阅读说明：本技术 一种利用d-葡萄糖生物合成d-阿洛糖的方法 (Method for biosynthesizing D-allose by using D-glucose ) 是由 李灏 郑灵洁 范立海 于 2020-11-26 设计创作，主要内容包括：一种利用D-葡萄糖生物合成D-阿洛糖的方法属于大肠杆菌代谢工程领域,旨在利用生物体合成D-阿洛糖,由于D-阿洛糖在自然界中含量极少,且提取率低,所以在工程大肠杆菌中合成D-阿洛糖是一个值得研究的内容。在大肠杆菌中引入三个外源基因gi、dpe、rpiB最终将D-葡萄糖异构合成D-阿洛糖。为了稳定产量,在途径构建时,敲除了FruA、ptsg、pfkB、glk、mak、pfkA六个基因,并且过表达了Galp,进而在D-阿洛糖合成时,能够实现多种碳水化合物的共利用,而且使得甘油用于菌种生长,D-葡萄糖以更高的转化率合成D-阿洛糖。(A method for biosynthesizing D-allose by using D-glucose belongs to the field of escherichia coli metabolic engineering, and aims to synthesize D-allose by using organisms. Introducing three exogenous genes gi, dpe and rpiB into escherichia coli to finally isomerize D-glucose into D-allose. In order to stabilize the yield, six genes of FruA, ptsg, pfkB, glk, mak and pfkA are knocked out during pathway construction, Galp is overexpressed, and further, during D-allose synthesis, the co-utilization of various carbohydrates can be realized, glycerol is used for strain growth, and D-allose is synthesized by D-glucose at a higher conversion rate.)

1. A method for biosynthesizing D-allose from glucose, which comprises the following steps: constructing and expressing a pathway for synthesizing D-allose from D-glucose: glucose isomerase gene (gi), psicose epimerase gene (dpe), and ribose-5-phosphate isomerase gene (rpiB), enabling E.coli to synthesize D-psicose; on the basis, Galp is over-expressed, ptsG and FruA are knocked out to eliminate ccr effect, then glk and mak are knocked out, and a D-glucose and D-fructose consumption pathway is cut off; finally, pfka and pfkb were knocked out in order to prevent carbon flux loss.

2. The method for the biosynthesis of D-allose from glucose according to claim 1, wherein said step of: in step (1) the gi gene is from Streptomyces, with the sequence references NCBI, dpe, rpiB from Agrobacterium and Clostridium respectively.

3. The method for the biosynthesis of D-allose from glucose according to claim 1, further comprising the steps of: inoculating the product of claim 1 into LB test tube for overnight culture, culturing in M9 culture medium for 14-16h, inoculating by fermentation, and fermenting for 72-90 h.

Technical Field

The invention belongs to the field of escherichia coli metabolic engineering, particularly relates to a method for synthesizing D-allose, and particularly relates to an engineering bacterium for producing allose by combining glucose and glycerol as well as a construction method and a path thereof.

Background

D-allose is an aldohexose, a rare monosaccharide, a white tasteless crystalline solid, a non-caloric sugar, and no toxicity. Chemical formula CH₂OH(CHOH)₄CHO, isolated from the leaves of an African shrub, butThe extraction rate is extremely low, so the artificial synthesis of D-allose becomes more urgent. D-allose is soluble in water but hardly soluble in methanol, and is a C-3 epimer of glucose. D-allose exists mainly in cyclic form, and is composed of beta-D-allo-1.5-pyranose (77.5%), alpha-D-allo-1.5-pyranose (14%), beta-D-allo-1.4-furanose (5%) and alpha-D-allo-1.4-furanose (3.5%), wherein beta-D-allo-1.5-pyranose is the main component.

In recent years, D-allose has attracted much attention because of its many pharmaceutical activities, and it inhibits carcinogenesis, particularly under oxidative stress conditions, and can inhibit the proliferation of a variety of cancer cell lines, including cervical cancer, hepatocellular carcinoma, ovary, head and neck, skin, and prostate cancer. And the combination of D-allose and radiation in the treatment of cancer increases the cure rate of tumors. D-allose can also be used as an antioxidant, and has certain potential as a therapeutic agent in pharmaceutical preparations and a functional component in formula foods. D-allose acts as an anti-inflammatory agent, inhibiting ischemia-reperfusion injury, inhibiting segmental neutrophil production, and reducing platelet count. The use of D-allose in combination with low dose FK506 significantly increased allograft survival while reducing tissue damage. In addition, dextran has a cryoprotective effect on cells. Therefore, the D-allose has a certain application prospect in surgical operation and transplantation.

