A xylose isomerase obtained based on an ancestral sequence construction method and its application

Abstract

The present invention discloses xylose isomerases obtained based on an ancestral sequence construction method and their applications. These xylose isomerases are obtained based on the ancestral sequence construction method and have any of the amino acid sequences ID NO. 1 to SEQ ID NO. 4. Expression of these xylose isomerases alone or in combination can confer upon yeast cells the ability to convert xylose to xylulose, thereby conferring upon host cells the ability to convert xylose to other products. The present invention also relates to the application of these four xylose isomerases in yeast production of chemicals such as ethanol using xylose as a substrate. When expressed in yeast cells such as Saccharomyces cerevisiae, these xylose isomerases can confer upon a host cell that originally lacks the ability to convert xylose to xylulose this ability, conferring upon the host cell the ability to produce chemicals such as ethanol using xylose or lignocellulose hydrolysate, which are rich in xylose.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a xylose isomerase obtained based on an ancestral sequence construction method and its application. The amino acid sequence of the xylose isomerase does not come from nature, but is reconstructed by a computer algorithm based on a known xylose isomerase amino acid sequence. Background Art

[0002] Xylose is the most abundant pentose in nature and the main sugar component besides glucose in lignocellulosic raw materials such as straw. The efficient conversion of xylose into chemicals such as ethanol and oil through microbial metabolic pathways is an important direction for the refining of lignocellulosic raw materials such as corn straw. Many microorganisms, such as Saccharomyces cerevisiae, have a complete xylulose metabolic system. Xylulose generates 5-phosphate xylulose under the action of xylulokinase, enters the non-oxidative pentose phosphate pathway, and can be further converted into a variety of chemicals. Therefore, how to convert xylose into xylulose has become the key to xylose utilization. There are two main xylose metabolic pathways in nature. The first is the xylose reductase-xylitol dehydrogenase pathway present in fungi such as Pichia pastoris. The NADPH-preferring xylose reductase (XR) reduces xylose to xylitol, and the NAD+-dependent xylitol dehydrogenase (XDH) oxidizes xylitol to xylulose ( Bioprocess and biosystems engineering,2020,43(8):1509-1519 The second is the xylose isomerase pathway that exists in bacteria and a few fungi. Xylose isomerase (XI) can directly convert xylose into xylulose ( Critical Reviews in Biotechnology,2022,42(5):693-712 ).

[0003] Yeast strains such as Saccharomyces cerevisiae are an important type of industrial microbial strain. Since the 1990s, there have been a large number of studies on the expression of xylose isomerase in Saccharomyces cerevisiae. However, the xylose isomerases expressed in the early stages were inactive, possibly due to protein misfolding, post-translational modification, disulfide bond formation, and inappropriate intracellular pH. It was not until 1996 that xylose isomerase (TheXI) from Thermus thermophilus was actively expressed in Saccharomyces cerevisiae for the first time, but its optimum temperature was 85°C, and it only maintained 4% activity at 30°C, limiting its further application ( Applied and Environmental Microbiology,1996,62(12):4648-51 In 2003, Kuype et al. reported that xylose isomerase (PirXI) from the anaerobic fungus Piromycess p.E2 showed high activity in Saccharomyces cerevisiae and has since been widely used in the construction of xylose metabolizing yeast ( FEMS Yeast Research,2003,4(1):69-78 To date, only a small amount of xylose isomerase active in Saccharomyces cerevisiae has been found.

