HK1237370B - Microbial ergothioneine biosynthesis - Google Patents

Description

Microbial ergothioneine biosynthesis

Statement of Support for Submission of Sequence Listing

A paper copy of the sequence listing and a computer readable form of the sequence listing comprising a file named "32559-12_ST25.txt" having a size of 19,443 bytes (as measured in MICROSOFT WINDOWS® EXPLORER) are provided herein and are incorporated herein by reference. The sequence listing consists of SEQ ID NOs: 1-16.

Public background

The present disclosure generally relates to methods for ergothioneine biosynthesis. More specifically, the present disclosure relates to methods for microbial ergothioneine biosynthesis.

Ergothioneine (ET) is a histidine betaine derivative with a sulfhydryl group attached to the _C2 atom of the imidazole ring. As a thiol tautomer, ET is a very stable antioxidant with unique properties. Unlike glutathione and ascorbic acid, ET can scavenge oxidative species that are not free radicals. ET is a natural compound produced in actinomycetes such as Mycobacterium smegmatis and filamentous fungi such as Neurospora crassa . Other bacterial species, such as Bacillus subtilis, Escherichia coli, Proteus vulgaris , and Streptococcus , as well as fungi belonging to the Ascomycetes and Deuteromycetes , cannot produce ergothioneine. Animals and plants also cannot produce ergothioneine and must obtain it from dietary sources or, in the case of plants, from their environment.

Although the function of ET in microbial cells is not fully understood, it is believed to be crucial in human physiology. Humans absorb ET from dietary sources, and ET accumulates in specific tissues and cells such as the liver, kidneys, central nervous system, and red blood cells. A specific cation transporter (OCTN1) has been shown to have a high affinity for ET in humans, and overactivity and deficiency of this transporter exert negative effects on human cells.

The biosynthesis of ET has been detected in some mycobacterial fungi, however, the exact metabolic pathway has not been completed or has only been partially confirmed. Seebeck reconstructed mycobacterial ergothioneine biosynthesis in vitro using Escherichia coli to express formylglycine-generating enzyme-like protein (EgtB), glutamine transamidase (EgtC), histidine methyltransferase (EgtD) and an unrelated β-lyase from Erwinia tasmaniensis that replaced pyridoxal-5-phosphate binding protein (EgtE) (because recombinant production of soluble EgtE protein failed) (see, J. Am. Chem. Soc. 2010, 132:6632-6633).

Up to now, only 3 kinds of genes encoding EgtB, EgtC and EgtD have been identified for in vitro production of thioneine.The putative gene of EgtE is still not characterized in vitro or in vivo.Up to now, although there is the attempt of various biotransformations, it has not yet been reported to use the above-mentioned gene to produce the microorganism of the engineered mycobacterium thioneine metabolic pathway in Escherichia coli.In addition, although various fungi and mycobacterium sources can be used for thioneine extraction, output is too low, so that it can not be commercially used in the industrial production of thioneine.Therefore, there is the demand for producing thioneine.

Public Overview

The present disclosure generally relates to engineered host cells and methods for producing ergothioneine. More specifically, the present disclosure relates to engineered host cells and methods for microbial ergothioneine biosynthesis using the engineered host cells.

In one aspect, the present disclosure relates to a transformed host cell for producing ergothioneine, comprising a nucleic acid sequence encoding EgtB, a nucleic acid sequence encoding EgtC, a nucleic acid sequence encoding EgtD, and a nucleic acid sequence encoding EgtE.

In another aspect, the present disclosure relates to a method for producing ergothioneine. The method includes cultivating a host cell, wherein the host cell is transformed with a nucleic acid sequence encoding EgtB, a nucleic acid sequence encoding EgtC, a nucleic acid sequence encoding EgtD, and a nucleic acid sequence encoding EgtE; inducing the host cell to express the nucleic acid sequence encoding EgtB, the nucleic acid sequence encoding EgtC, the nucleic acid sequence encoding EgtD, and the nucleic acid sequence encoding EgtE; and collecting ergothioneine.

In another aspect, the present disclosure relates to an expression vector for producing ergothioneine, comprising a nucleic acid sequence encoding an amino acid sequence selected from the group consisting of EgtB, EgtC, EgtD, and EgtE.

BRIEF DESCRIPTION OF THE DRAWINGS

The present disclosure will be better understood when considering the following detailed description thereof, and features, aspects and advantages other than those described above will become apparent. Such detailed description refers to the following drawings, in which:

FIG1A is a map of a vector containing the EgtD and EgtB genes, as discussed in Example 1.

FIG. 1B is a map of a vector containing the EgtC and EgtE genes, as discussed in Example 1.

2 is a graph illustrating that ET is produced only in strains containing all four genes, as discussed in Example 2. EI, empty vector cells induced with IPTG; SI, strain containing four genes induced with IPTG; Ck+, sample supplemented with 20 mg/L ergothioneine.

3A and 3B are graphs showing the HPLC retention time and UV spectrum of a 100 mg/L ergothioneine standard, as discussed in Example 2.

4A and 4B are graphs showing HPLC retention time and UV spectra of ergothioneine produced in E. coli transformed with nucleic acid sequences encoding EgtB, EgtC, EgtD, and EgtE, as discussed in Example 2.

Figure 5 is a graph showing the time course of ergothioneine production in engineered E. coli cells and empty vector control cells, as discussed in Example 3. EI, empty vector control induced with IPTG; SI, strain containing EgtB, EgtC, EgtD, and EgtE induced with IPTG.

Figure 6 is a graph showing transformed E. coli strains fed with various substrates and cofactors: None, no substrate or cofactor added; His, histidine; Met, methionine; Cys, cysteine; Fe, iron Fe ⁺⁺ .

While the present disclosure is susceptible to various changes and alternative forms, specific embodiments thereof have been shown by way of example in the drawings and are described in detail herein below. However, it should be understood that the description of specific embodiments is not intended to limit the disclosure to encompass all changes, equivalents, and alternatives that fall within the spirit and scope of the disclosure as defined by the appended claims.

Details

The term "complementary" is used according to its ordinary and conventional meaning as understood by those of ordinary skill in the art and is used, without limitation, to describe the relationship between nucleotide bases that are capable of hybridizing to each other. For example, with respect to DNA, adenosine is complementary to thymine, and cytosine is complementary to guanine. Thus, the present technology also includes isolated nucleic acid fragments that are complementary to the complete sequences reported in the accompanying sequence listing, as well as those substantially similar nucleic acid sequences.

The terms "nucleic acid" and "nucleotide" are used according to their respective ordinary and conventional meanings as understood by those of ordinary skill in the art and are used without limitation to refer to deoxyribonucleotides or ribonucleotides and polymers thereof in single-stranded or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified or degenerate variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the explicitly indicated sequence.

The term "isolated" is used according to its ordinary and conventional meaning as understood by those of ordinary skill in the art and, when used in the context of an isolated nucleic acid or isolated polypeptide, is used without limitation to refer to a nucleic acid or polypeptide that has been removed from its natural environment by the hand of man and is therefore not a product of nature. An isolated nucleic acid or polypeptide may exist in a purified form or may exist in a non-natural environment, such as, for example, in a transgenic host cell.

As used herein, the terms "incubating" and "incubation" refer to the process of mixing two or more chemical or biological entities (such as a compound and an enzyme) and allowing them to interact under conditions favorable to the production of a steviol glycoside composition.