However, the source of D-allose mainly comes from chemical synthesis and biosynthesis, and since chemical synthesis has many disadvantages, such as complicated synthesis route, many byproducts, great environmental pollution, and high difficulty in separation and purification, biosynthesis is more competitive when synthesizing allose, and microbial fermentation for producing allose is low in cost and environmentally friendly.

However, when D-glucose is used as the sole carbon source, the yield of D-allose in E.coli is relatively low, since glucose is responsible for both the production of cell biomass and the production of D-allose. Previous reports indicate that, after knocking out phosphorylated specific permeases, glucose uptake utilization can be reduced, but the biomass of E.coli is affected, so additional carbon sources are required to compensate for the deficit in cell growth. Due to the experimental approach, glycerin, a three-carbon substance downstream of the glycolysis pathway, is selected to supplement deficiency, and the glycerin has a simple structure and a clear metabolic pathway. When glucose and glycerol coexist as a carbon source, wild escherichia coli preferentially consumes the glucose, so that the co-utilization of the glucose and the glycerol is realized, and the bacteria can efficiently produce the D-allose by utilizing the glucose still has great challenge.

Disclosure of Invention

In view of the above, the present invention aims to provide an engineering bacterium for producing D-allose, a construction method and an application thereof, and utilizes gene regulation to balance cell metabolism, and D-glucose is used for synthesizing D-allose while glycerol is used for growth.

In order to achieve the purpose, the specific technical scheme of the invention is as follows:

d-glucose is utilized to synthesize D-allose in a metabolic engineering strain Escherichia coli JM109(DE3) (as shown in figure 1), three genes required by a main pathway are firstly constructed, then a pathway for the D-glucose to be consumed for growth is knocked out according to the metabolic pathway, and simultaneously glucose effect is relieved, so that the situation of polysaccharide co-utilization is realized, most of D-glucose is used for synthesizing the D-allose, the glycerol consumption mainly supports cell growth, and the non-phosphorylation pathway of the D-glucose entering the cell is optimized.

The invention provides an engineering bacterium for producing D-allose, which comprises a host bacterium and a plasmid vector transferred into the host bacterium, wherein the plasmid vector integrates a gene of exogenous genes glucose isomerase (gi), a gene of D-psicose epimerase (dpe), a gene of ribose-5-phosphate isomerase (rpiB) and a gene of over-expressed endogenous genes galactose permease (Galp).

Wherein the host bacterium is a wild or modified bacterium or fungus, and the preferred host bacterium of the invention is Escherichia coli.

Based on the above, D-allose is synthesized from D-glucose in the metabolic engineering bacterium JM109(DE3), and 3 genes required for expression are first constructed, and then the following genes are sequentially knocked out: the gene of pts pathway (FruA) for transporting fructose phosphorylation, the gene of pts pathway (ptsg) for transporting glucose, the gene of glucokinase (glk), 6-phosphofructokinase I, 6-phosphofructokinase II and mannose fructokinase (mak) ensure that most of D-glucose is converted into D-allose, and glycerol is added into the culture medium for growth of the engineering bacteria so as not to influence the growth of the engineering bacteria. Under the condition of not influencing the growth of the strains, the strains can produce D-allose as much as possible.

A method for biosynthesizing D-allose from glucose and glycerol, which comprises the following steps: under the condition of coexistence of glycerol and glucose, co-utilization of carbohydrate can be realized, inhibition of glucose and fructose on glycerol utilization is eliminated by knocking out ptsG gene and FruA gene, and a non-phosphorylation way of glucose entering cells is improved by over-expressing endogenous gene Galp gene, so that glucose can enter cells more quickly for subsequent synthesis reaction.

Further, the method for biosynthesizing D-allose by using D-glucose comprises the following steps:

(1) constructing and expressing genes for synthesizing the D-allose pathway, including gi, dpe, rpiB;

(2) constructing and over-expressing a gene Galp for transferring D-glucose in a non-phosphate way;

(3) knocking out genes of glycolysis pathway, namely glk, pgi, mak and pfkA/B, knocking out ptsG and FruA genes to relieve ccr effect.

