CN116814452A

CN116814452A - Saccharomyces cerevisiae genetically engineered bacterium and application thereof in production of lycopene

Info

Publication number: CN116814452A
Application number: CN202211126636.1A
Authority: CN
Inventors: 王淑洁; 丘璨瑜; 颜梦燎; 董宇亮; 赵山岑
Original assignee: BGI Shenzhen Co Ltd
Current assignee: BGI Shenzhen Co Ltd
Priority date: 2022-09-16
Filing date: 2022-09-16
Publication date: 2023-09-29

Abstract

The application discloses a saccharomyces cerevisiae genetically engineered bacterium and application thereof in lycopene production. The Saccharomyces cerevisiae genetically engineered bacterium is Saccharomyces cerevisiae L-SCR (1), and the preservation number of the Saccharomyces cerevisiae genetically engineered bacterium in China center for type culture collection is CCTCC No: m20211141. Experiments prove that the Saccharomyces cerevisiae L-SCR (1) has extremely low fermentation oxygen demand and low energy consumption, and the fermentation medium and the feed supplement medium have simple components and convenient preparation, can produce lycopene without adding an inducer and an inhibitor, and provide a simple and energy-saving fermentation culture mode for large-scale industrialized fermentation of lycopene. Saccharomyces cerevisiae62L-SCR (1) has important application value in the production of lycopene.

Description

Saccharomyces cerevisiae genetically engineered bacterium and application thereof in production of lycopene

Technical Field

The application belongs to the technical field of biology, and particularly relates to a saccharomyces cerevisiae genetically engineered bacterium and application thereof in lycopene production.

Background

Lycopene is a carotenoid, and is mainly found in ripe fruits of fruits such as tomatoes, watermelons, guavas and the like. The chemical structure of the compound contains 11 unsaturated conjugated double bonds and 2 unconjugated double bonds, is one of the compounds with the strongest antioxidant capacity in natural existence, and has various physiological activities of scavenging free radicals, preventing tumor cell proliferation and cancer, protecting cardiovascular system, enhancing human immunity and the like. In addition, the lycopene can be used as a natural harmless edible pigment, and can be widely applied to various industries such as food and medicine, cosmetics, medical cosmetology and the like, and has wide market prospect. At present, lycopene is mainly obtained by three methods of plant extraction, chemical synthesis and microbial synthesis. The plant extraction and chemical synthesis methods consume a large amount of organic reagents, have high cost and are easy to cause environmental pollution. The microbial synthesis method has the advantages of environmental friendliness, low cost, high yield and the like, and becomes the research direction with the market prospect.

At present, lycopene has been successfully and efficiently produced in a plurality of microorganisms such as escherichia coli, blakeslea trispora, saccharomyces cerevisiae and the like. Saccharomyces cerevisiae is a well-known safe and non-toxic (GRAS) eukaryotic mode microorganism that has been successfully engineered to produce natural products such as artemisinin, resveratrol, paclitaxel, and the like. Compared with other microorganisms, the saccharomyces cerevisiae thallus has high vitamin and protein content, does not contain toxicity, and has food safety; the modified starch has low pH tolerance and higher tolerance to different environments; as eukaryotes have similar transcription and translation mechanisms as plants, the species sources of heterologous expression enzymes are widened, and the strain is an ideal strain for biosynthesis of lycopene. However, in the prior report, the yield of the lycopene synthesized by the saccharomyces cerevisiae is still far lower than that of the escherichia coli, so that the high yield of the lycopene still faces a great challenge by optimizing a construction strategy and a metabolic regulation method of the lycopene synthesized by the saccharomyces cerevisiae.