The term "degenerate variant" refers to a nucleic acid sequence having a residue sequence that differs from a reference nucleic acid sequence by one or more degenerate codon substitutions. Degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is replaced with mixed bases and/or deoxyinosine residues. A nucleic acid sequence and all its degenerate variants will express the same amino acid or polypeptide.

The terms "polypeptide," "protein," and "peptide" are used according to their respective ordinary and conventional meanings as understood by those of ordinary skill in the art; the three terms are sometimes used interchangeably and are used without limitation to refer to polymers of amino acids or amino acid analogs, regardless of their size or function. Although "protein" is often used to refer to relatively large polypeptides and "peptide" is often used to refer to small polypeptides, the use of these terms in the art overlaps and varies. As used herein, the term "polypeptide" refers to peptides, polypeptides, and proteins, unless otherwise indicated. When referring to polynucleotide products, the terms "protein," "polypeptide," and "peptide" are used interchangeably herein. Thus, exemplary polypeptides include polynucleotide products, naturally occurring proteins, homologs, orthologs, paralogs, fragments, and other equivalents, variants, and analogs of the foregoing.

The terms "polypeptide fragment" and "fragment" when used with respect to a reference polypeptide are used according to their ordinary and customary meaning by those of ordinary skill in the art and are used without limitation to refer to polypeptides in which amino acid residues are deleted compared to the reference polypeptide itself, but in which the remaining amino acid sequence is generally identical to the corresponding positions in the reference polypeptide. Such deletions can occur at the amino terminus or carboxyl terminus, or both, of the reference polypeptide.

The term "functional fragment" of a polypeptide or protein refers to a peptide fragment that is a portion of the full-length polypeptide or protein and has substantially the same biological activity as the full-length polypeptide or protein, or performs substantially the same function as the full-length polypeptide or protein (e.g., performs the same enzymatic reaction).

The terms "variant polypeptide," "modified amino acid sequence," or "modified polypeptide," used interchangeably, refer to an amino acid sequence that differs from a reference polypeptide in one or more amino acids (e.g., by one or more amino acid substitutions, deletions, and/or additions). In one aspect, the variant is a "functional variant" that retains some or all of the capabilities of the reference polypeptide.

The term "functional variant" also includes conservatively substituted variants. The term "conservatively substituted variant" refers to a peptide with an amino acid sequence that is different from a reference peptide by one or more conservative amino acid substitutions, and maintains some or all of the activities of the reference peptide. A "conservative amino acid substitution" is a substitution of an amino acid residue with a functionally similar residue. Examples of conservative substitutions include one non-polar (hydrophobic) residue such as isoleucine, valine, leucine or methionine replacing another non-polar (hydrophobic) residue; one charged or polar (hydrophilic) residue replacing another charged or polar (hydrophilic) residue, such as between arginine and lysine, between glutamine and asparagine, between threonine and serine; one basic residue such as lysine or arginine replacing another basic residue; or one acidic residue such as aspartic acid or glutamic acid replacing another acidic residue; or one aromatic residue (such as phenylalanine, tyrosine or tryptophan) replacing another aromatic residue. It is expected that such substitutions have little or no effect on the apparent molecular weight or isoelectric point of a protein or polypeptide. The phrase "conservatively substituted variants" also includes peptides in which residues are substituted with chemically derivatized residues, provided that the resulting peptide retains some or all of the activity of the reference peptide as described herein.

The term "variant" in connection with the polypeptides of the present technology also includes functionally active polypeptides having an amino acid sequence that is at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% and even 100% identical to the amino acid sequence of the reference polypeptide.

The term "homologous" with all its grammatical forms and spelling variations refers to the relationship between the polynucleotides or polypeptides with a "common evolutionary origin", including polynucleotides or polypeptides from superfamily and homologous polynucleotides or proteins from different species (Reeck et al., Cell 50:667, 1987). Such polynucleotides or polypeptides have sequence homology, as reflected by their sequence similarity, whether in terms of percentage identity or in terms of the presence of specific amino acids or motifs in conserved positions. For example, two homologous polypeptides can have at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% and even 100% identical amino acid sequence.

"Percent (%) amino acid sequence identity," with respect to variant polypeptide sequences of the present technology, refers to the percentage of amino acid residues in the candidate sequence that are identical with the amino acid residues in a reference polypeptide (such as, for example, SEQ ID NO: 6), after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity (and not considering any conservative substitutions as part of the sequence identity).

Alignment for the purpose of determining percent amino acid sequence identity can be accomplished in various ways within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm required to achieve maximum alignment over the full length of the compared sequences. For example, % amino acid sequence identity can be determined using the sequence comparison program NCBI-BLAST2. The NCBI-BLAST2 sequence comparison program can be downloaded from ncbi.nlm.nih.gov. NCBI BLAST2 uses several search parameters, all of which are set to default values, including, for example, unmask yes; chain = all; expected number of occurrences: 10; minimum low complexity length = 15/5; multipass e-value = 0.01; multipass constant = 25; dropoff of final gap alignment = 25; and scoring matrix = BLOSUM62. Where NCBI-BLAST2 is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be expressed as a given amino acid sequence A having or comprising a particular % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: the fraction X/Y multiplied by 100, where X is the number of amino acid residues scored as identical matches by the sequence alignment program NCBI-BLAST2 in this program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be understood that when the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A.

In this sense, the technology for determining amino acid sequence "similarity" is well known in the art. Generally, "similarity" refers to the exact amino acid and amino acid comparison of two or more polypeptides at the appropriate position, wherein the amino acid is identical or has similar chemical and/or physical properties, such as charge or hydrophobicity. Then the so-called "percent similarity" can be determined between the polypeptide sequences compared. The technology for determining nucleic acid and amino acid sequence identity is also well known in the art, and includes determining the nucleotide sequence of the mRNA of the gene (usually via a cDNA intermediate), and determining the amino acid sequence encoded therein, and comparing it with the second amino acid sequence. Generally, "identity" refers to the exact nucleotide and nucleotide or amino acid and amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Two or more polynucleotide sequences can be compared by determining their "percent identity", as two or more amino acid sequences. The available programs in the Wisconsin Sequence Analysis Package, Version 8 (available from Genetics Computer Group, Madison, Wis.), such as the GAP program, can calculate the identity between two polynucleotides and the identity and similarity between two polypeptide sequences, respectively. Other programs for calculating identity or similarity between sequences are known to those skilled in the art.

An amino acid position that "corresponds to" a reference position refers to a position that is aligned with a reference sequence, as identified by aligning the amino acid sequences. Such alignment can be performed manually or by using well-known sequence alignment programs such as ClustalW2, Blast 2, etc.

Unless otherwise indicated, the percent identity of two polypeptide or polynucleotide sequences refers to the percentage of identical amino acid residues or nucleotides over the entire length of the shorter of the two sequences.

"Coding sequence" is used according to its ordinary and customary meaning as understood by those of ordinary skill in the art and is used without limitation to refer to a DNA sequence that encodes a specific amino acid sequence.

"Suitable regulatory sequences" are used according to their ordinary and conventional meaning as understood by those of ordinary skill in the art and are used without limitation to refer to nucleotide sequences that are located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence and that influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, translation leader sequences, introns, and polyadenylation recognition sequences.

