KR20220110412A - Novel protein variant and a method for producing L-lysine using the same - Google Patents

Abstract

The present application relates to a novel protein variant, a Corynebacterium glutamicum strain containing the variant, and a method for producing L-lysine using the strain. According to the present invention, it is possible to produce L-lysine in high yield compared to microorganisms having conventional unmodified polypeptides.

Description

Novel protein variant and L-lysine production method using the same {Novel protein variant and a method for producing L-lysine using the same}

The present application relates to a novel protein variant, a Corynebacterium glutamicum strain comprising the variant, and a method for producing L- lysine using the strain.

In order to produce L-amino acids and other useful substances, various studies are being conducted for the development of high-efficiency production microorganisms and fermentation process technology. For example, a target substance-specific approach such as increasing the expression of a gene encoding an enzyme involved in L-lysine biosynthesis or removing a gene unnecessary for biosynthesis is mainly used (WO2008-082181 A1).

However, as the demand for L-lysine increases, there is still a need for research to increase the effective production capacity of L-lysine.

One object of the present application is to provide a protein variant selected from the group consisting of the following (1) to (14):

(1) an endogenous membrane transport protein variant consisting of the amino acid sequence set forth in SEQ ID NO: 1 in which phenylalanine, an amino acid corresponding to position 220 of SEQ ID NO: 3, is substituted with cysteine;

(2) DNA polymerase III gamma and tau subunit variants consisting of the amino acid sequence set forth in SEQ ID NO: 5 in which proline, an amino acid corresponding to position 43 of SEQ ID NO: 7, is substituted with leucine;

(3) a protein variant consisting of the amino acid sequence shown in SEQ ID NO: 9, in which cysteine, an amino acid corresponding to position 33 of SEQ ID NO: 11, is substituted with serine;

(4) a transcriptional anti-termination protein variant consisting of the amino acid sequence set forth in SEQ ID NO: 13 in which asparagine, an amino acid corresponding to position 210 of SEQ ID NO: 15, is substituted with aspartic acid;

(5) an ABC transporter ATP-binding protein variant consisting of the amino acid sequence shown in SEQ ID NO: 17 in which valine, an amino acid corresponding to position 199 of SEQ ID NO: 19, is substituted with methionine;

(6) a malate dehydrogenase variant consisting of the amino acid sequence set forth in SEQ ID NO: 21 in which threonine, an amino acid corresponding to position 272 of SEQ ID NO: 23, is substituted with isoleucine;

(7) a primosome assembly protein variant consisting of the amino acid sequence shown in SEQ ID NO: 25, in which arginine, an amino acid corresponding to position 304 of SEQ ID NO: 27, is substituted with glutamine;

(8) a type II citrate synthase variant consisting of the amino acid sequence shown in SEQ ID NO: 29, in which alanine, an amino acid corresponding to position 169 of SEQ ID NO: 31, is substituted with valine;

(9) a membrane protein variant consisting of the amino acid sequence set forth in SEQ ID NO: 33 in which glutamic acid, an amino acid corresponding to position 294 of SEQ ID NO: 35, is substituted with lysine;

(10) a mycothione reductase variant consisting of the amino acid sequence set forth in SEQ ID NO: 37 in which proline, which is the amino acid corresponding to position 358 of SEQ ID NO: 39, is substituted with serine;

(11) a Co/Zn/Cd efflux system component variant consisting of the amino acid sequence set forth in SEQ ID NO: 41 in which glycine, an amino acid corresponding to position 130 of SEQ ID NO: 43, is substituted with aspartic acid;

(12) a DAHP synthase variant consisting of the amino acid sequence set forth in SEQ ID NO: 45 in which glycine, an amino acid corresponding to position 382 of SEQ ID NO: 47, is substituted with cysteine;

(13) an N-succinyldiaminopimelate aminotransferase variant consisting of the amino acid sequence set forth in SEQ ID NO: 49, in which proline, an amino acid corresponding to position 209 of SEQ ID NO: 51, is substituted with leucine; and

(14) A helicase variant consisting of the amino acid sequence shown in SEQ ID NO: 53, wherein tyrosine, which is an amino acid corresponding to position 592 of SEQ ID NO: 55, is substituted with phenylalanine.

Another object of the present application is to provide a polynucleotide encoding the variant of the present application.

Another object of the present application is to include a polynucleotide encoding the protein variants of (a) and (b) or the variants (protein variants of (a) and/or (b) below), and L-lysine It is to provide a strain with a production capacity, Corynebacterium glutamicum ( Corynebacterium glutamicum ):

(a) a helicase variant consisting of the amino acid sequence set forth in SEQ ID NO: 53 in which tyrosine, an amino acid corresponding to position 592 of SEQ ID NO: 55, is substituted with phenylalanine, and

(b) at least one protein variant selected from the group consisting of the following (1) to (13):

(1) an endogenous membrane transport protein variant consisting of the amino acid sequence set forth in SEQ ID NO: 1 in which phenylalanine, an amino acid corresponding to position 220 of SEQ ID NO: 3, is substituted with cysteine;

(2) DNA polymerase III gamma and tau subunit variants consisting of the amino acid sequence set forth in SEQ ID NO: 5 in which proline, an amino acid corresponding to position 43 of SEQ ID NO: 7, is substituted with leucine;

(3) a protein variant consisting of the amino acid sequence shown in SEQ ID NO: 9, in which cysteine, an amino acid corresponding to position 33 of SEQ ID NO: 11, is substituted with serine;

(4) a transcriptional anti-termination protein variant consisting of the amino acid sequence set forth in SEQ ID NO: 13 in which asparagine, an amino acid corresponding to position 210 of SEQ ID NO: 15, is substituted with aspartic acid;

(5) an ABC transporter ATP-binding protein variant consisting of the amino acid sequence shown in SEQ ID NO: 17 in which valine, an amino acid corresponding to position 199 of SEQ ID NO: 19, is substituted with methionine;

(6) a malate dehydrogenase variant consisting of the amino acid sequence set forth in SEQ ID NO: 21 in which threonine, an amino acid corresponding to position 272 of SEQ ID NO: 23, is substituted with isoleucine;

(7) a primosome assembly protein variant consisting of the amino acid sequence shown in SEQ ID NO: 25, in which arginine, an amino acid corresponding to position 304 of SEQ ID NO: 27, is substituted with glutamine;

(8) a type II citrate synthase variant consisting of the amino acid sequence shown in SEQ ID NO: 29, in which alanine, an amino acid corresponding to position 169 of SEQ ID NO: 31, is substituted with valine;

(9) a membrane protein variant consisting of the amino acid sequence set forth in SEQ ID NO: 33 in which glutamic acid, an amino acid corresponding to position 294 of SEQ ID NO: 35, is substituted with lysine;

(10) a mycothione reductase variant consisting of the amino acid sequence set forth in SEQ ID NO: 37 in which proline, which is the amino acid corresponding to position 358 of SEQ ID NO: 39, is substituted with serine;

(11) a Co/Zn/Cd efflux system component variant consisting of the amino acid sequence set forth in SEQ ID NO: 41 in which glycine, an amino acid corresponding to position 130 of SEQ ID NO: 43, is substituted with aspartic acid;

(12) a DAHP synthase variant consisting of the amino acid sequence set forth in SEQ ID NO: 45 in which glycine, an amino acid corresponding to position 382 of SEQ ID NO: 47, is substituted with cysteine; and

(13) An N-succinyldiaminopimelate aminotransferase mutant consisting of the amino acid sequence shown in SEQ ID NO: 49, wherein proline, which is the amino acid corresponding to position 209 of SEQ ID NO: 51, is substituted with leucine.

Another object of the present application is to provide a method for producing L-lysine, comprising culturing the Corynebacterium glutamicum strain of the present application in a medium.

A detailed description of this is as follows. Meanwhile, each description and embodiment disclosed in the present application may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in this application fall within the scope of this application. In addition, it cannot be seen that the scope of the present application is limited by the specific descriptions described below. In addition, a number of papers and patent documents are referenced throughout this specification and their citations are indicated. The disclosure contents of the cited papers and patent documents are incorporated herein by reference in their entirety to more clearly describe the level of the technical field to which the present invention pertains and the content of the present invention.

One aspect of the present application is to provide a protein variant selected from the group consisting of the following (1) to (14):

(1) an endogenous membrane transport protein variant consisting of the amino acid sequence set forth in SEQ ID NO: 1 in which phenylalanine, an amino acid corresponding to position 220 of SEQ ID NO: 3, is substituted with cysteine;

(2) DNA polymerase III gamma and tau subunit variants consisting of the amino acid sequence set forth in SEQ ID NO: 5 in which proline, an amino acid corresponding to position 43 of SEQ ID NO: 7, is substituted with leucine;

(3) a protein variant consisting of the amino acid sequence shown in SEQ ID NO: 9, in which cysteine, an amino acid corresponding to position 33 of SEQ ID NO: 11, is substituted with serine;

(4) a transcriptional anti-termination protein variant consisting of the amino acid sequence set forth in SEQ ID NO: 13 in which asparagine, an amino acid corresponding to position 210 of SEQ ID NO: 15, is substituted with aspartic acid;

(5) an ABC transporter ATP-binding protein variant consisting of the amino acid sequence shown in SEQ ID NO: 17 in which valine, an amino acid corresponding to position 199 of SEQ ID NO: 19, is substituted with methionine;

(6) a malate dehydrogenase variant consisting of the amino acid sequence set forth in SEQ ID NO: 21 in which threonine, an amino acid corresponding to position 272 of SEQ ID NO: 23, is substituted with isoleucine;

(7) a primosome assembly protein variant consisting of the amino acid sequence shown in SEQ ID NO: 25, in which arginine, an amino acid corresponding to position 304 of SEQ ID NO: 27, is substituted with glutamine;

(8) a type II citrate synthase variant consisting of the amino acid sequence shown in SEQ ID NO: 29, in which alanine, an amino acid corresponding to position 169 of SEQ ID NO: 31, is substituted with valine;

(9) a membrane protein variant consisting of the amino acid sequence set forth in SEQ ID NO: 33 in which glutamic acid, an amino acid corresponding to position 294 of SEQ ID NO: 35, is substituted with lysine;

(10) a mycothione reductase variant consisting of the amino acid sequence set forth in SEQ ID NO: 37 in which proline, which is the amino acid corresponding to position 358 of SEQ ID NO: 39, is substituted with serine;

(11) a Co/Zn/Cd efflux system component variant consisting of the amino acid sequence set forth in SEQ ID NO: 41 in which glycine, an amino acid corresponding to position 130 of SEQ ID NO: 43, is substituted with aspartic acid;

(12) a DAHP synthase variant consisting of the amino acid sequence set forth in SEQ ID NO: 45 in which glycine, an amino acid corresponding to position 382 of SEQ ID NO: 47, is substituted with cysteine;

(13) an N-succinyldiaminopimelate aminotransferase variant consisting of the amino acid sequence set forth in SEQ ID NO: 49, in which proline, an amino acid corresponding to position 209 of SEQ ID NO: 51, is substituted with leucine; and

(14) A helicase variant consisting of the amino acid sequence shown in SEQ ID NO: 53, wherein tyrosine, which is an amino acid corresponding to position 592 of SEQ ID NO: 55, is substituted with phenylalanine.

The variant of the present application has or comprises the amino acid sequence set forth in SEQ ID NO: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, or 53 selected from the group consisting of Or, it may consist essentially of the amino acid sequence.

In addition, the variants of the present application include SEQ ID NOs: 3, 7, Based on the amino acid sequence of 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, or 55, respectively, 220, 43, 33, 210, 199, 272, 304 The amino acids corresponding to positions 169, 294, 358, 130, 382, 209, or 592 are cysteine, leucine, serine, aspartic acid, methionine, isoleucine, glutamine, valine, lysine, serine, aspartic acid, cysteine, leucine, or phenylalanine, and is selected from the group consisting of SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, and 53 having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% homology or identity to a disclosed amino acid sequence; It may comprise an amino acid sequence. Each amino acid sequence of the variants of the present application, positions, and correspondences to amino acid types corresponding to positions are shown in Table 1 below. In addition, as long as it is an amino acid sequence having such homology or identity and exhibiting efficacy corresponding to the variant of the present application, variants having an amino acid sequence in which some sequences are deleted, modified, substituted, conservatively substituted or added are also included within the scope of the present application. is self-evident

amino acid sequence The amino acid sequence as a reference for counting positions location type of amino acid One SEQ ID NO: 1 SEQ ID NO: 3 220 cysteine 2 SEQ ID NO: 5 SEQ ID NO: 7 43 leucine 3 SEQ ID NO: 9 SEQ ID NO: 11 33 serine 4 SEQ ID NO: 13 SEQ ID NO: 15 210 aspartic acid 5 SEQ ID NO: 17 SEQ ID NO: 19 199 methionine 6 SEQ ID NO: 21 SEQ ID NO: 23 272 isoleucine 7 SEQ ID NO: 25 SEQ ID NO: 27 304 glutamine 8 SEQ ID NO: 29 SEQ ID NO: 31 169 valine 9 SEQ ID NO: 33 SEQ ID NO: 35 294 lysine 10 SEQ ID NO: 37 SEQ ID NO: 39 358 serine 11 SEQ ID NO: 41 SEQ ID NO: 43 130 aspartic acid 12 SEQ ID NO: 45 SEQ ID NO: 47 382 cysteine 13 SEQ ID NO: 49 SEQ ID NO: 51 209 leucine 14 SEQ ID NO: 53 SEQ ID NO: 55 592 phenylalanine

For example, sequence additions or deletions, naturally occurring mutations, silent mutations or conservation within the N-terminus, C-terminus and/or within the amino acid sequence that do not alter the function of the variants of the present application It is a case of having an enemy substitution.

The term “conservative substitution” means substituting an amino acid for another amino acid having similar structural and/or chemical properties. Such amino acid substitutions may generally occur based on similarity in the polarity, charge, solubility, hydrophobicity, hydrophilicity and/or amphipathic nature of the residues. Typically, conservative substitutions may have little or no effect on the activity of the protein or polypeptide.

As used herein, the term "variant" means that one or more amino acids are conservatively substituted and/or modified so that they differ from the amino acid sequence before the mutation of the variant, but have functions or properties. refers to a polypeptide that is maintained. Such variants can generally be identified by modifying one or more amino acids in the amino acid sequence of the polypeptide and evaluating the properties of the modified polypeptide. That is, the ability of the variant may be increased, unchanged, or decreased compared to the polypeptide before the mutation. In addition, some variants may include variants in which one or more portions, such as an N-terminal leader sequence or a transmembrane domain, have been removed. Other variants may include variants in which a portion is removed from the N- and/or C-terminus of the mature protein. The term “variant” may be used interchangeably with terms such as mutant, modified, mutant polypeptide, mutated protein, mutant and mutant (in English, modified, modified polypeptide, modified protein, mutant, mutein, divergent, etc.) and, as long as it is a term used in a mutated sense, it is not limited thereto. For the purposes of the present application, the variant is at position 220, 43, or 33 of the amino acid sequence of SEQ ID NO: 3, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, or 55 , 210, 199, 272, 304, 169, 294, 358, 130, 382, 209, or 592 amino acids corresponding to positions phenylalanine, proline, cysteine, asparagine, valine, respectively threonine, arginine, alanine, glutamic acid, proline, glycine, glycine, proline, or tyrosine is cysteine, leucine, serine, aspartic acid, methionine, isoleucine, glutamine, valine, lysine, serine, aspartic acid, cysteine, leucine, or phenylalanine It may be a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, or 53, each substituted with . Table 2 below shows the correspondence between amino acid sequences, mutation positions, amino acids before substitution corresponding to the positions, and types of amino acids after substitution of the variants of the present application.

amino acid sequence The amino acid sequence as a reference for counting positions mutation position Type of amino acid before substitution Substituted Amino Acid Types One SEQ ID NO: 1 SEQ ID NO: 3 220 phenylalanine cysteine 2 SEQ ID NO: 5 SEQ ID NO: 7 43 proline leucine 3 SEQ ID NO: 9 SEQ ID NO: 11 33 cysteine serine 4 SEQ ID NO: 13 SEQ ID NO: 15 210 asparagine aspartic acid 5 SEQ ID NO: 17 SEQ ID NO: 19 199 valine methionine 6 SEQ ID NO: 21 SEQ ID NO: 23 272 threonine isoleucine 7 SEQ ID NO: 25 SEQ ID NO: 27 304 arginine glutamine 8 SEQ ID NO: 29 SEQ ID NO: 31 169 alanine valine 9 SEQ ID NO: 33 SEQ ID NO: 35 294 glutamic acid lysine 10 SEQ ID NO: 37 SEQ ID NO: 39 358 proline serine 11 SEQ ID NO: 41 SEQ ID NO: 43 130 glycine aspartic acid 12 SEQ ID NO: 45 SEQ ID NO: 47 382 glycine cysteine 13 SEQ ID NO: 49 SEQ ID NO: 51 209 proline leucine 14 SEQ ID NO: 53 SEQ ID NO: 55 592 tyrosine phenylalanine

In addition, variants may include deletions or additions of amino acids that have minimal effect on the properties and secondary structure of the polypeptide. For example, a signal (or leader) sequence involved in protein translocation may be conjugated to the N-terminus of the mutant, either co-translationally or post-translationally. The variants may also be conjugated with other sequences or linkers for identification, purification, or synthesis.

When culturing the Corynebacterium glutamicum strain, including the variant of the protein of the present application, it is possible to produce a high yield of L-lysine compared to microorganisms having an existing unmodified polypeptide.

As used herein, the term 'homology' or 'identity' refers to the degree of similarity between two given amino acid sequences or nucleotide sequences and may be expressed as a percentage. The terms homology and identity can often be used interchangeably.

Sequence homology or identity of a conserved polynucleotide or polypeptide is determined by standard alignment algorithms, with default gap penalties established by the program used may be used. Substantially homologous or identical sequences are generally capable of hybridizing with all or part of a sequence under moderate or high stringent conditions. It is apparent that hybridization also includes hybridization with polynucleotides containing common codons or codons taking codon degeneracy into account in the polynucleotide.

Whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can be determined, for example, by Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444, using a known computer algorithm such as the "FASTA" program. or, as performed in the Needleman program (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (version 5.0.0 or later) of the EMBOSS package, Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) can be used to determine (GCG program package (Devereux, J., et al, Nucleic Acids) Research 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL 215]: 403 (1990); Guide to Huge Computers, Martin J. Bishop , [ED.,] Academic Press, San Diego, 1994, and [CARILLO ETA/.] (1988) SIAM J Applied Math 48: 1073).For example, BLAST of the National Center for Biotechnology Information Database, or ClustalW can be used to determine homology, similarity or identity.

Homology, similarity or identity of polynucleotides or polypeptides is described, for example, in Smith and Waterman, Adv. Appl. Math (1981) 2:482, see, for example, Needleman et al. (1970), J Mol Biol. can be determined by comparing sequence information using a GAP computer program such as 48:443. In summary, a GAP program can be defined as the total number of symbols in the shorter of the two sequences divided by the number of similarly aligned symbols (ie, nucleotides or amino acids). Default parameters for the GAP program are: (1) a binary comparison matrix (containing values of 1 for identity and 0 for non-identity) and Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation , pp. 353-358 (1979), Gribskov et al (1986) Nucl. Acids Res. 14: weighted comparison matrix of 6745 (or EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap (or a gap open penalty of 10, a gap extension penalty of 0.5); and (3) no penalty for end gaps.

As an example of the present application, the variant of the present application is an integral membrane transport protein; DNA polymerase III subunits gamma and tau; a polypeptide comprising the amino acid sequence of SEQ ID NO: 11 and/or a protein encoded by the NCgl3069 gene; transcription antitermination protein; ABC transporter ATP-binding protein; malate dehydrogenase; primosome assembly protein; type II citrate synthase; membrane protein (TerC); mycothione reductase; Co/Zn/Cd efflux system component; DAHP synthase (3-deoxy-D-arabinoheptulosonate-7-phosphate synthase; DAHP synthase); N-succinyldiaminopimelate aminotransferase; And/or it may have the activity of helicase (Helicase).

As used herein, the term "integral membrane transport protein" refers to the formation of macromolecules, small molecules, and ions, such as other proteins across biological membranes. It is a membrane protein that is involved in migration and is attached to a biological membrane.Specifically, the integral membrane transport protein of the present application can be used interchangeably as an integral membrane transport protein.In the present application, the intrinsic membrane transport protein is known The sequence can be obtained from GenBank of NCBI, which is the base, for example, it can be GenBank Accession No. YP_227155.1, WP_011015489.1. Specifically, it can be a polypeptide having an intrinsic membrane transport protein activity encoded by the NCgl2816 gene. However, it is not limited thereto.

As used herein, the term "DNA polymerase Ⅲ subunits gamma and tau" is a major enzyme responsible for DNA replication, and the DNA polymerase Ⅲ tau subunit is a core (alpha ( alpha), epsilon, and theta (consisting of chains) bind to dimerization of the core, and DNA polymerase III gamma subunit is a clamp loader that obtains the beta subunit to the lagging strand. Specifically, the DNA polymerase Ⅲ gamma and tau subunit of the present application may be used interchangeably as DNA polymerase Ⅲ gamma and tau subunits, or DNA polymerase Ⅲ subunit gamma/tau. Polymerase III gamma and tau subunits can be sequenced from GenBank of NCBI, which is a known database, for example, GenBank Accession No. YP_224542.1, WP_011013499.1. Specifically, the dnaZX gene (or NCgl0239) gene) encoded by DNA polymerase III gamma and tau subunit activity, but is not limited thereto.

As used herein, the term "transcription antitermination protein" is a transcription complex, capable of interacting with the termination factor Rho and RNA polymerase, transcription termination and/or anti-termination It is a polypeptide that is a transcriptional elongation factor involved in Specifically, the transcriptional anti-termination protein of the present application may be used interchangeably as a transcription termination/antitermination protein, NusG protein, or NusG. In the present application, the transcriptional anti-termination protein can be sequenced from GenBank of NCBI, a known database, for example, GenBank Accession No. It may be WP_011013678.1, YP_224775.1. Specifically, it may be a polypeptide having transcriptional anti-termination protein activity encoded by the nusG gene (or NCgl0458 gene), but is not limited thereto.

