CN108192853B - Method for promoting microbial cells to transport glucose, xylose and arabinose and application of method in fermentation of bio-based products - Google Patents

Abstract

The invention discloses a method for promoting microbial cells to transport glucose, xylose and arabinose and application thereof in fermentation of bio-based products. The five transport proteins provided by the invention have the capacity of transporting glucose, xylose or arabinose. The method for promoting the microbial cell transfer is to introduce the transfer protein into a microbial strain, and the obtained recombinant microbial strain obtains or improves the transfer capacity of glucose, xylose and arabinose, so that the glucose, xylose and arabinose can be used for producing fuel ethanol and other bio-based fermentation products.

Description

Method for promoting microbial cells to transport glucose, xylose and arabinose and application of method in fermentation of bio-based products

The application is a divisional application of the Chinese patent application with the application number of CN201510081668.8, which is applied on 15/2/2015.

Technical Field

The invention belongs to the technical field of biology, and discloses a method for promoting microbial cells to transport glucose, xylose and arabinose. Mainly relates to a transporter (GLT-1, XYT-1, XAT-1, LAT-1 and MtLAT-1), a coding gene, a glucose, xylose or arabinose transport capacity obtained or improved by introducing a microbial strain, and application of the transporter in producing bio-based products by microbial fermentation.

Background

Biomass is the largest renewable resource on earth and is also the most widely distributed carbohydrate. Facing the energy crisis and resource shortage, the production of biological energy by renewable biomass provides hope for sustainable development of human beings. The biomass mainly comprises cellulose, hemicellulose and lignin, and degradation products mainly comprise glucose, xylose, arabinose and other monosaccharides and partial oligosaccharides.

In order to increase the utilization of biomass degradants, researchers have conducted metabolic engineering on a variety of microorganisms. The engineering saccharomyces cerevisiae has the characteristics of easy culture, high yield, higher tolerance to metabolic inhibitors and ethanol, clear research background and simple genetic operation, and becomes a main strain for performing ethanol fermentation by using biomass degradation products. However, since many of the microorganisms important in the fermentation industry, such as Saccharomyces cerevisiae, cannot utilize xylose and arabinose, the efficient use of biomass degradation products for fermentation production of bio-based chemicals (ethanol, butanol, etc.) is limited. Subsequently, although researchers have made efforts to construct engineered strains (e.g., engineered s.cerevisiae) that are genetically engineered to be able to ferment bio-based chemicals such as ethanol using xylose and arabinose as carbon sources, there still remain many problems, such as unbalanced intracellular redox metabolism, slow pentose metabolism, lack of effective pentose transporters, and the like. In addition, the carbon source repression effect exists in almost all microorganisms, so that the engineering strains (such as the engineering saccharomyces cerevisiae) can not utilize various components in the total sugar at the same time in the process of fermenting by utilizing the mixed sugar of the biomass degradation products, thereby prolonging the fermentation time and reducing the fermentation efficiency. When the mixed sugar is used for fermentation, when the glucose is used up, the ethanol content in the fermentation liquid reaches a higher concentration, and the fermentation rate of pentose is greatly reduced. Recent studies have shown that the repression effect of glucose occurs mainly in sugar transport, and therefore, the search for xylose or arabinose transporters has been a hot spot in order to increase the utilization rate of total sugar by Saccharomyces cerevisiae.

In the field of five-carbon sugar transporters, studies have been focused on the transport of five-carbon sugars using a broad-spectrum Transporter, such as hexose Transporter, as a substrate, and studies on a Specific five-carbon sugar Transporter (pentase Specific Transporter) have just started. In 2011, the laboratory of the teaching of Zhao Hui nationality of the university of Illinois, USA identified two xylose-specific transporters (An25 and Xyp29) from Neurospora crassa and Pichia pastoris, respectively (Du et al 2010.discovery and characterization of novel d-xylose-specific transporters from Neurospora crassa and Pichia stipitis. mol biosystem, 6(11): 2150-. As one of the main components of hemicellulose, the utilization of arabinose is crucial for the complete conversion and utilization of lignocellulose, and international studies on arabinose transporters have been increasingly involved, in 2011, the Finland VTT scientist Richard laboratory cloned two arabinose-specific transport transporters (LAT1, LAT2) from the yeast Ambrosiozyme monospora, but the arabinose transporters had not high affinity (Verho et al 2011.cloning of two genes (LAT1,2) encoding specific L-arabinanosomes transporters of the L-arabinanosomes fertilization yeast, applied Biochem Biotechnology, 164(5):604, 611).

Unlike bacterial xylooligosaccharide transporters, sugar transporters of filamentous fungi can divide the transporters into primary active transporters (primary transporters) and secondary transporters (secondary transporters) depending on the source of the transport energy. The primary active transporter realizes a transport process by utilizing energy released by ATP hydrolysis, photon absorption, electron flow, substrate decarboxylation or methyl transfer reaction and the like, and is typically represented by an ATP Binding Cassette (ABC) superfamily; the secondary transporters utilize electrochemical osmotic potential due to the difference of the concentration of the substance inside and outside the membrane to transport the substrate, and typically represent the major auxiliary transporters superfamily (MFS) (see the references: Sun forest peak, Wang Jia Wei, Yan Ning, MFS superfamily, research on the structure and molecular mechanism of transporters, Life sciences, Vol.23, No. 11, 1052-1056.). MFS are further classified into simple transporters (uniporters), also known as Facilitated Diffusion proteins (diffusing proteins), symporters (symporters) and antiporters (antiporters), depending on the transport mechanism. Uniporter, which relies on a substrate concentration gradient to drive transport, transporters mainly play a role in facilitating transport. Symporter transports two or more substrates simultaneously in the same direction, and takes the electrochemical gradient of one substrate as the driving force, commonly comprising sugar/H +, glucose/Na +, phosphate/H +, nucleotide/H +, and nitrate/H +, etc. Antiporter, the cooperative transport of two or more substrates in the reverse direction, has the same driving force source as Symporter, and many of the antiporters in this category are drag/H + antiporters. Sugar transporters in fungi are mainly of the Uniporter and Symporter/H + type (reduce et al 2012.A collection of programs for the student of transport protein evolution. FEBS J,279(11): 2036-. It has been reported as early as 1974 that Neurospora bacteria coordinate the transport of protons while actively transporting sugars into cells (Slayman et al 1974. polarization of the plasma membrane of Neurospora reduced active transport of glucose: evidence for a proton-dependent transport system. Proc Natl Acad Sci USA 71(5): 1935. 1931939.), but the role of which sugar transporters at the molecular level leads to apparent potential changes has not been elucidated so far. In addition, since Neurospora species can grow on various dried wood fibers in nature, it is expected that the sugar transporter family has high functional diversity. Meanwhile, in the long-term evolution adaptation process, cells face different growth environments, such as acidity-alkalinity, sufficient carbon and deficient carbon, and a good strategy is provided for coordinating the transport work among the transporters, so that the transporters can efficiently absorb external nutrition. The comprehensive and systematic understanding of the power types and biochemical characteristics of the sugar transporters is of great significance for researching the environmental adaptability of filamentous fungi and cleaning the mechanism of lignocellulose utilization by cellulose degrading bacteria. Furthermore, the efficient sugar transport and absorption mode has certain guiding significance for transforming engineering bacteria (including yeast, aspergillus and trichoderma) to fully utilize residual sugar in the culture medium for fermentation and improve the conversion utilization rate of sugar.

Disclosure of Invention

In a first aspect of the invention, there is provided an isolated sugar transporter polypeptide selected from the group consisting of:

(a) a polypeptide having an amino acid sequence as set forth in any one of SEQ ID No.10 (MtLAT-1), 8(LAT-1), 6(XAT-1), 4(XYT-1), or 2 (GLT-1);

(b) 10, 8, 6, 4 or 2 by substitution, deletion or addition of one or more amino acid residues, or by addition of a signal peptide sequence, or a derivative polypeptide having pentose and/or hexose transport activity;

(c) a derivative polypeptide having the sequence of the polypeptide of (a) or (b);

(d) a derivative polypeptide having an amino acid sequence homology of 85% or more (preferably 90% or more, more preferably 95% or more, 98%) to any of the amino acid sequences set forth in SEQ ID No.10, 8, 6, 4, or 2, and having pentose and/or hexose transport activity;

the pentose and/or hexose transport activity means that pentose and/or hexose is transported from outside the cell to inside the cell.

In another preferred embodiment, the derivative polypeptide described in (d) is derived from one or more of the following strains: myceliophthora thermophila (Myceliophthora thermophila), Chaetomium globosum (Chaetomium globosum), corynespora chrysosporium (Podospora anserina), pyricularia oryzae (Magnaporthe oryzae), Gibberella zeae (Gibberella zeae), Fusarium oxysporum (Fusarium oxysporum), Penicillium chrysogenum (Penicillium chrysogenum), Aspergillus terreus (Aspergillus terreus), Gibberella flagellata (necatria haematococcus), Thielavia spinosa (Thielavia terrestris), Trichoderma viride (Trichoderma virens), phaeochaetomium carnosum (neochaetosa), Aspergillus niger (Aspergillus niger), Trichoderma schoerei (Trichoderma bresei), pichia stipitis (pichia stipitis), pichia pastoris (pichia lactis), and saccharomyces lactis (kluyveris).

In another preferred embodiment, the cell is a microbial cell.

In another preferred embodiment, the polypeptide has an activity of transporting a saccharide selected from one or more of the following from outside the cell to inside the cell: arabinose, xylose, and glucose.

In another preferred embodiment, the pentose comprises arabinose, or xylose;

in another preferred embodiment, the hexose comprises glucose.

In another preferred embodiment, the polypeptide having the sequence as shown in SEQ ID No.10 has the activity of transporting arabinose from outside the cell to inside the cell; and/or

A polypeptide having the sequence shown in SEQ ID No.8 and having the activity of transporting arabinose from the outside of cells to the inside of cells; and/or

A polypeptide having the sequence as shown in SEQ ID No.6, having the activity of transporting xylose and/or arabinose from outside the cell to inside the cell; and/or

A polypeptide having a sequence as set forth in SEQ ID No.4, having an activity of transporting xylose from outside the cell to inside the cell; and/or

The polypeptide with the sequence shown in SEQ ID No.2 has the activity of transferring glucose from the outside of cells to the inside of cells.

In a second aspect of the invention, there is provided an isolated polynucleotide, said polynucleotide being a sequence selected from the group consisting of:

(A) a nucleotide sequence encoding a polypeptide according to the first aspect of the invention;

(B) a nucleotide sequence encoding a polypeptide set forth in any one of SEQ ID No.10, 8, 6, 4, or 2;

(C) a nucleotide sequence set forth in any one of SEQ ID No.9, 7, 5, 3, or 1;

(D) a nucleotide sequence complementary to the nucleotide sequence of any one of (A) to (C).

In a third aspect of the invention, there is provided a vector comprising a polynucleotide according to the second aspect of the invention.

In another preferred embodiment, the vector comprises an expression vector, a shuttle vector and an integration vector.

In a fourth aspect of the invention, there is provided a host cell comprising a vector according to the third aspect of the invention and/or having integrated into its chromosome an exogenous polynucleotide according to the second aspect of the invention.

In another preferred embodiment, the host cell comprises a eukaryotic or prokaryotic cell, preferably a eukaryotic cell.

In another preferred embodiment, the host cell expresses one or more polypeptides according to the first aspect of the invention.

In another preferred embodiment, the host cell comprises a yeast (Saccharomyces) genus, a Kluyveromyces genus, a Clostridium genus, or a filamentous fungus.

In another preferred embodiment, the genus Saccharomyces includes Saccharomyces cerevisiae, Saccharomyces mansoniae, Saccharomyces monacinis, Saccharomyces bayanus, Saccharomyces pastorianus, Saccharomyces carlsbergensis, Schizosaccharomyces pombe, Saccharomyces pombe;

the Kluyveromyces (Kluyveromyces) includes Kluyveromyces marxianus (Kluyveromyces marxianus), Kluyveromyces lactis (Kluyveromyces lactis), Kluyveromyces fragilis (Kluyveromyces fragilis), Pichia stipitis (Pichia stipites), Candida shehatae (Candida shehatae), Candida tropicalis (Candida tropicalis), and Zymomonas mobilis (Zymomonas mobilis);

the genus Clostridium (Clostridium sp.) includes Clostridium (Clostridium thermocellum), Clostridium beijerinckii (Clostridium beijerinckii), Clostridium acetobutylicum (Clostridium acetobutylicum), thermoacetobacter (Moorella thermoacetica), Escherichia coli (Escherichia coli), Klebsiella oxytoca (Klebsiella oxytoca), anaerobacterium (Thermoanaerobacterium saccharolyticum), or Bacillus subtilis;

the filamentous fungi include Thermomyces thermophilus (Sporotrichum thermophile), Neurospora crassa (Neurospora crassa).

In a fifth aspect, the invention provides a use of a polypeptide according to the first aspect of the invention, a polynucleotide according to the second aspect of the invention, a vector according to the third aspect of the invention or a host cell according to the fourth aspect of the invention, (i) for the transport of pentoses and/or hexoses from outside the cell to inside the cell; (ii) is used for preparing ethanol.

In a sixth aspect of the present invention, there is provided a method for producing ethanol and/or for promoting the transport of pentoses and/or hexoses by a host cell, comprising the steps of: culturing the host cell according to the fourth aspect of the present invention in the presence of pentose or hexose.

In another preferred embodiment, the method further comprises the step of separating and purifying ethanol in the culture.

In another preferred embodiment, when the host cell of the fourth aspect of the invention expresses a polypeptide as shown in SEQ ID No.10 or 8, the pentose sugar is arabinose.

In another preferred embodiment, when the host cell according to the fourth aspect of the present invention expresses the polypeptide of SEQ ID No.6, the pentose sugar is arabinose, and/or xylose.

In another preferred embodiment, when the host cell according to the fourth aspect of the present invention expresses the polypeptide of SEQ ID No.4, the pentose sugar is xylose.

In another preferred embodiment, when the host cell according to the fourth aspect of the present invention expresses the polypeptide of SEQ ID No.2, the hexose is glucose.

The seventh aspect of the present invention is a method for preparing a recombinant ethanol fermentation strain, comprising the steps of: transferring the vector of the third aspect of the invention into an original strain to obtain a recombinant ethanol fermentation strain.

In another preferred embodiment, the ethanol fermentation activity of the recombinant ethanol fermentation strain is 1.2-5 times, preferably 1.5-2 times that of the starting strain.

In another preferred embodiment, the recombinant ethanol fermentation strain is a strain using pentose (such as arabinose and/or xylose) and/or hexose (such as glucose) as a carbon source.

In another preferred embodiment, the starting strain comprises Saccharomyces cerevisiae, such as Saccharomyces cerevisiae BSW2AP and Saccharomyces cerevisiae EBY.VW4000, preferably Saccharomyces cerevisiae BSW2AP

The purpose of this patent is to provide five novel sugar transporters, the specific nucleotide sequences and the amino acid residue sequences of the encoded proteins thereof are as follows

a. The nucleotide sequence of the glucose transporter gene GLT-1(NCU01633, derived from Neurospora crassa) is shown as SEQ ID NO.1 in the sequence table;

the amino acid residue sequence of the protein coded by the glucose transporter gene GLT-1(NCU01633, derived from Neurospora crassa) is shown as SEQ ID NO.2 in the sequence table;

b. the nucleotide sequence of the xylose transporter gene XYT-1(NCU05627, derived from Neurospora crassa) is shown as SEQ ID NO.3 in the sequence table;

the amino acid residue sequence of the protein coded by the xylose transporter gene XYT-1(NCU05627, derived from Neurospora crassa) is shown as SEQ ID NO.4 in the sequence table

c. The nucleotide sequence of the xylose and arabinose transporter gene XAT-1(NCU01132, derived from Neurospora crassa) is shown as SEQ ID NO.5 in the sequence table;

the amino acid residue sequence of the protein coded by the xylose and arabinose transporter gene XAT-1(NCU01132, which is derived from Neurospora crassa) is shown as SEQ ID NO.6 in the sequence table;

d. the nucleotide sequence of arabinose transporter gene LAT-1(NCU02188, derived from Neurospora crassa) is shown as SEQ ID NO.7 in the sequence table;

the amino acid residue sequence of the arabinose transporter gene LAT-1(NCU02188, which is derived from Neurospora crassa) coding protein is shown as SEQ ID NO.8 in the sequence table;

e. the nucleotide sequence of the arabinose transporter gene MtLAT-1(MYCTH _95427, derived from myceliophthora thermophila, Myceliophora thermophila) is shown as SEQ ID NO.9 in the sequence table;

the amino acid residue sequence of the arabinose transporter gene MtLAT-1(MYCTH _95427, from myceliophthora thermophila) coding protein is shown as SEQ ID NO.10 in the sequence table.

