WO2023213294A1

WO2023213294A1 - Xylose isomerase and use thereof

Info

Publication number: WO2023213294A1
Application number: PCT/CN2023/092250
Authority: WO
Inventors: 金明杰; 陈思同; 许召贤
Original assignee: 南京理工大学
Priority date: 2022-05-05
Filing date: 2023-05-05
Publication date: 2023-11-09

Abstract

A xylose isomerase and a use thereof, the xylose isomerase comprising xylose isomerase with high activity expression in yeast cells, xylose isomerase obtained by modifying the N-terminus of the protein sequence, and xylose isomerase obtained based on an ancestral sequence construction method; the single expression or combination expression of same can endow the yeast cell with the capability of converting xylose into xylulose, so that the host cell is endowed with the capability of converting xylose into other products. The present invention also relates to a use of four types of xylose isomerase in the production of chemicals such as ethanol from yeast by using xylose as a substrate. When expressed in yeast cells such as saccharomyces cerevisiae, the xylose isomerase enables a host which does not have the capability of converting xylose to xylulose to acquire said conversion capability, and endows the host cell with the capability of producing chemicals such as ethanol by using xylose or raw materials rich in xylose such as lignocellulose hydrolysate.

Description

Xylose isomerase and its applications

Technical field

The invention belongs to the field of biotechnology, and in particular relates to a xylose isomerase and the application of the xylose isomerase to endow host cells with the production of various fermentation products using xylose or lignocellulose hydrolyzate.

Background technique

The over-exploitation and use of fossil resources not only causes the stock of such unsustainable resources to continue to decrease, but also causes massive emissions of carbon dioxide and causes problems such as global warming and frequent occurrence of extreme climates. Therefore, humans began to look for alternative resources to fossil resources. Lignocellulosic biomass is the most abundant renewable organic resource on the earth. The use of lignocellulose to produce fuels or chemicals such as ethanol plays an important role in alleviating the energy crisis, reducing environmental pollution and the greenhouse effect (Vu et al., Science of The Total Environment,2020,743:140630).

According to the different structural units, lignocellulose is mainly divided into three main components: cellulose, hemicellulose and lignin. After pretreatment and enzymatic hydrolysis of lignocellulosic biomass, its cellulose and hemicellulose components are hydrolyzed into two main monosaccharides, glucose and xylose, respectively. Converting glucose and xylose into chemicals such as ethanol is one of the core technologies of biorefinery. Many microorganisms, such as Saccharomyces cerevisiae, can utilize glucose in lignocellulose hydrolyzate to ferment to produce a variety of chemicals including ethanol. However, wild Saccharomyces cerevisiae lacks the ability to convert xylose, resulting in xylose in lignocellulose. Cannot be efficiently converted into target chemicals. Considering the high proportion of hemicellulose components in lignocellulosic biomass, if xylose hydrolyzed from hemicellulose can also be converted into related products by microorganisms, lignocellulose can be used as raw material to produce chemicals such as ethanol. The conversion rate has been greatly improved (Lee et al., Current Opinion in Biotechnology, 2021, 67:15-25).

Many microorganisms, such as Saccharomyces cerevisiae, have a complete xylulose metabolic system. Xylulose generates xylulose 5-phosphate under the action of Chemicals. Therefore, how to convert xylose into xylulose has become the key to the utilization of xylose. There are currently two main pathways for converting xylose into xylulose found in microorganisms. The first is the xylose reductase-xylitol dehydrogenase pathway present in fungi such as Pichia pastoris. During the conversion process of this pathway There is a large amount of accumulation of by-product xylitol, which reduces the yield of the target product (Cunha et al., Biotechnology for Biofuels, 2019, 12(1):1-14). The second microbial xylose utilization pathway is the xylose isomerase pathway found mostly in bacteria. This pathway involves only one key enzyme, xylose isomerase, which can directly isomerize xylose into xylulose without relying on cofactors (Hou et al., Journal of Bioscience and Bioengineering, 2016, 121 (2):160-165; Brat et al.,Applied and Environmental Microbiology, 2009,75(8):2304-2311). However, currently only very few xylose isomerases can show activity in Saccharomyces cerevisiae, limiting the application of xylose metabolic pathways based on xylose isomerase.

Some studies have tried to express xylose isomerase genes in yeast cells such as Saccharomyces cerevisiae, but most of the expressed xylose isomerases are inactive. It is speculated that the reasons may be misfolding of the protein, post-translational modifications, and disulfide bonds. form. Amino acid sequence analysis of xylose isomerase actively expressed in Saccharomyces cerevisiae found some conserved sites for substrate binding and metal ion binding, but this was not a sufficient condition for active expression in Saccharomyces cerevisiae. Xylose isomerases currently reported to be active in Saccharomyces cerevisiae include Piromyces sp.E2, Orpinomyces sp.ukk1, Termite gut from fungi (unspecified), bacterial Thermus thermophilus, Clostridium phytofermentans, Soil-xym1(unspecified), Soil-xym2(unspecified), Bacteroides stercoris, Ruminococcus flavefaciens, Prevotella ruminicola, Burkholderia cenocepacia, Bacteroides vulgatus, Bovine rumen(unspecified), Sorangium cellulosum, Uncultured Lachnospira sp.clone XI58444 and Passalid beetle gut—8054_2 (unspecified). However, only Piromyces sp.E2, Clostridium phytofermentans and Bovine rumen (unspecified) show high activity in yeast cells such as Saccharomyces cerevisiae. Discover more xylose isomerases that are active in yeast cells such as Saccharomyces cerevisiae. The conversion of xylose, especially the conversion of xylose in lignocellulosic resources, is of great significance.

In response to the problem that only a few xylose isomerases show activity in yeast, previous studies conducted amino acid sequence analysis on xylose isomerases that can be actively expressed in Saccharomyces cerevisiae and found some substrate binding and metal ion binding. A conserved site, but is not a sufficient condition for active expression in yeast. At present, it is not possible to analyze the key factors for the active expression of xylose isomerase in yeast. Therefore, protein engineering is used to empower xylose isomerase, which currently cannot be expressed in yeast, to enable it to be active in yeast. The ability to express.

In addition, there are two main methods for discovering xylose isomerase that are commonly used at present. The first method 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. constitutive enzyme. Another method is to sequence environmental metagenomes where xylose isomerase gene sequences may exist, infer possible xylose isomerases based on the sequencing results, and then conduct in vitro synthesis and yeast in vivo expression screening of related gene sequences. For example, the metagenomic sequencing of the intestinal microorganisms of cattle feces and Ordontotaenius disjunctus deduced 92 and 182 putative XIs respectively, from which LacXI active in Saccharomyces cerevisiae was screened ( 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 environmental samples rich in such xylose-metabolizing microorganisms limits the speed of discovery of more xylose isomerase and the discovery of highly active xylose isomerase.