"Promoter" is used according to its common and conventional meaning as understood by those of ordinary skill in the art, and is used to refer to a DNA sequence that is capable of controlling the expression of a coding sequence or functional RNA without limitation. Typically, the coding sequence is located at the 3' end of the promoter sequence. The promoter can be derived in its entirety from a natural gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It will be understood by those skilled in the art that different promoters can direct the expression of a gene in different cell types or at different developmental stages or in response to different environmental conditions. Promoters that cause a gene to be expressed in most cell types at most times are generally referred to as "constitutive promoters." It is further recognized that, since in most cases the exact boundaries of regulatory sequences have not yet been fully determined, DNA fragments of different lengths can have the same promoter activity.

The term "operably linked" refers to the association of nucleic acid sequences on a single nucleic acid fragment such that the function of one nucleic acid sequence is affected by the other. For example, a promoter is operably linked to a coding sequence when the promoter is capable of affecting the expression of the coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter). A coding sequence can be operably linked to a regulatory sequence in either sense or antisense orientation.

As used herein, the term "expression" is used according to its ordinary and conventional meaning as understood by those of ordinary skill in the art and is used without limitation to refer to the transcription and stable accumulation of sense (mRNA) or antisense RNA of nucleic acid fragments derived from the present technology. "Overexpression" refers to the production of a gene product in a transgenic or recombinant organism that exceeds the level of production in a normal or non-transformed organism.

"Transformation" is used according to its common and conventional meaning as understood by those of ordinary skill in the art, and is used to refer to the transfer of a polynucleotide into a target cell without limitation. The polynucleotide transferred can be incorporated into the genome or chromosomal DNA of the target cell, resulting in genetically stable inheritance, or it can replicate independently of the host chromosome. A host organism containing the transformed nucleic acid fragment is referred to as a "transgenic" or "recombinant" or "transformed" organism.

When used in conjunction with host cells herein, the terms "transformed," "transgenic," and "recombinant" are used according to their common and conventional meanings as understood by those of ordinary skill in the art, and are used without limitation to refer to cells of a host organism, such as a plant or microbial cell, into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule can be stably integrated into the genome of the host cell, or the nucleic acid molecule can exist as an extrachromosomal molecule. Such extrachromosomal molecules can replicate autonomously. Transformed cells, tissues, or subjects are understood to encompass not only the end product of the transformation process, but also their transgenic progeny.

When used herein in conjunction with polynucleotides, the terms "recombinant," "heterologous," and "exogenous" are used according to their ordinary and conventional meanings as understood by those of ordinary skill in the art, and are used without limitation to refer to a polynucleotide (e.g., a DNA sequence or gene) that is derived from a source that is foreign to a particular host cell, or, if derived from the same source, is modified from its original form. Thus, a heterologous gene in a host cell includes a gene that is endogenous to the particular host cell but has been modified, for example, by using site-directed mutagenesis or other recombinant techniques. The term also includes non-naturally occurring multiple copies of a naturally occurring DNA sequence. Thus, the term refers to a DNA segment that is foreign or heterologous to the cell, or homologous to the cell, but at a location or in a form that does not normally occur within the host cell.

Similarly, the terms "recombinant," "heterologous," and "exogenous," when used herein in conjunction with a polypeptide or amino acid sequence, mean a polypeptide or amino acid sequence that is derived from a source foreign to the particular host cell, or, if derived from the same source, is modified from its original form. Thus, a recombinant DNA segment can be expressed in a host cell to produce a recombinant polypeptide.

The terms "plasmid," "vector," and "cassette" are used according to their ordinary and conventional meanings as understood by those of ordinary skill in the art and are used without limitation to refer to extrachromosomal elements that often carry genes that are not part of the central metabolism of the cell and are typically in the form of circular double-stranded DNA molecules. Such elements can be linear or circular autonomously replicating sequences, genome integrating sequences, phages, or nucleotide sequences derived from single-stranded or double-stranded DNA or RNA from any source, in which multiple nucleotide sequences have been joined or recombined into a unique construct capable of introducing a promoter fragment and the DNA sequence of a selected gene product, along with appropriate 3' non-translated sequences, into a cell. A "transformation cassette" refers to a specific vector that contains a foreign gene and, in addition to the foreign gene, has elements that promote the transformation of a specific host cell. An "expression cassette" refers to a specific vector that contains a foreign gene and, in addition to the foreign gene, has elements that allow for enhanced expression of the gene in a foreign host.

Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described, for example, in Sambrook, J., Fritsch, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual, 2nd ed.; Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y., 1989 (hereinafter "Maniatis"); and Silhavy, T. J., Bennan, M. L., and Enquist, L. W. Experiments with Gene Fusions; Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y., 1984; and Ausubel, F. M. et al., In Current Protocols in Molecular Biology, published by Greene Publishing and Wiley-Interscience, 1987; each of which is hereby incorporated by reference in its entirety to the extent it is consistent herewith.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, preferred materials and methods are described below.

According to the present disclosure, methods for producing thioneine and host cells having genes encoding EgtB, EgtC, EgtD and EgtE that can be used to produce thioneine have been developed. Surprisingly and unexpectedly, thioneine production pathways have been reproduced in in vitro microbial production systems.

Engineered host cells for the production of ergothioneine

In one aspect, the present disclosure relates to an engineered host cell. The engineered host cell comprises a nucleic acid sequence encoding EgtB, a nucleic acid sequence encoding EgtC, a nucleic acid sequence encoding EgtD, and a nucleic acid sequence encoding EgtE.

EgtB (or iron(II)-dependent oxidoreductase EgtB) catalyzes the oxidative sulfidation of histidine trimethylolate (N-α,N-α,N-α-trimethyl-L-histidine) via the addition of oxygen and γ-glutamyl-cysteine to the histidine trimethylolate.

A suitable EgtB can be, for example, a Mycobacterium EgtB. A particularly suitable EgtB can be, for example, an EgtB nucleic acid sequence encoding an amino acid sequence that is at least 95% identical to the amino acid sequence provided in SEQ ID NO: 2. In another aspect, a particularly suitable EgtB can be, for example, an EgtB nucleic acid sequence encoding an amino acid sequence that is at least 96% identical to the amino acid sequence provided in SEQ ID NO: 2. In another aspect, a particularly suitable EgtB can be, for example, an EgtB nucleic acid sequence encoding an amino acid sequence that is at least 97% identical to the amino acid sequence provided in SEQ ID NO: 2. In another aspect, a particularly suitable EgtB can be, for example, an EgtB nucleic acid sequence encoding an amino acid sequence that is at least 98% identical to the amino acid sequence provided in SEQ ID NO: 2. In another aspect, a particularly suitable EgtB can be, for example, an EgtB nucleic acid sequence encoding an amino acid sequence that is at least 99% identical to the amino acid sequence provided in SEQ ID NO: 2. In another aspect, a particularly suitable EgtB can be, for example, an EgtB nucleic acid sequence encoding an amino acid sequence that is 100% identical to the amino acid sequence provided in SEQ ID NO: 2.

EgtC (or amidohydrolase EgtC) catalyzes the hydrolysis of the gamma-glutamyl amide bond from N-(γ-glutamyl)-[N(α), N(α), N(α)-trimethyl-L-histidyl]-cysteine sulfoxide to produce histidine trimethylcysteine sulfoxide.