[0004] There are two main methods for discovering xylose isomerase that are commonly used. The first is to directly amplify the xylose isomerase gene from xylose metabolizing microorganisms in nature or an environment rich in such microorganisms, and then express and screen the obtained gene in Saccharomyces cerevisiae to obtain active xylose isomerase. For example, OrpXI (which is active in Saccharomyces cerevisiae) has been amplified from rumen fungi Orpinomyces, anaerobic bacteria Bacteroides stercoris HJ-15 and Bifidobacterium longum MG1, Ruminococcus flavefaciens, xylan-degrading microorganisms Prevotella spp., and mammalian intestinal Bacteroidetes group. Applied Microbiology and Biotechnology,2009,82(6):1067-78 )、BasXI( Applied Microbiology and Biotechnology,2011,92(1):77-84 )、RumXI( Journal of Industrial Microbiology& Biotechnology,2012,39(11):1597-604 )、PreXI( Biotechnology for Biofuels,2013,6: 84 )、BavXI( Microbial cell factories,2015,14(1):1-14 Another method is to sequence the environmental metagenomes that may contain xylose isomerase gene sequences, infer possible xylose isomerases based on the sequencing results, and then synthesize the relevant gene sequences in vitro and screen for expression in yeast. For example, sequencing the intestinal microbial metagenomes of cattle feces and the beetle (Ordontotaenius disjunctus) inferred 92 and 182 putative XIs, respectively, from which LacXI ( Applied Microbiology and Biotechnology,2019,103(23):9465-9477 ) and PasXI( Scientific Reports,2021,11(1): 4766 However, this method, which relies on xylose-metabolizing microorganisms or on environmental samples rich in such xylose-metabolizing microorganisms, limits the speed of discovering more xylose isomerases and the discovery of highly active xylose isomerases.

[0005] The idea of deriving reasonable approximations of ancient / ancestral protein sequences from known extant protein sequences was first proposed around 1963 ( Acta chem scand,1963,17:S9-S16). However, ancestral sequence construction has been a theoretical concept for a long time. In recent years, with the advancement of bioinformatics, the increasing number of protein sequences and the progress of molecular biology, proteins encoded by ancestral sequences can be molecularly cloned in the laboratory, and have gradually become a powerful means to study the relationship between enzyme sequence, structure and function. At present, ancestral enzyme construction can generally be divided into the following steps: collection of nucleic acid / amino acid sequences of modern enzymes, multiple sequence alignment, phylogenetic tree construction, computer speculation of ancestral enzyme sequences, gene cloning, and characterization of enzymatic properties. This method is widely used to study the adaptability and evolutionary mechanism of molecules to changing environmental conditions on a planetary time scale. As enzymes play an increasingly important role in the field of biocatalysis, this method has gradually become a powerful means to study the relationship between enzyme sequence, structure and function ( Current Opinion in Structural Biology, 2021, 69:131-141; Briefings in bioinformatics,2021,22(4):bbaa337 The majority of xylose isomerases known to be active in Saccharomyces cerevisiae originate from Firmicutes and Bacteroidetes, which are located on two different branches of the xylose isomerase phylogenetic tree. The ancestors of Firmicutes and Bacteroidetes likely possessed the ability to convert xylose to xylulose in Saccharomyces cerevisiae. However, over time, various mutations arose during gene amplification and transfer, leading to changes in the amino acid sequence that could increase, decrease, or eliminate XI activity when expressed in Saccharomyces cerevisiae. Using ancestral sequence construction methods, ancestral XI sequences from Firmicutes and Bacteroidetes have been artificially constructed, and these artificially constructed XI ancestral sequences are likely to exhibit activity in Saccharomyces cerevisiae. Summary of the Invention

[0006] In view of the deficiencies of the prior art, the present invention provides a xylose isomerase obtained based on an ancestral sequence construction method and its application.

[0007] The object of the present invention is achieved through the following technical solution: an artificially constructed xylose isomerase, characterized in that its amino acid sequence is one of the following amino acid sequences:

[0008] (1) the amino acid sequences shown in SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, and SEQ ID NO. 4;

[0009] (2) An amino acid sequence in which one or more amino acids are added, deleted, substituted, or inserted into the amino acid sequence shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, or SEQ ID NO.4;

[0010] (3) An amino acid sequence having 70% or greater identity with the amino acid sequence represented by any one of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, and SEQ ID NO. 4.

