Application of coleus microcoleus in production of glycosylglycerol

Abstract

The invention relates to a method for producing glycosylglycerol. The method comprises the following steps: S1, culturing trichocephalus microcoleus to a logarithmic phase; and S2, adding glycerol and glucose into the culture environment of the coleus microcoleus to induce the coleus microcoleus to accumulate the glycosylglycerol. According to the invention, by optimizing the culture of the coleus microcoleus BL0902, the production of the glycosylglycerol by using the strain is realized, and on the basis, the strain is subjected to metabolic engineering transformation, so that the yield of the glycosylglycerol is greatly improved by the obtained engineering strain.

Description

Technical Field

[0001] The present invention belongs to the field of microalgae metabolic engineering, and specifically relates to the application of Leptolyngbya in the production of glucosylglycerol. Background Art

[0002] Glucosylglycerol (GG) is a class of glycoside compounds formed by the linkage of glycerol molecules and glucose molecules through glycosidic bonds. Cyanobacteria produce GG under salt stress conditions as a compatible solute to maintain the osmotic pressure balance inside and outside the cells. GG can also be synthesized during the fermentation processes of some higher plants and certain microorganisms. GG has various physical and chemical properties and biological activities, such as low sweetness, low hygroscopicity, high water retention capacity, excellent biocompatibility, etc., and shows great application potential in the fields of food, cosmetics, health products, pharmaceuticals, etc.

[0003] The production methods of glucosylglycerol include chemical methods, enzymatic methods, and microbial fermentation methods, etc. Chemical methods can catalyze the synthesis of glucosylglycerol from sugars such as glucose and trehalose and polyols. However, the conversion efficiency of chemical synthesis is relatively low, and the products are mostly mixtures, and the subsequent separation and purification steps are very complex. Compared with chemical methods, the enzymatic production of GG has higher efficiency. Among them, the transglycosylation activity of α-glucosidase can be used to synthesize a GG mixture from maltose and glycerol, and sucrose phosphorylase can be used to synthesize 2-O-α-GG from sucrose and glycerol. Compared with chemical and enzymatic methods, using cyanobacterial cell factories to produce GG has a series of advantages, such as producing GG products with specific configurations, avoiding the use of metal catalysts, and releasing intracellular GG into the external environment through osmotic treatment.

[0004] Arthrospira (also known as Spirulina), as a cyanobacterium that can be mass-cultured, is considered an ideal microalgae for GG production due to its advantages such as fast growth rate, strong tolerance, and easy harvesting. Through techniques such as large-scale cultivation of Arthrospira, salt stress-induced accumulation of GG, and non-destructive extraction, the industrial production of GG has been achieved. However, Arthrospira is difficult to be genetically engineered, and it is very difficult to significantly increase the yield of GG through metabolic engineering means.

[0005] The growth rate of Leptolyngbya BL0902 under outdoor conditions can be comparable to that of Arthrospira. It can tolerate 0.5 M NaCl, 32 mM urea, and high light, and grows strongly at 22°C to 40°C. It shows good stability in large outdoor pond cultivation, and a stable genetic operation system has been established. Therefore, if it can be used to produce GG, it will have more advantages. However, there is currently no report in this field on using this strain to produce GG. Summary of the Invention

[0006] To solve the above problems, the present invention provides the use of Leptolyngbya tenera in the production of glucosylglycerol.

[0007] The present invention also provides a method for producing glucosylglycerol, which includes the step of culturing Leptolyngbya tenera to accumulate glucosylglycerol.

[0008] In a specific embodiment, the method includes the following steps:

[0009] S1: Culturing Leptolyngbya tenera to the logarithmic phase;

[0010] S2: Inducing the Leptolyngbya tenera to accumulate glucosylglycerol.

[0011] In a specific embodiment, in S2, the Leptolyngbya tenera is induced to accumulate glucosylglycerol by adding glycerol and glucose to the culture environment of the Leptolyngbya tenera.

[0012] In a specific embodiment, the working concentration of glycerol is 5 mM, and the working concentration of glucose is 5 mM.

[0013] In a specific embodiment, the Leptolyngbya tenera is Leptolyngbya tenera BL0902 or its mutant strain or engineered strain.

[0014] In a specific embodiment, the Leptolyngbya tenera is Leptolyngbya tenera BL0902 overexpressing a glucose transporter.

[0015] In a specific embodiment, the amino acid sequence of the glucose transporter is as shown in SEQ ID NO: 1.

[0016] In a specific embodiment, the nucleic acid coding sequence of the glucose transporter is as shown in SEQ ID NO: 2.

[0017] The present invention optimizes the culture of Leptolyngbya tenera BL0902, realizes the production of glucosylglycerol using this strain, and on this basis, performs metabolic engineering transformation on this strain, and the resulting engineered bacteria greatly improve the yield of glucosylglycerol. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is the growth curve of Leptolyngbya tenera BL0902 under different culture conditions.

[0019] Figure 2 It is the time curve of the GG accumulation amount of Leptolyngbya tenera BL0902 under different culture conditions.

[0020] Figure 3 It is the growth curve of Leptolyngbya tenera BL0902 overexpressing gtr under different culture conditions.

