An engineered algae that produces high yield and secretes isomaltulose and trehalulose and a preparation method thereof
By transferring sucrose transporter and isomerase frames into cyanobacteria, the method addresses high sugar input issues in isomaltulose and trehalulose production, achieving efficient and cost-effective synthesis and secretion of these sugars using CO2 as a carbon source.
Patent Information
- Application Number
- CN202210878243.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-07-25
- Publication Date
- 2024-12-13
- Estimated Expiration
- 2042-07-25
AI Technical Summary
Current methods for producing isomaltulose and trehalulose using heterotrophic microorganisms require high sugar raw material inputs, increasing economic and environmental costs, while autotrophic cyanobacteria offer a promising but underutilized platform for efficient and green synthesis.
Transfer a sucrose transporter and sucrose isomerase expression frames into cyanobacteria, such as Synechococcus PCC7942, to enhance the synthesis and secretion of isomaltulose and trehalulose, utilizing CO2 as a carbon source and reducing sugar dependency.
The engineered cyanobacteria achieve high yields of isomaltulose and trehalulose, with concentrations up to 570-776 mg/L and 139-326 mg/L extracellularly, significantly reducing sugar input and production costs.
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Figure CN115927135B_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of genetic engineering and bioengineering, and specifically relates to an engineered algae for producing isomaltulose and trehalulose, and a construction method and application thereof. Background Art
[0002] Isomaltulose, also known as palatinose, is a reducing disaccharide and an isomer of sucrose. It is composed of glucose and fructose bonded by α-1,6-glycosidic bonds and exists naturally in honey and sugarcane juice. It has similar physical and sensory properties to sucrose, and its sweetness is only half of that of sucrose. In addition, isomaltulose has the characteristics of low energy value and low digestion and absorption rate. It can slowly release glucose into the blood and does not stimulate the production of insulin, thereby avoiding the sudden rise and fall of glucose levels, which is beneficial for diabetic patients. At the same time, it is not easily used by mutans streptococci that form dental plaque, which can avoid the occurrence of dental caries. Trehalulose is also a reducing disaccharide, and its functional properties are basically similar to those of isomaltulose. Therefore, as healthy food additives, isomaltulose and trehalulose have broad market application prospects in the food industry.
[0003] At present, the commonly used method for producing isomaltulose and trehalulose at home and abroad is the sucrose isomerization method based on biocatalysis. With heterotrophic microorganisms such as Escherichia coli, yeast, and Bacillus subtilis as the chassis, under the action of sucrose isomerase, microorganisms can isomerize sucrose into isomaltulose and trehalulose. However, when using the above chassis for industrial production and fermentation, a large amount of sugar raw materials such as glucose and sucrose need to be added to increase the accumulation of target products, which will increase the economic and environmental costs of the sugar industry. Autotrophic microorganisms such as cyanobacteria can use CO2 as the only carbon source to achieve cell growth. Compared with heterotrophic microorganisms, it can save fermentation costs and at the same time play the role of carbon fixation, emission reduction and clean production. Therefore, it has also received more and more attention from the scientific community.
[0004] Cyanobacteria are considered to be one of the most promising microbial chassis for photosynthetic biomanufacturing due to their simple structure, rapid growth, ease of genetic manipulation, and high photosynthetic efficiency. With the rapid development of synthetic biology and metabolic engineering techniques, it has been realized that cyanobacteria can be used as photosynthetic platforms to synthesize a variety of natural or non-natural metabolites, including sugars, alcohols, acids, alkanes, alkenes, ketones, fatty acids, fatty alcohols, and terpenes. However, there are no reports on the commercial production of isomaltulose and trehalulose using cyanobacteria. Therefore, using cyanobacteria as a chassis to develop efficient and green new isomaltulose and trehalulose synthesis technologies is of great significance to improving product competitiveness and market acceptance. Summary of the invention
[0005] In previous studies, we found that after the expression frame of the sucrose transporter was transferred into Synechococcus PCC7942, the algae cells secreted a large amount of sucrose. Theoretically, sucrose isomerase can use sucrose as a substrate to convert sucrose into isomaltulose. When the sucrose transporter is expressed in the algae cells and causes sucrose to be secreted, the amount of sucrose in the cells decreases. Even if the enzymes related to isomaltulose synthesis are transferred into the algae cells, the efficiency of the cells in synthesizing isomaltulose should also decrease.
[0006] However, unexpectedly, we found that after the expression frame of sucrose transporter was transferred into the cell, and then the expression frame of sucrose isomerase was transferred, the synthesis efficiency of isomaltulose was not reduced, but increased, and even reached the extracellular isomaltulose content of hundreds of milligrams; and in addition to isomaltulose, the content and proportion of trehalulose were greatly increased. Therefore, we are expected to achieve commercial production of isomaltulose and trehalulose.
