Use of mutant gene of zmnst2 and mutant thereof in production of bioethanol by means of corn stover fermentation

Abstract

The present invention relates to the technical field of genetic engineering and breeding. Provided is the use of a mutant gene of ZmNST2 and a mutant thereof in the production of bioethanol by means of corn stover fermentation. The mutant gene of ZmNST2 is obtained via molecular genetic means. Additionally, by means of the determination of lignin content and ethanol fermentation level, it is found that the mutant gene of ZmNST2 is capable of reducing the lignin content in corn, reducing the content of fermentation inhibitors and improving the hydrolysis rate of cellulase, such that the yield and efficiency in the production of bioethanol by means of corn stover fermentation can be improved. The provided mutant gene of ZmNST2 plays a critical role in the use of same in the production of bioethanol by means of corn stover fermentation, and is an important resource for cultivating new corn varieties that have high ethanol yield by means of fermentation.

Description

Application of a ZmNST2 mutant gene and its mutant in bioethanol production by corn straw fermentation Technical Field

[0001] The present invention relates to the technical field of genetic engineering breeding, and in particular to the application of a ZmNST2 mutant gene and a mutant thereof in the production of bioethanol by fermentation of corn straw. Background Art

[0002] Energy is essential for human survival. Fossil energy, as the primary energy source, has made tremendous contributions to the development of human society. Currently, the use of fossil energy not only has adverse environmental impacts, such as the intensification of smog and worsening water and soil pollution, but is also becoming increasingly scarce, posing a significant threat to human survival. Therefore, the search for a sustainable and green energy source is urgent. Bioethanol, due to its green and renewable nature, has become a hot topic in energy research. First-generation bioethanol production primarily uses sugars and starches from grain as fermentation feedstock. Initially, this approach addressed the problem of stockpiled grain. However, the rapid development of grain-based bioethanol production has not only consumed large quantities of old grain but also led to the use of new grain, which not only impacts my country's food security but also violates the concept of sustainable development. Second-generation bioethanol is produced using lignocellulosic biomass, the most abundant natural, renewable carbohydrate resource. One of its raw materials is crop straw. This bioethanol offers significant advantages in industrial production, such as stable yields, and the potential to alleviate the atmospheric and environmental pollution caused by the discarding and incineration of straw during traditional production processes.

[0003] The production of cellulosic ethanol from lignocellulose primarily involves enzymatic hydrolysis of the cellulose and hemicellulose produced after pretreatment, followed by fermentation of fermentable sugars (primarily glucose and xylose) to produce ethanol. Lignocellulose is primarily composed of cellulose, hemicellulose, and lignin. Its structure is characterized by densely packed cellulose within, lignin surrounding the exterior, and hemicellulose connecting the fibers in the middle. The lignin coating provides support and protection for the surface of the lignocellulose fibers. However, due to their high hydrophobicity, degree of polymerization, and chemical stability, the cellulase enzymes used for hydrolysis have difficulty hydrolyzing the cellulose and hemicellulose within. Dilute acid hydrolysis is the most commonly used chemical pretreatment method for enzymatic hydrolysis of lignocellulose. This pretreatment dissolves most of the hemicellulose and disrupts the lignin structure, increasing the cellulose hydrolysis rate and thus the amount of ethanol produced by fermentation. However, during pretreatment, soluble inhibitors such as furfural and phenolic compounds are generated, which typically inhibit fermentation and reduce ethanol yield. Corn is a widely cultivated crop in my country. Its higher straw biomass compared to crops like wheat and rice makes it a promising candidate for lignocellulosic ethanol production. Discovering the genes that affect ethanol production from corn straw fermentation is of great significance for breeding new corn varieties with high fermentation ethanol yields, preparing bioethanol, energy sustainability, and environmental improvement.