In addition, the protein with homology of over 75 percent with the whole length or local structural domain of the amino acid residue sequence of the five transport proteins is also included; the protein with the homology of more than 75% is from the following bacteria: myceliophthora thermophila (Myceliophthora thermophila), Chaetomium globosum (Chaetomium globosum), corynespora chrysosporium (Podospora anserina), pyricularia oryzae (Magnaporthe oryzae), Gibberella zeae (Gibberella zeae), Fusarium oxysporum (Fusarium oxysporum), Penicillium chrysogenum (Penicillium chrysogenum), Aspergillus terreus (Aspergillus terreus), Gibberella flagellata (necatria haematococcus), Thielavia spinosa (Thielavia terrestris), Trichoderma viride (Trichoderma virens), phaeochaetomium carnosum (neochamyces carnosa), Aspergillus niger (Aspergillus niger), Trichoderma schoerei (Trichoderma bresei), pichia pastoris (pichia stipitis), pichia lactis (kluyveris, etc.

The microorganisms used include, but are not limited to, the following: saccharomyces cerevisiae (Saccharomyces cerevisiae), Saccharomyces cerevisiae (Saccharomyces monangis), Saccharomyces bayanus (Saccharomyces bayanus), Saccharomyces pastorianus (Saccharomyces pastorianus), Saccharomyces carlsbergensis (Saccharomyces pombe), Kluyveromyces (Kluyveromyces sp.), Kluyveromyces lactis (Saccharomyces lactis), Kluyveromyces fragilis (Kluyveromyces fragilis), Pichia stipitis (Pichia stipitis), Thermomyces thermophilus (Sporhizomorph), Candida utilis (Candida albicans), Candida utilis (Candida utilis), Clostridium trichothecoides (Clostridium trichoderma), Clostridium trichoderma (Clostridium trichoderma), Clostridium thermocellum (Clostridium thermocellum), Clostridium thermocellum), Clostridium thermocellum (Clostridium thermocellum), Clostridium thermocellum (Clostridium thermocellum), Clostridium thermocellum (Clostridium thermocellum) and Bacillus thermocellum (Clostridium thermocellum) strain, Escherichia coli (Escherichia coli), Klebsiella oxytoca (Klebsiella oxytoca), anaerobic Bacillus (Thermoanaerobacterium saccharolyticum), and Bacillus subtilis (Bacillus subtilis).

In another aspect, the invention provides a method for obtaining or increasing the utilization of glucose, xylose or arabinose by a microorganism, comprising introducing the transporter into a microbial cell (as listed above), wherein the resulting engineered strain of the microorganism can transport glucose, xylose or arabinose from outside the microorganism to inside the cell, thereby increasing the efficiency of glucose, xylose or arabinose utilization by the microorganism and the capacity of the microorganism to produce a bio-based product by fermentation.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 is a map of the green fluorescence localization of sugar transporters in Saccharomyces cerevisiae;

FIG. 2 is a physical map of recombinant expression plasmid pRS426-LAT carrying GLT-1 gene;

FIG. 3 is a measurement of GLT-1 transport of glucose;

FIG. 4 shows the growth of GLT-1 containing Saccharomyces cerevisiae EGLT on glucose plates;

FIG. 5 is a measurement of xylose transport capacity by XYT-1;

FIG. 6A is a XAT-1 assay for xylose transport capacity;

FIG. 6B is a XAT-1 assay for arabinose transport capacity;

FIG. 7 is a measurement of the capacity of LAT-1 for arabinose transport;

FIG. 8 is an assay of LAT-1 versus arabinose transport type;

FIG. 9 is a measurement of the arabinose transport capacity by MtLAT-1;

FIG. 10 is an assay of the type of arabinose transport by MtLAT-1;

FIG. 11 is a plot of the growth of Saccharomyces cerevisiae XXYT carrying XYT-1 on xylose;

FIG. 12 is a physical map of a recombinant expression plasmid p426LAT carrying LAT-1 gene;

FIG. 13 is a physical map of recombinant expression plasmid p426MtLAT carrying the MtLAT-1 gene;

FIG. 14 is a graph showing the growth curve (A) on arabinose and the L-arabinose consumption curve (B) for Saccharomyces cerevisiae BSWLAT expressing LAT-1 or MtLAT-1 under aerobic conditions;

FIG. 15 is a graph showing the growth curve (A) on arabinose, L-arabinose consumption curve (B) and ethanol production curve (C) for Saccharomyces cerevisiae BSWLAT carrying LAT-1 or MtLAT-1 under anaerobic conditions.

Detailed Description

The present inventors have conducted extensive and intensive studies and, for the first time, have found and identified several sugar transporters capable of imparting pentose or hexose (particularly pentose-specific) utilizing ability to microbial cells. The protein can be used for fermenting microorganisms such as yeast and the like by taking arabinose and xylose as carbon sources, so that the utilization of the carbon sources by the microorganisms is not influenced by the repression effect of glucose, and thus, bio-based products such as ethanol and the like can be obtained more efficiently and more economically. On the basis of this, the present invention has been completed.

Definition of

As used herein, the terms "active polypeptide", "polypeptide of the invention and its derived polypeptides", "transporter of the invention", "pentose and/or hexose transporter", "polypeptide of SEQ ID No.:10, 8, 6, 4, or 2", all refer to polypeptides of MtLAT-1(SEQ ID No.:10), LAT-1(SEQ ID No.:8), XAT-1(SEQ ID No.:6), XYT-1(SEQ ID No.:4), or GLT-1(SEQ ID No.:2) and their derived polypeptides having the activity of transporting pentose and/or hexose from outside the cell to inside the cell.

As used herein, the term "pentose" refers to sugars containing 5 carbon atoms, typically, the pentose includes pentaaldose (e.g., ribose, lyxose, arabinose, xylose) and ketopentose (e.g., ribulose, xylulose), preferably, the pentose includes arabinose, xylose, and the like, useful for microbial fermentation.

As used herein, the term "hexose" refers to a sugar containing 6 carbon atoms, and typically, the hexose includes glucose, galactose, mannose, fructose, and the like. Preferably, the hexose is a six-carbon sugar such as glucose, which can be used for microbial fermentation.

Isolated polypeptides and encoding polynucleotides

As used herein, "isolated polypeptide" means that the polypeptide is substantially free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated. One skilled in the art can purify the polypeptide using standard protein purification techniques. Substantially pure polypeptides are capable of producing a single major band on a non-reducing polyacrylamide gel. The purity of the polypeptide can be further analyzed by amino acid sequence.

The active polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide. The polypeptides of the invention may be naturally purified products, or chemically synthesized products, or produced from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, plants) using recombinant techniques. Depending on the host used in the recombinant production protocol, the polypeptides of the invention may be glycosylated or may be non-glycosylated. The polypeptides of the invention may or may not also include an initial methionine residue.

The invention also includes fragments, derivatives and analogues of the polypeptides. As used herein, the terms "fragment," "derivative," and "analog" refer to a polypeptide that retains substantially the same biological function or activity as the polypeptide.

A polypeptide fragment, derivative or analogue of the invention may be (i) a polypeptide in which one or more conserved or non-conserved amino acid residues, preferably conserved amino acid residues, are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a polypeptide in which the mature polypeptide is fused to another compound, such as a compound that increases the half-life of the polypeptide, e.g. polyethylene glycol, or (iv) a polypeptide in which an additional amino acid sequence is fused to the sequence of the polypeptide (e.g. a leader or secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or a fusion protein with an antigenic IgG fragment). Such fragments, derivatives and analogs are within the purview of those skilled in the art in view of the teachings herein.

The polypeptide of the present invention has an activity of transporting pentose and/or hexose from outside to inside of a cell, and preferably, the polypeptide has an activity of transporting one or more sugars selected from the group consisting of: arabinose, xylose, and glucose.

For example:

the polypeptide with the sequence shown in SEQ ID NO.10 or the derivative polypeptide thereof has the activity of transporting arabinose from the outside of cells to the inside of cells; and/or

The polypeptide with the sequence shown in SEQ ID No.8 or the derivative polypeptide thereof has the activity of transferring arabinose from the outside of cells to the inside of cells; and/or

A polypeptide having a sequence as shown in SEQ ID No.6 or a polypeptide derived therefrom, having an activity of transporting xylose and/or arabinose from outside the cell to inside the cell; and/or

A polypeptide having a sequence as shown in SEQ ID No.4 or a polypeptide derived therefrom, having an activity of transporting xylose from outside the cell to inside the cell; and/or

The polypeptide with the sequence shown in SEQ ID NO.2 or the derivative polypeptide thereof has the activity of transporting glucose from the outside of cells to the inside of cells.

The preferred sequence of the polypeptide of the invention is that shown in SEQ ID No.10, 8, 6, 4, or 2, and the term also includes variants and derivatives of these polypeptides having the same or similar function as the polypeptide shown. These variants include (but are not limited to): deletion, insertion and/or substitution of one or more (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10) amino acids, and addition of one or several (usually up to 20, preferably up to 10, more preferably up to 5) amino acids at the C-terminus and/or N-terminus. For example, in the art, substitutions with amino acids of similar or similar properties will not generally alter the function of the protein. Also, for example, the addition of one or several amino acids at the C-terminus and/or N-terminus does not generally alter the function of the protein. The invention also provides analogues or active derivatives of said polypeptides. These analogs may differ from the native polypeptide of the invention by amino acid sequence differences, by modifications that do not affect the sequence, or by both. These polypeptides include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by irradiation or exposure to mutagens, site-directed mutagenesis, or other known molecular biological techniques. Analogs also include analogs having residues other than the natural L-amino acids (e.g., D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (e.g., beta, gamma-amino acids). It is to be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above.

Modified (generally without altering primary structure) forms include: chemically derivatized forms of the polypeptide, such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those resulting from glycosylation modifications in the synthesis and processing of the polypeptide or in further processing steps. Such modification may be accomplished by exposing the polypeptide to an enzyme that performs glycosylation, such as a mammalian glycosylase or deglycosylase. Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides modified to increase their resistance to proteolysis or to optimize solubility.

The polynucleotide of the present invention may be in the form of DNA or RNA. The form of DNA includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand. The sequence of the coding region encoding the mature polypeptide may be identical to the sequence of the coding region shown in SEQ ID NO.1 or may be a degenerate variant. As used herein, "degenerate variant" refers in the present invention to nucleic acid sequences that differ in the sequence of the coding region as set forth in SEQ ID No.9, 7, 5, 3, or 1.

A polynucleotide encoding a mature polypeptide of SEQ ID No.10, 8, 6, 4, or 2 comprising: a coding sequence encoding only the mature polypeptide; the coding sequence for the mature polypeptide and various additional coding sequences; the coding sequence (and optionally additional coding sequences) as well as non-coding sequences for the mature polypeptide.

The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, and may also include additional coding and/or non-coding sequences.

The present invention also relates to variants of the above polynucleotides which encode polypeptides having the same amino acid sequence as the present invention or fragments, analogs and derivatives of the polypeptides. The variant of the polynucleotide may be a naturally occurring allelic variant or a non-naturally occurring variant. These nucleotide variants include substitution variants, deletion variants and insertion variants. As is known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, without substantially altering the function of the polypeptide encoded thereby.

The present invention also relates to polynucleotides which hybridize to the sequences described above and which have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides hybridizable under stringent conditions (or stringent conditions) with the polynucleotides of the present invention. In the present invention, "stringent conditions" mean: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 XSSC, 0.1% SDS, 60 ℃; or (2) adding denaturant during hybridization, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42 deg.C, etc.; or (3) hybridization occurs only when the identity between two sequences is at least 90% or more, preferably 95% or more. And, the polynucleotides that hybridize encode polypeptides having the same biological functions and activities as the mature polypeptides set forth in SEQ ID No.10, 8, 6, 4, or 2.

The full-length nucleotide sequence of the polypeptide of the present invention or a fragment thereof can be obtained by PCR amplification, recombination, or artificial synthesis. For PCR amplification, primers can be designed based on the nucleotide sequences disclosed herein, particularly open reading frame sequences, and the sequences can be amplified using commercially available cDNA libraries or cDNA libraries prepared by conventional methods known to those skilled in the art as templates. When the sequence is long, two or more PCR amplifications are often required, and then the amplified fragments are spliced together in the correct order.

Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into a cell, and then obtaining the sequence of interest from the propagated host cell by conventional methods.

A method of amplifying DNA/RNA using PCR technology is preferably used to obtain the gene of the present invention. Particularly, when it is difficult to obtain a full-length cDNA from a library, it is preferable to use the RACE method (RACE-cDNA terminal rapid amplification method), and primers used for PCR can be appropriately selected based on the sequence information of the present invention disclosed herein and synthesized by a conventional method. The amplified DNA/RNA fragments can be isolated and purified by conventional methods, such as by gel electrophoresis.

Carrier

The invention also relates to vectors comprising the polynucleotides of the invention, as well as genetically engineered host cells encoded with the vector or polypeptide coding sequences of the invention, and methods for producing the polypeptides of the invention by recombinant techniques.

In the present invention, a polynucleotide sequence encoding a polypeptide of the present invention is inserted into a recombinant expression vector. The term "recombinant expression vector" refers to a bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus, or other vectors well known in the art. Any plasmid or vector may be used as long as it can replicate and is stable in the host. An important feature of expression vectors is that they generally contain an origin of replication, a promoter, a marker gene and translation control elements.

Methods well known to those skilled in the art can be used to construct expression vectors containing a DNA sequence encoding a polypeptide of the present invention and appropriate transcription/translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence may be operably linked to a suitable promoter in an expression vector to direct mRNA synthesis. Representative examples of such promoters are: lac or trp promoter of E.coli; a lambda phage PL promoter; eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, LTRs of retrovirus, and other known promoters capable of controlling gene expression in prokaryotic or eukaryotic cells or viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

Furthermore, the expression vector preferably comprises one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance and Green Fluorescent Protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for E.coli.

Vectors comprising the appropriate DNA sequences described above, together with appropriate promoter or control sequences, may be used to transform appropriate host cells to enable expression of the protein.

The host cell of the present invention is preferably a host cell which has an ability to utilize sugars and is capable of further producing a bio-based fermentation product such as ethanol. Preferably including Saccharomyces cerevisiae (Saccharomyces cerevisiae), Saccharomyces cerevisiae (Saccharomyces monangis), Saccharomyces bayanus (Saccharomyces bayanus), Saccharomyces pastorianus (Saccharomyces pastorianus), Saccharomyces carlsbergensis (Saccharomyces carlsbergensis), Saccharomyces cerevisiae (Saccharomyces pombe), Kluyveromyces marxianus (Kluyveromyces marxianus), Kluyveromyces lactis (Kluyveromyces lactis), Kluyveromyces fragilis (Kluyveromyces fragilis), Pichia stipitis (Pichia stipitis), Thermus thermophilus (Sporomyces thermophilus), Candida sheilans (Candida sheila), Candida rugosa (Clostridium trichothecoides), Clostridium trichothecoides (Clostridium trichoderma), Clostridium trichoderma (Clostridium trichoderma), Clostridium (Clostridium strain (Clostridium trichoderma), Clostridium (Clostridium strain (Clostridium trichoderma), Clostridium trichoderma strain (Clostridium strain (Clostridium thermoanaerobacteosporium), Bacillus strain (Clostridium thermoanaerobium), Bacillus strain (Clostridium thermoanaerobium), Bacillus strain (Clostridium strain, Bacillus, Klebsiella oxytoca (Klebsiella oxytoca), anaerobic Bacillus (Thermoanaerobacterium saccharolyticum) or Bacillus subtilis (Bacillus subtilis)

It will be clear to one of ordinary skill in the art how to select appropriate vectors, promoters, enhancers and host cells.