Suitable EgtC can be, for example, mycobacterial EgtC. Particularly suitable EgtC can be, for example, an EgtC nucleic acid sequence encoding an amino acid sequence that is at least 95% identical to the amino acid sequence provided in SEQ ID NO: 4. In another aspect, a particularly suitable EgtC can be, for example, an EgtC nucleic acid sequence encoding an amino acid sequence that is at least 96% identical to the amino acid sequence provided in SEQ ID NO: 4. In another aspect, a particularly suitable EgtC can be, for example, an EgtC nucleic acid sequence encoding an amino acid sequence that is at least 97% identical to the amino acid sequence provided in SEQ ID NO: 4. In another aspect, a particularly suitable EgtC can be, for example, an EgtC nucleic acid sequence encoding an amino acid sequence that is at least 98% identical to the amino acid sequence provided in SEQ ID NO: 4. In another aspect, a particularly suitable EgtC can be, for example, an EgtC nucleic acid sequence encoding an amino acid sequence that is at least 99% identical to the amino acid sequence provided in SEQ ID NO: 4. In another aspect, a particularly suitable EgtC can be, for example, an EgtC nucleic acid sequence encoding an amino acid sequence that is 100% identical to the amino acid sequence provided in SEQ ID NO: 4.

EgtD (or histidine-specific methyltransferase EgtD) catalyzes the methylation of histidine to form N-α,N-α,N-α-trimethyl-L-histidine (also known as hercynine). Histidine and α-N,N-dimethylhistidine are preferred substrates.

A suitable EgtD can be, for example, a Mycobacterium EgtD. A particularly suitable EgtD can be, for example, an EgtD nucleic acid sequence encoding an amino acid sequence that is at least 95% identical to the amino acid sequence provided in SEQ ID NO: 6. In another aspect, a particularly suitable EgtD can be, for example, an EgtD nucleic acid sequence encoding an amino acid sequence that is at least 96% identical to the amino acid sequence provided in SEQ ID NO: 6. In another aspect, a particularly suitable EgtD can be, for example, an EgtD nucleic acid sequence encoding an amino acid sequence that is at least 97% identical to the amino acid sequence provided in SEQ ID NO: 6. In another aspect, a particularly suitable EgtD can be, for example, an EgtD nucleic acid sequence encoding an amino acid sequence that is at least 98% identical to the amino acid sequence provided in SEQ ID NO: 6. In another aspect, a particularly suitable EgtD can be, for example, an EgtD nucleic acid sequence encoding an amino acid sequence that is at least 99% identical to the amino acid sequence provided in SEQ ID NO: 6. In another aspect, a particularly suitable EgtD can be, for example, an EgtD nucleic acid sequence encoding an amino acid sequence that is 100% identical to the amino acid sequence provided in SEQ ID NO: 6.

EgtE (or pyridoxal phosphate-dependent protein EgtE) is believed to catalyze the removal of pyruvate, ammonia, and oxygen to produce ergothioneine.

A suitable EgtE can be, for example, a mycobacterium EgtE. A particularly suitable EgtE can be, for example, an EgtE nucleic acid sequence encoding an amino acid sequence that is at least 95% identical to the amino acid sequence provided in SEQ ID NO: 8. In another aspect, a particularly suitable EgtE can be, for example, an EgtE nucleic acid sequence encoding an amino acid sequence that is at least 96% identical to the amino acid sequence provided in SEQ ID NO: 8. In another aspect, a particularly suitable EgtE can be, for example, an EgtE nucleic acid sequence encoding an amino acid sequence that is at least 97% identical to the amino acid sequence provided in SEQ ID NO: 8. In another aspect, a particularly suitable EgtE can be, for example, an EgtE nucleic acid sequence encoding an amino acid sequence that is at least 98% identical to the amino acid sequence provided in SEQ ID NO: 8. In another aspect, a particularly suitable EgtE can be, for example, an EgtE nucleic acid sequence encoding an amino acid sequence that is at least 99% identical to the amino acid sequence provided in SEQ ID NO: 8. In another aspect, a particularly suitable EgtE can be, for example, an EgtE nucleic acid sequence encoding an amino acid sequence that is 100% identical to the amino acid sequence provided in SEQ ID NO: 8.

Suitable host cells may be, for example, bacterial cells and yeast cells. Suitable bacterial cells may be, for example, Escherichia coli.

Suitable yeast cells may be, for example, Saccharomyces and Pichia . Particularly suitable yeast may be, for example, Saccharomyces cerevisiae. Particularly suitable Pichia may be, for example, Pichia pastoris.

The nucleic acid sequences encoding EgtB, EgtC, EgtD and EgtE are cloned into expression vectors under the control of promoters known to those skilled in the art. Suitable promoters can be, for example, constitutively active promoters and inducible promoters known to those skilled in the art. Suitable inducible promoters are well known to those skilled in the art and can be, for example, chemical inducers, nutrients are added, nutrient depletion and physical or physiological chemical factors change, such as, for example, pH changes and temperature induction. Suitable chemical inducers can be, for example, isopropyl β-D-1-thiogalactopyranoside (IPTG) inducible promoters and antibiotic inducible promoters known to those skilled in the art. Particularly suitable chemical inducible promoters can be, for example, isopropyl β-D-1-thiogalactopyranoside (IPTG) inducible promoters known to those skilled in the art. Other suitable inducible promoters can be, for example, temperature inducible promoters known to those skilled in the art, such as, for example, pL and pR lambda phage promoters.

Particularly suitable expression vectors are shown in Figures 1A and 1 B. Other suitable expression vectors are known to the person skilled in the art and may be, for example, pET vectors, pCDF vectors, pRSF vectors and Duet vectors.

Method for producing ergothioneine

In another aspect, the present disclosure relates to a method for producing ergothioneine. The method includes cultivating a host cell, wherein the host cell is transformed with a nucleic acid sequence encoding EgtB, a nucleic acid sequence encoding EgtC, a nucleic acid sequence encoding EgtD, and a nucleic acid sequence encoding EgtE; inducing the host cell to express the nucleic acid sequence encoding EgtB, the nucleic acid sequence encoding EgtC, the nucleic acid sequence encoding EgtD, and the nucleic acid sequence encoding EgtE; and collecting ergothioneine.

The method may further comprise adding a substrate to the culture. A suitable amount of substrate may be, for example, from about 1 mM to about 20 mM. Particularly suitable substrates may be, for example, histidine, methionine, cysteine, γ-glutamylcysteine, and combinations thereof.

In another embodiment, the method can include adding a cofactor to the culture. A suitable amount of the cofactor can be, for example, about 0.05 mM to about 0.4 mM. A particularly suitable cofactor can be, for example, iron (II) (Fe ⁺⁺ ).

Suitable host cells may be, for example, bacterial cells and yeast cells. Suitable bacterial cells may be, for example, Escherichia coli.

Suitable yeast cells may be, for example, Saccharomyces and Pichia . Particularly suitable yeast may be, for example, Saccharomyces cerevisiae. Particularly suitable Pichia may be, for example, Pichia pastoris.

In one embodiment, the host cell can produce about 10 mg to about 30 mg of ergothioneine per liter.

The present disclosure will be more fully understood upon consideration of the following non-limiting examples.