Furthermore, the idea of deriving relatively 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 have enabled proteins encoded by ancestral sequences to be molecularly cloned in the laboratory, gradually becoming a powerful means to study the relationship between enzyme sequence, structure and function. At present, the construction of ancestral enzymes can usually 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 adaptation and evolutionary mechanisms of molecules to changing environmental conditions on planetary time scales. 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 ). Most of the xylose isomerases known to be active in S. cerevisiae are from the phyla Firmicutes and Bacteroidetes, which are located in two different branches of the xylose isomerase phylogenetic tree. The ancestors of Firmicutes and Bacteroidetes may have had the ability to convert xylose to xylulose in Saccharomyces cerevisiae, but over time, various mutations continued to occur during the process of gene amplification and transfer, resulting in amino acid Sequence changes may increase, decrease, or eliminate the activity of XI when expressed in S. cerevisiae. The XI ancestral sequences of Firmicutes and Bacteroidetes were artificially constructed through the method of ancestral sequence construction. These artificially constructed XI ancestral sequences are likely to be active in Saccharomyces cerevisiae.

Contents of the invention

In view of the shortcomings of the existing technology, the present invention provides xylose isomerase and its applications.

The purpose of the present invention is achieved through the following technical solutions:

In a first aspect of the present invention, a xylose isomerase expressed with high activity in yeast cells is provided, and its amino acid sequence is one of the following amino acid sequences:

(1) The amino acid sequence shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4;

(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 and SEQ ID NO.4;

(3) Having an amino acid sequence that is 70% or more identical to the amino acid sequence shown in any one of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, and SEQ ID NO. 4.

Further, its nucleotide sequence is one of the following nucleotide sequences:

(1) The nucleotide sequence shown in SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.8;

(2) The nucleotide sequence shown in SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.8 has one or more nucleotides added, deleted, substituted, or inserted Nucleotide sequence;

(3) A nucleotide that has more than 70% 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 sequence;

(4) Due to the degeneracy of the genetic code, the nucleotide sequence is different from the nucleotide sequence shown in SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.8.

In a second aspect of the present invention, a xylose isomerase modified at the N-terminus of the protein sequence is provided, and its amino acid sequence is one of the following amino acid sequences:

(1) The amino acid sequence shown in SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, and SEQ ID NO.13;

(2) One or more amino acid sequences shown in SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, and SEQ ID NO.13 are added, deleted, substituted or inserted the amino acid sequence of an amino acid;

(3) Having an identity of more than 70% with the amino acid sequence shown in any one of SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, and SEQ ID NO.13 amino acid sequence.

Further, its nucleotide sequence is one of the following nucleotide sequences:

(1) The nucleotide sequence shown in SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, and SEQ ID NO.18;

(2) The nucleotide sequence shown in SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, and SEQ ID NO.18 has one or more added, deleted, substituted or inserted A nucleotide sequence of multiple nucleotides;

(3) Have more than 70% similarity with the nucleotide sequence shown in any one of SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, and SEQ ID NO.18 Identity of nucleotide sequence;

(4) Due to the degeneracy of the genetic code, it is different from the core of the nucleotide sequences shown in SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, and SEQ ID NO.18. nucleotide sequence.

In a third aspect of the present invention, a xylose isomerase obtained based on the ancestral sequence construction method is provided, and its amino acid sequence is one of the following amino acid sequences:

(1) The amino acid sequence shown in SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.21, and SEQ ID NO.22;

(2) Amino acid sequences in which one or more amino acids are added, deleted, substituted or inserted into the amino acid sequences shown in SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.21 and SEQ ID NO.22;

(3) Having an amino acid sequence that is 70% or more identical to the amino acid sequence shown in any one of SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, and SEQ ID NO. 22.

Further, its nucleotide sequence is one of the following nucleotide sequences:

(1) The nucleotide sequence shown in SEQ ID NO.23, SEQ ID NO.24, SEQ ID NO.25, and SEQ ID NO.26;

(2) The nucleotide sequence shown in SEQ ID NO.23, SEQ ID NO.24, SEQ ID NO.25, and SEQ ID NO.26 has one or more nucleotides added, deleted, substituted, or inserted Nucleotide sequence;

(3) A nucleotide having more than 70% identity with the nucleotide sequence shown in any one of SEQ ID NO.23, SEQ ID NO.24, SEQ ID NO.25, and SEQ ID NO.26 sequence;

(4) Due to the degeneracy of the genetic code, the nucleotide sequence is different from the nucleotide sequence shown in SEQ ID NO.23, SEQ ID NO.24, SEQ ID NO.25, and SEQ ID NO.26.

Furthermore, the expression of xylose isomerase in the above three aspects can all give the host cell the ability to convert xylose into xylulose, thereby giving the host cell the ability to assimilate xylose. The host cell is Saccharomyces cell (Saccharomyces cell). ), Yarrowia, Candida, Pichia, Schizosaccharomyces, Hansenula, Kluyveromyces.

Further, the host cell is preferably a Saccharomyces cerevisiae cell.

Further, 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 integrated and expressed in the host;

(3) The xylose isomerase gene is expressed both free and integrated in the host.

Furthermore, the yeast cells may be wild strains or yeast cells that have undergone one or more genetic modifications.

In a third aspect of the present invention, an application of xylose isomerase in the above three aspects is provided. The application is specifically: the xylose isomerase endows host cells with the ability to utilize xylose or lignocellulose hydrolyzate to produce multiple Fermented products, including xylulose, fructose, ethanol, butanol, microbial lipids, free fatty acids, furfural, lactic acid, succinic acid, citric acid, propionic acid, 3-hydroxypropionic acid, adipic acid, xylulose-5 -Phosphoric acid, isoprene, polyhydroxyalkanoate, lysine, glutamic acid, phenylalanine, alanine, vanillic acid, vanillin.

