Xylose isomerase obtained based on ancestor sequence construction method and application thereof

Abstract

The invention discloses xylose isomerase obtained based on an ancestor sequence construction method and application of the xylose isomerase. The xylose isomerase is obtained on the basis of an ancestor sequence construction method and has an amino acid sequence shown in SEQ ID NO.3, the capacity of converting xylose into xylulose can be endowed to yeast cells through independent expression or combined expression of the xylose isomerase, and then the capacity of converting xylose into other products is endowed to host cells. The invention also relates to an application of the xylose isomerase in producing chemicals such as ethanol and the like by using xylose as a substrate. When the xylose isomerase is expressed in yeast cells such as saccharomyces cerevisiae and the like, a host which does not have the capacity of converting xylose into xylulose originally can obtain the conversion capacity, and host cells are endowed with the capacity of producing chemicals such as ethanol by using xylose-rich raw materials such as xylose or lignocellulose hydrolysate and the like.

Description

[0001] This application is a divisional application of application number 202310040197.0, application date January 13, 2023, and invention name “A xylose isomerase obtained based on an ancestral sequence construction method and its application”. Technical Field

[0002] 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 an application thereof. 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

[0003] Xylose is the most abundant pentose in nature and the main sugar component in wood fiber raw materials such as straw besides glucose. The effective conversion of xylose into chemicals such as ethanol and oil through microbial metabolic pathways is an important direction for the refining of wood fiber 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 (xylose reductase, XR) reduces xylose to xylitol, and the NAD+-dependent xylitol dehydrogenase (xylitol dehydrogenase, XDH) oxidizes xylitol to xylulose ( Bioprocess and biosystemsengineering,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 inBiotechnology,2022,42(5):693-712 ).

[0004] Yeast strains such as Saccharomyces cerevisiae are an important class of industrial microbial strains. 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 stage were inactive, which may be 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 first actively expressed in Saccharomyces cerevisiae, but its optimum temperature was 85°C, and it only maintained 4% activity at 30°C, limiting further applications ( Applied and EnvironmentalMicrobiology,1996,62(12):4648-51In 2003, Kuype et al. reported that xylose isomerase (PirXI) from the anaerobic fungus Piromyces sp. 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 ). So far, only a small amount of xylose isomerase actively expressed in Saccharomyces cerevisiae has been found.

[0005] 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 andBiotechnology, 2009,82(6): 1067-78 )、BasXI( Applied Microbiology and Biotechnology,2011,92(1):77-84 )、RumXI( Journal ofIndustrial Microbiology& Biotechnology,2012,39(11):1597-604 )、PreXI( Biotechnology forBiofuels,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 feces of cattle and the intestinal microbial metagenomes of the Pasaly 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 that relies on xylose-metabolizing microorganisms or relies 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.

[0006] 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 long been a theoretical concept. In recent years, with the advancement of bioinformatics, the increasing number of protein sequences and the progress of molecular biology, the 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 inference 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 inbioinformatics,2021,22(4):bbaa337 ). Most of the xylose isomerases known to be active in Saccharomyces cerevisiae come from Firmicutes and Bacteroidetes, which are located in two different branches of the xylose isomerase phylogenetic tree. The ancestors of Firmicutes and Bacteroidetes may have the ability to convert xylose to xylulose in Saccharomyces cerevisiae, but over time, various mutations continue to occur during gene amplification and transfer, resulting in changes in the amino acid sequence, which may increase, decrease or eliminate the activity of XI when expressed in Saccharomyces cerevisiae. The ancestral sequences of XI of Firmicutes and Bacteroidetes were artificially constructed by the ancestral sequence construction method, and these artificially constructed ancestral sequences of XI are likely to be active in Saccharomyces cerevisiae. Summary of the invention

[0007] 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.

[0008] The object of the present invention is achieved through the following technical scheme: an artificially constructed xylose isomerase, characterized in that its amino acid sequence is one of the following amino acid sequences:

[0009] (1) the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4;

[0010] (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;

[0011] (3) An amino acid sequence having 70% or more 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.

[0012] Furthermore, the nucleotide sequence is one of the following nucleotide sequences:

[0013] (1) The nucleotide sequences shown in SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8;

[0014] (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;

[0015] (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;

[0016] (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.

