阅读说明：本技术 一种利用起皱假单胞菌ⅱ型合酶生产pha的方法 (Method for producing PHA (polyhydroxyalkanoate) by utilizing pseudomonas puckeri type II synthase ) 是由 胡风庆 赵小慧 辛知纯 于 2021-02-26 设计创作，主要内容包括：本发明涉及PHA生物合成领域,具体涉及一种新型生产PHA的方法。具体实施方案是：以起皱假单胞菌为出发菌株,利用起皱假单胞菌pBBR1MCS2-phaC1、pBBR1MCS2-phaC2的表达质粒,选用果糖、甘油、月桂酸、葡萄糖酸钠、油酸钠等不同碳源验证起皱假单胞菌pBBR1MCS2-phaC2合酶功能活性,结果表明重组菌可以利用油酸钠为碳源产生了PHA。从而保证了今后嵌合酶的成功构建,嵌合PHA合酶可以扩展其底物的广泛性,以提高PHA产量,极大的促进PHA的广泛应用。(The invention relates to the field of PHA biosynthesis, in particular to a novel PHA production method. The specific implementation scheme is as follows: the pseudomonas corrugating is used as an initial strain, expression plasmids of pseudomonas corrugating pBBR1MCS2-phaC1 and pBBR1MCS2-phaC2 are used, different carbon sources such as fructose, glycerol, lauric acid, sodium gluconate and sodium oleate are selected to verify the functional activity of the pseudomonas corrugating pBBR1MCS2-phaC2 synthase, and the result shows that the recombinant strain can generate PHA by using sodium oleate as a carbon source. Thus ensuring the successful construction of the chimeric enzyme in the future, and the chimeric PHA synthase can expand the universality of the substrate thereof so as to improve the PHA yield and greatly promote the wide application of the PHA.)

1. A method for producing PHA by using Pseudomonas corrugator type II synthase is characterized by comprising the following steps:

1) selecting single colony of Pseudomonas corrugate cultured on LB agar medium at 30 deg.c for 12 hr, inoculating to 10ml LB medium, and shake culturing at 30 deg.c and 200rpm for 8-12 hr to obtain seed liquid;

2) inoculating the seed liquid into a mineral salt fermentation culture medium in an inoculation amount of 5%, and performing shake culture for 72h at 30 ℃ and 200 rpm;

3) after the culture is finished, washing and collecting thalli;

4) the wet cells obtained were frozen overnight in a freezer at-20 ℃ and dried to constant cell weight.

2. The process for PHA production by using Pseudomonas puckeri type II synthase according to claim 1, wherein LB medium is prepared as follows: collecting 5g/l yeast extract, 10g/l tryptone, and 10g/l NaCl, subpackaging, and sterilizing at 121 deg.C for 15-20 min.

3. The process for PHA production by using Pseudomonas corrugator type II synthase according to claim 2, wherein said LB agar medium is prepared by the following method: adding 15g/l agar powder into 5g/l yeast extract, 10g/l tryptone, and 10g/l NaCl, subpackaging, and sterilizing at 121 deg.C for 15-20 min.

4. The process for the production of PHA of claim 3, wherein said mineral salt fermentation medium consists of component I, component II, component III and component IV;

the component I is as follows: 180g/l of disodium hydrogen phosphate dodecahydrate and 30g/l of dipotassium hydrogen phosphate;

the component II is as follows: 1g/l of ammonium sulfate and 0.41g/l of magnesium sulfate;

the component III is as follows: 5g/l of ferric ammonium citrate (17%) and 2g/l of calcium chloride;

the component IV is as follows: 20mg/l of nickel chloride, 10mg/l of copper sulfate, 30mg/l of sodium molybdate, 30mg/l of manganese chloride, 200mg/l of cobalt chloride, 100mg/l of zinc sulfate and 300mg/l of boric acid.