The method is detailed as follows:

further, the gene gi gene in step (1) is derived from Streptomyces, herein artificially synthesized, with the sequence references NCBI, dpe, rpiB derived from Agrobacterium tumefaciens and Clostridium thermocellum, respectively, with reference to NCBI number. The construction and expression of the pathway for the synthesis of D-allose from D-glucose involves the synthesis of key genes, wherein a glucose isomerase gene (gi) is chemically synthesized, and D-glucose is directly isomerized into D-fructose; a psicose epimerase gene (dpe) that isomerizes D-fructose to D-psicose; and a ribose-5-phosphate isomerase gene (rpiB) that isomerizes psicose to D-psicose. The gene is amplified and then connected with a vector (pETDuet-1) to construct a recombinant plasmid pETDuet-1-dpe-rpiB-gi which is needed by people, and the recombinant plasmid is transfected into Escherichia coli JM109(DE3) to obtain a strain capable of synthesizing D-allose.

Further, the Galp gene overexpressed in the step (2) is a gene of Escherichia coli itself for expressing a membrane protein of non-phosphorylated transport glucose. The Galp gene was overexpressed. In order to improve the efficiency of the non-phosphate pathway of D-glucose transport, a Galp gene was constructed to express, and the non-phosphorylation efficiency was further improved after the ccr effect was released. The gene was ligated with a vector (pRSFDuet-1) to construct the plasmid pRSFDuet-1-Galp required by us, and the plasmid was transfected into the strain harboring the recombinant plasmid pETDuet-1-dpe-rpiB-gi and constructed.

Further, the sequence of continuous gene knockout in the unified strain in step (3) must be FruA and ptsG, and after knockout, the ccr effect is relieved, so that the co-utilization of glucose and glycerol can be realized. Then, pfka, glk, mak and pfkb were knocked out. To block the consumption of glucose and fructose. Knock-out of the relevant gene in the metabolic pathway. Red homologous recombination is utilized to knock out FruA and ptsG (phosphorylation pathways of D-glucose and D-fructose entering cells), the two genes are knocked out, the ccr effect is relieved, the efficiency of non-phosphorylation pathways can be improved, then glk and mak are knocked out subsequently, the pathways consuming D-glucose and D-fructose are cut off, the target product is produced in a more focused mode, and finally pfka and pfkb are knocked out in order to prevent carbon flow loss, so that the pathways are further perfected, and the product yield is improved.

Compared with the prior art, the invention has the beneficial effects that:

according to the invention, cheap glucose and glycerol are utilized, the co-utilization of various carbohydrates is realized by knocking out key genes and overexpressing specific genes, and the consumption ways of glucose and fructose are cut off subsequently, so that the glucose is provided for production, and the co-metabolism of glycerol and glucose and the growth of glycerol are provided due to the elimination of ccr effect. In the original strain, however, this was not done. The invention constructs and improves the yield of D-allose, and the yield is obviously improved by further modifying the way.

Drawings

FIG. 1 shows the construction of the pathway for biosynthesis of D-allose and the basic metabolic pathways for glucose and fructose.

FIG. 2 is a schematic diagram of the construction of a synthetic D-allose vector and the construction of a vector overexpressing a non-phosphate transport pathway gene Galp.

FIG. 3 is a diagram of ptsg, pfkB, glk, mak, pfkA, FruA gene knockout validation gels.

Detailed Description

The present invention is further illustrated by the following examples, which are intended to be illustrative only and are not intended to limit the scope of the invention.

Example 1

The kinds of the medium are as follows

LB culture medium:

peptone 10g/L

Yeast powder 5g/L

Sodium chloride 10g/L

M9 medium:

the constructed D-allose-synthesizing strain JM109(DE3) -pETDuet-1-dpe-rpiB-gi-pRSFDuet-1-Galp, (plasmid construction is shown in FIG. 1) was stored at-80 ℃ and, after taking out, it was left at ordinary temperature, 4. mu.l was taken out on a clean bench and inoculated into 4 ml of sterilized LB tube, and ampicillin and kanamycin, which were one thousandth of the volume of the tube medium, were added and cultured overnight for 14 to 16 hours.

Then inoculating the bacteria into a shake flask LB culture medium with one thousandth of ampicilin and kanamycin, wherein the inoculation amount is about one thousandth of the culture volume, culturing for 48-72 h in a shaking table at 180-200rpm under the condition of 26-37 ℃, and ensuring that the control group JM109(DE3) -pETDuet-1-pRSFDuet-1 has no change with the experimental group OD600 and has extremely low allose production rate, thereby indicating that the pathway modification is effective and does not influence the growth of the strains.