[0007] Based on the above findings, the present invention provides a method for preparing engineered algae with high yield of isomaltulose and trehalulose, comprising the steps of transferring a sucrose transporter expression frame and a sucrose isomerase expression frame into chassis cyanobacteria, wherein the sucrose isomerase is a sucrose isomerase capable of converting sucrose into isomaltulose or trehalulose, and the chassis cyanobacteria are all cyanobacteria capable of producing sucrose, and may be cyanobacteria capable of producing sucrose directly isolated from the environment, or cyanobacteria capable of producing sucrose after genetic modification.
[0008] In a specific embodiment, the chassis cyanobacteria are Synechococcus, Synechocystis, and Anabaena. For example, we found through experiments that Synechococcus PCC7942, Synechocystis PCC6803, Synechococcus UTEX2973, Anabaena PCC 7120, and Synechococcus PCC7002 can all achieve the production of isomaltulose and trehalulose through the method of the present invention.
[0009] In a specific embodiment, in the sucrose transporter expression frame, the protein sequence of the sucrose transporter is shown as SEQ ID NO:10.
[0010] We tested most of the sucrose isomerases we could obtain that had the ability to convert sucrose into isomaltulose or trehalulose, including sucrose isomerases from Serrati, Erwinina, Klebsiella, Pantoea dispers, and Protaminobacter rubrum, and found that they could significantly increase the content of isomaltulose and trehalulose.
[0011] In a specific embodiment, in the sucrose isomerase expression cassette, the sucrose isomerase is derived from Erwiniarhapontici NX-5, Raoultella terrigena or Klebsiella sp. LX3, or a variant thereof.
[0012] In a specific embodiment, in the sucrose isomerase expression frame, the protein sequence of sucrose isomerase is shown as one of SEQ ID NOs: 1, 4, and 7.
[0013] In the above gene expression frame, the promoter used to drive the expression of sucrose transporter and sucrose isomerase is not particularly limited, as long as it can strongly express the target gene in the target algae strain. For example, in Synechococcus PCC7942, the promoters that can be used include but are not limited to: constitutive strong promoters such as cpcB1 promoter, cpcB560 promoter, rbcL promoter, and inducible strong promoters such as lac promoter and trc promoter.
[0014] In a specific embodiment, the nucleic acid sequence of the sucrose isomerase is shown in one of SEQ ID NO: 2, 3, 5, 6, 8, and 9.
[0015] The present invention also provides cyanobacteria obtained by the method with high yield of isomaltulose and trehalulose.
[0016] The present invention unexpectedly enables the algal cells to have the function of high production and secretion of isomaltulose and trehalulose by transferring the sucrose transporter expression frame and the sucrose isomerase expression frame into Synechococcus. Preliminary culture shows that the engineered algae strain of the present invention contains up to 570-776 mg / L of isomaltulose and 139-326 mg / L of trehalulose in the culture through secretion, which are extremely high concentrations. After certain culture optimization, it can be used for commercial production. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] Figure 1 This is the plasmid map of pYW01.
[0018] Figure 2 This is the plasmid map of pYW02.
[0019] Figure 3 This is the plasmid map of pYW03.
[0020] Figure 4 Figure 1 is the plasmid map of the plasmid carrying the sucrose transporter expression cassette.
[0021] Figure 5 These are electrophoresis photos of the amplified products after amplification of the NS1 site (left) and NS3 (right) of the engineered algae strain.
[0022] Figure 6 The peak diagram of isomaltulose A and trehalulose B standards detected by HPLC.
[0023] Figure 7 The peak diagram of the supernatant of the engineered algal strain culture was detected by HPLC.
[0024] Figure 8 A is the growth curve of the engineered algae strains FL92, YW07, YW08, and YW09, as well as the statistical graphs of intracellular and extracellular sucrose content B, intracellular and extracellular isomaltulose content C, and intracellular and extracellular trehalulose content D. DETAILED DESCRIPTION
[0025] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.
[0026] 1. Algae strains used
[0027] The present invention aims to transform an engineered algae strain that can efficiently synthesize isomaltulose using Synechococcus PCC7942 (Syn7942) as a chassis algae for commercial production. Syn7942 is a model algae for cyanobacteria research, has been widely used, and can be obtained by the public in a variety of ways.
[0028] 2. Plasmid Construction
[0029] 1) The sucrose isomerase sequence was found from the genome of Erwinia rhapontici NX-5 (the protein sequence is shown in SEQ ID NO: 1, and the nucleic acid sequence is shown in SEQ ID NO: 2). The sequence obtained after codon optimization is shown in SEQ ID NO: 3. The codon-optimized sequence was connected to the promoter P cpcB1 Downstream, the NX-5 sucrose isomerase gene expression cassette is constructed and the gene expression cassette is linked to the resistance fragment Km r Connect and insert between the upstream and downstream homology arms of a neutral site (NS1) of Syn7942, and integrate into pUC19 to obtain plasmid pYW01 ( Figure 1 ).
