CN114645009B - Fructokinase-disabled cyanobacteria and its use for secretion and production of fructose - Google Patents
Fructokinase-disabled cyanobacteria and its use for secretion and production of fructose Download PDFInfo
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Abstract
The invention relates to application of a cyanobacterium fructokinase disabling agent in preparation of cyanobacteria with fructose secretion capacity and high fructose yield, a reagent or a fructokinase function antagonist for knocking out fructokinase genes corresponding to fructokinase in the cyanobacteria, and application of the cyanobacterium fructokinase disabling agent in production of fructose. The invention knocks out fructokinase in a carbon circulation path of cyanobacteria, and the mutant strain unexpectedly obtains the capability of producing fructose in large quantity and secreting the fructose outside cells, so that the fructose yield is improved by hundreds of times, the possibility of industrially producing the fructose by using cyanobacteria is provided, and the foundation is provided for further metabolic engineering modification by taking cyanobacteria as a chassis algae strain.
Description
Technical Field
The present invention relates to the field of synthetic biology of cyanobacteria, and more particularly to fructokinase-disabled cyanobacteria and their use for secretion and production of fructose.
Background
Fructose is widely applied in the industries of food, medicine, chemical industry and the like, and can be particularly used for preventing diseases such as diabetes, obesity, decayed tooth and the like. Currently, fructose products on the market are in the form of fructose syrup and crystalline fructose. The production of high fructose syrup is to use starch as main raw material, through chemical catalytic synthesis route, firstly, utilizing alpha-amylase and saccharifying enzyme to hydrolyze starch into glucose, then utilizing glucose isomerase to convert glucose into high fructose syrup containing 42% fructose and 58% glucose. Crystalline fructose (fructose with purity more than 97%) is fine powder crystal, and the production is based on fructose-fructose syrup, and the product is extracted from a glucose-fructose mixed system, and the product is prepared by a series of operations of chromatographic separation, enrichment, concentration, crystallization, screening and the like, and has the advantages of complicated production process, lower conversion rate, higher grain consumption and higher equipment requirement. Therefore, developing a novel efficient and green fructose synthesis technology has important significance for reducing the economic and environmental cost of the fructose industry and improving the competitiveness and market acceptance of the product.
Cyanobacteria is a prokaryotic microorganism for carrying out plant type oxygen-releasing photosynthesis, and compared with microalgae and higher plants, cyanobacteria has the advantages of simple structure, rapid growth, convenient genetic transformation and the like, so that the cyanobacteria becomes a potential photosynthetic platform for bioenergy and bio-based chemicals. However, no potential high fructose cyanobacteria have been found in the current studies, nor has cyanobacteria been found to have the ability to secrete fructose outwards. N iederho ltmeyer and the like, by transferring a hexose transporter G/f gene into Synechococcus PCC7942, a fructose secretion pathway is formed so that the bacterium can secrete fructose outwards, but even under optimal conditions, the produced extracellular fructose content is only 28.8mg/L at maximum, and the fructose level decreases with increasing cell density, which does not meet the requirements of industrial production (Niederholmeyer, H., wolfstadter, B.T., savage, D.F., silver, P.A., and Way, J.C., engineering cyanobacteria to synthesize and export hydrophilic products. Application. Environ. Microbiol.,2010.76 (11): p.3462-3466).
Thus, there is a need for a new method for obtaining the ability of cyanobacteria to produce fructose in large quantities and to secrete it outwards.
Disclosure of Invention
During the research of a plurality of cyanobacteria such as Synechococcus PCC7942, synechococcus PCC7002 and the like, fructokinase is commonly found in cyanobacteria, when the enzyme is knocked out, the mutant strain unexpectedly shows extremely high fructose yield, and basically all fructose is secreted outside cells, so that the fructose content in a culture system is hundreds of milligrams per liter.
Based on the above findings, the present invention provides the use of a cyanobacterial fructokinase disabling agent for the preparation of cyanobacteria having a fructose secretion function or having a high fructose yield. In the present invention, the fructokinase disabling agent means an agent capable of disabling fructokinase activity in cyanobacteria, for example, a nucleic acid construct capable of completely deleting fructokinase gene in cyanobacteria or a nucleic acid construct capable of partially deleting or partially mutating fructokinase gene in cyanobacteria to cause it to lose fructokinase function. Such nucleic acid constructs may be DNA and/or RNA. The fructokinase disabling agent may also be an antagonist of fructokinase function, for example by adding an antagonist of fructokinase function to the culture or by expressing a gene having antagonistic fructokinase function in cyanobacteria.
