CN109971664B - Bacterial strain for high yield of astaxanthin and application thereof - Google Patents

Bacterial strain for high yield of astaxanthin and application thereof Download PDF

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CN109971664B
CN109971664B CN201910329007.0A CN201910329007A CN109971664B CN 109971664 B CN109971664 B CN 109971664B CN 201910329007 A CN201910329007 A CN 201910329007A CN 109971664 B CN109971664 B CN 109971664B
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元英进
贾斌
靳瑾
齐丹丹
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Tianjin University
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Abstract

The invention relates to the field of bioengineering, in particular to a strain for high-yield astaxanthin and application thereof. Compared with the existing single mutagenesis technology, the method for simultaneously carrying out two mutagenesis on the astaxanthin-producing strains of the saccharomyces cerevisiae generates more kinds of mutations, and the yeast strains with obviously improved astaxanthin yield can be screened from a large amount of libraries generated by mutation.

Description

Bacterial strain for high yield of astaxanthin and application thereof
Technical Field
The invention relates to the field of bioengineering, in particular to a strain and application thereof.
Background
Astaxanthin is a natural carotenoid, has the functions of resisting oxidation, resisting tumors, enhancing immunity, preventing cardiovascular diseases and the like, and has great market value in the industries of food, medicine and cosmetics (as shown in figure 1). Astaxanthin protects the membrane structure by quenching singlet state, scavenging oxygen free radicals to prevent chain reaction, and inhibiting lipid peroxidation, thereby preventing oxidative damage. Unlike other carotenoids, astaxanthin binds both the inner and outer membranes of cells, and it scavenges both the intracellular and outer membrane free radicals.
In 2009 Ukibe et al first synthesized astaxanthin in Saccharomyces cerevisiae using bacterially derived crtZ/W at a yield of 29. mu.g/g. Aiming at the problems of more intermediate metabolites and low astaxanthin yield, researchers begin to screen the sources of CrtZ and CrtW with stronger substrate specificity, thereby achieving the purpose of reducing the intermediate metabolites. The Wang combined screening of 8 CrtW sources and 9 CrtZ sources shows that the combined astaxanthin yield of the CrtW from brevundimonas and the CrtZ from alcaligenes is the highest, but a large amount of intermediate metabolites are still accumulated in the product. The introduction of the exogenous metabolic pathway inevitably interacts with the endogenous metabolic pathway of the cell, influences the whole metabolic level of the cell, and controls the cell globally, which is one of the keys of high astaxanthin yield.
Therefore, the strain for high yield of astaxanthin has important practical significance.
Disclosure of Invention
In view of the above, the present invention provides a strain and its use. The invention utilizes ARTP mutagenesis to change the overall metabolic level of cells, thereby improving the yield of target metabolites.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a bacterial strain, the preservation number of which is CGMCC No. 17267.
On the basis of the research, the invention also provides the application of the strain in improving the yield of the metabolite of the strain; the metabolites include primary metabolites and secondary metabolites.
In some embodiments of the invention, the metabolite is astaxanthin.
In some embodiments of the invention, the strain is a yeast.
The invention also provides a construction method of the strain, constructs the strain integrated with geranylgeranyl pyrophosphate (GGPP) synthase gene crtE, phytoene synthetase gene crtYB, phytoene dehydrogenase gene crtI, beta-carotene hydroxylase gene crtZ and beta-carotene ketolase gene crtW, and carries out plasma mutagenesis and SCRAMBLE mutagenesis.
In some embodiments of the invention, the strain is yeast, the genes crtE, crtbb and crtI are Leu tagged and integrated into the synthetic type V chromosome, and the genes crtZ and crtW are His tagged and integrated into the delta site.
On the basis of the research, the invention also provides a production method of astaxanthin, which comprises the steps of inoculating the strain with the preservation number of CGMCC No.17267 into a culture medium, fermenting and culturing, and collecting fermentation liquor.
In some embodiments of the invention, the medium comprises a seed medium and a fermentation medium; the seed culture medium comprises 40/L glucose, 6.7g/L yeast nitrogen source and 2g/L amino acid deletion mixed powder; the fermentation medium comprises 40/L glucose, 20g/L peptone and 10g/L yeast extract powder.