[0030] 2) The sucrose isomerase sequence was found from the genome of Raoultella terrigena (the protein sequence is shown in SEQ ID NO:4, and the nucleic acid sequence is shown in SEQ ID NO:5). The sequence obtained after codon optimization is shown in SEQ ID NO:6. The codon-optimized sequence was connected to the promoter P cpcB1 Downstream, the RT sucrose isomerase gene expression cassette is constructed and the gene expression cassette is linked to the resistance fragment Km rConnect and insert between the upstream and downstream homology arms of a neutral site (NS1) of Syn7942, and integrate into pUC19 to obtain plasmid pYW02 ( Figure 2 ).
[0031] 3) The sucrose isomerase sequence was found from the genome of Klebsiella sp. LX3 (the protein sequence is shown in SEQ ID NO:7, and the nucleic acid sequence is shown in SEQ ID NO:8). The sequence obtained after codon optimization is shown in SEQ ID NO:9. The codon-optimized sequence was connected to the promoter P cpcB1 Downstream, the LX3 sucrose isomerase gene expression cassette is constructed and the gene expression cassette is linked to the resistance fragment Km r The plasmid pYW03 ( Figure 3 ).
[0032] 3. Transformation and Screening of Cyanobacteria
[0033] 1) A plasmid carrying an expression cassette of a sucrose transporter (CscB) (amino acid sequence as shown in SEQ ID NO: 10, nucleic acid sequence as shown in SEQ ID NO: 11) was introduced into Syn7942 (the plasmid map is shown in FIG4 ), and the expression cassette was inserted between the upstream and downstream homology arms of a neutral site (NS3), thereby obtaining an engineered algae strain FL92;
[0034] 2) pYW01, pYW02, and pYW03 were respectively transferred into the above-mentioned engineered algae strain FL92 to obtain three positive algae strains YW07, YW08, and YW09 ( Figure 5 ).
[0035] 4. Engineering algae to synthesize and secrete isomaltulose and trehalulose
[0036] The column photoreactor used was a round-bottom glass tube made of ordinary glass with a diameter of 3 cm and a length of 20 cm. The total liquid volume of the reactor could reach 100 mL, and 65 mL was filled during the experiment. The seed liquid was a BG11 culture in the logarithmic growth phase (supplemented with corresponding antibiotics: kanamycin, 10 μg / mL; chloramphenicol: 5 μg / mL), and the initial inoculation concentration OD 730 1, medium was BG11 (with antibiotics added), at 30°C, 150 μmol photons m -2 s -1 , and culture was carried out under the condition of passing mixed gas (3% CO2 + 97% air).
[0037] Take 1 mL of the algae liquid of the engineered strain during the culture process, centrifuge at 13000 rpm for 10 min, transfer the supernatant after centrifugation into another clean 1.5 mL EP tube, and use liquid chromatography to determine the extracellular sugar content. Use Agilent 1260 high performance liquid chromatograph, HPX-87H sugar analysis column. The mobile phase is 5 mM H2SO4 solution, and the flow rate is 0.5 mL / min. Figure 6 A shows the peak diagram of isomaltulose standard (400 mg / L), and the peak time is about 9.306 min; Figure 6 B shows the peak diagram of trehalulose standard (200 mg / L), with the peak time at about 9.676 min; Figure 7 The peaks of the supernatant of the genetically engineered strain culture were determined by liquid chromatography. The elution time of isomaltulose was 9.307 min and the elution time of trehalulose was 9.684 min, which were consistent with those of the standard.
[0038] The extracellular sucrose, isomaltulose, and trehalulose contents as well as the intracellular isomaltulose and trehalulose were quantitatively analyzed.
[0039] The results are as follows Figure 8 As shown in Figure 2, the growth rates of the four algae strains were similar from day 0 to day 6, but the cell concentration of FL92 was higher than that of the other three algae strains at day 8 to 10. Figure 8 A). After 6 days of culture, the extracellular sucrose secretion of FL92 reached 860 mg / L, while the extracellular sucrose secretion of YW07, YW08, and YW09 was only 63, 41, and 21 mg / L, respectively ( Figure 8 B) This indicates that when algal cells express both sucrose transporters and sucrose isomerase, sucrose isomerase can convert sucrose into its isomerized form before sucrose transporters, and in this case, the conversion rates of sucrose from the three isomerases from different sources are as high as 92%, 95%, and 97%, respectively.
