recovered with an organic solvent or Alkaline treatment It is recovered by extraction, preferably the organic solvent is chloroform, 1,4- Dioxa It is selected from methyl tetrahydrofuran, ethylene carbonate, acetone, and ethyl acetate. [Effects of the Invention]

[0025] By employing the above means, the beneficial effects of the present invention are as follows.

[0026] Mallet, natural na S ynechococcus elongatu s(Using cyanobacteria (also referred to as "cyanobacteria" in this text) as a starting strain, we successfully synthesized PLA using the metabolic pathway of Synechococcus by introducing heterologous D-lactate dehydrogenase (LDH), propionyl coenzyme A transferase (PCT), and polyhydroxy fatty acid ester synthase (PHA) using genetic engineering techniques. Next, the present invention systematically carried out different metabolic engineering strategies, including promoter optimization of the rate-limiting step enzyme, construction of self-recycling of acetyl coenzyme A, and reorientation of carbon flux. Specifically, the promoters of the rate-limiting step enzymes PCT and PHA were optimized, acetyl coenzyme A synthase was overexpressed, acetate kinase expression was weakened, and acetyl coenzyme A carboxylase, a key enzyme in the fatty acid pathway, was overexpressed. β-Ketoacyl-ACP Synthetic enzymes II Furthermore, knockdown of the β-ketoacyl-ACP synthase III gene further improved the yield of PLA.

[0027] At the same time, the present invention relates to the conditions for introducing CO2 into the culture medium and the light illumination. Shooting This invention explores high-density culture (HDC) strategies, including the exploration of specific conditions. The high-density fermentation strategy allows for easy adjustment of light intensity and the use of different light types in combination (combination of red and white light). The invention enables automatic replenishment and stirring during condensation and fermentation, with a stirring speed of 200 rpm for the culture conditions. This invention further improves PLA yield through the exploration of culture and fermentation conditions.

[0028] This invention discloses the production of plastic from carbon dioxide using a cyanobacterial cell factory.

[0029] This invention relates to natural cyanobacteria ( Synechococcus elongatus By modifying the system and adopting a high-density fermentation strategy, CO2 and light ShootingThis invention allows for the direct use of conditions to produce PLA, significantly increasing the biomass of cyanobacteria. It solves the problem of conventional PLA production relying solely on sugar-derived raw materials, further reducing production and collection costs, and simultaneously addressing the issues of plastic pollution and excessive greenhouse gas CO2 emissions. Furthermore, it can be used for the production of biodegradable PLA, possessing enormous potential for industrial applications and accelerating the commercialization of PLA production by cyanobacteria. Additionally, this invention enables the very simple and easy collection of cyanobacterial cells using an amphoteric electrolyte polyacrylamide flocculant. [Brief explanation of the drawing]

[0030] [Figure 1] This is a schematic diagram of a PLA synthesis pathway and metabolic engineering modification strategy according to one embodiment of the present invention. [Figure 2] Figure 2A shows the hydrogen spectrum and carbon spectrum of PLA synthesized in Example 7 of the present invention, Figure 2B shows the carbon spectrum, Figure 2C shows the structural formula of PLA, and Figure 2D shows the 1H-1H two-dimensional COSY diagram. [Modes for carrying out the invention]

[0031] One aspect of the present invention is, Synechococcus elongatus The present invention provides a genetically engineered bacterial strain, characterized in that by integrating the coding sequence of an exogenous D-lactate dehydrogenase gene, an exogenous propionyl coenzyme A transferase gene, and an exogenous polyhydroxy fatty acid ester synthase gene into the genome of the genetically engineered bacterial strain, the genetically engineered bacterial strain can express exogenous D-lactate dehydrogenase, exogenous propionyl coenzyme A transferase, and exogenous polyhydroxy fatty acid ester synthase.

[0032] In some implementations, the coding sequence of the exogenous D-lactate dehydrogenase gene is 、LActobacillus bulgaricu s The coding sequence of the exogenous propionyl coenzyme A transferase gene is derived from the above. 、C Losridium propionicus m The coding sequence of the exogenous polyhydroxy fatty acid ester synthase gene originates from Pseudomonas.

[0033] In the present invention, the coding sequences of the D-lactate dehydrogenase gene, the propionyl coenzyme A transferase gene, and the polyhydroxy fatty acid ester synthase gene may be wild-type or appropriately modified. The present invention has discovered that the yield of PLA is improved by introducing mutations into the gene coding sequences of the three enzymes, as described in the literature "Yang et al., Biotechnology & Bioengineering, 2010, 105(1):150-160." and "Li, C., Tao, F., Ni, J. et al. Enhancing the light-driven production of D-lactate by engineering cyanobacterium using a combinational strategy. Sci Rep 5, 9777 (2015)." The entire contents of the above literature are incorporated into this text by reference.

[0034] In this text, the term "exogenous" as used when referring to genes, coding sequences, proteins, enzymes, etc., refers to substances that do not belong to a particular strain of bacteria in their natural state. For example, an exogenous gene is the introduction of a gene from a particular strain of bacteria from an external source, and this exogenous gene may be an existing gene in the genome of the strain or a gene not present in the genome. In one embodiment of the present invention, if necessary, from an external source Synechococcus elongatus The gene is overexpressed by introducing an existing acetyl coenzyme A synthase gene into the bacterial strain genome.