[0011] Furthermore, the nucleotide sequence is one of the following nucleotide sequences:

[0012] (1) The nucleotide sequences shown in SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, and SEQ ID NO. 8;

[0013] (2) A nucleotide sequence in which one or more nucleotides are added, deleted, substituted, or inserted into the nucleotide sequence shown in SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, or SEQ ID NO.8;

[0014] (3) a nucleotide sequence having 70% or more identity with the nucleotide sequence shown in any one of SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, and SEQ ID NO. 8;

[0015] (4) A nucleotide sequence that is different from the nucleotide sequences shown in SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, and SEQ ID NO. 8 due to the degeneracy of the genetic code.

[0016] Furthermore, the amino acid sequence of the xylose isomerase does not come from nature, but is reconstructed by a computer algorithm based on the known amino acid sequence of xylose isomerase.

[0017] Furthermore, the expression of the xylose isomerase can endow the host cell with the ability to convert xylose into xylulose, thereby endowing the host cell with the ability to assimilate xylose. The host cell is Saccharomyces cerevisiae, Yarrowia, Candida, Pichia, Schizosaccharomyces, Hansenula, or Kluyveromyces.

[0018] Furthermore, the xylose isomerase is expressed in the host in one of the following ways:

[0019] (1) The xylose isomerase gene is linked to the host's episomal plasmid and expressed episomally in the host;

[0020] (2) The xylose isomerase gene is integrated into the chromosome of the host cell and expressed in the host;

[0021] (3) The xylose isomerase gene is expressed both episomally and integratedly in the host.

[0022] Furthermore, the xylose isomerase can be expressed alone in the host strain or co-expressed in the host cell. The co-expression forms include SEQ ID NO.5+SEQ ID NO.6, SEQ ID NO.5+SEQ ID NO.7, SEQ ID NO.5+SEQ ID NO.8, SEQ ID NO.6+SEQ ID NO.7, SEQ ID NO.6+SEQ ID NO.8, SEQ ID NO.7+SEQ ID NO.8, SEQ ID NO.5+SEQ ID NO.6+SEQ ID NO.7, SEQ ID NO.5+SEQ ID NO.6+SEQ ID NO.8, SEQ ID NO.5+SEQ ID NO.7+SEQ ID NO.8, SEQ ID NO.6+SEQ ID NO.7+SEQ ID NO.8, SEQ ID NO.5+SEQ ID NO.6+SEQ ID NO.8, SEQ ID NO.5+SEQ ID NO.7+SEQ ID NO.8, SEQ ID NO.6+SEQ ID NO.7+SEQ ID NO.8, and SEQ ID NO.5+SEQ ID NO.6+SEQ ID NO.7+SEQ ID NO.8.

[0023] Furthermore, the yeast cell is a wild strain or a yeast cell that has undergone one or more genetic modifications.

[0024] An application of the above-mentioned xylose isomerase, which specifically comprises the following steps: the xylose isomerase enables host cells to utilize xylose or lignocellulose hydrolyzate to produce a variety of fermentation products, including xylulose, fructose, ethanol, butanol, microbial oils and fats, free fatty acids, furfural, lactic acid, succinic acid, citric acid, propionic acid, 3-hydroxypropionic acid, adipic acid, xylulose-5-phosphate, isoprene, polyhydroxyalkanoate, lysine, glutamic acid, phenylalanine, alanine, vanillic acid, and vanillin.