Blakeslea trispora is the only strain for industrially producing lycopene at present. The Blakeslea trispora can synthesize beta-carotene by itself, lycopene is formed after passing through a mevalonate pathway, and then the lycopene is catalyzed into beta-carotene by lycopene cyclase. Therefore, in order to obtain lycopene, inhibitors are needed to inhibit the activity of lycopene cyclase in the fermentation process, and the process of lycopene cyclization is controlled to achieve the aim of accumulating lycopene. In addition, the addition of lipid substances (such as vegetable oil and fatty acid) to the culture medium has been proved to be effective in promoting the accumulation of carotenoids, especially the promotion effect of fatty acids such as oleic acid, linoleic acid and linolenic acid is more remarkable. Vereschagina et al, in which Blakeslea trispora is cultivated by fermentation in a medium rich in linolenic acid, have significantly increased lycopene production. In recent years, research on synthesizing lycopene by using saccharomyces cerevisiae as a host has been greatly developed, lian and the like modularly regulate and control the flux of both an MVA path and a heterologous synthetic lycopene path by regulating gene integration copy number, so that the lycopene yield is improved to 11.2mg/g (DCW), and Chen and the like use D- (+) -galactose to induce saccharomyces cerevisiae to ferment lycopene, and the highest yield reaches 55.56mg/g (DCW).

The Blakeslea trispora fermentation process requires addition of lycopene cyclase inhibitors, otherwise lycopene cannot be accumulated in large quantities, and the yield is drastically reduced from shake flask fermentation to fermentation tank amplification fermentation, and cannot reach the yield in shake flask fermentation, so that high yield of a large-scale fermentation tank cannot be realized. Lycopene causes stress on the growth of Saccharomyces cerevisiae to limit the final yield, and the lycopene Saccharomyces cerevisiae strain reported at present is added with inducers such as galactose when the lycopene grows to a plateau phase, and a synthesis path is started to synthesize lycopene. However, galactose price is about ten times the price of glucose, increasing the fermentation step and cost.

Disclosure of Invention

The object of the present application is to produce lycopene.

The application firstly protects Saccharomyces cerevisiae L-SCR (1), the strain is preserved in China center for type culture collection (CCTCC for short, address: university of Wuhan, china) in 2021, and the preservation number is CCTCC No: m20211141. Saccharomyces cerevisiae62L-SCR (1) is fully named Saccharomyces cerevisiae L-SCR (1) CCTCC No: m20211141, saccharomyces cerevisiae62L-SCR (1) or Saccharomyces cerevisiae strain 62L-SCR (1).

The application also provides a microbial inoculum containing Saccharomyces cerevisiae L-SCR (1) (as an active ingredient). The use of the microbial inoculum can be used for producing lycopene.

The preparation method of the microbial inoculum can comprise the following steps: and (3) inoculating Saccharomyces cerevisiae L-SCR (1) to a bacterial culture medium, and culturing to obtain bacterial liquid, namely the bacterial agent.

In the preparation method, the OD of the bacterial liquid _600nm Can be 6-7.

The bacterial culture medium may be a seed culture medium or a fermentation culture medium.

The preparation method of the seed culture medium comprises the following steps: dissolving 18-22g (such as 18-20g, 20-22g, 18g, 20g or 22 g) peptone, 18-22g (such as 18-20g, 20-22g, 18g, 20g or 22 g) glucose and 8-12g (such as 8-10g, 10-12g, 8g, 10g or 12 g) yeast extract in 1L distilled water, and naturally pH.

The preparation method of the fermentation medium comprises the following steps: 18-22g (such as 18-20g, 20-22g, 18g, 20g or 22 g) peptone, 18-22g (such as 18-20g, 20-22g, 18g, 20g or 22 g) glucose and 8-12g (such as 8-10g, 10-12g, 8g, 10g or 12 g) yeast extract and 50-70 μl (such as 50-60 μl, 60-70 μl, 50 μl, 60 μl or 70 μl) linoleic acid are dissolved in 1L distilled water, and the pH is natural.

In the preparation method of the microbial inoculum, the culture conditions can be as follows: culturing at 18-25deg.C (such as 18-20deg.C, 20-25deg.C, 18deg.C, 20deg.C or 25deg.C), 200-400rpm (such as 200-300rpm, 300-400rpm, 200rpm, 300rpm or 400 rpm).