As used herein, the term "ABC transporter ATP-binding protein" refers to a transmembrane protein that uses the energy obtained by hydrolyzing ATP to move and/or transport various substrates across the membrane, and Polypeptides with biological functions such as repair of unrelated DNA and RNA. Specifically, the ABC transporter ATP-binding protein of the present application may be used in combination as the ABC transporter ATP-binding protein. In the present application, the ABC transporter ATP-binding protein may have its sequence obtained from GenBank of NCBI, a known database, for example, GenBank Accession No. It may be WP_011013802.1, YP_224957.1. Specifically, it may be a polypeptide having an ABC transporter ATP-binding protein activity encoded by the NCgl0636 gene, but is not limited thereto.

As used herein, the term "N-succinyldiaminopimelate aminotransferase" refers to L-glutamate and N-succinyl-2-amino-6-ketopimelate as substrates, 2-oxoglutarate and N-succinyl-L,L It is an enzyme involved in the lysine biosynthetic pathway to produce -2,6-diaminopimelate. Specifically, the N-succinyldiaminopimelate aminotransferase of the present application can be used in combination with N-succinyldiaminopimelate aminotransferase. The N-succinyldiaminopimelate aminotransferase sequence can be obtained from GenBank of NCBI, a known database, for example, GenBank Accession No. WP_011014123.1, YP_225395.1. Specifically, NCgl1058 It may be a polypeptide having N-succinyldiaminopimelate aminotransferase activity encoded by the gene (or dapC gene), but is not limited thereto.

As used herein, the term "malate dehydrogenase" is an enzyme that reversibly catalyzes the oxidation of malate (malic acid) to oxaloacetic acid and the reduction of NAD+ to NADH in this process. The malate dehydrogenase of the application can be used interchangeably with malate dehydrogenase, MDH, or malate dehydrogenase In the present application, the malate dehydrogenase sequence can be obtained from NCBI's GenBank, a known database. and may be, for example, GenBank Accession No. WP_011015079.1, YP_226625.1. Specifically, it may be a polypeptide having malate dehydrogenase activity encoded by NCgl2297 gene (or mdh gene), but is not limited thereto. does not

As used herein, the term "primosome assembly protein" refers to a protein having a helicase activity and involved in the DNA replication process. Specifically, the primosome assembly protein of the present application may be used interchangeably as primosome assembly protein, PriA protein, PriA, or primosomal protein N'. In the present application, the sequence of the primosome assembly protein can be obtained from GenBank of NCBI, a known database, for example, GenBank Accession No. It may be WP_011014474.1, YP_225886.1. Specifically, it may be a polypeptide having a primosome assembly protein activity encoded by the NCgl1540 gene (or priA gene), but is not limited thereto.

As used herein, the term "helicase" is a polypeptide capable of separating a DNA double helix or a strand of a self-associated RNA molecule. Specifically, the helicase of the present application may be used in combination with a helicase. In the present application, the helicase sequence can be obtained from GenBank of NCBI, a known database, for example, GenBank Accession No. It can be WP_011014500.1, YP_225922.1. Specifically, it may be a polypeptide having a helicase activity encoded by the NCgl1575 gene, but is not limited thereto.

Type II citrate synthase of the present application may be used in combination with integral type II citrate synthase, citrate synthase, or citrate synthase. In the present application, the type II citrate synthase may have its sequence obtained from GenBank of NCBI, which is a known database, for example, GenBank Accession No. It may be YP_225121.1 or WP_011013914.1. Specifically, it may be a polypeptide having a type II citrate synthase activity encoded by the gltA gene (or NCgl0795 gene), but is not limited thereto.

The membrane protein of the present application may be used interchangeably as a membrane protein, a membrane protein, TerC, or a TerC protein. In the present application, the membrane protein sequence can be obtained from GenBank of NCBI, which is a known database, for example, GenBank Accession No. It may be YP_226209.1 or WP_011014789.1. Specifically, it may be a polypeptide having a membrane protein activity encoded by the terC gene (or NCgl1892 gene), but is not limited thereto.

Mycothione reductase of the present application may be used in combination with mycothione reductase, or mycothione reductase. In the present application, the mycothione reductase sequence can be obtained from GenBank of NCBI, a known database, for example, GenBank Accession No. It may be YP_226245.1 or WP_011014816.1. Specifically, it may be a polypeptide having mycothione reductase activity encoded by the mtr gene (or NCgl1928 gene), but is not limited thereto.

The Co/Zn/Cd efflux system component of the present application may be used in combination as a Co/Zn/Cd efflux system component. In the present application, the Co/Zn/Cd efflux system component can obtain its sequence from NCBI's GenBank, a known database, for example, GenBank Accession No. It may be YP_226309.1, WP_011014862.1. Specifically, it may be a polypeptide having a Co/Zn/Cd efflux system component activity encoded by the NCgl1992 gene, but is not limited thereto.

DAHP synthase of the present application is 3-deoxy-D-arabinoheptulosonic acid 7-phosphate synthase, 3-deoxy-D-arabinoheptulosonic acid 7-phosphate synthase, 3-deoxy-D-arabinoheptulosonate -7-phosphate synthase, or DAHP synthase may be used in combination. In the present application, the sequence of the DAHP synthase can be obtained from GenBank of NCBI, a known database, for example, GenBank Accession No. It may be YP_226420.1, WP_011014936.1. Specifically, it may be a polypeptide having DAHP synthase activity encoded by the aroG gene (or NCgl2098 gene), but is not limited thereto.

As an example of the present application, the mutant of the present application may have an activity to increase L-lysine production capacity compared to the wild-type polypeptide.

the endogenous membrane transport protein; DNA polymerase III gamma and tau subunits; a protein encoded by the NCgl3069 gene; transcriptional anti-termination protein; ABC transporter ATP-binding protein; malate dehydrogenase; primosome assembly protein; type II citrate synthase; membrane protein; mycothion reductase; Co/Zn/Cd effluent system components; DAHP synthase; N-succinyldiaminopimelate aminotransferase; And/or the amino acid sequence of the wild-type polypeptide of helicase and an example of the nucleic acid sequence encoding the same are shown in Table 3 below.

protein amino acid sequence a nucleic acid sequence encoding a protein endogenous membrane transport protein SEQ ID NO: 3 SEQ ID NO: 4 DNA Polymerase III Gamma and Tau Subunits SEQ ID NO: 7 SEQ ID NO: 8 Protein encoded by the NCgl3069 gene SEQ ID NO: 11 SEQ ID NO: 12 transcriptional anti-termination protein SEQ ID NO: 15 SEQ ID NO: 16 ABC transporter ATP-binding protein SEQ ID NO: 19 SEQ ID NO: 20 malate dehydrogenase SEQ ID NO: 23 SEQ ID NO: 24 primosome assembly protein SEQ ID NO: 27 SEQ ID NO: 28 Type II citrate synthase SEQ ID NO: 31 SEQ ID NO: 32 membrane protein SEQ ID NO: 35 SEQ ID NO: 36 mycothion reductase SEQ ID NO: 39 SEQ ID NO: 40 Co/Zn/Cd effluent system components SEQ ID NO: 43 SEQ ID NO: 44 DAHP synthase SEQ ID NO: 47 SEQ ID NO: 48 N-succinyldiaminopimelate aminotransferase SEQ ID NO: 51 SEQ ID NO: 52 helicase SEQ ID NO: 55 SEQ ID NO: 56

As used herein, the term “corresponding to” refers to an amino acid residue at a position listed in a polypeptide, or an amino acid residue similar to, identical to, or homologous to a residue listed in a polypeptide. Identifying an amino acid at a corresponding position may be determining a specific amino acid in a sequence that refers to a specific sequence. As used herein, "corresponding region" generally refers to a similar or corresponding position in a related protein or reference protein.

For example, any amino acid sequence is aligned with SEQ ID NO: 3, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, or 55, based on which the amino acid sequence is Each amino acid residue in the sequence is identified by reference to the numeric position of the amino acid residue corresponding to the amino acid residue of SEQ ID NO: 3, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, or 55 can be numbered. For example, a sequence alignment algorithm such as that described in this application can identify the position of an amino acid, or a position at which modifications, such as substitutions, insertions, or deletions, occur compared to a query sequence (also referred to as a "reference sequence").

Such alignments include, for example, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), the Needle program in the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al. , 2000), Trends Genet. 16: 276-277), etc., but is not limited thereto, and a sequence alignment program known in the art, a pairwise sequence comparison algorithm, etc. may be appropriately used.

Another aspect of the present application is to provide a polynucleotide encoding the variant of the present application.

As used herein, the term "polynucleotide" refers to a DNA or RNA strand of a certain length or longer as a polymer of nucleotides in which nucleotide monomers are linked in a long chain by covalent bonds, and more specifically, encoding the variant. polynucleotide fragments.

The polynucleotide encoding the variant of the present application has a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, or 53 may include As an example of the present application, the polynucleotide of the present application may have or include the sequence of SEQ ID NO: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, or 54 have. In addition, the polynucleotide of the present application may consist of, or consist essentially of, the sequence of SEQ ID NO: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, or 54 . In another example, the polynucleotide of the present application is SEQ ID NO: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, or 54, respectively, in the nucleic acid sequence set forth above. 659, 128, 97, 628, 595, 815 based on the nucleic acid sequence of 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, or 56 The bases corresponding to positions 911, 506, 880, 1072, 389, 1144, 626, or 1775, respectively, are G, T, A, G, A, T, A, T, A , T, A, T, T, or T, and at least a nucleic acid sequence set forth in SEQ ID NO: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, or 54 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% or more homology or identity of a nucleic acid sequence. can In addition, if the sequence encoding a polypeptide or protein having such homology or identity and exhibiting efficacy corresponding to the variant of the present application, a polynucleotide having a nucleic acid sequence in which some sequences are deleted, modified, substituted, conservatively substituted or added is also It is obvious that they are included within the scope of the present application. Table 4 below shows the correspondence of each nucleic acid sequence, position, and base type corresponding to the position of the polynucleotide of the present application.

nucleic acid sequence Nucleic acid sequence as a reference for counting positions location base type One SEQ ID NO: 2 SEQ ID NO: 4 659 G 2 SEQ ID NO: 6 SEQ ID NO: 8 128 T 3 SEQ ID NO: 10 SEQ ID NO: 12 97 A 4 SEQ ID NO: 14 SEQ ID NO: 16 628 G 5 SEQ ID NO: 18 SEQ ID NO: 20 595 A 6 SEQ ID NO: 22 SEQ ID NO: 24 815 T 7 SEQ ID NO: 26 SEQ ID NO: 28 911 A 8 SEQ ID NO: 30 SEQ ID NO: 32 506 T 9 SEQ ID NO: 34 SEQ ID NO: 36 880 A 10 SEQ ID NO: 38 SEQ ID NO: 40 1072 T 11 SEQ ID NO: 42 SEQ ID NO: 44 389 A 12 SEQ ID NO: 46 SEQ ID NO: 48 1144 T 13 SEQ ID NO: 50 SEQ ID NO: 52 626 T 14 SEQ ID NO: 54 SEQ ID NO: 56 1775 T

In consideration of codon degeneracy or preferred codons in organisms that want to express the variants of the present application, the polynucleotides of the present application are various in the coding region within the range that does not change the amino acid sequence of the variants of the present application. Deformation can be made. Specifically, the polynucleotide of the present application has at least 70% homology or identity with the sequence of SEQ ID NO: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, or 54 , 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% of the base sequence, or SEQ ID NO: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, or 54 has at least 70%, at least 75%, at least 80%, at least 85% homology or identity to the sequence of 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% of the nucleotide sequence may consist or consist essentially of, but is not limited thereto. At this time, in the sequence having the homology or identity, 220, 43, 33 of SEQ ID NO: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, or 53 The codons encoding the amino acids corresponding to positions 210, 199, 272, 304, 169, 294, 358, 130, 382, 209, or 592, respectively, are cysteine, one of the codons encoding leucine, serine, aspartic acid, methionine, isoleucine, glutamine, valine, lysine, serine, aspartic acid, cysteine, leucine, or phenylalanine.

In addition, the polynucleotide of the present application can be included without limitation as long as it can hybridize under stringent conditions with a probe that can be prepared from a known gene sequence, for example, a sequence complementary to all or part of the polynucleotide sequence of the present application. can The “stringent condition” refers to a condition that enables specific hybridization between polynucleotides. These conditions are described in J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; F.M. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, 9.50-9.51, 11.7-11.8). For example, polynucleotides with high homology or identity, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or a condition in which polynucleotides having 99% or more homology or identity hybridize with each other, and polynucleotides having lower homology or identity do not hybridize, or 60 ° C., which is a washing condition of conventional Southern hybridization, 1×SSC, 0.1% SDS, specifically 60°C, 0.1×SSC, 0.1% SDS, more specifically 68°C, 0.1×SSC, 0.1% SDS at a salt concentration and temperature equivalent to once, specifically twice The conditions for washing to 3 times can be enumerated.

Hybridization requires that two nucleic acids have complementary sequences, although mismatch between bases is possible depending on the stringency of hybridization. The term “complementary” is used to describe the relationship between nucleotide bases capable of hybridizing to each other. For example, with respect to DNA, adenine is complementary to thymine and cytosine is complementary to guanine. Accordingly, the polynucleotides of the present application may also include substantially similar nucleic acid sequences as well as isolated nucleic acid fragments complementary to the overall sequence.

Specifically, a polynucleotide having homology or identity with the polynucleotide of the present application can be detected using the hybridization conditions including a hybridization step at a Tm value of 55°C and using the above-described conditions. In addition, the Tm value may be 60 °C, 63 °C, or 65 °C, but is not limited thereto and may be appropriately adjusted by those skilled in the art according to the purpose.

The appropriate stringency for hybridizing the polynucleotides depends on the length of the polynucleotides and the degree of complementarity, and the variables are well known in the art (eg, J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold). Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; see F.M. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, 9.50-9.51, 11.7-11.8).

Another aspect of the present application is to provide a vector comprising the polynucleotide of the present application. The vector may be an expression vector for expressing the polynucleotide in a host cell, but is not limited thereto.

In the present application, "vector" refers to a DNA preparation comprising a base sequence of a polynucleotide encoding the target polypeptide operably linked to a suitable expression control region (or expression control sequence) so that the target polypeptide can be expressed in a suitable host. may include The expression control region may include a promoter capable of initiating transcription, an optional operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence regulating the termination of transcription and translation. After transformation into an appropriate host cell, the vector can replicate or function independently of the host genome, and can be integrated into the genome itself.

The vector used in the present application is not particularly limited, and any vector known in the art may be used. Examples of commonly used vectors include plasmids, cosmids, viruses and bacteriophages in a natural or recombinant state. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A may be used as phage vectors or cosmid vectors, and pDZ-based, pBR-based, and pUC-based plasmid vectors may be used. , pBluescript II-based, pGEM-based, pTZ-based, pCL-based, pET-based and the like can be used. Specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors and the like can be used.

For example, a polynucleotide encoding a target polypeptide may be inserted into a chromosome through a vector for intracellular chromosome insertion. The insertion of the polynucleotide into the chromosome may be performed by any method known in the art, for example, homologous recombination, but is not limited thereto. It may further include a selection marker (selection marker) for confirming whether the chromosome is inserted. The selection marker is used to select cells transformed with the vector, that is, to determine whether a target nucleic acid molecule is inserted, and selectable phenotypes such as drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of surface polypeptides. Markers to be given can be used. In an environment treated with a selective agent, only the cells expressing the selectable marker survive or exhibit other expression traits, so that the transformed cells can be selected.

In the present application, the term "transformation" means introducing a vector including a polynucleotide encoding a target polypeptide into a host cell or microorganism so that the polypeptide encoded by the polynucleotide can be expressed in the host cell. The transformed polynucleotide may include all of them regardless of whether they are inserted into the chromosome of the host cell or located outside the chromosome, as long as they can be expressed in the host cell. In addition, the polynucleotide includes DNA and/or RNA encoding a target polypeptide. The polynucleotide may be introduced in any form as long as it can be introduced and expressed into a host cell. For example, the polynucleotide may be introduced into a host cell in the form of an expression cassette, which is a gene construct including all elements necessary for self-expression. The expression cassette may include a promoter operably linked to the polynucleotide, a transcription termination signal, a ribosome binding site, and a translation termination signal. The expression cassette may be in the form of an expression vector capable of self-replication. In addition, the polynucleotide may be introduced into a host cell in its own form and operably linked to a sequence required for expression in the host cell, but is not limited thereto.

In addition, as used herein, the term “operably linked” means that a promoter sequence that initiates and mediates transcription of a polynucleotide encoding the target variant of the present application and the polynucleotide sequence are functionally linked.

Another aspect of the present application, Corynebacterium glutamicum ( Corynebacterium glutamicum ) It is to provide a strain comprising the mutant of the present application or the polynucleotide of the present application.

An example is a protein variant of the following (a) and (b) or a polynucleotide encoding the variant (eg, a polypeptide encoding the variant (a), a polypeptide encoding the variant (b), and/or The (a) and (b) encoding a polypeptide comprising), Corynebacterium glutamicum ( Corynebacterium glutamicum ) It can provide a strain:

(a) a helicase variant consisting of the amino acid sequence set forth in SEQ ID NO: 53 in which tyrosine, an amino acid corresponding to position 592 of SEQ ID NO: 55, is substituted with phenylalanine, and

(b) at least one protein variant selected from the group consisting of the following (1) to (13):

(1) an endogenous membrane transport protein variant consisting of the amino acid sequence set forth in SEQ ID NO: 1 in which phenylalanine, an amino acid corresponding to position 220 of SEQ ID NO: 3, is substituted with cysteine;

(2) DNA polymerase III gamma and tau subunit variants consisting of the amino acid sequence set forth in SEQ ID NO: 5 in which proline, an amino acid corresponding to position 43 of SEQ ID NO: 7, is substituted with leucine;

(3) a protein variant consisting of the amino acid sequence shown in SEQ ID NO: 9, in which cysteine, an amino acid corresponding to position 33 of SEQ ID NO: 11, is substituted with serine;

(4) a transcriptional anti-termination protein variant consisting of the amino acid sequence set forth in SEQ ID NO: 13 in which asparagine, an amino acid corresponding to position 210 of SEQ ID NO: 15, is substituted with aspartic acid;

(5) an ABC transporter ATP-binding protein variant consisting of the amino acid sequence shown in SEQ ID NO: 17 in which valine, an amino acid corresponding to position 199 of SEQ ID NO: 19, is substituted with methionine;

(6) a malate dehydrogenase variant consisting of the amino acid sequence set forth in SEQ ID NO: 21 in which threonine, an amino acid corresponding to position 272 of SEQ ID NO: 23, is substituted with isoleucine;

(7) a primosome assembly protein variant consisting of the amino acid sequence shown in SEQ ID NO: 25, in which arginine, an amino acid corresponding to position 304 of SEQ ID NO: 27, is substituted with glutamine;

(8) a type II citrate synthase variant consisting of the amino acid sequence shown in SEQ ID NO: 29, in which alanine, an amino acid corresponding to position 169 of SEQ ID NO: 31, is substituted with valine;

(9) a membrane protein variant consisting of the amino acid sequence set forth in SEQ ID NO: 33 in which glutamic acid, an amino acid corresponding to position 294 of SEQ ID NO: 35, is substituted with lysine;

(10) a mycothione reductase variant consisting of the amino acid sequence set forth in SEQ ID NO: 37 in which proline, which is the amino acid corresponding to position 358 of SEQ ID NO: 39, is substituted with serine;

(11) a Co/Zn/Cd efflux system component variant consisting of the amino acid sequence set forth in SEQ ID NO: 41 in which glycine, an amino acid corresponding to position 130 of SEQ ID NO: 43, is substituted with aspartic acid;

(12) a DAHP synthase variant consisting of the amino acid sequence set forth in SEQ ID NO: 45 in which glycine, an amino acid corresponding to position 382 of SEQ ID NO: 47, is substituted with cysteine; and

(13) An N-succinyldiaminopimelate aminotransferase mutant consisting of the amino acid sequence shown in SEQ ID NO: 49, wherein proline, which is the amino acid corresponding to position 209 of SEQ ID NO: 51, is substituted with leucine.

The strain of the present application includes a variant polypeptide of the present application (eg, a variant of (a) and (b) above), a polynucleotide encoding the polypeptide (eg, a polypeptide encoding the variant (a) above; (b) a polypeptide encoding the variant, and/or a polypeptide encoding (a) and (b) above), or a vector comprising the polynucleotide of the present application (eg, (a) and/or ( a vector comprising a polynucleotide encoding b); and/or a vector comprising a polypeptide comprising the variant (a) and a vector comprising the variant (b)).

As used herein, the term "strain (or microorganism)" includes both wild-type microorganisms and microorganisms in which genetic modification has occurred naturally or artificially. As a result, a specific mechanism is weakened or enhanced as a microorganism, and may be a microorganism including genetic modification for the production of a desired polypeptide, protein or product.

The strain of the present application is a variant of the present application (eg, the variants of (a) and (b) above), the polynucleotide of the present application (eg, the polypeptide encoding the variant (a) above, the (b) Polypeptides encoding variants, and/or polypeptides encoding (a) and (b) above) and a vector comprising a polynucleotide of the present application (eg, encoding (a) and/or (b) above) A vector comprising a polynucleotide, and/or a strain comprising at least one of (a) a vector comprising a polypeptide comprising a variant and (b) a vector comprising a variant); a strain modified to express a variant of the present application or a polynucleotide of the present application; a variant of the present application, or a strain expressing the polynucleotide of the present application (eg, a recombinant strain); Or it may be a strain having the mutant activity of the present application (eg, a recombinant strain), but is not limited thereto.

The strain of the present application may be a strain having L-lysine-producing ability.

The strain of the present application naturally comprises (i) an integral membrane transport protein; (ii) DNA polymerase Ⅲ subunits gamma and tau; (iii) a polypeptide comprising the amino acid sequence of SEQ ID NO: 11 and/or a protein encoded by the NCgl3069 gene; (iv) transcription antitermination protein; (v) ABC transporter ATP-binding protein; (vi) malate dehydrogenase; (vii) primosome assembly protein; (viii) type II citrate synthase; (ⅸ) membrane protein (TerC); (x) mycothione reductase; (xi) Co/Zn/Cd efflux system component; (xii) DAHP synthase (3-deoxy-D-arabinoheptulosonate-7-phosphate synthase; DAHP synthase); (xiii) N-succinyldiaminopimelate aminotransferase; And/or (xiv) a microorganism having helicase activity and/or L-lysine-producing ability, or one or more proteins selected from the group consisting of (i) to (xiv) activity and/or A mutant of the present application (eg, a mutant of (a) and (b) above) or a polynucleotide encoding the same (or a vector including the polynucleotide) is introduced into the parent strain without L-lysine-producing ability, and / Or it may be a microorganism to which L-lysine-producing ability is imparted, but is not limited thereto.