[0035] The present invention further improves PLA yield by further optimizing the promoter that controls the transcription of exogenous genes. Preferably, the coding sequence of the D-lactate dehydrogenase gene is P trc Promoter, P psba Promoter or P cpc560 It is controlled by a promoter. More preferably, the coding sequence of the propionyl coenzyme A transferase gene is P trc Promoter, P psba Promoter or P cpc560 It is controlled by a promoter. More preferably, the coding sequence of the polyhydroxy fatty acid ester synthase gene is P trc Promoter, P psba Promoter or P cpc560 It is controlled by the promoter.

[0036] The present invention has discovered that by further integrating the coding sequence of an exogenous acetyl coenzyme A synthase gene into the genome of the genetically engineered bacterial strain, the genetically engineered bacterial strain can express the exogenous acetyl coenzyme A synthase gene, and thus, the PLA yield can be further improved.

[0037] The present invention further weakens the expression of acetate kinase and / or acetyl coenzyme A carboxylase, a key enzyme in the fatty acid pathway. β-Ketoacyl-ACP Synthetic enzymes II We discovered that knocking down the β-ketoacyl-ACP synthase III gene further improves PLA yield.

[0038] Therefore, preferably, the expression of the acetate kinase gene in the genetically engineered bacterial strain is knocked down. This is achieved by integrating an exogenous sRNA coding sequence capable of suppressing the expression of the acetate kinase gene into the genome of the bacterial strain.

[0039] More preferably, the acetyl coenzyme A carboxylase gene of the genetically engineered bacterial strain, β-Ketoacyl-ACP Synthetic enzymes IIThe expression of the gene and / or the β-ketoacyl-ACP synthase III gene is knocked down. This is because the expression of the acetyl coenzyme A carboxylase gene, β-Ketoacyl-ACP Synthetic enzymes II This is achieved by integrating the coding sequence of an exogenous sRNA that can suppress the expression of the gene and / or the β-ketoacyl-ACP synthase III gene.

[0040] In several specific implementation methods, the genetically engineered bacterial strain is Synechococcus elongatus This is the PCC 7942 strain.

[0041] Another aspect of the present invention provides a method for producing polylactic acid using carbon dioxide, 1) Any of the items listed in (1)-(11) Synechococcus elongatus The steps include providing a genetically engineered bacterial strain, 2) Introducing carbon dioxide into light Shooting The steps include culturing the genetically engineered bacterial strain under certain conditions, 3) Growth of the genetically engineered bacterial strain optical density The process includes the step of collecting and drying the genetically engineered bacterial strain when it reaches its maximum value, and recovering the polylactic acid within the strain.

[0042] Preferably, in step 2), the amount of carbon dioxide permeated is 1.5 vvm. More preferably, the volume ratio of introduced carbon dioxide to air is 1:(1-10), and most preferably 5%.

[0043] Preferably, in step 2), the light illumination Shooting The intensity is 125 μmol photon quantum m -2 s -1 From 1000 μmol of photons m -2 s -1 Less than, and more preferably, the light Shooting The intensity is 125-500 μmol photons m -2 s -1 And most preferably 500 μmol of photons m -2 s -1 That is the case.

[0044] Furthermore, the present invention states that it is undesirable for the genetically engineered bacterial strain to have too much light during the early stages of growth. In such cases, preferably 150 μmol photons m -2 s -1 The following light intensities, most preferably 125 μmol photons m -2 s -1 Select the light intensity, and as the strain grows, gradually increase the light intensity to 500 μmol photons m -2 s -1 We discovered that raising the temperature to this level allows for optimal growth of the bacterial strain.

[0045] The present invention further explores culture media used for culturing the aforementioned strains, and finds that a culture medium with the following composition can improve the biomass of the genetically engineered strains. The culture medium comprises sodium carbonate, sodium nitrate, dipotassium hydrogen phosphate, magnesium sulfate, calcium chloride, citric acid, ferric ammonium citrate, ethylenediaminetetraacetic acid, boric acid, manganese chloride, zinc sulfate, sodium molybdate, copper sulfate, and cobalt nitrate. More preferably, in addition to the above composition, Na2CO3, MgSO4·7H2O, NaNO3, KH2PO4, and trace elements. compounds Add the following: Preferably, the trace elements compounds The selected element is chosen from H3BO3, MnCl2·7H2O, ZnSO4·7H2O, Na2MoO4·2H2O, CuSO4·5H2O, and Co(NO3)2·6H2O.

[0046] The present invention has found that the final concentration of sodium carbonate in the culture medium is preferably 0.25-1.5 g / L.

[0047] Preferably, in step 2), the genetically engineered bacterial strain is cultured in the presence of an inducer, thereby further improving the PLA yield. The inducer is preferably one or more selected from IPTG and theophylline.