5. The method of claim 4, wherein the step 3) comprises collecting the mycelia by centrifugation at 8000rpm for 10min after the culture, washing the mycelia precipitate with distilled water, centrifuging at 8000rpm for 10min, collecting the mycelia by washing the mycelia with 95% ethanol, and centrifuging at 8000rpm for 10min to collect the mycelia.

6. The process as claimed in claim 5, wherein the mineral salt fermentation medium contains a carbon source selected from the group consisting of glycerol, lauric acid, sodium gluconate, fructose and sodium oleate.

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to a method for producing PHA by using Pseudomonas corrugate II-type synthase.

Background

Hydroxy fatty acid ester (PHA) is an intracellular energy and carbon source storage substance synthesized by many bacteria under conditions of nutrient imbalance. PHA has good biocompatibility, biodegradability, hydrophobicity and unique properties brought by different functional groups, is used as a tissue engineering material drug sustained-release material, is a biological material with good development potential, but the existing biosynthetic PHA has the problems of low yield, high cost, single monomer component and the like, and cannot meet the requirements of various fields on novel biological materials, and researchers in academia, industry and the like try to solve the key technical problem by various methods. The PHA synthesis needs the participation of various enzymes, and the modification of PHA synthase by utilizing a molecular biology technology to change the substrate specificity, improve the catalytic activity, change the monomer composition and obtain novel PHA is the current research hotspot.

Because the homology of the pseudomonas corrugate 388 II type synthase (phaC2) and the pseudomonas putida KT2442II type synthase (phaC2) is 73.1 percent, and the pseudomonas putida KT2442 and the pseudomonas corrugate 388 can use various carbon sources to synthesize PHA, the chimeric enzyme is suitable to be constructed. Pseudomonas putida KT2442 phaC2 has been successfully cloned and expressed, while Pseudomonas corrugata phaC2 has been studied less, and the functional activity of the expression plasmid of Pseudomonas corrugata phaC2 has not been successfully verified, and if the functional activity is obtained, a type II chimeric enzyme can be constructed.

The construction of the chimeric enzyme can fuse the characteristics of different strains of PHA synthase, expand the substrate range, and change the monomer components of the product PHA, thereby generating novel PHA. The method has great theoretical and practical significance for reducing the production cost of PHA and synthesizing novel PHA in future, and can greatly promote the wide application of PHA.

Disclosure of Invention

In order to solve the above problems, the present invention provides a method for producing PHA by constructing a chimeric enzyme type II from Pseudomonas corrugating, in order to solve the problems of low PHA production and high production cost.

In order to achieve the purpose, the invention adopts the following technical scheme:

a method for producing PHA using pseudomonas puckeri type II synthase, comprising the steps of:

1) selecting single colony of Pseudomonas corrugate cultured on LB agar medium at 30 deg.c for 12 hr, inoculating to 10ml LB medium, and shake culturing at 30 deg.c and 200rpm for 8-12 hr to obtain seed liquid;

2) inoculating the seed liquid into a mineral salt fermentation culture medium in an inoculation amount of 5%, and performing shake culture for 72h at 30 ℃ and 200 rpm;

3) after the culture is finished, washing and collecting thalli;

4) the wet cells obtained were frozen overnight in a freezer at-20 ℃ and dried to constant cell weight.

In the method for producing PHA by using the Pseudomonas corrugating strain II type synthase, the preparation method of the LB culture medium is as follows: collecting 5g/l yeast extract, 10g/l tryptone, and 10g/l NaCl, subpackaging, and sterilizing at 121 deg.C for 15-20 min.

The method for producing PHA by using Pseudomonas corrugator type II synthase comprises the following steps: adding 15g/l agar powder into 5g/l yeast extract, 10g/l tryptone, and 10g/l NaCl, subpackaging, and sterilizing at 121 deg.C for 15-20 min.

