Engineering strain for producing xylitol through one-step whole-cell transformation of corncob xylan

Abstract

The invention discloses an engineering strain for producing xylitol through one-step whole-cell transformation of corncob xylan. The engineering strain comprises a recombinant escherichia coli strain for catalyzing the corncob xylan to produce the xylitol through a one-step method and a recombinant saccharomyces cerevisiae strain for catalyzing the corncob xylan to produce the xylitol through a one-step method. The invention provides a novel method for producing xylitol through double-enzyme synergistic effect and catalysis of corncob xylan by using double-enzyme co-expression engineering strain whole cells for the first time, the corncob xylan is hydrolyzed by using engineering strain cells, enzyme purification is omitted, the step of purifying xylose is simplified, the loss of xylose is reduced, and the yield of xylitol is increased. Xylose produced by engineering strain cell enzymolysis is directly used for producing xylitol. Wherein the content of xylitol produced by conversion of the engineering strain 2 by taking 10g / L of corncob xylan as a substrate is 7.65 g / L, and the yield of xylitol is 76.50%.

Description

Technical Field

[0001] The invention relates to the fields of bioengineering technology and the biosynthesis technology of natural compounds, and in particular to an engineering strain for producing xylitol by whole-cell conversion of corncob xylan in a one-step method. Background Art

[0002] Corn is one of the main food crops in the world, and its byproduct corncob resources are also very rich. Corncob is rich in xylan and is an ideal raw material for extracting xylan. Xylan is one of the most abundant polysaccharides in nature besides cellulose, and is composed of β-D-pyranose residues connected by β-1,4-glycosidic bonds. The research results of Wang Hai et al. show that the main components of monosaccharides in xylan from corncobs are xylose, arabinose and a small amount of glucose, which are mainly straight-chain xylan with few branches; the monosaccharide components in alcohol-insoluble corncob xylan are mainly xylose, arabinose, a small amount of glucose and galactose, but its arabinose is slightly higher than the former, and there are straight-chain and branched xylans with more branches.

[0003] Xylitol (C 5 H 10 O 5) is also called pentose, which is produced by hydrogenation reduction of D-xylose to xylitol. The sweetness and hygroscopicity of xylitol are similar to those of sucrose, and it is a natural functional sweetener. It is considered a high value-added chemical because of its wide range of uses and large market demand at home and abroad. In addition to being used as a sweetener in the food processing industry, xylitol also has multiple pharmacological effects such as anti-diabetes, prevention of dental caries, improvement of liver function, prevention of otitis media, and regulation of intestinal function. The U.S. Department of Energy (DOE) has calculated that the global xylitol market is 200,000 metric tons per year, and the value of the global xylitol industry is expected to reach US$6.93 billion by 2027. The current methods for producing xylitol mainly include direct extraction, chemical synthesis, and biotransformation. The direct extraction method is mainly extracted directly from plant raw materials by solvent extraction, but the method has low yield, high energy consumption, and high cost. Chemical synthesis is currently a commonly used method in industry. Under high temperature and high pressure hydrogenation conditions, xylose from various lignocellulose sources is hydrogenated to xylitol using nickel as a catalyst. However, this method is gradually being replaced by bioconversion due to its harsh process conditions, high production costs, and environmental non-friendliness. The biological method uses microorganisms to metabolize xylose through the xylose isomerase pathway or the xylose reductase-xylitol dehydrogenase pathway to reduce xylose to xylitol. Currently, natural microorganisms that use xylose fermentation to produce xylitol include bacteria, actinomycetes, molds, and yeasts. Among them, yeast (especially Candida) has the best conversion ability, which can reach 84.5%. In addition to natural xylose fermentation strains, recombinant engineered bacteria constructed using xylose reductase genes can also ferment and produce xylitol. Since recombinant Saccharomyces cerevisiae cannot metabolize xylose, the conversion rate of xylitol is relatively high, reaching 86%-100%, but the concentration of xylose substrate used is relatively low. Du et al. treated with acid (0.5% H 2 SO 4 and 1.5% H 3 PO 4 , 128℃ for 1h) corncob pulp as raw material, using Kluyveromyces marxianus CICC 1727-5 for two-stage fermentation to produce xylitol and ethanol, the maximum concentration of xylitol was 24.2g / L, and the yield was 0.82g / g. Candida tropicalis K2 screened out in the study of Singh et al. can produce xylitol with 100g / L xylose and 20g / L glycerol as co-substrates, with a yield of 0.90g / g; the yield of xylitol produced with corncob hydrolysate as substrate is 0.62g / g.