As an example, the strain of the present application is transformed with a vector containing a polynucleotide encoding the polynucleotide of the present application or a variant of the present application (eg, the variants of (a) and (b) above), As a cell or microorganism expressing a variant of, for the purpose of the present application, the strain of the present application may include all microorganisms capable of producing L-lysine, including the variant of the present application. For example, the strain of the present application is a polynucleotide encoding a variant of the present application in a natural wild-type microorganism or a microorganism producing L-lysine (eg, the (a) polypeptide encoding the variant, the (b) The polypeptide encoding the variant, and/or the polypeptide encoding the above (a) and (b)) may be a recombinant strain in which the protein variant is expressed, thereby increasing L-lysine-producing ability. In one example, the strain of the present application is a recombinant strain having an increased L-lysine production capacity compared to a protein variant or a strain that does not contain a polynucleotide encoding the variant (eg, Corynebacterium glutamicum). can be The recombinant strain having an increased ability to produce L-amino acids is a natural wild-type microorganism or a microorganism in which one or more proteins selected from the group consisting of (i) to (xiv) are unmodified microorganisms (ie, (i) to (xiv) ) a microorganism expressing one or more wild-type proteins selected from the group consisting of; for example, SEQ ID NO: 3, 7, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, or 55 A microorganism having an increased L-lysine-producing ability compared to (a microorganism comprising a polypeptide and/or a polynucleotide encoding the same), but is not limited thereto. For example, the non-modified microorganism, which is the target strain to compare whether the increase in the L-lysine production ability, is ATCC13032 strain and / or Corynebacterium glutamicum CJ3P (US 9556463 B2; the entire document is incorporated herein by reference) ), but is not limited thereto.

For example, the recombinant strain with increased production capacity has an L-lysine production capacity of about 1% or more, about 2.5% or more, about 5% or more, about 6% or more, about 7% compared to the parent strain or unmodified microorganism before mutation. or more, about 8% or more, about 9% or more, about 10% or more, about 10.5% or more, about 11% or more, about 11.5% or more, about 12% or more, about 12.5% or more, about 13% or more, about 13.5% or more, about 14% or more, about 14.5% or more, about 15% or more, about 15.5% or more, about 16% or more, about 16.5% or more, about 17% or more, about 17.5% or more, about 18% or more, about 18.5% or more, about 19% or more, about 19.5% or more, about 20% or more, about 20.5% or more, about 21% or more, about 21.5% or more, about 22% or more, about 22.5% or more, about 23% or more, about 23.5% or more, about 24% or more, about 24.5% or more, about 25% or more, about 25.5% or more, about 26% or more, about 26.5% or more, about 27% or more, about 27.5% or more, about 28% or more, about 28.5% or more, about 29% or more, about 29.5% or more, about 30% or more, about 31% or more, about 32% or more, about 33% or more, about 34% or more, or about 35% or more (the upper limit is not particularly limited, For example, it may be about 200% or less, about 150% or less, about 100% or less, about 50% or less, about 45% or less, about 40% or less, or about 35% or less). In another example, the recombinant strain with increased production capacity has an L-lysine production capacity of about 1.1 times or more, about 1.12 times or more, about 1.13 times or more, 1.15 times or more, 1.16 times or more, compared to the parent strain or unmodified microorganism before mutation. , 1.17 times or more, 1.18 times or more, 1.19 times or more, about 1.2 times or more, 1.25 times or more, or about 1.3 times or more (the upper limit is not particularly limited, for example, about 10 times or less, about 5 times or less, about 3 times or less, or about 2 times or less) may be increased. The term “about” is a range including all of ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc. not limited

As used herein, the term "unmodified microorganism" does not exclude a strain containing a mutation that can occur naturally in a microorganism, it is a wild-type strain or a natural-type strain itself, or a genetic variation caused by natural or artificial factors. It may mean the strain before being changed. For example, the unmodified microorganism may refer to a strain in which the transcriptional anti-termination protein variant described herein is not introduced or before it is introduced. The "unmodified microorganism" may be used interchangeably with "strain before modification", "microbe before modification", "unmodified strain", "unmodified strain", "unmodified microorganism" or "reference microorganism".

As another example of the present application, the microorganism of the present application is Corynebacterium glutamicum ( Corynebacterium glutamicum ), Corynebacterium crudilactis ( Corynebacterium crudilactis ), Corynebacterium deserti ( Corynebacterium deserti ), Cory Nebacterium efficiens ( Corynebacterium efficiens ), Corynebacterium callunae ), Corynebacterium stationis , Corynebacterium stationis ), Corynebacterium singulare ( Corynebacterium singulare ), Corynebacterium halo Tolerans ( Corynebacterium halotolerans ), Corynebacterium striatum ( Corynebacterium striatum ), Corynebacterium ammoniagenes ( Corynebacterium ammoniagenes ), Corynebacterium pollutisoli ( Corynebacterium pollutisoli ), Corynebacterium imitans ( Corynebacterium imitans ) imitans ), Corynebacterium testudinoris ) or Corynebacterium flavescens ).

As used herein, the term “weakened” of a polypeptide is a concept that includes both reduced or no activity compared to intrinsic activity. The attenuation may be used interchangeably with terms such as inactivation, deficiency, down-regulation, decrease, reduce, attenuation, and the like.

The attenuation is when the activity of the polypeptide itself is reduced or eliminated compared to the activity of the polypeptide possessed by the original microorganism due to mutation of the polynucleotide encoding the polypeptide, etc. When the overall polypeptide activity level and/or concentration (expression amount) in the cell is lower than that of the native strain due to (translation) inhibition, etc., when the expression of the polynucleotide is not made at all, and/or when the expression of the polynucleotide is Even if there is no activity of the polypeptide, it may also be included. The “intrinsic activity” refers to the activity of a specific polypeptide originally possessed by the parent strain, wild-type or unmodified microorganism before transformation when the trait is changed due to genetic mutation caused by natural or artificial factors. This may be used interchangeably with “activity before modification”. “Inactivation, deficiency, reduction, downregulation, reduction, attenuation” of the activity of a polypeptide compared to the intrinsic activity means that the activity of the specific polypeptide originally possessed by the parent strain or unmodified microorganism before transformation is lowered.

Attenuation of the activity of such a polypeptide may be performed by any method known in the art, but is not limited thereto, and may be achieved by application of various methods well known in the art (eg, Nakashima N et al., Bacterial cellular engineering by genome editing and gene silencing. Int J Mol Sci. 2014;15(2):2773-2793, Sambrook et al. Molecular Cloning 2012, etc.).

Specifically, the attenuation of the polypeptide of the present application is

1) deletion of all or part of a gene encoding a polypeptide;

2) modification of the expression control region (or expression control sequence) to reduce the expression of the gene encoding the polypeptide;

3) modification of the amino acid sequence constituting the polypeptide such that the activity of the polypeptide is eliminated or attenuated (eg, deletion/substitution/addition of one or more amino acids on the amino acid sequence);

4) modification of the gene sequence encoding the polypeptide such that the activity of the polypeptide is eliminated or attenuated (eg, one or more nucleobases on the nucleotide sequence of the polypeptide gene to encode a polypeptide modified such that the activity of the polypeptide is eliminated or attenuated) deletion/replacement/addition of);

5) modification of the nucleotide sequence encoding the initiation codon or 5'-UTR region of the gene transcript encoding the polypeptide;

6) introduction of an antisense oligonucleotide (eg, antisense RNA) that complementarily binds to the transcript of said gene encoding the polypeptide;

7) adding a sequence complementary to the Shine-Dalgarno sequence in front of the Shine-Dalgarno sequence of the gene encoding the polypeptide to form a secondary structure that cannot be attached to the ribosome;

8) addition of a promoter transcribed in the opposite direction to the 3' end of the open reading frame (ORF) of the gene sequence encoding the polypeptide (Reverse transcription engineering, RTE); or

9) It may be a combination of two or more selected from 1) to 8) above, but is not particularly limited thereto.

for example,

1) The deletion of a part or all of the gene encoding the polypeptide may be the removal of the entire polynucleotide encoding the endogenous target polypeptide in the chromosome, replacement with a polynucleotide in which some nucleotides are deleted, or replacement with a marker gene.

In addition, the above 2) modification of the expression control region (or expression control sequence), deletion, insertion, non-conservative or conservative substitution, or a combination thereof, mutation in the expression control region (or expression control sequence) occurs, or weaker replacement with an active sequence. The expression control region includes, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence regulating the termination of transcription and translation.

In addition, 3) the base sequence modification encoding the start codon or 5'-UTR region of the gene transcript encoding the polypeptide is, for example, a base encoding another start codon having a lower polypeptide expression rate than the intrinsic start codon It may be substituted with a sequence, but is not limited thereto.

In addition, the modification of the amino acid sequence or polynucleotide sequence of 4) and 5) above is a deletion, insertion, non-conservative or conservative substitution of the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide to weaken the activity of the polypeptide. Or a combination thereof may result in sequence mutation, or replacement with an amino acid sequence or polynucleotide sequence improved to have weaker activity or an amino acid sequence or polynucleotide sequence improved to have no activity, but is not limited thereto. For example, by introducing a mutation in the polynucleotide sequence to form a stop codon, the expression of a gene may be inhibited or attenuated, but is not limited thereto.

6) The introduction of an antisense oligonucleotide (eg, antisense RNA) that complementarily binds to the transcript of the gene encoding the polypeptide is described, for example, in Weintraub, H. et al., Antisense-RNA as a molecular tool. for genetic analysis, Reviews - Trends in Genetics, Vol. 1(1) 1986].

7) Addition of a sequence complementary to the Shine-Dalgarno sequence in front of the Shine-Dalgarno sequence of the gene encoding the polypeptide to form a secondary structure that cannot be attached to the ribosome is mRNA translation It may make it impossible or slow it down.

8) the addition of a promoter transcribed in the opposite direction to the 3' end of the open reading frame (ORF) of the gene sequence encoding the polypeptide (Reverse transcription engineering, RTE) is an antisense complementary to the transcript of the gene encoding the polypeptide It may be to attenuate activity by making nucleotides.

As used herein, the term “enhancement” of a polypeptide activity means that the activity of the polypeptide is increased compared to the intrinsic activity. The reinforcement may be used interchangeably with terms such as activation, up-regulation, overexpression, and increase. Herein, activation, enhancement, up-regulation, overexpression, and increase may include all of those exhibiting an activity that was not originally possessed, or exhibiting an improved activity compared to an intrinsic activity or an activity prior to modification. The “intrinsic activity” refers to the activity of a specific polypeptide originally possessed by the parent strain or unmodified microorganism before the transformation when the trait is changed due to genetic mutation caused by natural or artificial factors. This may be used interchangeably with “activity before modification”. “Enhancement”, “up-regulation”, “overexpression” or “increase” in the activity of a polypeptide compared to its intrinsic activity means that the activity and/or concentration (expression) of a specific polypeptide originally possessed by the parent strain or unmodified microorganism before transformation. amount), which means improved.

The enrichment can be achieved by introducing an exogenous polypeptide, or by enhancing the activity and/or concentration (expression amount) of the endogenous polypeptide. Whether or not the activity of the polypeptide is enhanced can be confirmed from the increase in the level of activity, expression level, or the amount of product excreted from the polypeptide.

The enhancement of the activity of the polypeptide can be applied by various methods well known in the art, and is not limited as long as it can enhance the activity of the target polypeptide compared to the microorganism before modification. Specifically, it may be one using genetic engineering and/or protein engineering well known to those skilled in the art, which is a routine method of molecular biology, but is not limited thereto (eg, Sitnicka et al. Functional Analysis of Genes. Advances in Cell). Biology 2010, Vol. 2. 1-16, Sambrook et al. Molecular Cloning 2012, etc.).

Specifically, the enrichment of the polypeptide of the present application is

1) increasing the intracellular copy number of a polynucleotide encoding the polypeptide;

2) replacing the gene expression control region on the chromosome encoding the polypeptide with a sequence with strong activity;

3) modification of the nucleotide sequence encoding the initiation codon or 5'-UTR region of the gene transcript encoding the polypeptide;

4) modification of the amino acid sequence of said polypeptide to enhance polypeptide activity;

5) modification of the polynucleotide sequence encoding the polypeptide to enhance the activity of the polypeptide (eg, modification of the polynucleotide sequence of the polypeptide gene to encode a polypeptide that has been modified to enhance the activity of the polypeptide);

6) introduction of a foreign polypeptide exhibiting the activity of the polypeptide or a foreign polynucleotide encoding the same;

7) codon optimization of the polynucleotide encoding the polypeptide;

8) by analyzing the tertiary structure of the polypeptide to select an exposed site for modification or chemical modification; or

9) It may be a combination of two or more selected from 1) to 8) above, but is not particularly limited thereto.

More specifically,

1) The increase in the intracellular copy number of the polynucleotide encoding the polypeptide is achieved by introduction into the host cell of a vector to which the polynucleotide encoding the polypeptide is operably linked, which can replicate and function independently of the host it may be Alternatively, the polynucleotide encoding the polypeptide may be achieved by introducing one copy or two or more copies into a chromosome in a host cell. The introduction into the chromosome may be performed by introducing a vector capable of inserting the polynucleotide into the chromosome in the host cell into the host cell, but is not limited thereto. The vector is the same as described above.

2) Replacing the gene expression control region (or expression control sequence) on the chromosome encoding the polypeptide with a sequence with strong activity is, for example, deletion, insertion, non-conservative or Conservative substitution or a combination thereof may result in a mutation in the sequence, or replacement with a sequence having a stronger activity. The expression control region is not particularly limited thereto, but may include a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence controlling the termination of transcription and translation. As an example, the original promoter may be replaced with a strong promoter, but is not limited thereto.

Examples of known strong promoters include CJ1 to CJ7 promoters (US 7662943 B2), lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, tet promoter, gapA promoter, SPL7 promoter, SPL13 (sm3) promoter (US Patent US 10584338 B2), O2 promoter (US Patent US 10273491 B2), tkt promoter, yccA promoter, etc., but is not limited thereto.

3) Modification of the nucleotide sequence encoding the start codon or 5'-UTR region of the gene transcript encoding the polypeptide is, for example, a nucleotide sequence encoding another start codon having a higher expression rate of the polypeptide compared to the intrinsic start codon. It may be a substitution, but is not limited thereto.

The modification of the amino acid sequence or polynucleotide sequence of 4) and 5) above may include deletion, insertion, non-conservative or conservative substitution of the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide to enhance the activity of the polypeptide; A combination thereof may result in sequence mutation, or replacement with an amino acid sequence or polynucleotide sequence improved to have stronger activity or an amino acid sequence or polynucleotide sequence improved to increase activity, but is not limited thereto. The replacement may be specifically performed by inserting a polynucleotide into a chromosome by homologous recombination, but is not limited thereto. In this case, the vector used may further include a selection marker for confirming whether or not the chromosome is inserted. The selection marker is the same as described above.

6) The introduction of the foreign polynucleotide exhibiting the activity of the polypeptide may be the introduction of the foreign polynucleotide encoding the polypeptide exhibiting the same/similar activity as the polypeptide into a host cell. The foreign polynucleotide is not limited in its origin or sequence as long as it exhibits the same/similar activity as the polypeptide. The method used for the introduction can be performed by appropriately selecting a known transformation method by those skilled in the art, and the introduced polynucleotide is expressed in a host cell to generate a polypeptide and increase its activity.

7) Codon optimization of the polynucleotide encoding the polypeptide is codon-optimized so that the transcription or translation of the endogenous polynucleotide is increased in the host cell, or the transcription and translation of the foreign polynucleotide is optimized in the host cell. It may be that its codons are optimized so that the

8) Selecting an exposed site by analyzing the tertiary structure of the polypeptide and modifying or chemically modifying it, for example, compares the sequence information of the polypeptide to be analyzed with a database in which sequence information of known proteins is stored to determine the degree of sequence similarity. Accordingly, it may be to determine a template protein candidate, check the structure based on this, and select an exposed site to be modified or chemically modified and modified or modified.

Such enhancement of polypeptide activity is to increase the activity or concentration of the corresponding polypeptide relative to the activity or concentration of the polypeptide expressed in the wild-type or unmodified microbial strain, or increase the amount of product produced from the polypeptide. However, the present invention is not limited thereto.

Modification of some or all of the polynucleotide in the microorganism of the present application (eg, modification for encoding the protein variant described above) is (a) homologous recombination using a vector for chromosomal insertion in the microorganism or engineered nuclease (e.g., CRISPR) -Cas9) and/or (b) induced by light and/or chemical treatment such as ultraviolet and radiation, but not limited thereto. The method for modifying part or all of the gene may include a method by DNA recombination technology. For example, by injecting a nucleotide sequence or a vector containing a nucleotide sequence homologous to a target gene into the microorganism to cause homologous recombination, a part or all of the gene may be deleted. The injected nucleotide sequence or vector may include a dominant selection marker, but is not limited thereto.

In the microorganism of the present application, variants, polynucleotides, L-lysine, and the like are as described in the other aspects above.

Another aspect of the present application is a variant of the present application (eg, a protein variant of (a) and (b) above) or a polynucleotide of the present application (eg, a polypeptide encoding the variant (a) above) , The (b) polypeptide encoding the variant, and / or the (a) and (b) comprising the step of culturing a Corynebacterium glutamicum strain comprising the polypeptide in a medium, L- A method for producing amino acids is provided.

In one example, it may provide a method for producing L- amino acids, comprising the step of culturing the Corynebacterium glutamicum strain of the present application in a medium.

The L-amino acid production method of the present application is a medium of the Corynebacterium glutamicum (or Corynebacterium glutamicum comprising the mutant of the present application or the polynucleotide of the present application or the vector of the present application) of the present application. It may include the step of culturing in

In addition, the L-amino acid of the present application may be L-lysine.

In the present application, the term "cultivation" means growing the Corynebacterium glutamicum strain of the present application under moderately controlled environmental conditions. The culture process of the present application may be performed according to a suitable medium and culture conditions known in the art. Such a culture process can be easily adjusted and used by those skilled in the art according to the selected strain. Specifically, the culture may be a batch, continuous and/or fed-batch, but is not limited thereto.

As used herein, the term "medium" refers to a material in which nutrients required for culturing the Corynebacterium glutamicum strain of the present application are mixed as a main component, and nutrients including water essential for survival and growth and growth factors. Specifically, any medium and other culture conditions used for culturing the Corynebacterium glutamicum strain of the present application may be used without any particular limitation as long as it is a medium used for culturing conventional microorganisms, but the Corynebacterium glutamicum of the present application Lium glutamicum strain can be cultured while controlling the temperature, pH, etc. under aerobic conditions in a conventional medium containing an appropriate carbon source, nitrogen source, phosphorus, inorganic compound, amino acid and / or vitamin and the like.

Specifically, the culture medium for the Corynebacterium sp. strain can be found in the literature ["Manual of Methods for General Bacteriology" by the American Society for Bacteriology (Washington D.C., USA, 1981)].

In the present application, the carbon source includes carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, maltose, and the like; sugar alcohols such as mannitol and sorbitol; organic acids such as pyruvic acid, lactic acid, citric acid and the like; Amino acids such as glutamic acid, methionine, lysine, and the like may be included. In addition, natural organic nutrient sources such as starch hydrolyzate, molasses, blackstrap molasses, rice winter, cassava, sugar cane offal and corn steep liquor can be used, specifically glucose and sterilized pre-treated molasses (i.e., converted to reducing sugar). molasses) may be used, and other appropriate amounts of carbon sources may be variously used without limitation. These carbon sources may be used alone or in combination of two or more, but is not limited thereto.

Examples of the nitrogen source include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, anmonium carbonate, and ammonium nitrate; Amino acids such as glutamic acid, methionine, glutamine, and organic nitrogen sources such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolyzate, fish or degradation products thereof, defatted soybean cake or degradation products thereof, etc. can be used These nitrogen sources may be used alone or in combination of two or more, but is not limited thereto.

The phosphorus may include potassium first potassium phosphate, second potassium phosphate, or a sodium-containing salt corresponding thereto. As the inorganic compound, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, etc. may be used, and in addition, amino acids, vitamins and/or suitable precursors may be included. These components or precursors may be added to the medium either batchwise or continuously. However, the present invention is not limited thereto.

In addition, during the culture of the Corynebacterium glutamicum strain of the present application, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc. may be added to the medium in an appropriate manner to adjust the pH of the medium. In addition, during culturing, an antifoaming agent such as fatty acid polyglycol ester may be used to suppress bubble formation. In addition, in order to maintain the aerobic state of the medium, oxygen or oxygen-containing gas may be injected into the medium, or nitrogen, hydrogen or carbon dioxide gas may be injected without or without gas to maintain anaerobic and microaerobic conditions, it is not

In the culture of the present application, the culture temperature may be maintained at 20 to 45° C., specifically, 25 to 40° C., and may be cultured for about 10 to 160 hours, but is not limited thereto.

The L-amino acid produced by the culture of the present application may be secreted into the medium or may remain in the cell.

The L-amino acid production method of the present application includes the steps of preparing the Corynebacterium glutamicum strain of the present application, preparing a medium for culturing the strain, or a combination thereof (regardless of the order, in any order) ), for example, prior to the culturing step, may further include.

The method for producing L-amino acids of the present application may further include recovering L-amino acids from the culture medium (the culture medium) or the Corynebacterium glutamicum strain. The recovering step may be further included after the culturing step.

The recovery may be to collect the desired L-amino acid using a suitable method known in the art according to the culture method of the microorganism of the present application, for example, a batch, continuous or fed-batch culture method, etc. . For example, centrifugation, filtration, treatment with a crystallized protein precipitating agent (salting out method), extraction, ultrasonic disruption, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity Various chromatography such as island chromatography, HPLC, or a combination thereof may be used, and a desired L-amino acid may be recovered from a medium or a microorganism using a suitable method known in the art.

In addition, the L-amino acid production method of the present application may include an additional purification step. The purification may be performed using a suitable method known in the art. In one example, when the L-amino acid production method of the present application includes both the recovery step and the purification step, the recovery step and the purification step are performed continuously or discontinuously, regardless of the order, or integrated into one step may be performed, but is not limited thereto.

In the method of the present application, variants, polynucleotides, vectors, strains, and the like are as described in the other aspects above.

