CN110358756B

CN110358756B - Construction of protein toggle switch and application thereof in fermentation production of xylonic acid by mixed sugar

Info

Publication number: CN110358756B
Application number: CN201910670796.4A
Authority: CN
Inventors: 陈修来; 高聪; 刘立明; 罗秋玲; 刘佳
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2019-07-24
Filing date: 2019-07-24
Publication date: 2021-03-02
Anticipated expiration: 2039-07-24
Also published as: CN110358756A

Abstract

The invention discloses a construction of a protein toggle switch and application thereof in fermentation production of xylonic acid by mixed sugar, belonging to the technical field of bioengineering. The protein level toggle switch constructed by the invention has the advantages of short response time and high regulation precision because regulation and control occur at the protein level and the principle is cutting and induced degradation triggered by protease. By utilizing the strategy, when the concentration ratio of the glucose to the xylose in the substrate is more than 1, the carbon metabolism repression phenomenon can be effectively relieved. When the ratio of glucose: when the ratio of xylose is 2:1, the introduction of the toggle switch can improve the yield of the xylonic acid by 0.79 time compared with a control group. The tool has good application prospect when cheap cellulose hydrolysate is used as a substrate to produce high value-added chemicals.

Description

Construction of protein toggle switch and application thereof in fermentation production of xylonic acid by mixed sugar

Technical Field

The invention relates to construction of a protein toggle switch and application thereof in fermentation production of xylonic acid by mixed sugar, belonging to the technical field of bioengineering.

Background

Toggle switches are a broad class of metabolic regulation switches that share the common feature of being able to control gene expression as the environment (inducer or other sensing substrate) changes. In 2000, Gardner et al described a toggle switch constructed on a plasmid that toggles back and forth between two promoter expressions in response to external signals. The switch consists of two repressors and two conserved promoters. Each promoter is repressed by a repressor of expression from the other promoter. Thus, the presence or absence of the inducer is controlled to switch the switch between "on" and "off" to control the expression of the downstream gene. Kobayashi et al designed a toggle switch consisting of the PL promoter, Ptrc promoter, lacI gene and λ cl gene. Based on the switch of Kobayashi et al, anesias et al designed a toggle switch that senses population density. Intercellular communication is accomplished by a population of signaling molecules, which are autoinducers. Homoserine Lactone (AHL) is one such autoinducer, and its concentration is proportional to the number of cells in the environment. The expression gene of AHL is designed into the toggle switch, and the toggle switch capable of sensing population density is constructed. The density of the bacteria determines the AHL concentration, and the AHL concentration determines LacI expression and further determines the expression of a target gene. The dynamic regulation of gene expression is realized, and the problem that the cell growth is influenced due to gene knockout, so that the yield is influenced is solved. For example, Tsuruno et al, applied toggle switches to the production of 3-hydroxypropionic acid, achieved higher yields and yields than traditional knock-out methods.

In conclusion, the toggle switch is a very important metabolic flow regulation tool and has wide application. However, the existing toggle switch control methods focus on regulation at the transcriptional level, i.e., by regulating the transcriptional expression level of the target pathway enzymes, changes in metabolic flux are controlled. When some specific chemicals are produced, a synthesis path is highly coupled with the central metabolism of the bacteria, and the transcription level is used for controlling and regulating the flow of a target path, and meanwhile, the physiological effect of the strain is greatly influenced. In order to overcome the above problems, it is necessary to directly regulate the protein concentration level of pathway enzymes without changing the transcription strength of the synthesis pathway of a target compound. Protein level toggle switches have the advantage of not affecting the expression of the target metabolic pathway, but currently there are few reports of toggle switch tools at the protein level.