Transformation of a host cell with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. The following DNA transfection methods may be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, etc.

The obtained transformant can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culturing is performed under conditions suitable for growth of the host cell. After the host cells have been grown to an appropriate cell density, the selected promoter is induced by suitable means (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period of time.

The recombinant polypeptide obtained by the method can be expressed on a cell membrane in a transmembrane mode.

Applications of

A novel recombinant strain can be prepared by using the polynucleotide encoding the transporter of the present invention, and the original strain which does or does not have the pentose or hexose transport ability can be used for the strain, so that the new or stronger pentose or hexose transport ability is endowed to the original strain by introducing the vector of the present invention, and the utilization rate of a carbon source is improved.

The methods used in the following examples are conventional unless otherwise specified, and specific procedures can be found in: molecular Cloning: A Laboratory Manual (Sambrook, J., Russell, David W., Molecular Cloning: A Laboratory Manual, 3rd edition, 2001, NY, Cold Spring Harbor).

The percentage concentrations are expressed as mass percentages unless otherwise specified.

The various biological materials described in the examples are obtained by way of experimental acquisition for the purposes of this disclosure and should not be construed as limiting the source of the biological material of the invention. In fact, the sources of the biological materials used are wide and any biological material that can be obtained without violating the law and ethics can be used instead as suggested in the examples.

The primers used were all synthesized by Kingzhi Biotech, Inc.

In the present invention, glucose, xylose and arabinose were purchased from sigma reagent;

metaphenylhydrazone chlorocarbonyl cyanide (CCCP) was purchased from sigma;

isotopically labeled glucose, xylose and arabinose were purchased from Radiolabeled chemical co.

Example 1 GLT-1 is a glucose transporter protein enabling microorganisms to acquire the ability to transport and utilize glucose

Construction of GLT-1 gene expression vector

The encoding reading frame of the GLT-1 gene was PCR amplified from Neurospora crassa cDNA using primers GLT-F (SEQ ID NO. 5'-CGCGGATCCATGGGTCTCTTCTCGAAAAAGTC-3') (SEQ ID NO. 11) and GLT-R (SEQ ID NO. 5'-CCGGAATTCCTAAACCTCTCCATGGCTTGAGG-3') (SEQ ID NO. 12) in the following PCR reaction system: 5 XPhusion HF buffer 10 u L, 10MMdNTPs 1u L, GLT-F2.5 u L, GLT-R2.5 u L, cDNA 1u L, Phusion DNA polymerase0.5 u L, water 32.5 u L. The PCR reaction conditions are as follows: firstly, the temperature is 98 ℃ for 30 s; then the temperature is 98 ℃ for 10s, the temperature is 65 ℃ for 30s, the temperature is 72 ℃ for 1.5min, and 35 cycles are carried out; finally, the temperature is 72 ℃ for 10min, and the temperature is 4 ℃ for 10 min. After the PCR reaction was completed, the PCR product and the plasmid pRS426-PGK1[ which was constructed according to the reference (Galazka, j.m., et al, 2010.Cellodextrin transport in yeast for improved bioorganic production. science.330,84-86 ]) were double-digested using restriction enzymes BamHI and EcoRI, and the two double-digested products were ligated, the ligated product was identified by digestion with restriction enzymes, and then sequenced, and the sequencing result showed that the nucleotide sequence of the GLT-1 gene was represented by sequence 1 in the sequence listing, indicating that a recombinant expression plasmid carrying the GLT-1 gene with the correct sequence insertion position was obtained, which was designated as pRS426-GLT, and its physical map was represented by fig. 2. Plasmids pRS426-GLT and pRS426-PGK1 were transformed into Saccharomyces cerevisiae EBY.VW4000(Wieczorke, R., et al.,1999. Current knock-out of at least 20transporter genes is required to block up take of hexoses in Saccharomyces cerevisiae FEBS Lett.464,123-128.) and named EGLT and E426, respectively.

Measurement of glucose transport by GLT-1

1. Separately picking single clones of EGLT and a control strain E426, inoculating the single clones in 10mL SC-URA medium (formulation: 6.7g/L of amino-free yeast nitrogen source, 1.4g/L of yeast synthetic deletion medium supplement, 20g/L of maltose, and 20mg/L of each of leucine, histidine and tryptophan) with 2% maltose as a carbon source, and culturing at 30 ℃ overnight (10-12 hours) until the cell concentration is 1.5-2.0(OD 600);

2. after centrifugation to collect the cells (4000rpm,5min), the cells were washed three times with ice-cold assay buffer (100mM Tris-Citrate buffer pH 5.0) and resuspended to an OD600 of 20;

3. the cells were aliquoted into 1.5mL centrifuge tubes (100 ul/tube), three of which were dried to weigh their dry weight, and the remainder were placed on ice for use.

4. Before the reaction, the thalli is placed for 5min at 30 ℃.

5. After reaction for 120 seconds, 50ul of isotope-labeled glucose solutions (sugar concentration: 400mM, 250mM, 100mM, 50mM, 10mM, 5mM) were added to 100ul of the cell suspension, and 1mL of ice water was added to terminate the reaction,

6. cells were collected by centrifugation at 10000rpm for 1min, washed twice with ice water, centrifuged and the supernatant removed.

7. The cells were reselected with 500mL of 0.1mM NaOH, transferred to a vial containing 3mL of Ultima Gold scanning fluid, the amount of radioactivity was measured, and the amount of transport per unit cell dry weight per unit time was calculated.

The final result is shown in fig. 3. Vw4000 lacks 17 hexose transporters and 3 maltose/glucose transporters, and loses the ability to transport glucose. Recombinant yeast EGLT restored the transport capacity for glucose due to the introduction of GLT-1, where the affinity Km for glucose of GLT was 18.42. + -. 3.38mM and the maximum transport rate was 30.75. + -. 1.34mmol/h/gram DCW as shown in FIG. 3.

Third, GLT-1 makes the microorganism have the ability to utilize glucose

1. Separately picking monoclonal antibodies of EGLT and a control strain E426, inoculating the monoclonal antibodies into 10mL SC-URA culture medium (formula: 6.7g/L of amino-free yeast nitrogen source, 1.4g/L of yeast synthetic deletion medium supplement, 20g/L of maltose, and 20mg/L of leucine, histidine and tryptophan respectively) with 2% maltose as a carbon source, and culturing at 30 ℃ overnight (10-12 hours) until the thallus concentration is 1.5-2.0;

2. after the thalli is collected by centrifugation (4000rpm,5min), the thalli is washed three times by ice-precooled double-distilled water and is resuspended until the OD600 is 2;

3. isocratic dilution of bacterial suspension (10)⁰,10^-1,10^-2,10^-3,10^-4,10^-5)；

4. The yeast diluted in an equal gradient was spotted on SC-URA plates (formulation: 6.7g/L of amino-free yeast nitrogen source, 1.4g/L of yeast synthesis deletion medium supplement, 20g/L of maltose, 20mg/L of each of leucine, histidine and tryptophan, 2% agar) using 2% maltose as a carbon source, and cultured in an incubator at 30 ℃.

The results are shown in FIG. 4, the ability of recombinant yeast (EGLT) to utilize glucose rapidly compared to the control strain (E426). The introduction of GLT-1 was demonstrated that Saccharomyces cerevisiae restored the ability to grow on glucose as a carbon source.

In summary, GLT-1 has high transport capacity (30.75. + -. 1.34mmol/h/gram DCW) and affinity (Km 18.42. + -. 3.38mM) for glucose, and GLT-1 can recover the ability to utilize glucose as a carbon source after being introduced into Saccharomyces cerevisiae.

Example 2 XYT-1 is a xylose transporter, enabling microorganisms to acquire the ability to transport xylose

Construction of XYT-1 gene expression vector

The coding reading frame of XYT-1 gene was PCR amplified from Neurospora crassa cDNA using primers XYT-F (SEQ ID NO: 5'-GGACTAGTATGGTTCTGGGGAAAAAGTCAATC-3') and XYT-R (SEQ ID NO: 5'-CCCAAGCTTCTAAACCCTATGGTTAATAACCTT-3') (SEQ ID NO: 14) in the PCR reaction system: 5 XPisuion HF buffer 10 u L, 10mM dNTPs 1u L, XYT-F2.5 u L, XYT-R2.5 u L, cDNA 1u L, Phusion DNA polymerase0.5 u L, water 32.5 u L. The PCR reaction conditions are as follows: firstly, the temperature is 98 ℃ for 30 s; then the temperature is 98 ℃ for 10s, 60 ℃ for 30s and 72 ℃ for 1.5min, and 35 cycles are carried out; finally, the temperature is 72 ℃ for 10min, and the temperature is 4 ℃ for 10 min. After the PCR reaction is finished, the PCR product and a plasmid pRS426-PGK1[ the plasmid construction is according to the reference (Galazka, J.M., et al, 2010. Cellodextrina transport in yeast for improved bio-production. science.330,84-86 ]) are subjected to double digestion by using restriction enzymes SpeI and HindIII, the two double digestion products are connected, the connection products are subjected to enzyme digestion identification by using the restriction enzymes, and then sequencing is carried out, and the sequencing result shows that the nucleotide sequence of the XYT-1 gene is shown as a sequence 3 in a sequence table, and the obtained sequence and the inserted position of the recombinant expression plasmid carrying the XYT-1 gene are correct and are named as pRS 426-XYT. Plasmids pRS426-XYT and pRS426-PGK1 were transformed into Saccharomyces cerevisiae EBY.VW4000(Wieczorke, R., et al.,1999. Current knock-out of at least 20transporter genes is required to be up to block up of hexoses in Saccharomyces cerevisiae FEBS Lett.464,123-128.) and designated as EXYT and E426, respectively.

Second, measurement of xylose transport by XYT-1

The transport capacity of XYT-1 for xylose was determined in example 1, and the results are shown in FIG. 5. Due to the introduction of XYT-1, the recombinant yeast EXYT has xylose transport capacity, the affinity Km of XYT-1 for xylose is 7.58 + -0.60 mM, and the maximum transport rate is 49.61 + -1.20 μmol/h/gram DCW.

Example 3, XAT-1 is a xylose and arabinose transporter enabling microorganisms to acquire the ability to transport xylose and arabinose

Firstly, construction of XAT-1 Gene expression vector

The coding reading frame of XAT-1 gene was PCR amplified from Neurospora crassa cDNA using primers XAT-F (SEQ ID NO. 5'-CGCGGATCCATGAAGCCATTTCTGGGGCTC-3') (SEQ ID NO. 15) and XAT-R (SEQ ID NO. 5'-CCCAAGCTTCTACGACTCCCGATTACCTCCAT-3') (SEQ ID NO. 16) in the following PCR reaction system: 5 XPhusion HF buffer 10 u L, 10mM dNTPs 1u L, XAT-F2.5 u L, XAT-R2.5 u L, cDNA 1u L, Phusion DNA polymerase0.5 u L, water 32.5 u L. The PCR reaction conditions are as follows: firstly, the temperature is 98 ℃ for 30 s; then the temperature is 98 ℃ for 10s, the temperature is 65 ℃ for 30s, the temperature is 72 ℃ for 1.5min, and 35 cycles are carried out; finally, the temperature is 72 ℃ for 10min, and the temperature is 4 ℃ for 10 min. After the PCR reaction, the PCR product and the plasmid pRS426-PGK1[ which was constructed according to the reference (Galazka, J.M., et al.,2010. Cellodextrins transport in yeast for improved bioorganic production. science.330,84-86 ]) were double-digested using restriction enzymes BamHI and HindIII, the two double-digested products were ligated, the ligated product was identified by restriction enzyme digestion and then sequenced, and the result of the sequencing showed that the nucleotide sequence of XAT-1 gene was shown as sequence 5 in the sequence listing, indicating that a recombinant expression plasmid carrying XAT-1 gene with the sequence and the correct insertion position was obtained and named pRS 426-XAT. Plasmids pRS426-XAT and pRS426-PGK1 were transformed into Saccharomyces cerevisiae EBY.VW4000(Wieczorke, R., et al.,1999. Current knock-out of at least 20transporter genes is required to block up take of hexoses in Saccharomyces cerevisiae FEBS Lett.464,123-128.) and named EXAT and E426, respectively.

Di, XAT-1 determination of xylose and arabinose transport

XAT-1 see example 1 for the determination of xylose and arabinose transport capacity, the results are shown in FIG. 6. Due to the introduction of XAT-1, the recombinant yeast EXAT has the transport capacity of xylose and arabinose, wherein the affinity Km of XAT-1 to xylose is 18.17 +/-3.23 mM, and the maximum transport rate is 54.11 +/-3.83 mu mol/h/gram DCW (FIG. 6A); XAT-1 had an affinity for arabinose, Km, of 61.93. + -. 17.68mM and a large transport rate of 65.84. + -. 11.76. mu. mol/h/gram DCW (FIG. 6B).

Example 4 LAT-1 is an arabinose transporter enabling microorganisms to acquire the ability to transport arabinose

Firstly, construction of LAT-1 gene expression vector

The coding reading frame of the LAT-1 gene was PCR amplified from Neurospora crassa cDNA using primers ELAT-F (SEQ ID NO. 5'-CGCGGATCCATGGGGCTCGGGCTTAAGCTAC-3') (SEQ ID NO. 17) and ELAT-R (SEQ ID NO. 5'-CGGAATTCCTAAACCTTCTCATGCTCATGCAC-3') (SEQ ID NO. 18) in the following PCR reaction system: 5 XPhusion HF buffer 10 u L, 10mM dNTPs 1u L, ELAT-F2.5 u L, ELAT-R2.5 u L, cDNA 1u L, Phusion DNA polymerase0.5 u L, water 32.5 u L. The PCR reaction conditions are as follows: firstly, the temperature is 98 ℃ for 30 s; then the temperature is 98 ℃ for 10s, the temperature is 65 ℃ for 30s, the temperature is 72 ℃ for 1.5min, and 35 cycles are carried out; finally, the temperature is 72 ℃ for 10min, and the temperature is 4 ℃ for 10 min. After the PCR reaction, the PCR product and the plasmid pRS426-PGK1[ which was constructed according to the reference (Galazka, J.M., et al.,2010. Cellodextrins transport in yeast for improved bioorganic production. science.330,84-86 ]) were double-digested using restriction enzymes BamHI and HindIII, the two double-digested products were ligated, the ligated product was identified by restriction enzymes, and then sequenced, and the result of the sequencing showed that the nucleotide sequence of LAT-1 gene was shown as sequence 7 in the sequence listing, indicating that a recombinant expression plasmid carrying LAT-1 gene with the sequence and the correct insertion position was obtained, which was designated pRS 426-LAT. Plasmids pRS426-LAT and pRS426-PGK1 were transformed into Saccharomyces cerevisiae EBY.VW4000(Wieczorke, R., et al.,1999. Current knock-out of at least 20transporter genes is required to block up take of hexoses in Saccharomyces cerevisiae FEBS Lett.464,123-128.) and named ELAT and E426, respectively.

Second, LAT-1 determination of arabinose transport

The ability of LAT-1 to transport xylose and arabinose was determined as described in example 1, and the results are shown in FIG. 7. Due to the introduction of LAT-1, the recombinant yeast ELAT has the transport capacity of arabinose, the affinity Km of LAT-1 to arabinose is 25.12 +/-2.98 mM, and the maximum transport rate is 116.7 +/-4.06 mmol/h/gram DCW.

Determination of the type of LAT-1 transport

The sugar transporters in the fungi are mainly of the Uniporter and Symporter/H + type, the Uniporter drives the transport by depending on the concentration gradient of a substrate, and the transporters mainly play a role in assisting the transportation. Symporter/H +, transports saccharide and H + simultaneously in the same direction, and takes the electrochemical gradient of H + as driving force. The following experiments were designed to identify the type of LAT-1 transport:

wherein CCCP (metaphenylhydrazone chlorocarbonyl cyanide) has the function of destroying the proton gradient of cells, so that the transport capacity of LAT-1 on arabinose is measured under different CCCP concentrations, and the transport type of LAT-1 is identified.

