阅读说明：本技术 一种提高水溶性藻青素外源合成产量的方法 (Method for improving exogenous synthesis yield of water-soluble phycocyanin ) 是由 林凌 陈明明 朱国萍 于 2019-11-27 设计创作，主要内容包括：本发明公开了一种提高水溶性藻青素外源合成产量的方法,属于生物基因工程、微生物发酵技术领域。该方法以TB培养基为对照,依据大肠杆菌诱导表达载体系统,使藻青素合成酶合成藻青素,选用特定的培养基组分和培养温度,使得水溶性藻青素合成产量获得大幅提高本发明生产的水溶性藻青素,具有比对照培养产生的水溶性藻青素产量高2.2倍的特性,为水溶性藻青素生产提供了更高效的培养条件。(The invention discloses a method for improving exogenous synthetic yield of water-soluble phycocyanin, and belongs to the technical field of biological genetic engineering and microbial fermentation. The method uses TB culture medium as a reference, synthesizes phycocyanin by phycocyanin synthetase according to an escherichia coli induced expression vector system, selects specific culture medium components and culture temperature, greatly improves the synthesis yield of water-soluble phycocyanin produced by the method, has the characteristic of 2.2 times higher than the yield of the water-soluble phycocyanin produced by the reference culture, and provides more efficient culture conditions for the production of the water-soluble phycocyanin.)

1. A method for improving exogenous synthesis yield of water-soluble phycocyanin is characterized in that: producing water-soluble phycocyanin by using recombinant escherichia coli, inoculating escherichia coli activated bacterial liquid for producing phycocyanin into a fermentation culture medium for improving the yield of water-soluble phycocyanin, and performing induced culture at the temperature of 35 ℃ for 30 hours; the recombinant strain used by the recombinant escherichia coli is Escherichia coli BL21 host bacteria, and the strain comprises a pCOLADuet-1 expression vector and an phycocyanin synthetase gene CphA derived from Thermoascus coli BP-1_BPThe numbering of the gene is GenBank No: NC-004113, wherein the fermentation medium comprises 2.4% of yeast powder, 1mM of aspartic acid and 1mM of lysine, 4mL/L of glycerol and 17mM KH₂PO₄And 72mM K₂HPO₄Phosphate buffer solution.

2. The method for increasing the yield of exogenously synthesized water-soluble phycocyanin according to claim 1, wherein said method comprises the steps of: in the culture medium for improving the yield of the water-soluble phycocyanin, the carbon source is glycerol, yeast powder and amino acid; the main nitrogen source is yeast powder; the inorganic salt is a phosphate.

3. The method for increasing the yield of exogenously synthesized water-soluble phycocyanin according to claim 1, wherein said method comprises the steps of: the preparation method of the activated bacterial liquid comprises the steps of inoculating a recombinant escherichia coli single colony to an LB liquid culture medium, and performing shake culture for 16 hours at the temperature of 37 ℃ and the rotating speed of 180rpm to obtain the activated bacterial liquid.

4. The method for increasing the yield of exogenously synthesized water-soluble phycocyanin according to claim 1, wherein said method comprises the steps of: inoculating the activated bacterial liquid and the fermentation medium according to the volume ratio of 1:100, pre-culturing for 2-3h under the conditions that the temperature is 37 ℃ and the rotating speed is 180rpm, and then performing induction culture for 30 h.

5. The method for increasing the yield of exogenously synthesized water-soluble phycocyanin according to claim 4, wherein said method comprises the steps of: inoculating the activated bacterial liquid and the fermentation culture medium according to the volume ratio of 1:100, and pre-culturing for 2-3h under the conditions that the temperature is 37 ℃ and the rotating speed is 180rpm until the OD of the bacterial liquid is₆₀₀When the concentration is 0.4-0.5, 1M isopropyl- β -D-thiogalactoside (IPTG) with concentration of 0.01% is added, and the mixture is cultured for 30h under the conditions that the temperature is 35 ℃ and the rotation speed is 180 rpm.