Xylonic acid is an important chemical intermediate, can be used for pH regulators, cement binders and the like, and is listed as one of 30 most promising biological building block compounds in reports of the U.S. department of energy. At present, xylonic acid is produced by utilizing an engineering escherichia coli biological method, and pure xylose is mainly used as a substrate for fermentation. For example, in the invention patent with the application number of CN201210539717.4, 30g/L D-xylose is used as a substrate for fermentation, and 27.3g/L xylonic acid can be produced by 48 hours of fermentation. The pure xylose is adopted as the substrate for fermentation, so that the production cost of the product is greatly increased. However, when xylonic acid is produced by fermenting low-value cellulose hydrolysate (rich in glucose and xylose), the high concentration of glucose in the substrate can greatly influence the uptake and utilization of xylose by escherichia coli. This phenomenon is called the catabolite repression effect (CCR), i.e.when the fermentation system contains a preferentially utilized carbon source (glucose), the strain will preferentially utilize glucose and prevent uptake of a less preferentially utilized carbon source (xylose). At present, the research on the production of xylonic acid by using corn cob hydrolysate as a substrate exists, but the xylose content used in a conversion system is far higher than the glucose content (40g/L xylose +4g/L glucose). The literature reports 32.04g/L xylose +51.57g/L glucose in rice hull hydrolysates (Chang, Z.; Liu D.; Yang, Z.; Wu, J.; Zhuang, W.; Niu, H.; Ying, H.Effectient xylolite Production from Cornstalk Hydrolysate Using Engineered Escherichia coli J. agricultural Food Chem 2018,66(50), 13209.). Therefore, in the actual fermentation production at present, the xylonic acid fermentation system needs to supplement glucose in order to maintain the cell homeostasis, but the glucose concentration must be controlled at a lower concentration so as to avoid the CCR phenomenon and influence the intake of xylose by the bacteria. As disclosed in the Bioresource Technology study of Huaiwei Liu et al (see Liu, H.; Valdehuesa, K.N.; Nisola, G.M.; Ramos, K.R.; Chung, W.J.high yield process of D-xylonic acid from D-xylonic using engineered Escherichia coli Bioresource Technology 2012,115,244.), glucose was continuously supplemented to a final concentration of 0.4-0.8g/L in the fermentation of xylonic acid, which was complicated and costly. Therefore, the development of a tool capable of artificially regulating the utilization conditions of bacteria on different substrates in a xylose and glucose mixed sugar system has important significance for the research on the production of xylonic acid by taking cheap cellulose hydrolysate as the substrate.

Disclosure of Invention

The protein degradation seed and the protease recognition cleavage short peptide are added at the N terminal of the target protein in a fusion expression mode, and when the protease is not expressed, the target protein can stably exist in the bacterial cell and can exert biological activity. When the protease is expressed, the protease recognizes and cleaves the specific protease recognition short peptide, so that the originally hidden protein degradant is exposed at the N terminal of the target protein. Because bacteria generally have an N-terminal degradation mechanism, target proteins of naked protein degradants can be recognized by an intracellular protein degradation machine and are finally degraded into short peptides, and the biological activity cannot be performed.

In order to construct a toggle switch, the invention provides a protease cascade degradation strategy, namely, the idea of degrading protease by protease is utilized to regulate and control the abundance of intracellular protease and target protein. The specific operation comprises the following steps: first, in the first layer of regulation, the N-terminus of the target protein a is modified with a specific recognition site and a specific proteolytic degradation site of protease a, the N-terminus of the target protein B is modified with a specific recognition site and a specific proteolytic degradation site of protease B, and at the same time, the N-terminus of protease a is modified with a specific recognition site and a specific proteolytic degradation site of protease B. And the target protein b keeps stable and has biological activity. When protease B is further expressed, protease A and target protein B are degraded, and target protein a is allowed to accumulate in the cell and exert its biological activity again.

The first purpose of the invention is to provide a protein level toggle switch, which comprises a target protein a, a target protein B, a protease A and a protease B, wherein the N terminal of the target protein a is modified with a specific recognition site of the protease A and a protein degradation molecule; the N-terminal of the target protein B is modified with a specific recognition site of the protease B and a protein degradation molecule, and the N-terminal of the protease A is modified with a specific recognition site of the protease B and a protein degradation molecule; wherein, the protease A cuts and degrades the target protein a, and the protease B cuts and degrades the target protein B.