[0004] Most of the existing technologies are about the optimization of the process methods or devices for industrial preparation of xylitol, and some natural strains and engineered bacteria that ferment xylose to produce xylitol. Among them, most of the engineered bacteria either use pure xylose or a xylose / glucose mixture as a substrate, or use agricultural waste hydrolyzate as a substrate to ferment and produce xylitol. The disadvantage of using xylose as a raw material is the high production cost. Using agricultural waste hydrolyzate as a raw material requires the raw material to be pretreated with high temperature and acid, and the process is relatively complicated. Summary of the invention

[0005] The present invention aims at the problem of high loss and low yield of producing xylitol in two steps in the prior art (i.e. using agricultural waste to separate and purify xylose and then using it to produce xylitol), and provides a method for producing xylitol in one step by catalyzing the hydrolysis of corn cob xylan using a double enzyme co-expression recombinant engineering strain.

[0006] To achieve the above purpose, the technical solution provided by the present invention is as follows:

[0007] The engineered strains for one-step whole-cell conversion of corncob xylan to produce xylitol are a recombinant E. coli strain E. coli-pSE-spbgl5-T5-xyl1 that catalyzes the one-step production of xylitol from corncob xylan and a recombinant Saccharomyces cerevisiae strain INVSc1-PGK-spbgl5-ADH1-xyl1 that catalyzes the one-step production of xylitol from corncob xylan.

[0008] A recombinant Escherichia coli strain E. coli-pSE-spbgl5-T5-xyl1 catalyzes the one-step production of xylitol from corncob xylan. A gene encoding β-glucosidase SPBGL5 and a gene encoding xylose reductase XYL1 are introduced into E. coli together to construct a recombinant E. coli containing a secretory double expression cassette of SPBGL5, so that the two genes can be expressed simultaneously in one E. coli host, thereby obtaining an engineered strain 1, namely E. coli-pSE-spbgl5-T5-xyl1.

[0009] Preferably, the construction method of the engineered strain 1 is to design XYL1F and XYL1R primer pairs to amplify the expression cassette fragment containing the xylose reductase XYL1 gene, and use the In-Fusion technology to clone and recombinantly clone the expression cassette fragment containing the xylose reductase XYL1 gene into the pSE-spbgl5 plasmid; then use the designed XS5-1F and XS5-1R primer pairs to use the reverse PCR technology and the In-Fusion technology to further transform and construct the SPBGL5 secretory double expression cassette vector pSE-spbgl5-T5-xyl1, and finally transfer it into Escherichia coli E.coli JM109 to obtain the engineered strain 1.

[0010] A recombinant Saccharomyces cerevisiae strain INVSc1-PGK-spbgl5-ADH1-xyl1 is disclosed for catalyzing the one-step production of xylitol from corncob xylan. β-glucosidase SPBGL5 and xylose reductase XYL1 are co-expressed in a yeast plasmid pESC-ura, and a co-expression vector is obtained after optimizing the promoter. The obtained co-expression vector is transferred into Saccharomyces cerevisiae INVSc1 to obtain an engineered strain 2, i.e., INVSc1-PGK-spbgl5-ADH1-xyl1.

[0011] Preferably, the promoter is a PGK promoter or an ADH1 promoter.

[0012] Preferably, the construction method of the engineered strain 2 is to design primer pairs of XYL1F, XYL1R and SPBGL5-1F, SPBGL5-1R, optimize the promoter using In-Fusion technology, construct a co-expression vector PGK-spbgl5-ADH1-xyl1, and transfer it into Saccharomyces cerevisiae INVSc1 to obtain engineered strain 2.

[0013] The engineering strain 1 and the engineering strain 2 as described above are used to catalyze the conversion of corncob xylan into xylitol in a one-step process.

[0014] The present invention provides two engineering strains 1 and 2 co-expressing beta-glucosidase SPBGL5 and xylose reductase XYL1, and a method for catalyzing and hydrolyzing corncob xylan by using a recombinant saccharomyces cerevisiae strain to produce xylitol by one-step fermentation and catalytic hydrolysis.

