WO2021017154A1 - Escherichia coli lipopolysaccharide simplified engineering bacteria and application thereof for high yield of phb - Google Patents

Abstract

Provided are Escherichia coli lipopolysaccharide simplified engineering bacteria and an application thereof for high yield of PHB. A gmhD gene and 14 genes of a core polysaccharide gene cluster on an Escherichia coli genome are knocked out, and 11 genes of an O-antigen gene cluster on the Escherichia coli genome are knocked out, so that the capability of synthesizing PHB by Escherichia coli W3110, DH5α, and JM109 can be significantly improved, and the PHB yield of recombinant Escherichia coli is increased by 1.8-54.7 times.

Description

E. coli lipopolysaccharide simplified engineering bacteria and its application of high-yield PHB technical field

[0001] The present invention relates to E. coli lipopolysaccharide simplified engineering bacteria and applications of high-yield PHB, belonging to the fields of genetic engineering and fermentation engineering.

Background technique

[0002] Polyhydroxyalkanoates PH A (Polyhydroxyalkanoates PH A) is a kind of biodegradable polymer synthesized by microorganisms and possessing multiple material properties. It has broad application prospects in the fields of medicine, materials and environmental protection. Polyhydroxy fatty acid esters are widely present in microbial cells and are mainly used as carbon sources and energy storage carriers. The higher the C:N ratio in the growth environment, the more favorable the synthesis of PHA. PHA exists in the form of hydrophobic particles in the cell, and its content can exceed 90% of the dry cell weight under certain conditions. According to different monomer types and polymerization methods, PHA has a series of diverse material properties from hard and brittle hard plastics to soft elastomers. Since PHA can be synthesized from renewable resources as raw materials, it can be completely degraded by bacteria and other organisms after entering the natural world. Substituting conventional petroleum-based plastics can alleviate the serious "white pollution" problem, which has attracted widespread attention from the scientific and industrial communities around the world.

[0003] Poly-3-hydroxybutyrate (PHB) is a type of PHA, and E. coli is commonly used in the industrial production of PHB.

PHB is an intracellular product, and PHB particles are the carbon source storage material in the cell, which exist in the cytoplasm in the form of insoluble microspherical particles. PHB synthesis is subject to many adjustments, such as the C:N ratio. When carbon is excessive and nitrogen is lacking, PHB is easier to synthesize. When other C sources in the cell are metabolized, or amino acid transport enzyme activity is low, PHB synthesis will be affected. The key to the synthesis of PHB by Escherichia coli is to balance the product and cell growth, not only to make the cell grow well, but also to enhance the metabolic flow of its synthesis pathway, and to increase the yield by controlling the expression level of the product formation pathway. The yield of PHB synthesized by the original E. coli strain is generally 0~50% of the dry cell weight. 5 See reports that mainly improve PHB by optimizing fermentation conditions, optimizing metabolic pathways, increasing intracellular coenzyme concentration, and optimizing expression plasmids. Synthesis.

Summary of the invention

technical problem

[0004] The current PHB output cannot meet the needs of industrial production. Therefore, to provide a new The successful method is of great significance for further improving the synthesis of PHB.

The solution to the problem

Technical solutions

[0005] The first objective of the present invention is to provide a recombinant Escherichia coli, knock out (a) or (b), and express (3-ketothiolase, acetyl-CoA reductase and PHB synthetase encoding Gene; Among them, (a) is the ADP-L-glycerol-D-mannose-heptose-6-epimerase gm/zD gene on the E. coli genome: (b) is the E. coli core sugar gene cluster gmhD waaQ

[0006] In one embodiment, the amino acid sequence of the ADP-L-glycerol-D-mannose-heptose-6-epimerase GmhD is shown in SEQ ID NO.1.

[0007] In one embodiment, the core sugar gene cluster contains 14 genes, which are respectively waaQ, waaG, waaP, waaS, waaB, waaO, waaR, waaY, waaZ, waaU, waaL, waaC, waaF, gmdD

The NCBI accession numbers of its sequence are "BAE77660.1", "BAE77661.1", "BAE77662.1", "BAE77663.1", "BAE77664.1", "BAE77665.1", "BAE77666.1" , "BAE77667.1"

, "BAE77668.1", "BAE77669.1", "BAE77670.1", "BAE77671.1", "BAE77672. r, "BAE77673.1".

[0008] In one embodiment, the protein ID of the 3-ketothiolase is QBK40993.1; the protein ID of the acetyl-CoA reductase is QBK40994.1; the protein of the PHB synthetase

The ID is QBK40992.1.

[0009] In one embodiment, the E. coli is E. coli W3110, E. coli DH5a, or E. coli JM109.

[0010] In one embodiment, the recombinant E. coli is WJW01, which is obtained by knocking out the core sugar gene cluster in E. coli W3110.

[0011] In one embodiment, the recombinant E. coli is modified on the basis of WJW01 as follows: knock out the 0-antigen gene cluster on the E. coli genome, and name the obtained strain as WJW02.

[0012] In one embodiment; the 0-antigen gene cluster is

[0013] In one embodiment, the 0-antigen gene cluster

Contains 11 genes, namely wbbL::IS5, wbbK, wbbJ, wbbl, wzy, gif, wzx, rmlC , RmlA, rmlD, rmlB, their serial registration numbers are "BAA15873.1", "BAA15874.1", "B AA15875.1", "BAA15876.1", "BAA15877.1", "BAA15878.1", "BAA15879.1"

, "BAA15880.1", "BAA15881.1", "BAA15882.1", "BAA15883.1", "BAA1588 4.1".

[0014] The second object of the present invention is to provide a method for constructing the recombinant Escherichia coli, the method knocks out (a) or (b) gene, and encodes (3-ketothiolase gene, code The acetyl-CoA reductase gene and the gene encoding PHB synthetase are separately or jointly linked to the plasmid; among them, (a) is the ADP-L-glycerol-D-mannose-heptose-6-episotropy on the E. coli genome Construct enzyme gm/zD gene: (b) is the core sugar gene of Escherichia coli gmhD waaQ

[0015] In one embodiment, the plasmid is pDXW-8.