The beneficial effects of the present invention are:

The invention discloses a variety of new amino acid sequences and nucleotide sequences of xylose isomerase that can be expressed with high activity in yeast cells. Four of the xylose isomerases are from Acetanaerobacterium elongatum, Bacterium J10, Hallella seregens, and Streptobacillus canis strains, and the remaining xylose isomerases are from artificial construction (including N-terminal modification of protein sequences and construction methods based on ancestral sequences). Expression alone or in combination 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 invention also relates to the application of these four xylose isomerase enzymes in the production of chemicals such as ethanol by yeast using xylose as a substrate. 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 enable the host cell to use xylose or lignocellulose to hydrolyze The ability to produce chemicals such as ethanol from xylose-rich feedstocks such as liquids.

Description of the drawings

Figure 1 is a histogram of the components of the fermentation broth after fermentation of the recombinant Saccharomyces cerevisiae CRD3AE, CRD3BJ, CRD3HS, and CRD3SC with an initial 40g/L xylose as the carbon source for 192 hours when four xylose isomerases are expressed freely in Saccharomyces cerevisiae.

Figure 2 is a fermentation curve diagram of recombinant Saccharomyces cerevisiae CRD5AE, CRD5BJ, CRD5HS and CRD5SC fermented with an initial 40g/L xylose when four xylose isomerases are integrated into the chromosome of Saccharomyces cerevisiae. (a) is the fermentation curve of CRD4AE. Figure, (b) is the fermentation curve of CRD4BJ, (c) is the fermentation curve of CRD4HS, (d) is the fermentation curve of CRD4SC.

Figure 3 is a fermentation curve of recombinant Saccharomyces cerevisiae CRD5AE, CRD5BJ, CRD5HS, and CRD5SC fermented in a mixed sugar medium with an initial 80g/L glucose and 40g/L xylose when four xylose isomerases are integrated into the Saccharomyces cerevisiae chromosome. Among them, (a) is the fermentation curve of CRD4AE, (b) is the fermentation curve of CRD4BJ, (c) is the fermentation curve of CRD4HS, and (d) is the fermentation curve of CRD4SC.

Figure 4 is a fermentation curve diagram of four xylose isomerases integrated into the chromosome of Saccharomyces cerevisiae and domesticated to ferment the recombinant Saccharomyces cerevisiae CRD5AE, CRD5BJ, CRD5HS, and CRD5SC in an initial 40g/L xylose medium, wherein, (a ) is the fermentation curve of CRD4AE, (b) is the fermentation curve of CRD4BJ, (c) is the fermentation curve of CRD4HS, and (d) is the fermentation curve of CRD4SC.

Figure 5 is a graph showing the fermentation curve of the recombinant Saccharomyces cerevisiae CRD5AE, CRD5BJ, CRD5HS, and CRD5SC after four xylose isomerases were integrated into the chromosome of Saccharomyces cerevisiae and domesticated using an initial culture medium of 80g/L glucose and 40g/L xylose. , (a) is the fermentation curve of CRD4AE, (b) is the fermentation curve of CRD4BJ, (c) is the fermentation curve of CRD4HS, (d) is the fermentation curve of CRD4SC.

Figure 6 is a fermentation experiment curve diagram of recombinant Saccharomyces cerevisiae CRD5AE, CRD5BJ, CRD5HS and CRD5SC using corn straw hydrolyzate pretreated with 30% (w/w) substrate concentration DLCA (ch) as the substrate, where (a) is The fermentation experiment curve of CRD4AE, (b) is the fermentation experiment curve of CRD4BJ, (c) is the fermentation experiment curve of CRD4HS, (d) is the fermentation experiment curve of CRD4SC.

Figure 7 is a fermentation experiment curve diagram of recombinant Saccharomyces cerevisiae CRD5AE, CRD5BJ, CRD5HS and CRD5SC using corn cobs pretreated with 30% (w/w) substrate concentration DLCA (sa) as the substrate, where (a) is CRD4AE Fermentation experiment curve graph, (b) is the fermentation experiment curve graph of CRD4BJ, (c) is the fermentation experiment curve graph of CRD4HS, (d) is the fermentation experiment curve graph of CRD4SC.

Figure 8 Schematic diagram of sequence alignment of xylose isomerases from different strains.

Figure 9 shows the original XI (NeoXI, AnaXI and RhiXI) and N-terminal modified XI (NeoXI-1, NeoXI-2, AnaXI-1, AnaXI-2 and RhiXI) from Neocallimastix californiae, Anaeromyces robustus and Rhizoclosmatium globosum ) was expressed freely in Saccharomyces cerevisiae, and the histogram of the fermentation broth composition of the recombinant yeast after fermentation for 96 hours with the initial 40g/L xylose as the carbon source.

Figure 10 is a display diagram of xylose isomerase obtained based on the ancestral sequence construction method. The ①②③④ in the figure respectively represent four computer-inferred xylose isomerases with ancestral sequences, numbered AncXI-1, AncXI-2, and AncXI -3. AncXI-4, its protein sequence is SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4.

Figure 10 is a histogram of the fermentation broth components after fermentation of recombinant Saccharomyces cerevisiae CRD3A1, CRD3A2, CRD3A3, and CRD3A4 with an initial 40g/L xylose as the carbon source for 96 hours when four xylose isomerases are expressed freely in Saccharomyces cerevisiae.

Detailed ways

The plasmids and strains cited in the following examples are only used to further illustrate the present invention and do not limit the essence of the present invention. In fact, using the nucleotide sequence discovered in the present invention, those skilled in the art can obtain a variety of other genetically engineered strains with the ability to convert xylose into xylulose, which cannot deviate from the spirit and ideas of the present invention. Unless otherwise stated, the percentages in the examples are mass percentages.

Example 1: Free expression of four xylose isomerases expressed with high activity in yeast cells in Saccharomyces cerevisiae

1.1. Construction of free expression vector

GenScript Biotechnology Co., Ltd. was entrusted 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 inserted into the Saccharomyces cerevisiae episomal expression vector respectively. The specific steps are: insert the G418 resistance gene into the SmaI-SalI site of the Saccharomyces cerevisiae episomal expression vector pESC-URA to obtain the G418_pESC-URA plasmid; Then insert the Saccharomyces cerevisiae promoter TDH3 sequence into the KpnI-NheI site of the G418_pESC-URA plasmid to obtain the TDH3_G418_pESC-URA plasmid; finally, the synthesized SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, The macromolecule nucleotide fragment corresponding to SEQ ID NO.8 was inserted into the NheI site of TDH3_G418_pESC-URA plasmid to obtain xylose isomerase free expression vectors pESC-AE, pESC-BJ, pESC-HS, and pESC-SS. In these Saccharomyces cerevisiae free expression vectors, the 5' side of the xylose isomerase gene is the TDH3 promoter and the 3' side is the CYC1 terminator.