Another aspect of the present application is a variant of the present application (for example, the variants of (a) and (b)), a polynucleotide encoding the variant (for example, the (a) variant encoding the variant) Polypeptides, polypeptides encoding the variants (b), and/or polypeptides encoding (a) and (b) above), vectors comprising the polynucleotides (eg, (a) and/or (b) above) ) a vector comprising a polynucleotide encoding the; Lium glutamicum strain; the culture medium; Or to provide a composition for the production of L- amino acids comprising a combination of two or more of them.

The composition of the present application may further include any suitable excipients commonly used in compositions for the production of amino acids, and these excipients may be, for example, preservatives, wetting agents, dispersing agents, suspending agents, buffering agents, stabilizing agents or isotonic agents, etc. However, the present invention is not limited thereto.

In the composition of the present application, variants, polynucleotides, vectors, strains, media and L-amino acids are the same as those described in the other aspects above.

<110> CJ CheilJedang Corporation <120> Novel protein variant and a method for producing L-lysine using the same <130> DPP20210468KR <160> 56 <170> enPatentIn 3.0 <210> 1 <211> 433 <212> PRT <213> Artificial Sequence <220> <223> (F220C)_integral membrane transport protein <400> 1 Met Thr Thr Ala Val Asp Gln Asn Ser Pro Pro Lys Gln Gln Leu Asn 1 5 10 15 Lys Arg Val Leu Leu Gly Ser Leu Ser Gly Ser Val Ile Glu Trp Phe 20 25 30 Asp Phe Leu Val Tyr Gly Thr Val Ala Ala Leu Val Phe Asn Lys Met 35 40 45 Tyr Phe Pro Ser Gly Asn Glu Phe Leu Ser Thr Ile Leu Ala Tyr Ala 50 55 60 Ser Phe Ser Leu Thr Phe Phe Phe Arg Pro Ile Gly Gly Val Ile Phe 65 70 75 80 Ala His Ile Gly Asp Arg Ile Gly Arg Lys Lys Thr Leu Phe Ile Thr 85 90 95 Leu Met Leu Met Gly Gly Gly Thr Val Ala Ile Gly Leu Leu Pro Asp 100 105 110 Tyr Asn Ala Ile Gly Ile Trp Ala Pro Ile Leu Leu Met Phe Leu Arg 115 120 125 Ile Leu Gln Gly Ile Gly Ile Gly Gly Glu Trp Gly Gly Ala Leu Leu 130 135 140 Leu Ala Tyr Glu Tyr Ala Pro Lys Lys Gln Arg Gly Leu Tyr Gly Ala 145 150 155 160 Val Pro Gln Met Gly Ile Ser Leu Gly Met Leu Leu Ala Ala Gly Val 165 170 175 Ile Ser Leu Leu Thr Leu Met Pro Glu Asp Gln Phe Leu Thr Trp Gly 180 185 190 Trp Arg Ile Pro Phe Val Gly Ser Ile Leu Leu Val Phe Ile Gly Leu 195 200 205 Phe Ile Arg Asn Gly Leu Asp Glu Thr Pro Glu Cys Lys Arg Ile Arg 210 215 220 Asp Ser Gly Gln Gln Val Lys Met Pro Leu Lys Glu Val Leu Thr Lys 225 230 235 240 Tyr Trp Pro Ala Val Leu Val Ser Ile Gly Ala Lys Ala Ala Glu Thr 245 250 255 Gly Pro Phe Tyr Ile Phe Gly Thr Tyr Ile Val Ala Tyr Ala Thr Asn 260 265 270 Phe Leu Asn Ile Arg Asp Asn Ile Val Leu Leu Ala Val Ala Cys Ala 275 280 285 Ala Leu Val Ala Thr Ile Trp Met Pro Leu Phe Gly Ser Phe Ser Asp 290 295 300 Arg Val Asn Arg Ala Val Leu Tyr Arg Ile Cys Ala Ser Ala Thr Ile 305 310 315 320 Val Leu Ile Val Pro Tyr Tyr Leu Val Leu Asn Thr Gly Glu Ile Trp 325 330 335 Ala Leu Phe Ile Thr Thr Val Ile Gly Phe Gly Ile Leu Trp Gly Ser 340 345 350 Val Asn Ala Ile Leu Gly Thr Val Ile Ala Glu Asn Phe Ala Pro Glu 355 360 365 Val Arg Tyr Thr Gly Ala Thr Leu Gly Tyr Gln Val Gly Ala Ala Leu 370 375 380 Phe Gly Gly Thr Ala Pro Ile Ile Ala Ala Trp Leu Phe Glu Ile Ser 385 390 395 400 Gly Gly Gln Trp Trp Pro Ile Ala Val Tyr Val Ala Ala Cys Cys Leu 405 410 415 Leu Ser Val Ile Ala Ser Phe Phe Ile Gln Arg Val Ala His Gln Glu 420 425 430 Asn <210> 2 <211> 1302 <212> DNA <213> Artificial Sequence <220> <223> (F220C)_NCgl2816 <400> 2 atgacaaccg cagtagatca aaactcaccg cccaagcagc aactcaacaa gcgcgtcctg 60 ctgggcagct tgagtggcag cgttatcgaa tggttcgact tcctggttta cggaaccgtc 120 gccgcgctgg tcttcaacaa gatgtacttc cccagcggca acgagttcct ctccacaatc 180 ctggcgtacg catccttctc cctgaccttc ttcttccgcc ccattggtgg cgtcatcttc 240 gcccacatcg gcgaccgcat tgggcgtaag aagaccctgt tcatcacctt gatgctcatg 300 ggtggcggca ccgtcgccat tggtttgctg cccgactaca acgccatcgg catttgggca 360 ccaatccttc tgatgttcct ccgcattttg cagggcatcg gaattggcgg cgaatggggt 420 ggcgcactgc tcctggcata cgaatacgct ccaaagaagc agcgtgggct ctacggcgca 480 gttcctcaaa tgggcatttc cctgggcatg ctgcttgcag ctggcgtgat ctctctgctc 540 accctcatgc cggaagatca gttcctcacc tggggctggc gcatcccatt cgtcggatcc 600 atcctcctag tgttcatcgg cctgttcatc cgaaacggcc ttgatgaaac ccccgagtgc 660 aagcgtatcc gcgattccgg ccagcaggta aagatgcctc tgaaggaagt tctgaccaag 720 tactggccag ccgttctggt ctccatcggc gcaaaagctg ccgagaccgg ccccttctac 780 atcttcggca cctacatcgt tgcttacgca accaacttcc tgaacatccg cgacaacatt 840 gtccttctgg cagttgcttg cgccgccctc gttgccacca tctggatgcc actgttcgga 900 tccttctccg accgcgtcaa ccgtgcagtg ctctacagga tctgtgcatc cgcaaccatc 960 gtgctgattg tcccttacta cttggtcctc aacaccggcg aaatttgggc actgtttatc 1020 actaccgtga ttggcttcgg catcctctgg ggtagcgtca acgcaatcct cggaaccgtc 1080 atcgcagaaa acttcgcacc tgaggtccgc tacaccggcg ctaccctggg taccaagtc 1140 ggagcagcac tcttcggcgg taccgcaccc attatcgcag catggctgtt cgaaatctcc 1200 ggcggacaat ggtggccaat cgccgtctac gtcgctgcat gttgccttct ctctgtgatc 1260 gcctcgttct tcatccaacg cgtcgcgcac caagagaact aa 1302 <210> 3 <211> 433 <212> PRT <213> Artificial Sequence <220> <223> (WT)_integral membrane transport protein <400> 3 Met Thr Thr Ala Val Asp Gln Asn Ser Pro Pro Lys Gln Gln Leu Asn 1 5 10 15 Lys Arg Val Leu Leu Gly Ser Leu Ser Gly Ser Val Ile Glu Trp Phe 20 25 30 Asp Phe Leu Val Tyr Gly Thr Val Ala Ala Leu Val Phe Asn Lys Met 35 40 45 Tyr Phe Pro Ser Gly Asn Glu Phe Leu Ser Thr Ile Leu Ala Tyr Ala 50 55 60 Ser Phe Ser Leu Thr Phe Phe Phe Arg Pro Ile Gly Gly Val Ile Phe 65 70 75 80 Ala His Ile Gly Asp Arg Ile Gly Arg Lys Lys Thr Leu Phe Ile Thr 85 90 95 Leu Met Leu Met Gly Gly Gly Thr Val Ala Ile Gly Leu Leu Pro Asp 100 105 110 Tyr Asn Ala Ile Gly Ile Trp Ala Pro Ile Leu Leu Met Phe Leu Arg 115 120 125 Ile Leu Gln Gly Ile Gly Ile Gly Gly Glu Trp Gly Gly Ala Leu Leu 130 135 140 Leu Ala Tyr Glu Tyr Ala Pro Lys Lys Gln Arg Gly Leu Tyr Gly Ala 145 150 155 160 Val Pro Gln Met Gly Ile Ser Leu Gly Met Leu Leu Ala Ala Gly Val 165 170 175 Ile Ser Leu Leu Thr Leu Met Pro Glu Asp Gln Phe Leu Thr Trp Gly 180 185 190 Trp Arg Ile Pro Phe Val Gly Ser Ile Leu Leu Val Phe Ile Gly Leu 195 200 205 Phe Ile Arg Asn Gly Leu Asp Glu Thr Pro Glu Phe Lys Arg Ile Arg 210 215 220 Asp Ser Gly Gln Gln Val Lys Met Pro Leu Lys Glu Val Leu Thr Lys 225 230 235 240 Tyr Trp Pro Ala Val Leu Val Ser Ile Gly Ala Lys Ala Ala Glu Thr 245 250 255 Gly Pro Phe Tyr Ile Phe Gly Thr Tyr Ile Val Ala Tyr Ala Thr Asn 260 265 270 Phe Leu Asn Ile Arg Asp Asn Ile Val Leu Leu Ala Val Ala Cys Ala 275 280 285 Ala Leu Val Ala Thr Ile Trp Met Pro Leu Phe Gly Ser Phe Ser Asp 290 295 300 Arg Val Asn Arg Ala Val Leu Tyr Arg Ile Cys Ala Ser Ala Thr Ile 305 310 315 320 Val Leu Ile Val Pro Tyr Tyr Leu Val Leu Asn Thr Gly Glu Ile Trp 325 330 335 Ala Leu Phe Ile Thr Thr Val Ile Gly Phe Gly Ile Leu Trp Gly Ser 340 345 350 Val Asn Ala Ile Leu Gly Thr Val Ile Ala Glu Asn Phe Ala Pro Glu 355 360 365 Val Arg Tyr Thr Gly Ala Thr Leu Gly Tyr Gln Val Gly Ala Ala Leu 370 375 380 Phe Gly Gly Thr Ala Pro Ile Ile Ala Ala Trp Leu Phe Glu Ile Ser 385 390 395 400 Gly Gly Gln Trp Trp Pro Ile Ala Val Tyr Val Ala Ala Cys Cys Leu 405 410 415 Leu Ser Val Ile Ala Ser Phe Phe Ile Gln Arg Val Ala His Gln Glu 420 425 430 Asn <210> 4 <211> 1302 <212> DNA <213> Artificial Sequence <220> <223> (WT)_NCgl2816 <400> 4 atgacaaccg cagtagatca aaactcaccg cccaagcagc aactcaacaa gcgcgtcctg 60 ctgggcagct tgagtggcag cgttatcgaa tggttcgact tcctggttta cggaaccgtc 120 gccgcgctgg tcttcaacaa gatgtacttc cccagcggca acgagttcct ctccacaatc 180 ctggcgtacg catccttctc cctgaccttc ttcttccgcc ccattggtgg cgtcatcttc 240 gcccacatcg gcgaccgcat tgggcgtaag aagaccctgt tcatcacctt gatgctcatg 300 ggtggcggca ccgtcgccat tggtttgctg cccgactaca acgccatcgg catttgggca 360 ccaatccttc tgatgttcct ccgcattttg cagggcatcg gaattggcgg cgaatggggt 420 ggcgcactgc tcctggcata cgaatacgct ccaaagaagc agcgtgggct ctacggcgca 480 gttcctcaaa tgggcatttc cctgggcatg ctgcttgcag ctggcgtgat ctctctgctc 540 accctcatgc cggaagatca gttcctcacc tggggctggc gcatcccatt cgtcggatcc 600 atcctcctag tgttcatcgg cctgttcatc cgaaacggcc ttgatgaaac ccccgagttc 660 aagcgtatcc gcgattccgg ccagcaggta aagatgcctc tgaaggaagt tctgaccaag 720 tactggccag ccgttctggt ctccatcggc gcaaaagctg ccgagaccgg ccccttctac 780 atcttcggca cctacatcgt tgcttacgca accaacttcc tgaacatccg cgacaacatt 840 gtccttctgg cagttgcttg cgccgccctc gttgccacca tctggatgcc actgttcgga 900 tccttctccg accgcgtcaa ccgtgcagtg ctctacagga tctgtgcatc cgcaaccatc 960 gtgctgattg tcccttacta cttggtcctc aacaccggcg aaatttgggc actgtttatc 1020 actaccgtga ttggcttcgg catcctctgg ggtagcgtca acgcaatcct cggaaccgtc 1080 atcgcagaaa acttcgcacc tgaggtccgc tacaccggcg ctaccctggg taccaagtc 1140 ggagcagcac tcttcggcgg taccgcaccc attatcgcag catggctgtt cgaaatctcc 1200 ggcggacaat ggtggccaat cgccgtctac gtcgctgcat gttgccttct ctctgtgatc 1260 gcctcgttct tcatccaacg cgtcgcgcac caagagaact aa 1302 <210> 5 <211> 775 <212> PRT <213> Artificial Sequence <220> <223> (P43L)_DNA polymerase III subunits gamma and tau <400> 5 Met Ala Leu Tyr Ser Lys Tyr Arg Pro Ala Ser Phe Gly Glu Leu Val 1 5 10 15 Gly Gln Ser Gln Val Thr Asp Pro Leu Ser Ala Ala Leu Asp Ser Gly 20 25 30 Arg Ile Asn His Ala Tyr Leu Phe Ser Gly Leu Arg Gly Cys Gly Lys 35 40 45 Thr Ser Ser Ala Arg Ile Leu Ala Arg Ser Leu Asn Cys Val Glu Gly 50 55 60 Pro Thr Ser Thr Pro Cys Gly Val Cys Asn Ser Cys Val Ala Leu Ala 65 70 75 80 Pro Gly Gly Pro Gly Thr Leu Asp Val Thr Glu Leu Asp Ala Ala Ser 85 90 95 Asn Asn Gly Val Asp Asp Met Arg Glu Leu Arg Glu Arg Ala Asn Tyr 100 105 110 Ala Pro Ala Glu Ser Arg Tyr Arg Val Phe Ile Ile Asp Glu Ala His 115 120 125 Met Ile Ser Thr Gln Gly Phe Asn Ala Leu Leu Lys Ile Val Glu Glu 130 135 140 Pro Pro Ala His Leu Ile Phe Ile Phe Ala Thr Thr Glu Pro Asp Lys 145 150 155 160 Met Ile Gly Thr Ile Arg Ser Arg Thr His Asn Tyr Pro Phe Arg Leu 165 170 175 Leu Thr Pro Gly Asp Met Arg Lys Val Leu Lys Asn Ala Val Asp Gly 180 185 190 Glu Gly Val His Val Asp Asp Ser Val Tyr Pro Leu Val Ile Arg Ala 195 200 205 Gly Gly Gly Ser Pro Arg Asp Ser Leu Ser Ile Leu Asp Gln Leu Ile 210 215 220 Ala Gly Ser Gly Pro Glu Gly Leu Thr Tyr Glu Arg Ala Leu Pro Leu 225 230 235 240 Leu Gly Val Thr Ser Phe Thr Leu Ile Asp Asp Ser Ile His Ala Leu 245 250 255 Ala Ser Lys Asp Asn Ala Ser Met Phe Thr Thr Ile Asp Asn Val Ile 260 265 270 Glu Glu Gly Leu Glu Pro Arg Arg Phe Thr Ile Asp Leu Leu Asp Arg 275 280 285 Leu Arg Asp Leu Met Val Leu Gln Ala Val Pro Glu Ala Leu Asn Leu 290 295 300 Gly Leu Val Asp Ala Pro Thr Asp Arg Ala Asp Ala Leu Ile Glu Gln 305 310 315 320 Ser Thr Leu Phe Lys Gly Asn Glu Leu Ala Asn Leu Ala Ser Met Val 325 330 335 Asn Ser Gly Leu Asp Asp Met Arg Gly Ala Thr Ser Pro Arg Leu Leu 340 345 350 Leu Glu Ile Leu Cys Ala Arg Leu Leu Leu Ala Ser Asn Thr Val Ala 355 360 365 Gly Pro Ala Val Ser Ser Ser Thr Asp Ala Ala Pro Ala Ala Thr Pro 370 375 380 Gly Gly Leu Thr Gly Ile Ala Ala Ala Arg Ala Lys Ala Arg Glu Tyr 385 390 395 400 Gly Gln Lys Lys Ala Ala Pro Ala Pro Ala Pro Thr Pro Ala Pro Glu 405 410 415 Pro Val Arg Glu Gln Ser Leu Ala Pro Thr Pro Glu Pro Thr Pro Ala 420 425 430 Ala Glu Pro Thr Ser Gln Pro Ala Pro Glu Pro Glu Pro Ala Arg Glu 435 440 445 Pro Val Val Glu Val Arg Glu Ala Ser Val Glu Lys Gln Pro Ala Ser 450 455 460 Ser Asp Pro Leu Glu Thr Ile Arg Ser Arg Trp Ser Glu Leu Arg Asn 465 470 475 480 Ile Val Glu Lys Gln Ser Val Arg Thr Ser Ile Met Leu Thr Glu Ala 485 490 495 Arg Val Leu Gly Leu Arg Gly Asp Thr Leu Val Leu Gly His Ser Thr 500 505 510 Gly Ala Leu Ala Ala Arg Leu Asn Ala Ala Asp His Asn Gly Ile Leu 515 520 525 Val Lys Val Leu Ala Glu Glu Thr Gly Leu Gln Leu Lys Val Glu Cys 530 535 540 Ile Val Gly Thr Asn Pro Ala Glu Ala Gly Phe Thr Ala Gln Pro Ala 545 550 555 560 Val Gln Lys Ser Thr Trp Asn Pro Asn Tyr Asp Ser Lys Pro Ala Thr 565 570 575 Pro Ser Ala Pro Ala Gln Pro Gln Thr Pro Lys Gln Glu Ser Val Pro 580 585 590 Thr Glu Pro Glu Glu Pro Glu Ser Ser Ala Glu Thr Ser Gly Trp Gly 595 600 605 Gln Pro Val Lys Ile Gly Gly Pro Ala Pro Glu Ala Gln Thr Pro Thr 610 615 620 Pro Pro Pro Ala Pro Val Val Pro Ala Thr Pro Ala Ala Pro Ala Ala 625 630 635 640 Gly Ala Ala Lys Pro Ala Trp Lys Glu Arg Val Glu Gln Ala Ala Ala 645 650 655 Asn Ala Ala Gln Gln Arg Gln His Arg Gln Gly Ser Ser Glu Pro Phe 660 665 670 Glu Arg Gly Val Pro Leu Pro Pro Glu Pro Asp Leu Pro Pro Asp Pro 675 680 685 Tyr Gly Tyr Pro Ala Asp Glu Gly Phe Pro Glu Arg Asn Gln Gly Phe 690 695 700 Gln Gln Gln Pro Ala Pro Pro Val Glu Gln Pro Ala Pro Glu Pro Ala 705 710 715 720 Thr Glu Pro Val Pro Ala Val Ala Pro Glu Pro Glu Val Ser Gln Leu 725 730 735 Ser Glu Glu Glu Gln Leu Ile Arg Glu Ala Asp Glu Glu Pro Gly Glu 740 745 750 Met Asp Arg Arg Asp Ala Lys Thr Ile Ala Met Glu Leu Leu Ala Gln 755 760 765 Glu Leu Gly Ala Lys Pro Leu 770 775 <210> 6 <211> 2328 <212> DNA <213> Artificial Sequence <220> <223> (P43L)_dnaZX <400> 6 gtggctttgt atagcaagta tcgaccggca agttttggtg aactagttgg gcagtcgcaa 60 gtgactgacc ctctgtccgc agctttggat agcgggcgca tcaaccatgc gtaccttttt 120 tcgggtctgc gtggttgtgg caagacgtcg tcggcacgca tccttgcccg gtccctcaac 180 tgcgtggaag gcccaacttc cacgccgtgt ggggtgtgta attcttgcgt agcgctggcc 240 ccgggtggcc ctggaaccct tgatgtaaca gagctcgacg ccgcgagtaa caatggtgtc 300 gatgacatgc gtgaactgcg cgaacgcgcg aactacgccc cggcggaatc tcgctaccgc 360 gtgttcatca ttgacgaagc ccacatgatc agtacccaag gcttcaacgc cttgctgaaa 420 atcgttgaag agccaccagc gcacctgatt ttcatcttcg ccaccaccga gccggacaaa 480 atgatcggta cgatccgttc ccgcacgcac aattacccat tccgcctgct caccccaggg 540 gatatgcgca aagtgctgaa aaatgcggtc gatggcgaag gcgtccacgt cgacgattcc 600 gtttacccac tggtcatccg cgccggcggc ggcagccccc gcgacagcct ctccatcctc 660 gaccagctca tcgccggctc gggcccggag ggcttgacat atgagcgcgc cttgccgctg 720 ctcggtgtca caagcttcac gcttatcgac gattcgatcc atgcccttgc atctaaagac 780 aacgcaagca tgttcaccac gatcgataac gtcatcgaag aaggcctcga accgcgacgc 840 ttcacgatcg acctcctcga