[0048] Step 3) involves collecting the genetically engineered bacterial strain by mixing the culture medium containing the polymer electrolyte flocculant and the genetically engineered bacterial strain for 5 minutes, letting it stand for 5 minutes, discarding the supernatant after standing, and collecting the precipitate, wherein the polymer electrolyte flocculant is a polyacrylamide flocculant.

[0049] Freeze-drying can be used to dry the genetically engineered bacterial strains.

[0050] In step 3), the polylactic acid in the genetically engineered bacterial strain is recovered with an organic solvent or Alkaline treatment It can be recovered by extraction.

[0051] The organic solvents used are chloroform, 1,4- Dioxa It is selected from methyl tetrahydrofuran, ethylene carbonate, acetone, and ethyl acetate.

[0052] Alkaline treatment The extraction method may include the steps of (a) providing dried bacterial cells rich in polymer particles, (b) grinding the dried cell particles, (c) dissolving the ground cell powder obtained from step (b) in an alkaline aqueous solution (pH 8-10), (d) homogenizing the polymer-rich alkaline solution, (e) separating non-target polymer fragments remaining in the cells to obtain a primary product, and (f) filtering and drying the concentrate to obtain polymer particles.

[0053] Harvesting microalgae biomass on an industrial scale is a techno-economic bottleneck in the algae biomass industry, further complicated by the small cell size of microalgae and the concentration of biomass diluted in cultures. The harvesting process requires the removal of large amounts of water, resulting in high process energy and cost, accounting for approximately 30% of the total cost of biomass production. Since PLA is an internal component of the cells, PLA particles and cells can be collected together by centrifugation. This invention discovers that using a flocculant allows for faster and simpler cell collection, which has significant potential industrial value.

[0054] In one specific embodiment of the present invention, the method for producing polylactic acid using carbon dioxide is: (1) The steps include synthesizing the target gene using primers from a denatured D-lactate dehydrogenase gene, a denatured propionyl coenzyme A transferase gene, and a denatured polyhydroxy fatty acid ester synthase gene, performing double enzymatic cleavage of the target gene and vector, purifying the gene, transforming susceptible E. coli cells, and obtaining a recombinant plasmid, (2) The steps of extracting the DNA of the recombinant plasmid, introducing it into the cyanobacterium S. elongatus PCC 7942, extracting and verifying it to obtain a genetically engineered cell strain, (3) The genetically engineered cell strain is cultured in a culture medium, carbon dioxide is introduced into the medium, and light is shone. Shooting The process includes the steps of collecting by centrifugation under certain conditions, then freeze-drying to recover and obtain PLA.

[0055] This invention relates to the conditions for introducing CO2 into the culture medium and the light illumination. Shooting This invention explores high-density culture (HDC) strategies, including the exploration of specific conditions. The high-density fermentation strategy achieves simple adjustment of light intensity and the use of different light types in combination (combination of red and white light). The invention allows for automatic replenishment and stirring during condensation and fermentation, with a stirring speed of 200 rpm for the culture conditions. This invention further improves PLA yield through the exploration of culture and fermentation conditions.

[0056] Example The technical details of the present invention will be further explained based on the examples. The following examples are illustrative and not limiting, and the scope of the present invention cannot be limited by these examples. 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 through commercial channels.

[0057] Dissolve the following in BG-11 liquid medium (1×): NaNO3 1.5g, K2HPO4·3H2O 0.04g, MgSO4·7H2O 0.075g, CaCl2·2H2O 0.036g, citric acid 0.006g, iron ammonium citrate 0.006g, EDTA 0.001g, Na2CO3 0.02g, and metal ion mother liquor 1mL (g / L: boric acid 2.86, MnCl2·4H2O 1.81, ZnSO4·7H2O 0.222, Na2MoO4·2H2O 0.39, CuSO4·5H2O 0.079, Co(NO3)2·6H2O 0.0494), and adjust the volume in 1L of distilled water. Sterilize by steam at 121℃ under high temperature and pressure for 20min.

[0058] BG-11 Solid Medium: Add 1.6-2.0% (w / v) agar powder to BG-11 liquid medium. Sterilize by steam at 121°C under high pressure for 20 minutes.

[0059] Culture method: The strain culture method for producing PLA involves taking S. elongatus PCC 7942 cells grown in the exponential stage, diluting them in a 300 mL Erlenmeyer flask containing 100 mL of BG-11 liquid medium, and adjusting the final cell concentration. optical density 730nm Set the ratio to 0.05 and add 20 mg / L of spectinomycin to the culture medium. optical density 730nm Once the cells have grown to 0.4-0.6 mm, induce cell growth by adding 1 mmol / L isopropyl-β-D-thiogalactoside (IPTG). Take a 2 mL sample daily and measure cell growth. After each sampling, replenish the Erlenmeyer flask with an equal volume of sterilized BG-11 liquid medium.