1.2. Transformation of free expression vectors and screening of transformants

Plasmids pESC-AE, pESC-BJ, pESC-HS, and pESC-SS with xylose isomerase genes 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 (400μg/mLG418), untransformed cells cannot be screened in these Grow on plates. Using a single colony on the plate as a template, the corresponding xylose isomerase gene was amplified by PCR and sequenced. The transformants containing the corresponding xylose isomerase gene plasmid were identified and named CRD3AE, CRD3BJ, CRD3HS, and CRD3SC respectively.

1.3. Determination of xylose utilization ability of recombinant strains

Yeasts CRD3AE, CRD3BJ, CRD3HS, and CRD3SC were cultured overnight in YPD (2% peptone, 1% yeast extract, 2% glucose) medium, and then transferred to YPX (2% peptone, 1% yeast) with an initial OD ₆₀₀ of 1.0. extract, 4% xylose) medium, 30°C, 150rpm for anaerobic culture. High-performance liquid chromatography (HPLC) was used to determine the xylose and ethanol concentrations in the culture medium. Yeast growth was monitored using a UV spectrophotometer by measuring _OD600 at a wavelength of 600 nm.

Result 1:

As shown in Figure 1, the initial xylose concentration of YPX medium is 40g/L. When Saccharomyces cerevisiae CRD3AE, CRD3BJ, CRD3HS and CRD3SC are cultured in it for 192 hours, the remaining xylose contents in the medium are 24.02, 8.84, 8.09, respectively. 9.67g/L xylose, accompanied by the growth of bacterial cells and the production of ethanol. 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, allowing it to use xylose to grow and generate ethanol. .

Example 2: Four xylose isomerase genes expressed with high activity in yeast cells are integrated and expressed in the chromosome of Saccharomyces cerevisiae

2.1. Construction of chromosome integration system based on Crispr-Cas9 system

The G418 resistance gene was cloned into the HindIII-EcoRI site of the pML104 vector to obtain plasmid pML-G418. Query the 20 bp target sequence of Saccharomyces cerevisiae delta sequence through http://crispr.dbcls.jp/, construct it to the 5' end of the sgRNA expression cassette of pML-G418 plasmid, and obtain the pML-delta plasmid. Using the Saccharomyces cerevisiae genome as a template, PCR amplified the upstream and downstream fragments of the delta sequence, TDH3 promoter, and CYC1 terminator. Overlapping PCR obtained the upstream fragment containing the delta sequence, TDH3 promoter, xylose isomerase gene, CYC1 terminator, Gene fragment of the downstream segment of the delta sequence, transform it and plasmid pML-delta into Saccharomyces cerevisiae CRD3, transfer to YPX liquid medium (400 μg/mL G418), and culture at 30°C and 150 rpm until the culture medium becomes slightly turbid.

2.2. Screening of Saccharomyces cerevisiae with chromosomally integrated xylose isomerase gene

Take the yeast grown in the YPX liquid medium and spread it on the YPX plate for culture, and culture it statically at 30°C until a single colony appears. Pick a single colony on the YPX plate into yeast lysis buffer, treat it at 85°C for 30 minutes, and then use it as a template to PCR amplify the corresponding xylose isomerase gene to obtain PCR products of corresponding sizes, and sequence to determine xylose isomerism. The enzyme genes are integrated into the yeast chromosome. The yeast containing the xylose isomerase nucleotide fragments corresponding to SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.8 were named CRD4AE, CRD4BJ, CRD4HS, and CRD4SC.

2.3. Determination of xylose utilization by CRD4AE, CRD4BJ, CRD4HS and CRD4SC yeasts

Yeasts CRD4AE, CRD4BJ, CRD4HS, and CRD4SC were cultured overnight in YPD liquid medium (2% peptone, 1% yeast extract, 2% glucose) at 30°C and 150 rpm as seed liquid, and then inserted into YPDX with an initial OD ₆₀₀ of 1.0. (2% peptone, 1% yeast extract, 8% glucose, 4% xylose), YPX (2% peptone, 1% yeast extract, 4% xylose) medium, 30°C, 150rpm for anaerobic fermentation experiment. High-performance liquid chromatography (HPLC) was used to determine the xylose and ethanol concentrations in the culture medium. Yeast growth was monitored using a UV spectrophotometer by measuring _OD600 at a wavelength of 600 nm.

Result 2:

When fermented with YPX (2% peptone, 1% yeast extract, 4% xylose) medium, CRD4HS, CRD4BJ, CRD4AE, and CRD4SC consumed 40g/L xylose at 72h, 84h, 108h, and 108h respectively, and their ethanol The yields were 0.38, 0.38, 0.40 and 0.39g ethanol/g xylose respectively (Figure 2). When fermented in YPDX (2% peptone, 1% yeast extract, 8% glucose, 4% xylose) medium, the four recombinant S. cerevisiae yeasts CRD4HS, CRD4BJ, CRD4AE, and CRD4SC all consumed glucose completely within 12 hours. About 10g/L xylose is utilized in 120h (Figure 3).

Example 3: Improving the ability of CRD5HS, CRD5BJ, CRD5AE, and CRD5SS to utilize xylose through strain domestication

The yeasts CRD4HS, CRD4BJ, CRD4AE, and CRD4SC were continuously subcultured in YPX (2% peptone, 1% yeast extract, 4% xylose) medium. The xylose utilization rate and growth rate continued to increase with the passage, and finally we obtained Stable domesticated yeasts were named CRD5HS, CRD5BJ, CRD5AE, and CRD5SC.

CRD5HS, CRD5BJ, CRD5AE, and CRD5SC yeasts were cultured overnight in YPX (2% peptone, 1% yeast extract, ₄ % xylose) medium, and inserted into YPDX (2% peptone, 1% Yeast extract, 8% glucose, 4% xylose), YPX (2% peptone, 1% yeast extract, 4% xylose) culture medium, 30°C, 150 rpm for anaerobic fermentation experiments. High-performance liquid chromatography (HPLC) was used to determine the xylose and ethanol concentrations in the culture medium. Yeast growth was monitored using a UV spectrophotometer by measuring _OD600 at a wavelength of 600 nm.