[0017] 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.

[0018] 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, and Kluyveromyces.

[0019] Furthermore, the xylose isomerase is expressed in the host in one of the following ways:

[0020] (1) The xylose isomerase gene is connected to the host's episomal plasmid and expressed episomally in the host;

[0021] (2) The xylose isomerase gene is integrated into the chromosome of the host cell and expressed in the host;

[0022] (3) The xylose isomerase gene is expressed both episomally and integratedly in the host.

[0023] Further, the xylose isomerase can be expressed in a host strain alone or in a host cell together. The forms of co-expression 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.6+SEQ ID NO.7+SEQ ID NO.8, SEQ ID NO.5+SEQ ID NO.6+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.7+SEQ ID NO.8.

[0024] Furthermore, the yeast cell is a wild strain or a yeast cell that has undergone one or more genetic modifications.

[0025] An application of the above-mentioned xylose isomerase, which specifically includes: 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, polyhydroxyalkanoates, lysine, glutamic acid, phenylalanine, alanine, vanillic acid, and vanillin.

[0026] The beneficial effect of the present invention is that the present invention discloses the amino acid sequence and nucleotide sequence of four new xylose isomerases that can be highly actively expressed in yeast cells. These four xylose isomerases are all artificially constructed, and their single expression or combined expression can give yeast cells the ability to convert xylose into xylulose, thereby giving host cells the ability to convert xylose into other products. The present invention also relates to the application of these four xylose isomerases in yeast using xylose as a substrate to produce chemicals such as ethanol. When the xylose isomerase is expressed in yeast cells such as saccharomyces cerevisiae, it can enable a host that originally does not have the ability to convert xylose into xylulose to obtain the conversion ability, and give the host cell the ability to use xylose or lignocellulose hydrolyzate and other xylose-rich raw materials to produce ethanol and other chemicals. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1It is a display diagram of xylose isomerase 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, and their protein sequences are SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4;

[0028] Figure 2 This is a bar graph of the fermentation broth composition of recombinant Saccharomyces cerevisiae CRD3A1, CRD3A2, CRD3A3, and CRD3A4 after fermentation for 96 hours with an initial 40 g / L xylose as the carbon source when the four xylose isomerases were freely expressed in Saccharomyces cerevisiae. DETAILED DESCRIPTION

[0029] The plasmids and strains described in the following examples are only used to further explain the present invention and are not intended to limit the essence of the present invention. In fact, using the nucleotide sequences discovered by the present invention, a person skilled in the art can obtain a variety of other genetically engineered strains capable of converting xylose into xylulose, which do not deviate from the spirit and concept of the present invention. Unless otherwise specified, the percentages in the examples are percentages by mass.

[0030] The present application constructs four artificial ancestral sequences of xylose isomerase by collecting homologous sequence sets, multiple sequence alignment of sequence sets, phylogenetic tree construction and inferring ancestral sequences with computer tools. These four xylose isomerases have great differences in amino acid sequence from all currently published xylose isomerases (200,000 possible xylose isomerase sequences have been published in the NCBI database, and these xylose isomerase sequences are all obtained by sequencing environmental or microbial samples). And through gene synthesis, gene expression, enzyme activity test and fermentation experiments, it is proved that the constructed four xylose isomerase sequences can give Saccharomyces cerevisiae strains a higher xylose utilization ability in Saccharomyces cerevisiae.

[0031] The present application provides an artificially constructed xylose isomerase, the amino acid sequence of which is one of the following amino acid sequences:

[0032] (1) the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4;

[0033] (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;

[0034] (3) An amino acid sequence having 70% or more 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.

[0035] Specifically, its nucleotide sequence is one of the following nucleotide sequences:

[0036] (1) The nucleotide sequences shown in SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8;

[0037] (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;

[0038] (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;

[0039] (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.

[0040] 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.

[0041] 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 cell is Saccharomyces cerevisiae, Yarrowia, Candida, Pichia, Schizosaccharomyces, Hansenula, and Kluyveromyces.