[0016] In one embodiment, the method includes a gene cluster phaCAB containing (3-ketothiolase, acetyl-CoA reductase and PHB synthase encoding genes)

Connect to plasmid pDXW-8 to obtain recombinant plasmid pDXW-S-z^aCAS, and then transform the recombinant plasmid into cells.

[0017] In one embodiment, the gene cluster

The NCBI accession number of the gene sequence is MH558939.1.

[0018] A method for producing poly-3-hydroxybutyrate (PHB), the method inoculates the recombinant E. coli into a fermentation medium, and uses glucose as a substrate for fermentation production.

[0019] In one embodiment, the fermentation medium contains: 20 g/L glucose, 17.1 g/L Na ₂ HP0 ₄ · 12H ₂ 0, 3 g/L KH ₂ P0 ₄ , 0.5 g/L NaCl, Add 1 mM MgS0 ₄ , 0.1 mM CaCl ₂ ) 10 mg/mL vitamin Bl.

[0020] In one embodiment, the fermentation specifically includes: transferring the seed solution of the recombinant bacteria to a fermentation medium, and adding kanamycin and the inducer IPTG (isopropyl thiogalactoside ) After fermentation.

[0021] The present invention also provides applications of the method in the fields of pharmaceutical preparation, materials or environmental protection.

[0022] In one embodiment, the application is to apply the produced PHB as a high molecular polymer in the field.

The beneficial effects of the invention

Beneficial effect [0023] Gene knockout can significantly improve the ability of E. coli W3110, DH5a and JM109 to synthesize PHB. After fermentation for 24 h and 48 h, the PHB volume yield of WJW00/pDXW-8-p/wCAS was increased to 2.24 g/L and 2.13 g/L, 3.95 and 3.66 times higher than the control strain WSl 10/pDXWI/^aCAS;

WJDOO/pDXW-8- /^aCAS cell synthesis PHB dry weight ratio increased by about 80%, conversion rate increased by about 1.9 times; WJJOO/pDXW-8- phaCAB

The dry weight ratio of PHB synthesized by cells is increased by about 75%, and the conversion rate is increased by about 1.8 times.

[0024] (2) Knock out 14 genes of the core sugar gene cluster on the E. coli genome to obtain mutant WJW01, continue to knock out 11 genes from the 0-antigen gene cluster on the E. coli genome to obtain mutant WJW02, and then P HB The three synthesized genes were respectively transformed into simplified strains WJW01 and WJW02, and the resulting recombinant strains WJ W01/pBHR68 and WJW02/pBHR68 could synthesize PHB up to 80.6% and 82.0% of the dry cell weight, respectively, which are wild-type control bacteria W3110/pBHR68 (1.5%) 53.7 and 54.7 times.

Brief description of the drawings

Description of the drawings

[0025] Figure 1: Schematic diagram of knockout of large fragments of E. coli.

[0026] Figure 2: Construction of engineering bacteria WJWOO.

Observed by laser confocal fiberscope. DIC: differential interference contrast, that is, differential interference contrast under white light conditions; FITC: fluorescence contrast, fluorescence contrast under excitation light 488 nm after Nile Red staining; Merged: the effect of superimposing pictures under DIC and FITC conditions, That is, the imaging under white light conditions and fluorescent conditions are superimposed.

Observation of the ultrathin section under electron microscope.

[0029] Figure 5: Shaking flask fermentation of Escherichia coli \¥3110/?0 \¥-8- /? 045 and \^\¥00/?0 \¥-8- /? 045 synthesized?1^.

[0030] Fig. 6: Recombinant strains DHSa/pDXW-S-z^aCAS, WJD00/pDXW-8-phaCAB

, JM109/pDXW-8- ;? 045 and WJro0/pDXW-8-;? 045 shake flask fermentation of cell synthesis PHB

[0031] Figure 7: Construction of plasmid pDTW202-cat [0032] FIG. 8: Construction of LPS Simplified Strain WJW02

[0033] Figure 9: PCR verification of the construction of WJW02; Lane 1: Knockout gene cluster waaQ-gmhD in W3110; Lane 2: Knockout gene cluster wbbL-rmffi in W3110; Lanes 3 and 4: Wild bacteria W3110 control.

[0034] Figure 10: Electron microscopic observation of PHB synthesized by recombinant strains W3110/pBHR68 and WJW00/pBHR68. a and b are the laser confocal fiber microscope observation of PHB particles synthesized by strains W3110/pBHR68 and WJW00/pBHR68, respectively. DIC: differential interference

Contrast, that is, differential interference contrast under white light conditions; FITC: fluorescence contrast, fluorescence contrast under excitation light 488 nm after Nile red staining; Merged: the effect of superimposing images under DIC and FITC conditions, that is, white light and Image overlay under fluorescent conditions. c and d are strain W

Ultrathin section electron microscope observation of 3110/pBHR68 and WJW00/pBHR68 synthetic PHB particles.

[0035] Figure 11: Shake flask fermentation of synthetic PHB by recombinant strains WJW01/pBHR68, WJW02/pBHR68 and control strain W3110/pBHR68.

[0036] Biological Material

[0037] Wild-type E. coli W3110 was purchased from ATCC, and the deposit number is ATCC39936.

[0038] Escherichia coli WJW00 (genotype E.coli W3110AgmhD) has been included in the patent "A genetically engineered strain producing Kdo2-li pidA and its construction method and application" (Publication No.: CN103820377A, Publication Date: 2014.05.28) It is disclosed that it was obtained by knocking out the key gene gm/zD for LD-heptose synthesis in the core sugar in wild-type E. coli W3110.

[0039] Table 1 Sequence Listing

[0040] [Table 1]

Invention embodiment

Embodiments of the invention

[0041] (1) Electron microscopic observation of Escherichia coli

[0042] ① Cell Crystal Violet Staining and Optical Microscope Observation: Centrifuge the fermentation broth, and take 0.5 pL of the bacterial cell and apply it on a clean glass slide, fix and dry, and drop a drop of 0.1% crystal violet for 1 min After rinsing and drying, drip cedar oil, observe with an optical microscope and oil lens, and take pictures.