Result 3:

When fermented in YPX (2% peptone, 1% yeast extract, 4% xylose) medium, the domesticated strain CRD5HS, CRD5BJ had 7.54 and 5.09g/L xylose remaining in 14h, and basically used 40g/L xylose in 16h. CRD5AE and CRD5SC had 11.87 and 10.76g/L xylose remaining at 14h, and basically utilized 40g/L xylose at 18h (Figure 4). When fermented in YPDX (2% peptone, 1% yeast extract, 8% glucose, 4% xylose) medium, CRD5HS, CRD5BJ, CRD5AE, and CRD5SC consumed 80g/L glucose in 14 hours. At 10h, xylose began to be utilized, and 4.45, 4.02, 25.21, and 20.40g/L xylose remained at 18h (Figure 5). The above results show that the recombinant Saccharomyces cerevisiae strain introduced with four xylose isomerases can utilize xylose significantly faster after strain domestication.

Example 4: Fermentation of CRD5HS, CRD5BJ, CRD5AE, and CRD5SC yeasts using DLCA(ch) corn straw hydrolyzate as substrate

4.1. DLC(ch) pretreatment of corn straw:

DLC (ch) pretreatment is carried out as described in the literature (Chen et al., Green Chemistry, 2021, 23:4828-4839). Specifically, the corn straw is first washed until the color of the washing water is close to colorless, and the washed straw is Put it in an oven at 60°C and dry it until the moisture is 10%-20%. DLC (ch) pretreatment (densifying lignocellulosic biomass with calcium hydroxide, calcium hydroxide-assisted densification pretreatment) is performed on the dried corn stalk, that is, the calcium hydroxide solution is first evenly sprayed onto the corn stalk, and the hydroxide The addition amounts of calcium and water were 0.15 and 0.5g/g corn straw respectively, and then the straw was granulated using a granulator. The corn straw prepared into granular form is dried and stored at room temperature until use.

4.2. DLCA(ch) corn straw hydrolysis:

Before enzymatic hydrolysis, DLC (ch) corn straw was first further processed using a high-temperature sterilization pot. The conditions were: the straw substrate concentration was 25% (w/w) and the reaction was performed at 121°C for 60 minutes. After the temperature of the DLC(ch) corn straw to be treated drops to room temperature, use sulfuric acid to adjust the pH to neutral, and dry it in a fume hood until the moisture content is about 10%. DLCA(ch) corn stover with a substrate concentration of 30% (w/w) was used for hydrolysis, and the cellulase was CTec2 (87 mg protein/mL), the enzyme dosage is 20 mg protein/g dextran. Straw and cellulase were added in two batches, that is, 50% of the mass of straw and cellulase was initially added, and the remaining straw and cellulase were added after 4 hours. The hydrolysis conditions were pH 4.8, 50°C, 250 rpm for 72 h.

4.3. DLCA (ch) corn straw enzyme hydrolyzate fermentation:

CRD5HS, CRD5BJ, CRD5AE, and CRD5SC yeasts were seed cultured in YPX (2% peptone, 1% yeast extract, 4% xylose) medium at 30°C and 150 rpm. The cultured seed liquid was added with 30% (w/w) substrate concentration DLCA (ch) corn straw hydrolyzate with an initial OD ₆₀₀ of 2.0, and 5g/L yeast powder and 10g/L peptone were added to adjust the pH to 5.5. Anaerobic fermentation experiments were conducted at 30°C and 150 rpm.

Result 4:

Figure 6 shows the changes in glucose, xylose and ethanol concentrations when the recombinant strains CRD5HS, CRD5BJ, CRD5AE and CRD5SC containing xylose isomerase were fermented using DLCA(ch) corn straw hydrolyzate. The initial glucose and xylose concentrations in DLCA(ch) corn straw hydrolyzate were 116.31 and 42.90g/L respectively. All four recombinant yeast strains consumed all glucose within 24 hours. At 120h, CRD5HS, CRD5BJ, CRD5AE, and CRD5SC strains consumed 39.86, 34.46, 31.40, and 20.86g/L xylose respectively, accompanied by 73.72, 71.00, 67.68, and 65.85g/L ethanol production. The above results indicate that Saccharomyces cerevisiae containing xylose isomerase can utilize glucose and xylose in corn stover hydrolyzate to ferment and generate ethanol.

Example 5: Fermentation of CRD5HS, CRD5BJ, CRD5AE, CRD5SC yeast using DLCA (sa) corn cob hydrolyzate as substrate

5.1. DLC(sa) preprocessing:

DLC (sa) pretreatment is carried out as described in the literature (Yuan et al., Renewable Energy, 2022, 182:377-389). Specifically, the corn cobs are first washed until the color of the washing water is close to colorless, and the washed corn is The core is placed in an oven at 60°C and dried until the moisture content is 10%-20%. The dried corn cobs are subjected to DLC (sa) pretreatment (densifying lignocellulosic biomass with sulfuric acid, sulfuric acid-assisted densification pretreatment), that is, firstly, the sulfuric acid solution is evenly sprayed on the straw, and the amounts of sulfuric acid and water are respectively 0.075, 0.5g/g corn cob, and then use a granulator to pellet the corn cob. The corn cobs prepared into granules are dried and stored at room temperature until use.

5.2. Hydrolysis of DLCA(sa) corn cob:

Before enzymatic hydrolysis, the DLC (sa) corncob was first further processed using a high-temperature sterilization pot. The conditions were: the straw substrate concentration was 30% (w/w) and the reaction was carried out at 121°C for 20 minutes. After the temperature of the DLC(ch) corncob to be treated drops to room temperature, use calcium hydroxide to adjust the pH to neutral, and dry it in a fume hood until the moisture content is about 10%. Hydrolysis was performed using DLCA (sa) corncob at a substrate concentration of 30% (w/w), in which the cellulase used was CTec2 (87 mg protein/mL), the enzyme dosage is 20 mg protein/g dextran. Straw and cellulase were added in two batches, that is, 50% of the mass of straw and cellulase was initially added, and the remaining straw and cellulase were added after 4 hours. The hydrolysis conditions were pH 4.8, 50°C, 250 rpm for 72 h.

5.3. Fermentation of DLCA(sa) corn cob enzyme hydrolyzate:

CRD5HS, CRD5BJ, CRD5AE, and CRD5SC yeasts were seed cultured in YPX (2% peptone, 1% yeast extract, 4% xylose) medium at 30°C and 150 rpm. The cultured seed liquid was added into 30% (w/w) substrate concentration DLCA (sa) corncob enzyme hydrolyzate with an initial OD ₆₀₀ of 2.0, and 5g/L yeast powder and 10g/L protein were added. Adjust the pH to 5.5 and conduct anaerobic fermentation experiments at 30°C and 150rpm.