While the present invention does not specifically demonstrate experimental evidence other than the use of homologous double crossover knockouts, those skilled in the art, after reading the present disclosure and appreciating the spirit of the present application, are fully able to obtain such a demonstration: after disabling the fructokinase, cyanobacteria are able to produce fructose in large quantities and transport it outside the cell. The person skilled in the art can realize fructokinase disablement by any technique existing and to be present, but these should be covered within the scope of the present invention.
The present invention also provides a cyanobacteria having a high fructose yield, in which the fructokinase is disabled. The cyanobacteria is one or more of Synechococcus, synechocystis, anabaena, arthrospira, nostoc, microcystis, and Sphingeum.
The invention also provides application of the fructokinase-disabled cyanobacteria in fructose production.
Fructokinase failure means that fructokinase no longer has the function of phosphorylating fructose. We disable fructokinase by deletion, mutation or expression of antagonists of the fructokinase. Cyanobacteria that are not able to phosphorylate the produced fructose, i.e. fructose is produced in large quantities and transported outside the cell. The person skilled in the art can realize fructokinase disablement by any technique existing and to be present, but these should be covered within the scope of the present invention.
The invention also provides a method for causing cyanobacteria to secrete fructose or produce fructose, comprising the step of disabling a fructokinase in said cyanobacteria.
In a specific embodiment, the fructokinase of said cyanobacterium is disabled by knocking out the fructokinase gene in said cyanobacterium.
In a specific embodiment, the knockout is a homologous double crossover knockout.
In a specific embodiment, the cyanobacteria is one of Synechococcus, synechocystis, anabaena, arthrospira, nostoc, microcystis, and Sphingesium.
The invention knocks out fructokinase in a carbon circulation path of cyanobacteria, and the mutant strain unexpectedly obtains the capability of producing fructose in large quantity and secreting the fructose outside cells, so that the fructose yield is improved by hundreds of times, the possibility of industrially producing the fructose by using cyanobacteria is provided, and the foundation is provided for further metabolic engineering modification by taking cyanobacteria as a chassis algae strain.
Drawings
FIG. 1 is a plasmid map of pJS 11.
FIG. 2 is a plasmid map of pYDO 8.
FIG. 3 is an electrophoretogram of PCR amplification products for the fructokinase knockout site, wherein the left panel is an electrophoretogram of amplification products of wild-type PCC7942 (WT) and knockout strain JS05, and the right panel is an electrophoretogram of amplification products of wild-type PCC7002 (WT) and knockout strain YD08.
FIG. 4 shows HPLC peak profiles for saccharide standards.
FIG. 5 is a graph showing the culture growth curve (A), extracellular fructose content (B) and intracellular fructose content (C) of wild-type PCC7942 (WT) and knockout strain JS05.
FIG. 6 is a statistical plot of the culture growth curve (A) and extracellular fructose content (B) of wild-type PCC7002 (WT) and knockout strain YD08.
Detailed Description
The principles and features of the present invention are described below with reference to the drawings, the examples are illustrated for the purpose of illustrating the invention and are not to be construed as limiting the scope of the invention.
1. The used algae strain
Both cyanobacteria Synechococcus PCC7942 and cyanobacteria Synechococcus PCC7002 were routinely used as model strains for related experimental studies. Wherein, the amino acid sequence of the fructokinase gene of PCC7942 is shown as SEQ ID NO.1, and the nucleic acid sequence is shown as SEQ ID NO. 2; the amino acid sequence of the fructokinase gene of PCC7002 is shown as SEQ ID NO.3, and the nucleic acid sequence is shown as SEQ ID NO. 4.
2. Plasmid construction
2.1 construction of PCC7942 fructokinase knockout plasmid pJS11: primers were designed based on the fructokinase gene of PCC7942 and its upstream and downstream sequences in the genome, and the PCC7942 genome was used as a template for amplification to obtain the upstream homology arm (FK-N) and the downstream homology arm (FK-C) of the fructokinase, and the chloramphenicol resistance fragment was inserted between the upstream homology arm and the downstream homology arm of the fructokinase, and was integrated into pUC19 backbone to obtain recombinant plasmid pJS11, the plasmid map of which is shown in FIG. 1.
2.2 construction of PCC7002 fructokinase knockout plasmid pYD08: primers are designed according to the fructokinase gene of PCC7002 and the upstream and downstream sequences thereof in the genome, the PCC7002 genome is used as a template for amplification to obtain an upstream homology arm (FK-upstream) and a downstream homology arm (FK-downstream) of the fructokinase, a kanamycin resistance fragment is inserted in the middle, and a fructokinase knockout plasmid pYD08 is obtained, and a plasmid map is shown in figure 2.