[0043] ② Observation by laser confocal fiber microscope: Take 0.5 mL of fermented bacteria, and wash the bacteria twice with pH 7.4 phosphate buffer to prepare Nile red dye solution

Nile red, dissolved in 1/ of DMSO), resuspend the bacterial solution and place it in 37 (staining in a dark room for 30 min. Wrap the EP tube with aluminum foil to avoid exposure to light. After staining, use pre-cooled pH 7.4 again Wash the bacteria with phosphate buffer solution three times, try to open the operation in the dark. In a dark room, draw 10 pL of bacteria solution, place it on a glass slide and cover it with a cover glass, press it with your thumb for more than 5 s, and then seal the edge with nail polish After the nail polish is dried, the light is turned off and the laser confocal fiberscope is observed in the dark. First, place the glass slide upside down on the LCFM stage, find the cell area under coarse adjustment, and then align the area to adjust the focus and excitation wavelength to 480nm. The LCFM is magnified by a certain magnification for observation. If there is a flow phenomenon in the cells during the observation process, the sample should be prepared again, and special attention should be paid to the operation of the edge sealing. The better the fixed degree of the cells, the better and the clearer the imaging. Fix the emitted light and adjust it well Magnification, multi-view photography.

[0044] ③ Observation of ultra-thin section with electron microscope: Take phosphotungstic acid negative staining method. After collecting the cells, wash the cells with PBS (pH 7.4) and fix them with 2.5% glutaraldehyde (the solvent is PBS with a final concentration of 0.1 M, pH 7.4). Gloves and masks are required. After 5 minutes, use a toothpick to pick up the bacteria mass, so that the fixing solution can fully fix the bacteria, then fill the fixing solution, and seal to prevent the bacteria from being scattered during transportation. Then it was dehydrated, embedded and ultra-thin sectioned, and finally negatively stained for electron microscope observation.

[0045] (2) Method for measuring PHB yield: Pour the fermentation broth into a centrifuge tube and stand still for 2 hours, discard the supernatant, freeze the bacteria, and freeze-dry. Weigh a certain amount of freeze-dried bacteria for methyl esterification, use the reported conventional methyl esterification method for methyl esterification, and finally perform conventional gas chromatography quantification.

[0046] (3) Fermentation parameter determination method

[0047] ① Determination of OD and pH: Adjust zero with deionized water and adjust the dilution factor to the OD _ measured in the cuvette between 0.2 and 0.8. After the reading is stable, record the reading and multiply by the dilution factor to obtain the OD.

[0048] The pH of the fermentation sample just taken is directly measured, and the direct reading is measured with a pH meter. [0049] ②Residual sugar determination: The SBA-40C biosensor quantifies the glucose concentration through the changes in the electrical signal reflected by the binding of glucose molecules and the receptor on the reaction membrane; the determination method is to centrifuge the sample at 10,000 rpm for 2 min, and 20 The W supernatant is added to the system to be tested with the total system being HXXHd and shaken vigorously; accurately draw 25 pi standard sample (containing lg/L glucose) into the injection hole quickly, wait for the instrument to balance, and wait for the indicator light After not flickering, repeat more than 3 stitches, all of which are basically the same, and the calibration is deemed to be completed; Accurately draw 25 pi sample into the injection hole quickly, record the display index of the instrument, and divide by 2 to get the residual sugar value.

[0050] ③ Extraction and determination of PHB: Take PHB synthetic cells with different fermentation times, centrifuge to collect 5 mL of cells, wash twice with pH 7.4 PBS buffer, after centrifugal collection, transfer the cells to a pre-weighed centrifuge In the tube, wrap the tube with plastic wrap and pierce a number of small holes so that the water can evaporate and prevent the bacteria from spraying out. It is freeze-dried in a vacuum freeze dryer for 48 hours to completely dry. Generally, the bottom of the tube is at room temperature. , It means that it is completely dry, or if you flick the bottom of the tube with your fingers, the bacteria can disperse and easily detach from the tube wall, it is considered as completely dry. Weigh the weight and calculate the dry weight.

[0051] Weigh 1-10 mg of completely dried bacterial cells, and at the same time weigh about 10 mg of the PHB standard sample and transfer it to an accurately weighed esterification tube in order to accurately calculate the weighed sample weight. Then carry out the esterification operation, add 2 mL methanol (contains 3% sulfuric acid) and 2 mL chloroform, add the cap of the esterification tube and close it tightly. During this time, ultrasound can help the sample to disperse and make the esterification more complete. Boiling water bath 6

h above. After about half an hour, it can be observed that the PHB standard sample generally melts rapidly in powder form. If the morphology and dissolution of the standard product after half an hour are not observed, the whole set of reagents should be replaced. The deterioration of methanol and chloroform will cause the esterification to fail to proceed normally, and replacing the newly opened reagents can often solve the above problems. After six hours of boiling water After the bath, after cooling sufficiently in a fume hood, carefully open the esterification tube and add 1 mL of deionized water. At this time, you need to tighten the lid and vigorously shake until the system is fully mixed. At this time, place the esterification tube in Let stand in a ventilated place for more than 3 hours to separate the phases. After the phase separation, the upper layer is the water phase, and the lower layer is the organic phase. Add an appropriate amount of the organic phase to the gas phase sample bottle, close the lid and keep it sealed, and store at -80°C .

[0052] Gas chromatography is an accurate and sensitive means of quantifying the content of PHB. Gas quantification uses Shimadzu GC 2010 gas chromatography, Agilent DB WAX 30m-0.32mm gas chromatography column and flame ionization detector, and the injection temperature is 250°C. Different amounts of commercial PHB were used as standard samples to draw the standard curve of each standard sample.

[0053] Example 1 Construction of engineering bacteria WJW00

[0054] Gene knockout method: A combined site-specific recombination method is adopted to knock out large fragment gene clusters, such as As shown in Figure 1, at one end of the target gene cluster was introduced by Red recombination

Then introduce the cat-lox fragment by Red recombination at the other end, and then express it by Cre enzyme, recognize the sites toe L and toe /?, perform site recombination, and use AMP resistance screening to obtain Kan, Cm-free through resistance verification For the resistant transformants, PCR was further used to verify the genotype to confirm that the transformants are correct. The simplified method of using FRT sites is similar. FRT sites are introduced at both ends of the target gene cluster (there can be two), and then Flp enzyme expression is used to identify the FRT sites, and the correct knockout mutants can be obtained through resistance screening. . The Flp enzyme can continuously recognize the FRT site until the farthest FRT site that does not contain essential genes is recombined.