In one embodiment of the invention, the protease comprises tobacco etch virus protease TEVp, tobacco vein mottle virus protease TVMVp, sunflower mild mosaic virus protease SuMMVp or hepatitis c virus protease HICVp.

In one embodiment of the invention, the target protein a is xylulokinase xylB, the target protein B is ptsI, which is a key enzyme of the PTS system, protease A is TVMVp, and protease B is TEVp.

The second purpose of the invention is to provide a genetic engineering strain, which is an overexpression plasmid containing pTet-tev and pTrcHisA- (tvF) Ccxylb- (teF) tvmv- (teF) ptsI is expressed in E.coli BL21(DE3) which knocks out or silences gene XylA coding xylose isomerase, gene XylB coding xylulokinase and ptsI coding PTS system.

The pTet-33tev plasmid (GenBank accession number: MK238517), TEVp mutant containing six mutation points (T17S, L56V, N68D, I77V, S135G, S219V), and the key enzyme ptsI of the TEVp degradable PTS system.

The pTrcHisA- (tvF) CcxyB- (teF) tvmv- (teF) ptsI plasmid (GenBank access number: MK258728) contains three genes, namely a Caulobacter crescentis (Caulobacter crescentus) xylose dehydrogenase gene CcxyB containing a protease TVMVp recognition site and a phenylalanine degradation seed F, a protease gene tvmv containing a protease TEVp recognition site and a phenylalanine degradation seed F, and a ptsI gene of a coding PTS system containing a protease TEVp recognition site and a phenylalanine degradation seed F.

The third purpose of the invention is to provide a method for producing xylonic acid under the condition of mixed sugar substrates, which is to use the genetic engineering strain for fermentation.

In one embodiment of the invention, the mixed sugar is a mixed system of xylose and glucose, and the concentration ratio of the glucose to the xylose is not lower than 1, so as to simulate the ratio of the glucose to the xylose in the cellulose hydrolysate.

In one embodiment of the invention, the ratio of the concentration of glucose to xylose is 1 to 5.

In one embodiment of the invention, the fermentation conditions are: inoculating the genetic engineering bacteria into an LB culture medium, culturing at 36-38 ℃ and 200-220rpm for 8-14 h; inoculating the obtained seed culture solution into TB culture medium at an inoculation amount of 1-10%, fermenting at constant temperature of 36-38 deg.C and fermenting at 200-220 rpm.

In one embodiment of the invention, 0.1-0.5mM IPTG is added at the beginning of the fermentation for induction for 6-10h, and when the cell concentration in the fermentation broth reaches OD600 ═ 0.6-0.8, 180 ng/mL anhydrotetracycline is added for induction.

The invention also provides application of the protein toggle switch or the genetic engineering bacteria in the fields of food, pharmacy and chemical industry.

Has the advantages that: the protein level toggle switch constructed by the invention has the advantages of short response time and high regulation precision because regulation and control occur at the protein level and the principle is cutting and induced degradation triggered by protease. By utilizing the strategy, when the concentration ratio of the glucose to the xylose in the substrate is more than 1, the carbon metabolism repression phenomenon can be effectively relieved. When the ratio of glucose: when the ratio of xylose is 2:1, the introduction of the toggle switch can improve the yield of the xylonic acid by 0.79 time compared with a control group. The tool has good application prospect when cheap cellulose hydrolysate is used as a substrate to produce high value-added chemicals.

Drawings

FIG. 1: orthogonal cleavage test of proteases TEVp and TVMVp.

FIG. 2: protease cascade degradation test.

FIG. 3: the protein toggle switch applies a schematic diagram of mixed sugar fermentation to produce xylonic acid.

FIG. 4: the addition time of the inducer is optimized.

FIG. 5: and (4) comparing fermentation of the xylonic acid under different substrate proportion conditions.

Detailed Description

Preparation of a fermentation sample: taking a fermentation liquid sample, centrifuging for 5min by a centrifuge with the weight of 12,000g, taking supernate, diluting by a certain multiple, filtering by a water system membrane with the weight of 0.22.m, and taking the filtrate for liquid chromatography analysis.