ccgcctccgc gacttgatgg tcctccaagc cgtcccagaa 900 gcactaaacc tcgggcttgt cgacgccccc accgaccgcg ccgacgccct aatcgaacaa 960 tccaccctgt tcaaaggcaa cgagctcgcc aacctagcct ccatggtgaa ctccggactc 1020 gacgacatgc gtggtgccac ctcaccgcgc ctccttctgg aaatcctctg cgcccgactg 1080 ctcctggcaa gcaataccgt ggcaggtcca gcggtcagta gttcgactga cgctgcgcct 1140 gcagctactc cgggtggtct cactggtatt gctgctgccc gcgcgaaagc acgggagtat 1200 ggacagaaga aggcagctcc agctcctgca ccaactcctg cgcccgagcc agtgcgcgaa 1260 cagtctcttg caccaacgcc tgaaccaacg ccagcggctg aacctacatc tcagcccgcg 1320 ccggaaccgg aacccgccag ggaaccagtg gtggaagtgc gggaggcgag cgtcgaaaag 1380 cagcctgcaa gcagtgatcc cctcgaaacc attcgaagcc gctggtcaga gctgcgcaac 1440 atcgtggaaa aacaaagtgt gcgcacctca atcatgctga ccgaagcgcg agttttggga 1500 ctgcgaggcg ataccctcgt gctcggtcac agcaccgggg cgttggctgc gcgtttgaac 1560 gctgctgatc acaacggaat tttggtcaag gtgttggctg aggaaactgg tctgcagctc 1620 aaggtcgaat gcattgtggg cacgaaccca gccgaagctg gatttaccgc ccagcctgct 1680 gtgcaaaaga gcacgtggaa ccccaactac gactccaaac cagccactcc aagtgcgcct 1740 gcgcagcctc agacgcctaa gcaagaatcc gtgcccaccg agccagagga gcctgagagt 1800 tcggctgaaa cttcgggatg ggggcaaccg gtaaaaatcg gtgggccagc acctgaggca 1860 cagactccaa cgcctccgcc tgctccggtg gttccggcta ctcctgctgc tccggctgcg 1920 ggtgctgcga aaccagcgtg gaaggaacgc gtcgaacaag ccgcggcaaa tgcagcgcaa 1980 caacgtcagc atcgccaggg cagctccgag cccttcgagc ggggtgttcc gctgccacca 2040 gaacctgacc tgccaccgga cccctatgga tacccagctg acgagggttt ccccgaacga 2100 aaccaagggt tccagcaaca gccagcgcct cccgttgagc aaccagctcc tgagcctgcg 2160 acggagccag ttccagcagt tgcgcctgag ccggaggtca gccagctatc tgaagaagag 2220 cagctgatcc gcgaagcaga cgaagagcca ggtgaaatgg atcgtcgtga tgcgaaaacc 2280 atcgcgatgg aactgcttgc tcaggaactc ggcgcgaaac ctttgtaa 2328 <210> 7 <211> 775 <212> PRT <213> Artificial Sequence <220> <223> (WT)_DNA polymerase III subunits gamma and tau <400> 7 Met Ala Leu Tyr Ser Lys Tyr Arg Pro Ala Ser Phe Gly Glu Leu Val 1 5 10 15 Gly Gln Ser Gln Val Thr Asp Pro Leu Ser Ala Ala Leu Asp Ser Gly 20 25 30 Arg Ile Asn His Ala Tyr Leu Phe Ser Gly Pro Arg Gly Cys Gly Lys 35 40 45 Thr Ser Ser Ala Arg Ile Leu Ala Arg Ser Leu Asn Cys Val Glu Gly 50 55 60 Pro Thr Ser Thr Pro Cys Gly Val Cys Asn Ser Cys Val Ala Leu Ala 65 70 75 80 Pro Gly Gly Pro Gly Thr Leu Asp Val Thr Glu Leu Asp Ala Ala Ser 85 90 95 Asn Asn Gly Val Asp Asp Met Arg Glu Leu Arg Glu Arg Ala Asn Tyr 100 105 110 Ala Pro Ala Glu Ser Arg Tyr Arg Val Phe Ile Ile Asp Glu Ala His 115 120 125 Met Ile Ser Thr Gln Gly Phe Asn Ala Leu Leu Lys Ile Val Glu Glu 130 135 140 Pro Pro Ala His Leu Ile Phe Ile Phe Ala Thr Thr Glu Pro Asp Lys 145 150 155 160 Met Ile Gly Thr Ile Arg Ser Arg Thr His Asn Tyr Pro Phe Arg Leu 165 170 175 Leu Thr Pro Gly Asp Met Arg Lys Val Leu Lys Asn Ala Val Asp Gly 180 185 190 Glu Gly Val His Val Asp Asp Ser Val Tyr Pro Leu Val Ile Arg Ala 195 200 205 Gly Gly Gly Ser Pro Arg Asp Ser Leu Ser Ile Leu Asp Gln Leu Ile 210 215 220 Ala Gly Ser Gly Pro Glu Gly Leu Thr Tyr Glu Arg Ala Leu Pro Leu 225 230 235 240 Leu Gly Val Thr Ser Phe Thr Leu Ile Asp Asp Ser Ile His Ala Leu 245 250 255 Ala Ser Lys Asp Asn Ala Ser Met Phe Thr Thr Ile Asp Asn Val Ile 260 265 270 Glu Glu Gly Leu Glu Pro Arg Arg Phe Thr Ile Asp Leu Leu Asp Arg 275 280 285 Leu Arg Asp Leu Met Val Leu Gln Ala Val Pro Glu Ala Leu Asn Leu 290 295 300 Gly Leu Val Asp Ala Pro Thr Asp Arg Ala Asp Ala Leu Ile Glu Gln 305 310 315 320 Ser Thr Leu Phe Lys Gly Asn Glu Leu Ala Asn Leu Ala Ser Met Val 325 330 335 Asn Ser Gly Leu Asp Asp Met Arg Gly Ala Thr Ser Pro Arg Leu Leu 340 345 350 Leu Glu Ile Leu Cys Ala Arg Leu Leu Leu Ala Ser Asn Thr Val Ala 355 360 365 Gly Pro Ala Val Ser Ser Ser Thr Asp Ala Ala Pro Ala Ala Thr Pro 370 375 380 Gly Gly Leu Thr Gly Ile Ala Ala Ala Arg Ala Lys Ala Arg Glu Tyr 385 390 395 400 Gly Gln Lys Lys Ala Ala Pro Ala Pro Ala Pro Thr Pro Ala Pro Glu 405 410 415 Pro Val Arg Glu Gln Ser Leu Ala Pro Thr Pro Glu Pro Thr Pro Ala 420 425 430 Ala Glu Pro Thr Ser Gln Pro Ala Pro Glu Pro Glu Pro Ala Arg Glu 435 440 445 Pro Val Val Glu Val Arg Glu Ala Ser Val Glu Lys Gln Pro Ala Ser 450 455 460 Ser Asp Pro Leu Glu Thr Ile Arg Ser Arg Trp Ser Glu Leu Arg Asn 465 470 475 480 Ile Val Glu Lys Gln Ser Val Arg Thr Ser Ile Met Leu Thr Glu Ala 485 490 495 Arg Val Leu Gly Leu Arg Gly Asp Thr Leu Val Leu Gly His Ser Thr 500 505 510 Gly Ala Leu Ala Ala Arg Leu Asn Ala Ala Asp His Asn Gly Ile Leu 515 520 525 Val Lys Val Leu Ala Glu Glu Thr Gly Leu Gln Leu Lys Val Glu Cys 530 535 540 Ile Val Gly Thr Asn Pro Ala Glu Ala Gly Phe Thr Ala Gln Pro Ala 545 550 555 560 Val Gln Lys Ser Thr Trp Asn Pro Asn Tyr Asp Ser Lys Pro Ala Thr 565 570 575 Pro Ser Ala Pro Ala Gln Pro Gln Thr Pro Lys Gln Glu Ser Val Pro 580 585 590 Thr Glu Pro Glu Glu Pro Glu Ser Ser Ala Glu Thr Ser Gly Trp Gly 595 600 605 Gln Pro Val Lys Ile Gly Gly Pro Ala Pro Glu Ala Gln Thr Pro Thr 610 615 620 Pro Pro Pro Ala Pro Val Val Pro Ala Thr Pro Ala Ala Pro Ala Ala 625 630 635 640 Gly Ala Ala Lys Pro Ala Trp Lys Glu Arg Val Glu Gln Ala Ala Ala 645 650 655 Asn Ala Ala Gln Gln Arg Gln His Arg Gln Gly Ser Ser Glu Pro Phe 660 665 670 Glu Arg Gly Val Pro Leu Pro Pro Glu Pro Asp Leu Pro Pro Asp Pro 675 680 685 Tyr Gly Tyr Pro Ala Asp Glu Gly Phe Pro Glu Arg Asn Gln Gly Phe 690 695 700 Gln Gln Gln Pro Ala Pro Pro Val Glu Gln Pro Ala Pro Glu Pro Ala 705 710 715 720 Thr Glu Pro Val Pro Ala Val Ala Pro Glu Pro Glu Val Ser Gln Leu 725 730 735 Ser Glu Glu Glu Gln Leu Ile Arg Glu Ala Asp Glu Glu Pro Gly Glu 740 745 750 Met Asp Arg Arg Asp Ala Lys Thr Ile Ala Met Glu Leu Leu Ala Gln 755 760 765 Glu Leu Gly Ala Lys Pro Leu 770 775 <210> 8 <211> 2328 <212> DNA <213> Artificial Sequence <220> <223> (WT)_dnaZX <400> 8 gtggctttgt atagcaagta tcgaccggca agttttggtg aactagttgg gcagtcgcaa 60 gtgactgacc ctctgtccgc agctttggat agcgggcgca tcaaccatgc gtaccttttt 120 tcgggtccgc gtggttgtgg caagacgtcg tcggcacgca tccttgcccg gtccctcaac 180 tgcgtggaag gcccaacttc cacgccgtgt ggggtgtgta attcttgcgt agcgctggcc 240 ccgggtggcc ctggaaccct tgatgtaaca gagctcgacg ccgcgagtaa caatggtgtc 300 gatgacatgc gtgaactgcg cgaacgcgcg aactacgccc cggcggaatc tcgctaccgc 360 gtgttcatca ttgacgaagc ccacatgatc agtacccaag gcttcaacgc cttgctgaaa 420 atcgttgaag agccaccagc gcacctgatt ttcatcttcg ccaccaccga gccggacaaa 480 atgatcggta cgatccgttc ccgcacgcac aattacccat tccgcctgct caccccaggg 540 gatatgcgca aagtgctgaa aaatgcggtc gatggcgaag gcgtccacgt cgacgattcc 600 gtttacccac tggtcatccg cgccggcggc ggcagccccc gcgacagcct ctccatcctc 660 gaccagctca tcgccggctc gggcccggag ggcttgacat atgagcgcgc cttgccgctg 720 ctcggtgtca caagcttcac gcttatcgac gattcgatcc atgcccttgc atctaaagac 780 aacgcaagca tgttcaccac gatcgataac gtcatcgaag aaggcctcga accgcgacgc 840 ttcacgatcg acctcctcga ccgcctccgc gacttgatgg tcctccaagc cgtcccagaa 900 gcactaaacc tcgggcttgt cgacgccccc accgaccgcg ccgacgccct aatcgaacaa 960 tccaccctgt tcaaaggcaa cgagctcgcc aacctagcct ccatggtgaa ctccggactc 1020 gacgacatgc gtggtgccac ctcaccgcgc ctccttctgg aaatcctctg cgcccgactg 1080 ctcctggcaa gcaataccgt ggcaggtcca gcggtcagta gttcgactga cgctgcgcct 1140 gcagctactc cgggtggtct cactggtatt gctgctgccc gcgcgaaagc acgggagtat 1200 ggacagaaga aggcagctcc agctcctgca ccaactcctg cgcccgagcc agtgcgcgaa 1260 cagtctcttg caccaacgcc tgaaccaacg ccagcggctg aacctacatc tcagcccgcg 1320 ccggaaccgg aacccgccag ggaaccagtg gtggaagtgc gggaggcgag cgtcgaaaag 1380 cagcctgcaa gcagtgatcc cctcgaaacc attcgaagcc gctggtcaga gctgcgcaac 1440 atcgtggaaa aacaaagtgt gcgcacctca atcatgctga ccgaagcgcg agttttggga 1500 ctgcgaggcg ataccctcgt gctcggtcac agcaccgggg cgttggctgc gcgtttgaac 1560 gctgctgatc acaacggaat tttggtcaag gtgttggctg aggaaactgg tctgcagctc 1620 aaggtcgaat gcattgtggg cacgaaccca gccgaagctg gatttaccgc ccagcctgct 1680 gtgcaaaaga gcacgtggaa ccccaactac gactccaaac cagccactcc aagtgcgcct 1740 gcgcagcctc agacgcctaa gcaagaatcc gtgcccaccg agccagagga gcctgagagt 1800 tcggctgaaa cttcgggatg ggggcaaccg gtaaaaatcg gtgggccagc acctgaggca 1860 cagactccaa cgcctccgcc tgctccggtg gttccggcta ctcctgctgc tccggctgcg 1920 ggtgctgcga aaccagcgtg gaaggaacgc gtcgaacaag ccgcggcaaa tgcagcgcaa 1980 caacgtcagc atcgccaggg cagctccgag cccttcgagc ggggtgttcc gctgccacca 2040 gaacctgacc tgccaccgga cccctatgga tacccagctg acgagggttt ccccgaacga 2100 aaccaagggt tccagcaaca gccagcgcct cccgttgagc aaccagctcc tgagcctgcg 2160 acggagccag ttccagcagt tgcgcctgag ccggaggtca gccagctatc tgaagaagag 2220 cagctgatcc gcgaagcaga cgaagagcca ggtgaaatgg atcgtcgtga tgcgaaaacc 2280 atcgcgatgg aactgcttgc tcaggaactc ggcgcgaaac ctttgtaa 2328 <210> 9 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> (C33S)_NCgl3069_amino acid <400> 9 Met Ala Val Ala Lys Asp Val Leu Ser Phe Ser Arg Pro Ile Pro Ala 1 5 10 15 Leu His Ala Ile Ile Gly Ala Leu Gly Trp Phe Gly Met Phe Ala Val 20 25 30 Ser Leu Trp Ser Thr Leu 35 <210> 10 <211> 117 <212> DNA <213> Artificial Sequence <220> <223> (C33S)_NCgl3069_nucleic acid <400> 10 gtggctgtcg caaaagacgt gcttagtttt tcacgaccca ttcccgcact tcacgccatc 60 atcggcgcgc ttggatggtt tggaatgttc gcagttagtc tgtggagcac gctgtaa 117 <210> 11 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> (WT)_NCgl3069_amino acid <400> 11 Met Ala Val Ala Lys Asp Val Leu Ser Phe Ser Arg Pro Ile Pro Ala 1 5 10 15 Leu His Ala Ile Ile Gly Ala Leu Gly Trp Phe Gly Met Phe Ala Val 20 25 30 Cys Leu Trp Ser Thr Leu 35 <210> 12 <211> 117 <212> DNA <213> Artificial Sequence <220> <223> (WT)_NCgl3069_nucleic acid <400> 12 gtggctgtcg caaaagacgt gcttagtttt tcacgaccca ttcccgcact tcacgccatc 60 atcggcgcgc ttggatggtt tggaatgttc gcagtttgtc tgtggagcac gctgtaa 117 <210> 13 <211> 318 <212> PRT <213> Artificial Sequence <220> <223> (N210D)_NusG <400> 13 Met Ser Asp Glu Asn Ile Asn Glu Phe Glu Gln Asp Glu Asp Leu Asn 1 5 10 15 Phe Gly Ala Ser Phe Ser Asp Glu Phe Ala Asp Asp Asp Phe Asp Ala 20 25 30 Glu Ala Asp Val Glu Ala Asp Ala Ala Ala Glu Ala Ser Ala Leu Glu 35 40 45 Ala Glu Gln Asp Leu Glu Glu Glu Thr Leu Asp Ala Pro Glu Glu Ala 50 55 60 Ala Glu Glu Ala Pro Ala Ala Ala Glu Ser Glu Ala Pro Val Glu Glu 65 70 75 80 Asp Glu Glu Ala Asp Ser Leu Ala Gln Ala Ala Ala Ala Leu Gly Asp 85 90 95 Thr Asp Glu Gln Asp Ala Asp Ala Glu Tyr Lys Ala Arg Leu Arg Lys 100 105 110 Phe Thr Arg Glu Leu Lys Lys Gln Pro Gly Val Trp Tyr Ile Ile Gln 115 120 125 Cys Tyr Ser Gly Tyr Glu Asn Lys Val Lys Ala Asn Leu Asp Met Arg 130 135 140 Ala Gln Thr Leu Glu Val Glu Asp Asp Ile Phe Glu Val Val Val Pro 145 150 155 160 Ile Glu Gln Val Thr Glu Ile Arg Asp Gly Lys Arg Lys Leu Val Lys 165 170 175 Arg Lys Leu Leu Pro Gly Tyr Val Leu Val Arg Met Asp Met Asn Asp 180 185 190 Arg Val Trp Ser Val Val Arg Asp Thr Pro Gly Val Thr Ser Phe Val 195 200 205 Gly Asp Glu Gly Asn Ala Thr Pro Val Lys His Arg Asp Val Ala Lys 210 215 220 Phe Leu Met Pro Gln Glu Gln Ala Val Val Thr Gly Glu Ala Ala Ala 225 230 235 240 Ala Ala Ala Glu Gly Glu Gln Val Val Ala Met Pro Thr Asp Thr Lys 245 250 255 Lys Pro Gln Val Ala Val Asp Phe Thr Val Gly Glu Ala Val Thr Ile 260 265 270 Leu Thr Gly Ala Phe Ala Ser Val Ser Ala Thr Ile Ser Ser Ile Asp 275 280 285 Pro Glu Leu Gln Lys Leu Glu Val Leu Val Ser Ile Phe Gly Arg Glu 290 295 300 Thr Pro Val Asp Leu Ser Phe Asp Gln Val Glu Lys Val Ser 305 310 315 <210> 14 <211> 957 <212> DNA <213> Artificial Sequence <220> <223> (N210D)_nusG <400> 14 atgagcgatg agaacattaa cgagtttgag caggacgagg atctgaactt cggcgcgagc 60 tttagtgatg aattcgcaga tgacgatttc gatgcagaag cagacgtaga agcagatgct 120 gctgcagagg cctctgccct ggaagctgag caggatctgg aagaagagac cctagatgct 180 ccagaagaag ccgcagaaga agctcctgct gctgcagagt ccgaagctcc agtagaagag 240 gacgaagagg ctgacagcct tgctcaggcg gctgctgcac ttggtgacac cgatgagcag 300 gacgcggatg cagagtacaa ggctcgtctg cgtaagttca ctcgtgagct gaagaagcag 360 cctggtgttt ggtacatcat tcagtgctac tccggctacg agaacaaggt gaaggcgaac 420 cttgacatgc gtgctcagac ccttgaggtt gaggatgaca tctttgaggt tgttgttcct 480 atcgagcagg tcactgagat ccgtgatggt aagcgcaagc tggttaagcg taagttgctg 540 ccgggctacg ttttggtccg catggacatg aatgaccgcg tgtggtctgt tgttcgcgat 600 acacctggtg tgaccagctt tgtgggtgac gagggcaatg caactcctgt gaagcaccgc 660 gatgttgcga agttcttgat gcctcaggag caggctgttg tcaccggtga ggctgctgct 720 gcggctgccg agggtgagca ggttgtggct atgcctaccg ataccaagaa gcctcaggtt 780 gctgtggact tcactgttgg tgaggctgtg accattctga ctggtgcttt cgcttctgtt 840 tctgcaacga tttcttctat cgatcctgag ctgcagaagc tggaagtttt ggtgtccatc 900 tttggtcgtg aaactcctgt tgatctcagc ttcgaccagg ttgagaaggt tagctag 957 <210> 15 <211> 318 <212> PRT <213> Artificial Sequence <220> <223> (WT)_NusG <400> 15 Met Ser Asp Glu Asn Ile Asn Glu Phe Glu Gln Asp Glu Asp Leu Asn 1 5 10 15 Phe Gly Ala Ser Phe Ser Asp Glu Phe Ala Asp Asp Asp Phe Asp Ala 20 25 30 Glu Ala Asp Val Glu Ala Asp Ala Ala Ala Glu Ala Ser Ala Leu Glu 35 40 45 Ala Glu Gln Asp Leu Glu Glu Glu Thr Leu Asp Ala Pro Glu Glu Ala 50 55 60 Ala Glu Glu Ala Pro Ala Ala Ala Glu Ser Glu Ala Pro Val Glu Glu 65 70 75 80 Asp Glu Glu Ala Asp Ser Leu Ala Gln Ala Ala Ala Ala Leu Gly Asp 85 90 95 Thr Asp Glu Gln Asp Ala Asp Ala Glu Tyr Lys Ala Arg Leu Arg Lys 100 105 110 Phe Thr Arg Glu Leu Lys Lys Gln Pro Gly Val Trp Tyr Ile Ile Gln 115 120 125 Cys Tyr Ser Gly Tyr Glu Asn Lys Val Lys Ala Asn Leu Asp Met Arg 130 135 140 Ala Gln Thr Leu Glu Val Glu Asp Asp Ile Phe Glu Val Val Val Pro 145 150 155 160 Ile Glu Gln Val Thr Glu Ile Arg Asp Gly Lys Arg Lys Leu Val Lys 165 170 175 Arg Lys Leu Leu Pro Gly Tyr Val Leu Val Arg Met Asp Met Asn Asp 180 185 190 Arg Val Trp Ser Val Val Arg Asp Thr Pro Gly Val Thr Ser Phe Val 195 200 205 Gly Asn Glu Gly Asn Ala Thr Pro Val Lys His Arg Asp Val Ala Lys 210 215 220 Phe Leu Met Pro Gln Glu Gln Ala Val Val Thr Gly Glu Ala Ala Ala 225 230 235 240 Ala Ala Ala Glu Gly Glu Gln Val Val Ala Met Pro Thr Asp Thr Lys 245 250 255 Lys Pro Gln Val Ala Val Asp Phe Thr Val Gly Glu Ala Val Thr Ile 260 265 270 Leu Thr Gly Ala Phe Ala Ser Val Ser Ala Thr Ile Ser Ser Ile Asp 275 280 285 Pro Glu Leu Gln Lys Leu Glu Val Leu Val Ser Ile Phe Gly Arg Glu 290 295 300 Thr Pro Val Asp Leu Ser Phe Asp Gln Val Glu Lys Val Ser 305 310 315 <210> 