Example 2

Pathway engineering was carried out on the engineered bacterium underpan cell JM109(DE3), and D-fructose was used as an important intermediate product, and because of its position in the glycolytic pathway, the metabolic pathway of D-fructose was also of interest when the process of the metabolic pathway of D-glucose was concerned. Therefore, the gene needs to be knocked out in FruA and ptsG, after knocking out, the ccr effect is relieved, and the co-utilization of glucose and glycerol can be realized. Then, pfka, glk, mak and pfkb were knocked out. To block the consumption of glucose and fructose. Knock-out of the relevant gene in the metabolic pathway. FruA and ptsG (phosphorylation pathways of D-glucose and D-fructose entering cells) are knocked out by virtue of Red homologous recombination, and the purpose of knocking out the two genes is to relieve the ccr effect and improve the efficiency of a non-phosphorylation pathway.

Primers for gene knock-outs are shown in Table 1 below.

After the knockout was completed (the verification chart is shown in FIG. 3), the density of bacteria was substantially unchanged when the medium was cultured in the medium containing only glucose and fructose, respectively, indicating that the knockout was effective. Meanwhile, the strain is cultured in an LB culture medium, and the strain can grow, so that the next operation can be carried out. The medium composition was adjusted.

During the construction of the pathway, the first half of the glycolytic pathway is knocked out, and the three-carbon compound is not modified backwards, so that glycerol is selected as a carbon source for the growth of the strain. A comparison experiment is carried out on a control group (glucose is 5-15 g/L) and an experimental group (containing 5-15 g/L of glucose and 5-30 g/L of glycerol), and the feasibility is proved.

Embodiment 3

Firstly, preparing a strain carrying a corresponding plasmid, inoculating 4 microliter of the strain (JM109DE 3/six-knocking JM109DE3) into an LB test tube, and culturing at 37 ℃ and 180rmp overnight; amplifying, inoculating 20 microliters of seed liquid into a small shake flask containing 20mL of LB culture medium, and culturing for two hours until the OD is about 0.6-0.8; taking out the shake flask and carrying out ice bath for 10min to ensure the activity of the cells; subpackaging, wherein each tube is subpackaged with 1mL into 16 centrifuge tubes with 1.5mL, then centrifuging at 6000rmp for 4min at 4 ℃, and discarding the supernatant; combining tubes, suspending the precipitate with 800 microliters of 10% glycerol, combining 4 tubes gradually with 1 tube, centrifuging at 6000rmp for 4min, and removing the supernatant; washing bacteria, suspending each tube with 800 microliters of 10% glycerol, centrifuging at 6000rmp at 4 ℃ for 4min, and removing supernatant; repeating the previous step; finally, 4 tubes were suspended in 100. mu.l of 10% glycerol and made competent.

Adding 2 microliter of plasmid (pETDuet-1-dpe-rpiB-gi/pRSFDuet-1-Galp) into the competence, and gently suspending; adding the suspension into a precooled electric rotating cup, and quickly adding the suspension into a precooled 1mL LB centrifugal tube after electric shock; after the electrotransformation is finished, putting the mixture into a shaking table for resuscitation for 1h, wherein the culture temperature is 37 ℃; and then the process is carried out.

Coating 100 microliters of bacterial liquid on the poured flat plate until the bacterial liquid is dried; centrifuging for 1min at 12000rpm, and discarding the supernatant to obtain 100 microliters; culturing for 14-16h overnight, performing colony PCR (polymerase chain reaction) verification after a monoclonal bacterium is produced, selecting half of the bacteria for performing bacterium P, and verifying to obtain a correct recon; and the other half of the bacteria are stored in a refrigerator, after verification, the bacteria are picked into an LB test tube to be cultured overnight, and the glycerin tube is stored.

The constructed six knock-out strain JM109(DE3) -pETDuet-1-dpe-rpiB-gi-pRSFDuet-1-Galp for synthesizing D-allose and a control group JM109(DE3) -pETDuet-1-dpe-rpiB-gi-pRSFDuet-1-Galp and JM109(DE3) -pETDuet-1-pRSFDuet-1 were inoculated into LB tubes with corresponding resistance for overnight culture, after being cultured in M9 medium (containing 5 to 15g/L glucose and 5 to 30g/L glycerol) for 14 to 16 hours, fermentation inoculation was carried out for 72 to 90 hours, D-fructose was consumed while producing 3.5g/L with a conversion rate of about 50%, D-allose was produced from fructose to 1.2g/L with a conversion rate of about 10%, the building and putting into the system play a role.