[0042] Specifically, the xylose isomerase is expressed in the host in one of the following ways:

[0043] (1) The xylose isomerase gene is connected to the host's episomal plasmid and expressed episomally in the host;

[0044] (2) The xylose isomerase gene is integrated into the chromosome of the host cell and expressed in the host;

[0045] (3) The xylose isomerase gene is expressed both episomally and integratedly in the host.

[0046] Specifically, the xylose isomerase can be expressed in a host strain alone or in a host cell together. The forms of co-expression 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).

[0047] Specifically, the yeast cell may be a wild strain, or a yeast cell that has undergone one or more genetic modifications.

[0048] The present application also provides an application of the above-mentioned xylose isomerase, which specifically includes: 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, polyhydroxyalkanoates, lysine, glutamic acid, phenylalanine, alanine, vanillic acid, and vanillin.

[0049] The present application discloses the amino acid sequence and nucleotide sequence of four new xylose isomerases that can be highly actively expressed in yeast cells. These four xylose isomerases are all artificially constructed, and their single expression or combined expression can endow yeast cells with the ability to convert xylose into xylulose, thereby endowing host cells with the ability to convert xylose into other products. The present invention also relates to the application of these four xylose isomerases in yeast using xylose as a substrate to produce chemicals such as ethanol. When the xylose isomerase is expressed in yeast cells such as Saccharomyces cerevisiae, it can enable a host that originally does not have the ability to convert xylose into xylulose to acquire the conversion ability, and endow the host cell with the ability to produce chemicals such as ethanol using xylose or lignocellulose hydrolyzate, etc., which are rich in xylose.

[0050] The following is a specific description with reference to embodiments.

[0051] Example 1: Construction of xylose isomerase sequence

[0052] 1.1. Acquisition of xylose isomerase data

[0053] Using the BLAST function in NCBI, the amino acid sequence XI from Piromyces sp. E2 was used as a template to perform BLAST on the amino acid sequences in the NCBI database, and the 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, and the identity threshold was set to 73% to obtain non-redundant amino acid sequences.

[0054] 1.2. Construction of the phylogenetic tree of xylose isomerase

[0055] MEGA 11 software was used to load the screened amino acid sequences and the XI sequences reported in the literature to be actively expressed in Saccharomyces cerevisiae, and multiple sequence alignment was performed using the ClustalX function. The sequences with conserved amino acid sites were retained (metal binding residues H102, D105, E233, K235, E269, H272, D297, D308, D310, D340 and residues W50, F61, F146, W140, W189 around the substrate pocket (amino acid positions were marked according to the XI amino acid sequence from Piromyces sp. E2)). The phylogenetic tree was constructed using Fastree, and the model used was the maximum likelihood method with a bootstrap value of 1000. The obtained phylogenetic tree was optimized using iTOL (https: / / itol.embl.de / ), and the species and length information of the XI sequence were obtained from the NCBI database.

[0056] 1.3 Artificial construction of xylose isomerase

[0057] Select all existing XIs evolved from the specified nodes in the phylogenetic tree constructed by the currently known xylose isomerase sequences, and load the amino acid sequence information into MEGA 11 software. Use the ClustalX function to align the amino acid sequences, delete the Graps in the alignment results, and construct the phylogenetic tree of the processed amino acid sequences by the maximum likelihood method. Use the CodeML function of pamlX to infer the ancestral sequence. First, load the processed amino acid sequence alignment result file and phylogenetic tree file, and modify the software parameters (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). Run the software to obtain the ancestral sequence of each node in the generated phylogenetic tree.

[0058] Result 1:

[0059] From the 250,000 amino acid sequences in the NCBI database, 867 sequences were obtained. A phylogenetic tree was constructed using these 867 XIs and 16 XIs reported to be highly active in Saccharomyces cerevisiae. Figure 1 As shown, the phylogenetic tree obtained has 9 major evolutionary branches, and most of the 16 XIs reported in the literature that are active in Saccharomyces cerevisiae are from Bacteroidetes (branch IX) and Firmicutes (branch IV). Figure 1 The amino acid sequences of the ancestral sequences of the designated nodes in the phylogenetic tree are shown in SEQ ID NO. 1-4. In order to compare the similarity of these deduced amino acid sequences with the existing XI, the BLAST function of NCBI was used, and it was found that the maximum Per.Ident between them and the most similar XI was 82.15%-90.14% (Table 1). Its amino acid sequence is completely different from the currently known XI.