[0055] Engineering bacteria WJW00 has been published in the SCI paper "Construction and Characterization of an

&c/zer/c/z/a coZ/ Mutant Producing Kdo ₂ -Lipid A" released (Publication date: March 13, 2014). The specific construction method has been recorded in the patent publication number CN103820377B.

[0056] Example 2 Construction of engineering strains W3110/pDXW-8-p/wCAS and WJW00/pDXW-8-p/wCAS [0057] (1) Construction of plasmids

[0058] Plasmid pDXW-8-;? 045 has been in the article "Ma, W., Wang, J., Li, Y... et.al.

Poly(3-hydroxybutyrate-co-3-hydroxy valerate) co-produced with L-isoleucine in Corynebacterium glutamicum WM001 [J]. Microb Cell Fact, 2018, 17(1): 93

10.1186/s 12934-018-0942-7)) published in (public date: 2018-12-31). The specific construction process is as follows:

[0059] Using Roche eutrophic bacteria genome NC_008313.1 as a template, using primers; ^045 /; ^045-11 amplification to obtain the p/wCAS gene cluster (phaCAB

For gene clusters, please refer to the accession number https://www.ncbi.nlm.nih.gOv/nuccore/MH558939.l), with restriction enzymes Eco/?I and XM [digest PCR products, vector pDXW-8 (patent: An Escherichia coli-Corynebacterium shuttle type inducible expression vector PDXW-8 and its construction method, publication date: 2010-04-14) It was also digested with Eco/? After the digested product was purified, T4 ligase was used at 22°C Ligation overnight, transforming into E. coli DH5a, screening correct transformants, obtaining correct transformants DHSa/pDXW-S-p/wCAS, and extracting plasmid to obtain correct recombinant plasmid pDXW-8-phaCAB.

[0060] Table 2 Primer sequence list

[0061] [Table 2]

(2) Preparation of Competent State

[0063] E. coli W3110 (ATCC39936) and the engineered bacteria WJW 00 constructed in Example 1 were respectively inoculated in LB liquid medium, 37° C., 200 rpm overnight culture. Transfer 2% inoculum to 50 mL LB liquid culture medium, culture at 37°C, 200 rpm to OD _=0.5, and transfer the culture solution to a pre-cooled 50 mL centrifuge tube after ice bath for half an hour, 4°C, The bacteria were collected by centrifugation at 8000 rpm for 10 min. The pellet was washed 3 times with pre-cooled 0.01 M CaCl ₂ and finally suspended with 1 mL 0.01 M CaCl ₂ and mixed with 1 m 30% glycerol. Each tube was aliquoted to 200 pL. Pre-chilled sterile EP tube.

(3) Conversion

[0065] 100-200 ng of plasmid pDXW-S-z^aCAS was added to competent cells, mixed well, and ice bathed for 30 minutes.

min, heat shock at 42°C for 90s, ice bath for 2~3 min, add 1 mL LB medium for recovery, incubate at 37°C for 2h, spread 3(H _lg /mL kanamycin LB solid plate, 37°C Cultivate, pick the transformant and cultivate the seed liquid in the LB liquid medium containing 3(H _lg /mL kanamycin) to obtain the recombinant strains W3110/pDXW-8-phaCAB and WJW00/pDXW-8-phaCAB

[0066] Example 3 Qualitative observation of PHB production by engineering bacteria fermentation

[0067] LB medium composition: yeast powder 5g/L, tryptone 10 g/L and NaCl 10 g/L.

[0068] The composition of M9G medium: 20g/L glucose (Glucose), 17.1g/L disodium hydrogen phosphate dodecahydrate

(Na ₂ HP0 ₄ 12H ₂ 0), 3g/L potassium dihydrogen phosphate (KH ₂ PO ₄ ), 0.5 g/L sodium chloride (NaCl)

, Add 1mM magnesium sulfate (MgS0 ₄ ), 0.1 mM calcium chloride (CaCl ₂ ), 10mg/mL vitamin B1 (VBj.

[0069] (1) Seed liquid culture

[0070] Picking out recombinant bacteria separately

To 25mL LB medium, and add 3 (Hig/mL kanamycin, 37 (, 200 rpm, culture for 5 h to mid-log phase. [0071] (2) Fermentation to synthesize PHB

[0072] Culture method: The seed solution (OD _=about 1.8) was transferred to the conventional PHB fermentation medium M9G according to the initial OD _=0.25, and kanamycin was added to make the final concentration 3 (Hig/mL, 37 .(:, 200

rpm, fermentation ⁴ 8 h.

[0073] Induction conditions: Add IPTG at a final concentration of 0.5 mM at 0 hours.

[0074] (3) Qualitative observation of intracellular synthesis of PHB particles

[0075] A variety of electron microscopy methods were used to observe the synthesis of PHB particles in the fermentation cells. The cells are stained with Nile Red, and the excitation light is 488

Green fluorescence can be emitted under the nm laser confocal microscope. The more fluorescence, the more PHB content. As shown in Figure 3, the control bacteria W3110/pDXW-8-phaCAB

There is little green fluorescence, indicating that there are fewer PHB particles synthesized, and WJWOO/pDXW-S-z^CAS cells synthesize many PHB particles, and almost all cells have green fluorescence, and the cell volume becomes larger.

[0076] Further observation with ultra-thin section electron microscope, as shown in Figure 4, WJW00/pDXW-8-/^aCAS cells synthesize more PHB particles, and the cell volume is significantly larger, about 5-10 times larger; WJW00 /pDXW-8- cell membrane thickness is only about 11 nm, while the control bacteria W3110/pDXW-8-p/wCAS is about 13 nm, indicating that the membrane wall of the mutant strain WJW00 is more flexible, which is conducive to the accumulation of PHB particles in the cell .

[0077] Example 4 Quantitative determination of PHB produced by engineering bacteria fermentation

[0078] The same fermentation method as in Example 3 was adopted, and the recombinant strain WSl 10/pDXW-S-p/wCAS

Fermented with WJWOO/pDXW-S-p/wCAS, and sampled 2 mL at different time points to determine the corresponding fermentation parameters.