[0025] The present invention has the beneficial effect of disclosing the amino acid and nucleotide sequences of four novel xylose isomerases that can be expressed at high activity in yeast cells. These four xylose isomerases are all artificially constructed, and their expression alone or in combination can confer upon yeast cells the ability to convert xylose to xylulose, thereby conferring upon host cells the ability to convert xylose to other products. The present invention also relates to the use of these four xylose isomerases in yeast for producing chemicals such as ethanol using xylose as a substrate. When expressed in yeast cells such as Saccharomyces cerevisiae, these xylose isomerases can enable a host cell that originally lacks the ability to convert xylose to xylulose to acquire this conversion ability, thereby conferring upon the host cell the ability to produce chemicals such as ethanol using xylose-rich feedstocks such as xylose or lignocellulose hydrolysate. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1This is a diagram showing xylose isomerases obtained based on the ancestral sequence construction method, wherein ①②③④ in the figure represent four computer-inferred xylose isomerases with ancestral sequences, numbered AncXI-1, AncXI-2, AncXI-3, and AncXI-4, respectively, and their protein sequences are SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4;

[0027] Figure 2 This is a bar graph of the fermentation broth composition of recombinant Saccharomyces cerevisiae CRD3A1, CRD3A2, CRD3A3, and CRD3A4 when the four xylose isomerases were expressed in Saccharomyces cerevisiae using an initial xylose of 40 g / L as the carbon source for 96 hours; DETAILED DESCRIPTION

[0028] The plasmids and strains described in the following examples are intended only to further illustrate the present invention and are not intended to limit its essence. Indeed, using the nucleotide sequences discovered in this invention, those skilled in the art can generate a variety of other genetically engineered strains capable of converting xylose to xylulose, without departing from the spirit and principles of the present invention. Unless otherwise noted, percentages in the examples are by mass.

[0029] This application has constructed four artificial ancestral sequences of xylose isomerase by collecting homologous sequence sets, performing multiple sequence alignments of these sequences, constructing phylogenetic trees, and inferring ancestral sequences using computer tools. These four xylose isomerases exhibit significant amino acid sequence differences from all currently published xylose isomerases (the NCBI database currently has 200,000 possible xylose isomerase sequences, all of which were sequenced from environmental or microbial samples). Gene synthesis, gene expression, enzyme activity testing, and fermentation experiments have demonstrated that the four constructed xylose isomerase sequences can confer high xylose utilization capacity in Saccharomyces cerevisiae strains.

[0030] The present application provides an artificially constructed xylose isomerase, the amino acid sequence of which is one of the following amino acid sequences:

[0031] (1) the amino acid sequences shown in SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, and SEQ ID NO. 4;

[0032] (2) An amino acid sequence in which one or more amino acids are added, deleted, substituted, or inserted into the amino acid sequence shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, or SEQ ID NO.4;

[0033] (3) An amino acid sequence having 70% or greater identity with the amino acid sequence represented by any one of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, and SEQ ID NO. 4.

[0034] Specifically, its nucleotide sequence is one of the following nucleotide sequences:

[0035] (1) The nucleotide sequences shown in SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, and SEQ ID NO. 8;

[0036] (2) A nucleotide sequence in which one or more nucleotides are added, deleted, substituted, or inserted into the nucleotide sequence shown in SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, or SEQ ID NO.8;

[0037] (3) a nucleotide sequence having 70% or more identity with the nucleotide sequence shown in any one of SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, and SEQ ID NO. 8;

[0038] (4) A nucleotide sequence that is different from the nucleotide sequences shown in SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, and SEQ ID NO. 8 due to the degeneracy of the genetic code.

[0039] Specifically, the amino acid sequence of the xylose isomerase does not come from nature, but is reconstructed by a computer algorithm based on the known amino acid sequence of xylose isomerase.

[0040] Specifically, the expression of the xylose isomerase can endow the host cell with the ability to convert xylose into xylulose, thereby endowing the host cell with the ability to assimilate xylose. The host cells are Saccharomyces cerevisiae, Yarrowia, Candida, Pichia, Schizosaccharomyces, Hansenula, and Kluyveromyces.

[0041] Specifically, the xylose isomerase is expressed in the host in one of the following ways:

[0042] (1) The xylose isomerase gene is linked to the host's episomal plasmid and expressed episomally in the host;

[0043] (2) The xylose isomerase gene is integrated into the chromosome of the host cell and expressed in the host;

[0044] (3) The xylose isomerase gene is expressed both episomally and integratedly in the host.