6. The method for increasing the yield of exogenously synthesized water-soluble phycocyanin according to claim 1, wherein said method comprises the steps of: further comprises a phycocyanin purification step, wherein according to the characteristics of the escherichia coli thallus, the bacteria collection condition is 5500rpm, and the centrifugation is carried out for 10 min; drying the collected thalli, adding hydrochloric acid with the pH value of 1.4 according to the dry weight of the thalli of 10-20mL/g, oscillating for 12 hours under the conditions that the temperature of a shaking table is 30 ℃ and the rotating speed is 140rpm, centrifuging the thalli lysate for 30 minutes at the rotating speed of 9000rpm, collecting supernatant, adding 5mL0.1M hydrochloric acid into the precipitate again, shaking for 1 hour at 30 ℃, and centrifuging and collecting the supernatant; mixing the supernatants obtained in the two times, adding NaOH to adjust the pH value to 7.0, centrifuging the neutral turbid solution at a rotating speed of 9000rpm for 30 minutes, collecting the precipitate as insoluble phycocyanin, adding 2 times of volume of glacial ethanol into the supernatant, centrifuging at the rotating speed of 9000rpm for 30 minutes again, and collecting the precipitate as water-soluble phycocyanin.

Technical Field

The invention relates to the technical field of biological genetic engineering and microbial fermentation, in particular to a method for improving the exogenous synthetic yield of water-soluble phycocyanin and application thereof.

Background

Phycocyanin (CGP) is a non-ribosomally synthesized polymer of amino acids found in most cyanobacteria and some heterotrophic bacteria. It comprises a poly-L-aspartic acid (Asp) backbone with a plurality of arginines (Arg) attached through their amino groups to the side chains formed at the carboxyl group of each aspartic acid. Arg and Asp are usually present in the polymer in equimolar amounts. The natural phycocyanin has the molecular weight of 25kDa to 100kDa and has wide polydispersity. Phycocyanin in its natural state can be classified into two forms according to its physical properties: insoluble and water-soluble phycocyanin. The complex, which is called water-soluble phycocyanin soluble in neutral solution, can be precipitated by adding ethanol. Insoluble phycocyanin is only dissolved in a solution with pH <2.0 or pH >9.0, and precipitates are generated under neutral conditions. Phycocyanin, which is a nitrogen and carbon storage compound, is considered to be a component of most cyanobacterial particle inclusions. In recent years, attention has been drawn to applications in the fields of food, medicine, cosmetics, nutrition, agriculture, and the like.

The mainstream trend of phycocyanin synthesis at present is to perform heterologous expression on phycocyanin synthetase CphA or directly perform in vitro synthesis of phycocyanin. The main hosts for heterologous expression include various bacilli, tobacco and yeast. Wherein, the Escherichia coli has a series of advantages of clear genetic background, complete carrier receptor system, rapid growth, simple culture, stable recombinant and the like, and is widely researched as a receptor. Since the enzyme which can decompose phycocyanin, that is, phycocyanin enzyme can be artificially removed by means of gene knockout and the activity of the enzyme is inhibited in E.coli, a large amount of phycocyanin is deposited in cells. Experiments show that the molecular weight of the phycocyanin separated from the exogenous expression system is different from that of the phycocyanin produced by cyanobacteria. The phycocyanin separated from the exogenous expression system generally has water-soluble phycocyanin of about 0-25kDa and insoluble phycocyanin of 25-33 kDa.

The heat stability and biocompatibility of phycocyanin are studied in recent years, the elastic compression modulus of phycocyanin is 560MPa, and the phycocyanin has heat stability below 200 ℃, in the aspect of biocompatibility, water-soluble phycocyanin has no toxicity to Chinese hamster ovary CHO cells, CHO cells show higher cell density on a film formed by phycocyanin, and after a serum-containing culture medium is removed, the cell morphology can be maintained for 72 h.

Although phycocyanin synthetic vectors are wide in range and numerous in research field, due to limited yield, CphA is adopted by taking escherichia coli as a vector at present₆₃₀₈Exogenous expression of phycocyanin of gene, 50-100mg/g of water-soluble phycocyanin yield, and 300mg/g of insoluble phycocyanin accounting for 200-300mg/g of dry weight of thallus, using Escherichia coli as vector and adopting CphA₆₈₀₃The phycocyanin of the gene is exogenously expressed, the yield of the water-soluble phycocyanin is mostly 30-50mg/g of the dry weight of the bacteria, and the insoluble phycocyanin accounts for 100-200mg/g of the dry weight of the bacteria. CphA used in the present invention_BPThe gene can reach the yield of water-soluble phycocyanin accounting for 160mg/g of dry cell weight under the condition of not reducing the yield of insoluble phycocyanin after the culture condition is changed.

Disclosure of Invention

The technical problem solved by the invention is as follows: provides a method for improving the yield of water-soluble phycocyanin heterologous synthesis, which is used for overcoming the defect of low yield of water-soluble phycocyanin in the prior art.