Determination of xylonic acid content: the DEAN high performance liquid chromatograph (equipped with ultraviolet visible detector) adopts BerleAminex HPX-87H (300 × 7.8mm, 9 μ M) chromatographic column, and the mobile phase is H with concentration of 0.005M₂SO₄Filtering the mobile phase with 0.22 μm filter membrane, ultrasonic degassing at flow rate of 0.6mL/min and column temperature of 35 deg.C, and detecting at ultraviolet detection wavelength of 210 nm.

Seed culture medium components: LB culture medium, the ingredients include peptone 10g/L, yeast powder 5g/L, sodium chloride 10 g/L.

Fermentation medium components: the specific components of the TB culture medium comprise 24g/L of yeast extract, 12g/L of peptone, 9.4g/L of dipotassium phosphate and 2.2g/L of potassium dihydrogen phosphate. After sterilization, 10-20g/L of glucose and 10g/L of xylose are added.

The whole knockout of XylA encoding xylose isomerase, XylB encoding xylulokinase and ptsI encoding PTS system gene in e.coli BL21(DE3) was achieved by CRISPR-Cas9 technology.

Example 1: protease orthogonal assay

Recognition sites of proteases TEVp and TVMVp and a phenylalanine degradant are added at the N terminal of the reporter gene respectively. The report plasmids are respectively co-expressed with protease TEVp and TVMVp to construct four strains.

The above strains were cultured in LB medium, respectively, and when the cell concentration of the cells reached 0.8, an inducer was added. After further incubation for 12h, fluorescence measurements were performed using a SpectraMax M3 microplate reader. As shown in FIG. 1, only when the protease is co-expressed with the corresponding reporter protein, which also has a protease recognition site, a significant decrease in fluorescence intensity occurs, indicating that the cleavage by the protease is specific.

Example 2: protease cascade degradation experiments

In order to verify whether the protease has cascade degradation capability, mCherry red fluorescent protein is used as a reporter protein, and a protein recognition cleavage short peptide containing TVMVp and a phenylalanine degradation factor F are expressed at the N terminal of the reporter protein in a fusion manner to obtain (tvF) mCherry. Meanwhile, constructing a protease TVMVp of which the N terminal contains TEVp protein recognition cleavage short peptide and phenylalanine degradation factor F to obtain (teF) the TVMVp and the protease TEVp of which the N terminal is not genetically modified. The detection strain is placed in an LB culture medium for culture, and continuous fluorescence measurement is carried out by using a SpectraMax M3 microplate enzyme-labeling instrument. As shown in fig. 2, when (tvF) mCherry and (teF) TVMVp were co-expressed, the fluorescence intensity was low, indicating that the reporter protein was efficiently cleaved and degraded. Further, when TEVp was co-expressed, (tvF) mCherry and (teF) TVMVp, fluorescence was accumulated, indicating that (teF) TVMVp was degraded by TEVp and that (tvF) mCherry, which was originally cleaved and degraded, was re-accumulated for expression.

Example 3: construction of strain for producing xylonic acid chassis

As shown in fig. 3, e.coli BL21(DE3) was used as a chassis microorganism to combine knock-out the XylA gene encoding xylose isomerase and XylB gene encoding xylulokinase. The engineering strain can not grow by using single xylose as a carbon source. Furthermore, on the basis of the engineering strain, the PtsI gene is knocked out, and the strain cannot grow by taking glucose or xylose as a carbon source due to the blockage of a glucose transport system. The E.coli BL21(DE3) with the gene XylA coding xylose isomerase, the gene XylB coding xylulokinase and the ptsI gene coding PTS system knocked out is used as the host.