16 <211> 957 <212> DNA <213> Artificial Sequence <220> <223> (WT)_nusG <400> 16 atgagcgatg agaacattaa cgagtttgag caggacgagg atctgaactt cggcgcgagc 60 tttagtgatg aattcgcaga tgacgatttc gatgcagaag cagacgtaga agcagatgct 120 gctgcagagg cctctgccct ggaagctgag caggatctgg aagaagagac cctagatgct 180 ccagaagaag ccgcagaaga agctcctgct gctgcagagt ccgaagctcc agtagaagag 240 gacgaagagg ctgacagcct tgctcaggcg gctgctgcac ttggtgacac cgatgagcag 300 gacgcggatg cagagtacaa ggctcgtctg cgtaagttca ctcgtgagct gaagaagcag 360 cctggtgttt ggtacatcat tcagtgctac tccggctacg agaacaaggt gaaggcgaac 420 cttgacatgc gtgctcagac ccttgaggtt gaggatgaca tctttgaggt tgttgttcct 480 atcgagcagg tcactgagat ccgtgatggt aagcgcaagc tggttaagcg taagttgctg 540 ccgggctacg ttttggtccg catggacatg aatgaccgcg tgtggtctgt tgttcgcgat 600 acacctggtg tgaccagctt tgtgggtaac gagggcaatg caactcctgt gaagcaccgc 660 gatgttgcga agttcttgat gcctcaggag caggctgttg tcaccggtga ggctgctgct 720 gcggctgccg agggtgagca ggttgtggct atgcctaccg ataccaagaa gcctcaggtt 780 gctgtggact tcactgttgg tgaggctgtg accattctga ctggtgcttt cgcttctgtt 840 tctgcaacga tttcttctat cgatcctgag ctgcagaagc tggaagtttt ggtgtccatc 900 tttggtcgtg aaactcctgt tgatctcagc ttcgaccagg ttgagaaggt tagctag 957 <210> 17 <211> 269 <212> PRT <213> Artificial Sequence <220> <223> (V199M)_ABC transporter ATP-binding protein <400> 17 Met Thr Thr Asn His Gln Leu Ser Ala Glu Glu Ile Ser Leu Ala Tyr 1 5 10 15 Gly Glu Arg Thr Ile Ile Asp Ser Leu Ser Val Asp Ile Val Pro Gly 20 25 30 Lys Ile Thr Ser Ile Val Gly Pro Asn Gly Cys Gly Lys Ser Thr Leu 35 40 45 Leu Arg Ala Phe Ala Arg Leu Leu Lys Pro Ser Ala Gly Gln Ala Leu 50 55 60 Ile Asp Ala His Pro Leu Pro Ser Leu Pro Gly Lys Glu Leu Ala Arg 65 70 75 80 Met Leu Gly Leu Leu Pro Gln Ser Pro Thr Ala Pro Glu Gly Ile Val 85 90 95 Val Ala Asp Leu Val Gly Arg Gly Arg His Pro His Gln Gly Leu Met 100 105 110 Gly Arg Trp Ser Thr Arg Asp Tyr Glu Val Val Ala Gln Ala Leu Glu 115 120 125 Met Thr Asn Thr Thr Glu Leu Ala Glu Arg Pro Ile Asp Glu Leu Ser 130 135 140 Gly Gly Gln Arg Gln Arg Val Trp Ile Ala Met Ala Leu Ala Gln Glu 145 150 155 160 Thr Asp Ile Leu Leu Leu Asp Glu Pro Thr Thr Tyr Leu Asp Ile Ala 165 170 175 Asn Gln Leu Glu Val Leu Asp Leu Leu Thr Asp Leu Asn His Asn His 180 185 190 Gly Thr Thr Ile Val Met Met Leu His Glu Leu Leu Gly Leu Ala Ala Arg 195 200 205 Tyr Ser Asp His Leu Ile Ala Met Asn Ala Gly Lys Ile Tyr Ala Gln 210 215 220 Gly Thr Pro Thr Asn Val Ile Thr Glu Thr Met Met Ser Glu Val Phe 225 230 235 240 His Thr Asp Ala Arg Ile Ile Ala Asp Pro Val Ser Gly Ala Pro Leu 245 250 255 Val Met Pro Met Gly Arg His His Ile Thr Ala Leu His 260 265 <210> 18 <211> 810 <212> DNA <213> Artificial Sequence <220> <223> (V199M)_NCgl0636 <400> 18 gtgaccacca accatcaact atccgccgaa gaaatttccc tggcgtacgg cgagcgcacc 60 atcatcgatt cgctcagcgt cgacatcgtc cccggcaaaa tcacctccat cgtcggcccc 120 aacggatgcg gcaaatcaac gctgctgcgc gcctttgcgc gcctccttaa acctagcgcc 180 gggcaagcgc ttatcgacgc ccaccccctt ccttcactgc caggcaaaga actagctcgc 240 atgctcgggc tgttaccgca atcccccacc gcacctgaag gcatcgtcgt cgccgacctc 300 gtgggccgcg gccgccaccc ccaccaagga ctcatgggca gatggtccac acgcgactac 360 gaagtagtgg cccaagcatt ggaaatgacc aacaccaccg agcttgccga acgccccatc 420 gacgaactct ccggcggcca acgccaacgc gtctggatcg ccatggccct tgcccaagaa 480 acagacatcc tgcttcttga tgaacccacc acctacctcg acatcgccaa ccagctcgaa 540 gtgcttgatc tcctcactga cctcaaccac aaccacggca ccaccatcgt catgatgctc 600 cacgaattgg gacttgccgc acgttactct gaccacctca tcgccatgaa cgccggaaaa 660 atctacgccc aaggcacccc caccaacgtc atcaccgaaa ccatgatgag cgaggtcttc 720 cacaccgacg cccgcattat cgccgaccca gtctccggcg caccactggt catgcccatg 780 ggacgacacc acatcaccgc acttcactag 810 <210> 19 <211> 269 <212> PRT <213> Artificial Sequence <220> <223> (WT)_ABC transporter ATP-binding protein <400> 19 Met Thr Thr Asn His Gln Leu Ser Ala Glu Glu Ile Ser Leu Ala Tyr 1 5 10 15 Gly Glu Arg Thr Ile Ile Asp Ser Leu Ser Val Asp Ile Val Pro Gly 20 25 30 Lys Ile Thr Ser Ile Val Gly Pro Asn Gly Cys Gly Lys Ser Thr Leu 35 40 45 Leu Arg Ala Phe Ala Arg Leu Leu Lys Pro Ser Ala Gly Gln Ala Leu 50 55 60 Ile Asp Ala His Pro Leu Pro Ser Leu Pro Gly Lys Glu Leu Ala Arg 65 70 75 80 Met Leu Gly Leu Leu Pro Gln Ser Pro Thr Ala Pro Glu Gly Ile Val 85 90 95 Val Ala Asp Leu Val Gly Arg Gly Arg His Pro His Gln Gly Leu Met 100 105 110 Gly Arg Trp Ser Thr Arg Asp Tyr Glu Val Val Ala Gln Ala Leu Glu 115 120 125 Met Thr Asn Thr Thr Glu Leu Ala Glu Arg Pro Ile Asp Glu Leu Ser 130 135 140 Gly Gly Gln Arg Gln Arg Val Trp Ile Ala Met Ala Leu Ala Gln Glu 145 150 155 160 Thr Asp Ile Leu Leu Leu Asp Glu Pro Thr Thr Tyr Leu Asp Ile Ala 165 170 175 Asn Gln Leu Glu Val Leu Asp Leu Leu Thr Asp Leu Asn His Asn His 180 185 190 Gly Thr Thr Ile Val Met Val Leu His Glu Leu Gly Leu Ala Ala Arg 195 200 205 Tyr Ser Asp His Leu Ile Ala Met Asn Ala Gly Lys Ile Tyr Ala Gln 210 215 220 Gly Thr Pro Thr Asn Val Ile Thr Glu Thr Met Met Ser Glu Val Phe 225 230 235 240 His Thr Asp Ala Arg Ile Ile Ala Asp Pro Val Ser Gly Ala Pro Leu 245 250 255 Val Met Pro Met Gly Arg His His Ile Thr Ala Leu His 260 265 <210> 20 <211> 810 <212> DNA <213> Artificial Sequence <220> <223> (WT)_NCgl0636 <400> 20 gtgaccacca accatcaact atccgccgaa gaaatttccc tggcgtacgg cgagcgcacc 60 atcatcgatt cgctcagcgt cgacatcgtc cccggcaaaa tcacctccat cgtcggcccc 120 aacggatgcg gcaaatcaac gctgctgcgc gcctttgcgc gcctccttaa acctagcgcc 180 gggcaagcgc ttatcgacgc ccaccccctt ccttcactgc caggcaaaga actagctcgc 240 atgctcgggc tgttaccgca atcccccacc gcacctgaag gcatcgtcgt cgccgacctc 300 gtgggccgcg gccgccaccc ccaccaagga ctcatgggca gatggtccac acgcgactac 360 gaagtagtgg cccaagcatt ggaaatgacc aacaccaccg agcttgccga acgccccatc 420 gacgaactct ccggcggcca acgccaacgc gtctggatcg ccatggccct tgcccaagaa 480 acagacatcc tgcttcttga tgaacccacc acctacctcg acatcgccaa ccagctcgaa 540 gtgcttgatc tcctcactga cctcaaccac aaccacggca ccaccatcgt catggtgctc 600 cacgaattgg gacttgccgc acgttactct gaccacctca tcgccatgaa cgccggaaaa 660 atctacgccc aaggcacccc caccaacgtc atcaccgaaa ccatgatgag cgaggtcttc 720 cacaccgacg cccgcattat cgccgaccca gtctccggcg caccactggt catgcccatg 780 ggacgacacc acatcaccgc acttcactag 810 <210> 21 <211> 328 <212> PRT <213> Artificial Sequence <220> <223> (T272I)_Malate dehydrogenase <400> 21 Met Asn Ser Pro Gln Asn Val Ser Thr Lys Lys Val Thr Val Thr Gly 1 5 10 15 Ala Ala Gly Gln Ile Ser Tyr Ser Leu Leu Trp Arg Ile Ala Asn Gly 20 25 30 Glu Val Phe Gly Thr Asp Thr Pro Val Glu Leu Lys Leu Leu Glu Ile 35 40 45 Pro Gln Ala Leu Gly Gly Ala Glu Gly Val Ala Met Glu Leu Leu Asp 50 55 60 Ser Ala Phe Pro Leu Leu Arg Asn Ile Thr Ile Thr Ala Asp Ala Asn 65 70 75 80 Glu Ala Phe Asp Gly Ala Asn Ala Ala Phe Leu Val Gly Ala Lys Pro 85 90 95 Arg Gly Lys Gly Glu Glu Arg Ala Asp Leu Leu Ala Asn Asn Gly Lys 100 105 110 Ile Phe Gly Pro Gln Gly Lys Ala Ile Asn Asp Asn Ala Ala Asp Asp 115 120 125 Ile Arg Val Leu Val Val Gly Asn Pro Ala Asn Thr Asn Ala Leu Ile 130 135 140 Ala Ser Ala Ala Ala Pro Asp Val Pro Ala Ser Arg Phe Asn Ala Met 145 150 155 160 Met Arg Leu Asp His Asn Arg Ala Ile Ser Gln Leu Ala Thr Lys Leu 165 170 175 Gly Arg Gly Ser Ala Glu Phe Asn Asn Ile Val Val Trp Gly Asn His 180 185 190 Ser Ala Thr Gln Phe Pro Asp Ile Thr Tyr Ala Thr Val Gly Gly Glu 195 200 205 Lys Val Thr Asp Leu Val Asp His Asp Trp Tyr Val Glu Glu Phe Ile 210 215 220 Pro Arg Val Ala Asn Arg Gly Ala Glu Ile Ile Glu Val Arg Gly Lys 225 230 235 240 Ser Ser Ala Ala Ser Ala Ala Ser Ser Ala Ile Asp His Met Arg Asp 245 250 255 Trp Val Gln Gly Thr Glu Ala Trp Ser Ser Ala Ala Ile Pro Ser Ile 260 265 270 Gly Ala Tyr Gly Ile Pro Glu Gly Ile Phe Val Gly Leu Pro Thr Val 275 280 285 Ser Arg Asn Gly Glu Trp Glu Ile Val Glu Gly Leu Glu Ile Ser Asp 290 295 300 Phe Gln Arg Ala Arg Ile Asp Ala Asn Ala Gln Glu Leu Gln Ala Glu 305 310 315 320 Arg Glu Ala Val Arg Asp Leu Leu 325 <210> 22 <211> 987 <212> DNA <213> Artificial Sequence <220> <223> (T272I)_NCgl2297 <400> 22 atgaattccc cgcagaacgt ctccaccaag aaggtcaccg tcaccggcgc agctggtcaa 60 atctcttatt cactgttgtg gcgcatcgcc aacggtgaag tattcggcac cgacacccct 120 gtagaactga aacttctgga gatccctcag gctcttggcg gggcagaggg tgtggctatg 180 gaacttctgg attctgcctt ccccctcctg cgaaacatca ccatcaccgc ggatgccaat 240 gaggcattcg acggcgctaa tgcggcgttt ttggtcggtg cgaagcctcg cggaaaaggc 300 gaagagcgcg cagatttgct ggctaacaac ggcaagattt tcggacctca aggtaaagct 360 atcaatgaca acgccgcaga tgacattcgt gtcctagttg ttggaaaccc agcgaacacc 420 aacgcgttga ttgcttcagc tgcggcccca gatgttccag catcccgctt caacgcaatg 480 atgcgccttg atcacaaccg tgcgatctcc cagctggcca ccaagcttgg ccgtggatct 540 gcggaattta acaacattgt ggtctgggga aatcactccg caacccagtt cccagacatc 600 acctacgcaa ccgttggtgg agaaaaggtc actgacctgg ttgatcacga ttggtatgtg 660 gaggagttca ttcctcgcgt ggctaaccgt ggcgctgaaa tcattgaggt ccgtggaaag 720 tcttctgcag cttctgcagc atcctctgcg attgatcaca tgcgcgattg ggtacagggc 780 accgaggcgt ggtcctctgc ggcaattcct tccatcggtg catacggcat tcctgagggc 840 atttttgtcg gtctgccaac cgtatcccgc aacggtgagt gggaaatcgt tgaaggcctg 900 gagatttccg atttccagcg cgcccgcatc gacgcgaatg ctcaggaatt gcaggccgag 960 cgcgaggcag tgcgcgactt gctctaa 987 <210> 23 <211> 328 <212> PRT <213> Artificial Sequence <220> <223> (WT)_Malate dehydrogenase <400> 23 Met Asn Ser Pro Gln Asn Val Ser Thr Lys Lys Val Thr Val Thr Gly 1 5 10 15 Ala Ala Gly Gln Ile Ser Tyr Ser Leu Leu Trp Arg Ile Ala Asn Gly 20 25 30 Glu Val Phe Gly Thr Asp Thr Pro Val Glu Leu Lys Leu Leu Glu Ile 35 40 45 Pro Gln Ala Leu Gly Gly Ala Glu Gly Val Ala Met Glu Leu Leu Asp 50 55 60 Ser Ala Phe Pro Leu Leu Arg Asn Ile Thr Ile Thr Ala Asp Ala Asn 65 70 75 80 Glu Ala Phe Asp Gly Ala Asn Ala Ala Phe Leu Val Gly Ala Lys Pro 85 90 95 Arg Gly Lys Gly Glu Glu Arg Ala Asp Leu Leu Ala Asn Asn Gly Lys 100 105 110 Ile Phe Gly Pro Gln Gly Lys Ala Ile Asn Asp Asn Ala Ala Asp Asp 115 120 125 Ile Arg Val Leu Val Val Gly Asn Pro Ala Asn Thr Asn Ala Leu Ile 130 135 140 Ala Ser Ala Ala Ala Pro Asp Val Pro Ala Ser Arg Phe Asn Ala Met 145 150 155 160 Met Arg Leu Asp His Asn Arg Ala Ile Ser Gln Leu Ala Thr Lys Leu 165 170 175 Gly Arg Gly Ser Ala Glu Phe Asn Asn Ile Val Val Trp Gly Asn His 180 185 190 Ser Ala Thr Gln Phe Pro Asp Ile Thr Tyr Ala Thr Val Gly Gly Glu 195 200 205 Lys Val Thr Asp Leu Val Asp His Asp Trp Tyr Val Glu Glu Phe Ile 210 215 220 Pro Arg Val Ala Asn Arg Gly Ala Glu Ile Ile Glu Val Arg Gly Lys 225 230 235 240 Ser Ser Ala Ala Ser Ala Ala Ser Ser Ala Ile Asp His Met Arg Asp 245 250 255 Trp Val Gln Gly Thr Glu Ala Trp Ser Ser Ala Ala Ile Pro Ser Thr 260 265 270 Gly Ala Tyr Gly Ile Pro Glu Gly Ile Phe Val Gly Leu Pro Thr Val 275 280 285 Ser Arg Asn Gly Glu Trp Glu Ile Val Glu Gly Leu Glu Ile Ser Asp 290 295 300 Phe Gln Arg Ala Arg Ile Asp Ala Asn Ala Gln Glu Leu Gln Ala Glu 305 310 315 320 Arg Glu Ala Val Arg Asp Leu Leu 325 <210> 24 <211> 987 <212> DNA <213> Artificial Sequence <220> <223> (WT)_NCgl2297 <400> 24 atgaattccc cgcagaacgt ctccaccaag aaggtcaccg tcaccggcgc agctggtcaa 60 atctcttatt cactgttgtg gcgcatcgcc aacggtgaag tattcggcac cgacacccct 120 gtagaactga aacttctgga gatccctcag gctcttggcg gggcagaggg tgtggctatg 180 gaacttctgg attctgcctt ccccctcctg cgaaacatca ccatcaccgc ggatgccaat 240 gaggcattcg acggcgctaa tgcggcgttt ttggtcggtg cgaagcctcg cggaaaaggc 300 gaagagcgcg cagatttgct ggctaacaac ggcaagattt tcggacctca aggtaaagct 360 atcaatgaca acgccgcaga tgacattcgt gtcctagttg ttggaaaccc agcgaacacc 420 aacgcgttga ttgcttcagc tgcggcccca gatgttccag catcccgctt caacgcaatg 480 atgcgccttg atcacaaccg tgcgatctcc cagctggcca ccaagcttgg ccgtggatct 540 gcggaattta acaacattgt ggtctgggga aatcactccg caacccagtt cccagacatc 600 acctacgcaa ccgttggtgg agaaaaggtc actgacctgg ttgatcacga ttggtatgtg 660 gaggagttca ttcctcgcgt ggctaaccgt ggcgctgaaa tcattgaggt ccgtggaaag 720 tcttctgcag cttctgcagc atcctctgcg attgatcaca tgcgcgattg ggtacagggc 780 accgaggcgt ggtcctctgc ggcaattcct tccaccggtg catacggcat tcctgagggc 840 atttttgtcg gtctgccaac cgtatcccgc aacggtgagt gggaaatcgt tgaaggcctg 900 gagatttccg atttccagcg cgcccgcatc gacgcgaatg ctcaggaatt gcaggccgag 960 cgcgaggcag tgcgcgactt gctctaa 987 <210> 25 <211> 688 <212> PRT <213> Artificial Sequence <220> <223> (R304Q)_primosome assembly protein <400> 25 Met Ala Lys Thr Arg Val Pro Ala Pro Glu Lys Ser Val Ala Arg Val 1 5 10 15 Leu Pro Leu Leu Gly Leu Pro His Leu Asp Arg Leu Phe Asp Tyr Arg 20 25 30 Ile Ser Glu Asp Gln His Asp Asp Val Gln Pro Gly Val Arg Val Arg 35 40 45 Val Arg Phe Gly Gly Arg Leu Val Asp Ala Ile Val Met Ser Arg Thr 50 55 60 Ala Gln Thr Ser His Glu Gly Lys Leu Met Trp Leu Asp Arg Val Ile 65 70 75 80 Ser Pro Ile Val Val Tyr Pro Pro Gln Thr Ala Lys Leu Ile Glu Gln 85 90 95 Leu Ser Asp Arg Tyr Gly Gly Val Arg Ser Asp Leu Ile Arg Ser Ala 100 105 110 Leu Pro Ala Arg His Ala Gly Ala Glu Glu Ala Asp Thr Ser Thr Ser 115 120 125 Trp Glu Ser Leu Gly Glu Val Lys Glu Pro Asp Leu Ser Ser Trp Ser 130 135 140 Ala Tyr Gln His Gly Gln Ser Phe Val Asp Ala Val Leu Ala Gly Thr 145 150 155 160 Thr Ala Arg Ala Ser Trp Gln Ile Ala Pro Gly Asp Asp Trp Ala Leu 165 170 175 Ala Leu Ala Ser Leu Ala Val Lys Val Val Lys Asp Gly Gly Gly Ala 180 185 190 Leu Leu Val Val Pro Asp Gln Arg Asp Leu Asp Arg Leu Glu Ala Ala 195 200 205 Leu Arg Gly Leu Val Ala Ala Lys Gln Ile Thr Val Leu Asn Ser Gly 210 215 220 Leu Gly Pro Gln Ala Arg Tyr Arg Arg Phe Leu Ser Val Leu Ser Gly 225 230 235 240 Gln Gly Arg Leu Ile Ile Gly Thr Arg Ser Ala Ala Phe Ala Pro Val 245 250 255 Lys Asp Leu Lys Leu Ala Val Ile Leu Asn Asp Gly Asp Asp Asn Leu 260 265 270 Val Asp Pro Arg Ala Pro Tyr Ala His Ala Arg Glu Val Leu Thr Thr 275 280 285 Arg Ser Ser Leu Glu Ala Ser Ser Leu Ile Ile Ala Gly His Ala Gln 290 295 300 Thr Ala Glu Thr Gln Leu Leu Val Glu Ser Gly Trp Met His Asn Leu 305 310 315 320 Ile Ala Pro Arg Asp Thr Ile Arg Thr Arg Met Pro Arg Ile Gln Ala 325 330 335 Val Gly Asp Ser Asp Phe Gln Met Glu Arg Asp Pro Met Ala Arg Ser 340 345 350 Ala Arg Leu Pro Gly Ile Ala Phe His Ala Val Arg Ser Ala Leu Glu 355 360 365 Arg Asp Gln Pro Ala Leu Ile Gln Val Pro Arg Lys Gly Tyr Val Pro 370 375 380 Thr Leu Ala Cys Gly