[0004] Summary of the Invention

[0005] The present invention aims to provide a ZmNST2 mutant gene and its mutants for use in bioethanol production from corn straw fermentation, addressing the aforementioned problems of the prior art. The present invention provides a ZmNST2 mutant gene that reduces lignin content, reduces fermentation inhibitors, and improves cellulase hydrolysis and fermentation ethanol yield. This mutant gene plays a key role in the production of bioethanol from corn straw and represents an important resource for this purpose.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a mutant gene of ZmNST2, wherein the nucleotide sequence of the mutant gene is shown as SEQ ID No: 1 or SEQ ID No: 2.

[0008] The present invention also provides a biological material containing the mutant gene of ZmNST2, wherein the biological material comprises an expression vector or a recombinant bacterium.

[0009] The present invention also provides a use of the mutant gene or biological material of ZmNST2 in creating a corn mutant, wherein the mutant gene or biological material of ZmNST2 is introduced into a corn material to obtain the corn mutant.

[0010] The present invention also provides a use of the mutant gene or biological material or corn mutant of ZmNST2 in the production of bioethanol by fermentation of corn straw.

[0011] Furthermore, the mutant gene, biological material or corn mutant of ZmNST2 improves the efficiency of producing bioethanol by fermentation of corn straw.

[0012] Furthermore, by reducing the content of lignin in corn straw, the efficiency of fermentation to produce bioethanol is improved.

[0013] Furthermore, the efficiency of bioethanol production by fermentation is improved by reducing the content of fermentation inhibitors after corn straw pretreatment.

[0014] Furthermore, by increasing the hydrolysis rate of cellulase in corn straw, the efficiency of fermentation to produce bioethanol is improved.

[0015] The present invention also provides an application of the mutant gene of ZmNST2 or the expression vector or recombinant bacteria in molecular breeding of corn.

[0016] The present invention discloses the following technical effects:

[0017] The present invention discovered that the zmnst2 mutant exhibits a pronounced limp leaf phenotype compared to wild-type corn plants. Lignin and ethanol fermentation level measurements revealed that the zmnst2 mutant exhibited reduced lignin content, fewer fermentation inhibitors, and improved cellulase degradation and ethanol fermentation capacity compared to the wild-type. Furthermore, the present invention obtained a mutant gene of ZmNST2 through molecular genetics and found that this mutant gene can reduce the lignin content in corn straw, resulting in fewer fermentation inhibitors and an increased cellulase hydrolysis rate after corn straw pretreatment, thereby improving the quality and efficiency of bioethanol fermentation. Therefore, the ZmNST2 mutant gene provided by the present invention has important application value for cultivating new corn varieties with high fermentation ethanol yields, producing bioethanol, promoting energy sustainability, and improving the environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0019] Figure 1 shows the genetic structure of ZmNST2 and the plant phenotypes of its mutants. Figure A: The genetic structure of ZmNST2, with red lines marking the physical locations of the mutation sites in the zmnst2-1 and zmnst2-2 mutants. Figure B: The plant phenotypes of wild-type B73, the mutants zmnst2-1, zmnst2-2, and zmnst2-1 / zmnst2-2. Scale bar = 10 cm.

[0020] Figure 2 shows the lignin content staining of wild-type B73, mutants zmnst2-1, zmnst2-2, and zmnst2-1 / zmnst2-2, wherein the first, third, and fifth rows are overall images of roots, stem nodes, and leaf veins, respectively, with a scale bar of 1 mm; the second, fourth, and sixth rows are partial magnified images of roots, stem nodes, and leaf veins, respectively, with a scale bar of 0.5 mm;

[0021] Figure 3 shows the analysis of lignin and cellulose content in zmnst2; Figure A: Monolignin content in roots of wild-type B73, mutant zmnst2-1, and zmnst2-2 plants; B: Monolignin content in ear nodes of wild-type B73, mutant zmnst2-1, and zmnst2-2 plants; C: Monolignin content in ear leaves of wild-type B73, mutant zmnst2-1, and zmnst2-2 plants; D: Cellulose content in roots, ear nodes, and ear leaves of wild-type B73, mutant zmnst2-1, and zmnst2-2 plants; different letters indicate significant differences (P<0.05);