Result 5:

Figure 7 shows the changes in the concentrations of glucose, xylose and ethanol in the hydrolyzate when the recombinant strains CRD5HS, CRD5BJ, CRD5AE and CRD5SC containing xylose isomerase were fermented by DLCA (sa) corncob enzyme hydrolyzate. The initial glucose and xylose concentrations in the DLCA (sa) corncob hydrolyzate were 96.27g/L and 94.09g/L xylose respectively. All four recombinant yeast strains consumed all glucose within 24 hours. At 120 h, CRD5HS, CRD5BJ, CRD5AE, and CRD5SC strains consumed 69.04, 57.97, 45.04, and 25.04g/L xylose, respectively, accompanied by the production of 76.78, 69.38, 64.09, and 55.21g/L ethanol. The above results indicate that Saccharomyces cerevisiae containing xylose isomerase can utilize glucose and xylose in corn cob hydrolyzate to ferment and produce ethanol.

Compare the amino acid sequences of currently reported xylose isomerases that can be actively expressed in yeast and reported xylose isomerases that cannot be actively expressed in yeast. It was found that many inactive xylose isomerases have significantly different N-terminal amino acid sequences than active xylose isomerases. For example, relative to xylose isomerase active in yeast, RhiXI (xylose isomerase from Rhizoclosmatium globosum) and AnaXI (xylose isomerase from Anaeromyces robustus) lack approximately 37 and 22 N, respectively. terminal amino acids, while NeoXI (xylose isomerase from Neocallimastix californiae) has 11 more N-terminal amino acids (Figure 8). As we all know, protein synthesis starts from the N-terminus, and the sequence composition of the N-terminus of the protein affects the overall biological function of the protein (Biochemical Journal, 2018, 475(20): 3201-3219; Proteomics, 2015, 15(14): 2385- 2401;Scientific reports,2017,7(1):1-13.). For example, the N-terminal sequence affects the half-life of the protein and is related to the subcellular organelle localization of the protein. From this, it can be inferred that if we can By modifying the N-terminus of inactive xylose isomerase, it is very possible to obtain active xylose isomerase.

In this application, the concept of combinatorial synthetic biology is used to splice the N-terminal amino acid sequence from the active xylose isomerase to the N-terminal of the inactive xylose isomerase RhiXI and AnaXI. The combined recombinant xylose isomerase The constructase acquires the ability for active expression in yeast. In addition, xylose isomerase NeoXI has 11 more amino acids at the N-terminus than the reported active amino acids. By deleting these amino acids, the N-terminal truncated xylose isomerase obtained can also be used in yeast. Demonstrates high levels of energy.

Example 6: Obtaining and expressing xylose isomerase with N-terminal modification of protein sequence

6.1. Obtaining XI

In order to discover novel XIs active in Saccharomyces cerevisiae, we selected three uncharacterized XIs from the NCBI database, which were derived from Anaeromyces robustus (AnaXI), Neocallimastix californiae (NeoXI), and Rhizoclosmatium globosum (RhiXI). Qingke Biotechnology Co., Ltd. was entrusted to synthesize the isomerase nucleotide sequences respectively. Then, the three synthesized nucleotide macromolecules were inserted into the Saccharomyces cerevisiae episomal expression vector respectively. The specific steps are: insert the G418 resistance gene into the SmaI-SalI site of the Saccharomyces cerevisiae episomal expression vector pESC-URA to obtain the G418_pESC-URA plasmid; Then insert the Saccharomyces cerevisiae promoter TDH3 sequence into the KpnI-NheI site of the G418_pESC-URA plasmid to obtain the TDH3_G418_pESC-URA plasmid; finally, the synthesized SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, The macromolecule nucleotide fragments corresponding to SEQ ID NO.17 and SEQ ID NO.18 were inserted into the NheI site of the TDH3_G418_pESC-URA plasmid to obtain xylose isomerase free expression vectors pESC-Ana, pESC-Neo, and pESC-Rhi. In these free expression vectors of Saccharomyces cerevisiae, the 5' side of the xylose isomerase gene is the TDH3 promoter and the 3' side is the CYC1 terminator.

6.2. Expression of XI

Plasmids pESC-Ana, pESC-Neo, and pESC-Rhi with xylose isomerase genes 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/mLG418), and untransformed cells cannot grow on these plates. Using a single colony on the plate as a template, the corresponding xylose isomerase gene was amplified by PCR and sequenced. The transformants containing the corresponding xylose isomerase gene plasmid were identified and named CRD3Ana, CRD3Neo, and CRD3Rhi respectively. Transfer it to YPX40 medium for culture.

Result 6:

As shown in Figure 9, the initial xylose concentration of YPX medium was 40g/L. When Saccharomyces cerevisiae CRD3Ana, CRD3Neo, and CRD3Rhi were cultured in it for 96 hours, the strains did not utilize xylose for growth, indicating that these three xylose isomerases did not Ability to convert xylose in Saccharomyces cerevisiae.

Example 7: Mutation and expression of xylose isomerase gene

7.1. Mutation of xylose isomerase gene

Amino acid site modification was performed on AnaXI, NeoXI, and RhiXI. The specific methods are shown in Table 1. The modified amino acid and nucleotide sequences are shown in SEQ ID NO.9-13 and SEQ ID NO.14-18.

Table 1 Mutation methods of AnaXI, NeoXI, and RhiXI

7.2. Transformation of free expression vectors and screening of transformants

Plasmids pESC-Ana1, pESC-Ana2, pESC-Neo1, pESC-Neo2, and pESC-Rhi with xylose isomerase genes were transformed into diploid Saccharomyces cerevisiae CRD3 (ATCC 26603, MATa/α, ΔGre3, pho13:: TPI1p ^- Cells cannot grow on these plates. Using a single colony on the plate as a template, the corresponding xylose isomerase gene was amplified by PCR and sequenced. The transformants containing the corresponding xylose isomerase gene plasmid were identified and named respectively CRD3Ana1, CRD3Ana2, CRD3Neo1, CRD3Neo2, and CRD3Rhi1.

7.3. Determination of xylose utilization ability of recombinant strains

Yeasts CRD3Ana-1, CRD3Ana-2, CRD3Neo-1, CRD3Neo-2, and CRD3Rhi-1 were cultured in YPD (2% peptone, 1% yeast extract, 2% glucose) medium overnight, and then the initial OD ₆₀₀ was 1.0 Transfer to YPX (2% peptone, 1% yeast extract, 4% xylose) medium, and conduct anaerobic culture at 30°C and 150 rpm. High-performance liquid chromatography (HPLC) was used to determine the xylose and ethanol concentrations in the culture medium. Yeast growth was monitored using a UV spectrophotometer by measuring _OD600 at a wavelength of 600 nm.