In the method for producing PHA by using the pseudomonas puckeri strain II-type synthase, the mineral salt fermentation medium consists of a component I, a component II, a component III and a component IV;

the component I is as follows: 180g/l of disodium hydrogen phosphate dodecahydrate and 30g/l of dipotassium hydrogen phosphate;

the component II is as follows: 1g/l of ammonium sulfate and 0.41g/l of magnesium sulfate;

the component III is as follows: 5g/l of ferric ammonium citrate (17%) and 2g/l of calcium chloride;

the component IV is as follows: 20mg/l of nickel chloride, 10mg/l of copper sulfate, 30mg/l of sodium molybdate, 30mg/l of manganese chloride, 200mg/l of cobalt chloride, 100mg/l of zinc sulfate and 300mg/l of boric acid.

The method for producing PHA by using the pseudomonas puckeri strain II-type synthase comprises the step 3) of centrifuging at 8000rpm for 10min after the culture is finished, collecting thalli, washing thalli precipitates with distilled water, centrifuging at 8000rpm for 10min, collecting thalli, washing the thalli with 95% ethanol, centrifuging at 8000rpm for 10min, and collecting the thalli.

In the method for producing PHA by using the Pseudomonas puckeri strain II-type synthase, the mineral salt fermentation medium contains a carbon source, and the carbon source is one of glycerol, lauric acid, sodium gluconate, fructose and sodium oleate.

The invention has the beneficial effects that: proves a novel strain for producing and synthesizing PHA, and the recombinant strain of pBBR1MCS2-phaC2 with sodium oleate as a carbon source can be used for producing PHA, thereby providing a foundation for the construction of the next step of chimeric enzyme and the continuous research and development of producing PHA.

Drawings

FIG. 1 is a gas chromatography analysis of medium and long chain PHA standards.

Figure 2 is a GC map of r.eutropha PHB-4 after fermentation with sodium oleate.

FIG. 3 GC map of R.eutropha PHB-4 containing empty vector after fermentation with sodium oleate.

Fig. 4 is a GC map of recombinant r.eutropha PHB-4-phaC2 after fermentation with sodium oleate.

FIG. 5 is a GC spectrum of medium-long chain PHA standard

FIG. 6 is a GC map of empty vector-containing R.eutropha PHB-4 of control group after fermentation with sodium oleate

FIG. 7 R.eutropha PHB of control group^-4GC map of synthase deletion mutant strain after fermentation by using sodium oleate

FIG. 8 is a GC map of recombinant R.eutropha PHB-4-phaC2 after fermentation with sodium oleate

Detailed Description

Example 1 production of PHA Using Pseudomonas rugosa type II synthase with sodium oleate as a carbon Source

Strain: pseudomonas corrugator pBBR1MCS2-phaC2

Culture medium:

LB medium: 5g/l yeast extract, 10g/l tryptone and 10g/l NaCl, subpackaging, and sterilizing at 121 deg.C for 15-20 min.

LB agar medium (pH7.0-7.2): adding the above components, adding 15g/l agar powder, subpackaging, and sterilizing at 121 deg.C for 15-20 min.

3. Mineral salt fermentation medium:

TABLE 1 table of mineral salt fermentation Medium components

The culture method comprises the following steps: culturing Pseudomonas corrugata on LB agar culture medium at 30 deg.C for about 12h, selecting single colony, inoculating in 10ml LB culture medium, shaking at 30 deg.C and 200rpm for 8-12h, and using as seed liquid. And adding 1% of sodium oleate into the component II as a carbon source to prepare a mineral salt fermentation medium, inoculating the mineral salt fermentation medium to recombinant bacteria (from the laboratory for storage) of pBBR1MCS2-phaC2 in an inoculation amount of 5%, and shaking at 30 ℃ for 72 hours at 200 rpm. After the culture is finished, the thalli is collected by centrifugation at 8000rpm for 10min, the thalli sediment is washed by distilled water, the thalli is collected by centrifugation at 8000rpm for 10min, then the thalli is washed by 95% ethanol, and the thalli is collected by centrifugation at 8000rpm for 10 min. The wet cells obtained were frozen overnight in a freezer at-20 ℃ and dried to constant cell weight.