Asn Cys Arg Thr Pro Ala Arg Cys Arg His Cys 385 390 395 400 Asn Gly Pro Val Gly Leu Pro Gln Gly Ser Ser Asp Leu Ala Gly Val 405 410 415 Pro Thr Cys Arg Trp Cys Gly Arg Pro Asp Ser Arg Phe Lys Cys Gln 420 425 430 Asn Cys Gly Ser Pro Lys Leu Arg Ala Val Val Leu Gly Thr Glu Arg 435 440 445 Thr Ala Glu Glu Leu Gly Arg Ala Phe Pro Ser Val Arg Val Ile Thr 450 455 460 Ser Gly Gly Asn Lys Val Val Asp Ser Val Glu Asn Arg Ala Ser Ile 465 470 475 480 Val Val Ser Thr Pro Gly Ala Glu Pro Phe Val Ala Asn Ser Pro Glu 485 490 495 Arg Pro Glu Lys Ser Glu Lys Pro Glu His Lys Gly Ala Tyr Gly Ala 500 505 510 Leu Leu Leu Leu Asp Thr Trp Ala Leu Met Gly Arg Gln Asp Leu Arg 515 520 525 Ala Met Glu Asp Ala Leu His Lys Trp Ala Ala Ala Ala Thr Leu Val 530 535 540 His Ser His Leu His Gln Gly Gln Val Ile Val Val Ala Asp Pro Ser 545 550 555 560 Phe Pro Ala Val Gln Ser Leu Ile Arg Trp Asp Met Ala Gly Ala Ala 565 570 575 Ala Gln Glu Leu Ala Ser Arg Arg Glu Val Met Phe Pro Ser Val 580 585 590 His Met Ala Ala Ile Asp Gly Ala Thr Ala Ala Leu Glu Ser Phe Leu 595 600 605 Asp Leu Ala Glu Leu Pro Asp His Ala Glu Val Leu Gly Pro Val Asp 610 615 620 Leu Pro Pro Gly Val Ser Leu Pro Gly Glu Tyr Asp Glu Gln Arg Phe 625 630 635 640 Gly Pro Pro Gln Arg Leu Leu Ile Arg Thr Pro Leu Gly Pro Arg Ser 645 650 655 Glu Leu Gly Arg Ala Leu Arg Ser Ala Gln Val Ala Arg Ala Val Arg 660 665 670 Lys Asn Asp Leu Pro Leu Arg Ile Gln Met Asp Pro Ile His Ile Gly 675 680 685 <210> 26 <211> 2067 <212> DNA <213> Artificial Sequence <220> <223> (R304Q)_NCgl1540 <400> 26 atggcaaaaa cccgcgtccc cgctcctgaa aagtcggtgg cgcgggtttt acctcttttg 60 gggttacctc acctggatcg actgtttgat taccgcatca gcgaagacca acacgatgat 120 gtgcaacctg gcgtgcgggt gcgcgtgcgt tttggtggac gtttagttga tgccatcgtg 180 atgtcacgca ccgcgcaaac ctcgcacgag ggaaagctga tgtggctgga tcgggtgatt 240 tcgccgatcg tggtgtatcc acctcaaaca gcaaagctaa ttgagcaact cagtgatcgc 300 tatggcgggg tacgttcgga tctcatccgt tcggcgctac cggcgcggca tgctggggca 360 gaagaggcag atacctccac gtcgtgggag tcattgggtg aggttaaaga acccgattta 420 tcgtcgtggt ctgcgtatca gcatggtcaa tcatttgttg acgccgtctt ggcgggaaca 480 actgcgcggg cgtcatggca gattgctccc ggagatgatt gggcgctggc tttggcttct 540 ttggcggtca aggttgtcaa agacggcggc ggagcgcttc tcgtagtgcc tgatcagcgc 600 gatctcgacc gcttggaagc tgcgcttcga ggtttggttg cggcgaaaca aatcactgtg 660 cttaattcag gtcttggtcc gcaggcacga tatcggcgtt tcctatcggt actcagtggg 720 cagggacgac tgattattgg aaccagaagt gccgcttttg cacccgtgaa ggatctgaaa 780 ctggccgtca ttttaaatga tggcgacgac aacctcgttg atcctagagc gccctatgcc 840 cacgccaggg aagtgctgac cacgcgttcc agtttggaag caagctcctt gattattgcg 900 ggacatgcgc agaccgcgga aacccaattg ctggtggaat cgggatggat gcacaatctc 960 atcgcaccga gggataccat tcgcactagg atgccgcgta ttcaggcagt gggcgattcc 1020 gatttccaga tggaacgcga tccaatggcc cgatcagcgc ggctgcctgg cattgcgttt 1080 catgcggtgc gcagcgcctt agaacgtgat caaccagcgc ttatccaggt accaaggaaa 1140 ggctacgtgc caaccttggc gtgtggaaac tgccgcaccc cagcgcggtg ccggcactgt 1200 aatgggcctg tgggacttcc ccagggaagc tctgatctag cgggagtgcc cacttgccga 1260 tggtgcggac gccctgattc gcggtttaag tgccaaaact gcggctctcc aaaactgcgt 1320 gctgtggtgc tgggaacgga acgcacagca gaagaactgg gccgcgcgtt cccgtctgtg 1380 cgggtaatta cctctggtgg caacaaggtg gtggattcgg tggaaaaccg agccagcatt 1440 gtggtgtcca cgccaggtgc agaacctttt gtggccaact cgccggagag accagagaaa 1500 tcggagaaac cagagcacaa gggcgcttac ggtgccttgt tattgctgga tacctgggcg 1560 ttgatgggtc ggcaagatct gcgcgccatg gaggacgcgc tgcacaagtg ggcagcggcg 1620 gccacgctgg tgcattctca tctgcaccag ggtcaagtca tcgtggttgc agatccatcg 1680 tttcctgctg tgcaatcgtt gattcggtgg gatatggcag gtgctgcagc gcaagagttg 1740 gctagccgac gcgaggttat gttcccgcct tctgtacaca tggcagcaat cgatggggct 1800 accgctgcat tggaaagttt cttggatttg gcggagcttc ccgatcatgc tgaagtcctc 1860 ggccctgttg atctgccacc gggtgtgagt ttgcctggtg aatatgatga gcagcgcttt 1920 ggtccgccgc agcgccttct catcagaact ccattgggac cgcggtctga gttgggtaga 1980 gcgctgcgct cagcccaggt ggcgcgtgcg gtgaggaaga atgatttgcc gttgcgtatt 2040 cagatggatc cgattcacat cggataa 2067 <210> 27 <211> 688 <212> PRT <213> Artificial Sequence <220> <223> (WT)_primosome assembly protein <400> 27 Met Ala Lys Thr Arg Val Pro Ala Pro Glu Lys Ser Val Ala Arg Val 1 5 10 15 Leu Pro Leu Leu Gly Leu Pro His Leu Asp Arg Leu Phe Asp Tyr Arg 20 25 30 Ile Ser Glu Asp Gln His Asp Asp Val Gln Pro Gly Val Arg Val Arg 35 40 45 Val Arg Phe Gly Gly Arg Leu Val Asp Ala Ile Val Met Ser Arg Thr 50 55 60 Ala Gln Thr Ser His Glu Gly Lys Leu Met Trp Leu Asp Arg Val Ile 65 70 75 80 Ser Pro Ile Val Val Tyr Pro Pro Gln Thr Ala Lys Leu Ile Glu Gln 85 90 95 Leu Ser Asp Arg Tyr Gly Gly Val Arg Ser Asp Leu Ile Arg Ser Ala 100 105 110 Leu Pro Ala Arg His Ala Gly Ala Glu Glu Ala Asp Thr Ser Thr Ser 115 120 125 Trp Glu Ser Leu Gly Glu Val Lys Glu Pro Asp Leu Ser Ser Trp Ser 130 135 140 Ala Tyr Gln His Gly Gln Ser Phe Val Asp Ala Val Leu Ala Gly Thr 145 150 155 160 Thr Ala Arg Ala Ser Trp Gln Ile Ala Pro Gly Asp Asp Trp Ala Leu 165 170 175 Ala Leu Ala Ser Leu Ala Val Lys Val Val Lys Asp Gly Gly Gly Ala 180 185 190 Leu Leu Val Val Pro Asp Gln Arg Asp Leu Asp Arg Leu Glu Ala Ala 195 200 205 Leu Arg Gly Leu Val Ala Ala Lys Gln Ile Thr Val Leu Asn Ser Gly 210 215 220 Leu Gly Pro Gln Ala Arg Tyr Arg Arg Phe Leu Ser Val Leu Ser Gly 225 230 235 240 Gln Gly Arg Leu Ile Ile Gly Thr Arg Ser Ala Ala Phe Ala Pro Val 245 250 255 Lys Asp Leu Lys Leu Ala Val Ile Leu Asn Asp Gly Asp Asp Asn Leu 260 265 270 Val Asp Pro Arg Ala Pro Tyr Ala His Ala Arg Glu Val Leu Thr Thr 275 280 285 Arg Ser Ser Leu Glu Ala Ser Ser Leu Ile Ile Ala Gly His Ala Arg 290 295 300 Thr Ala Glu Thr Gln Leu Leu Val Glu Ser Gly Trp Met His Asn Leu 305 310 315 320 Ile Ala Pro Arg Asp Thr Ile Arg Thr Arg Met Pro Arg Ile Gln Ala 325 330 335 Val Gly Asp Ser Asp Phe Gln Met Glu Arg Asp Pro Met Ala Arg Ser 340 345 350 Ala Arg Leu Pro Gly Ile Ala Phe His Ala Val Arg Ser Ala Leu Glu 355 360 365 Arg Asp Gln Pro Ala Leu Ile Gln Val Pro Arg Lys Gly Tyr Val Pro 370 375 380 Thr Leu Ala Cys Gly Asn Cys Arg Thr Pro Ala Arg Cys Arg His Cys 385 390 395 400 Asn Gly Pro Val Gly Leu Pro Gln Gly Ser Ser Asp Leu Ala Gly Val 405 410 415 Pro Thr Cys Arg Trp Cys Gly Arg Pro Asp Ser Arg Phe Lys Cys Gln 420 425 430 Asn Cys Gly Ser Pro Lys Leu Arg Ala Val Val Leu Gly Thr Glu Arg 435 440 445 Thr Ala Glu Glu Leu Gly Arg Ala Phe Pro Ser Val Arg Val Ile Thr 450 455 460 Ser Gly Gly Asn Lys Val Val Asp Ser Val Glu Asn Arg Ala Ser Ile 465 470 475 480 Val Val Ser Thr Pro Gly Ala Glu Pro Phe Val Ala Asn Ser Pro Glu 485 490 495 Arg Pro Glu Lys Ser Glu Lys Pro Glu His Lys Gly Ala Tyr Gly Ala 500 505 510 Leu Leu Leu Leu Asp Thr Trp Ala Leu Met Gly Arg Gln Asp Leu Arg 515 520 525 Ala Met Glu Asp Ala Leu His Lys Trp Ala Ala Ala Ala Thr Leu Val 530 535 540 His Ser His Leu His Gln Gly Gln Val Ile Val Val Ala Asp Pro Ser 545 550 555 560 Phe Pro Ala Val Gln Ser Leu Ile Arg Trp Asp Met Ala Gly Ala Ala 565 570 575 Ala Gln Glu Leu Ala Ser Arg Arg Glu Val Met Phe Pro Ser Val 580 585 590 His Met Ala Ala Ile Asp Gly Ala Thr Ala Ala Leu Glu Ser Phe Leu 595 600 605 Asp Leu Ala Glu Leu Pro Asp His Ala Glu Val Leu Gly Pro Val Asp 610 615 620 Leu Pro Pro Gly Val Ser Leu Pro Gly Glu Tyr Asp Glu Gln Arg Phe 625 630 635 640 Gly Pro Pro Gln Arg Leu Leu Ile Arg Thr Pro Leu Gly Pro Arg Ser 645 650 655 Glu Leu Gly Arg Ala Leu Arg Ser Ala Gln Val Ala Arg Ala Val Arg 660 665 670 Lys Asn Asp Leu Pro Leu Arg Ile Gln Met Asp Pro Ile His Ile Gly 675 680 685 <210> 28 <211> 2067 <212> DNA <213> Artificial Sequence <220> <223> (WT)_NCgl1540 <400> 28 atggcaaaaa cccgcgtccc cgctcctgaa aagtcggtgg cgcgggtttt acctcttttg 60 gggttacctc acctggatcg actgtttgat taccgcatca gcgaagacca acacgatgat 120 gtgcaacctg gcgtgcgggt gcgcgtgcgt tttggtggac gtttagttga tgccatcgtg 180 atgtcacgca ccgcgcaaac ctcgcacgag ggaaagctga tgtggctgga tcgggtgatt 240 tcgccgatcg tggtgtatcc acctcaaaca gcaaagctaa ttgagcaact cagtgatcgc 300 tatggcgggg tacgttcgga tctcatccgt tcggcgctac cggcgcggca tgctggggca 360 gaagaggcag atacctccac gtcgtgggag tcattgggtg aggttaaaga acccgattta 420 tcgtcgtggt ctgcgtatca gcatggtcaa tcatttgttg acgccgtctt ggcgggaaca 480 actgcgcggg cgtcatggca gattgctccc ggagatgatt gggcgctggc tttggcttct 540 ttggcggtca aggttgtcaa agacggcggc ggagcgcttc tcgtagtgcc tgatcagcgc 600 gatctcgacc gcttggaagc tgcgcttcga ggtttggttg cggcgaaaca aatcactgtg 660 cttaattcag gtcttggtcc gcaggcacga tatcggcgtt tcctatcggt actcagtggg 720 cagggacgac tgattattgg aaccagaagt gccgcttttg cacccgtgaa ggatctgaaa 780 ctggccgtca ttttaaatga tggcgacgac aacctcgttg atcctagagc gccctatgcc 840 cacgccaggg aagtgctgac cacgcgttcc agtttggaag caagctcctt gattattgcg 900 ggacatgcgc ggaccgcgga aacccaattg ctggtggaat cgggatggat gcacaatctc 960 atcgcaccga gggataccat tcgcactagg atgccgcgta ttcaggcagt gggcgattcc 1020 gatttccaga tggaacgcga tccaatggcc cgatcagcgc ggctgcctgg cattgcgttt 1080 catgcggtgc gcagcgcctt agaacgtgat caaccagcgc ttatccaggt accaaggaaa 1140 ggctacgtgc caaccttggc gtgtggaaac tgccgcaccc cagcgcggtg ccggcactgt 1200 aatgggcctg tgggacttcc ccagggaagc tctgatctag cgggagtgcc cacttgccga 1260 tggtgcggac gccctgattc gcggtttaag tgccaaaact gcggctctcc aaaactgcgt 1320 gctgtggtgc tgggaacgga acgcacagca gaagaactgg gccgcgcgtt cccgtctgtg 1380 cgggtaatta cctctggtgg caacaaggtg gtggattcgg tggaaaaccg agccagcatt 1440 gtggtgtcca cgccaggtgc agaacctttt gtggccaact cgccggagag accagagaaa 1500 tcggagaaac cagagcacaa gggcgcttac ggtgccttgt tattgctgga tacctgggcg 1560 ttgatgggtc ggcaagatct gcgcgccatg gaggacgcgc tgcacaagtg ggcagcggcg 1620 gccacgctgg tgcattctca tctgcaccag ggtcaagtca tcgtggttgc agatccatcg 1680 tttcctgctg tgcaatcgtt gattcggtgg gatatggcag gtgctgcagc gcaagagttg 1740 gctagccgac gcgaggttat gttcccgcct tctgtacaca tggcagcaat cgatggggct 1800 accgctgcat tggaaagttt cttggatttg gcggagcttc ccgatcatgc tgaagtcctc 1860 ggccctgttg atctgccacc gggtgtgagt ttgcctggtg aatatgatga gcagcgcttt 1920 ggtccgccgc agcgccttct catcagaact ccattgggac cgcggtctga gttgggtaga 1980 gcgctgcgct cagcccaggt ggcgcgtgcg gtgaggaaga atgatttgcc gttgcgtatt 2040 cagatggatc cgattcacat cggataa 2067 <210> 29 <211> 437 <212> PRT <213> Artificial Sequence <220> <223> (A169V)_type II citrate synthase <400> 29 Met Phe Glu Arg Asp Ile Val Ala Thr Asp Asn Asn Lys Ala Val Leu 1 5 10 15 His Tyr Pro Gly Gly Glu Phe Glu Met Asp Ile Ile Glu Ala Ser Glu 20 25 30 Gly Asn Asn Gly Val Val Leu Gly Lys Met Leu Ser Glu Thr Gly Leu 35 40 45 Ile Thr Phe Asp Pro Gly Tyr Val Ser Thr Gly Ser Thr Glu Ser Lys 50 55 60 Ile Thr Tyr Ile Asp Gly Asp Ala Gly Ile Leu Arg Tyr Arg Gly Tyr 65 70 75 80 Asp Ile Ala Asp Leu Ala Glu Asn Ala Thr Phe Asn Glu Val Ser Tyr 85 90 95 Leu Leu Ile Asn Gly Glu Leu Pro Thr Pro Asp Glu Leu His Lys Phe 100 105 110 Asn Asp Glu Ile Arg His His Thr Leu Leu Asp Glu Asp Phe Lys Ser 115 120 125 Gln Phe Asn Val Phe Pro Arg Asp Ala His Pro Met Ala Thr Leu Ala 130 135 140 Ser Ser Val Asn Ile Leu Ser Thr Tyr Tyr Gln Asp Gln Leu Asn Pro 145 150 155 160 Leu Asp Glu Ala Gln Leu Asp Lys Val Thr Val Arg Leu Met Ala Lys 165 170 175 Val Pro Met Leu Ala Ala Tyr Ala His Arg Ala Arg Lys Gly Ala Pro 180 185 190 Tyr Met Tyr Pro Asp Asn Ser Leu Asn Ala Arg Glu Asn Phe Leu Arg 195 200 205 Met Met Phe Gly Tyr Pro Thr Glu Pro Tyr Glu Ile Asp Pro Ile Met 210 215 220 Val Lys Ala Leu Asp Lys Leu Leu Ile Leu His Ala Asp His Glu Gln 225 230 235 240 Asn Cys Ser Thr Ser Thr Val Arg Met Ile Gly Ser Ala Gln Ala Asn 245 250 255 Met Phe Val Ser Ile Ala Gly Gly Ile Asn Ala Leu Ser Gly Pro Leu 260 265 270 His Gly Gly Ala Asn Gln Ala Val Leu Glu Met Leu Glu Asp Ile Lys 275 280 285 Ser Asn His Gly Gly Asp Ala Thr Glu Phe Met Asn Lys Val Lys Asn 290 295 300 Lys Glu Asp Gly Val Arg Leu Met Gly Phe Gly His Arg Val Tyr Lys 305 310 315 320 Asn Tyr Asp Pro Arg Ala Ala Ile Val Lys Glu Thr Ala His Glu Ile 325 330 335 Leu Glu His Leu Gly Gly Asp Asp Leu Leu Asp Leu Ala Ile Lys Leu 340 345 350 Glu Glu Ile Ala Leu Ala Asp Asp Tyr Phe Ile Ser Arg Lys Leu Tyr 355 360 365 Pro Asn Val Asp Phe Tyr Thr Gly Leu Ile Tyr Arg Ala Met Gly Phe 370 375 380 Pro Thr Asp Phe Phe Thr Val Leu Phe Ala Ile Gly Arg Leu Pro Gly 385 390 395 400 Trp Ile Ala His Tyr Arg Glu Gln Leu Gly Ala Ala Gly Asn Lys Ile 405 410 415 Asn Arg Pro Arg Gln Val Tyr Thr Gly Asn Glu Ser Arg Lys Leu Val 420 425 430 Pro Arg Glu Glu Arg 435 <210> 30 <211> 1314 <212> DNA <213> Artificial Sequence <220> <223> (A169V)_gltA <400> 30 atgtttgaaa gggatatcgt ggctactgat aacaacaagg ctgtcctgca ctaccccggt 60 ggcgagttcg aaatggacat catcgaggct tctgagggta acaacggtgt tgtcctgggc 120 aagatgctgt ctgagactgg actgatcact tttgacccag gttatgtgag cactggctcc 180 accgagtcga agatcaccta catcgatggc gatgcgggaa tcctgcgtta ccgcggctat 240 gacatcgctg atctggctga gaatgccacc ttcaacgagg tttcttacct acttatcaac 300 ggtgagctac caaccccaga tgagcttcac aagtttaacg acgagattcg ccaccacacc 360 cttctggacg aggacttcaa gtcccagttc aacgtgttcc cacgcgacgc tcacccaatg 420 gcaaccttgg cttcctcggt taacattttg tctacctact accaggacca gctgaaccca 480 ctcgatgagg cacagcttga taaggtaacc gttcgcctca tggcaaaggt tccaatgctg 540 gctgcgtacg cacaccgcgc acgcaagggt gctccttaca tgtacccaga caactccctc 600 aatgcgcgtg agaacttcct gcgcatgatg ttcggttacc caaccgagcc atacgagatc 660 gacccaatca tggtcaaggc tctggacaag ctgctcatcc tgcacgctga ccacgagcag 720 aactgctcca cctccaccgt tcgtatgatc ggttccgcac aggccaacat gtttgtctcc 780 atcgctggtg gcatcaacgc tctgtccggc ccactgcacg gtggcgcaaa ccaggctgtt 840 ctggagatgc tcgaagacat caagagcaac cacggtggcg acgcaaccga gttcatgaac 900 aaggtcaaga acaaggaaga cggcgtccgc ctcatgggct tcggacaccg cgtttacaag 960 aactacgatc cacgtgcagc aatcgtcaag gagaccgcac acgagatcct cgagcacctc 1020 ggtggcgacg atcttctgga tctggcaatc aagctggaag aaattgcact ggctgatgat 1080 tacttcatct cccgcaagct ctacccgaac gtagacttct acaccggcct gatctaccgc 1140 gcaatgggct tcccaactga cttcttcacc gtattgttcg caatcggtcg tctgccagga 1200 tggatcgctc actaccgcga gcagctcggt gcagcaggca acaagatcaa ccgcccacgc 1260 caggtctaca ccggcaacga atcccgcaag ttggttcctc gcgaggagcg ctaa 1314 <210> 31 <211> 437 <212> PRT <213> Artificial Sequence <220> <223> (WT)_type II citrate synthase <400> 31 Met Phe Glu Arg