[0079] (1) Determination of fermentation OD _ and dry cell weight

[0080] The results showed that: after fermentation for 24 h and 48 h, WJWOO/pDXWI/^aCAS fermentation OD _

Reached 9.0 and 9.8, while the control strain WS110/pDXWI/^aCAS only 5.6 and 6.1 (Figure 5a), the dry cell weight increased by 47.9% and 67.0% (Figure 5b), but the cell sugar consumption only increased by 30.4% and 34.3% (Figure 5c).

[0081] (2) PHB production measurement: The results showed that the increase in dry cell weight was mainly due to WJWOO/pDXW-8-

/^aCAS Intracellular PHB particles accumulate more. At 24 h and 48 h, WJWOO/pDXW-8- p/wCAS can synthesize PHB up to 67.8% and 63.4% of the dry cell weight, respectively, while the wild control bacteria W3110/pDXW- 8-

; At the same time, the volume output is also significantly increased to 2.24 g/L and 2.13 g/L, which are 3.95 and 3.66 times higher than that of WSl lO/pDXWI/^aCAS (Figure 5e); the conversion coefficient of glucose to PHB reached 0.224 g/g and 0.185 g/g, while WSl lO/pDXWI The conversion coefficients of /^aCAS are only 0.063 g/g and 0.053 g/g (Figure 5f). It can be seen that knockout of gm/zD gene can significantly improve the ability of E. coli W3110 to synthesize PHB.

[0082] Table 3 Engineering bacteria WJWOO/pDXW-8-p/wCAS fermentation production PHB

[0083]

6h 24k micro

w3iio wmm w3iio wiwm w3iio wiwm

: Set parameters ^:

/pDXW-E- /pIIKW-S- /pDXW-S- /pDXW-S- /pDXW-E~ /pDKW-^

[0084] Example 5 Modification of other E. coli fermentation to produce PHB

[0085] (1) Construction of engineering bacteria WJDOO and WJJOO

[0086] With reference to the construction method of the engineered strain WJWOO in Example 1, the gm/zD genes in E. coli DH5a and JM109 were knocked out to obtain gm/zD mutant strains WJDOO and WJJOO.

[0087] (2) Construction of engineering strains DHSa/pDXW-S-z^aCAS, WJDOO/pDXW-S-z^aCAS

[0088] Referring to the method of Example 2, the plasmid pDXW-8 containing PHB synthesis-related genes-

They were respectively transformed into wild-type strain DH5a and mutant strain WJDOO to obtain recombinant strain DHSa/pDXW-S-z^aC^

, WJDOO/pDXW-8-phaCAB _^

[0089] (3) Construction of engineering strain JMHW/pDXW-S-z^aCAS, WJK) 0/pDXW-8-; 045

[0090] Referring to the method of Example 2, the plasmid pDXW-8 containing PHB synthesis-related genes-

They were respectively transformed into wild-type strain JM109 and mutant strain WJJOO to obtain recombinant strains JMHW/pDXW-S-z^aCAS and WJJOO/pDXW-8-phaCAB

[0091] (4) Fermentation of strains to produce PHB

[0092] According to the same fermentation method as in Example 3, the recombinant bacteria obtained in step (2) and step (3) were subjected to Fermentation and production of PHB Fermentation parameters were determined for shake flask fermentation of each recombinant strain.

[0093] Table 4 Recombinant bacteria fermentation 24h to produce PHB

[0094] [Table 3]

[0095] Table 5 Recombinant bacteria fermentation 48h to produce PHB

[0096] [Table 4]

(1) Determination of fermentation parameters of recombinant strain WJDOO/pDXWI/^aCAS

[0098] The results showed that after 24 h and 48 h in shake flask fermentation, the dry cell weight of recombinant strain WJDOO/pDXW-S-p/wCAS cells reached 3.98 g/L and 4.42 g/L (Figure 6a) The synthesized PHB reached 78.3% and 78.6% of the dry cell weight (Figure 6b) The conversion coefficients of glucose into PHB were 0.37 g/g and 0.39 g/g (Figure 6c); while DH5a/pDXW -8- p / wCAS dry cell weight only ² .33 g / L and ^{2. 4} 9 g / L (FIG. 6A) PHB yield of ^39.4% and 3.3% ⁴ (FIG. 6b) conversion of glucose coefficient of PHB They are 0.14 g/g and 0.12 g/g (Figure 6c). Compared with the control bacteria Dfi5a/pDXW-8-, the WJDOO/pDXW-S-z^aCAS cell synthesis PHB dry weight ratio increased by about 80%, and the conversion rate increased by about 1.9 times.

[0099] (2) Determination of fermentation parameters of recombinant strain WJJOO/pDXW-S-z^aCAS

[0100] After shaking flask fermentation for 24 h and 48 h, the recombinant strain WHOO/pDXW-S-p/wCAS

The dry cell weight of the cells reached 4.93 g/L and 5.51

g/L (Figure 6a) The synthesized PHB reached 84.3% and 84.8% of the dry cell weight (Figure 6b) The conversion coefficients of glucose into PHB were 0.36 g/g and 0.37 g/g (Figure 6c)

The dry cell weight is only 2.42 g/L and 2.57 g/L (Figure 6a) PHB yields are 46.2% and 48.3% (Figure 6b) The conversion coefficients of glucose to PHB are 0.15 g/g and 0.13

g/g (Figure 6c). Compared with the control strain JMHW/pDXW-S-z^aCAS, WJJOO/pDXW-8-phaCAB cells synthesize PHB dry weight ratio increased by about 75%, and the conversion rate increased by about 1.8 times.