1. Selecting ELAT monoclonal, inoculating into 10mL SC-URA medium (formulation: 6.7g/L nitrogen source without amino yeast, 1.4g/L yeast synthetic deletion medium supplement, 20g/L maltose, leucine, histidine and tryptophan each 20mg/L) with 2% maltose as carbon source, culturing at 30 deg.C overnight (10-12 hr) to thallus concentration of 1.5-2.0(OD 600);

2. after centrifugation to collect the cells (4000rpm,5min), the cells were washed three times with ice-cold assay buffer (100mM Tris-Citrate buffer pH 5.0) and resuspended to an OD600 of 20;

3. the cells were dispensed into 1.5mL centrifuge tubes (100 ul/tube), 50. mu.L each, three of which were dried to dry weight, and the remainder were placed on ice for use.

4. mu.L of CCCP (final concentrations: 0. mu.M, 0.5. mu.M, 1.0. mu.M, 25. mu.M, 50. mu.M, respectively) was added thereto at different concentrations before the reaction, and the mixture was left in an incubator at 30 ℃ for 10 min.

5. 50ul of the cell suspension was added to 50ul of a 50mM isotope-labeled arabinose solution, and after reacting at 30 ℃ for 120 seconds, 1mL of ice water was added to terminate the reaction,

6. cells were collected by centrifugation at 10000rpm for 1min, washed twice with ice water, centrifuged and the supernatant removed.

7. The cells were reselected with 500mL of 0.1mM NaOH, transferred to a vial containing 3mL of Ultima Gold scanning fluid, and the amount of radioactivity was measured, and the amount of transport per unit dry weight per unit time was calculated.

The experimental result is shown in FIG. 8, the transport capacity of ELAT to arabinose gradually decreases with the increase of CCCP concentration, which indicates that the transport type of LAT-1 is Symporter/H +, when the concentration of CCCP is 25 μ M respectively, the transport rate of LAT-1 to arabinose decreases to the lowest, and at this time, LAT-1 transports arabinose by relying on the arabinose concentration gradient inside and outside the cell as the power. Experiments prove that when the transport type of the transport protein is Symporter/H +, the transport protein has a larger transport rate and better application prospect.

Example 5 MtLAT-1 is an arabinose transporter enabling microorganisms to acquire the ability to transport arabinose

First, construction of MtLAT-1 gene expression vector

The coding reading frame of the MtLAT-1 gene was PCR amplified from the myceliophthora thermophila cDNA using primers MtLAT-F (SEQ ID NO. 5'-CGCGGATCCATGAAGCTGCCCACGATTTAC-3') (SEQ ID NO. 19) and MtLAT-R (SEQ ID NO. 5'-CCGGAATTCTTAAACCTTCTCCTGCTCGCC-3') (SEQ ID NO. 20) in the following PCR reaction scheme: 5 XPisuion HF buffer 10 u L, 10mM dNTPs 1u L, MtLAT-F2.5 u L, MtLAT-R2.5 u L, cDNA 1u L, Phusion DNA polymerase0.5 u L, water 32.5 u L. The PCR reaction conditions are as follows: firstly, the temperature is 98 ℃ for 30 s; then, the temperature is 10s at 98 ℃, 30s at 67 ℃, 1.5min at 72 ℃ and 35 cycles; finally, the temperature is 72 ℃ for 10min, and the temperature is 4 ℃ for 10 min. After the PCR reaction was completed, the PCR product and the plasmid pRS426-PGK1[ which was constructed according to the reference (Galazka, J.M., et al.,2010.Cellodextrin transport in yeast for improved bioorganic production. science.330,84-86 ]) were double-digested and the two double-digested products were ligated, the ligated product was identified by digestion with restriction enzymes and then sequenced, and the result of the sequencing showed that the nucleotide sequence of MtLAT-1 gene was represented by SEQ ID NO.9 in the sequence listing, indicating that a recombinant expression plasmid carrying the MtLAT-1 gene having the correct sequence and insertion position was obtained and named pRS 426-MtLAT. Plasmids pRS426-MtLAT and pRS426-PGK1 were transformed into Saccharomyces cerevisiae EBY.VW4000(Wieczorke, R., et al.,1999. Current knock-out of at least 20transporter genes is required to be up to block up of hexoses in Saccharomyces cerevisiae FEBS Lett.464,123-128.) and named EMtLAT and E426, respectively.

Determination of Di, MtLAT-1 arabinose transport

The ability of MtLAT-1 to transport arabinose was determined as described in example 1, and the results are shown in FIG. 9. Due to the introduction of MtLAT-1, the recombinant yeast EMtLAT has the capability of transporting xylose and arabinose, wherein the affinity Km of the MtLAT-1 to the arabinose is 10.29 +/-0.35 mM, and the large transport rate is 10.29 +/-3.6 mmol/h/gram DCW.

Determination of type of Tri, MtLAT-1 transport

The experimental method is described in example 4, the experimental result is shown in FIG. 10, and the experiment shows that the transport type of MtLAT-1 to arabinose is Symporter/H +; the transport rate of MtLAT-1 decreased with increasing CCCP concentration, and decreased to a minimum when CCCP was 25. mu.M.

Example 6 XYT-1 promotes Saccharomyces cerevisiae xylose growth and ethanol fermentation

Plasmids pRS426-XYT and pRS426-PGK1 described in example 2 were transformed into Saccharomyces cerevisiae EBY.VW4000(Wieczorke, R., et al, 1999. Current knock-out of at least 20transporter genes is required to block up of hexoses in Saccharomyces cerevisiae. FEBS Lett.464, 123-128) containing the Pichia xylose metabolic pathway, which were designated as XXYT and X426, respectively.

XXYT and control strain X426 monoclonals were picked up separately, inoculated into 10mL SC-URA medium (formulation: 6.7g/L without amino yeast nitrogen source, 1.4g/L yeast synthetic deletion medium supplement, 20g/L maltose, leucine, histidine and tryptophan each 20mg/L) with 2% maltose as carbon source, cultured overnight at 30 ℃ (10-12 hours), and then transferred into 40mL SC-URA medium with 2% xylose as carbon source, respectively, cultured in 100mL triangular flasks (250rpm, 30 ℃) at a final OD600 of 1, and the OD600 was measured at each time.

The results are shown in FIG. 11: XXYT has the ability to grow on xylose relative to the control strain X426. The results show that XYT-1 has the capacity of transporting xylose and simultaneously can allow saccharomyces cerevisiae to regain the capacity of utilizing xylose as a carbon source for growth.

Example 7 promotion of Saccharomyces cerevisiae arabinose growth and ethanol fermentation by LAT-1 and MtLAT-1

LAT-1 is an arabinose transporter, and the purpose of this example is to determine the function of LAT-1 and its effect on recombinant Saccharomyces cerevisiae by the expression of LAT-1 gene in Saccharomyces cerevisiae.

Construction of LAT-1 and MtLAT-1 gene expression vector

The coding reading frame of the resistance gene KanMX of geneticin G418 was PCR amplified from plasmid pUG-6 (Guelder, U, et al, 2002.A second set of loxP marker cassettes for Cre-mediated multiple gene cloning in cloning year. nucleic Acids Res.30(6)) using primers KanMX-F (sequence: 5'-GGGAATTCCATATGGATCTGTTTAGCTTGCCTCGTC-3') (SEQ ID No.:21) and KanMX-R (sequence: 5'-ATGGGCCCCGACACTGGATGGCGGCGTTAG-3') (SEQ ID No.:22) in the reaction scheme: 5 XPisuion HF buffer 10 u L, 10mM dNTPs 1u L, KanMX-F2.5 u L, KanMX-R2.5 u L, pUG6 plasmid 1u L, Phusion DNA polymerase0.5 u L, water 32.5 u L. The PCR reaction conditions are as follows: firstly, the temperature is 98 ℃ for 30 s; then, the temperature is 10s at 98 ℃, 30s at 60 ℃ and 1.0min at 72 ℃ for 35 cycles; finally, the temperature is 72 ℃ for 10min, and the temperature is 4 ℃ for 10 min. After the PCR reaction was completed, the PCR product and the plasmid pRS426-PGK1[ the plasmid construction was slightly modified according to the reference (Galazka, j.m., et al, 2010. cellodexrin transport in yeast for improved bio-fuel production.science.330,84-86 ]) were subjected to double digestion using restriction enzymes NdeI and ApaI, and the two double digestion products were ligated, and the ligation products were subjected to restriction enzyme digestion and identification using restriction enzymes, followed by sequencing verification, and the recombinant expression plasmid of KanMX gene was named p426KanMX 4. The coding reading frame of the LAT-1 gene was PCR amplified from Neurospora crassa cDNA using primers ELAT-F (SEQ ID NO: 5'-CGCGGATCCATGGGGCTCGGGCTTAAGCTAC-3') and ELATF-R (SEQ ID NO: 5'-CGGAATTCCTAAACCTTCTCATGCTCATGCAC-3') (SEQ ID NO: 18) in the following PCR reaction system: 5 XPisuion HF buffer 10 u L, 10mM dNTPs 1u L, LAT-F2.5 u L, LAT-R2.5 u L, cDNA 1u L, Phusion DNA polymerase0.5 u L, water 32.5 u L. The PCR reaction conditions are as follows: firstly, the temperature is 98 ℃ for 30 s; then the temperature is 98 ℃ for 10s, 64 ℃ for 30s and 72 ℃ for 1.5min, and 35 cycles are carried out; finally, the temperature is 72 ℃ for 10min, and the temperature is 4 ℃ for 10 min. After the PCR reaction is finished, the PCR product and the plasmid pRS426-PGK1URA are subjected to double enzyme digestion by using restriction enzymes SpeI and EcoRI, KanMX is subjected to double enzyme digestion, the two double enzyme digestion products are connected, the connection product is subjected to enzyme digestion identification by using the restriction enzymes, sequencing is performed, the sequencing result shows that the nucleotide sequence of the LAT-1 gene is shown as a sequence 7 in a sequence table, the obtained sequence and the recombinant expression plasmid carrying the LAT-1 gene with the correct insertion position are named as p426LAT, and the physical map of the plasmid is shown as figure 12.

The reading frame encoding the MtLAT-1 gene was PCR amplified from the myceliophthora thermophila cDNA using primers MtLAT-F (sequence: 5'-CGCGGATCCATGAAGCTGCCCACGATTTAC-3') (SEQ ID NO.:19) and MtLATF-R (sequence: 5'-CCGGAATTCTTAAACCTTCTCCTGCTCGCCGAC-3') (SEQ ID NO.:20) in the PCR reaction regime: 5 XPisuion HF buffer 10 u L, 10mM dNTPs 1u L, LAT-F2.5 u L, LAT-R2.5 u L, cDNA 1u L, Phusion DNA polymerase0.5 u L, water 32.5 u L. The PCR reaction conditions are as follows: firstly, the temperature is 98 ℃ for 30 s; then the temperature is 98 ℃ for 10s, 64 ℃ for 30s and 72 ℃ for 1.5min, and 35 cycles are carried out; finally, the temperature is 72 ℃ for 10min, and the temperature is 4 ℃ for 10 min. After the PCR reaction is finished, the PCR product and the plasmid pRS426-PGK1URA are subjected to double enzyme digestion by using restriction enzymes BamHI and EcoRI, KanMX is subjected to double enzyme digestion, the two double enzyme digestion products are connected, the connection product is subjected to enzyme digestion identification by using the restriction enzymes, then sequencing is carried out, the sequencing result shows that the nucleotide sequence of the MtLAT-1 gene is shown as a sequence 7 in a sequence table, the obtained recombinant expression plasmid which has the correct sequence and insertion position and carries the MtLAT-1 gene is named as p426MtLAT, and the physical map of the plasmid is shown as figure 13.

Plasmids p426LAT, p426MtLAT and p426kanmx were transformed into Saccharomyces cerevisiae BSW2AP (Wang, et al, 2013.Improvement of L-Arabidopsis Fermentation by modification the Metabolic Pathway and Transport in Saccharomyces cerevisiae. biomed Res Int.).

Secondly, LAT-1 and MtLAT-1 promote the growth of the recombinant Saccharomyces cerevisiae in L-arabinosine.

Single colonies of Saccharomyces cerevisiae transformants containing p426LAT, p426MtLAT and p426kanmx, respectively, were picked up, inoculated into 50mL of SC-URA-LEU medium (formulation: 6.7G/L of amino-free yeast nitrogen source, 1.4G/L of yeast synthesis deletion medium supplement, 20G/L of maltose, 20mg/L of each of histidine and tryptophan, with 400. mu.g/mL of G418 added) containing 1.5% L-arabinose and 0.5% D-glucose as carbon sources, cultured overnight at 30 ℃ (10-12 hours), then transferred into 40mL of SC-URA-LEU medium (with 400. mu.g/mL of G418 added) containing 2% L-arabinose as carbon source, and finally OD600 was 1.0, cultured in 250mL of triangular flask (aerobic culture) and 100mL of oxygen flask (anaerobic culture with limited oxygen) (250rpm,25 ℃), samples were taken at each time and their OD600 was measured. The results are shown in FIGS. 14 and 15.

In FIG. 14, the strain BSW2AP over-expressed LAT-1 and MtLAT-1, which had a faster growth rate under aerobic conditions relative to the control strain, also exhibited a significantly faster L-arabinose consumption rate. It is demonstrated that LAT-1 and MtLAT-1 can enhance the utilization of arabinose by microorganisms and promote the growth of microorganisms under aerobic conditions.

In FIG. 15, strain BSW2AP over-expressed LAT-1 and MtLAT-1 and exhibited a faster growth rate under oxygen-limited conditions relative to the control strain, and the L-arabinose consumption rate and ethanol production rate were also significantly increased. The fact that under the condition of oxygen limitation, LAT-1 and MtLAT-1 can enhance the utilization of arabinose by microorganisms, promote the growth of the microorganisms and utilize the production of ethanol is demonstrated.