[0045] Specifically, the xylose isomerase can be expressed alone in the host strain or co-expressed in the host cell. The co-expression forms include SEQ ID NO.5+SEQ ID NO.6 (combination 1), SEQ ID NO.5+SEQ ID NO.7 (combination 2), SEQ ID NO.5+SEQ ID NO.8 (combination 3), SEQ ID NO.6+SEQ ID NO.7 (combination 4), SEQ ID NO.6+SEQ ID NO.8 (combination 5), SEQ ID NO.7+SEQ ID NO.8 (combination 6), SEQ ID NO.5+SEQ ID NO.6+SEQ ID NO.7 (combination 7), SEQ ID NO.5+SEQ ID NO.6+SEQ ID NO.8 (combination 8), SEQ ID NO.5+SEQ ID NO.7+SEQ ID NO.8 (combination 9), SEQ ID NO.6+SEQ ID NO.7+SEQ ID NO.8 (combination 10), and SEQ ID NO.5+SEQ ID NO.6+SEQ ID NO.7+SEQ ID NO.8 (combination 11).

[0046] Specifically, the yeast cell can be a wild strain, or a yeast cell that has undergone one or more genetic modifications.

[0047] The present application also provides an application of the above-mentioned xylose isomerase, which specifically comprises the following steps: the xylose isomerase enables host cells to utilize xylose or lignocellulose hydrolysate to produce a variety of fermentation products, including xylulose, fructose, ethanol, butanol, microbial oils and fats, free fatty acids, furfural, lactic acid, succinic acid, citric acid, propionic acid, 3-hydroxypropionic acid, adipic acid, xylulose-5-phosphate, isoprene, polyhydroxyalkanoate, lysine, glutamic acid, phenylalanine, alanine, vanillic acid, and vanillin.

[0048] This application discloses the amino acid and nucleotide sequences of four novel xylose isomerases that can be expressed at high activity in yeast cells. These four xylose isomerases are all artificially constructed. Their expression alone or in combination can confer upon yeast cells the ability to convert xylose to xylulose, thereby endowing host cells with the ability to convert xylose to other products. The present invention also relates to the use of these four xylose isomerases in yeast cells to produce chemicals such as ethanol using xylose as a substrate. When expressed in yeast cells such as Saccharomyces cerevisiae, these xylose isomerases can enable hosts that originally lack the ability to convert xylose to xylulose to acquire this conversion ability, thereby endowing host cells with the ability to produce chemicals such as ethanol using xylose or lignocellulose hydrolysate, which are rich in xylose.

[0049] The following is a detailed description with reference to the embodiments.

[0050] Example 1: Construction of xylose isomerase sequence

[0051] 1.1. Acquisition of xylose isomerase data

[0052] Using the BLAST function in NCBI, the amino acid sequence XI from Piromyces sp. E2 was used as a template to perform a BLAST against amino acid sequences in the NCBI database. Amino acid sequences with a score greater than 300 and a length of 370-470 were downloaded. The obtained amino acid sequences were clustered using CD-HIT with an identity threshold of 73% to obtain non-redundant amino acid sequences.

[0053] 1.2. Construction of the phylogenetic tree of xylose isomerase

[0054] MEGA 11 software was used to load the selected amino acid sequences and XI sequences reported to be active in Saccharomyces cerevisiae. Multiple sequence alignment was performed using ClustalX. Sequences with conserved amino acid positions were retained (metal-binding residues H102, D105, E233, K235, E269, H272, D297, D308, D310, and D340, as well as residues W50, F61, F146, W140, and W189 surrounding the substrate pocket (amino acid positions are indicated based on the XI amino acid sequence from Piromyces sp. E2)). A phylogenetic tree was constructed using Fastree, using the maximum likelihood model with a bootstrap value of 1000. The resulting phylogenetic tree was optimized using iTOL (https: / / itol.embl.de / ). Species and length information for the XI sequences were obtained from the NCBI database.