[0015] The present invention provides two engineering strains for directly converting corncob xylan to produce xylitol, one is an engineering strain 1 of a prokaryotic Escherichia coli engineering strain, and the other is an engineering strain 2 of a eukaryotic saccharomyces cerevisiae engineering strain. The present invention firstly uses a preliminary experiment to catalyze and hydrolyze 10 g / L corncob xylan with a recombinant Escherichia coli strain whole cell to prepare a xylose hydrolysate, and adds tropical Candida tropicalis to the hydrolysate for fermentation to produce xylitol. After 48 hours of fermentation, when the xylose concentration in the hydrolysate is 6.94 g / L, the maximum yield of xylitol is 2.95 g / L. After verifying its feasibility, the xylose reductase XYL1 from tropical Candida tropicalis and the above-mentioned β-glucosidase SPBGL5 are cloned into the engineering strain to synergistically catalyze and hydrolyze corncob xylan to produce xylitol in one step. First, the engineered strain of Escherichia coli was used to successfully construct the recombinant strain and then optimize its secretory expression to obtain the engineered strain 1 with co-expression of the SPBGL5 secretory double expression cassette. Since the recombinant Saccharomyces cerevisiae cannot metabolize xylose, it can reduce the loss of xylose in the metabolic process. Based on the above research, the engineered strain of Saccharomyces cerevisiae was further constructed, and the co-expressed engineered strain 2 was obtained after optimizing the promoter. The above two engineered strains are the engineered strains that catalyze the one-step production of xylitol from corncob xylan.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] (1) The present invention provides for the first time a novel method for producing xylitol from corncob xylan by catalyzing the whole cell of a dual-enzyme co-expressing engineered strain, using the engineered strain cells to hydrolyze corncob xylan, eliminating the need for enzyme purification, simplifying the steps of purifying xylose, and reducing the loss of xylose. The xylitol produced by the enzymatic hydrolysis of the engineered strain cells is directly used to produce xylitol. The engineered strain 2 can produce 7.65 g / L of xylitol by using 10 g / L of corncob xylan as a substrate, and the xylitol yield is 76.50%.

[0018] (2) The two enzymes involved in the present invention have clear physicochemical properties, can synergistically hydrolyze corn cob xylan and be co-expressed in the same host. The whole-cell hydrolysis method is safer, more environmentally friendly, and easier to operate than the existing traditional separation method. In the process of cell-catalyzed hydrolysis of corn cob xylan to produce xylitol, it has the characteristics of mild reaction conditions, environmental friendliness, high conversion rate, and great application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1This is a denaturing polyacrylamide gel electrophoresis (SDS-PAGE) diagram of the purified β-glucosidase SPBGL5 in Example 1 of the present invention; wherein M is a protein molecular weight standard, and 1 is purified SPBGL5.

[0020] Figure 2 This is a HPAEC analysis chart of the hydrolysis product of corncob xylan hydrolyzed by β-glucosidase SPBGL5 used in Example 1 of the present invention; wherein a is the xylose standard sample, b is the control group, and c is the SPBGL5 experimental group.

[0021] Figure 3 Schematic diagram of the construction of recombinant plasmids of engineering strain 1 and engineering strain 2 in Example 2 of the present invention; wherein, engineering strain 1 comprises the co-expression vector pSE-spbgl5-T5-xyl1; and engineering strain 2 comprises the co-expression vector PGK-spbgl5-ADH1-xyl1.

[0022] Figure 4 The figures are HPAEC analysis graphs of the production of xylitol by catalytic hydrolysis of corn cob xylan by two engineering strains 1 and 2 in Example 3 of the present invention; wherein A is the HPAEC detection graph of the fermentation broth of engineering strain 1, a is the xylitol standard, b is the xylose standard, c is the control group, and d is the experimental group of engineering strain 1; B is the HPACE detection graph of the fermentation broth of engineering strain 2, a is the xylitol standard, b is the xylose standard, c is the control group, and d is the experimental group of engineering strain 2.

[0023] Figure 5 The figure is a schematic diagram of the results of fermenting corn cob xylan with a substrate concentration of 10 g / L to produce xylitol by the engineered strain 2 in Example 4 of the present invention using a microaerobic intermittent fed-batch fermentation method; wherein the curve xylitol (glucose) represents the production of xylitol during the cell fermentation process when 2 g / L glucose is used as an auxiliary carbon source; the curve xylitol (glucose) represents the consumption of xylose during the cell fermentation process when 2 g / L glucose is used as an auxiliary carbon source; the curve xylitol (galactose) represents the production of xylitol during the cell fermentation process when 2 g / L galactose is used as an auxiliary carbon source; the curve xylitol (galactose) represents the consumption of xylose during the cell fermentation process when 2 g / L galactose is used as an auxiliary carbon source. DETAILED DESCRIPTION

[0024] The specific implementation is described in detail below in conjunction with the accompanying drawings, but it should be understood that the scope of protection of the present invention is not limited by the specific implementation. Unless otherwise specified, the raw materials, reagents, etc. used in the examples are all commercially available, and corncob xylan is purchased from MacLean. The β-glucosidase SPBGL5 gene used in the present invention is from patent ZL201710695489.2. The main Escherichia coli E.coli JM109 and Saccharomyces cerevisiae INVSc 1 are commercially available.