A method for producing PHA by using a Pseudomonas corrugating strain II synthase firstly compares the homology of the Pseudomonas corrugating 388 II synthase (phaC2) with other strain gene sequences:

TABLE 2 comparison of sequence homologies of PHA producer strains

Note:

pseudomonas oleovorans (P. olevorans) 14682 type I synthase (phbC) GenBank AF422800

2 Pseudomonas putida (P.putida) type II synthase KT2442(phaC1) GenBank AF150670

3 Pseudomonas putida (P.putida) type II synthase KT2442(phaC2) GenBank AF150670

4 Pseudomonas corrugator (P.corrugata)388 type II synthase (phaC1) GenBank GU966686

Pseudomonas corrugator (P.corrugata)388 II type synthase (phaC2) GenBank GU966687

6 Aeromonas hydrophila (A. hydrophila) WQI type synthase (phaC) GenBank AY665302

The table shows the sequence homology comparison of the PHA-producing strains in the laboratory, and the most homologous gene sequences are that the gene sequences of the Pseudomonas corrugating 388 II type synthase (phaC2) and the Pseudomonas putida KT2442II type synthase (phaC2) are 73.1%, the Pseudomonas putida KT2442 phaC2 (from the bioscience and technology system of the university of Qinghua) has been successfully cloned and expressed, and the research on the Pseudomonas corrugating phaC2 is less, but the laboratory has successfully cloned and applied for GeneBank numbers (GU966686, GU966687), and at the same time, the expression plasmid of the Pseudomonas corrugating phaC2 is successfully constructed, the functional activity of the expression plasmid is not successfully verified, and only the functional activity of the expression plasmid is verified, namely the type II chimeric enzyme can be constructed, so as to produce PHA.

Comparative example 1 production of PHA from empty vector R. eutropha PHB-4 with sodium oleate as carbon Source

The culture method comprises the following steps: culturing Pseudomonas corrugata on LB agar culture medium at 30 deg.C for about 12h, selecting single colony, inoculating in 10ml LB culture medium, shaking at 30 deg.C and 200rpm for 8-12h, and using as seed liquid. Then 1% sodium oleate is added into the component II as a carbon source to prepare a mineral salt fermentation culture medium, and the mixture is inoculated into the R.eutropha PHB-4 (stored in the laboratory) of an empty carrier in an inoculation amount of 5%, and the shaking table is used for 30 ℃, 200rpm and 72 hours. After the culture is finished, the thalli is collected by centrifugation at 8000rpm for 10min, the thalli sediment is washed by distilled water, the thalli is collected by centrifugation at 8000rpm for 10min, then the thalli is washed by 95% ethanol, and the thalli is collected by centrifugation at 8000rpm for 10 min. The wet cells obtained were frozen overnight in a freezer at-20 ℃ and dried to constant cell weight.

Comparative example 2 production of PHA Using R.eutropha PHB-4 synthase-deficient mutant Strain with sodium oleate as carbon Source

The culture method comprises the following steps: culturing Pseudomonas corrugata on LB agar culture medium at 30 deg.C for about 12h, selecting single colony, inoculating in 10ml LB culture medium, shaking at 30 deg.C and 200rpm for 8-12h, and using as seed liquid. And adding 1% of sodium oleate into the component II as a carbon source to prepare a mineral salt fermentation culture medium, inoculating 5% of the culture medium to the R.eutropha PHB-4 synthase deletion mutant strain (stored in a laboratory), and shaking at 30 ℃ for 200rpm for 72 h. After the culture is finished, the thalli is collected by centrifugation at 8000rpm for 10min, the thalli sediment is washed by distilled water, the thalli is collected by centrifugation at 8000rpm for 10min, then the thalli is washed by 95% ethanol, and the thalli is collected by centrifugation at 8000rpm for 10 min. The wet cells obtained were frozen overnight in a freezer at-20 ℃ and dried to constant cell weight.