In some embodiments of the invention, the fermentation culture is in particular: selecting the strain, inoculating the strain in a seed culture medium, culturing at 30 ℃ and 250rpm for 14-16 h, and taking the initial thallus concentration OD600Transferring the strain to seed culture medium at 0.2, culturing at 30 deg.C and 250rpm to middle logarithmic growth phase, and determining initial strain concentration OD600The strain was inoculated in a fermentation medium at 0.1 ℃ and cultured at 250rpm at 30 ℃.
In the long-term evolution process of natural organisms, small-scale DNA mutation and large-scale DNA structural variation have important significance on the evolution and the function change of biological phenotypes, the large-scale DNA structural variation can generate the copy of genes and even the copy of chromosomes to strengthen a certain local metabolic pathway or biochemical function, the small-scale DNA mutation can generate an effect in the genes to change the molecular function of proteins and even possibly change the action substrates of enzymes, and the small-scale DNA mutation acting on non-coding regions can influence the expression level of the genes. The project opens up a new idea, combines the traditional physical chemical mutagenesis and the artificial genome rearrangement for the first time, designs and establishes a new strategy of the yeast genome DNA evolution at the multi-scale level, realizes the global disturbance and regulation of the cell metabolism and strengthens the synthetic capacity of the astaxanthin. Compared with the existing single mutagenesis technology, the method for simultaneously carrying out two mutagenesis on the astaxanthin-producing strains of the saccharomyces cerevisiae generates more kinds of mutations, and the yeast strains with obviously improved astaxanthin yield can be screened from a large amount of libraries generated by mutation.
Biological preservation Instructions
Biological material: SyBE _ Sc 3071009; and (3) classification and naming: saccharomyces cerevisiae (Saccharomyces cerevisiae); the microbial culture is preserved in China general microbiological culture Collection center (CGMCC) at 27.02.2019, and the preservation center addresses are as follows: the institute of microbiology, national academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, Beijing; the preservation number is CGMCC No. 17267.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 shows a scheme for the synthesis of astaxanthin using recombinant Saccharomyces cerevisiae;
FIG. 2 shows a gene structure diagram of the carotene synthesis pathway in the method;
FIG. 3 shows a structural diagram of astaxanthin synthesis pathway genes in the present method;
FIG. 4 shows a flow chart of iterative evolutionary genome rearrangement and plasma mutagenesis;
FIG. 5 is a graph showing a comparison of astaxanthin shake flask production by a single mutagenized strain, a single rearranged strain and a mutagenized combined rearranged strain.
Detailed Description
The invention discloses a bacterial strain and application thereof, and can be realized by appropriately improving process parameters by referring to the content in the text by a person skilled in the art. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The invention aims to overcome the defects in the prior art and provide an evolution method of a recombinant saccharomyces cerevisiae strain for producing astaxanthin.
A second object of the invention is to provide a mutagenized Saccharomyces cerevisiae strain with high astaxanthin production.
The technical scheme of the invention is summarized as follows:
an evolution method of a saccharomyces cerevisiae strain producing astaxanthin, comprising the following steps: (1) constructing a saccharomyces cerevisiae strain with synthetic chromosomes for producing astaxanthin, and providing high-quality host cells for producing astaxanthin; (2) the plasma mutagenesis is combined with the SCRaMbLE and simultaneously the saccharomyces cerevisiae for producing the astaxanthin is subjected to continuous mutagenesis for 30 days to obtain a high-yield mutagenesis strain AS30(CGMCC NO: 17267).
The invention has the advantages that:
compared with the existing single mutagenesis technology, the method for simultaneously carrying out two mutagenesis on the astaxanthin-producing strains of the saccharomyces cerevisiae generates more kinds of mutations, and the yeast strains with obviously improved astaxanthin yield can be screened from a large amount of libraries generated by mutation.
The strain provided by the invention and the raw materials, starting strains and reagents used in the application of the strain can be purchased from the market.