[0040] In addition, the extracellular isomaltulose production of YW07, YW08, and YW09 reached 776, 768, and 570 mg / L, respectively. Figure 8 C), the extracellular trehalulose production reached 139, 158, and 326 mg / L respectively ( Figure 8D), the ratios of extracellular isomaltulose to extracellular trehalulose were 85%:15%, 83%:17%, and 63%:37%, respectively, indicating that among the three isomerases, the isomaltulose yield of the sucrose isomerase gene derived from Erwiniarhapontici NX-5 was higher (i.e., the proportion of sucrose as a substrate converted into isomaltulose was high, and the proportion of by-product trehalulose was low); the intracellular isomaltulose contents of YW07, YW08, and YW09 were 7, 10.6, and 4.6 mg / L, respectively, i.e., the extracellular secretion rates of isomaltulose were 99%, 98%, and 99%, respectively. Through these results, we not only discovered for the first time that sucrose transporter has the function of secreting isomaltulose and trehalulose to the extracellular space, but also that it seems to have a higher transport priority for isomaltulose and trehalulose than sucrose. This results in the sucrose production of the engineered algae strain being reduced to less than 1 / 10 of the original amount, while the production of isomaltulose and trehalulose is much higher than that of sucrose.
[0041] These results show that by systematic metabolic engineering of the model chassis Sny7942 (expression of isomaltulose synthase, expression of transporter), the engineered algae strain can efficiently isomerize the sucrose in the cell into isomaltulose and trehalulose and preferentially secrete both of them to the extracellular space. Among them, when the target product is isomaltulose, the sucrose isomerase gene from Erwinia rhapontici NX-5 has the best expression effect, with isomaltulose production reaching 776 mg / L, the extracellular secretion rate of isomaltulose being 99%, and the ratio of extracellular isomaltulose to extracellular trehalulose being 85%:15%.
[0042] In the embodiments of the present invention, for the purpose of illustration, some specific promoters and specific sucrose transporters and sucrose isomerase protein and nucleic acid coding sequences are listed, but these specific promoters, proteins and nucleic acid coding sequences should not be used as limitations of the present invention. Those skilled in the art can design existing or future promoters and proteins or nucleic acid coding sequences with the same or similar functions on their own after reading this application and understanding its spirit, form the required gene expression cassette, and transfer it into the corresponding cyanobacteria to obtain an engineered algae strain that can produce high yields and secrete isomaltulose and trehalulose, and such promoters and protein and nucleic acid coding sequences should also be covered under the protection scope of the present invention.
[0043] It should also be noted that although Syn7942 is used as the chassis algae in the present invention, the use of other similar cyanobacteria (such as Synechococcus PCC7002, Synechocystis PCC6803, etc.) also has the same or similar effects. Those skilled in the art, after reading this application and understanding its spirit, can select existing or existing cyanobacteria as chassis algae to construct an engineered algae strain with high yield and secretion of isomaltulose and trehalulose, and such chassis algae should also be covered under the protection scope of the present invention.
[0044] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims
1. A method for preparing an engineered algae that produces high yield and secretes isomaltulose and trehalulose, characterized in that: The method comprises the steps of transferring a sucrose transporter expression frame and a sucrose isomerase expression frame into the chassis cyanobacteria, wherein the sucrose isomerase is a sucrose isomerase capable of converting sucrose into isomaltulose or trehalulose, and the chassis cyanobacteria is a cyanobacteria capable of producing sucrose; In the sucrose transporter expression frame, the protein sequence of the sucrose transporter is shown in SEQ ID NO: 10; The chassis cyanobacteria are selected from Synechococcus.
2. The method according to claim 1, characterized in that The chassis cyanobacteria are cyanobacteria that are directly separated from the environment and are capable of producing sucrose, or cyanobacteria that are genetically modified to be capable of producing sucrose.
3. The method according to claim 2, characterized in that The chassis cyanobacteria are selected from Synechococcus PCC7942, Synechococcus UTEX2973, and Synechococcus PCC7002.
4. The method according to claim 1, characterized in that: In the sucrose isomerase expression frame, sucrose isomerase is derived from Serratia ( Serrati ), Erwinia ( Erwinina ), Klebsiella spp. Klebsiella ), Pantoea dispersa ( Pantoea dispers ), Spermatozoa rubrum ( Protaminobacter rubrum ) of sucrose isomerase.
5. The method according to claim 4, characterized in that The sucrose isomerase is derived from Erwinia rhapontici NX-5 , Raoultella terrigena or Klebsiella sp. LX3 of sucrose isomerase.
6. The method according to claim 5, characterized in that In the sucrose isomerase expression frame, the protein sequence of sucrose isomerase is shown in one of SEQ ID NOs: 1, 4, and 7.
7. The method according to claim 6, characterized in that The nucleic acid sequence of the sucrose isomerase is shown in one of SEQ ID NOs: 2, 3, 5, 6, 8, and 9.
8. A cyanobacterium that produces high yield and secretes isomaltulose and trehalulose, characterized in that: It is prepared by the method according to any one of claims 1 to 7.