Result 7:

As shown in Figure 9, the initial xylose concentration of YPX medium is 40g/L. When Saccharomyces cerevisiae CRD3Ana-1, CRD3Ana-2, CRD3Neo-1, CRD3Neo-2, and CRD3Rhi-1 are cultured in it for 96 hours, the xylose consumed The amounts were 11.55, 10.63, 18.61, 17.26, and 7.32g/L xylose respectively, and were accompanied by the growth of bacterial cells and the production of ethanol. This result shows that after the five xylose isomerases involved in this laboratory were expressed in S. cerevisiae, they all gave S. cerevisiae the ability to convert xylose into xylulose, allowing it to use xylose to grow and generate ethanol. .

XI can only function normally when forming dimers or tetramers, and the N-terminal sequence plays an important role in stabilizing the diagonal dimers formed by subunits A and D, and subunits B and C. Taking subunits B and C of AnaXI and AnaXI-1 as an example, the amino acids interacting between the two subunits are in the form of sticks, mainly coming from the N-terminus and C-terminus of XI. The N-terminal sequence of AnaXI is short, and only His4, Tyr5 and adjacent subunits interact. In AnaXI-1 that extends the N-terminal sequence, Lys17, Asp18, Lys20, Asn21, Pro22, Leu23, His26, and Tyr27 all interact with adjacent subunits, and there are multiple amino acids and adjacent subunits. Amino acids form hydrogen bonds, which contribute to the stability of the tetrameric structure. After adding the N-terminal sequence to RhiXI, RhiXI-1 gained activity in Saccharomyces cerevisiae, probably for this reason. The redundant N-terminal structure of NeoXI may affect the spatial structure of the tetramer, resulting in the inability to convert xylose. To our knowledge, this is the first time that inactive XI has been changed into active XI by modifying the amino acid sequence.

This application can construct four artificial ancestral sequences of xylose isomerase by collecting homologous sequence sets, multiple sequence alignments of sequence sets, phylogenetic tree construction and computer tools to infer ancestral sequences. sequence and all currently published xylose isomerases (200,000 possible xylose isomerase sequences have been published in the NCBI database. These xylose isomerase sequences were obtained by sequencing environmental or microbial samples. ) show great differences in sequence. And through gene synthesis, gene expression, enzyme activity testing and fermentation experiments, it was proved that the four constructed xylose isomerase sequences can endow the S. cerevisiae strain with higher xylose utilization ability in Saccharomyces cerevisiae.

Example 8: Construction of xylose isomerase sequence obtained based on ancestral sequence construction method

8.1. Obtaining xylose isomerase data

Use the BLAST function in NCBI and use the XI amino acid sequence from Piromyces sp. CD-HIT was used to perform cluster analysis on the obtained amino acid sequences, and the identity threshold was set to 73% to obtain non-redundant amino acid sequences.

8.2. Construction of phylogenetic tree of xylose isomerase

Use MEGA 11 software to load the screened amino acid sequences and XI sequences reported in the literature that can be actively expressed in Saccharomyces cerevisiae, and perform multiple sequence alignment through the ClustalX function. Retain conserved sequences of amino acid sites (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 Indicated according to the amino acid sequence of XI from Piromyces sp.E2)). Use Fastree to construct a phylogenetic tree, the model used is the maximum likelihood method, and the bootstrap value is 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.

8.3. Artificial construction of xylose isomerase

Select all existing XIs evolved from the specified nodes in the phylogenetic tree constructed with currently known xylose isomerase sequences, and load the amino acid sequence information into MEGA 11 software. Amino acid sequence alignment was performed through the ClustalX function, Graps in the alignment results were deleted, and a phylogenetic tree of the processed amino acid sequences was constructed using the maximum likelihood method. Use the CodeML function of pamlX to speculate on ancestral sequences. 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 sequences of each node in the generated phylogenetic tree.

Result 8:

867 sequences were obtained from the 250,000 amino acid sequences in the NCBI database, and a phylogenetic tree was constructed using these 867 XIs and 16 XIs that have been reported to be highly active in Saccharomyces cerevisiae. As shown in Figure 10, the obtained phylogenetic tree has 9 main evolutionary branches. Most of the XIs reported to be active in Saccharomyces cerevisiae in 16 literatures come from Bacteroidetes (branch IX) and Firmicutes (branch IV). ). Construct the ancestral sequence of the specified node in the phylogenetic tree in Figure 11, and its amino acid sequence is shown in SEQ ID NO.19-22. In order to compare the similarity between these deduced amino acid sequences and existing XIs, NCBI's BLAST function was used, and it was found that the maximum Per.Ident between them and their respective most similar XIs was 82.15%-90.14% (Table 2). Its amino acid sequence is completely different from the currently known XI.

Table 2 Comparison results between the amino acid sequences of four xylose isomerases and the amino acid sequences of currently known proteins

Example 9: Four xylose isomerase genes are expressed episomally in Saccharomyces cerevisiae

9.1. Construction of free expression vector

GenScript Biotechnology Co., Ltd. was entrusted to synthesize the xylose isomerase nucleotide sequences of SEQ ID NO.23, SEQ ID NO.24, SEQ ID NO.25, and SEQ ID NO.26 respectively. Then, the four synthesized nucleotide macromolecules were inserted into the Saccharomyces cerevisiae episomal expression vector respectively. The specific steps are: insert the G418 resistance gene into the SmaI-SalI site of the Saccharomyces cerevisiae episomal expression vector pESC-URA to obtain the G418_pESC-URA plasmid; Then insert the Saccharomyces cerevisiae promoter TDH3 sequence into the KpnI-NheI site of the G418_pESC-URA plasmid to obtain the TDH3_G418_pESC-URA plasmid; finally, the synthesized SEQ ID NO.23, SEQ ID NO.24, SEQ ID NO.25, The macromolecule nucleotide fragment corresponding to SEQ ID NO.26 was inserted into the NheI site of the TDH3_G418_pESC-URA plasmid to obtain xylose isomerase free expression vectors pESC-Anc1, pESC-Anc2, pESC-Anc3, and pESC-Anc4. In these Saccharomyces cerevisiae free expression vectors, the 5' side of the xylose isomerase gene is the TDH3 promoter and the 3' side is the CYC1 terminator.