[0060] Example 1: Introduction of the metabolic pathway for PLA synthesis

[0061] As shown in Figure 1, based on the coding sequences of the already reported modified D-lactate dehydrogenase gene (ldhD), propionyl coenzyme A transferase gene (pct), and polyhydroxy fatty acid ester synthase gene (pha) (see "Yang et al., Biotechnology & Bioengineering, 2010, 105(1):150-160." and "Li, C., Tao, F., Ni, J. et al., Enhancing the light-driven production of D-lactate by engineering cyanobacterium using a combinational strategy., Sci Rep 5, 9777 (2015)"), the sequences are arranged in the order of IdhD, ​​pct, and pha, and RBS (SEQ ID) is used between the two genes. By inserting the sequence NO.19:AAGAAGGAGATATACC, a full-length sequence consisting of the modified gene coding sequences of the three enzymes described above is artificially synthesized. Then, using the following primers, the synthesized sequence is used as a template for PCR amplification to obtain a recombinant gene fragment containing the three key enzymes. SEQ ID NO.1: 5- ACTCGAG ATGAGCAACAAGTCAAATGATGAA-3; SEQ ID NO.2: 5- GGATCCT CTAGGACTTCATTTCTTTCAGGCC-3.

[0062] The recombinant gene and pAMMCS12 were double-enzyme-cleaved with XhoI and BamHI, respectively, and after purification, ligated overnight at 16°C using T4 DNA ligase. E. coli susceptible cells were transformed by ligation product chemistry. The transformation solution was plated with spectinomycin (50 μg / mL) LB, the plasmid was extracted, and the constructed recombinant plasmid was validated by double-enzyme-cleavage and named pAM-ldhD-pct-pha. Sequencing was completed by Shanghai Paimonuo Biotechnology Co., Ltd.

[0063] The extracted recombinant plasmid DNA utilizes the principle of homologous recombination through spontaneous transformation to target genes. Synechococcus elongatus PCC 7942 (from the Global Bioresource Center, ATCC) was integrated into the chromosome. After extracting the genome of the recombinant transformant, the success of the insertion was confirmed by PCR amplification validation using validation primers SEQ ID NO.3: 5-ACCTGGAATTGGTTGAAGG-3 and SEQ ID NO.4: 5-ACAGCCAAGCTTGCATGC-3, and the PYLW01 strain was obtained. The yield of the obtained PYLW01 strain, measured according to the method described in Example 7, was 0.8 mgg. -1 It's DCW.

[0064] Example 2: Optimization of the rate-limiting step enzyme promoter

[0065] P trc Following the promoter, the entire gene was synthesized, and primers SEQ ID NO.5: 5-CGACTGCACGGTGCACCA-3 and SEQ ID NO.6: 5-CATGGTCTGTTTCCTGTGTGAA-3 were designed, and PCR amplification was performed. trc Get a promoter.

[0066] P psba Following the promoter, the entire gene was synthesized, and primers SEQ ID NO.7: 5-TATCAATAAGTATTAGGTATATGG-3 and SEQ ID NO.8: 5-ATGTATTTGTCGATGTTCAGAT-3 were designed, and PCR amplification was performed. psba Get a promoter.

[0067] P cpc560 Following the promoter, the entire gene was synthesized, and primers SEQ ID NO.9: 5- ACCTGTAGAGAAGAGTCCCT-3 and SEQ ID NO.10: 5-TGAATTAATCTCCTACTTGACTTTA-3 were designed, and PCR amplification was performed. cpc560 Get a promoter.

[0068] P obtained by PCR amplificationtrc First, take 1 μL each of the promoter sequence and the three gene coding sequences of ldhD, pct, and pha, and then perform duplicate derivatization PCR to remove P trc -ldhD fragment, P trc -pct fragment, and P trc -pha fragments are formed, and then the three fragments are sequentially ligated into the pAM vector using EcoRI, BamHI, and XhoI enzyme cleavage enzymes, resulting in the pAM-P vector. trc -ldhD-P trc -pct-P trc - Obtain pha.

[0069] P obtained by PCR amplification psba First, take 1 μL each of the promoter sequence and the three gene coding sequences of ldhD, pct, and pha, and then perform duplicate derivatization PCR to remove P psba -ldhD fragment, P psba -pct fragment, and P psba -pha fragments are formed, and then these three fragments are sequentially ligated into the pAM vector using EcoRI, BamHI, and XhoI enzyme cleavage enzymes, resulting in the pAM-P vector. psba -ldhD-P psba -pct-P psba - Obtain pha.

[0070] P obtained by PCR amplification cpc560 First, take 1 μL each of the promoter sequence and the three gene coding sequences of ldhD, pct, and pha, and then perform duplicate derivatization PCR to remove P cpc560 -ldhD fragment, P cpc560 -pct fragment, and P cpc560 -pha fragments are formed, and then these three fragments are sequentially ligated into the pAM vector using EcoRI, BamHI, and XhoI enzyme cleavage enzymes, resulting in the pAM-P vector. cpc560 -ldhD-P cpc560 -pct-P cpc560 - Obtain pha.

[0071] Example 3: Construction of genetically engineered bacterial strains PYLW02, PYLW03, and PYLW004

[0072] Vector pAM-P trc -ldhD-P trc -pct-P trc -pha, by natural conversion Synechococcus elongatus The PYLW02 strain was obtained by integrating it into chromosomal neutral site I of PCC 7942.