[0025] Example 1

[0026] Hydrolysis of corncob xylan by purified β-glucosidase SPBGL5

[0027] (1) Add IPTG with a final concentration of 0.5 mM, induce and culture the constructed recombinant E. coli JM109 / pSE-spbgl5 at 30°C and 180 rpm for 10 h, collect the induced cultured bacteria, break the cells and purify them to obtain the corresponding pure enzyme solution, take the desalted protein and perform SDS-PAGE analysis, and find a protein band of the expected target size; Figure 1 It can be seen that the molecular weight of the purified recombinant β-glucosidase SPBGL5 is about 87 kDa;

[0028] (2) Analysis of products of corncob xylan hydrolyzed by β-glucosidase SPBGL5: Corncob xylan with a final concentration of 0.5% (w / v) was used as substrate, pure enzyme solution was added at a concentration of 60 μg / mL, and the reaction was carried out at pH 6.0 and 50°C for 6 h. The reaction was terminated by boiling, and the reaction solution was subjected to HPAEC analysis. Inactivated β-glucosidase SPBGL5 was added as a control, and commercially purchased xylose was used as a standard.

[0029] HPAEC test results Figure 2 As shown, the results showed that the product of corncob xylan hydrolyzed by β-glucosidase SPBGL5 was only xylose, and the xylose conversion rate of corncob xylan with a final concentration of 0.5% (w / v) after hydrolysis was 80.58%.

[0030] Example 2

[0031] The primers used to construct the co-expression engineering strain are summarized in Table 1. Unless otherwise specified, PCR amplification, enzyme digestion, T 4 DNA ligase ligation and In-Fusion enzyme ligation steps were performed according to the operation manual. The construction method operation steps are as follows:

[0032] Table 1 Primers used in the construction of engineering strains

[0033]

[0034]

[0035] (1) Design primer pairs XYL1F and XYL1R, and clone and recombinant the gene encoding xylose reductase XYL1 into the pSE-spbgl5 plasmid using the In-Fusion technology. Use the In-Fusion technology to design primer pairs XS5-1F and XS5-1R to further transform and construct a double expression cassette co-expression recombinant plasmid pSE-spbgl5-T5-xyl1 containing secretory SPBGL5, and transform it into E. coli JM109 to obtain the engineered strain 1, namely E. coli-pSE-spbgl5-T5-xyl1.

[0036] (2) Design primer pairs of XYL1F, XYL1R and SPBGL5-1F, SPBGL5-1R, and optimize the promoter using In-Fusion technology to construct the co-expression recombinant plasmid PGK-spbgl5-ADH1-xyl1, which was then transferred into Saccharomyces cerevisiae INVSc1 to obtain engineered strain 2, INVSc1-PGK-spbgl5-ADH1-xyl1. The schematic diagram of the recombinant plasmid is shown in Figure 2. Figure 3 shown.

[0037] Example 3

[0038] The engineered strain 1 and the engineered strain 2 constructed in Example 2 were used to catalyze the hydrolysis of corncob xylan to produce xylitol. The specific operation steps are as follows:

[0039] (1) The engineered strain 1 was fermented in TB medium containing 10 g / L corncob xylan for 48 h, and the components of the fermentation broth were detected using HPAEC (experimental group).

[0040] (2) The engineered strain 2 was fermented in YDP medium containing 10 g / L corncob xylan, and the components of the fermentation broth were detected using HPAEC (experimental group).

[0041] Detection and analysis:

[0042] (1) HPAEC analysis conditions:

[0043] Instruments and equipment: Dionex ICS-5000 series high-performance ion chromatograph;

[0044] Chromatographic column: CarboPac PA100 Analytical Column (4×250 mm);

[0045] Detection conditions: detection wavelength 210nm, column temperature 40℃, flow rate 1mL / min;

[0046] Mobile phase A: ultrapure water, mobile phase B: 0.5mol / L NaOH, mobile phase C: 0.5mol / L NaOAC and 0.08mol / L NaOH. Gradient elution: 0-10min (15% B, 2% C); 10-20min (15-50% B, 2-20% C); 20-25min (50-15% B, 20-2% C); 25-30min (15% B, 2% C), flow rate 1mL / min, detection time 30min;

[0047] (2) Results

[0048] like Figure 4 As shown, HPAEC was used to analyze the fermentation broth in the step of the above Example 3. The xylitol production of the engineered strain 1 in the TB medium containing 10 g / L corncob xylan after fermentation for 48 h was 4.23 g / L. After optimizing the fermentation conditions, the xylitol content of 10 g / L corncob xylan fermented for 48 h at an IPTG concentration of 0.5 mM and 30°C was the highest, which was 6.54 g / L, and the corresponding yield was 65.40%.