[0022] Figure 4 shows the analysis of ethanol fermentation indicators of zmnst2; Figure A: the content of acid-insoluble lignin, glucan, and xylan in the straw of wild-type B73 and mutant zmnst2-1; B: the content of glucose and xylose in the straw of wild-type B73 and mutant zmnst2-1 after dilute acid pretreatment; C: the content of fermentation inhibitor phenolic compounds (AT 280 ) and the contents of furfural and 5-hydroxymethylfurfural, the toxic substances; D: ethanol content 1 produced by co-fermentation of glucose and xylose in the straws of wild-type B73 and mutant zmnst2-1, and ethanol content 2 produced by glucose fermentation alone after cellulose hydrolysis by cellulase; E: hydrolysis rate of cellulase in the straws of wild-type B73 and mutant zmnst2-1; *P<0.05; **P<0.005; ***P<0.001, ns indicates no significant difference. DETAILED DESCRIPTION

[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0024] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0025] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0026] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0027] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0028] Example 1

[0029] 1. Experimental Materials and Methods

[0030] 1 Source of Materials

[0031] Mutants zmnst2-1 and zmnst2-2 were purchased from the maize mutant library (https: / / elabcaas.cn / memd / public / index.html# / , with original mutant numbers EMS4-134f89 and EMS4-1ad22e, respectively). Prior to this study, no functional studies of the zmnst2 mutant had been reported. This study investigated the zmnst2 mutant for the first time, and the mutant gene of ZmNST2 was identified using molecular genetics. Details are as follows.

[0032] 2 Experimental methods

[0033] 2.1 Lignin staining

[0034] During field cultivation, the zmnst2 mutant exhibited a noticeably softer leaf phenotype compared to the wild-type B73, suggesting a potential reduction in lignin content. The total lignin content in roots, ear nodes, and leaf veins of wild-type B73, zmnst2-1, zmnst2-2, and the zmnst2-1 / zmnst2-2 (F1 generation hybrids of zmnst2-1 and zmnst2-2) mutant was observed using the Wiesner staining method. First, tissue samples were cut into uniform slices approximately 1 mm in diameter using a razor blade. The slices were then dechlorophyllized in 75% alcohol and subsequently placed in Wiesner staining solution until color developed. The slices were then observed under a stereomicroscope and photographed for preservation.

[0035] 2.2 Determination of lignin and cellulose content

[0036] 2.2.1 Extraction of cell wall

[0037] First, the sample was air-dried and ground into a powder. 70 mg of the powder was added to 1.5 mL of 70% ethanol and heated in a 70°C water bath, after which the supernatant was discarded. 1.5 mL of chloroform:methanol (v / v = 1:1) was added and the sample was resuspended by vortexing. 1 mL of acetone was then added, and the sample was resuspended by vortexing and the supernatant was discarded. This step was repeated several times until the supernatant was colorless. The treated sample was then dried at 35°C to obtain the cell wall.

[0038] 2.2.2 Determination of lignin content

[0039] Cell wall samples from different tissues (about 20 mg of root and leaf tissue, about 10 mg of stem node tissue) were weighed and analyzed by GC-MS using the thioacid hydrolysis method.

[0040] 2.2.3 Determination of cellulose content

[0041] About 5 mg of cell wall sample was weighed and treated with 1 mL of acetic acid and nitric acid solution (acetic acid: nitric acid: water = 8:1:2, v / v) to hydrolyze cellulose into glucose. The glucose content (representing the cellulose content) was then determined using the anthrone colorimetric method.