3. Construction of transgenic algae strains
Plasmid pJS11 was transformed into wild-type PCC7942 to give algal strain JS05. The method comprises the following steps:
1) 1mL logarithmic phase of growth (OD) 730 PCC7942 culture of about 1), cells were collected by centrifugation at 5000g for 5min, washed 2 times with fresh BG11 medium, and discardedSupernatant, cell pellet was resuspended in 250 μl BG11 solution;
2) 2. Mu.L of plasmid pJS11 (plasmid concentration 100. Mu.g/mL) was added to the resuspended algae solution;
3) Wrapping the EP added with the plasmid by using tinfoil paper, and incubating for 20 hours at a temperature of 30 ℃ by using a shaking table;
4) The incubated transformation products were plated on BG11 plates with corresponding resistance (chloramphenicol: 5. Mu.g/mL), 30 ℃, 100. Mu. Mol of photons m -2 s -1 Culturing under the condition that transformants grow out in 4-5 days, picking up the transformants and streaking on a fresh BG11 plate (chloramphenicol: 5. Mu.g/mL); completely separated algae strains are obtained through screening.
Plasmid pYD08 was transferred into wild-type PCC7002 to obtain algal strain YD08. The method comprises the following steps:
1) 1mL of logarithmic phase (OD) 730 About 1) Synechococcus PCC7002, 5000g centrifuged for 5min to collect the cells, and the cells were washed 2 times with fresh A+ medium, the supernatant was discarded, and the cell pellet was resuspended in 250. Mu.L of non-anti-A+ medium;
2) 3000ng of plasmid pYD08 (concentration 100. Mu.g/mL) was added to the resuspended algae solution;
3) Wrapping the EP added with the plasmid by using tinfoil paper, and incubating for 20 hours at a temperature of 30 ℃ by using a shaking table;
4) The incubated transformation products were plated on a+ plates with corresponding resistance (kanamycin: 50. Mu.g/mL), 30 ℃, 100. Mu. Mol of photons m -2 s -1 Culturing under the condition that transformants grow out in 5-7 days, picking up the transformants and streaking on a fresh A+ plate (kanamycin: 50. Mu.g/mL); completely separated algae strains are obtained through screening.
As shown in fig. 3, JS05 and YD08, their corresponding Fructokinase (FK) was completely knocked out.
4. JS05 secretes fructose
A column type photo-reactor is used, the column type photo-reactor is a round bottom glass tube made of common glass with the diameter of 3cm and the length of 20cm, the total liquid loading amount of the reactor can reach 100mL, and 65mL of liquid is loaded in the experimental process. The seed solution is BG11 culture in logarithmic growth phase (supplemented with corresponding chloromycetin: 5 μg/mL), initial inoculation concentration OD 730 2, 150. Mu. Mol of photons m at 30 DEG C -2 s -1 Introducing mixed gas (3% CO) 2 +97% air), or 150mM NaCl is added to the culture medium at the initial stage of the culture.
The growth of the strain is monitored in the column type illumination culture process of JS05, a strain growth curve is drawn, and the extracellular fructose content and the intracellular fructose content are quantitatively analyzed.
Fructose content analysis the HPLC method was used: taking 1mL of algae liquid of the engineering strain in the culture process, centrifuging at 13000rpm for 10min, transferring the supernatant after centrifugation into another clean EP tube with the volume of 1.5mL, and measuring the extracellular fructose content; the extracellular extracted fructose was diluted and then detected by liquid chromatography (using Agilent high performance liquid chromatograph 1260 equipped with differential detector, using HPX-87H sugar analytical column with mobile phase of 5mM H) 2 SO 4 The solution, flow rate 0.5 mL/min), showed, by preliminary experiments, that fructose peaked around 20min (FIG. 4).
As shown in FIG. 5, the growth curves of the mutant strain JS05 cultured in the normal BG11 medium and the BG11 medium supplemented with 150mM NaCl were not greatly different. In the early stage of culture, the saline culture medium can cause the algae strain to accumulate higher intracellular fructose content (6-12 days), but by 14 days, the intracellular fructose content of each group has no significant difference, which indicates that the intracellular fructose content accumulation gradually reaches a limit value along with the extension of the culture time.