[0073] Vector pAM-P psba -ldhD-P psba -pct-P psba -pha, by natural conversion Synechococcus elongatus The PYLW03 strain was obtained by integrating it into chromosomal neutral site I of PCC 7942.

[0074] Vector pAM-P cpc560 -ldhD-P cpc560 -pct-P cpc560 -pha, by natural conversion Synechococcus elongatus The PYLW04 strain was obtained by integrating it into chromosomal neutral site I of PCC 7942.

[0075] The yield of the obtained PYLW02 strain, measured according to the method described in Example 7, was the highest at 5.6 mgg. -1 It's DCW.

[0076] Example 4: Overexpression of acetyl coenzyme A synthase

[0077] Synechococcus elongatus Primers based on the PCC 7942 genome (NC_007595.1) sequence: SEQ ID NO.11:5- ACTAGT ATGGCTGCACCTGTCACGAA-3; SEQ ID NO.12:5- GGATCC Design TTAGTCACTGCTTTCCAGAAACAC-3.

[0078] Recombinant genes acsA (GenBank: ABB57382.1) and pBA3031M were digested using SpeI and BamHI double enzymes, respectively, purified, and then ligated overnight at 16°C using T4 DNA ligase. E. coli susceptible cells were transformed by the ligation product chemistry method. Kanamycin (50 μg / mL) LB plates were plated with the transformation solution, plasmids were extracted, and the constructed recombinant plasmid was validated by double enzyme digestion and named pBA-acsA. Sequencing was completed by Shanghai Paimonuo Biotechnology Co., Ltd.

[0079] The extracted recombinant plasmid DNA utilizes the principle of homologous recombination through natural transformation to target genes. Synechococcus elongatus The recombinant transformant genome is integrated into the neutral site III chromosome of PCC 7942. After extracting the genome of the recombinant transformant, the success of the insertion is confirmed by PCR amplification validation using the validation primers SEQ ID NO.3: 5-ACCTGGAATTGGTTGAAGG-3 and SEQ ID NO.4: 5-ACAGCCAAGCTTGCATGC-3, and the PYLW05 strain is obtained. The yield of the obtained PYLW05 strain, measured according to the method described in Example 7, was 6.5 mgg. -1 It's DCW.

[0080] Example 5: Knockdown of the acetate kinase gene

[0081] Synechococcus elongatus Based on the gene sequence of acetate kinase on the PCC 7942 genome, a 24 bp antisense RNA was designed, and the full-length sRNA sequence of acetate kinase (shown as SEQ ID NO. 20) was artificially synthesized based on the sequences of Micc and Trbcl, which are the sRNA backbone.

[0082] Primer SEQ ID NO.15:5- GGATCC TATCAATAAGTATTAGGTATATGG-3, SEQ ID NO.16:5- GAATTCGCTGTCGAAGTTGAACAT-3 was designed, the full-length sRNA sequence was amplified, and the full-length sRNA sequence and the recombinant plasmid pBA-acsA obtained in Example 4 were double-enzyme-cleaved with BamHI and EcoRI, respectively. After purification, the plasmids were ligated overnight at 16°C using T4 DNA ligase. E. coli susceptible cells were transformed by the ligation product chemistry method. Kanamycin (50 μg / mL) LB plates were plated with the transformation solution, the plasmid was extracted, and the constructed recombinant plasmid was validated by double-enzyme-cleavage and named pBA-acsA-as-ackA. Sequencing was completed by Shanghai Paimonuo Biotechnology Co., Ltd.

[0083] The extracted recombinant plasmid DNA utilizes the principle of homologous recombination through natural transformation to target genes. Synechococcus elongatus The PYLW06 strain is integrated into the neutral site III chromosome of PCC 7942. The yield of the obtained PYLW06 strain, measured according to the method described in Example 7, was 9.8 mgg. -1 It's DCW.

[0084] Example 6: Acetyl coenzyme A carboxylase gene, β-Ketoacyl-ACP Synthetic enzymes II Continue knocking down the β-ketoacyl-ACP synthase III gene.

[0085] Synechococcus elongatus Acetyl coenzyme A carboxylase gene on the PCC 7942 genome, β-Ketoacyl-ACP Synthetic enzymes II Based on the sequences of the β-ketoacyl-ACP synthase III gene, 24 bp antisense RNAs were designed for each gene, and based on the sequences of Micc and Trbcl, which are the sRNA backbone, the acetyl coenzyme A carboxylase gene was designed. β-Ketoacyl-ACP Synthetic enzymes II The gene, specifically the full-length sRNA sequence of β-ketoacyl-ACP synthase III (shown in SEQ ID NO. 21), is artificially synthesized.