BSWLAT has a faster growth after 50 hours relative to the control strain BSW426, indicating that under oxygen-limited conditions, expression of LAT-1 promotes the metabolism of the yeast by fermenting Saccharomyces cerevisiae with arabinose.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing

<110> institute of biotechnology for Tianjin industry of Chinese academy of sciences

<120> a method for promoting microbial cells to transport glucose, xylose and arabinose and a bio-based product thereof

Use in fermentation

<130> P2018-0213

<150> 201410051718.3

<151> 2014-02-16

<160> 22

<170> PatentIn version 3.5

<210> 1

<211> 1599

<212> DNA

<213> Neurospora crassa (Neurospora crassa)

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atg ggt ctc ttc tcg aaa aag tcg gct gcg ccg cag acc caa tca caa 48

Met Gly Leu Phe Ser Lys Lys Ser Ala Ala Pro Gln Thr Gln Ser Gln

1 5 10 15

gat gag atc gat ctc gct gct gag cag aag gtc act ttc cgt gcc gtc 96

Asp Glu Ile Asp Leu Ala Ala Glu Gln Lys Val Thr Phe Arg Ala Val

20 25 30

ttc ctc ggt gtt gtc gcc tcc gta ggt ggc ttc atg ttt ggc tac gtc 144

Phe Leu Gly Val Val Ala Ser Val Gly Gly Phe Met Phe Gly Tyr Val

35 40 45

agt ggt caa att tct ggt ttc ttc gac atg gaa gac ttc ggt cgt cgg 192

Ser Gly Gln Ile Ser Gly Phe Phe Asp Met Glu Asp Phe Gly Arg Arg

50 55 60

ttc ggt aac tac caa gac gcg gat ggc tgg gtc ttc tca gca tac cgc 240

Phe Gly Asn Tyr Gln Asp Ala Asp Gly Trp Val Phe Ser Ala Tyr Arg

65 70 75 80

cag ggt gct att gtc gcc cta ctc cct gct ggt gcc ctt ctt ggt tcg 288

Gln Gly Ala Ile Val Ala Leu Leu Pro Ala Gly Ala Leu Leu Gly Ser

85 90 95

ctc gtt gcc ggt aga att gcg gat acc ctt ggt cgc cgt atc gcc atc 336

Leu Val Ala Gly Arg Ile Ala Asp Thr Leu Gly Arg Arg Ile Ala Ile

100 105 110

tct gcg tcc gcc ctt ttc tcc tgc atc gga aca att atc gag atc gcc 384

Ser Ala Ser Ala Leu Phe Ser Cys Ile Gly Thr Ile Ile Glu Ile Ala

115 120 125

tcc acc acg cac tgg gcc cag ttt gcg gtc ggt cgt ctt atc acc ggt 432

Ser Thr Thr His Trp Ala Gln Phe Ala Val Gly Arg Leu Ile Thr Gly

130 135 140

att ggt atc ggt gct ctc tcc gtc gtc gtc ccg atg tac cag tct gag 480

Ile Gly Ile Gly Ala Leu Ser Val Val Val Pro Met Tyr Gln Ser Glu

145 150 155 160

tcc gcg ccc gcc atc ctc cgt ggt atc ctc gtc tcg tgc tac cag ctc 528

Ser Ala Pro Ala Ile Leu Arg Gly Ile Leu Val Ser Cys Tyr Gln Leu

165 170 175

ttc atc act ctt ggt atc tgg acc gct gag atg atc aac tac ggt act 576

Phe Ile Thr Leu Gly Ile Trp Thr Ala Glu Met Ile Asn Tyr Gly Thr

180 185 190

cac gac ctc agc aac tcc gcc tct tgg cgt att ccc aac ggt atc tcc 624

His Asp Leu Ser Asn Ser Ala Ser Trp Arg Ile Pro Asn Gly Ile Ser

195 200 205

ttc ctc tgg gct ttg gtt ctc ggt ggc gga ata ttg ttc ctt cct gag 672

Phe Leu Trp Ala Leu Val Leu Gly Gly Gly Ile Leu Phe Leu Pro Glu

210 215 220

tct ccc cgt tat gcc tac cgt gtt ggt cgc gag gac gag gct cgc aac 720

Ser Pro Arg Tyr Ala Tyr Arg Val Gly Arg Glu Asp Glu Ala Arg Asn

225 230 235 240

acc att gcc cgc ctt gcc ggt ctc gag ccc agc gcc cgc tct gtc aac 768

Thr Ile Ala Arg Leu Ala Gly Leu Glu Pro Ser Ala Arg Ser Val Asn

245 250 255

atg caa atc gat gag atc cgt atg aag ctt gag gag gag aag gct ggt 816

Met Gln Ile Asp Glu Ile Arg Met Lys Leu Glu Glu Glu Lys Ala Gly

260 265 270

gcc gac acc aag tgg tac gag atc ttc gga cct gct ctg ttg cgc cgc 864

Ala Asp Thr Lys Trp Tyr Glu Ile Phe Gly Pro Ala Leu Leu Arg Arg

275 280 285

acc ctt atc ggt atc att ctt cag tct ggc cag cag ctt act ggt gcc 912

Thr Leu Ile Gly Ile Ile Leu Gln Ser Gly Gln Gln Leu Thr Gly Ala

290 295 300

aac ttc ttc ttc tac tac gga acc acg att ttc aag gct act ggt ctt 960

Asn Phe Phe Phe Tyr Tyr Gly Thr Thr Ile Phe Lys Ala Thr Gly Leu

305 310 315 320

agc gac tct tac gtt acc cag atc att ctt ggt tcc gtc aac gct gga 1008

Ser Asp Ser Tyr Val Thr Gln Ile Ile Leu Gly Ser Val Asn Ala Gly

325 330 335

tgc act gtt gct ggt ctc tgg gtt gtc aag aat gtt ggc cgc cgt aag 1056

Cys Thr Val Ala Gly Leu Trp Val Val Lys Asn Val Gly Arg Arg Lys

340 345 350

gcc ctc atc ggt ggt gcc ctc tgg atg acc atg tgc ttc ttg gtc tac 1104

Ala Leu Ile Gly Gly Ala Leu Trp Met Thr Met Cys Phe Leu Val Tyr

355 360 365

tct ttc gtc gga aga ttt gtg ctc gac ccc gtc aac ccg gct agc act 1152

Ser Phe Val Gly Arg Phe Val Leu Asp Pro Val Asn Pro Ala Ser Thr

370 375 380

cct cag gcc ggc aac gtc ctc att gtc ttc tcc tgc ttc ttc atc gtc 1200

Pro Gln Ala Gly Asn Val Leu Ile Val Phe Ser Cys Phe Phe Ile Val

385 390 395 400

gcc ttt gcc acc act tgg ggt cct ctc gtc tgg gcc gtc gtt gct gag 1248

Ala Phe Ala Thr Thr Trp Gly Pro Leu Val Trp Ala Val Val Ala Glu

405 410 415

ctc tac cct gct cgc tac cgt gct cct gcc atg gcc ttg gcc acc gct 1296

Leu Tyr Pro Ala Arg Tyr Arg Ala Pro Ala Met Ala Leu Ala Thr Ala

420 425 430

tcc aac tgg ctg tgg aac ttc ctc atg tcc ctc ttc acg cgc ccc atc 1344

Ser Asn Trp Leu Trp Asn Phe Leu Met Ser Leu Phe Thr Arg Pro Ile

435 440 445

acc gac tcc att ggc tac ttc tat ggc ttg gtg ttc gcc gga tgc tgc 1392

Thr Asp Ser Ile Gly Tyr Phe Tyr Gly Leu Val Phe Ala Gly Cys Cys

450 455 460

ctt gcc ctc gcc gct ttc gtt tgg ctc ttt gtg atc gag tcc aag gac 1440

Leu Ala Leu Ala Ala Phe Val Trp Leu Phe Val Ile Glu Ser Lys Asp

465 470 475 480

cgc acc ctt gag gag atc gag acc atg tac aac cag aag gtc agc cct 1488

Arg Thr Leu Glu Glu Ile Glu Thr Met Tyr Asn Gln Lys Val Ser Pro

485 490 495

agg cac tcc acc cac tgg cac gct gag gtc cct tcg gga ccg cgg gat 1536

Arg His Ser Thr His Trp His Ala Glu Val Pro Ser Gly Pro Arg Asp

500 505 510

gcg gag gag aag ccc gag gtt cac agt ggt tct gcg aca acc tca agc 1584

Ala Glu Glu Lys Pro Glu Val His Ser Gly Ser Ala Thr Thr Ser Ser

515 520 525

cat gga gag gtt tag 1599

His Gly Glu Val

530

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<213> Neurospora crassa (Neurospora crassa)

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Met Gly Leu Phe Ser Lys Lys Ser Ala Ala Pro Gln Thr Gln Ser Gln

1 5 10 15

Asp Glu Ile Asp Leu Ala Ala Glu Gln Lys Val Thr Phe Arg Ala Val

20 25 30

Phe Leu Gly Val Val Ala Ser Val Gly Gly Phe Met Phe Gly Tyr Val

35 40 45

Ser Gly Gln Ile Ser Gly Phe Phe Asp Met Glu Asp Phe Gly Arg Arg

50 55 60

Phe Gly Asn Tyr Gln Asp Ala Asp Gly Trp Val Phe Ser Ala Tyr Arg

65 70 75 80

Gln Gly Ala Ile Val Ala Leu Leu Pro Ala Gly Ala Leu Leu Gly Ser

85 90 95

Leu Val Ala Gly Arg Ile Ala Asp Thr Leu Gly Arg Arg Ile Ala Ile

100 105 110

Ser Ala Ser Ala Leu Phe Ser Cys Ile Gly Thr Ile Ile Glu Ile Ala

115 120 125

Ser Thr Thr His Trp Ala Gln Phe Ala Val Gly Arg Leu Ile Thr Gly

130 135 140

Ile Gly Ile Gly Ala Leu Ser Val Val Val Pro Met Tyr Gln Ser Glu

145 150 155 160

Ser Ala Pro Ala Ile Leu Arg Gly Ile Leu Val Ser Cys Tyr Gln Leu

165 170 175

Phe Ile Thr Leu Gly Ile Trp Thr Ala Glu Met Ile Asn Tyr Gly Thr

180 185 190

His Asp Leu Ser Asn Ser Ala Ser Trp Arg Ile Pro Asn Gly Ile Ser

195 200 205

Phe Leu Trp Ala Leu Val Leu Gly Gly Gly Ile Leu Phe Leu Pro Glu

210 215 220

Ser Pro Arg Tyr Ala Tyr Arg Val Gly Arg Glu Asp Glu Ala Arg Asn

225 230 235 240

Thr Ile Ala Arg Leu Ala Gly Leu Glu Pro Ser Ala Arg Ser Val Asn

245 250 255

Met Gln Ile Asp Glu Ile Arg Met Lys Leu Glu Glu Glu Lys Ala Gly

260 265 270

Ala Asp Thr Lys Trp Tyr Glu Ile Phe Gly Pro Ala Leu Leu Arg Arg

275 280 285

Thr Leu Ile Gly Ile Ile Leu Gln Ser Gly Gln Gln Leu Thr Gly Ala

290 295 300

Asn Phe Phe Phe Tyr Tyr Gly Thr Thr Ile Phe Lys Ala Thr Gly Leu

305 310 315 320

Ser Asp Ser Tyr Val Thr Gln Ile Ile Leu Gly Ser Val Asn Ala Gly

325 330 335

Cys Thr Val Ala Gly Leu Trp Val Val Lys Asn Val Gly Arg Arg Lys

340 345 350

Ala Leu Ile Gly Gly Ala Leu Trp Met Thr Met Cys Phe Leu Val Tyr

355 360 365

Ser Phe Val Gly Arg Phe Val Leu Asp Pro Val Asn Pro Ala Ser Thr

370 375 380

Pro Gln Ala Gly Asn Val Leu Ile Val Phe Ser Cys Phe Phe Ile Val

385 390 395 400

Ala Phe Ala Thr Thr Trp Gly Pro Leu Val Trp Ala Val Val Ala Glu

405 410 415

Leu Tyr Pro Ala Arg Tyr Arg Ala Pro Ala Met Ala Leu Ala Thr Ala

420 425 430

Ser Asn Trp Leu Trp Asn Phe Leu Met Ser Leu Phe Thr Arg Pro Ile

435 440 445

Thr Asp Ser Ile Gly Tyr Phe Tyr Gly Leu Val Phe Ala Gly Cys Cys

450 455 460

Leu Ala Leu Ala Ala Phe Val Trp Leu Phe Val Ile Glu Ser Lys Asp

465 470 475 480

Arg Thr Leu Glu Glu Ile Glu Thr Met Tyr Asn Gln Lys Val Ser Pro

485 490 495

Arg His Ser Thr His Trp His Ala Glu Val Pro Ser Gly Pro Arg Asp

500 505 510

Ala Glu Glu Lys Pro Glu Val His Ser Gly Ser Ala Thr Thr Ser Ser

515 520 525

His Gly Glu Val

530

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<213> Neurospora crassa (Neurospora crassa)

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atg gtt ctg ggg aaa aag tca atc aaa atc aat ggc gcc gac att ggc 48