In addition to the active ingredient, the microbial agent may also include a carrier. The carrier may be a solid carrier or a liquid carrier. The solid support may be a mineral material, a plant material or a polymeric compound. The mineral material may be at least one of clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica, and diatomaceous earth. The plant material may be at least one of corn flour, soy flour and starch. The polymer compound may be polyvinyl alcohol. The liquid carrier may be an organic solvent, vegetable oil, mineral oil, or water. The organic solvent may be decane and/or dodecane. In the microbial inoculum, the active ingredient may be present in the form of living cells being cultured, a fermentation broth of living cells, a filtrate of a cell culture, or a mixture of cells and filtrate. The formulation of the composition can be various formulations, such as liquid, emulsion, suspending agent, powder, granule, wettable powder or water dispersible granule.

Surfactants (such as Tween 20, tween 80, etc.), binders, stabilizers (such as antioxidants), pH regulators, etc. can also be added into the microbial inoculum according to the need.

The use of Saccharomyces cerevisiae L-SCR (1) or any of the above-mentioned microbial agents for the production of lycopene also falls within the scope of the present application.

The use of Saccharomyces cerevisiae L-SCR (1) or any of the above-mentioned microbial agents for the preparation of a product for the production of lycopene also falls within the scope of the present application.

The application also protects a product comprising Saccharomyces cerevisiae L-SCR (1); the function of the product may be to produce lycopene.

The application also provides a method for producing lycopene, which can be fermenting and culturing Saccharomyces cerevisiae L-SCR (1), and obtaining lycopene from fermentation broth.

In the above method, the fermentation temperature may be 18-25deg.C (such as 18-20deg.C, 20-25deg.C, 18 deg.C, 20 deg.C or 25deg.C).

In the above method, the steps of the fermentation culture Saccharomyces cerevisiae L-SCR (1) are as follows: inoculating Saccharomyces cerevisiae L-SCR (1) to a fermentation medium, fermenting at 18-25deg.C (such as 18-20deg.C, 20-25deg.C, 18 deg.C, 20 deg.C or 25deg.C) for 72-160h (such as 72-88h, 88-94h, 94-113h, 113-127h, 127-149h, 149-155h, 155-159h, 159-175h, 72h, 88h, 94h, 113h, 127h, 149h, 155h, 159h or 175 h); feeding with a feeding medium is started when the pH value of the fermentation liquid is reduced to 5.80-6.20 (such as 5.80-6.00, 6.00-6.20, 5.80, 6.00 or 6.20), and the feeding speed can be 5-8mL/h/L (such as 5-6mL/h/L, 6-8mL/h/L, 5mL/h/L, 6mL/h/L, 7mL/h/L or 8 mL/h/L); the pH value is maintained at 6.00+/-0.05 after the material is fed.

In the above method, the solute and concentration of the fermentation medium may be 18-22g/L (e.g., 18-20g/L, 20-22g/L, 18g/L, 20g/L or 22 g/L) peptone, 18-22g/L (e.g., 18-20g/L, 20-22g/L, 18g/L, 20g/L or 22 g/L) glucose and 8-12g/L (e.g., 8-10g/L, 10-12g/L, 8g/L, 10g/L or 12 g/L) yeast extract and 50-70. Mu.L (e.g., 50-60. Mu.L, 60-70. Mu.L, 50. Mu.L/L, 60. Mu.L or 70. Mu.L) linoleic acid, and the solvent may be water, and the pH is natural.

In the method, the solute and the concentration of the solute in the feed medium can be 480-520g/L (such as 480-500g/L, 500-520g/L, 480g/L, 500g/L or 520 g/L) glucose and 14-16g/L (such as 14-15g/L, 15-16g/L, 14g/L, 15g/L or 16 g/L) yeast extract, the solvent can be water, and the pH is natural.

Experiments prove that the Saccharomyces cerevisiae L-SCR (1) has extremely low fermentation oxygen demand and low energy consumption, and the fermentation medium and the feed supplement medium have simple components and convenient preparation, can produce lycopene without adding an inducer and an inhibitor, and provide a simple and energy-saving fermentation culture mode for large-scale industrialized fermentation of lycopene. Saccharomyces cerevisiae62L-SCR (1) has important application value in the production of lycopene.