[0049] The xylitol yield of the engineered strain 2 after fermentation in YPD medium containing 10 g / L corncob xylan was 4.56 g / L.

[0050] Saccharomyces cerevisiae INVSc1 harboring the plasmid pESC-ura without the spbgl5 and xyl1 genes was used as a control, and commercially purchased xylitol and xylose were used as standards.

[0051] Example 4

[0052] The growth of the engineered strains with different auxiliary carbon sources of the engineered strain 2 with higher xylitol production in Example 3 was optimized:

[0053] Different auxiliary carbon sources (2 g / L glucose, 2 g / L galactose) were added to the YDP medium to obtain YDP medium i. The engineered strain 2 was fermented in YDP medium i containing different carbon sources such as 10 g / L corncob xylan, and the fermentation broth components were detected using HPAEC.

[0054] The xylitol production of engineered strain 2 was the highest when glucose was used as the auxiliary carbon source, which was 4.56 g / L, followed by galactose as the auxiliary carbon source, with a xylitol production of 3.82 g / L. The fermentation conditions were optimized: in the first stage, in order to allow sufficient growth and exogenous gene expression of engineered strain 2, aerobic fermentation was used for the first 24 hours, and the rotation speed was set to 170 rpm; the second stage was the fermentation after 24 hours, using microaerobic fermentation, and the rotation speed was set to 120 rpm. Engineered strain 2 used glucose as an auxiliary carbon source and fermented xylitol at a rate of 7.65 g / L in a microaerobic intermittent feeding mode, with a corresponding yield of 76.50%. Figure 5 shown.

[0055] In summary, the present invention provides for the first time an engineering strain that uses a dual enzyme synergistic effect and a dual enzyme co-expressing Escherichia coli strain and a Saccharomyces cerevisiae strain to catalyze the hydrolysis of corncob xylan to produce xylitol. The corncob xylan is directly hydrolyzed by a one-step method, and the xylose in the hydrolyzate is directly converted to produce xylitol, which simplifies the purification process and reduces the loss of xylose. Among them, the engineering strain 2 uses 10g / L corncob xylan as a substrate for micro-aerobic intermittent feeding fermentation, and the yield of xylitol is 7.65g / L, and the conversion rate is 76.50%. The invention has the advantages of mild reaction conditions, environmental friendliness, cost and time savings, and great application potential.

[0056] The foregoing description of specific exemplary embodiments of the present invention is for the purpose of illustration and demonstration. These descriptions are not intended to limit the present invention to the precise form disclosed, and it is clear that many changes and variations can be made based on the above teachings. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the present invention and its practical application, so that those skilled in the art can realize and utilize various different exemplary embodiments of the present invention and various different selections and changes. The scope of the present invention is intended to be limited by the claims and their equivalents.

Claims

1. A recombinant Escherichia coli strain that catalyzes the one-step production of xylitol from corncob xylan, characterized in that: The gene encoding β-glucosidase SPBGL5 and the gene encoding xylose reductase XYL1 were introduced into Escherichia coli together to construct a recombinant Escherichia coli containing a secretory double expression cassette of SPBGL5, so that these two genes can be expressed simultaneously in one Escherichia coli host, thus obtaining an engineered strain 1, namely E. coli-pSE-spbgl5-T5-xyl1.

2. A recombinant Saccharomyces cerevisiae strain that catalyzes the one-step production of xylitol from corncob xylan, characterized in that: β-glucosidase SPBGL5 and xylose reductase XYL1 were co-expressed in yeast plasmid pESC-ura, and the promoter was optimized to obtain a co-expression vector, which was then transferred into Saccharomyces cerevisiae INVSc1 to obtain engineered strain 2, i.e., INVSc1-PGK-spbgl5-ADH1-xyl1.

3. Use of the recombinant Escherichia coli strain for catalyzing the one-step production of xylitol from corncob xylan as claimed in claim 1 and the recombinant Saccharomyces cerevisiae strain for catalyzing the one-step production of xylitol from corncob xylan as claimed in claim 2 in catalyzing the one-step conversion of corncob xylan into xylitol.

Images