Surprisingly, no extracellular secretion of fructose was observed for wild type, either with or without salt, but in JS05 we observed higher concentrations of extracellular fructose under both conditions, especially after 14 days of culture under salt conditions, the extracellular fructose content of the JS05 strain was up to 387mg/L. This suggests that the knockdown of fructokinase not only surprisingly greatly enhances the synthesis of fructose in the Synechococcus PCC7942, but also activates the extracellular secretion pathway of fructose.
5. YD08 synthesizes and secretes fructose
A column type photoreactor is used, and the column type photoreactor is a round bottom glass tube made of common glass with the diameter of 3cm and the length of 20 cm; total reactor chargeUp to 100mL, 65mL of liquid is filled in the experimental process. Seed solution is A+ culture in logarithmic growth phase (supplemented with corresponding antibiotic: kanamycin, 50. Mu.g/mL), initial inoculation concentration OD 730 1, 200. Mu. Mol of photons m at 30 DEG C - 2 s -1 Introducing mixed gas (3% CO) 2 +97% air). The detection method is the same as above.
The growth of the strain is monitored in the column type illumination culture process of YD08, a strain growth curve is drawn, and the extracellular fructose content is quantitatively analyzed.
As shown in FIG. 6, wild-type PCC7002 and fructokinase knockout strain YD08 were grown in A+ medium and 200. Mu. Mol photons m -2 s -1 The growth curves obtained by culture under the condition of (2) are not quite different. However, wild type did not produce extracellular fructose, whereas YD08 strain was cultured until the eighth day at extracellular fructose concentration was as high as 635mg/L. It can be seen that in Synechococcus PCC7002, the fructokinase was knocked out to promote the secretion of fructose by algal cells to the outside in a large amount.
In addition, we performed related experiments on synechococcus UTEX2973, synechocystis PCC6803, anabaena PCC7120, and the like, all obtained similar results. These experimental evidence demonstrate that knocking out fructokinase in a variety of cyanobacteria not only greatly increases its fructose production, but also enables it to secrete fructose outwards, and that the total fructose production in mutant strains can be hundreds of times that of wild-type.
Without being bound by the theory presented in the art, we speculate that the fructokinase in cyanobacteria can immediately phosphorylate the produced fructose, changing its conformation, and the conformationally modified phosphorylated fructose cannot be secreted extracellularly, but rather enters the corresponding carbon metabolism, thus limiting the yield of fructose in cyanobacteria. After we knocked out fructokinase, since fructose is no longer phosphorylated, this results in the produced fructose being present in a non-phosphorylated form, which on the one hand does not enter the metabolic form of phosphorylated fructose, and more importantly, the non-phosphorylated fructose is unexpectedly capable of being secreted extracellularly in large amounts, so that fructose is not accumulated intracellular, and the yield of fructose is greatly improved.
It should be noted that, in the examples of the present invention, we describe, for the purpose of illustration, that fructokinase in a specific cyanobacterium is knocked out by a specific method to obtain a cyanobacterial strain that does not contain fructokinase with an effective function, however, these specific methods should not be used to limit the scope of the present invention. The fructose secretion and yield of cyanobacteria can be greatly improved only by disabling fructokinase in cyanobacteria by the existing or existing technology.
The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.
Sequence listing
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Claims (8)
1. Use of a cyanobacterial fructokinase disabling agent, which is an agent for knockdown of a fructokinase gene corresponding to a fructokinase in cyanobacteria, or a nucleic acid construct capable of causing partial mutation of a fructokinase gene in cyanobacteria to cause loss of fructokinase function, for the preparation of cyanobacteria having a fructosecretion function or having a high fructosyl yield.
2. Cyanobacteria having a fructose secretion function or having a high fructose yield, wherein a fructokinase in the cyanobacteria is nonfunctional.
3. Use of cyanobacteria according to claim 2 for the production of fructose.
4. The use according to claim 3, wherein the cyanobacteria is one or more combinations of synechococcus, synechocystis, anabaena, arthrospira, nostoc, microcystis, and Sphingomonas.
5. A method for causing cyanobacteria to secrete fructose or produce fructose, comprising the step of disabling a fructokinase in the cyanobacteria.
6. The method of claim 5, wherein said cyanobacterium is freed from fructokinase by knocking out a fructokinase gene in said cyanobacterium.
7. The method of claim 6, wherein the knockout is a homologous double crossover knockout.
8. The method of claim 7, wherein the cyanobacteria is one of synechococcus, synechocystis, anabaena, arthrospira, nostoc, microcystis, and Sphingomonas.
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