Drawings

FIG. 1 is a schematic diagram of the construction flow of Saccharomyces cerevisiae strain 62L-SCR (1) (i.e., saccharomyces cerevisiae62L-SCR (1)).

FIG. 2 shows the assembly sequence of four enzymes and three promoters in the lycopene synthesis pathway.

FIG. 3 shows the first chromosomal rearrangement of rDNA25 to obtain integrated s.cerevisiae rDNA25-25.

FIG. 4 shows the acquisition of a highly stable Saccharomyces cerevisiae strain 62L-SCR (1) by chromosomal rearrangement of Saccharomyces cerevisiae 62L-SCR.

FIG. 5 shows the results of the colony concentration and lycopene dry weight content measurements in experiment one of example 2.

FIG. 6 shows the results of the colony concentration and lycopene dry weight content measurements in experiment two of example 2.

FIG. 7 shows the results of the colony concentration and lycopene dry weight content measurements in experiment III of example 2.

Detailed Description

The following detailed description of the application is provided in connection with the accompanying drawings that are presented to illustrate the application and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the application in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

EXAMPLE 1 preparation and preservation of Saccharomyces cerevisiae Strain 62L-SCR (1)

Through a large number of experiments, the inventor of the application uses haploid synthetic yeasts (specifically haploid yeasts (Syn 3,6,9R) containing right arm chromosomes of synthetic numbers 3,6 and 9) as chassis bacteria to perform multi-round strain optimization, so as to prepare and obtain a Saccharomyces cerevisiae strain 62L-SCR (1), and a specific construction flow is shown in figure 1.

The specific steps for preparing the saccharomyces cerevisiae strain 62L-SCR (1) are as follows:

1. four enzymes (CrtE, crtB, crtI and IdI, respectively) and promoters (CYC 1p, CCW12p and TDH3 p) in the lycopene synthesis pathway were assembled in the sequence shown in FIG. 2.

2. And (3) converting haploid synthetic saccharomycetes by a lithium acetate conversion method through matching the elements assembled in the step (1) with 250bp upstream and downstream of the selected integration site rDNA and Kmax labels, and obtaining initial integration type Saccharomyces cerevisiae rDNA25, which is called rDNA25 for short.

3. After the step 2 is completed, in order to increase the yield of lycopene synthesized by the strain, the initial integrated Saccharomyces cerevisiae rDNA25 is subjected to first chromosome rearrangement to obtain integrated Saccharomyces cerevisiae rDNA25-25, abbreviated as rDNA25-25 (see figure 3).

4. After the step 3 is completed, in order to improve the stability of lycopene pathway, the catalytic key enzyme crtI is integrated and the ypl062w gene is knocked out at the same time, so that a saccharomyces cerevisiae62L-SCR with again improved lycopene yield is obtained, and the saccharomyces cerevisiae is named as 62L-SCR.

5. After the step 4 is completed, the saccharomyces cerevisiae62L-SCR is subjected to chromosome rearrangement to obtain a saccharomyces cerevisiae strain 62L-SCR (1) with high stability (see FIG. 4).

Saccharomyces cerevisiae strain 62L-SCR (1) has been preserved in China center for type culture collection (CCTCC for short, address: university of Wuhan, china) for 09 and 06 days in 2021, with a preservation number of CCTCC No: m20211141. The Saccharomyces cerevisiae strain 62L-SCR (1) is totally named Saccharomyces cerevisiae62L-SCR (1), and is simply named Saccharomyces cerevisiae62L-SCR (1).

Example 2 use of Saccharomyces cerevisiae Strain 62L-SCR (1) in the production of lycopene

YPD plates: dissolving 20g peptone, 20g glucose and 10g yeast extract (product of OXOID Co., catalog number LP 0021) and 20g agar in a proper amount of water, then diluting to 1L with water, and naturally pH; sterilizing at 115deg.C for 15min, cooling to 55deg.C, introducing into sterile plate, and naturally cooling.

The solute of the seed culture medium and the concentration thereof are 20g/L peptone, 20g/L glucose and 10g/L yeast extract, the solvent is water, and the pH is natural.