[0086] Primer SEQ ID NO.17:5- GAATTCTATCAATAAGTATTAGGTATATGG-3, SEQ ID NO.18: 5- GAGCTC GCTGTCGAAGTTGAACAT-3 was designed, the full-length sRNA sequence was amplified, and the full-length sRNA sequence and the recombinant plasmid pBA-acsA-as-ackA obtained in Example 5 were double-enzyme-cleaved with EcoRI and SacI, respectively. After purification, the plasmids were ligated overnight at 16°C with T4 DNA ligase. The transformation solution was plated onto a kanamycin (50 μg / mL) LB plate, the plasmid was extracted, and the constructed recombinant plasmid was validated by double-enzyme-cleavage and named pBA-acsA-as-ackA-as-accC-as-fabF-as-fabH. Sequencing was completed by Shanghai Paimon Biotechnology Co., Ltd.

[0087] The extracted recombinant plasmid DNA utilizes the principle of homologous recombination through spontaneous transformation to target genes. Synechococcus elongatus The PYLW07 strain is integrated into the neutral site III chromosome of PCC 7942. The yield of the obtained PYLW06 strain, measured according to the method described in Example 7, was 15.0 mgg. -1 It's DCW.

[0088] Example 7: Preparation of PLA using recombinant cyanobacteria cells

[0089] The recombinant cyanobacterial cell strain constructed in Example 1 was incubated in BG-11 liquid medium at 30°C with 5% (v:v) carbon dioxide aeration and light exposure. Shooting A photon quantum with an intensity of 125 μmol m -2 s -1 under the conditions optical density 730nm Until it becomes 0.5, m IPTG at a final concentration of mol / L was added as an inducer, and growth was observed after induced expression. optical density When the pressure reaches its maximum value, the bacterial strain is collected by centrifugation. The collected strain is freeze-dried, and the polymer material collected within the strain is recovered using chloroform. The recovered polymer is then analyzed by nuclear magnetic resonance (NMR). The results prove that the obtained polymer material is PLA, and the results are shown in Figure 2.

[0090] For quantitative experiments to produce PLA from the strain, the yield of PLA was calculated by measuring its elemental content using gas chromatography-mass spectrometry (GC-MS). The method is similar to that described by Jung et al., Biotechnology & Bioengineering, 2010, 105(1):161-171.

[0091] Example 8: Investigating the effects of different CO2 concentrations on cyanobacteria growth.

[0092] Since cyanobacteria are bacteria that grow by utilizing CO2 as a carbon source, the concentration of introduced CO2 has a very significant impact on cyanobacteria. In order to develop a high-density fermentation strategy for cyanobacteria, this invention first investigates the effect of introducing different concentrations of CO2 into the culture medium during the culture process on the growth of cyanobacteria (other culture conditions are the same as in Example 7, using the PYLW07 strain). When the volume ratio of introduced CO2 to air is 1%, 2%, 5%, 7%, and 10%, the growth over 5 days is optical density These are 2.5, 3.0, 3.5, 3.1, and 2.8 respectively. If the volume ratio of CO2 to air is 5%, Synechococcus elongatus Growth OD in PCC 7942 strain 730nm It was found that the highest value is 3.5.

[0093] Example 9: Investigating the effect of light intensity on cyanobacteria growth

[0094] Based on the conditions of Example 8, the present invention further explores the effect of light intensity on the growth of cyanobacteria (using the PYLW07 strain under the optimal conditions of Example 8, with other culture conditions being the same as in Example 7). The present invention uses 125-1000 μmol photons m -2 s -1 We will test the light intensity. Light irradiation is 500 μmol photons m -2 s -1 When it increases, the growth of cyanobacteria optical densityreached its maximum, and it was found that when it exceeded 500 μmol photons m -2 s -1 , the growth rate decreased. When it reached 1000 μmol photons m -2 s -1 , the cyanobacteria were threatened by the strong light and stopped growing. When the light intensity (μmol photons m -2 s -1 ) was 125, 250, 500, and 750 respectively, the optical density of growth for 5 days was 2.0, 2.5, 3.5, and 2.2 respectively. However, for cyanobacteria, since light irradiation should not be too strong until optical density 730nm = 1.0, for cyanobacteria, until optical density 730 = 1.0, a light intensity of 125 μmol photons m -2 s -1 was selected, and as the light intensity increased to 500 μmol photons m -2 s -1 along with growth, growth could be optimized the most, and optical density 730nm could reach 3.5 within 5 days.

[0095] Example 10: Explore the influence of the medium on the growth of cyanobacteria

[0096] After finding the optimal light intensity for cyanobacteria growth in Example 9, by optimizing the medium, it was explored whether the optical density (under the optimal conditions of Example 9, other culture conditions are the same as in Example 7, using the PYLW07 strain) of growth could be further enhanced. In this invention, starting from the BG - 11 medium, referring to the composition of the 5×BG - 11 medium, the following conditions (L -1 ) were added to the basal medium of BG - 11: 1 g of Na2CO3, 5 g of MgSO4·7H2O, 5 g of NaNO3, 2 mL of KH2PO4 (1 mol / L), and BG11 trace elements compounds , BG11 trace elements compoundsThe following solutions (mM) are added: 0.4 H3BO3, 0.1 MnCl2·7H2O, 0.004 ZnSO4·7H2O, 0.01 Na2MoO4·2H2O, 0.003 CuSO4·5H2O, and 0.001 Co(NO3)2·6H2O. This culture medium is named MBG-11. This shows improved growth compared to before. optical density 730nm It improved from 1.5 to 4.0.