Comparative example 3 production of PHA Using Pseudomonas rugosa type II synthase Using Glycerol as carbon Source

The culture method comprises the following steps: culturing Pseudomonas corrugata on LB agar culture medium at 30 deg.C for about 12h, selecting single colony, inoculating in 10ml LB culture medium, shaking at 30 deg.C and 200rpm for 8-12h, and using as seed liquid. Then 1% of glycerol is added into the component II as a carbon source to prepare a mineral salt fermentation medium, and the components are respectively inoculated with 5% of inoculum size: empty vector R.eutropha PHB-4, control group R.eutropha PHB-4 synthase deletion mutant and recombinant R.eutropha PHB-4, shaker 30 ℃, 200rpm, 72 h. After the culture is finished, the thalli is collected by centrifugation at 8000rpm for 10min, the thalli sediment is washed by distilled water, the thalli is collected by centrifugation at 8000rpm for 10min, then the thalli is washed by 95% ethanol, and the thalli is collected by centrifugation at 8000rpm for 10 min. The wet cells obtained were frozen overnight in a freezer at-20 ℃ and dried to constant cell weight.

Comparative example 4 production of PHA Using Pseudomonas rugosa type II synthase Using lauric acid as carbon Source

The culture method comprises the following steps: culturing Pseudomonas corrugata on LB agar culture medium at 30 deg.C for about 12h, selecting single colony, inoculating in 10ml LB culture medium, shaking at 30 deg.C and 200rpm for 8-12h, and using as seed liquid. Adding 1% of lauric acid serving as a carbon source into the component II to prepare a mineral salt fermentation medium, and respectively inoculating with 5% of inoculum size: empty vector R.eutropha PHB-4, control group R.eutropha PHB-4 synthase deletion mutant and recombinant R.eutropha PHB-4, shaker 30 ℃, 200rpm, 72 h. After the culture is finished, the thalli is collected by centrifugation at 8000rpm for 10min, the thalli sediment is washed by distilled water, the thalli is collected by centrifugation at 8000rpm for 10min, then the thalli is washed by 95% ethanol, and the thalli is collected by centrifugation at 8000rpm for 10 min. The wet cells obtained were frozen overnight in a freezer at-20 ℃ and dried to constant cell weight.

Comparative example 5 production of PHA Using Pseudomonas rugosa type II synthase with sodium gluconate as carbon Source

The culture method comprises the following steps: culturing Pseudomonas corrugata on LB agar culture medium at 30 deg.C for about 12h, selecting single colony, inoculating in 10ml LB culture medium, shaking at 30 deg.C and 200rpm for 8-12h, and using as seed liquid. And adding 1% of sodium gluconate as a carbon source to the component II to prepare a mineral salt fermentation medium, and respectively inoculating with 5% of inoculum size: empty vector R.eutropha PHB-4, control group R.eutropha PHB-4 synthase deletion mutant and recombinant R.eutropha PHB-4, shaker 30 ℃, 200rpm, 72 h. After the culture is finished, the thalli is collected by centrifugation at 8000rpm for 10min, the thalli sediment is washed by distilled water, the thalli is collected by centrifugation at 8000rpm for 10min, then the thalli is washed by 95% ethanol, and the thalli is collected by centrifugation at 8000rpm for 10 min. The wet cells obtained were frozen overnight in a freezer at-20 ℃ and dried to constant cell weight.

Comparative example 6 production of PHA Using Pseudomonas rugosa type II synthase Using fructose as carbon Source

The culture method comprises the following steps: culturing Pseudomonas corrugata on LB agar culture medium at 30 deg.C for about 12h, selecting single colony, inoculating in 10ml LB culture medium, shaking at 30 deg.C and 200rpm for 8-12h, and using as seed liquid. And then adding 1% of fructose as a carbon source to the component II to prepare a mineral salt fermentation medium, and respectively inoculating with 5% of inoculum size: empty vector R.eutropha PHB-4, control group R.eutropha PHB-4 synthase deletion mutant and recombinant R.eutropha PHB-4, shaker 30 ℃, 200rpm, 72 h. After the culture is finished, the thalli is collected by centrifugation at 8000rpm for 10min, the thalli sediment is washed by distilled water, the thalli is collected by centrifugation at 8000rpm for 10min, then the thalli is washed by 95% ethanol, and the thalli is collected by centrifugation at 8000rpm for 10 min. The wet cells obtained were frozen overnight in a freezer at-20 ℃ and dried to constant cell weight.