Example

Example 1

In this example, the nucleic acid sequences of EgtB, EgtC, EgtD and EgtE were cloned into E. coli.

Specifically, the following sequences were obtained from GenBank (Accession No. NC 008596): Egt B: MSMEG_6249 (SEQ ID NO: 1); Egt C: MSMEG_6248 (SEQ ID NO: 3); Egt D: MSMEG_6247 (SEQ ID NO: 5); and Egt E: MSMEG_6246 (SEQ ID NO: 7). The genes were introduced into a vector under the control of an IPTG-inducible promoter.

To construct the ET pathway in E. coli, the primer pairs outlined in Table 1 were used to PCR amplify the EgtB , C , D , and E nucleic acid sequences from the genomic sequence of Mycobacterium smegmatis. All 5'-primers used for cloning included EcoRI and BglI restriction sites and a ribosome binding site (RBS), and all 3'-primers included BamHI-XhoI sites. The EgtD and EgtB sequences were cloned into the pConB7A vector (Figure 1A), and the EgtC and EgtE sequences were cloned into the pConA5K vector (Figure 1B). No sequence errors were identified in the cloned sequences. Empty vectors were prepared in the same manner. The constructs were then co-transformed into the E. coli strain BL21 (DE3).

Table 1. Primers used for gene cloning.

Example 2

In this example, ergothioneine was produced in an engineered microbial system.

Specifically, E. coli was transformed with the pConB7A vector and the pConA5K vector encoding EgtB, EgtC, EgtD, and EgtE as described in Example 1. To co-express the four genes ( EgtB , C , D , E ) in the E. coli system, transformants were grown in LB medium containing 100 mg/L ampicillin and 50 mg/L kanamycin at 37°C until an OD ₆₀₀ of ~0.6 was reached. Expression was induced by adding 0.2-0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG), and the cultures were further grown at 30°C or 37°C for 16-24 hours. Cells were harvested by centrifugation, and the supernatant and cell pellet were collected separately. The supernatant was centrifuged at 16,000 x g for 5 minutes for HPLC analysis. The pellet was resuspended in 1 ml of 50% methanol and sonicated for 1 minute (3 x 20 seconds). After centrifugation for 5 minutes at 16,000 × g, 5 μ l samples were analyzed by HPLC, as described below. The Escherichia coli transformed with the empty vector was treated in the same manner and analyzed by HPLC. The sample obtained from the Escherichia coli containing EgtB, EgtC, EgtD, EgtE gene induced by IPTG was mixed with 20 mg/L ergothioneine and analyzed by HPLC.

Samples were analyzed using a Dionex UPLC Ultimate 3000 (Sunnyvale, CA). Compounds were separated on an Atlantis HILIC silica column (3.0 μm particle size, diameter × length = 2.1 × 100 mm; Waters) with detection at 264 nm. The mobile phase consisted of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). The gradient program was 95% B in 1 minute, 40% B in 8 minutes, 95% B in 8.1 minutes, and terminated at 11 minutes. The flow rate was 0.6 ml/min, and the injection volume was 5 μl.

As shown in Figure 2, ET surprisingly accumulates only in the E. coli strain containing EgtB, EgtC, EgtD, EgtE sequences induced by IPTG ("SI"), successfully demonstrating the biosynthesis of ET in engineered E. coli. In contrast, the E. coli containing the empty vector induced by IPTG does not produce any ET ("EI"). In the ET-spiked samples, the ET peak from the E. coli strain containing EgtB, EgtC, EgtD and EgtE induced by IPTG overlaps with the added ergothioneine and demonstrates increased levels to account for the added ET ("Ck+").

Fig. 3 A and 3B illustrate the HPLC analysis of 100 mg/L thioneine standard substances.As shown in Figure 4 A, the retention time of ET from the coliform bacteria containing EgtB, EgtC, EgtD and EgtE overlaps with the retention time of thioneine standard substances (referring to Fig. 3 A).Except retention time, the UV spectrum at ET peak (referring to Fig. 4 B) also mates with thioneine standard substances (referring to Fig. 3 B).These results show, the peak from the engineered coliform bacteria expressing EgtB, EgtC, EgtD and EgtE corresponds to ET.

Example 3

In this example, the time course of ergothioneine production in an engineered microbial system was performed.

Specifically, E. coli was transformed with a vector containing the genes for EgtB, EgtC, EgtD, and EgtE as described in Example 1. Control E. coli cells included cells with an empty vector (no Egt gene) and an uninduced strain containing an Egt vector but not induced. The cells were grown at 30°C or 37°C as described in Example 2. Samples were taken at different time points from 0 hour to 20 hours. After ultrasonic treatment for 1 minute (3 x 20 seconds), the samples were centrifuged at 16,000 x g for 5 minutes, and 5 μl samples were analyzed by HPLC as discussed in Example 2.

As shown in Figure 5, HPLC analysis revealed that cells began to produce ET by about 1 hour after IPTG induction. The fastest increase in ET production was observed from about 3 hours to about 10 hours after IPTG induction. ET production slowed after 10 hours, but continued to be produced until at least 20 hours. At the same time, no ET was detected in the empty vector control throughout the entire time course. These results further indicate that ET is produced exclusively in E. coli strains engineered to express EgtB, EgtC, EgtD, and EgtE.

Example 4

In this example, feeding experiments were conducted to determine the effects on ergothioneine production in an engineered microbial system.

Without being bound by theory, it is believed that ET is synthesized from amino acids such as histidine (His), methionine (Met), and cysteine (Cys). The imidazole ring of ET is donated by His, which is then methylated to produce histidine betaine. Met is a building block for S-adenosylmethionine (SAM), which acts as a methyl donor. The sulfur atom is introduced from Cys.

In order to measure the impact of the thioneine production in the engineered intestinal bacteria, by substratum by several substrates and cofactors such as Fe ⁺⁺ feed to genetically modified Escherichia coli cells.After induction 3 hours, by 2 mM His, 4 mM Met, 4mM Cys and 0.2 mM Fe ⁺⁺ add to substratum, and cell is further cultivated 16 hours, 24 hours and 42 hours.With identical substrate or cofactor feeding control Escherichia coli culture (carrying empty vector).By HPLC analytical sample, as described in Example 2.

As shown in Figure 6, the feeding experiment revealed that the addition of Cys increased ET production by 17.3-44.4% over the three time points. This result indicates that Cys and its derivative γ-glutamylcysteine play an important role in the biosynthesis of ET. The control culture did not produce any ET.

Example 5

In this example, ergothioneine will be produced in an engineered Saccharomyces cerevisiae system.

To produce ET in Saccharomyces cerevisiae, the EgtB, C, D, and E genes were cloned into commercially available pESC vectors, such as pESC-His and pESC-Leu (Agilent Technologies). These vectors contain the GAL1 and GAL10 yeast promoters in opposite orientations, which allow the two genes to be introduced into yeast strains under the control of two repressible promoters. The two constructs were then co-transformed into Saccharomyces cerevisiae. To co-express the four genes (EgtB, C, D, and E) in yeast, the transformants were grown in a culture medium containing the amino acids histidine and leucine until an OD ₆₀₀ of ~0.4 was reached. Expression was induced by adding 2% galactose, and the cultures were further grown at 28°C or 30°C for 24-48 hours. The cells were harvested by centrifugation, and the supernatant and cell pellet were collected respectively. The supernatant was centrifuged at 12,000 x g for 5 minutes and analyzed by HPLC. The pellet was resuspended in 1 ml of 50% methanol and sonicated for 1 minute (3 x 20 seconds). After centrifugation at 12,000 x g for 5 minutes, 5 μl of the sample was injected into the HPLC. Yeast carrying the empty vector was transformed and analyzed in the same manner. The above construct can be finally integrated into the yeast genome and expressed under the control of a constitutive promoter such as the GPD promoter or the GAP promoter.