The solute of the fermentation medium and the concentration thereof are 20g/L peptone, 20g/L glucose, 10g/L yeast extract and 60 mu L/L linoleic acid, the solvent is water, and the pH is natural.

The solute of the feed medium and the concentration thereof are 500g/L glucose and 15g/L yeast extract, the solvent is water, and the pH is natural.

In this example, the procedure for detecting the dry weight of the cells was as follows: 10mL of the fermentation broth was centrifuged at 4000rpm for 5min, and the cells were collected with ddH ₂ O is washed twice; and then placing the thalli in a 75 ℃ oven for drying to constant weight, and weighing to obtain the dry weight of the thalli. Further obtaining the dry weight content of the thalli.

In this embodiment, the steps for detecting lycopene are as follows: firstly, 200 mu L of fermentation liquor is taken, centrifuged at 4000rpm for 5min, and the bacterial cells are collected by ddH ₂ O is washed twice; extracting thallus with acetone-petroleum ether extract (prepared by mixing acetone and petroleum ether at ratio of 10:1) to obtain supernatant until thallus has no red color, adding glass sand with diameter of 2mm into thallus, adding 500 μl acetone-petroleum ether extract, and shaking by vortexSwinging for about 1min, ultrasonic processing for 60min, centrifuging at 12000rpm for 1min, and collecting supernatant (placed in a dark place) and thallus; and finally, uniformly mixing the supernatant fluid extracted each time, filtering the mixture by using a 0.22 mu m organic filter membrane, and detecting lycopene by using HPLC. Further obtaining the dry weight content and the lycopene concentration of lycopene.

1. Experiment one

1.Saccharomyces cerevisiae strain 62L-SCR (1) was streaked on YPD plates and cultured at 30℃for 4-5 days to give several red colonies.

2. Inoculating 3-4 red colonies obtained in step 1 into shake flask (250 mL) containing 50mL seed culture medium, and culturing at 20deg.C for 4-5 days to obtain OD _600nm 6-7 of seed liquid.

3. Inoculating (10% of the inoculation amount) the seed solution obtained in the step 2 to a fermentation tank (3L in specification) filled with 2L of fermentation medium, and fermenting.

The fermentation conditions were as follows: temperature: 20 ℃; ventilation volume: 0.5-1vvm; rotational speed: 200-400rpm; the pH was 6.+ -. 0.05 (controlled with 2mol/L NaOH). At normal pressure, the amount of dissolved oxygen in the tank is calculated as dO2=100%.

During the fermentation, when the pH value of the fermentation liquid is reduced to 6 (about 48h of fermentation), the feeding is started by using a feeding culture medium, and the feeding speed is about 5-8mL/h/L according to the fermentation condition.

Sampling is performed periodically during fermentation, and colony concentrations (OD is used _600nm Indicated), dry weight content of thalli, wet weight content of thalli, dry weight content of lycopene and lycopene concentration.

The results of the partial detection are shown in FIG. 5 (dry weight content of lycopene, dry weight content, OD _600nm The concentration in the lycopene tank is lycopene concentration). The results showed that the colony concentration OD during fermentation _600nm The highest dry weight content of the thalli can reach 37.6g/L, the highest dry weight content of the lycopene can reach 34.5mg/g, and the highest lycopene concentration can reach 861.6mg/L.

2. Experiment two

During the fermentation, when fermentation is carried out for 24h, feeding is started by using a feeding culture medium, and the feeding speed is about 5-8mL/h/L according to the fermentation condition.

The results of the partial detection are shown in FIG. 6 (dry weight content of lycopene, dry weight content, OD _600nm The concentration in the lycopene tank is lycopene concentration). The results showed that the colony concentration OD during fermentation _600nm The highest content of the dry weight of the thalli can reach 39.0g/L, the highest content of the dry weight of the lycopene can reach 25.8mg/g, and the highest concentration of the lycopene can reach 917.76mg/L.