[0042] 2.3 Fermentation index determination

[0043] Corn straw was air-dried, crushed, and sieved, and 20-40 mesh corn straw powder was selected for use. The contents of acid-insoluble lignin, hemicellulose (glucan), and cellulose (xylan) were determined using the three-element assay method of the National Renewable Energy Laboratory of the United States. The straw powder was pretreated by dilute sulfuric acid hydrolysis, and the content of phenolic compounds in the hydrolyzate was determined by UV-visible spectrophotometry (AT 280), and the contents of furfural, 5-hydroxymethylfurfural, glucose, and xylose in the hydrolyzate were detected using a high-performance liquid chromatography. Pichia stipitis (CICC1960, purchased from the China Industrial Microbiological Culture Collection) was used to ferment the detoxified and decolorized hydrolyzed sugar solution to produce ethanol (ethanol 1), and the ethanol concentration was then detected using a high-performance liquid chromatography. The pretreated cellulose residue was first neutralized with NaHCO3, then rinsed with tap water, and finally the dried cellulose was hydrolyzed using cellulase. Saccharomyces cerevisiae P1 (screened and preserved by Zhang Qin's research group at Anhui University of Technology) was used to ferment the hydrolyzed sugar solution to produce ethanol (ethanol 2), and the ethanol concentration was then detected using a high-performance liquid chromatography.

[0044] 2. Experimental Results

[0045] 1 Phenotypes of zmnst2 mutants

[0046] ZmNST2 has three exons. As shown in Figure 1A, the mutants zmnst2-1 and zmnst2-2, respectively, have a single G-to-A mutation in the second exon of the ZmNST2 gene, 658 and 769 bp from the start codon, resulting in premature termination of amino acid translation. Other loss-of-function mutations within this region can reduce lignin content, reduce fermentation inhibitors, and increase cellulase hydrolysis, thereby enhancing bioethanol production from corn straw fermentation.

[0047] The nucleotide sequences of the ZmNST2 mutant genes of mutants zmnst2-1 and zmnst2-2 are shown in SEQ ID NOs: 1-2:

[0048] Mutant zmnst2-1 (SEQ ID NO: 1):

[0049] Mutant zmnst2-2 (SEQ ID NO: 2):

[0050] Compared to wild-type B73, the mutants zmnst2-1 and zmnst2-2 exhibited a soft leaf phenotype. Crossing zmnst2-1 and zmnst2-2 to obtain F1 lines and conducting genetic allelic testing revealed that zmnst2-1 / zmnst2-2 also exhibited soft leaves (Figure 1B), indicating that the ZmNST2 gene mutation causes changes in maize leaf firmness and firmness, and that zmnst2-1 and zmnst2-2 are allelic mutants of the ZmNST2 gene. Compared to wild-type B73, the mutants zmnst2-1, zmnst2-2, and zmnst2-1 / zmnst2-2 exhibited no significant differences in other morphologies.

[0051] 2 Lignin staining of zmnst2

[0052] One possible cause of maize leaf softening is reduced lignin content. Therefore, we first stained the roots, ear nodes, and ear veins of wild-type B73 and the mutants zmnst2-1, zmnst2-2, and zmnst2-1 / zmnst2-2 for total lignin (Figure 2). The results showed that total lignin staining in the roots, ear nodes, and ear veins of the zmnst2-1, zmnst2-2, and zmnst2-1 / zmnst2-2 mutants was significantly weaker than that of wild-type B73. This suggests that the ZmNST2 mutation reduces lignin content in the roots, nodes, and veins of maize plants, and that zmnst2-1 and zmnst2-2 are allelic mutants.

[0053] 3 Analysis of lignin and cellulose content in zmnst2

[0054] To further determine whether ZmNST2 regulates lignin accumulation, we measured the contents of G and S lignin monomers in roots (Figure 3A), ear nodes (Figure 3B), and ear leaves (Figure 3C) of wild-type B73, zmnst2-1, and zmnst2-2 mutants. The results showed that the contents of G and S lignin in the roots, ear nodes, and leaves of zmnst2-1 and zmnst2-2 were significantly lower than those of wild-type B73, while the S / G ratios did not differ significantly. We also analyzed the cellulose content in the roots, ear nodes, and leaves of wild-type B73, zmnst2-1, and zmnst2-2 mutants (Figure 3D). The results showed that the cellulose content in the roots, ear nodes, and leaves of zmnst2-1 and zmnst2-2 mutants was not significantly different from that of wild-type B73.