Met Val Leu Gly Lys Lys Ser Ile Lys Ile Asn Gly Ala Asp Ile Gly

1 5 10 15

gtc gaa gcc atc ctt ctc ggt gcc gtc acc gcg ata gga ggc ttc ctt 96

Val Glu Ala Ile Leu Leu Gly Ala Val Thr Ala Ile Gly Gly Phe Leu

20 25 30

ttc ggc tat gac acc ggt cag atc tcg ggc atg ctt cta ttc agc gac 144

Phe Gly Tyr Asp Thr Gly Gln Ile Ser Gly Met Leu Leu Phe Ser Asp

35 40 45

ttc aag aac aga ttt ggc cag atc acc caa cca gat gga tca aag gaa 192

Phe Lys Asn Arg Phe Gly Gln Ile Thr Gln Pro Asp Gly Ser Lys Glu

50 55 60

ttc gag tcc atc atc cag tca ctg cta gta tct ctt atg agt atc ggt 240

Phe Glu Ser Ile Ile Gln Ser Leu Leu Val Ser Leu Met Ser Ile Gly

65 70 75 80

aca ctc ctc ggt tct ttg act tca tca tac acc gcc acc tgg tgg ggc 288

Thr Leu Leu Gly Ser Leu Thr Ser Ser Tyr Thr Ala Thr Trp Trp Gly

85 90 95

cgc cgc aag tcc ctt aca ttc ggc gtc ggt ctc ttc atc atc ggc aac 336

Arg Arg Lys Ser Leu Thr Phe Gly Val Gly Leu Phe Ile Ile Gly Asn

100 105 110

atc att cag atc acc gca atg cac tcc tgg gta cac atg atg atg ggc 384

Ile Ile Gln Ile Thr Ala Met His Ser Trp Val His Met Met Met Gly

115 120 125

cgc ttc gtc gcc ggt cta ggc gtc ggt acc ctc tcc gtc ggt gtg ccc 432

Arg Phe Val Ala Gly Leu Gly Val Gly Thr Leu Ser Val Gly Val Pro

130 135 140

atg ttc caa tcc gag tgc tcc cct aga gaa atc cgt ggt gcc gtc gtc 480

Met Phe Gln Ser Glu Cys Ser Pro Arg Glu Ile Arg Gly Ala Val Val

145 150 155 160

gcc tct tac caa ctg ctc atc acc ttt ggt atc ctc atc gcc aac att 528

Ala Ser Tyr Gln Leu Leu Ile Thr Phe Gly Ile Leu Ile Ala Asn Ile

165 170 175

gtc aac tac ggc gtg cgc gag atc cag gag cag gac gct tcc tgg cgt 576

Val Asn Tyr Gly Val Arg Glu Ile Gln Glu Gln Asp Ala Ser Trp Arg

180 185 190

atc gtc att ggt ttg ggt atc ttt ttc tcg ctt ccg ctt ggc gtg gga 624

Ile Val Ile Gly Leu Gly Ile Phe Phe Ser Leu Pro Leu Gly Val Gly

195 200 205

gtg ctg ttg gtc ccc gaa agc cca agg tgg ctg gcg tcc aag gag gat 672

Val Leu Leu Val Pro Glu Ser Pro Arg Trp Leu Ala Ser Lys Glu Asp

210 215 220

tgg gag gga gcc cgc atg tcg atg gcg aga ctg aga ggc ttg aag cat 720

Trp Glu Gly Ala Arg Met Ser Met Ala Arg Leu Arg Gly Leu Lys His

225 230 235 240

gac ccg cat aac gag ctg gtg gag gat gat atg aag gag atg cgg gag 768

Asp Pro His Asn Glu Leu Val Glu Asp Asp Met Lys Glu Met Arg Glu

245 250 255

gtt ttg gag aaa gag cgg act gcg gcc gtg gga act tgg aag gag tgt 816

Val Leu Glu Lys Glu Arg Thr Ala Ala Val Gly Thr Trp Lys Glu Cys

260 265 270

ttc atc ccc aac aag aac ggt gtt ccc aag cag gtg tac cgc aca ttc 864

Phe Ile Pro Asn Lys Asn Gly Val Pro Lys Gln Val Tyr Arg Thr Phe

275 280 285

ctg ggt atc ggc atc cac ttc ctg caa cag tgg act ggc gtc aac tac 912

Leu Gly Ile Gly Ile His Phe Leu Gln Gln Trp Thr Gly Val Asn Tyr

290 295 300

ttc ttc tac tat ggc gcc acc atc ttc cag tcg gcg ggt atc aag gac 960

Phe Phe Tyr Tyr Gly Ala Thr Ile Phe Gln Ser Ala Gly Ile Lys Asp

305 310 315 320

ccc att caa aca cag ctc atc ctc ggc gct gtc aac gtc ttc tca acc 1008

Pro Ile Gln Thr Gln Leu Ile Leu Gly Ala Val Asn Val Phe Ser Thr

325 330 335

ctg ttc ggt ctc tgg gtc gtc gag cgc ttc gga cgc aga tgg ccg ctg 1056

Leu Phe Gly Leu Trp Val Val Glu Arg Phe Gly Arg Arg Trp Pro Leu

340 345 350

ttc atc ggt gct atc tgg cag gcg tct tgg ctg gcc gtg ttc gct tcc 1104

Phe Ile Gly Ala Ile Trp Gln Ala Ser Trp Leu Ala Val Phe Ala Ser

355 360 365

atg ggc acc gct ctc gaa ccc gac caa aac aag gcg tcg ggt att gtc 1152

Met Gly Thr Ala Leu Glu Pro Asp Gln Asn Lys Ala Ser Gly Ile Val

370 375 380

atg atc gtc tcg gcc gca ctc ttc atc gct tcc ttt gct tgc acc tgg 1200

Met Ile Val Ser Ala Ala Leu Phe Ile Ala Ser Phe Ala Cys Thr Trp

385 390 395 400

ggt ccc att gct tgg gtc gtc atc ggc gag agt ttc cct ctg cgc acc 1248

Gly Pro Ile Ala Trp Val Val Ile Gly Glu Ser Phe Pro Leu Arg Thr

405 410 415

cgt gcc aag cag gct tcg ctt gcc acg gct ggt aac tgg ctt gga aac 1296

Arg Ala Lys Gln Ala Ser Leu Ala Thr Ala Gly Asn Trp Leu Gly Asn

420 425 430

ttc atg atc tcc ttc ctc acc ccc ctc gcc acc gat ggc atc ggc tat 1344

Phe Met Ile Ser Phe Leu Thr Pro Leu Ala Thr Asp Gly Ile Gly Tyr

435 440 445

gcc tac ggc tac gtc ttt gta gcc acc aac atc atg ggc gca ctt ttg 1392

Ala Tyr Gly Tyr Val Phe Val Ala Thr Asn Ile Met Gly Ala Leu Leu

450 455 460

gtc tgg ttc ttc ctg tac gag tcc acc agc ttg tcc ctc gag aac gtc 1440

Val Trp Phe Phe Leu Tyr Glu Ser Thr Ser Leu Ser Leu Glu Asn Val

465 470 475 480

gac ctc atg tac agc gag ccg ggc atc aag ccg tgg aac tcg cac aag 1488

Asp Leu Met Tyr Ser Glu Pro Gly Ile Lys Pro Trp Asn Ser His Lys

485 490 495

tgg atg ccg cct ggc tac atc acg cgc atg cag cgc gat gat gag tac 1536

Trp Met Pro Pro Gly Tyr Ile Thr Arg Met Gln Arg Asp Asp Glu Tyr

500 505 510

ttc cac cat cct gag aag ggt ggt tcg gat ggt ctt gaa atc acg aat 1584

Phe His His Pro Glu Lys Gly Gly Ser Asp Gly Leu Glu Ile Thr Asn

515 520 525

gga agc aag gag atg cac ccc cac gag gag agg gaa gag aag gtt att 1632

Gly Ser Lys Glu Met His Pro His Glu Glu Arg Glu Glu Lys Val Ile

530 535 540

aac cat agg gtt tag 1647

Asn His Arg Val

545

<210> 4

<211> 548

<212> PRT

<213> Neurospora crassa (Neurospora crassa)

<400> 4

Met Val Leu Gly Lys Lys Ser Ile Lys Ile Asn Gly Ala Asp Ile Gly

1 5 10 15

Val Glu Ala Ile Leu Leu Gly Ala Val Thr Ala Ile Gly Gly Phe Leu

20 25 30

Phe Gly Tyr Asp Thr Gly Gln Ile Ser Gly Met Leu Leu Phe Ser Asp

35 40 45

Phe Lys Asn Arg Phe Gly Gln Ile Thr Gln Pro Asp Gly Ser Lys Glu

50 55 60

Phe Glu Ser Ile Ile Gln Ser Leu Leu Val Ser Leu Met Ser Ile Gly

65 70 75 80

Thr Leu Leu Gly Ser Leu Thr Ser Ser Tyr Thr Ala Thr Trp Trp Gly

85 90 95

Arg Arg Lys Ser Leu Thr Phe Gly Val Gly Leu Phe Ile Ile Gly Asn

100 105 110

Ile Ile Gln Ile Thr Ala Met His Ser Trp Val His Met Met Met Gly

115 120 125

Arg Phe Val Ala Gly Leu Gly Val Gly Thr Leu Ser Val Gly Val Pro

130 135 140

Met Phe Gln Ser Glu Cys Ser Pro Arg Glu Ile Arg Gly Ala Val Val

145 150 155 160

Ala Ser Tyr Gln Leu Leu Ile Thr Phe Gly Ile Leu Ile Ala Asn Ile

165 170 175

Val Asn Tyr Gly Val Arg Glu Ile Gln Glu Gln Asp Ala Ser Trp Arg

180 185 190

Ile Val Ile Gly Leu Gly Ile Phe Phe Ser Leu Pro Leu Gly Val Gly

195 200 205

Val Leu Leu Val Pro Glu Ser Pro Arg Trp Leu Ala Ser Lys Glu Asp

210 215 220

Trp Glu Gly Ala Arg Met Ser Met Ala Arg Leu Arg Gly Leu Lys His

225 230 235 240

Asp Pro His Asn Glu Leu Val Glu Asp Asp Met Lys Glu Met Arg Glu

245 250 255

Val Leu Glu Lys Glu Arg Thr Ala Ala Val Gly Thr Trp Lys Glu Cys

260 265 270

Phe Ile Pro Asn Lys Asn Gly Val Pro Lys Gln Val Tyr Arg Thr Phe

275 280 285

Leu Gly Ile Gly Ile His Phe Leu Gln Gln Trp Thr Gly Val Asn Tyr

290 295 300

Phe Phe Tyr Tyr Gly Ala Thr Ile Phe Gln Ser Ala Gly Ile Lys Asp

305 310 315 320

Pro Ile Gln Thr Gln Leu Ile Leu Gly Ala Val Asn Val Phe Ser Thr

325 330 335

Leu Phe Gly Leu Trp Val Val Glu Arg Phe Gly Arg Arg Trp Pro Leu

340 345 350

Phe Ile Gly Ala Ile Trp Gln Ala Ser Trp Leu Ala Val Phe Ala Ser

355 360 365

Met Gly Thr Ala Leu Glu Pro Asp Gln Asn Lys Ala Ser Gly Ile Val

370 375 380

Met Ile Val Ser Ala Ala Leu Phe Ile Ala Ser Phe Ala Cys Thr Trp

385 390 395 400

Gly Pro Ile Ala Trp Val Val Ile Gly Glu Ser Phe Pro Leu Arg Thr

405 410 415

Arg Ala Lys Gln Ala Ser Leu Ala Thr Ala Gly Asn Trp Leu Gly Asn

420 425 430

Phe Met Ile Ser Phe Leu Thr Pro Leu Ala Thr Asp Gly Ile Gly Tyr

435 440 445

Ala Tyr Gly Tyr Val Phe Val Ala Thr Asn Ile Met Gly Ala Leu Leu

450 455 460

Val Trp Phe Phe Leu Tyr Glu Ser Thr Ser Leu Ser Leu Glu Asn Val

465 470 475 480

Asp Leu Met Tyr Ser Glu Pro Gly Ile Lys Pro Trp Asn Ser His Lys

485 490 495

Trp Met Pro Pro Gly Tyr Ile Thr Arg Met Gln Arg Asp Asp Glu Tyr

500 505 510

Phe His His Pro Glu Lys Gly Gly Ser Asp Gly Leu Glu Ile Thr Asn

515 520 525

Gly Ser Lys Glu Met His Pro His Glu Glu Arg Glu Glu Lys Val Ile

530 535 540

Asn His Arg Val

545

<210> 5

<211> 1662

<212> DNA

<213> Neurospora crassa (Neurospora crassa)

<400> 5

atg aag cca ttt ctg ggg ctc aga ggt cag ccc ctc aac ctg gcc gtg 48

Met Lys Pro Phe Leu Gly Leu Arg Gly Gln Pro Leu Asn Leu Ala Val

1 5 10 15

ggc gca gtt gcc gga tgt gat ttt ctt ctt ttc gga tac gat cag ggt 96

Gly Ala Val Ala Gly Cys Asp Phe Leu Leu Phe Gly Tyr Asp Gln Gly

20 25 30

gtt atg ggc gga att ctg act ctc ccc gag ttc ctc ggt tac ttt gaa 144

Val Met Gly Gly Ile Leu Thr Leu Pro Glu Phe Leu Gly Tyr Phe Glu

35 40 45

caa atc aat cca gac gca ccg ggc ctc aca cca cac gaa tct tct atg 192

Gln Ile Asn Pro Asp Ala Pro Gly Leu Thr Pro His Glu Ser Ser Met

50 55 60

cga tct act tac caa ggc atc tcc gtc gct tcc tac aac ctg gga tgt 240

Arg Ser Thr Tyr Gln Gly Ile Ser Val Ala Ser Tyr Asn Leu Gly Cys

65 70 75 80

ttt atc ggc gcc atc atc act atc ttc att ggc aat ccc tgg ggc cgt 288

Phe Ile Gly Ala Ile Ile Thr Ile Phe Ile Gly Asn Pro Trp Gly Arg

85 90 95

aag aaa ata ata ttg ctc ggg act tct atc atg att gtc ggt gcc att 336

Lys Lys Ile Ile Leu Leu Gly Thr Ser Ile Met Ile Val Gly Ala Ile

100 105 110

ctt cag gcc agc gct act act cta ggc cat ttc atc att ggg cgt atc 384

Leu Gln Ala Ser Ala Thr Thr Leu Gly His Phe Ile Ile Gly Arg Ile

115 120 125

att acc ggc att gga aac gga gga aac aca tct act gtc cct act tgg 432

Ile Thr Gly Ile Gly Asn Gly Gly Asn Thr Ser Thr Val Pro Thr Trp

130 135 140

caa tcc gag act tcg aaa gca cac aag cga ggc aag atg gtc atg atc 480

Gln Ser Glu Thr Ser Lys Ala His Lys Arg Gly Lys Met Val Met Ile

145 150 155 160

gaa gga tct ctc gtc acc gcc ggc atc atg ttg agt tac tgg att gac 528

Glu Gly Ser Leu Val Thr Ala Gly Ile Met Leu Ser Tyr Trp Ile Asp

165 170 175

ctc ggg tta agt ttt gcc cct ggc tca gtt gct tgg cgc ttc cct ctt 576

Leu Gly Leu Ser Phe Ala Pro Gly Ser Val Ala Trp Arg Phe Pro Leu

180 185 190

gcc ttc cag atc atc ttc tgc atc ctc atc ctc atc ttt att ccc ttc 624

Ala Phe Gln Ile Ile Phe Cys Ile Leu Ile Leu Ile Phe Ile Pro Phe

195 200 205

ctg ccc gaa tca cct cgt tgg ttg gtt ttc aaa ggc cgc gac gcg gaa 672

Leu Pro Glu Ser Pro Arg Trp Leu Val Phe Lys Gly Arg Asp Ala Glu

210 215 220

gcc aag gaa atc ctg gcc gcc ctc aac gac gtg gaa ctt gac gat ccc 720

Ala Lys Glu Ile Leu Ala Ala Leu Asn Asp Val Glu Leu Asp Asp Pro

225 230 235 240

att gtc gac acc gag ttc cat ttc atc cac gat acg gtt gtc gag atg 768

Ile Val Asp Thr Glu Phe His Phe Ile His Asp Thr Val Val Glu Met

245 250 255

tcc aaa ggc agc ttc aag gac ctc ttc acc atg gac aag gac cgc aac 816

Ser Lys Gly Ser Phe Lys Asp Leu Phe Thr Met Asp Lys Asp Arg Asn

260 265 270

ttc cac cgc act ctg ctg gca tac ctc aac cag gtt ttc cag cag atc 864

Phe His Arg Thr Leu Leu Ala Tyr Leu Asn Gln Val Phe Gln Gln Ile

275 280 285

tct ggt ata aac ctc atc acc tac tat gcc gcc gtc atc tac agc gga 912

Ser Gly Ile Asn Leu Ile Thr Tyr Tyr Ala Ala Val Ile Tyr Ser Gly

290 295 300

ctt ggc atg tcc gac ttc ctg gcc cgt ctc ctc gcc gcc ctc aac ggc 960

Leu Gly Met Ser Asp Phe Leu Ala Arg Leu Leu Ala Ala Leu Asn Gly

305 310 315 320

acc gag tac ttc att gcc tcg tgg cca gcc gtc ttc ctt gtc gag cgc 1008

Thr Glu Tyr Phe Ile Ala Ser Trp Pro Ala Val Phe Leu Val Glu Arg

325 330 335

gtc ggc cgc cgc aag ctc atg ttg ttt ggt gcc atc ggc caa gcg gcc 1056

Val Gly Arg Arg Lys Leu Met Leu Phe Gly Ala Ile Gly Gln Ala Ala

340 345 350

acc atg gcc atc ctg gca ggc gtc aac tcc cgg cca gac gac aag cca 1104

Thr Met Ala Ile Leu Ala Gly Val Asn Ser Arg Pro Asp Asp Lys Pro

355 360 365

tac caa att gcg gga atc gtc ttt ctg ttc gtc ttc aac acg gtt ttc 1152

Tyr Gln Ile Ala Gly Ile Val Phe Leu Phe Val Phe Asn Thr Val Phe

370 375 380

gcg gtc ggc tgg ctc ggc atg tcc tgg ctc tac cca gcc gag atc gtg 1200

Ala Val Gly Trp Leu Gly Met Ser Trp Leu Tyr Pro Ala Glu Ile Val

385 390 395 400

ccg ctc cgc atc cgc gcc ccg gct aac gcg cta tcc acg tcg gcg aac 1248

Pro Leu Arg Ile Arg Ala Pro Ala Asn Ala Leu Ser Thr Ser Ala Asn

405 410 415

tgg atc ttc aac ttt atg gtt gtc atg atc act ccg gtt gcg ttc aac 1296

Trp Ile Phe Asn Phe Met Val Val Met Ile Thr Pro Val Ala Phe Asn

420 425 430

aaa atc aag tat cag acg tac atc atc ttc gcc gtc atc aac gcc ttt 1344

Lys Ile Lys Tyr Gln Thr Tyr Ile Ile Phe Ala Val Ile Asn Ala Phe

435 440 445

att gtt cca gtc gtc tat ttc ttc tat ccg gaa acg gcg tgc cgg tca 1392

Ile Val Pro Val Val Tyr Phe Phe Tyr Pro Glu Thr Ala Cys Arg Ser

450 455 460

cta gag gaa atg gat atg atc ttc cac aag gtt gat ggg tgg aag ggg 1440

Leu Glu Glu Met Asp Met Ile Phe His Lys Val Asp Gly Trp Lys Gly

465 470 475 480

tac ttc acg gta gtg cac cag gcc aag gtt gag cct aaa tgg tac gac 1488

Tyr Phe Thr Val Val His Gln Ala Lys Val Glu Pro Lys Trp Tyr Asp

485 490 495

aag gat ggc cag cgc att ggc ggg gct gac ttt gag aag act gct ggg 1536

Lys Asp Gly Gln Arg Ile Gly Gly Ala Asp Phe Glu Lys Thr Ala Gly

500 505 510

tac cag agc cac tcg att ccg gag tcg tct gga ttt gaa aag ccg acg 1584

Tyr Gln Ser His Ser Ile Pro Glu Ser Ser Gly Phe Glu Lys Pro Thr

515 520 525

aag gcg cat gtt gag tcg cct agg gcg gat gac ggg atc act tcg tcg 1632

Lys Ala His Val Glu Ser Pro Arg Ala Asp Asp Gly Ile Thr Ser Ser

530 535 540

agt agt gat gga ggt aat cgg gag tcg tag 1662

Ser Ser Asp Gly Gly Asn Arg Glu Ser

545 550

<210> 6

<211> 553

<212> PRT

<213> Neurospora crassa (Neurospora crassa)