In order to solve the technical problems, the technical scheme provided by the invention is as follows: a method for improving exogenous synthesis yield of water-soluble phycocyanin comprises producing water-soluble phycocyanin with recombinant Escherichia coli, inoculating activated Escherichia coli liquid for producing phycocyanin into fermentation culture medium for improving yield of water-soluble phycocyanin, and performing induced culture at 35 deg.C for 30 hr; the recombinant strain used by the recombinant Escherichia coli is Escherichia coli BL21 host bacteria, and the strain contains pCOLADuet-1 expression vector and phycocyanin synthetase gene CphA derived from Thermoynechococcus elongatus BP-1_BPThe numbering of the gene is GenBank No: NC-004113, wherein the fermentation medium comprises 2.4% of yeast powder, 1mM of aspartic acid and 1mM of lysine, 4mL/L of glycerol and 17mM of KH₂PO₄And 72mM K₂HPO₄Phosphate buffer solution.

Preferably, in the culture medium for increasing the yield of the water-soluble phycocyanin, the carbon source is glycerol, yeast powder and amino acid; the main nitrogen source is yeast powder; the inorganic salt is a phosphate.

Preferably, the preparation method of the activated bacterial liquid is to inoculate the recombinant escherichia coli single colony in an LB liquid culture medium, and shake-culture is carried out for 16h under the conditions that the temperature is 37 ℃ and the rotating speed is 180rpm, so as to obtain the activated bacterial liquid.

Preferably, the activated bacterium liquid and the fermentation medium are inoculated according to the volume ratio of 1:100, pre-cultured for 2-3h under the conditions that the temperature is 37 ℃ and the rotating speed is 180rpm, and then induced culture is carried out for 30 h.

Preferably, the activated bacterium liquid and the fermentation medium are inoculated according to the volume ratio of 1:100, and are pre-cultured for 2-3h under the conditions that the temperature is 37 ℃ and the rotating speed is 180rpm, and the OD of the bacterium liquid is to be obtained₆₀₀When the concentration is 0.4-0.5, 1M isopropyl- β -D-thiogalactoside (IPTG) with concentration of 0.01% is added, and the mixture is cultured for 30h under the conditions that the temperature is 35 ℃ and the rotation speed is 180 rpm.

Preferably, the method also comprises a phycocyanin purification step, wherein the bacteria collection condition is 5500rpm according to the characteristics of the escherichia coli thallus, and the centrifugation is carried out for 10 min; drying the collected thalli, adding hydrochloric acid with the pH value of 1.4 according to the dry weight of the thalli of 10-20mL/g, shaking for 12 hours under the conditions that the temperature of a shaking table is 30 ℃ and the rotating speed is 140rpm, centrifuging the thalli lysate for 30 minutes at the rotating speed of 9000rpm, collecting a supernatant, adding 5mL of 0.1M hydrochloric acid again into the precipitate, shaking for 1 hour at 30 ℃, and centrifuging and collecting the supernatant; mixing the supernatants obtained in the two times, adding NaOH to adjust the pH value to 7.0, centrifuging the neutral turbid solution at a rotating speed of 9000rpm for 30 minutes, collecting the precipitate as insoluble phycocyanin, adding 2 times of volume of glacial ethanol into the supernatant, centrifuging at the rotating speed of 9000rpm for 30 minutes again, and collecting the precipitate as water-soluble phycocyanin.

The method for exogenously synthesizing and analyzing the water-soluble phycocyanin comprises the following steps: constructing a recombinant strain, activating thalli, carrying out amplification culture, transferring to a specific culture medium, adding an inducer, carrying out bacterium collection and purification, calculating yield and carrying out liquid chromatography detection.

CphA used_BPThe gene, which is derived from Thermosynechococcus elonegatus BP-1, is disclosed in The A cyanophycin synthase from TheThe gene sequence of the rmosynecococcus elongatus BP-1catalyzes primer-independent cyanophyll synthesis is disclosed in GenBank public database, and the number is NC-004113 (2249112.. 2251802).

Reacting CphA_BPThe DNA fragment of the gene was ligated to plasmid pCOLADuet-1 and transformed into Escherichia coli BL21 host bacteria to obtain a recombinant strain. The method of activation and expansion culture is to recover the activity of the recombinant strain according to the growth of the recombinant strain and the characteristics of the plasmid pCOLADuet-1. Wherein, the LB culture medium comprises the following components:

LB liquid medium (100mL) (M/V): peptone 1.0%, yeast extract 0.5%, NaCl 1.0%.