[0055] 1.3 Artificial construction of xylose isomerase

[0056] All existing XIs that evolved from a specific node in the phylogenetic tree constructed from known xylose isomerase sequences were selected and their amino acid sequence information was loaded into MEGA 11 software. Amino acid sequences were aligned using ClustalX, and Graps were removed from the resulting alignment. A phylogenetic tree of the processed amino acid sequences was constructed using the maximum likelihood method. Ancestral sequence inference was performed using the CodeML function of pamlX. First, the processed amino acid sequence alignment file and phylogenetic tree file were loaded, and the software parameters were modified (ncatG: 8, Small Diff: 5e-6, amino acid rate file: Pamltest\paml4.9j\dat\wag.dat, fix blength: 2: fixed, model: 3: Empirical+F, RateAncestor). The software was run to obtain the ancestral sequence for each node in the generated phylogenetic tree.

[0057] Result 1:

[0058] From the 250,000 amino acid sequences in the NCBI database, 867 sequences were obtained. These 867 XIs and 16 XIs reported to be highly active in Saccharomyces cerevisiae were used to construct a phylogenetic tree. Figure 1 As shown, the phylogenetic tree obtained has 9 major evolutionary branches, and most of the 16 XIs reported in the literature to be active in Saccharomyces cerevisiae are from Bacteroidetes (branch IX) and Firmicutes (branch IV). Figure 1 The amino acid sequences of the ancestral sequences of designated nodes in the phylogenetic tree are shown in SEQ ID NOs. 1-4. To compare the similarity of these deduced amino acid sequences with existing XIs, the NCBI BLAST function was used, revealing that the maximum Per.Ident of these sequences to their respective most similar XIs ranged from 82.15% to 90.14% (Table 1). These amino acid sequences are completely different from currently known XIs.

[0059] Table 1 Comparison of the amino acid sequences of four xylose isomerases with the amino acid sequences of currently known proteins

[0060] ancestral sequence Maximum similarity to known proteins NCBI accession number of known protein Known sources of protein AncXI-1 90.14％ OUM58912.1 Piromyces sp.E2 AncXI-2 83.03％ WP_028668460.1 Runella zeae AncXI-3 82.15％ WP_092638076.1 Acetanaerobacterium elongatum AncXI-4 82.31％ WP_094546057.1 Petroclostridium xylanilyticum

[0061] Example 2: Episomal expression of four xylose isomerase genes in Saccharomyces cerevisiae

[0062] 2.1. Construction of episomal expression vector

[0063] GenScript Biotech Co., Ltd. was commissioned to synthesize the xylose isomerase nucleotide sequences of SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.8 respectively. Then, the four synthesized nucleotide macromolecules were respectively inserted into the free expression vector of Saccharomyces cerevisiae. The specific steps were as follows: the G418 resistance gene was inserted into the SmaI-SalI site of the Saccharomyces cerevisiae free expression vector pESC-URA to obtain the G418_pESC-URA plasmid; then the Saccharomyces cerevisiae promoter TDH3 sequence was inserted into the KpnI-NheI site of the G418_pESC-URA plasmid to obtain the TDH3_G418_pESC-URA plasmid; finally, the synthesized macromolecular nucleotide fragments corresponding to SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.8 were respectively inserted into the NheI site of the TDH3_G418_pESC-URA plasmid to obtain the xylose isomerase free expression vectors pESC-Anc1, pESC-Anc2, pESC-Anc3, and pESC-Anc4. In the obtained Saccharomyces cerevisiae episomal expression vectors, the 5' side of the xylose isomerase gene is the TDH3 promoter, and the 3' side is the CYC1 terminator.