<400> 6

Met Lys Pro Phe Leu Gly Leu Arg Gly Gln Pro Leu Asn Leu Ala Val

1 5 10 15

Gly Ala Val Ala Gly Cys Asp Phe Leu Leu Phe Gly Tyr Asp Gln Gly

20 25 30

Val Met Gly Gly Ile Leu Thr Leu Pro Glu Phe Leu Gly Tyr Phe Glu

35 40 45

Gln Ile Asn Pro Asp Ala Pro Gly Leu Thr Pro His Glu Ser Ser Met

50 55 60

Arg Ser Thr Tyr Gln Gly Ile Ser Val Ala Ser Tyr Asn Leu Gly Cys

65 70 75 80

Phe Ile Gly Ala Ile Ile Thr Ile Phe Ile Gly Asn Pro Trp Gly Arg

85 90 95

Lys Lys Ile Ile Leu Leu Gly Thr Ser Ile Met Ile Val Gly Ala Ile

100 105 110

Leu Gln Ala Ser Ala Thr Thr Leu Gly His Phe Ile Ile Gly Arg Ile

115 120 125

Ile Thr Gly Ile Gly Asn Gly Gly Asn Thr Ser Thr Val Pro Thr Trp

130 135 140

Gln Ser Glu Thr Ser Lys Ala His Lys Arg Gly Lys Met Val Met Ile

145 150 155 160

Glu Gly Ser Leu Val Thr Ala Gly Ile Met Leu Ser Tyr Trp Ile Asp

165 170 175

Leu Gly Leu Ser Phe Ala Pro Gly Ser Val Ala Trp Arg Phe Pro Leu

180 185 190

Ala Phe Gln Ile Ile Phe Cys Ile Leu Ile Leu Ile Phe Ile Pro Phe

195 200 205

Leu Pro Glu Ser Pro Arg Trp Leu Val Phe Lys Gly Arg Asp Ala Glu

210 215 220

Ala Lys Glu Ile Leu Ala Ala Leu Asn Asp Val Glu Leu Asp Asp Pro

225 230 235 240

Ile Val Asp Thr Glu Phe His Phe Ile His Asp Thr Val Val Glu Met

245 250 255

Ser Lys Gly Ser Phe Lys Asp Leu Phe Thr Met Asp Lys Asp Arg Asn

260 265 270

Phe His Arg Thr Leu Leu Ala Tyr Leu Asn Gln Val Phe Gln Gln Ile

275 280 285

Ser Gly Ile Asn Leu Ile Thr Tyr Tyr Ala Ala Val Ile Tyr Ser Gly

290 295 300

Leu Gly Met Ser Asp Phe Leu Ala Arg Leu Leu Ala Ala Leu Asn Gly

305 310 315 320

Thr Glu Tyr Phe Ile Ala Ser Trp Pro Ala Val Phe Leu Val Glu Arg

325 330 335

Val Gly Arg Arg Lys Leu Met Leu Phe Gly Ala Ile Gly Gln Ala Ala

340 345 350

Thr Met Ala Ile Leu Ala Gly Val Asn Ser Arg Pro Asp Asp Lys Pro

355 360 365

Tyr Gln Ile Ala Gly Ile Val Phe Leu Phe Val Phe Asn Thr Val Phe

370 375 380

Ala Val Gly Trp Leu Gly Met Ser Trp Leu Tyr Pro Ala Glu Ile Val

385 390 395 400

Pro Leu Arg Ile Arg Ala Pro Ala Asn Ala Leu Ser Thr Ser Ala Asn

405 410 415

Trp Ile Phe Asn Phe Met Val Val Met Ile Thr Pro Val Ala Phe Asn

420 425 430

Lys Ile Lys Tyr Gln Thr Tyr Ile Ile Phe Ala Val Ile Asn Ala Phe

435 440 445

Ile Val Pro Val Val Tyr Phe Phe Tyr Pro Glu Thr Ala Cys Arg Ser

450 455 460

Leu Glu Glu Met Asp Met Ile Phe His Lys Val Asp Gly Trp Lys Gly

465 470 475 480

Tyr Phe Thr Val Val His Gln Ala Lys Val Glu Pro Lys Trp Tyr Asp

485 490 495

Lys Asp Gly Gln Arg Ile Gly Gly Ala Asp Phe Glu Lys Thr Ala Gly

500 505 510

Tyr Gln Ser His Ser Ile Pro Glu Ser Ser Gly Phe Glu Lys Pro Thr

515 520 525

Lys Ala His Val Glu Ser Pro Arg Ala Asp Asp Gly Ile Thr Ser Ser

530 535 540

Ser Ser Asp Gly Gly Asn Arg Glu Ser

545 550

<210> 7

<211> 1557

<212> DNA

<213> Neurospora crassa (Neurospora crassa)

<400> 7

atg ggg ctc ggg ctt aag cta ccg aca atc tac aat gtc cac ctt gtg 48

Met Gly Leu Gly Leu Lys Leu Pro Thr Ile Tyr Asn Val His Leu Val

1 5 10 15

gct atc att gcc acc ttg ggt ggt gca ctc ttc ggc ttt gat atc tcc 96

Ala Ile Ile Ala Thr Leu Gly Gly Ala Leu Phe Gly Phe Asp Ile Ser

20 25 30

tcc atg tcc gcc atc gtc gtg acc gac caa tac ctc acc tac ttc aac 144

Ser Met Ser Ala Ile Val Val Thr Asp Gln Tyr Leu Thr Tyr Phe Asn

35 40 45

aac ccc cat gat atc atc caa gga gcc atc ggc tct gcc ctt gct gct 192

Asn Pro His Asp Ile Ile Gln Gly Ala Ile Gly Ser Ala Leu Ala Ala

50 55 60

ggc tcc gtc gtc ggt tcc gcc atc gcc ggt cct ctt tcc gac aag atc 240

Gly Ser Val Val Gly Ser Ala Ile Ala Gly Pro Leu Ser Asp Lys Ile

65 70 75 80

ggt cgt cgt gac tcc atc ttt ttc gcc tgc ttc ttc tgg ctc att ggt 288

Gly Arg Arg Asp Ser Ile Phe Phe Ala Cys Phe Phe Trp Leu Ile Gly

85 90 95

acc tcc gtc cag gtt gcc tgc aag aac tat ggc cag ctc atc gcc ggc 336

Thr Ser Val Gln Val Ala Cys Lys Asn Tyr Gly Gln Leu Ile Ala Gly

100 105 110

cgt gtg ctc aac ggc ttt acc gtc ggc atc act tcc tcc cag gtt ccc 384

Arg Val Leu Asn Gly Phe Thr Val Gly Ile Thr Ser Ser Gln Val Pro

115 120 125

gtg tac ctt gcc gag atc gcc aag gca gag aag cgt ggt tcc ttg gtc 432

Val Tyr Leu Ala Glu Ile Ala Lys Ala Glu Lys Arg Gly Ser Leu Val

130 135 140

atc atc cag caa ctc gcc atc gag ttt ggt atc ttg atc atg tac ttt 480

Ile Ile Gln Gln Leu Ala Ile Glu Phe Gly Ile Leu Ile Met Tyr Phe

145 150 155 160

atc ggc tac ggc tgt gcg tcg atc gag ggc cct gct tcg ttc cgg acc 528

Ile Gly Tyr Gly Cys Ala Ser Ile Glu Gly Pro Ala Ser Phe Arg Thr

165 170 175

gct tgg ggc att cag ttt atc cct tgc ttt ttc ctc atg gtc ggt ctt 576

Ala Trp Gly Ile Gln Phe Ile Pro Cys Phe Phe Leu Met Val Gly Leu

180 185 190

ccc ttc ttg cct agg tcg ccc aga tgg ctg gcc aag gtc ggt agg gac 624

Pro Phe Leu Pro Arg Ser Pro Arg Trp Leu Ala Lys Val Gly Arg Asp

195 200 205

cag gag gcc att gct gtc ctg gct aac atc cag gct gat ggc aac gtt 672

Gln Glu Ala Ile Ala Val Leu Ala Asn Ile Gln Ala Asp Gly Asn Val

210 215 220

gat gac ccg aga gtc gtt gct gag tgg gag gag att gtc acc gtt atg 720

Asp Asp Pro Arg Val Val Ala Glu Trp Glu Glu Ile Val Thr Val Met

225 230 235 240

aac gcc gag cgt gag gcc ggt aag gga tgg agg aag ttt gtc aag aac 768

Asn Ala Glu Arg Glu Ala Gly Lys Gly Trp Arg Lys Phe Val Lys Asn

245 250 255

ggc atg tgg aag cga acc atg gct ggc atg act gta cag gct tgg cag 816

Gly Met Trp Lys Arg Thr Met Ala Gly Met Thr Val Gln Ala Trp Gln

260 265 270

caa ctc gcc ggc gcc aac gta atc gtc tac tac cta acc tac atc gcc 864

Gln Leu Ala Gly Ala Asn Val Ile Val Tyr Tyr Leu Thr Tyr Ile Ala

275 280 285

caa atg gcc gga ctc aca ggc aac gtc gcc atg gta acc tcg ggc atc 912

Gln Met Ala Gly Leu Thr Gly Asn Val Ala Met Val Thr Ser Gly Ile

290 295 300

caa tac gcc gtt ttc atc atc ttc acc ggc gtc atg tgg ctc ttc atc 960

Gln Tyr Ala Val Phe Ile Ile Phe Thr Gly Val Met Trp Leu Phe Ile

305 310 315 320

gac aag acc ggt cgt cgc acc ctt tta gtt tac ggc gcc ttg gga atg 1008

Asp Lys Thr Gly Arg Arg Thr Leu Leu Val Tyr Gly Ala Leu Gly Met

325 330 335

gcc ttc tgc cac ttt gtc gtc ggc ggc gtc atg ggc gcg cac cac gac 1056

Ala Phe Cys His Phe Val Val Gly Gly Val Met Gly Ala His His Asp

340 345 350

aac gtt ccg gac ggc gtc ggc ggc aac gcc aac att gtc att agc gtg 1104

Asn Val Pro Asp Gly Val Gly Gly Asn Ala Asn Ile Val Ile Ser Val

355 360 365

cac aag ggc gcg ccc gcc aac acg gtc atc ctg ttc tcg tac ctg ctc 1152

His Lys Gly Ala Pro Ala Asn Thr Val Ile Leu Phe Ser Tyr Leu Leu

370 375 380

att gtc gtc tac gcc ttg acg ctc gct ccc gtc tgc tgg atc tac gcc 1200

Ile Val Val Tyr Ala Leu Thr Leu Ala Pro Val Cys Trp Ile Tyr Ala

385 390 395 400

gcc gag gtc tgg tcg ttg ggc act cgc gct acg ggc atg tcc atg gct 1248

Ala Glu Val Trp Ser Leu Gly Thr Arg Ala Thr Gly Met Ser Met Ala

405 410 415

gcc atg tcc aac tgg gtg ttc aac ttt gcg ctg ggc atg ttc acg ccg 1296

Ala Met Ser Asn Trp Val Phe Asn Phe Ala Leu Gly Met Phe Thr Pro

420 425 430

ccg gcg ttt gtc aat att acg tgg aag ctg ttt atc att ttc ggg gtg 1344

Pro Ala Phe Val Asn Ile Thr Trp Lys Leu Phe Ile Ile Phe Gly Val

435 440 445

ctt tgc gtc acg gcg gcg gtc tgg ttc tgg ttg ttt tac ccg gag acg 1392

Leu Cys Val Thr Ala Ala Val Trp Phe Trp Leu Phe Tyr Pro Glu Thr

450 455 460

tgt ggt aag acg ctg gag gag att gag atc ctg ttt ggt gat cag ggt 1440

Cys Gly Lys Thr Leu Glu Glu Ile Glu Ile Leu Phe Gly Asp Gln Gly

465 470 475 480

cct aag ccg tgg aag acg aag aag ggc gag tcg aga ctt acg gcg gag 1488

Pro Lys Pro Trp Lys Thr Lys Lys Gly Glu Ser Arg Leu Thr Ala Glu

485 490 495

att gag gct gtc aag gcg agg aag acg gtg gag cac gag att gag gtg 1536

Ile Glu Ala Val Lys Ala Arg Lys Thr Val Glu His Glu Ile Glu Val

500 505 510

cat gag cat gag aag gtt tag 1557

His Glu His Glu Lys Val

515

<210> 8

<211> 518

<212> PRT

<213> Neurospora crassa (Neurospora crassa)

<400> 8

Met Gly Leu Gly Leu Lys Leu Pro Thr Ile Tyr Asn Val His Leu Val

1 5 10 15

Ala Ile Ile Ala Thr Leu Gly Gly Ala Leu Phe Gly Phe Asp Ile Ser

20 25 30

Ser Met Ser Ala Ile Val Val Thr Asp Gln Tyr Leu Thr Tyr Phe Asn

35 40 45

Asn Pro His Asp Ile Ile Gln Gly Ala Ile Gly Ser Ala Leu Ala Ala

50 55 60

Gly Ser Val Val Gly Ser Ala Ile Ala Gly Pro Leu Ser Asp Lys Ile

65 70 75 80

Gly Arg Arg Asp Ser Ile Phe Phe Ala Cys Phe Phe Trp Leu Ile Gly

85 90 95

Thr Ser Val Gln Val Ala Cys Lys Asn Tyr Gly Gln Leu Ile Ala Gly

100 105 110

Arg Val Leu Asn Gly Phe Thr Val Gly Ile Thr Ser Ser Gln Val Pro

115 120 125

Val Tyr Leu Ala Glu Ile Ala Lys Ala Glu Lys Arg Gly Ser Leu Val

130 135 140

Ile Ile Gln Gln Leu Ala Ile Glu Phe Gly Ile Leu Ile Met Tyr Phe

145 150 155 160

Ile Gly Tyr Gly Cys Ala Ser Ile Glu Gly Pro Ala Ser Phe Arg Thr

165 170 175

Ala Trp Gly Ile Gln Phe Ile Pro Cys Phe Phe Leu Met Val Gly Leu

180 185 190

Pro Phe Leu Pro Arg Ser Pro Arg Trp Leu Ala Lys Val Gly Arg Asp

195 200 205

Gln Glu Ala Ile Ala Val Leu Ala Asn Ile Gln Ala Asp Gly Asn Val

210 215 220

Asp Asp Pro Arg Val Val Ala Glu Trp Glu Glu Ile Val Thr Val Met

225 230 235 240

Asn Ala Glu Arg Glu Ala Gly Lys Gly Trp Arg Lys Phe Val Lys Asn

245 250 255

Gly Met Trp Lys Arg Thr Met Ala Gly Met Thr Val Gln Ala Trp Gln

260 265 270

Gln Leu Ala Gly Ala Asn Val Ile Val Tyr Tyr Leu Thr Tyr Ile Ala

275 280 285

Gln Met Ala Gly Leu Thr Gly Asn Val Ala Met Val Thr Ser Gly Ile

290 295 300

Gln Tyr Ala Val Phe Ile Ile Phe Thr Gly Val Met Trp Leu Phe Ile

305 310 315 320

Asp Lys Thr Gly Arg Arg Thr Leu Leu Val Tyr Gly Ala Leu Gly Met

325 330 335

Ala Phe Cys His Phe Val Val Gly Gly Val Met Gly Ala His His Asp

340 345 350

Asn Val Pro Asp Gly Val Gly Gly Asn Ala Asn Ile Val Ile Ser Val

355 360 365

His Lys Gly Ala Pro Ala Asn Thr Val Ile Leu Phe Ser Tyr Leu Leu

370 375 380

Ile Val Val Tyr Ala Leu Thr Leu Ala Pro Val Cys Trp Ile Tyr Ala

385 390 395 400

Ala Glu Val Trp Ser Leu Gly Thr Arg Ala Thr Gly Met Ser Met Ala

405 410 415

Ala Met Ser Asn Trp Val Phe Asn Phe Ala Leu Gly Met Phe Thr Pro

420 425 430

Pro Ala Phe Val Asn Ile Thr Trp Lys Leu Phe Ile Ile Phe Gly Val

435 440 445

Leu Cys Val Thr Ala Ala Val Trp Phe Trp Leu Phe Tyr Pro Glu Thr

450 455 460

Cys Gly Lys Thr Leu Glu Glu Ile Glu Ile Leu Phe Gly Asp Gln Gly

465 470 475 480

Pro Lys Pro Trp Lys Thr Lys Lys Gly Glu Ser Arg Leu Thr Ala Glu

485 490 495

Ile Glu Ala Val Lys Ala Arg Lys Thr Val Glu His Glu Ile Glu Val

500 505 510

His Glu His Glu Lys Val

515

<210> 9

<211> 1575

<212> DNA

<213> myceliophthora thermophila (Myceliophora thermophila)