The thallus activating step comprises: under the aseptic condition of an ultra-clean workbench, a glycerol strain is streaked on an LB solid plate added with 0.1% kanamycin, and the plate is placed in a constant-temperature incubator at 37 ℃ for culture for 12 hours.

The expanded culture step of the thallus comprises the following steps: the activated recombinant strain was picked up as a single colony and inoculated into a Erlenmeyer flask containing 100mL of LB liquid medium. In addition, 0.1% kanamycin was added to the medium at 37 ℃ for 16 hours at 180 rpm.

Transferring to TB medium and specific medium, and culturing in constant temperature shaking table. Specific media components include: yeast powder, glycerol, phosphate buffer solution and amino acid. Furthermore, 0.1% kanamycin was added at the same time. The culture conditions were set as follows: the temperature was 35 ℃ and the rotational speed was 180 rpm.

Bacteria solution OD₆₀₀When the concentration is 0.4-0.5%, 0.01% IPTG is added and the culture is continued for 30 hours.

According to the characteristics of the Escherichia coli, the bacteria collection condition is 5500rpm, and centrifugation is carried out for 10 min. Collecting thallus, weighing dry weight, adding hydrochloric acid with pH value of 1.4 according to dry weight of 10-20mL/g, shaking and shaking for 12h at constant temperature of 30 ℃ and 140rpm of a shaking table.

According to the acidolysis purification method, water-soluble and insoluble phycocyanin is purified, and the yield is analyzed. The calculation method is the phycocyanin mass/thallus mass.

Has the advantages that:

a method for increasing the yield of water-soluble phycocyanin by exogenous synthesis can be used for the industrial mass production of water-soluble phycocyanin.

From the above, the invention provides a method for improving the exogenous synthesis yield of water-soluble phycocyanin and application thereof. The method uses TB culture medium as a reference, synthesizes phycocyanin by phycocyanin synthetase according to an escherichia coli induced expression vector system, selects specific culture medium components and culture temperature, greatly improves the synthesis yield of water-soluble phycocyanin produced by the method, has the characteristic of 2.2 times higher than the yield of the water-soluble phycocyanin produced by the reference culture, and provides more efficient culture conditions for the production of the water-soluble phycocyanin.

The invention discloses a method for improving exogenous synthetic yield of water-soluble phycocyanin, which comprises the step of taking escherichia coli as a carrier and adopting CphA_BPExogenous expression of phycocyanin, and selection of specific culture medium and culture temperature capable of raising the yield of water soluble phycocyanin. The specific culture medium only contains yeast powder with a fixed proportion as a carbon source and a nitrogen source required by thallus production, peptone components containing a large amount of complex amino acids and polypeptide molecules are removed to avoid interference of the peptone components on the synthesis efficiency of the water-soluble phycocyanin, and the added aspartic acid and lysine components provide exogenous substrates for the synthesis of the phycocyanin, so that the yield of the water-soluble phycocyanin is improved. According to the exogenous synthesis method of the water-soluble phycocyanin, the culture temperature is 35 ℃, and compared with culture conditions of 25 ℃ and 40 ℃, the yield of the water-soluble phycocyanin is improved by 20.6% and 44.5% respectively. At present, CphA is adopted by taking escherichia coli as a vector₆₈₀₃The phycocyanin of the gene is exogenously expressed, the yield of the water-soluble phycocyanin is mostly 30-50mg/g of the dry weight of the bacteria, and the insoluble phycocyanin accounts for 100-200mg/g of the dry weight of the bacteria. The invention can reach the water-soluble phycocyanin yield accounting for 160mg/g of the dry weight of the thallus without influencing the yield of insoluble phycocyanin, which is far more than the yield of the synthesized water-soluble phycocyanin reported in the prior art, which is a great breakthrough in the field and has obvious technical progressThe water-soluble phycocyanin is produced, and the economic benefit is good.

Drawings

The invention will be further explained with reference to the drawings.

FIG. 1 is an electrophoresis chart of the contents of the bacterial suspension in example 1.

FIG. 2 is a purity electrophoretogram of phycocyanin in examples 1, 2, 3, 4, 5, 6 and 7.

FIG. 3 is a diagram of the liquid chromatography detection of phycocyanin in example 1.

FIG. 4 is a diagram of the liquid chromatography detection of phycocyanin in example 2.

FIG. 5 is a diagram of the liquid chromatography detection of phycocyanin in example 5.

FIG. 6 is an electrophoresis chart of the contents of the bacterial suspension in example 7.

FIG. 7 is an electrophoretogram showing the purity of phycocyanin in example 7.

Detailed Description