The invention is further illustrated by the following examples:
example 1 construction of Saccharomyces cerevisiae strains producing astaxanthin with synthetic chromosomes
The specific process of constructing the astaxanthin-producing strain SyBE _ SC307118 by using the saccharomyces cerevisiae strain containing synthetic chromosomes V and X as an initial strain is as follows: the foreign genes include three genes for synthesizing lycopene and two genes for synthesizing astaxanthin: geranylgeranyl pyrophosphate (GGPP) synthase gene crtE, phytoene synthase gene crtbb and phytoene dehydrogenase gene crtI, and β -carotene hydroxylase gene crtZ and β -carotene ketolase gene crtW, the genes crtE, crtbb and crtI are integrated into synthetic type V chromosome with Leu as a tag (fig. 2), and the genes crtZ and crtW are integrated into delta site with His as a tag (fig. 3). The SCRaMbLE is started, plasmid Gal-Cre-URA is used, the plasmid is transformed into a synthetic type V chromosome saccharomyces cerevisiae body and a synthetic type X chromosome saccharomyces cerevisiae body through a lithium acetate method, after the transformation, an SC-URA solid plate (6.7 g/L of synthetic yeast nitrogen source YNB, 20g/L of glucose, 2g/L of mixed amino acid powder with single uracil deletion, 2% agar powder) is adopted for screening, and after the obtained transformant is subjected to pure culture, the correct strain is named as SyBE _ SC307118 (Control).
Example 2 plasma-bound SCRAMBLE Simultaneous mutagenesis (FIG. 4)
Experimental group (S + a): the strain SyBE _ SC307118 was cultured overnight in SC-URA liquid medium, fresh SC-URA-Gal medium was inoculated with OD ═ 1 every other day, 1ul/5ml of estrogen was added, and SCRaMbLE was turned on for 6 h. Taking 1mL of the bacterial solution after the SCRAMBLE, placing the bacterial solution in a 1.5mL centrifuge tube, centrifuging at 4500rpm for 1min, and using 1mL of ddH2O washing twice, taking 10ul to the metal sheet, and processing for 20 s. Putting the mutagenized bacteria liquid into a container with 1mL saline in 1.5mL EP tube; and (3) oscillating the EP tube on a vortex oscillator for 1min to completely dissolve the bacterial liquid attached to the metal sheet in the physiological saline, centrifuging at 4500rpm for 1min, transferring the centrifuged bacterial liquid to a fresh SC-URA culture medium, incubating overnight for resuscitation for 16-18h, wherein the bacterial liquid is the first day bacterial mixing bank, repeating every other day, and performing 30 days in total. Bacterial suspensions from day 10, day 20, and day 30 were diluted 10-fold and 100-fold, 100. mu.L was spread on Sc-Ura plates and 3 replicates were prepared. Cultured at 30 ℃ for 3 days, counted and observed for colony morphology and color. Yeast (AS30) SyBE _ Sc3071009, which had been obtained by iterative evolution of 30 th round of genome rearrangement in combination with plasma mutagenesis, was obtained.
Control group (a): culturing the strain SyBE _ SC307118 overnight, inoculating fresh SC-URA culture medium with OD of 0.1 every other day for 6h, taking 1mL of bacterial liquid cultured to logarithmic phase, placing in a 1.5mL centrifuge tube, centrifuging at 4500rpm for 1min, washing twice with 1mL physiological saline, and diluting with appropriate amount of distilled water to obtain OD600Bacterial suspension of 1. 10ul of the solution was put on a metal plate and processed for 20 seconds. Putting the bacterium liquid after the mutagenesis treatment into a 1.5mL EP tube filled with 1mL of physiological saline; oscillating the EP tube on a vortex oscillator for 1min to ensure that the bacterial liquid attached to the metal sheet is completely dissolved in physiological saline to form new bacterial suspension; centrifuging at 4500rpm for 1min, transferring the centrifuged bacteria solution into fresh SC-URA culture medium, incubating overnight for resuscitation for 16-18h, wherein the bacteria solution is the first day mixed bacteria library, repeating every other day for 30 days. Bacterial suspensions from day 10, day 20, and day 30 were diluted 10-fold and 100-fold, 100. mu.L was spread on Sc-Ura plates and 3 replicates were prepared. Cultured at 30 ℃ for 3 days, counted and observed for colony morphology and color. The ARTP parameter for this experiment was set as: output power 120W, processing distance 2mm, gas flow rate 10L/min. The time for ARTP plasma treatment of the sample was 20 s.