[0064] 2.2 Transformation of episomal expression vectors and screening of transformants

[0065] The plasmids pESC-Anc1, pESC-Anc2, pESC-Anc3, and pESC-Anc4 harboring the xylose isomerase gene were transformed into diploid Saccharomyces cerevisiae CRD3 (ATCC 26603, MATa / α, ΔGre3, pho13::TPI1p-XKS1-ADH1t-FBA1p-TKL1-FBA1t-PGK1p-RKI1-GAL2t, pyk2::TEF1p-GAL2 N376F -TEF1t-TDH3p-TAL1-PGI1t). Transformants were screened on YPD plates (400 μg / mL G418). Untransformed cells were unable to grow on these plates. Using single colonies from these plates as templates, the corresponding xylose isomerase genes were amplified by PCR and sequenced. Transformants containing the corresponding xylose isomerase gene plasmids were identified and named CRD3A1, CRD3A2, CRD3A3, and CRD3A4, respectively.

[0066] 2.3. Determination of the ability of recombinant strains to utilize xylose

[0067] Yeast CRD3A1, CRD3A2, CRD3A3, and CRD3A4 were cultured overnight in YPD (2% peptone, 1% yeast extract, and 2% glucose) medium, and then the initial OD 600The cells were transferred to YPX (2% peptone, 1% yeast extract, 4% xylose) medium at 1.0 and cultured at 30°C and 150 rpm. The xylose and ethanol concentrations in the culture medium were determined by high performance liquid chromatography (HPLC). The OD was measured at a wavelength of 600 nm using a UV spectrophotometer. 600 To monitor yeast growth.

[0068] Result 2:

[0069] like Figure 2 As shown, after 96 hours of culture in YPX medium with an initial xylose concentration of 40 g / L, Saccharomyces cerevisiae CRD3A1, CRD3A2, CRD3A3, and CRD3A4 consumed 12.43, 11.80, 12.70, and 8.84 g / L of xylose, respectively, accompanied by bacterial growth and ethanol production. These results demonstrate that the four xylose isomerases described in this study, when expressed in Saccharomyces cerevisiae, confer the ability to convert xylose to xylulose, enabling growth and ethanol production on xylose.

Claims

1. A xylose isomerase obtained based on the ancestral sequence construction method, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

1.

2. The xylose isomerase according to claim 1, characterized in that Its nucleotide sequence is shown in SEQ ID NO.

5.

3. The xylose isomerase according to claim 1 or 2, characterized in that The expression of the xylose isomerase can endow the host cell with the ability to convert xylose into xylulose, thereby endowing the host cell with the ability to assimilate xylose. The host cell is a Saccharomyces cerevisiae cell, Yarrowia, Candida, Pichia pastoris, Schizosaccharomyces, Hansenula or Kluyveromyces.

4. The xylose isomerase according to claim 1 or 2, characterized in that The xylose isomerase is expressed in the host in one of the following ways: (1) The xylose isomerase gene is linked to the host's episomal plasmid and expressed episomally in the host; (2) The xylose isomerase gene is integrated into the chromosome of the host cell and expressed in the host; (3) The xylose isomerase gene is expressed both episomally and integratedly in the host.

5. The xylose isomerase according to claim 3, characterized in that The host cell is a wild strain or a yeast that has undergone one or more genetic modifications.

6. Use of the xylose isomerase according to any one of claims 1 to 5 in preparing a fermentation product using xylose, characterized in that: The fermentation products are xylulose, ethanol, butanol, lactic acid, succinic acid, citric acid, 3-hydroxypropionic acid, xylulose-5-phosphate or isoprene.

7. Use of the xylose isomerase according to any one of claims 1 to 5 in preparing a fermentation product from a lignocellulose hydrolyzate, characterized in that: The fermentation products are xylulose, ethanol, butanol, lactic acid, succinic acid, citric acid, 3-hydroxypropionic acid, xylulose-5-phosphate or isoprene.

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