Example 2 demonstration of Pseudomonas corrugator type II synthase production of PHA Using sodium oleate as a carbon Source

The detection method comprises the following steps: the dry cell weight was weighed and analyzed by gas chromatography.

TABLE 3 Shake flask fermentation results for R.eutropha PHB-4 containing empty vector, R.eutropha PHB-4 synthase deletion mutant of control group and recombinant R.eutropha PHB-4 containing five carbon sources

Note: ND: not detected

Weighing the dry weight of the cells: three groups of cells were compared by weighing as above: the results of the recombinant strain containing empty vector R.eutropha PHB-4, control R.eutropha PHB-4 synthase deletion mutant strain and recombinant R.eutropha PHB-4 showed that only recombinant bacteria using sodium oleate as carbon source produced PHA, which accounted for about 1.25% of the dry weight of the cells. As can be seen from the results of the measurement of the dry cell weight, only the recombinant bacteria using sodium oleate as a carbon source produced PHA, and thus the gas chromatography analysis and detection of the R.eutropha PHB-4, R.eutropha PHB-4 synthase deletion mutant and the recombinant R.eutropha PHB-4-phaC2, which contained empty vectors and were fermented with sodium oleate, were continued.

Preparation of samples by gas chromatography: 50mg of the stem cells were placed in an esterification tube, 2ml of chloroform and 2ml of an esterification solution were added, and a cap was closed. Sealing and putting in an oven at 100 ℃ for esterification for 4 h. Taking out after esterification, cooling to room temperature, opening the cover, adding 1ml of sterile water, oscillating and uniformly mixing the organic phase of the water phase, standing and placing until the water phase and the chloroform phase are completely layered, and transferring the chloroform phase to another tube for storage for gas chromatography analysis.

FIG. 5 is a GC spectrum of medium-long chain PHA standard, wherein the GC spectrum shows that the standard PHA contains HO, HD, HDD.

Fig. 6 is a GC map of r.eutropha PHB-4 containing empty vector after fermentation with sodium oleate of the control group, and no PHA was detected.

FIG. 7 is R.eutropha PHB of control group^-4The synthase deletion mutant utilized the GC profile after fermentation with sodium oleate and no PHA was detected.

FIG. 8 is a GC map of recombinant R.eutropha PHB-4-phaC2 after fermentation with sodium oleate, which allows recombinant bacteria to successfully produce PHA with sodium oleate as a carbon source.

As a result: the recombinant bacterium with pBBR1MCS2-phaC2 can only produce PHA in a culture medium with sodium oleate as a carbon source, and the R.eutropha PHB-4 synthase deletion mutant and the recombinant bacterium without pBBR1MCS2-phaC2 cannot produce PHA, so that the successful construction of an expression plasmid and the function of a synthase gene are proved. The recombinant strain takes sodium oleate as a carbon source to produce PHA which consists of 3HO, 3HD and 3HDD and accounts for about 1.25 percent of the dry weight of cells.

In the invention, five carbon sources of sodium gluconate, glycerol, lauric acid, sodium oleate and fructose are fermented, and as a result, only the recombinant R.eutropha PHB-4 strain taking sodium oleate as a carbon source generates PHA, while the R.eutropha PHB-4 synthase deletion mutant strain of the control group and the strain containing the empty vector R.eutropha PHB-4 are fermented by utilizing the five carbon sources, and any PHA cannot be generated by GC detection, so that the function of a synthase gene is proved, and the recombinant strain pBBR1MCS2-phaC2 taking sodium oleate as a carbon source can be used for producing PHA.