Therefore, the concentration of fermented bacterial colony is increased, but the dry weight content of lycopene is reduced, and the bacterial strain grows too fast due to too early feeding, so that the bacterial strain carries out homologous recombination to lose lycopene synthesis pathway gene fragments, and more bacterial strains which do not produce lycopene are generated.

3. Experiment three

The fermentation conditions were as follows: temperature: 20 ℃; ventilation volume: 0.5-1vvm; rotational speed: 200-400rpm; the pH was 6.+ -. 0.05 (controlled with 2mol/L NaOH). The pressure was increased and the amount of dissolved oxygen in the tank was calculated as dO2=102%.

In the fermentation process, when the fermentation is carried out for 48 hours, feeding is started by using a feeding culture medium, and the feeding speed is about 5-8mL/h/L according to the fermentation condition.

The results of the partial detection are shown in FIG. 7 (dry weight content of lycopene, dry weight content, OD _600nm The concentration in the lycopene tank is lycopene concentration). The results showed that the colony concentration OD during fermentation _600nm The highest lycopene dry weight content can reach 35, the highest lycopene dry weight content can reach 37.4mg/g, and the highest lycopene concentration can reach 348.66mg/L.

It follows that the reduced concentration of fermented colonies and the increased dry lycopene content may inhibit bacterial strain growth and reproduction due to increased pressure, but are beneficial for intracellular lycopene metabolic accumulation.

In view of the above results, the Saccharomyces cerevisiae strain 62L-SCR (1) has extremely low fermentation oxygen demand (the dissolved oxygen amount in the tank is basically unchanged in the fermentation process), low energy consumption, simple components of the fermentation medium and the feed supplement medium, convenient preparation, and capability of producing lycopene in large quantity without adding an inducer and an inhibitor, thereby providing a simple and energy-saving fermentation culture mode for large-scale industrialized fermentation of lycopene. Therefore, the saccharomyces cerevisiae strain 62L-SCR (1) has important application value in the production of lycopene.

The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains.

Claims

1.Saccharomyces cerevisiae62L-SCR (1), its preservation number in China center for type culture collection is CCTCC No: m20211141.

2. A microbial inoculum comprising the Saccharomyces cerevisiae L-SCR (1) of claim 1.

3. The method for preparing the microbial inoculum of claim 2, comprising the following steps: inoculating Saccharomyces cerevisiae L-SCR (1) according to claim 1 to a bacterial culture medium, and culturing to obtain bacterial liquid, namely a bacterial agent.

4. Use of Saccharomyces cerevisiae L-SCR (1) according to claim 1 or a microbial agent according to claim 2 for the production of lycopene.

5. Use of a microbial inoculum according to claim 1 Saccharomyces cerevisiae L-SCR (1) or claim 2 in the manufacture of a product for use in the production of lycopene.

6. A product comprising the Saccharomyces cerevisiae L-SCR (1) of claim 1; the function of the product is to produce lycopene.

7. A process for producing lycopene comprising culturing Saccharomyces cerevisiae L-SCR (1) of claim 1 in fermentation to obtain lycopene from the fermentation broth.

8. The method according to claim 7, wherein: the fermentation temperature is 18-25 ℃.

9. The method according to claim 7, wherein: the steps of fermenting and culturing Saccharomyces cerevisiae L-SCR (1) according to claim 1 are as follows: inoculating Saccharomyces cerevisiae L-SCR (1) according to claim 1 to a fermentation medium, and fermenting at 18-25 ℃ for 72-160h; when the pH value of the fermentation liquid is reduced to 5.80-6.20, feeding is started by using a feeding culture medium, and the feeding speed is 5-8mL/h/L; the pH value is maintained at 6.00+/-0.05 after the material is fed.

10. The method according to claim 9, wherein:

the solute of the fermentation medium and the concentration thereof are 18-22g/L peptone, 18-22g/L glucose, 8-12g/L yeast extract and 50-70 mu L/L linoleic acid, the solvent is water, and the pH is natural;

the solute of the feed medium and the concentration thereof are 480-520g/L glucose and 14-16g/L yeast extract, the solvent is water, and the pH is natural.