[0060] Table 1 Comparison of the amino acid sequences of four xylose isomerases with the amino acid sequences of currently known proteins

[0061] ancestral sequence Maximum similarity to known proteins NCBI accession number of known proteins 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

[0062] Example 2: Free expression of four xylose isomerase genes in Saccharomyces cerevisiae

[0063] 2.1. Construction of episomal expression vector

[0064] GenScript Biotech Inc. 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 Saccharomyces cerevisiae free expression vector. 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.

[0065] 2.2 Transformation of episomal expression vector and screening of transformants

[0066] The plasmids pESC-Anc1, pESC-Anc2, pESC-Anc3, and pESC-Anc4 containing the xylose isomerase gene were transformed into diploid Saccharomyces cerevisiae CRD3 (ATCC 26603, MATa / α, △Gre3, pho13::TPI1p-XKS1-ADH1t-FBA1p-TKL1-FBA1t-PGK1p-RKI1-GAL2t, pyk2::TEF1p-GAL2 N376F -TEF1t-TDH3p-TAL1-PGI1t), and the transformants were screened on YPD plates (400μg / mL G418). Untransformed cells could not grow on these plates. Using the single colonies on the plates as templates, PCR amplified the corresponding xylose isomerase gene and sequenced it. The transformants containing the corresponding xylose isomerase gene plasmids were identified and named CRD3A1, CRD3A2, CRD3A3, and CRD3A4, respectively.

[0067] 2.3. Determination of the ability of recombinant strains to utilize xylose

[0068] Yeast CRD3A1, CRD3A2, CRD3A3, and CRD3A4 were cultured overnight in YPD (2% peptone, 1% yeast extract, 2% glucose) medium and then the initial OD 600The culture medium was transferred to YPX (2% peptone, 1% yeast extract, 4% xylose) 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.

[0069] Result 2:

[0070] like Figure 2 As shown, the initial xylose concentration of YPX medium was 40 g / L. After 96 hours of culture, the xylose content consumed by Saccharomyces cerevisiae CRD3A1, CRD3A2, CRD3A3, and CRD3A4 was 12.43, 11.80, 12.70, and 8.84 g / L, respectively, accompanied by bacterial growth and ethanol production. This result shows that the four xylose isomerases involved in this laboratory, after being expressed in Saccharomyces cerevisiae, all give Saccharomyces cerevisiae the ability to convert xylose into xylulose, so that it can use xylose to grow and produce ethanol.

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.

3.

2. The xylose isomerase according to claim 1, characterized in that The nucleotide sequence of its coding gene is shown as SEQ ID NO.

7.

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 Saccharomyces cerevisiae, Yarrowia, Candida, Pichia, Schizosaccharomyces, Hansenula, and Kluyveromyces.

4. The xylose isomerase according to claim 3, characterized in that The xylose isomerase is expressed in the host in one of the following ways: (1) The xylose isomerase gene is connected 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 xylose isomerase can be expressed alone in the host strain or expressed together in the host cell, wherein the forms of co-expression include SEQ ID NO.5 + SEQID NO.7, SEQ ID NO.6 + SEQ ID NO.7, SEQ ID NO.7 + SEQ ID NO.8, SEQ ID NO.5 + SEQID NO.6 +SEQ ID NO.7, SEQ ID NO.5 + SEQ ID NO.7 +SEQ ID NO.8, SEQ ID NO.6 + SEQID NO.7 + SEQ ID NO.8, SEQ ID NO.5 + SEQ ID NO.6 + SEQ ID NO.7 + SEQ ID NO.8, SEQ ID NO.5 + SEQ ID NO.6 + SEQ ID NO.7 + SEQ ID NO.

8.

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

7. Use of the xylose isomerase according to any one of claims 1 to 6, characterized in that: The specific application is: the xylose isomerase enables the host cell 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, polyhydroxyalkanoates, lysine, glutamic acid, phenylalanine, alanine, vanillic acid, and vanillin.

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