[0101] Example 6 Construction of LPS Simplified Strain WJW01

[0102] Lipopolysaccharide (LPS) includes three parts: lipid A, core sugar, and O-antigen. Lipid A is a highly conserved region, while core sugar and O-antigen are composed of different sugar molecules. The core sugar synthesis gene cluster contains 14 genes, namely waaQ, waaG, waaP, waaS, waaB, waaO, wa aR, waaY, waaZ, waaU, waaL, waaC, waaF, gmdD

. Continue to knock out the core sugar gene cluster on the basis of the strain WJW00 obtained in Example 1.

gmdD-waoFC:-

(1) Refer to the gm/zD gene knockout process in Example 1 to knock out the waaQ gene

[0104] 1) Obtaining MM gene knockout fragments: Obtained by chemical total synthesis or PCR stepwise amplification

The upper and lower homology arms of the gene, and the fern fragment with FRT site in the middle. The nucleotide sequence of the wg gene knockout fragment is shown in SEQ ID No.3. The MM knockout fragment was cloned into pBlueScript II SK(+), and the recombinant plasmid pBlueScript II SK(+)-waaQU was obtained

Using this plasmid as a template, the knockout fragment waaQU -F kan waaQD can be amplified [0105] 2) Preparation and electrotransformation of knockout competence: Refer to Example 1 for specific steps, the difference is:

[0106] Change 500-1000

Knockout fragments were added to competent cells, mixed, ice bath for 15 min, 1.5 kv electric shock for 5 ms, 30 ° (: incubate 2 11, spread 3 (Hig/mL kanamycin LB solid plate, 30 °C Culture, pick the transformants and culture them in LB liquid medium containing 30 pg/mL kanamycin and 100 pg/mL ampicillin, and verify the results by PCR.

[0107] 3) Mutant strain resistance screening: Inoculate the strains with positive PCR verification in LB liquid medium containing 3 (Hig/mL kanamycin), cultivate overnight at 42°C, and streak the culture medium 30

pg/mL kanamycin LB solid plate, cultured at 37 °C, pick a single colony to verify its sensitivity to ampicillin, and name the strain containing kanamycin resistance and ampicillin sensitive as WJW00△waaQ :: fam and preserve.

[0108] (2) Knockout of core sugar gene cluster

[0109] Step (1) The obtained strain \¥ 00 ^ and _{: /} ^ There are three ?111' sites on the genome, which are 1 FRT site and 1 FRT site left by gm/zD knockout

Knock out the two FRT sites at both ends of the introduced resistance marker.

[0110] Using the strain WJW00△ waaQ::kan

To make the starting strain competent, transfer the pCP20 plasmid (Cherepanov PP, Wackernagel W. Gene disruption in Escherichia coli: Tc ^R and Km ^R cassettes with the option of Flp-catalyzed excision of the antibiotic-resistance determinant. Gene, 1995, 158 (1): 9-14), 42 °C heat shock to express FLP enzyme, culture the positive strain in LB liquid medium containing 100 pg/mL ampicillin, add L-arabinose (final concentration of 30

mmol/L) induces the expression of Flp recombinase and mediates all FRT site-specific recombination, so after recombining the three FRT sites, not only the kanamycin resistance marker but also gmAZ) and gmAZ are deleted. All the genes between Li.

[0111] Using primers MM ag-U-F and gm/zD-D-R PCR to verify the selection of transformants, and PCR to verify the correct strain 42. (: After heat shock, streak the LB plate, pick a single colony to verify its sensitivity to ampicillin and kanamycin, select the strain sensitive to both antibiotics, name it WJW01 and save it.

[0112] Table 6 Primer sequence list [0113] [Table 5]

[0114] Example 8 Construction of LPS Simplified Strain WJW02

[0115] 0-Antigen synthesis gene cluster rfbBDACX- glf-wbbHIJKL

Contains 11 genes, namely wbbL::IS5, wbbK, wbbJ, wbbl, wzy, gif, wzx, rmlC, rmlA, rmlD, rmlB. On the basis of WJW01, the O-antigen synthesis gene cluster was further knocked out to obtain WJ W02. The specific operations are as follows:

[0116] (1) Construction of tool plasmid pDTW202-c

[0117] 0-Antigen gene cluster

The knockout of ZOJC L/? site-specific recombination method is adopted. Therefore, when constructing the E. coli genome streamlining system, a tool plasmid pDTW202-cat needs to be constructed. The specific construction process of the plasmid is:

[0118] 1) Using plasmid pDTW109 (refer to Tan Yanzhen’s master's degree thesis "Construction of Corynebacterium glutamicum Gene Knockout System" section 3.2.1, publication date: 2012.03.27) as a template, using primers ac-Mc-mouth Tac-M-cat-R, amplify the ac-M-cW gene cassette, and use the restriction site Smo [for single enzyme double cut to obtain resistant fragments for ligation.

[0119] 2) Using the tool plasmid pDTW202 plasmid (see Tan Yanzhen’s master's degree thesis "Construction of Corynebacterium glutamicum Gene Knockout System" section 2.4.2, publication date: 2012.03.27) as a template, using primers pDTW2 02- lox RE-F and pDTW202-lox LE-R, amplify the remaining plasmid fragments that do not contain the fam resistance gene fragment, and also use Smo [for single enzyme double digestion, and the amplified plasmid fragments have

Specific site

[0120] 3) The fragments obtained in step 1) and step 2) are ligated with T4 ligase to construct

The plasmid pDTW202-c of the ZOJC L-C-ZOJC/? gene cassette (see Figure 7) contains the chloramphenicol resistance gene a? with a strong priming ability, which can be used for gene cluster knockout selection markers. Enzyme digestion and PCR verification are correct.

[0121] (2)

nn gene knockout

[0122] 1) Obtaining of wbbLm nn gene knockout fragment [0123] Construction of wbbL gene knockout fragments: obtained by chemical total synthesis or PCR stepwise amplification

Knockout fragment. The upstream and downstream homology arms are respectively designed in the primers, using the tool plasmid _P DTW202 as the template, and the primers

Amplify directly to obtain a wbbL knockout fragment, which contains only loxL sites but not loxR sites. The nucleotide sequence of the wbbL gene knockout fragment is shown in SEQ ID No. 0.4.

[0124] Construction of rmffi knockout fragments: The rmffi knockout fragments are obtained by chemical total synthesis or PCR stepwise amplification. The upstream and downstream homology arms are respectively designed in the primers, using the tool plasmid pDTW202-cat as the template, and the primers

The rmlB knock-out fragment is obtained by incorporation and amplification, and the knock-out fragment contains only loxR sites but not loxL sites. The nucleotide sequence of rmlB gene knockout fragment is shown in SEQ ID No. 5.