9.2. Transformation of free expression vectors and screening of transformants

Plasmids pESC-Anc1, pESC-Anc2, pESC-Anc3, and pESC-Anc4 with xylose isomerase genes 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 (400μg/mLG418), untransformed cells cannot be screened in these Grow on plates. Using a single colony on the plate as a template, the corresponding xylose isomerase gene was amplified by PCR and sequenced. The transformants containing the corresponding xylose isomerase gene plasmid were identified and named CRD3A1, CRD3A2, CRD3A3, and CRD3A4 respectively.

9.3. Determination of xylose utilization ability of recombinant strains

Yeasts CRD3A1, CRD3A2, CRD3A3, and CRD3A4 were cultured in YPD (2% peptone, 1% yeast extract, 2% glucose) medium overnight, and then transferred to YPX (2% peptone, 1% yeast) with an initial OD ₆₀₀ of 1.0. extract, 4% xylose) culture medium, 30°C, 150rpm. High-performance liquid chromatography (HPLC) was used to determine the xylose and ethanol concentrations in the culture medium. Yeast growth was monitored using a UV spectrophotometer by measuring _OD600 at a wavelength of 600 nm.

Result 9:

As shown in Figure 11, the initial xylose concentration of YPX medium is 40g/L. When Saccharomyces cerevisiae CRD3A1, CRD3A2, CRD3A3, and CRD3A4 are cultured in it for 96 hours, the xylose contents consumed are 12.43, 11.80, 12.70, and 8.84g/L respectively. L, and is 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, allowing it to use xylose to grow and generate ethanol. .

Claims

A xylose isomerase expressed with high activity in yeast cells, characterized in that its amino acid sequence is one of the following amino acid sequences:

(1) The amino acid sequence shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4;

(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 and SEQ ID NO.4;

(3) Having an amino acid sequence that is 70% or more identical to the amino acid sequence shown in any one of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, and SEQ ID NO. 4.
The xylose isomerase expressed with high activity in yeast cells according to claim 1, characterized in that its nucleotide sequence is one of the following nucleotide sequences:

(1) The nucleotide sequence shown in SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.8;

(2) The nucleotide sequence shown in SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.8 has one or more nucleotides added, deleted, substituted, or inserted Nucleotide sequence;

(3) A nucleotide having more than 70% 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 sequence;

(4) Due to the degeneracy of the genetic code, the nucleotide sequence is different from the nucleotide sequence shown in SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.8.
A xylose isomerase modified at the N-terminus of a protein sequence, characterized in that its amino acid sequence is one of the following amino acid sequences:

(1) The amino acid sequence shown in SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, and SEQ ID NO.13;

(2) One or more amino acid sequences shown in SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, and SEQ ID NO.13 are added, deleted, substituted or inserted the amino acid sequence of an amino acid;

(3) Having an identity of more than 70% with the amino acid sequence shown in any one of SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, and SEQ ID NO.13 amino acid sequence.
The xylose isomerase according to claim 3 is characterized in that its nucleotide sequence is one of the following nucleotide sequences:

(1) The nucleotide sequence shown in SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, and SEQ ID NO.18;

(2) The nucleotide sequence shown in SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, and SEQ ID NO.18 has one or more added, deleted, substituted or inserted A nucleotide sequence of multiple nucleotides;

(3) Have more than 70% similarity with the nucleotide sequence shown in any one of SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, and SEQ ID NO.18 Identity of nucleotide sequence;

(4) Due to the degeneracy of the genetic code, it is different from the core of the nucleotide sequences shown in SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, and SEQ ID NO.18. nucleotide sequence.
A xylose isomerase obtained based on the ancestral sequence construction method, characterized in that its amino acid sequence is one of the following amino acid sequences:

(1) The amino acid sequence shown in SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.21, and SEQ ID NO.22;

(2) Amino acid sequences in which one or more amino acids are added, deleted, substituted or inserted into the amino acid sequences shown in SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.21 and SEQ ID NO.22;

(3) Having an amino acid sequence that is 70% or more identical to the amino acid sequence shown in any one of SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, and SEQ ID NO. 22.
The xylose isomerase according to claim 5, characterized in that its nucleotide sequence is one of the following nucleotide sequences: (1) SEQ ID NO.23, SEQ ID NO.24, SEQ ID NO. 25. The nucleotide sequence shown in SEQ ID NO.26;

(2) The nucleotide sequence shown in SEQ ID NO.23, SEQ ID NO.24, SEQ ID NO.25, and SEQ ID NO.26 has one or more nucleotides added, deleted, substituted, or inserted Nucleotide sequence;

(3) A nucleotide having more than 70% identity with the nucleotide sequence shown in any one of SEQ ID NO.23, SEQ ID NO.24, SEQ ID NO.25, and SEQ ID NO.26 sequence;

(4) Due to the degeneracy of the genetic code, the nucleotide sequence is different from the nucleotide sequence shown in SEQ ID NO.23, SEQ ID NO.24, SEQ ID NO.25, and SEQ ID NO.26.
The xylose isomerase according to any one of claims 1 to 6, characterized in that the expression of the xylose isomerase can give the host cell the ability to convert xylose into xylulose, thereby giving the host cell the ability to convert xylose into xylulose. The ability to assimilate xylose, the host cells are Saccharomyces, Yarrowia, Candida, Pichia, Schizosaccharomyces, Hansenula (Hansenula), Kluyveromyces.
The xylose isomerase according to claim 7, wherein the host cell is a Saccharomyces cerevisiae cell.
The xylose isomerase according to claim 7, characterized in that the expression mode of the xylose isomerase in the host is one of the following modes:

(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 integrated and expressed in the host;

(3) The xylose isomerase gene is expressed both free and integrated in the host.
The xylose isomerase according to claim 7, characterized in that the xylose isomerase is expressed in a host strain alone or in combination in a host cell.
The xylose isomerase according to claim 7, wherein the yeast cell is a wild bacterium or a yeast cell that has undergone one or more genetic modifications.
An application of the xylose isomerase according to claim 1, characterized in that the application is specifically: the xylose isomerase empowers the host cell to produce a variety of fermentation products using xylose or lignocellulose hydrolyzate, Including xylulose, fructose, ethanol, butanol, microbial lipids, free fatty acids, furfural, lactic acid, succinic acid, citric acid, propionic acid, 3-hydroxypropionic acid, adipic acid, xylulose-5-phosphate, isophosphate Pentadiene, polyhydroxyalkanoate, lysine, glutamic acid, phenylalanine, alanine, vanillic acid, vanillin.