The principle of the protein toggle switch for producing xylonic acid by fermenting mixed sugar is that under the condition that the mixed sugar (glucose and xylose) is used as a substrate, IPTG inducer is added firstly, pTrcHisA- (tvF) CcxyB- (teF) tvmv- (teF) ptsI is expressed in a host, TVMVp is expressed, the stable existence of key enzyme ptsI of a PTS system is ensured, xylose dehydrogenase is degraded, cells only utilize glucose to carry out biomass synthesis, and the synthesis of products is not carried out. When the biomass of the strain is increased to a certain stage, the inducer anhydrotetracycline is added to express pTet-33tev, and another protease TEVp is expressed, so that ptsI starts to degrade and xylose dehydrogenase is accumulated. The glucose utilization pathway is blocked and xylose enters the cell to begin product synthesis.

Example 4: introduction of protein toggle switch for testing mixed sugar fermentation xylonic acid

The addition time of the inducer anhydrotetracycline is optimized first. As shown in FIG. 4, anhydrotetracycline was added to the strain at a final concentration of 200ng/mL after 4h, 6h, 8h and 10h of culture, respectively. The result shows that when the strain is cultured for 6 hours and then the inducer is added, the yield of the xylonic acid is the highest.

Under the conditions of different proportions of glucose and xylose, the conditions of fermentation of xylonic acid by an experimental group introduced with a toggle switch and a control group not introduced with the toggle switch are respectively tested. As shown in fig. 5, when glucose: when xylose is 0.5, the strain produces metabolic load due to the expression of a plurality of proteases in an experimental group, and the accumulation amount of the xylonic acid is lower than that in a control group. However, with glucose: the ratio of xylose was increased, the carbon metabolism repression effect began to become significant, and the production of xylonic acid in the control group was significantly decreased at the same xylose concentration. In contrast, the experimental group maintained substantially unchanged the production of xylonic acid due to the elimination of the carbon metabolism repression effect. Finally, when the glucose in the fermentation medium: when xylose is 2, the control group can only accumulate 2.37g/L of xylonic acid after fermentation for 48 hours, while the experimental group can accumulate 4.25g/L of xylonic acid, so that the yield is improved by 79%.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. A protein toggle switch is characterized by comprising a target protein a, a target protein B, a protease A and a protease B, wherein the N-terminal of the target protein a is modified with a specific recognition site of the protease A and a protein degradation molecule; the N-terminal of the target protein B is modified with a specific recognition site of the protease B and a protein degradation molecule, and the N-terminal of the protease A is modified with a specific recognition site of the protease B and a protein degradation molecule; wherein, the protease A cuts and degrades the target protein a, and the protease B cuts and degrades the target protein B; the target protein a is xylulokinase xylB, and the target protein b is key enzyme ptsI of PTS system; the protease A is TVMVp, and the protease B is TEVp.

2. A genetic engineering strain is characterized in that an overexpression plasmid containing pTet-tev and pTrcHisA- (tvF) Ccxylb- (teF) tvmv- (teF) ptsI is expressed by taking E.coli BL21(DE3) of knocking out or silencing a gene XylA coding xylose isomerase, a gene XylB coding xylulokinase and a ptsI gene coding PTS system as a host.

3. A method for producing xylonic acid under the condition of mixed sugar substrate, which is characterized in that the genetically engineered strain of claim 2 is used for fermentation, and the mixed sugar is xylose and glucose.

4. The method of claim 3, wherein the ratio of glucose to xylose concentration is not less than 1.

5. The method of claim 3, wherein the fermentation conditions are: inoculating the genetic engineering bacteria into an LB culture medium, culturing at 36-38 ℃ and 200-220rpm for 8-14 h; inoculating the obtained seed culture solution into TB culture medium at an inoculation amount of 1-10%, fermenting at constant temperature of 36-38 deg.C and fermenting at 200-220 rpm.

6. The method as claimed in claim 5, wherein 0.1-0.5mM IPTG is added at the beginning of the fermentation for induction for 6-10h, and 180 ng/mL anhydrotetracycline is added when the cell concentration in the fermentation broth reaches OD600= 0.6-0.8.

7. Use of the protein toggle switch of claim 1 or the genetically engineered bacterium of claim 3 in the production of xylose.