Example 6

In this example, ergothioneine was produced in an engineered Pichia pastoris system.

To produce ET in Pichia pastoris, the EgtB, C, D, and E genes were cloned into the commercially available pPICZ or pGAPZ vectors (Invitrogen, Life Technologies). The pPICZ vector contains the methanol-regulated AOX1 promoter, while the pGAPZ vector has a constitutive glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter. Co-expression of the four genes (EgtB, C, D, and E) in the pPICZ vector is induced by 0.5-5% methanol. ET production was analyzed by HPLC using the same method described above.

In view of the foregoing, it will be seen that the several advantages of the present disclosure are achieved and other advantageous results are obtained. As various changes can be made in the above-described methods and systems without departing from the scope of the present disclosure, it is intended that all matter contained in the above description and shown in the accompanying drawings shall be interpreted as illustrative and not in a limiting sense.

When introducing elements of the present disclosure or various versions, embodiments, or aspects thereof, the articles "a," "an," "the," and "said" mean that there are one or more of the elements. The terms "comprising," "including," and "having" are intended to be inclusive and mean that there may be additional elements other than the listed elements.

Sequence Listing

<110> Conagen Inc.

Han, Jixiang

Chen, Hui

Yu, Oliver

<120> Microbial ergothioneine biosynthesis

<130> C1497.70015

<140> Not yet specified

<141> At the same time

<150> PCT/US2015/027977

<151> 2015-04-28

<160> 16

<170> PatentIn Version 3.5

<210> 1

<211> 1287

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 1

atgatcgcac gcgagacact ggccgacgag ctggccctgg cccgcgaacg cacgttgcgg 60

ctcgtggagt tcgacgacgc ggaactgcat cgccagtaca acccgctgat gagcccgctc 120

gtgtgggacc tcgcgcacat cgggcagcag gaagaactgt ggctgctgcg cgacggcaac 180

cccgaccgcc ccggcatgct cgcacccgag gtggaccggc tttacgacgc gttcgagcac 240

tcacgcgcca gccgggtcaa cctcccgttg ctgccgcctt cggatgcgcg cgcctactgc 300

gcgacggtgc gggccaaggc gctcgacacc ctcgacacgc tgcccgagga cgatccgggc 360

ttccggttcg cgctggtgat cagccacgag aaccagcacg acgagaccat gctgcaggca 420

ctcaacctgc gcgagggccc acccctgctc gacaccggaa ttcccctgcc cgcgggcagg 480

ccaggcgtgg caggcacgtc ggtgctggtg ccgggcggcc cgttcgtgct cggggtcgac 540

gcgctgaccg aaccgcactc actggacaac gaacggcccg cccacgtcgt ggacatcccg 600

tcgttccgga tcggccgcgt gccggtcacc aacgccgaat ggcgcgagtt catcgacgac 660

ggtggctacg accaaccgcg ctggtggtcg ccacgcggct gggcgcaccg ccaggaggcg 720

ggcctggtgg ccccgcagtt ctggaacccc gacggcaccc gcacccggtt cgggcacatc 780

gaggagatcc cgggtgacga acccgtgcag cacgtgacgt tcttcgaagc cgaggcctac 840

gcggcgtggg ccggtgctcg gttgcccacc gagatcgaat gggagaaggc ctgcgcgtgg 900

gatccggtcg ccggtgctcg gcgccggttc ccctggggct cagcacaacc cagcgcggcg 960

ctggccaacc tcggcggtga cgcacgccgc ccggcgccgg tcggggccta cccggcgggg 1020

gcgtcggcct atggcgccga gcagatgctg ggcgacgtgt gggagtggac ctcctcgccg 1080

ctgcggccgt ggcccggttt cacgccgatg atctacgagc gctacagcac gccgttcttc 1140

gagggcacca catccggtga ctaccgcgtg ctgcgcggcg ggtcatgggc cgttgcaccg 1200

ggaatcctgc ggcccagctt ccgcaactgg gaccacccga tccggcggca gatattctcg 1260

ggtgtccgcc tggcctggga cgtctga 1287

<210> 2

<211> 428

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 2

Met Ile Ala Arg Glu Thr Leu Ala Asp Glu Leu Ala Leu Ala Arg Glu

1 5 10 15

Arg Thr Leu Arg Leu Val Glu Phe Asp Asp Ala Glu Leu His Arg Gln

20 25 30

Tyr Asn Pro Leu Met Ser Pro Leu Val Trp Asp Leu Ala His Ile Gly

35 40 45

Gln Gln Glu Glu Leu Trp Leu Leu Arg Asp Gly Asn Pro Asp Arg Pro

50 55 60

Gly Met Leu Ala Pro Glu Val Asp Arg Leu Tyr Asp Ala Phe Glu His

65 70 75 80

Ser Arg Ala Ser Arg Val Asn Leu Pro Leu Leu Pro Pro Ser Asp Ala

85 90 95

Arg Ala Tyr Cys Ala Thr Val Arg Ala Lys Ala Leu Asp Thr Leu Asp

100 105 110

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

115 120 125

His Glu Asn Gln His Asp Glu Thr Met Leu Gln Ala Leu Asn Leu Arg

130 135 140

Glu Gly Pro Pro Leu Leu Asp Thr Gly Ile Pro Leu Pro Ala Gly Arg

145 150 155 160

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

165 170 175

Leu Gly Val Asp Ala Leu Thr Glu Pro His Ser Leu Asp Asn Glu Arg

180 185 190

Pro Ala His Val Val Asp Ile Pro Ser Phe Arg Ile Gly Arg Val Pro

195 200 205

Val Thr Asn Ala Glu Trp Arg Glu Phe Ile Asp Asp Gly Gly Tyr Asp

210 215 220

Gln Pro Arg Trp Trp Ser Pro Arg Gly Trp Ala His Arg Gln Glu Ala

225 230 235 240

Gly Leu Val Ala Pro Gln Phe Trp Asn Pro Asp Gly Thr Arg Thr Arg

245 250 255

Phe Gly His Ile Glu Glu Ile Pro Gly Asp Glu Pro Val Gln His Val

260 265 270

Thr Phe Phe Glu Ala Glu Ala Tyr Ala Ala Trp Ala Gly Ala Arg Leu

275 280 285

Pro Thr Glu Ile Glu Trp Glu Lys Ala Cys Ala Trp Asp Pro Val Ala

290 295 300

Gly Ala Arg Arg Arg Phe Pro Trp Gly Ser Ala Gln Pro Ser Ala Ala

305 310 315 320

Leu Ala Asn Leu Gly Gly Asp Ala Arg Arg Pro Ala Pro Val Gly Ala

325 330 335

Tyr Pro Ala Gly Ala Ser Ala Tyr Gly Ala Glu Gln Met Leu Gly Asp

340 345 350

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

355 360 365

Pro Met Ile Tyr Glu Arg Tyr Ser Thr Pro Phe Phe Glu Gly Thr Thr

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425

<210> 3

<211> 684

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 3

atgtgccggc atgtggcgtg gctgggcgcg ccgcggtcgt tggccgacct ggtgctcgac 60