<400> 9

atg aag ctg ccc acg att tac aat gtc cag ctc gtg gcc atc atc gcg 48

Met Lys Leu Pro Thr Ile Tyr Asn Val Gln Leu Val Ala Ile Ile Ala

1 5 10 15

acc ctc ggt ggc atg ctg ttc ggg ttc gac att tct tcc atg tcg gcc 96

Thr Leu Gly Gly Met Leu Phe Gly Phe Asp Ile Ser Ser Met Ser Ala

20 25 30

atc gtc gtg acc gac cag tac att gag tac ttc aac aac cct acc ggg 144

Ile Val Val Thr Asp Gln Tyr Ile Glu Tyr Phe Asn Asn Pro Thr Gly

35 40 45

gtt atc caa ggc gcc atc ggg tcg gct ctc gcg gcc ggc tcg gtc gtc 192

Val Ile Gln Gly Ala Ile Gly Ser Ala Leu Ala Ala Gly Ser Val Val

50 55 60

ggt tct gcc gtg gca ggt ccg ctg tcc gac tgg atg ggc cgg cgc gac 240

Gly Ser Ala Val Ala Gly Pro Leu Ser Asp Trp Met Gly Arg Arg Asp

65 70 75 80

tcc atc atg ttt gcc tgc ttg ttc tgg ctc gtc ggg acg gcg gtc cag 288

Ser Ile Met Phe Ala Cys Leu Phe Trp Leu Val Gly Thr Ala Val Gln

85 90 95

gtg gcg acc cag aac gtc ggc cag ctc atc gcc ggc cgc gtg ctc aac 336

Val Ala Thr Gln Asn Val Gly Gln Leu Ile Ala Gly Arg Val Leu Asn

100 105 110

ggc ttc acc gtc ggc atc acg tcg tcc cag gtg ccc gtc tac ctg gcc 384

Gly Phe Thr Val Gly Ile Thr Ser Ser Gln Val Pro Val Tyr Leu Ala

115 120 125

gag atc gcc aag gcc gag aag cgc ggc tcc atc gtc atc atc cag cag 432

Glu Ile Ala Lys Ala Glu Lys Arg Gly Ser Ile Val Ile Ile Gln Gln

130 135 140

ctg gcc gtc gag ttc ggc atc ctc atc atg tac ttc atc ggc tac ggc 480

Leu Ala Val Glu Phe Gly Ile Leu Ile Met Tyr Phe Ile Gly Tyr Gly

145 150 155 160

tgc gcc tcc atc gag ggc acc ggc tcc ttc cgc acc gcc tgg ggc acc 528

Cys Ala Ser Ile Glu Gly Thr Gly Ser Phe Arg Thr Ala Trp Gly Thr

165 170 175

cag ttc atc ccc tgc gtc ttc ctc atg ctc ggc ctc ccc ttc ctc ccg 576

Gln Phe Ile Pro Cys Val Phe Leu Met Leu Gly Leu Pro Phe Leu Pro

180 185 190

cgc tcg ccc cga tgg ctc gcc aag gtc ggc cgc gac aag gag gcc atc 624

Arg Ser Pro Arg Trp Leu Ala Lys Val Gly Arg Asp Lys Glu Ala Ile

195 200 205

gag acc ctc gcc aac atc cag gcc gac ggc aac acc cag gac tcc cgc 672

Glu Thr Leu Ala Asn Ile Gln Ala Asp Gly Asn Thr Gln Asp Ser Arg

210 215 220

gtc att gcc gag tgg gag gag atc cag acc gtc atg cag gcc gag cgc 720

Val Ile Ala Glu Trp Glu Glu Ile Gln Thr Val Met Gln Ala Glu Arg

225 230 235 240

gag gcc ggc cgc ggc tgg agg aag ttc ttg ctc aac ggc atg tgg aag 768

Glu Ala Gly Arg Gly Trp Arg Lys Phe Leu Leu Asn Gly Met Trp Lys

245 250 255

cgt acc ctg gcc ggc atg tcg gtc cag gcg tgg cag caa ctg gct ggc 816

Arg Thr Leu Ala Gly Met Ser Val Gln Ala Trp Gln Gln Leu Ala Gly

260 265 270

gcc aac gtg atc gtt tac tac ctg act tac atc gct cag atg gct ggt 864

Ala Asn Val Ile Val Tyr Tyr Leu Thr Tyr Ile Ala Gln Met Ala Gly

275 280 285

ctg acc ggc gac gtc gcc atg gtt acg tcg ggt atc caa tat gcc gtc 912

Leu Thr Gly Asp Val Ala Met Val Thr Ser Gly Ile Gln Tyr Ala Val

290 295 300

ttc atc gtc ttc acc ggc atc atg tgg ctc ttc atc gac aag acc ggc 960

Phe Ile Val Phe Thr Gly Ile Met Trp Leu Phe Ile Asp Lys Thr Gly

305 310 315 320

cgc cgc acg ctg ctc atc tgg ggc gcc ctc ggc atg ggc ttc tgc cac 1008

Arg Arg Thr Leu Leu Ile Trp Gly Ala Leu Gly Met Gly Phe Cys His

325 330 335

ttc gtc gtc ggc ggc gtc atg ggc gcg cac tcg acc tac cac ccc gag 1056

Phe Val Val Gly Gly Val Met Gly Ala His Ser Thr Tyr His Pro Glu

340 345 350

ggc gtg ggc aac ccg ccc aac ggc aac atc gtc atc gcc gtc aac aag 1104

Gly Val Gly Asn Pro Pro Asn Gly Asn Ile Val Ile Ala Val Asn Lys

355 360 365

ggc gcg ccg gcc aac acg gtc atc acc ttc tcg tac ctg ctc atc gtc 1152

Gly Ala Pro Ala Asn Thr Val Ile Thr Phe Ser Tyr Leu Leu Ile Val

370 375 380

gtc tac gcg ctg acg ctg gcc ccg gtc tgc tgg atc tac gcg gcc gag 1200

Val Tyr Ala Leu Thr Leu Ala Pro Val Cys Trp Ile Tyr Ala Ala Glu

385 390 395 400

gtc tgg tcg ctg ggc acg cgg gcg acg ggc atg tcc ctg gcg gcc atg 1248

Val Trp Ser Leu Gly Thr Arg Ala Thr Gly Met Ser Leu Ala Ala Met

405 410 415

agc aac tgg gtc ttc aac ttc gcc ctc ggc atg ttc acc ccg ccg ggc 1296

Ser Asn Trp Val Phe Asn Phe Ala Leu Gly Met Phe Thr Pro Pro Gly

420 425 430

ttc gtc aac atc acc tgg aag ctc ttc atc atc ttc ggc gtc ctc tgc 1344

Phe Val Asn Ile Thr Trp Lys Leu Phe Ile Ile Phe Gly Val Leu Cys

435 440 445

gtc acc gcc gcc gcc tgg ttc ttc ctg ctc tgc ccg gag acg tgc ggc 1392

Val Thr Ala Ala Ala Trp Phe Phe Leu Leu Cys Pro Glu Thr Cys Gly

450 455 460

aag acg ctc gag gag atc gag ctc ctc ttc tcc ggc ccg gac gcc ccg 1440

Lys Thr Leu Glu Glu Ile Glu Leu Leu Phe Ser Gly Pro Asp Ala Pro

465 470 475 480

cac ccc tgg aac acc agg aag ggc gac tcc cgc ctc gcc gcc gag atc 1488

His Pro Trp Asn Thr Arg Lys Gly Asp Ser Arg Leu Ala Ala Glu Ile

485 490 495

gcc gcc gtc gag gcc cgc cgg cgc gag aag acc gag gcc ggc gag gtg 1536

Ala Ala Val Glu Ala Arg Arg Arg Glu Lys Thr Glu Ala Gly Glu Val

500 505 510

gag gcc gtg ccc tcc gtc ggc gag cag gag aag gtt taa 1575

Glu Ala Val Pro Ser Val Gly Glu Gln Glu Lys Val

515 520

<210> 10

<211> 524

<212> PRT

<213> myceliophthora thermophila (Myceliophora thermophila)

<400> 10

Met Lys Leu Pro Thr Ile Tyr Asn Val Gln Leu Val Ala Ile Ile Ala

1 5 10 15

Thr Leu Gly Gly Met Leu Phe Gly Phe Asp Ile Ser Ser Met Ser Ala

20 25 30

Ile Val Val Thr Asp Gln Tyr Ile Glu Tyr Phe Asn Asn Pro Thr Gly

35 40 45

Val Ile Gln Gly Ala Ile Gly Ser Ala Leu Ala Ala Gly Ser Val Val

50 55 60

Gly Ser Ala Val Ala Gly Pro Leu Ser Asp Trp Met Gly Arg Arg Asp

65 70 75 80

Ser Ile Met Phe Ala Cys Leu Phe Trp Leu Val Gly Thr Ala Val Gln

85 90 95

Val Ala Thr Gln Asn Val Gly Gln Leu Ile Ala Gly Arg Val Leu Asn

100 105 110

Gly Phe Thr Val Gly Ile Thr Ser Ser Gln Val Pro Val Tyr Leu Ala

115 120 125

Glu Ile Ala Lys Ala Glu Lys Arg Gly Ser Ile Val Ile Ile Gln Gln

130 135 140

Leu Ala Val Glu Phe Gly Ile Leu Ile Met Tyr Phe Ile Gly Tyr Gly

145 150 155 160

Cys Ala Ser Ile Glu Gly Thr Gly Ser Phe Arg Thr Ala Trp Gly Thr

165 170 175

Gln Phe Ile Pro Cys Val Phe Leu Met Leu Gly Leu Pro Phe Leu Pro

180 185 190

Arg Ser Pro Arg Trp Leu Ala Lys Val Gly Arg Asp Lys Glu Ala Ile

195 200 205

Glu Thr Leu Ala Asn Ile Gln Ala Asp Gly Asn Thr Gln Asp Ser Arg

210 215 220

Val Ile Ala Glu Trp Glu Glu Ile Gln Thr Val Met Gln Ala Glu Arg

225 230 235 240

Glu Ala Gly Arg Gly Trp Arg Lys Phe Leu Leu Asn Gly Met Trp Lys

245 250 255

Arg Thr Leu Ala Gly Met Ser Val Gln Ala Trp Gln Gln Leu Ala Gly

260 265 270

Ala Asn Val Ile Val Tyr Tyr Leu Thr Tyr Ile Ala Gln Met Ala Gly

275 280 285

Leu Thr Gly Asp Val Ala Met Val Thr Ser Gly Ile Gln Tyr Ala Val

290 295 300

Phe Ile Val Phe Thr Gly Ile Met Trp Leu Phe Ile Asp Lys Thr Gly

305 310 315 320

Arg Arg Thr Leu Leu Ile Trp Gly Ala Leu Gly Met Gly Phe Cys His

325 330 335

Phe Val Val Gly Gly Val Met Gly Ala His Ser Thr Tyr His Pro Glu

340 345 350

Gly Val Gly Asn Pro Pro Asn Gly Asn Ile Val Ile Ala Val Asn Lys

355 360 365

Gly Ala Pro Ala Asn Thr Val Ile Thr Phe Ser Tyr Leu Leu Ile Val

370 375 380

Val Tyr Ala Leu Thr Leu Ala Pro Val Cys Trp Ile Tyr Ala Ala Glu

385 390 395 400

Val Trp Ser Leu Gly Thr Arg Ala Thr Gly Met Ser Leu Ala Ala Met

405 410 415

Ser Asn Trp Val Phe Asn Phe Ala Leu Gly Met Phe Thr Pro Pro Gly

420 425 430

Phe Val Asn Ile Thr Trp Lys Leu Phe Ile Ile Phe Gly Val Leu Cys

435 440 445

Val Thr Ala Ala Ala Trp Phe Phe Leu Leu Cys Pro Glu Thr Cys Gly

450 455 460

Lys Thr Leu Glu Glu Ile Glu Leu Leu Phe Ser Gly Pro Asp Ala Pro

465 470 475 480

His Pro Trp Asn Thr Arg Lys Gly Asp Ser Arg Leu Ala Ala Glu Ile

485 490 495

Ala Ala Val Glu Ala Arg Arg Arg Glu Lys Thr Glu Ala Gly Glu Val

500 505 510

Glu Ala Val Pro Ser Val Gly Glu Gln Glu Lys Val

515 520

<210> 11

<211> 32

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 11

cgcggatcca tgggtctctt ctcgaaaaag tc 32

<210> 12

<211> 32

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 12

ccggaattcc taaacctctc catggcttga gg 32

<210> 13

<211> 32

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 13

ggactagtat ggttctgggg aaaaagtcaa tc 32

<210> 14

<211> 33

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 14

cccaagcttc taaaccctat ggttaataac ctt 33

<210> 15

<211> 30

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 15

cgcggatcca tgaagccatt tctggggctc 30

<210> 16

<211> 32

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 16

cccaagcttc tacgactccc gattacctcc at 32

<210> 17

<211> 31

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 17

cgcggatcca tggggctcgg gcttaagcta c 31

<210> 18

<211> 32

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 18

cggaattcct aaaccttctc atgctcatgc ac 32

<210> 19

<211> 30

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 19

cgcggatcca tgaagctgcc cacgatttac 30

<210> 20

<211> 30

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 20

ccggaattct taaaccttct cctgctcgcc 30

<210> 21

<211> 36

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 21

gggaattcca tatggatctg tttagcttgc ctcgtc 36

<210> 22

<211> 30

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 22

atgggccccg acactggatg gcggcgttag 30

Claims (8)

1. A host cell having integrated into its chromosome an exogenous polynucleotide encoding a polypeptide having the activity of transporting arabinose from outside the cell to inside the cell as set forth in SEQ ID No.6, 8, and 10, said polypeptide being derived from Neurospora crassa (R) ((R))Neurospora crassa) Or myceliophthora thermophila: (Myceliophora thermophila) The host cell further comprises a polynucleotide encoding a polypeptide of SEQ ID No.2 having the activity of transporting glucose from outside the cell to inside the cellAcid, the polypeptide is derived from Neurospora crassa (A)Neurospora crassa) And/or the host cell comprises a vector comprising the polynucleotides of SEQ ID No.2, 6, 8, and 10, the host cell is yeast(s) ((R))Saccharomyces) Genus Kluyveromyces, Clostridium, or filamentous fungus.

2. Use of a host cell according to claim 1, wherein (i) the pentose sugars are transported from outside the cell to inside the cell; (ii) for the production of ethanol, the pentose is arabinose and/or xylose.

3. A method for producing ethanol and/or facilitating the transport of pentose sugars by a host cell, comprising the steps of: culturing the host cell of claim 1 in the presence of a pentose sugar which is arabinose and/or xylose.

4. The use according to claim 2 or the method according to claim 3, further comprising the step of separating and purifying the ethanol in the culture.

5. A method of producing a recombinant ethanol fermentation strain comprising the steps of: transferring a vector into an original strain to obtain a recombinant ethanol fermentation strain, wherein the vector comprises the polynucleotides shown in SEQ ID No.2, 6, 8 and 10.

6. The method of claim 5, wherein the recombinant ethanol fermentation strain has 1.2-5 times higher ethanol fermentation activity than the starting strain.

7. The method according to claim 5, wherein the recombinant ethanol-fermenting strain is a strain having pentose and/or hexose as a carbon source.

8. The method of claim 6, wherein the starting strain comprises Saccharomyces cerevisiae.

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