[0055] 4. Analysis of fermentation indicators of zmnst2

[0056] In addition, the lignin and cellulose contents of the straw of wild-type B73 and the zmnst2-1 mutant were further measured using the US Energy Laboratory's three-prime assay, where acid-insoluble lignin represents the amount of lignin insoluble in sulfuric acid, glucan represents the amount of cellulose, and xylan represents the amount of hemicellulose. The results showed that compared with wild-type B73, the acid-insoluble lignin and xylan contents in the zmnst2-1 mutant straw were reduced by 15.02% and 7.52%, respectively, while the glucan content remained unchanged (Figure 4A). This result is consistent with the results of lignin analysis using GC-MS and cellulose analysis using an anthrone colorimetric method. Given that lignin affects the binding of cellulase to cellulose and plays a crucial role in cellulosic ethanol production, and that the zmnst2-1 mutant had significantly lower lignin content than wild-type B73, ethanol fermentation parameters were measured and analyzed. The results showed that the content of glucose in zmnst2-1 decreased by 11.60% after dilute acid pretreatment compared with wild-type B73, while the content of xylose did not change (Figure 4B). 280 ) were significantly reduced in zmnst2-1, with the content of furfural, a toxic substance for fermentation, reduced by 50.60%, while the content of 5-hydroxymethylfurfural increased by 48.92% (Figure 4C). Measurements of ethanol production from fermentation substrates showed that the contents of ethanol produced by co-fermentation of glucose and xylose (ethanol 1) and ethanol produced solely by glucose fermentation after cellulose hydrolysis by cellulose hydrolases (ethanol 2) in the zmnst2-1 mutant increased by 91.89% and 13.82%, respectively, compared to wild-type B73 (Figure 4D). Measurements of the cellulase hydrolysis rate in the straw of wild-type B73 and mutant zmnst2-1 showed that the cellulase hydrolysis rate of zmnst2-1 was significantly increased by 25.34% compared to wild-type B73 (Figure 4E). These results indicate that although the mutation of ZmNST2 slightly reduces the content of glucose in the fermentation substance, the reduction in lignin content reduces the content of most fermentation inhibitors, while increasing the hydrolysis of cellulose by cellulose hydrolases, significantly improving the efficiency of fermentation to produce ethanol.

[0057] In summary, the present invention has identified a maize mutant with reduced lignin content and increased ethanol production during fermentation. Analysis of the results showed that the ZmNST2 mutation reduced lignin content, decreased fermentation inhibitors, and increased cellulase hydrolysis, thereby facilitating bioethanol production from corn straw fermentation. Therefore, this gene plays a key role in the application of bioethanol production from corn straw fermentation and is an important resource for developing new maize varieties with high fermentation ethanol yields.

[0058] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should fall within the scope of protection determined by the claims of the present invention.

Claims

1. A mutant gene of ZmNST2, characterized in that, The nucleotide sequence of the mutant gene is shown as SEQ ID No: 1 or SEQ ID No:

2.

2. A biological material containing the mutant gene of ZmNST2 described in claim 1, characterized in that, The biological material comprises an expression vector or a recombinant bacterium.

3. Use of the mutant gene of ZmNST2 as described in claim 1 or the biological material as described in claim 2 in creating maize mutants, characterized in that, The mutant gene of ZmNST2 or the biological material is introduced into a maize material to obtain the maize mutant.

4. Use of the mutant gene of ZmNST2 according to claim 1, the biological material according to claim 2, or the maize mutant according to claim 3 in the production of bioethanol by fermentation of maize straw.

5. The application according to claim 4, wherein The mutant gene, the biological material, or the maize mutant improves the efficiency of producing bioethanol by fermentation of maize straw.

6. The application according to claim 5, characterized in that, By reducing the lignin content in maize straw, thereby improving the efficiency of producing bioethanol by fermentation.

7. The application according to claim 5, characterized in that, By reducing the content of fermentation inhibitors in maize straw, thereby improving the efficiency of producing bioethanol by fermentation.

8. The application according to claim 5, characterized in that, By increasing the hydrolysis rate of cellulase in maize straw, thereby improving the efficiency of producing bioethanol by fermentation.

9. Use of the mutant gene of ZmNST2 according to claim 1 or the biological material according to claim 2 in maize molecular breeding.

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