ccgccgcagg gactgctggt gcagtcctac gcaccgcgac gacagaagca cggtctgatg 120

aacgccgacg gttggggcgc agggtttttc gacgacgagg gagtggcccg ccgctggcgc 180

agcgacaaac cgctgtgggg tgatgcgtcg ttcgcgtcgg tggcacccgc actacgcagt 240

cgttgcgtgc tggccgcggt gcgctcggcc accatcggca tgcccatcga accgtcggcg 300

tcggcgccgt tcagcgacgg gcagtggctg ctgtcgcaca acggcctggt cgaccgcggg 360

gtgctcccgt tgaccggtgc cgccgagtcc acggtggaca gcgcgatcgt cgcggcgctc 420

atcttctccc gtggcctcga cgcgctcggc gccaccatcg ccgaggtcgg cgaactcgac 480

ccgaacgcgc ggttgaacat cctggccgcc aacggttccc ggctgctcgc caccacctgg 540

ggggacacgc tgtcggtcct gcaccgcccc gacggcgtcg tcctcgcgag cgaaccctac 600

gacgacgatc ccggctggtc ggacatcccg gaccggcacc tcgtcgacgt ccgcgacgcc 660

cacgtcgtcg tgacacccct gtga 684

<210> 4

<211> 227

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 4

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

1 5 10 15

Leu Val Leu Asp Pro Pro Gln Gly Leu Leu Val Gln Ser Tyr Ala Pro

20 25 30

Arg Arg Gln Lys His Gly Leu Met Asn Ala Asp Gly Trp Gly Ala Gly

35 40 45

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

50 55 60

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

65 70 75 80

Arg Cys Val Leu Ala Ala Val Arg Ser Ala Thr Ile Gly Met Pro Ile

85 90 95

Glu Pro Ser Ala Ser Ala Pro Phe Ser Asp Gly Gln Trp Leu Leu Ser

100 105 110

His Asn Gly Leu Val Asp Arg Gly Val Leu Pro Leu Thr Gly Ala Ala

115 120 125

Glu Ser Thr Val Asp Ser Ala Ile Val Ala Ala Leu Ile Phe Ser Arg

130 135 140

Gly Leu Asp Ala Leu Gly Ala Thr Ile Ala Glu Val Gly Glu Leu Asp

145 150 155 160

Pro Asn Ala Arg Leu Asn Ile Leu Ala Ala Asn Gly Ser Arg Leu Leu

165 170 175

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

180 185 190

Val Val Leu Ala Ser Glu Pro Tyr Asp Asp Asp Pro Gly Trp Ser Asp

195 200 205

Ile Pro Asp Arg His Leu Val Asp Val Arg Asp Ala His Val Val Val

210 215 220

Thr Pro Leu

225

<210> 5

<211> 966

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 5

atgacgctct cactggccaa ctacctggca gccgactcgg ccgccgaagc actgcgccgt 60

gacgtccgcg cgggcctcac cgcggcaccg aagagtctgc cgcccaagtg gttctacgac 120

gccgtcggca gtgatctgtt cgaccagatc acccggctcc ccgagtatta ccccacccgc 180

accgaggcgc agatcctgcg gacccggtcg gcggagatca tcgcggccgc gggtgccgac 240

accctggtgg aactgggcag tggtacgtcg gagaaaaccc gcatgctgct cgacgccatg 300

cgcgacgccg agttgctgcg ccgcttcatc ccgttcgacg tcgacgcggg cgtgctgcgc 360

tcggccgggg cggcaatcgg cgcggagtac cccggtatcg agatcgacgc ggtatgtggc 420

gatttcgagg aacatctggg caagatcccg catgtcggac ggcggctcgt ggtgttcctg 480

gggtcgacca tcggcaacct gacacccgcg ccccgcgcgg agttcctcag tactctcgcg 540

gacacgctgc agccgggcga cagcctgctg ctgggcaccg atctggtgaa ggacaccggc 600

cggttggtgc gcgcgtacga cgacgcggcc ggcgtcaccg cggcgttcaa ccgcaacgtg 660

ctggccgtgg tgaaccgcga actgtccgcc gatttcgacc tcgacgcgtt cgagcatgtc 720

gcgaagtgga actccgacga ggaacgcatc gagatgtggt tgcgtgcccg caccgcacag 780

catgtccgcg tcgcggcact ggacctggag gtcgacttcg ccgcgggtga ggagatgctc 840

accgaggtgt cctgcaagtt ccgtcccgag aacgtcgtcg ccgagctggc ggaagccggt 900

ctgcggcaga cgcattggtg gaccgatccg gccggggatt tcgggttgtc gctggcggtg 960

cggtga 966

<210> 6

<211> 321

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 6

Met Thr Leu Ser Leu Ala Asn Tyr Leu Ala Ala Asp Ser Ala Ala Glu

1 5 10 15

Ala Leu Arg Arg Asp Val Arg Ala Gly Leu Thr Ala Ala Pro Lys Ser

20 25 30

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

35 40 45

Gln Ile Thr Arg Leu Pro Glu Tyr Tyr Pro Thr Arg Thr Glu Ala Gln

50 55 60

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

65 70 75 80

Thr Leu Val Glu Leu Gly Ser Gly Thr Ser Glu Lys Thr Arg Met Leu

85 90 95

Leu Asp Ala Met Arg Asp Ala Glu Leu Leu Arg Arg Phe Ile Pro Phe

100 105 110

Asp Val Asp Ala Gly Val Leu Arg Ser Ala Gly Ala Ala Ile Gly Ala

115 120 125

Glu Tyr Pro Gly Ile Glu Ile Asp Ala Val Cys Gly Asp Phe Glu Glu

130 135 140

His Leu Gly Lys Ile Pro His Val Gly Arg Arg Leu Val Val Phe Leu

145 150 155 160

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

165 170 175

Ser Thr Leu Ala Asp Thr Leu Gln Pro Gly Asp Ser Leu Leu Leu Gly

180 185 190

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

195 200 205

Ala Ala Gly Val Thr Ala Ala Phe Asn Arg Asn Val Leu Ala Val Val

210 215 220

Asn Arg Glu Leu Ser Ala Asp Phe Asp Leu Asp Ala Phe Glu His Val

225 230 235 240

Ala Lys Trp Asn Ser Asp Glu Glu Arg Ile Glu Met Trp Leu Arg Ala

245 250 255

Arg Thr Ala Gln His Val Arg Val Ala Ala Leu Asp Leu Glu Val Asp

260 265 270

Phe Ala Ala Gly Glu Glu Met Leu Thr Glu Val Ser Cys Lys Phe Arg

275 280 285

Pro Glu Asn Val Val Ala Glu Leu Ala Glu Ala Gly Leu Arg Gln Thr

290 295 300

His Trp Trp Thr Asp Pro Ala Gly Asp Phe Gly Leu Ser Leu Ala Val

305 310 315 320

Arg

<210> 7

<211> 1113

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 7

atgctcgcgc agcagtggcg tgacgcccgt cccaaggttg ccgggttgca cctggacagc 60

ggggcatgtt cgcggcagag cttcgcggtg atcgacgcga ccaccgcaca cgcacgccac 120

gaggccgagg tgggtggtta tgtggcggcc gaggctgcga cgccggcgct cgacgccggg 180

cgggccgcgg tcgcgtcgct catcggtttt gcggcgtcgg acgtggtgta caccagcgga 240

tccaaccacg ccatcgacct gttgctgtcg agctggccgg ggaagcgcac gctggcctgc 300

ctgcccggcg agtacgggcc gaatctgtct gccatggcgg ccaacggttt ccaggtgcgt 360

gcgctaccgg tcgacgacga cgggcgggtg ctggtcgacg aggcgtcgca cgaactgtcg 420

gcccatcccg tcgcgctcgt acacctcacc gcattggcaa gccatcgcgg gatcgcgcaa 480

cccgcggcag aactcgtcga ggcctgccac aatgcgggga tccccgtggt gatcgacgcc 540