[0097] Furthermore, to explore whether higher concentrations of Na2CO3 result in greater biomass, the present invention tests the effects of different concentrations of Na2CO3 on cyanobacteria growth, trying 0.25 g / L, 0.50 g / L, 1.0 g / L, and 1.5 g / L of Na2CO3, respectively. Experiments showed that growth over 5 days... optical density These are 3.5, 5.0, 7.0, and 3.0 respectively. When adding 1.0 g / L of Na2CO3, optical density 730nm The maximum score is 7.0.

[0098] Example 11: High-density fermentation of recombinant cyanobacterium PYLW07

[0099] From the experimental data of Example 7, the strain with the highest yield was PYLW07, and this strain will be used to explore a high-density fermentation strategy. When fermentation is performed using a shake flask, the basal medium for the PCC 7942 strain (BG-11 liquid medium) will be used. For the high-density culture strategy, genetically engineered PCC 7942 will be cultured at 30°C in improved BG-11 solid medium (i.e., MBG-11 liquid medium) using a homemade photobioreactor (published in a Chinese patent, patent number: ZL202220201535.5, the full text of which is incorporated into this text by reference), and will be light-illuminated in the early stages of growth. Shooting The intensity is 125 μmol photons m -2 s -1 And, after 2 days, 500 μmol photons m -2 s -1 Increase to 50 mL. For bubbling air culture containing 5% CO2 by volume, the gas is 50 mL min -1The fluid passes through a 5 μm pore size exhaust valve via bubbling at a flow rate. For PLA production, the PYLW07 strain was inoculated into 500 mL of MBG-11 liquid medium containing 20 mg / L of spectinomycin in an 800 mL flask, and CO2-rich air (5%, v / v) was continuously supplied. Next, optical density 730nm After the cells have grown to 0.4-0.6 mm, add 1 mmol / L of IPTG and 2 mmol / L of theophylline-inducing culture.

[0100] GC-MS detection of PLA yield revealed a final yield of 108 mg / L, equivalent to 23 mg / g DCW, approximately 270 times the yield of the initially constructed strain under shake flask culture conditions. The molecular weight of the prepared PLA (weight-average molecular weight Mw of 62.5 kDa and number-average molecular weight Mn of 32.8 kDa) is among the highest levels reported in the literature. Furthermore, a high-density culture strategy was employed, increasing the cell density tenfold compared to the same genetically engineered strain under shake flask culture conditions.

[0101] Example 12: Collection of recombinant cyanobacteria cells

[0102] To facilitate and rapidly collect cyanobacterial cells, this invention attempts to explore the effect of spontaneous sedimentation using a polymer electrolyte flocculant. Polyacrylamide flocculants are dissolved in water at room temperature in ratios of 1:300 (w / w) and 5:100 (w / w), respectively. Next, the dissolved polyacrylamide flocculant is added to a fermented broth cultured at high density at a ratio of 1.5% (v / v). The culture is mixed and stirred for 2 minutes. Then, the culture is allowed to settle by gravity for 5-10 minutes. The flocculation efficiency is: optical density 730 The data was characterized by optical density measurements before and after sedimentation at wavelengths in the nm range, and the measurement results showed that the aggregation efficiency reached 90%.

[0103] Industrial applicability Harvesting microalgae biomass on an industrial scale is a techno-economic bottleneck in the algae biomass industry, and the situation is further complicated by the small cell size of microalgae and the concentration of biomass diluted in cultures. The harvesting process requires the removal of large amounts of water, resulting in high process energy and costs, accounting for approximately 30% of the total cost of biomass production. Since PLA is an internal component of the cells, PLA particles and cells can be collected together by centrifugation. This invention discovers that using a flocculant allows for faster and simpler collection of cells, which has significant potential industrial value.

[0104] The above description of embodiments is intended to enable those skilled in the art to understand and use the present invention. It will be apparent to those skilled in the art that various modifications can be readily made to these embodiments, and that the general principles described herein can be applied to other embodiments without creative effort. Therefore, the present invention is not limited to the above embodiments. Improvements and modifications made by those skilled in the art based on the principles of the present invention, without departing from the scope of the invention, should also be within the scope of the invention.

Claims

1. A genetically engineered strain of Synechococcus elongatus, characterized in that, by integrating the coding sequence of an exogenous D-lactate dehydrogenase gene, the coding sequence of an exogenous propionyl coenzyme A transferase gene, and the coding sequence of an exogenous polyhydroxy fatty acid ester synthase gene into the genome of the genetically engineered strain, the genetically engineered strain can express exogenous D-lactate dehydrogenase, exogenous propionyl coenzyme A transferase, and exogenous polyhydroxy fatty acid ester synthase.

2. The coding sequence of the D-lactate dehydrogenase gene is P ｔｒｃ Promoter, P ｐｓｂａ Promoter or P ｃｐｃ５６０ A genetically engineered bacterial strain according to claim 1, controlled by a promoter.