[0125] 2) Preparation and electrotransformation of knockout competence: Refer to Example 1 for specific steps, the difference is that 500-10

Add OOngwZ^L knock-out fragment to competent cells, mix well, ice bath for 15min, 1.5 kv electric shock for 5 ms, incubate at 30 °C for 2h, spread 30/mL kanamycin LB solid plate, 30

Cultivate at °C, pick the transformants and culture them in LB liquid medium containing 30 pg/mL kanamycin and 100 pg/mL ampicillin, using primers wbbL-F^W wbbL-R

PCR verification results. The correct strain was named WJW01AwbbL _:: loxL-kan.

[0126] In \^ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ 1 ^ _:: 1 («1 ^ 01 11 strain further knock out] 111 genes, 500-1000 ngnn knock-out fragment was added to competent cells, coated 3 (Hig /mL chloramphenicol LB solid plate, culture at 30°C, pick the transformants and culture in LB liquid medium containing 30 [xg/mL kanamycin and 30 [xg/mL chloramphenicol, using primers rml -F^W rml-R PCR verification result.

[0127] The PCR-positive strains were inoculated into LB liquid medium containing 30 pg/mL kanamycin and 30 pg/mL chloramphenicol, incubated overnight at 42°C, and the culture solution was streaked 3 (H _lg / mL kanamycin and 30

LB solid plate with pg/mL chloramphenicol, culture at 37°C, pick a single colony to verify its sensitivity to ampicillin, and will contain kanamycin-resistant and chloramphenicol-resistant strains that are sensitive to ampicillin Name it W31 10 A wbbL:: kanA rmlB:: cat and save it.

[0128] (3) O-Knockout of antigen synthesis gene cluster

[0129] Step ⑵ resulting strain _{\ ¥ 3110 ^ # :: ^^ /} ^ / 7 «:: there is a 1 (<< 1 _^ site and a loxR sites on the genome to make this experience as the original strain. Into the pKD-Cre plasmid (Master Han Yaning Graduation thesis "Construction of Escherichia coli Synthesizing Lipid A Molecules with Different Structures", publication date: 2013-12-31), expressing Cre enzyme, and culturing the positive strains in LB liquid medium containing 100 pg/mL ampicillin, The addition of L-arabinose (final concentration of 30 mmol/L) induces the expression of Cre recombinase, mediates lox L and lox R site-specific recombination, and not only deletes the kanamycin resistance marker and chloramphenicol resistance Mark, also delete all genes between wbbL^W rmlB. PCR verification was performed with primers wbbL-F^W rmlB-/?, the PCR-verified strain was heat shocked at 42 °C and streaked on an LB plate, and a single colony was picked to verify its resistance to ampicillin, kanamycin and chloramphenicol The strains sensitive to the three antibiotics were selected and named as WJ W02 and preserved.

[0130] The genotype of the simplified strain WJW02 was verified by PCR to be correct, but the fragment of the control wild type was too long and no band was amplified, indicating that the LPS gene cluster of the simplified strain WJW02 had been successfully deleted.

[0131] Table 7 Primer sequence list

[0132]

[Table 6]

Example 9 Construction of recombinant bacteria W3110/pBHR68, WJW01/pBHR68 and WJW02/pBHR68 [0134] (1) Construction of plasmid pBHR68

[0135] Plasmid pBHR68 is disclosed in SC Li Lunwen (Spiekermann, P., Rehm, B.H., Kalscheuer, R.,

Baumeister, D., Steinbuchel, A., 1999. A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds. Arch. Microbiol. 171 (2),

73-80, publication date: 1999.01.17), which contains

The gene cluster also contains ampicillin resistance markers, and the pkCAS gene cluster contains (3-ketothiolase, acetyl-CoA reductase and PHB synthase coding genes.

(2) Refer to Example 2 for the preparation method of competence.

[0137] (3) Transformation: 100-200 ng of plasmid pBHR68 was added to competent cells, and 100 pg/mL ampicillin LB solid plates were coated, 37

. (: Culture, pick the transformants and name them as W3110/pBHR68, WJW01/pBHR68 and WJW02/pBHR68 after verification.

[0138] Example 10 Fermentation of recombinant bacteria to produce PHB

[0139] The composition of LB medium: 5 g/L yeast powder, 10 g/L tryptone and 10 g/L NaCl.

[0140] Composition of M9G medium: 20 g/L glucose (Glucose), 17.1 g/L disodium hydrogen phosphate dodecahydrate

(Na ₂ HP0 ₄ 12H ₂ 0), 3 g/L potassium dihydrogen phosphate (KH ₂ PO ₄ ), 0.5 g/L sodium chloride (NaCl)

, Add 1 mM magnesium sulfate (MgS0 ₄ ), 0.1 mM calcium chloride (CaCl ₂ ), 10 mg/mL vitamin B 1 (VB j.

[0141] (1) Seed liquid culture

[0142] One loop of the recombinant bacteria W3110/pBHR68, WJW01/pBHR68, and WJW 02/pBHR68 obtained in Example 4 was picked up into 25 mL LB medium, and 100 pg/mL ampicillin 37 was added.

C. Incubate at 200 rpm for 5 h to mid-log phase.

(2) PHB synthesis by fermentation

[0144] Culture method: The seed solution (OD _6QQ = about 1.8) was transferred to the conventional PHB fermentation medium M9G according to the initial OD ₆₍₎₍₎ = 0.25, and ampicillin was added to make the final concentration 100 pg/mL, 31 (, 200 rpm, fermentation for 48 h.

(3) Qualitative observation of intracellular synthesis of PHB particles

[0146] Various electron microscopy methods were used to observe the synthesis of PHB particles in the fermentation cells. The cells are stained with Nile Red, and the excitation light is 488

Green fluorescence can be emitted under the nm laser confocal microscope. The more fluorescence, the more PHB content. As shown in Figures 10a and 10b, the control bacterium W3110/pBHR68 contains almost no green fluorescence, indicating that it is almost incompatible. The WJW02/pBHR68 cells synthesize many PHB particles, and almost all cells have green fluorescence, and the cell volume is significantly larger.

[0147] Further observation with ultrathin section electron microscopy, as shown in Figures 10d and 10c, WJW02/pBHR68 cells synthesize more PHB particles, and the cell volume is significantly larger, which can increase up to 25 times; WJW02/pBHR68 cell membrane wall thickness Only about 8-9 nm, while the control bacteria W3110/pBHR68 is 13

About nm, indicating that the membrane wall of the mutant strain WJW00 is more flexible, which is beneficial to the accumulation of PHB particles in the cell.