gcgcaggcgc tggggcatct ggactgcaat gtcggggccg acgcggtgta ctcatcgtcg 600

cgcaagtggc tcgccggccc gcgtggtgtc ggggtgctcg cggtgcggcc cgaactcgcc 660

gagcgtctgc aaccgcggat ccccccgtcc gactggccaa ttccgatgag cgtcttggag 720

aagctcgaac taggtgagca caacgcggcg gcgcgtgtgg gattctccgt cgcggttggt 780

gagcatctcg cagcagggcc cacggcggtg cgcgaacgac tcgccgaggt ggggcgtctc 840

tctcggcagg tgctggcaga ggtcgacggg tggcgcgtcg tcgaacccgt cgaccaaccc 900

accgcgatca ccacccttga gtccaccgat ggtgccgatc ccgcgtcggt gcgctcgtgg 960

ctgatcgcgg agcgtggcat cgtgaccacc gcgtgtgaac tcgcgcgggc accgttcgag 1020

atgcgcacgc cggtgctgcg aatctcgccg cacgtcgacg tgacggtcga cgaactggag 1080

cagttcgccg cagcgttgcg tgaggcgccc tga 1113

<210> 8

<211> 370

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic

<400> 8

Met Leu Ala Gln Gln Trp Arg Asp Ala Arg Pro Lys Val Ala Gly Leu

1 5 10 15

His Leu Asp Ser Gly Ala Cys Ser Arg Gln Ser Phe Ala Val Ile Asp

20 25 30

Ala Thr Thr Ala His Ala Arg His Glu Ala Glu Val Gly Gly Tyr Val

35 40 45

Ala Ala Glu Ala Ala Thr Pro Ala Leu Asp Ala Gly Arg Ala Ala Val

50 55 60

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

65 70 75 80

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

85 90 95

Thr Leu Ala Cys Leu Pro Gly Glu Tyr Gly Pro Asn Leu Ser Ala Met

100 105 110

Ala Ala Asn Gly Phe Gln Val Arg Ala Leu Pro Val Asp Asp Asp Gly

115 120 125

Arg Val Leu Val Asp Glu Ala Ser His Glu Leu Ser Ala His Pro Val

130 135 140

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

145 150 155 160

Pro Ala Ala Glu Leu Val Glu Ala Cys His Asn Ala Gly Ile Pro Val

165 170 175

Val Ile Asp Ala Ala Gln Ala Leu Gly His Leu Asp Cys Asn Val Gly

180 185 190

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

195 200 205

Gly Val Gly Val Leu Ala Val Arg Pro Glu Leu Ala Glu Arg Leu Gln

210 215 220

Pro Arg Ile Pro Pro Ser Asp Trp Pro Ile Pro Met Ser Val Leu Glu

225 230 235 240

Lys Leu Glu Leu Gly Glu His Asn Ala Ala Ala Arg Val Gly Phe Ser

245 250 255

Val Ala Val Gly Glu His Leu Ala Ala Gly Pro Thr Ala Val Arg Glu

260 265 270

Arg Leu Ala Glu Val Gly Arg Leu Ser Arg Gln Val Leu Ala Glu Val

275 280 285

Asp Gly Trp Arg Val Val Glu Pro Val Asp Gln Pro Thr Ala Ile Thr

290 295 300

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

305 310 315 320

Leu Ile Ala Glu Arg Gly Ile Val Thr Thr Ala Cys Glu Leu Ala Arg

325 330 335

Ala Pro Phe Glu Met Arg Thr Pro Val Leu Arg Ile Ser Pro His Val

340 345 350

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

355 360 365

Ala Pro

370

<210> 9

<211> 49

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 9

agaattcaaa agatctaaag gaggccatcc atgatcgcac gcgagacac 49

<210> 10

<211> 53

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 10

actcgagttt ggatcctcag acgtcccagg ccaggcggac acccgagaat atc 53

<210> 11

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 11

agaattcaaa agatctaaag gaggccatcc atgtgccggc atgtggcgtg 50

<210> 12

<211> 34

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 12

actcgagttt ggatcctcac aggggtgtca cgac 34

<210> 13

<211> 51

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 13

agaattcaaa agatctaaag gaggccatcc atgacgctct cactggccaa c 51

<210> 14

<211> 35

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 14

actcgagttt ggatcctcac cgcaccgcca gcgac 35

<210> 15

<211> 47

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 15

agaattcaaa agatctaaag gaggccatcc atgctcgcgc agcagtg 47

<210> 16

<211> 35

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic

<400> 16

actcgagttt ggatcctcag ggcgcctcac gcaac 35

Claims (9)

1. An engineered host cell for the production of ergothionein, wherein the engineered host cell is transformed with a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:2, the amino acid sequence of SEQ ID NO:4, the amino acid sequence of SEQ ID NO:6, and the amino acid sequence of SEQ ID NO:8, wherein the host cell is * Escherichia coli *, * Saccharomyces cerevisiae *, or * Pichia pastoris *. 2. The engineered host cell of claim 1, wherein the host cell is an Escherichia coli cell. 3. The engineered host cell of claim 1, wherein the host cell is selected from Saccharomyces cerevisiae cells and Pichia pastoris cells. 4. A method for producing ergothioneine, the method comprising: Culture the host cells according to any one of claims 1-3; Inducing the host cells to express nucleic acid sequences encoding EgtB, EgtC, EgtD, and EgtE; and Collect ergothioneine. 5. The method of claim 4, wherein during the culture of host cells, a substrate selected from histidine, methionine, cysteine, γ-glutamylcysteine, and combinations thereof is added to the culture. 6. The method of claim 4, wherein iron (II) is added to the culture during the culture of host cells. 7. The method of claim 4, wherein the host cell is an Escherichia coli cell. 8. The method of claim 4, wherein the host cell is selected from Saccharomyces cerevisiae cells and Pichia pastoris cells. 9. An expression vector for the production of ergothioneine, comprising a nucleic acid sequence encoding an amino acid sequence selected from EgtB, EgtC, EgtD, and EgtE, wherein the nucleic acid sequence encodes an amino acid sequence selected from the following amino acid sequences: the amino acid sequence of SEQ ID NO:2; the amino acid sequence of SEQ ID NO:4; the amino acid sequence of SEQ ID NO:6; and the amino acid sequence of SEQ ID NO:8.