3. The coding sequence of the propionyl coenzyme A transferase gene is P ｔｒｃ Promoter, P ｐｓｂａ Promoter or P ｃｐｃ５６０ A genetically engineered bacterial strain according to claim 1, controlled by a promoter.

4. The coding sequence of the polyhydroxy fatty acid ester synthase gene is P ｔｒｃ promoter, P ｐｓｂａ promoter or P ｃｐｃ５６０ The genetic engineering strain according to claim 1, which is controlled by a promoter.

5. The genetically engineered bacterial strain according to claim 1, wherein the coding sequence of the exogenous D-lactate dehydrogenase gene is derived from Lactobacillus bulgaricus, the coding sequence of the exogenous propionyl coenzyme A transferase gene is derived from Clostridium propionicum, and the coding sequence of the exogenous polyhydroxy fatty acid ester synthase gene is derived from Pseudomonas.

6. The genetically engineered bacterial strain according to claim 1, wherein the genetically engineered bacterial strain can express the exogenous acetyl coenzyme A synthase gene by integrating the coding sequence of the exogenous acetyl coenzyme A synthase gene into the genome of the genetically engineered bacterial strain.

7. The genetically engineered bacterial strain according to claim 1, wherein the expression of the acetate kinase gene in the genetically engineered bacterial strain is knocked down.

8. The genetically engineered bacterial strain according to claim 7, wherein the genome of the genetically engineered bacterial strain has a coding sequence for an exogenous sRNA that suppresses the expression of an acetate kinase gene, and the exogenous sRNA is small regulatory RNA.

9. The genetically engineered bacterial strain according to claim 1, wherein the expression of the acetyl coenzyme A carboxylase gene, the β-ketoacyl-ACP synthase II gene, and / or the β-ketoacyl-ACP synthase III gene of the genetically engineered bacterial strain is knocked down.

10. The genetically engineered bacterial strain according to claim 9, wherein the genome of the genetically engineered bacterial strain is integrated with coding sequences of exogenous sRNAs capable of suppressing the expression of the acetyl coenzyme A carboxylase gene, the β-ketoacyl-ACP synthase II gene, and / or the β-ketoacyl-ACP synthase III gene, and the exogenous sRNA is small regulatory RNA.

11. The genetically engineered bacterial strain is the Synechococcus elongatus PCC 7942 strain, according to any one of claims 1 to 10.

12. A method for producing polylactic acid using carbon dioxide, 1) The step of providing a genetically engineered strain of Synechococcus elongatus according to claim 1, 2) A step of introducing carbon dioxide and culturing the genetically engineered bacterial strain under light irradiation conditions, 3) A method for producing polylactic acid using carbon dioxide, comprising the steps of: when the growth optical density of the genetically engineered strain reaches its maximum value, collecting and drying the genetically engineered strain and recovering the polylactic acid within the genetically engineered strain.

13. The method according to claim 12, step 2) wherein the amount of carbon dioxide to be permeated is 1.5 vvm, and the volume ratio of introduced carbon dioxide to air is 1:(1-10).

14. Step 2) The intensity of the light irradiation is 125 μmol photon m －２ s －１ From 1000 μmol of photon quantum m －２ s －１ The method according to claim 12, wherein the result is less than [value missing].

15. The method according to claim 12, step 2) culturing the genetically engineered bacterial strain in a culture medium containing sodium carbonate, sodium nitrate, dipotassium hydrogen phosphate, magnesium sulfate, calcium chloride, citric acid, ferric ammonium citrate, ethylenediaminetetraacetic acid, boric acid, manganese chloride, zinc sulfate, sodium molybdate, copper sulfate, and cobalt nitrate.

16. In addition to the composition of the culture medium, Na ２ CO ３ MgSO ４ 7H ２ O, NaNO ３ 7KH ２ PO ４ and trace element compounds are added, and the trace element compounds are H ３ BO ３ , MnCl ２ 7H ２ O, ZnSO ４ 7H ２ O, Na ２ MoO ４ ・2H ２ O, CuSO ４ ・5H ２ O and Co(NO) ３ ) ２ 6H ２ The method according to claim 15, selected from O.

17. The method according to claim 15 or 16, wherein the final concentration of sodium carbonate in the culture medium is 0.25-1.5 g / L.

18. The method according to claim 12, wherein in step 2) the genetically engineered bacterial strain is cultured in the presence of one or more substances selected from the group consisting of IPTG and theophylline.

19. The method according to claim 12, wherein step 3) collecting the genetically engineered bacterial strain comprises mixing a culture medium containing a polymer electrolyte flocculant and the genetically engineered bacterial strain for 5 minutes, letting it stand for 5 minutes, discarding the supernatant after standing and collecting the precipitate, wherein the polymer electrolyte flocculant is a polyacrylamide flocculant.

20. The method according to claim 12, wherein in step 3) the polylactic acid in the genetically engineered bacterial strain is recovered with an organic solvent or by an alkaline extraction method, and the organic solvent is selected from chloroform, 1,4-dioxane, methyl-tetrahydrofuran, ethylene carbonate, acetone, and ethyl acetate.

Images