(3) Quantitative determination of the intracellular synthesis of PHB in engineering bacteria

[0149] Fermentation of recombinant bacteria W3110/pBHR68, WJW01/pBHR68 and WJW02/pBHR68, and sampling at different time points 2

mL, determine the corresponding fermentation parameters. The results showed that the fermentation curves of WJW01/pBHR68 and WJW02/pBHR68 had a higher OD _6(X) than the wild control W3110/pBHR68 (Figure 11a), which increased by 1.92 times and 2.10 times respectively at 24 h; WJW01/pBHR68 and WJW02/pBHR68 The dry cell weight is also higher than that of the wild control W3110/p BHR68 (Figure 1 ib). After 24 hours of fermentation, it can be increased by 2.2 times and 2.5 times, respectively; WJW01/pBHR68 and WJW02/pBHR68 can synthesize PHB up to 80.6 of the dry cell weight, respectively. % And 82.0% (Figure 11c), which are 53.7 and 54.7 times of the wild-type control strain W3110/pBHR68 (1.5%) (Figure 11c); the conversion rate of WJW01/pB HR68 and WJW02/pBHR68 using glucose to synthesize PHB can reach 0.32, respectively g/g and 0.34 g/g, compared with the wild control W3110/pBHR68 (0.002 g/g) can increase 159 times and 169 times, respectively. The above results indicate that 14 genes in the lipopolysaccharide core sugar gene cluster have been knocked out and the 0-antigen synthesis gene has been knocked out

PHB capabilities.

[0150] Table 8 Shake flask fermentation of strains (24 h)

[0151] [Table 7]

Claims

Claims

[Claim 1] A recombinant Escherichia coli characterized by knocking out (a) or (b) and expressing (3-ketothiolase, acetyl-CoA reductase and PHB synthase coding genes;

Among them, (a) is the ADP-L-glycerol-D-mannose-heptose-6-episomeric enzyme gm/zD gene on the E. coli genome: ⑹ is the E. coli core sugar gene cluster gmhD-waaQ

[Claim 2] The recombinant E. coli according to claim 1, wherein the ADP-L-glycerol

The amino acid sequence of -D-mannose-heptose-6-epimerase GmhD is shown in SEQ ID No. 1

[Claim 3] The recombinant Escherichia coli according to claim 1, wherein the core sugar gene cluster contains 14 genes, respectively waaQ, waaG, waaP, waaS, waaB, waaO, waaR, waaY, waaZ , waaU, waaL, waaC, waaF, gmdD, the NCBI accession numbers of its sequence are "BAE77660.1", "BAE77661.1", "BAE77662.1", "BAE77663.1", "BAE77664.1", " BAE77665.1", "BAE77666.1"

, "BAE77667.1", "BAE77668.1", "BAE77669.1", "BAE77670.1", "BAE77671.1", "BAE77672.1,,,"BAE77673.1".

[Claim 4] The recombinant E. coli according to claim 1, wherein the protein ID of the (3-ketothiolase) is QBK40993.1; the protein ID of the acetyl-CoA reductase is QBK40994 .1; The protein ID of the PHB synthase is QBK40992.1.

[Claim 5] The recombinant Escherichia coli according to any one of claims 1 to 4, wherein the Escherichia coli is Escherichia coli W3110, Escherichia coli DH5a or Escherichia coli JM109.

[Claim 6] The recombinant E. coli according to claim 1, wherein the 0-antigen gene curtain is wbbL-rmlB.

[Claim 7] The recombinant Escherichia coli according to claim 6, wherein the 0-antigen gene cluster wW?L-rmIB contains 11 genes, respectively wbbL::IS5, wbbK, wbbJ, wbbl, wzy, gif, wzx, rmlC, rmlA, rmlD, rmlB, the serial registration numbers are "BAA15873.1", "BAA15874.1", "BAA15875.1", "BAA15876.1", "B AA15877.1" , "BAA15878.1", "BAA15879.1", "BAA15880.1", "BA A15881.1", "BAA15882.1", "BAA15883.1", "BAA15884.1".

[Claim 8] A method for constructing the recombinant Escherichia coli described in claim 1, characterized in that the (a) or (b) gene is knocked out, and the gene encoding (3-ketothiolase, the gene encoding acetyl The gene of coenzyme A reductase and the gene encoding PHB synthetase are separately or jointly linked to the plasmid; among them, (a) is the ADP-L-glycerol-D-mannose-heptose-6-epimer on the E. coli genome Enzyme gm/zD gene: ⑹ is the E. coli core sugar gene cluster gm/zD-waaQ.

[Claim 9] The method of claim 8, wherein the plasmid is _P DXW-8.

[Claim 10] The method according to claim 8 or 9, characterized in that the method connects the gene cluster phaCAB containing (3-ketothiolase, acetyl-CoA reductase and PHB synthase encoding genes) On the plasmid pDXW-8, the recombinant plasmid pDXW-S-z^aCAS is obtained, and then the recombinant plasmid is transformed into the cell.

[Claim 11] A method for producing poly-3-hydroxybutyrate, characterized in that the method inoculates the recombinant Escherichia coli into a fermentation medium, and uses glucose as a substrate for fermentation production.

[Claim 12] The method of claim 11, wherein the fermentation medium contains: 10

~30 g/L glucose, 15~20 g/L Na ₂ HPO ₄ , 1~5 g/L KH ₂ PO ₄ , 0.2-0.8 g/L NaCl, 0.5-1.5 mM MgS0 ₄ , 0.05-0.2 mM CaCl ₂ ) 5~15 mg/mL Vitamin Bl.

[Claim 13] The method according to claim 11 or 12, wherein the fermentation is specifically: transferring the seed liquid of the recombinant bacteria to a fermentation medium, and adding kanamycin and an inducer IP TG (isopropyl thiogalactoside) is then fermented.

[Claim 14] The application of the recombinant E. coli according to any one of claims 1 to 7 or the method according to any one of claims 11 to 13 in the fields of pharmaceutical preparation, materials or environmental protection.

[Claim 15] The application according to claim 14, characterized in that the produced poly-3-hydroxybutyrate is used as a polymer in the field.