[0021] Figure 4 Time curve of GG accumulation in Leptolyngbya sp. BL0902 overexpressing gtr under different culture conditions. Detailed implementation manners

[0022] The principles and features of the present invention will be described below in conjunction with the accompanying drawings. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0023] 1. Cultivation of Leptolyngbya sp. BL0902 and detection of GG

[0024] Inoculate algal cells into 200 ml of Zarrouk medium in a 400 ml Erlenmeyer flask to make the initial OD 730 be 0.05. Place it in a shaker at 30 °C, 30 μmol photons m -2 s -1 and 135 rpm for cultivation. The OD 730 of the seed culture reaches 0.8.

[0025] Transfer the seed culture to a 100 ml Erlenmeyer flask and add 5 mM glycerol or 5 mM glucose, and place it in an incubator at 30 °C, 30 μmol photons m -2 s -1 for static cultivation. Take 1 ml of samples every 12 h to monitor cell growth and GG production.

[0026] Take 1 ml of the liquid culture, centrifuge at 13300 rpm for 5 min. Filter the supernatant, concentrate it by vacuum centrifugation for detecting the extracellular GG content. Resuspend the cell pellet in an ethanol-aqueous solution containing 80% and place it in a metal bath at 65 °C for 4 h, then centrifuge at 13000 rpm for 10 min. Take 200 μL of the supernatant and concentrate it by vacuum centrifugation for detecting intracellular GG.

[0027] 2. Optimize the culture conditions for Leptolyngbya sp. BL0902 to produce glucosylglycerol

[0028] Compare the growth of Leptolyngbya sp. BL0902 and the accumulation of glucosylglycerol under the conditions of adding exogenous glycerol, exogenous glucose, and adding both glycerol and glucose in Zarrouk medium.

[0029] The growth is as Figure 1 shown. Compared with the normal conditions, the growth of the algal strain has no significant change after adding 5 mM glycerol exogenously, while the growth of the algal strain increases by 44.8% and 42.1% respectively when only 5 mM glucose is added and when both 5 mM glycerol and 5 mM glucose are added.

[0030] The situation of GG accumulation is as Figure 2As shown, under the condition of adding 5 mM glycerol and 5 mM glucose externally, the accumulation amount of glucosylglycerol in the algal strain was the highest at 36 h, about 19.0 mg / L. When only 5 mM glucose or only 5 mM glycerol was added externally, the highest accumulation amount of glucosylglycerol within 48 h was about 16.1 mg / L and 14.6 mg / L respectively. Without adding glycerol and glucose, the highest accumulation amount of glucosylglycerol in the algal strain within 48 h was about 3.8 mg / L.

[0031] Thus, it can be seen that externally adding glycerol and glucose in ZarrouK medium can significantly increase the GG accumulation amount of the algal strain.

[0032] 3. Effect of overexpressing glucose transporter gene gtr on Leptolyngbya boryana BL0902

[0033] Clone its glucose transporter gene gtr from Synechocystis sp. PCC 6803 (the amino acid sequence is shown in SEQ ID NO:1, and the nucleic acid sequence is shown in SEQ ID NO:2), and overexpress it in Leptolyngbya boryana BL0902. Detect the growth situation and GG accumulation amount of the engineered bacteria.

[0034] The growth situation is as Figure 3 shown. The effects of externally adding glycerol or glucose on the gtr overexpressing mutant strain are the same as those of the control strain, that is, after externally adding 5 mM glycerol, the growth of the algal strain has no significant change, while when only 5 mM glucose is added and when 5 mM glycerol and 5 mM glucose are added simultaneously, the growth of the algal strain is significantly accelerated.

[0035] The situation of GG accumulation amount is as Figure 4 shown. Under the condition of externally adding 5 mM glycerol and 5 mM glucose, the accumulation amount of glucosylglycerol in the gtr overexpressing strain is the highest, and the yield at 36 h is about 29.1 mg / L. Compared with the control strain without the gtr gene transferred, the accumulation amount of glucosylglycerol in this gtr overexpressing mutant strain has increased by 53.2%.

[0036] Thus, it can be seen that overexpressing the glucose transporter gene gtr of Synechocystis sp. PCC 6803 can improve the ability of Leptolyngbya boryana BL0902 to absorb exogenous glucose to synthesize glucosylglycerol.

[0037] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. Application of Leptolyngbya sp. in the production of glycerol glucoside.

2. A method for producing glycerol glucoside, characterized in that: The method comprises the steps of culturing the fine-scaly filamentous algae and allowing it to accumulate glycerol glucoside.

3. The method according to claim 1, characterized in that The following steps are involved: S1: Cultivate the tenuissima to the logarithmic phase; S2: Inducing the tenuiscaphos algae to accumulate glycerol glucoside.

4. The method according to claim 3, characterized in that In S2, glycerol and glucose are added to the culture environment of the tricholoma to induce the tricholoma to accumulate glycerol glucoside.

5. The method according to claim 4, characterized in that The working concentration of glycerol was 5 mM and the working concentration of glucose was 5 mM.

6. The method according to claim 3, characterized in that: The tricholoma is tricholoma BL0902 or a mutant or engineered strain thereof.

7. The method according to claim 6, characterized in that The tricholoma is tricholoma BL0902 which overexpresses glucose transporter.

8. The method according to claim 7, characterized in that The amino acid sequence of the glucose transporter is shown in SEQ ID NO:

1.

9. The method according to claim 8, characterized in that The nucleic acid coding sequence of the glucose transporter is shown in SEQ ID NO:2.

Images