Control group (S): the strain SyBE _ SC307118 was cultured in SC-URA liquid medium overnight, fresh medium was inoculated with OD ═ 1 every other day, 1ul/5ml of estrogen was added, and SCRaMbLE was turned on for 6 h. Taking 1mL of the bacterial solution after the SCRAMBLE, placing the bacterial solution in a 1.5mL centrifuge tube, centrifuging at 4500rpm for 1min, and using 1mL of ddH2Washing twice with O, centrifuging at 4500rpm for 1min, transferring the centrifuged bacteria solution into fresh SC-URA culture medium, incubating overnight for resuscitation for 16-18h, wherein the bacteria solution is the firstThe bacterial bank is mixed in one day, and repeated every other day for 30 days. Bacterial suspensions from day 10, day 20, and day 30 were diluted 10-fold and 100-fold, 100. mu.L was spread on Sc-Ura plates and 3 replicates were prepared. Cultured at 30 ℃ for 3 days, counted and observed for colony morphology and color.
Example 3 comparison of astaxanthin shake flask yields of strains
Test materials: the strain test method comprises the following steps:
seed culture medium: 40/L glucose, 6.7g/L yeast nitrogen source and 2g/L amino acid deletion mixed powder;
fermentation medium: 40/L glucose, 20g/L peptone and 10g/L yeast extract powder.
The strains prepared in the experimental group (S + A), the control group (A) and the control group (S) prepared in example 2 were inoculated into 5mL of seed culture medium, cultured at 30 ℃ and 250rpm for 14-16 h, and then the initial cell concentration OD was obtained600Transferred to fresh 5mL seed medium at 0.2, cultured at 30 ℃ and 250rpm to the middle of logarithmic growth, and the initial cell concentration OD600The cells were inoculated into 50mL of each fermentation medium at 0.1, cultured at 30 ℃ and 250rpm, and the cell density (OD) was monitored during the fermentation600) And astaxanthin production (FIG. 5).
An astaxanthin quantification method comprises the following steps: two equal portions of the fermentation broth were taken, centrifuged at 4000g for 2min to collect the thallus, and washed twice with water. Placing one part of the thalli at 80 ℃ to dry to constant weight, and weighing to calculate the dry weight of the cells; the other part of the thallus is used for product extraction, and the specific method comprises the following steps: resuspending the cells with 3N HCl, boiling in a boiling water bath for 5min, and immediately ice-cooling for 5 min; centrifuging the crushed cells at 12000rpm at 4 ℃ for 4min, discarding the supernatant, washing with water for 2 times, adding acetone, and vortexing for 5 min; and finally, centrifugally collecting an acetone phase, filtering the acetone phase by using a filter membrane with the diameter of 2 mu m, and detecting the astaxanthin by using an ultraviolet liquid phase, wherein the detection wavelength of the astaxanthin is 470 nm.
AS shown in fig. 5, yeast (AS30) SyBE _ Sc3071009, which had been iteratively evolved from round 30 by genomic rearrangement in combination with plasma mutagenesis, produced significantly higher amounts of synthetic astaxanthin than either the single mutagenized strain (a) or the rearrangement (S).
In the long-term evolution process of natural organisms, small-scale DNA mutation and large-scale DNA structural variation have important significance on the evolution and the function change of biological phenotypes, the large-scale DNA structural variation can generate the copy of genes and even the copy of chromosomes to strengthen a certain local metabolic pathway or biochemical function, the small-scale DNA mutation can generate an effect in the genes to change the molecular function of proteins and even possibly change the action substrates of enzymes, and the small-scale DNA mutation acting on non-coding regions can influence the expression level of the genes. The project opens up a new idea, combines the traditional physical chemical mutagenesis and the artificial genome rearrangement for the first time, designs and establishes a new strategy of the yeast genome DNA evolution at the multi-scale level, realizes the global disturbance and regulation of the cell metabolism and strengthens the synthetic capacity of the astaxanthin.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims (4)

1. The strain is characterized in that the preservation number is CGMCC No. 17267.
2. Use of a strain according to claim 1 for increasing the production of a metabolite thereof;
the metabolite is astaxanthin.
3. The production method of astaxanthin is characterized in that a strain with the preservation number of CGMCC No.17267 is inoculated into a culture medium, fermented and cultured, and fermentation liquor is collected.
4. The production method according to claim 3, wherein the fermentation culture is in particular: selecting the strain, inoculating the strain in a seed culture medium, culturing at 30 ℃ and 250rpm for 14-16 h, and taking the initial thallus concentration OD600=0.2 transferring to seed culture medium, culturing to middle logarithmic growth phase under conditions of 30 deg.C and 250rpm, and using initial thallus concentration OD600=0.1 inoculating in fermentation medium, culturing at 30 deg.C and 250rpm。
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