Asp Ile Val Ala Thr Asp Asn Asn Lys Ala Val Leu 1 5 10 15 His Tyr Pro Gly Gly Glu Phe Glu Met Asp Ile Ile Glu Ala Ser Glu 20 25 30 Gly Asn Asn Gly Val Val Leu Gly Lys Met Leu Ser Glu Thr Gly Leu 35 40 45 Ile Thr Phe Asp Pro Gly Tyr Val Ser Thr Gly Ser Thr Glu Ser Lys 50 55 60 Ile Thr Tyr Ile Asp Gly Asp Ala Gly Ile Leu Arg Tyr Arg Gly Tyr 65 70 75 80 Asp Ile Ala Asp Leu Ala Glu Asn Ala Thr Phe Asn Glu Val Ser Tyr 85 90 95 Leu Leu Ile Asn Gly Glu Leu Pro Thr Pro Asp Glu Leu His Lys Phe 100 105 110 Asn Asp Glu Ile Arg His His Thr Leu Leu Asp Glu Asp Phe Lys Ser 115 120 125 Gln Phe Asn Val Phe Pro Arg Asp Ala His Pro Met Ala Thr Leu Ala 130 135 140 Ser Ser Val Asn Ile Leu Ser Thr Tyr Tyr Gln Asp Gln Leu Asn Pro 145 150 155 160 Leu Asp Glu Ala Gln Leu Asp Lys Ala Thr Val Arg Leu Met Ala Lys 165 170 175 Val Pro Met Leu Ala Ala Tyr Ala His Arg Ala Arg Lys Gly Ala Pro 180 185 190 Tyr Met Tyr Pro Asp Asn Ser Leu Asn Ala Arg Glu Asn Phe Leu Arg 195 200 205 Met Met Phe Gly Tyr Pro Thr Glu Pro Tyr Glu Ile Asp Pro Ile Met 210 215 220 Val Lys Ala Leu Asp Lys Leu Leu Ile Leu His Ala Asp His Glu Gln 225 230 235 240 Asn Cys Ser Thr Ser Thr Val Arg Met Ile Gly Ser Ala Gln Ala Asn 245 250 255 Met Phe Val Ser Ile Ala Gly Gly Ile Asn Ala Leu Ser Gly Pro Leu 260 265 270 His Gly Gly Ala Asn Gln Ala Val Leu Glu Met Leu Glu Asp Ile Lys 275 280 285 Ser Asn His Gly Gly Asp Ala Thr Glu Phe Met Asn Lys Val Lys Asn 290 295 300 Lys Glu Asp Gly Val Arg Leu Met Gly Phe Gly His Arg Val Tyr Lys 305 310 315 320 Asn Tyr Asp Pro Arg Ala Ala Ile Val Lys Glu Thr Ala His Glu Ile 325 330 335 Leu Glu His Leu Gly Gly Asp Asp Leu Leu Asp Leu Ala Ile Lys Leu 340 345 350 Glu Glu Ile Ala Leu Ala Asp Asp Tyr Phe Ile Ser Arg Lys Leu Tyr 355 360 365 Pro Asn Val Asp Phe Tyr Thr Gly Leu Ile Tyr Arg Ala Met Gly Phe 370 375 380 Pro Thr Asp Phe Phe Thr Val Leu Phe Ala Ile Gly Arg Leu Pro Gly 385 390 395 400 Trp Ile Ala His Tyr Arg Glu Gln Leu Gly Ala Ala Gly Asn Lys Ile 405 410 415 Asn Arg Pro Arg Gln Val Tyr Thr Gly Asn Glu Ser Arg Lys Leu Val 420 425 430 Pro Arg Glu Glu Arg 435 <210> 32 <211> 1314 <212> DNA <213> Artificial Sequence <220> <223> (WT)_gltA <400> 32 atgtttgaaa gggatatcgt ggctactgat aacaacaagg ctgtcctgca ctaccccggt 60 ggcgagttcg aaatggacat catcgaggct tctgagggta acaacggtgt tgtcctgggc 120 aagatgctgt ctgagactgg actgatcact tttgacccag gttatgtgag cactggctcc 180 accgagtcga agatcaccta catcgatggc gatgcgggaa tcctgcgtta ccgcggctat 240 gacatcgctg atctggctga gaatgccacc ttcaacgagg tttcttacct acttatcaac 300 ggtgagctac caaccccaga tgagcttcac aagtttaacg acgagattcg ccaccacacc 360 cttctggacg aggacttcaa gtcccagttc aacgtgttcc cacgcgacgc tcacccaatg 420 gcaaccttgg cttcctcggt taacattttg tctacctact accaggacca gctgaaccca 480 ctcgatgagg cacagcttga taaggcaacc gttcgcctca tggcaaaggt tccaatgctg 540 gctgcgtacg cacaccgcgc acgcaagggt gctccttaca tgtacccaga caactccctc 600 aatgcgcgtg agaacttcct gcgcatgatg ttcggttacc caaccgagcc atacgagatc 660 gacccaatca tggtcaaggc tctggacaag ctgctcatcc tgcacgctga ccacgagcag 720 aactgctcca cctccaccgt tcgtatgatc ggttccgcac aggccaacat gtttgtctcc 780 atcgctggtg gcatcaacgc tctgtccggc ccactgcacg gtggcgcaaa ccaggctgtt 840 ctggagatgc tcgaagacat caagagcaac cacggtggcg acgcaaccga gttcatgaac 900 aaggtcaaga acaaggaaga cggcgtccgc ctcatgggct tcggacaccg cgtttacaag 960 aactacgatc cacgtgcagc aatcgtcaag gagaccgcac acgagatcct cgagcacctc 1020 ggtggcgacg atcttctgga tctggcaatc aagctggaag aaattgcact ggctgatgat 1080 tacttcatct cccgcaagct ctacccgaac gtagacttct acaccggcct gatctaccgc 1140 gcaatgggct tcccaactga cttcttcacc gtattgttcg caatcggtcg tctgccagga 1200 tggatcgctc actaccgcga gcagctcggt gcagcaggca acaagatcaa ccgcccacgc 1260 caggtctaca ccggcaacga atcccgcaag ttggttcctc gcgaggagcg ctaa 1314 <210> 33 <211> 369 <212> PRT <213> Artificial Sequence <220> <223> (E294K)_TerC <400> 33 Met Glu Val Asn Leu Ala Thr Trp Leu Ile Thr Ile Ala Val Ile Ala 1 5 10 15 Gly Phe Phe Ile Phe Asp Phe Tyr Ser His Val Arg Thr Pro His Glu 20 25 30 Pro Thr Ile Lys Glu Ser Ala Trp Trp Ser Leu Phe Tyr Val Ala Leu 35 40 45 Ala Cys Val Phe Gly Val Phe Leu Trp Phe Ala Trp Gly Glu Pro Gly 50 55 60 Asn Pro His Gln His Gly Ile Glu Phe Phe Thr Gly Tyr Val Thr Glu 65 70 75 80 Lys Ala Leu Ser Val Asp Asn Leu Phe Ile Phe Ala Leu Ile Met Gly 85 90 95 Ser Phe Lys Ile Pro Arg Lys Tyr Gln Gln Lys Val Leu Leu Ile Gly 100 105 110 Ile Ala Leu Ala Leu Val Phe Arg Leu Ala Phe Ile Leu Ala Gly Ala 115 120 125 Ala Val Ile Glu Ala Trp Ser Asp Val Phe Tyr Ile Phe Ser Ile Trp 130 135 140 Leu Ile Tyr Thr Ala Val Lys Leu Leu Trp Asp Glu Val Arg Asp Thr 145 150 155 160 Pro Glu Thr Asp Pro Asn Asp Met Phe Ile Ile Lys Ala Leu Arg Lys 165 170 175 Val Ile Pro Val Thr Glu Gly Tyr His Gly Asp Lys Leu Thr His Arg 180 185 190 Phe Gly Gly Lys Leu His Leu Thr Pro Leu Phe Val Ala Leu Val Ser 195 200 205 Ile Gly Met Val Asp Leu Met Phe Ala Leu Asp Ser Ile Pro Ala Ile 210 215 220 Tyr Gly Ile Thr Thr Glu Pro Tyr Ile Val Phe Thr Thr Asn Ala Phe 225 230 235 240 Ala Leu Leu Gly Leu Arg Gln Met Tyr Phe Leu Leu Asp Gly Leu Leu 245 250 255 Asp Arg Leu Val Tyr Leu Pro Tyr Gly Leu Gly Leu Ile Leu Leu Phe 260 265 270 Ile Gly Ala Lys Leu Gly Leu His Ala Leu His Glu Asn Asn Leu Pro 275 280 285 Phe Ile Asn Gly Gly Lys Asn Val Ser Val Pro Glu Val Ser Thr Val 290 295 300 Phe Ser Leu Val Phe Ile Ile Gly Val Leu Thr Ile Thr Val Ile Ala 305 310 315 320 Ser Ile Ile Lys Asn Lys Arg Asp Glu Asn Gln Gly Gly Ile Pro Pro 325 330 335 Lys Trp Asn Ala Ala Lys Tyr Glu Gly Asp Asn Trp Glu Thr Gly Glu 340 345 350 Glu Pro Glu Thr Gly Ser Ala Thr Val Thr Asp Ile Ser Ser Glu Lys 355 360 365 Lys <210> 34 <211> 1110 <212> DNA <213> Artificial Sequence <220> <223> (E294K)_terC <400> 34 atggaagtaa acttagccac atggctaatc actatcgcag tgattgctgg cttcttcatt 60 ttcgatttct attcccacgt ccgcacccca cacgagccca ctatcaaaga atccgcatgg 120 tggagcctct tctacgtagc cctcgcctgt gttttcggcg tgttcctctg gtttgcttgg 180 ggcgagccag gtaacccaca ccagcacggc attgagttct tcaccggtta cgtgacagag 240 aaggcgttga gtgttgataa cctcttcatc ttcgcgctga tcatgggttc tttcaagatt 300 cctcgcaagt accagcagaa ggttctgctc atcggtatcg cgctggcact ggtcttccgc 360 ctggcattca tcctcgcagg tgctgcagtt atcgaagcct ggtccgatgt cttctacatc 420 ttctccatct ggctgatcta caccgctgtg aagctcctgt gggacgaagt tcgagacacc 480 cctgagaccg acccgaacga catgttcatc attaaggcgc tgcgcaaggt cattccggtt 540 actgagggct accacggcga caagctcact caccgcttcg gaggcaagct gcacctgacc 600 ccactgttcg ttgcacttgt atccatcggc atggttgacc tgatgttcgc actggactct 660 atcccagcga tttacggcat caccacggag ccttacatcg tgttcaccac taacgcattc 720 gccctgctgg gtctgcgcca aatgtacttc ctgcttgacg gcctgcttga ccgcctggtc 780 tacctgcctt atggcttggg tcttatcctg ctgttcattg gtgcaaagct tggtctgcat 840 gcactgcacg aaaacaacct gccattcatc aacggtggta aaaacgtctc cgtacctgag 900 gtttccaccg tgttctcctt ggtcttcatc attggtgtcc tgacaatcac cgtcatcgca 960 tccatcatca agaacaaacg cgacgaaaac cagggcggta ttccaccaaa gtggaacgct 1020 gcaaagtatg agggcgacaa ctgggagacc ggtgaggagc ctgaaaccgg atccgccacc 1080 gttaccgata tctcctcaga gaagaagtaa 1110 <210> 35 <211> 369 <212> PRT <213> Artificial Sequence <220> <223> (WT)_TerC <400> 35 Met Glu Val Asn Leu Ala Thr Trp Leu Ile Thr Ile Ala Val Ile Ala 1 5 10 15 Gly Phe Phe Ile Phe Asp Phe Tyr Ser His Val Arg Thr Pro His Glu 20 25 30 Pro Thr Ile Lys Glu Ser Ala Trp Trp Ser Leu Phe Tyr Val Ala Leu 35 40 45 Ala Cys Val Phe Gly Val Phe Leu Trp Phe Ala Trp Gly Glu Pro Gly 50 55 60 Asn Pro His Gln His Gly Ile Glu Phe Phe Thr Gly Tyr Val Thr Glu 65 70 75 80 Lys Ala Leu Ser Val Asp Asn Leu Phe Ile Phe Ala Leu Ile Met Gly 85 90 95 Ser Phe Lys Ile Pro Arg Lys Tyr Gln Gln Lys Val Leu Leu Ile Gly 100 105 110 Ile Ala Leu Ala Leu Val Phe Arg Leu Ala Phe Ile Leu Ala Gly Ala 115 120 125 Ala Val Ile Glu Ala Trp Ser Asp Val Phe Tyr Ile Phe Ser Ile Trp 130 135 140 Leu Ile Tyr Thr Ala Val Lys Leu Leu Trp Asp Glu Val Arg Asp Thr 145 150 155 160 Pro Glu Thr Asp Pro Asn Asp Met Phe Ile Ile Lys Ala Leu Arg Lys 165 170 175 Val Ile Pro Val Thr Glu Gly Tyr His Gly Asp Lys Leu Thr His Arg 180 185 190 Phe Gly Gly Lys Leu His Leu Thr Pro Leu Phe Val Ala Leu Val Ser 195 200 205 Ile Gly Met Val Asp Leu Met Phe Ala Leu Asp Ser Ile Pro Ala Ile 210 215 220 Tyr Gly Ile Thr Thr Glu Pro Tyr Ile Val Phe Thr Thr Asn Ala Phe 225 230 235 240 Ala Leu Leu Gly Leu Arg Gln Met Tyr Phe Leu Leu Asp Gly Leu Leu 245 250 255 Asp Arg Leu Val Tyr Leu Pro Tyr Gly Leu Gly Leu Ile Leu Leu Phe 260 265 270 Ile Gly Ala Lys Leu Gly Leu His Ala Leu His Glu Asn Asn Leu Pro 275 280 285 Phe Ile Asn Gly Gly Glu Asn Val Ser Val Pro Glu Val Ser Thr Val 290 295 300 Phe Ser Leu Val Phe Ile Ile Gly Val Leu Thr Ile Thr Val Ile Ala 305 310 315 320 Ser Ile Ile Lys Asn Lys Arg Asp Glu Asn Gln Gly Gly Ile Pro Pro 325 330 335 Lys Trp Asn Ala Ala Lys Tyr Glu Gly Asp Asn Trp Glu Thr Gly Glu 340 345 350 Glu Pro Glu Thr Gly Ser Ala Thr Val Thr Asp Ile Ser Ser Glu Lys 355 360 365 Lys <210> 36 <211> 1110 <212> DNA <213> Artificial Sequence <220> <223> (WT)_terC <400> 36 atggaagtaa acttagccac atggctaatc actatcgcag tgattgctgg cttcttcatt 60 ttcgatttct attcccacgt ccgcacccca cacgagccca ctatcaaaga atccgcatgg 120 tggagcctct tctacgtagc cctcgcctgt gttttcggcg tgttcctctg gtttgcttgg 180 ggcgagccag gtaacccaca ccagcacggc attgagttct tcaccggtta cgtgacagag 240 aaggcgttga gtgttgataa cctcttcatc ttcgcgctga tcatgggttc tttcaagatt 300 cctcgcaagt accagcagaa ggttctgctc atcggtatcg cgctggcact ggtcttccgc 360 ctggcattca tcctcgcagg tgctgcagtt atcgaagcct ggtccgatgt cttctacatc 420 ttctccatct ggctgatcta caccgctgtg aagctcctgt gggacgaagt tcgagacacc 480 cctgagaccg acccgaacga catgttcatc attaaggcgc tgcgcaaggt cattccggtt 540 actgagggct accacggcga caagctcact caccgcttcg gaggcaagct gcacctgacc 600 ccactgttcg ttgcacttgt atccatcggc atggttgacc tgatgttcgc actggactct 660 atcccagcga tttacggcat caccacggag ccttacatcg tgttcaccac taacgcattc 720 gccctgctgg gtctgcgcca aatgtacttc ctgcttgacg gcctgcttga ccgcctggtc 780 tacctgcctt atggcttggg tcttatcctg ctgttcattg gtgcaaagct tggtctgcat 840 gcactgcacg aaaacaacct gccattcatc aacggtggtg aaaacgtctc cgtacctgag 900 gtttccaccg tgttctcctt ggtcttcatc attggtgtcc tgacaatcac cgtcatcgca 960 tccatcatca agaacaaacg cgacgaaaac cagggcggta ttccaccaaa gtggaacgct 1020 gcaaagtatg agggcgacaa ctgggagacc ggtgaggagc ctgaaaccgg atccgccacc 1080 gttaccgata tctcctcaga gaagaagtaa 1110 <210> 37 <211> 465 <212> PRT <213> Artificial Sequence <220> <223> (P358S)_mycothione reductase <400> 37 Met Ser Glu Gln Pro Ala Ser Ile Lys His Tyr Asp Leu Ile Ile Ile 1 5 10 15 Gly Thr Gly Ser Gly Asn Ser Ile Pro Gly Pro Glu Phe Asp Asp Lys 20 25 30 Ser Ile Ala Ile Val Glu Lys Gly Ala Phe Gly Gly Thr Cys Leu Asn 35 40 45 Val Gly Cys Ile Pro Thr Lys Met Tyr Val Tyr Ala Ala Asp Ile Ala 50 55 60 Gln Glu Ile Gln Glu Ser Ala Arg Leu Gly Ile Asp Ala Thr Val Asn 65 70 75 80 Ser Val Asp Trp Pro Ser Ile Val Ser Arg Val Phe Asp Lys Arg Ile 85 90 95 Asp Leu Ile Ala Gln Gly Gly Glu Ala Tyr Arg Arg Gly Pro Glu Thr 100 105 110 Pro Asn Ile Asp Val Tyr Asp Met His Ala Ser Phe Val Asp Ser Lys 115 120 125 Thr Ile Ser Thr Gly Ile Ala Gly Gln Glu Gln Leu Ile Ser Gly Thr 130 135 140 Asp Ile Val Ile Ala Thr Gly Ser Arg Pro Tyr Ile Pro Glu Ala Ile 145 150 155 160 Ala Glu Ser Gly Ala Arg Tyr Tyr Thr Asn Glu Asp Ile Met Arg Leu 165 170 175 Ala Gln Gln Pro Glu Ser Leu Val Ile Val Gly Gly Gly Phe Ile Ala 180 185 190 Leu Glu Phe Ala His Val Phe Glu Ala Leu Gly Thr Lys Val Thr Ile 195 200 205 Leu Asn Arg Ser Asp Val Leu Leu Arg Glu Ala Asp Ala Asp Ile Ser 210 215 220 Ala Lys Ile Leu Glu Leu Ser Lys Lys Arg Phe Asp Val Arg Leu Ser 225 230 235 240 Thr Ala Val Thr Ala Val His Asn Lys Ala Asp Gly Gly Val Lys Ile 245 250 255 Ser Thr Asp Thr Gly Asp Asp Ile Glu Ala Asp Ile Leu Leu Val Ala 260 265 270 Thr Gly Arg Thr Pro Asn Gly Asn Gln Met Asn Leu Asp Ala Ala Gly 275 280 285 Ile Glu Met Asn Gly Arg Ser Ile Lys Val Asp Glu Phe Gly Arg Thr 290 295 300 Ser Val Glu Gly Val Trp Ala Leu Gly Asp Val Ser Ser Pro Tyr Lys 305 310 315 320 Leu Lys His Val Ala Asn Ala Glu Met Arg Ala Ile Lys His Asn Leu 325 330 335 Ala Asn Pro Asn Asp Leu Gln Lys Met Pro His Asp Phe Val Pro Ser 340 345 350 Ala Val Phe Thr Asn Ser Gln Ile Ser Gln Val Gly Met Thr Glu Gln 355 360 365 Glu Ala Arg Glu Ala Gly Leu Asp Ile Thr Val Lys Ile Gln Asn Tyr 370 375 380 Ser Asp Val Ala Tyr Gly Trp Ala Met Glu Asp Lys Asp Gly Phe Val 385 390 395 400 Lys Leu Ile Ala Asp Lys Asp Thr Gly Lys Leu Val Gly Ala His Ile 405 410 415 Ile Gly Ala Gln Ala Ser Thr Leu Ile Gln Gln Leu Ile Thr Val Met 420 425 430 Ala Phe Gly Ile Asp Ala Arg Glu Ala Ala Thr Lys Gln Tyr Trp Ile 435 440 445 His Pro Ala Leu Pro Glu Val Ile Glu Asn Ala Leu Leu Gly Leu Glu 450 455 460 Phe 465 <210> 38 <211> 1398 <212> DNA <213> Artificial Sequence <220> <223> (P358S)_mtr <400> 38 atgtctgagc agccagcttc cattaagcat tatgacctca tcatcattgg taccggctct 60 ggaaactcca ttcctggacc agagtttgat gataaatcaa ttgccatcgt ggaaaagggt 120 gctttcggcg gaacttgcct caatgtgggc tgcatcccta ccaagatgta cgtttacgct 180 gcggatatcg ctcaagaaat tcaggagtct gctcgcctgg gtatcgatgc gacggtcaac 240 agcgtggatt ggccttccat cgtcagccgc gttttcgaca agcgcatcga cctcatcgcg 300 caaggcggcg aggcttatcg acgtggcccc gaaactccaa acatcgatgt gtatgacatg 360 cacgcatcgt ttgttgattc caagacaatc tccactggta ttgccggcca agaacagctg 420 atcagcggta ctgacattgt aatcgcaacc ggctcccgcc cttacatccc tgaagctatt 480 gcagagtccg gcgcacgcta ctacaccaac gaagacatca tgcgcctggc acagcagcct 540 gaatctttgg tgattgttgg tggcggtttc atcgctttgg aatttgctca cgtttttgaa 600 gcgcttggca ccaaggtcac catcctcaac cgctctgacg tgctgctgcg cgaggcagat 660 gcagacatct ccgcgaaaat cctcgagctt tccaaaaagc gtttcgacgt ccgcctcagc 720 actgcggtca ccgcagtaca caacaaggcc gacggcggcg tgaagatctc caccgacacc 780 ggcgacgaca tcgaggcaga tattttgctc gttgccactg gtcgcacccc taacggcaac 840 caaatgaact tggacgccgc aggcatcgag atgaacggtc gttccatcaa ggttgatgaa 900 ttcggtcgca ccagtgttga aggcgtgtgg gcgcttggcg atgtctcctc cccttacaag 960 ctcaagcacg tagccaatgc ggaaatgcga gcaatcaagc acaaccttgc caaccctaat 1020 gacctgcaga agatgccaca tgatttcgtg ccatcagctg ttttcaccaa ctctcagatc 1080 tcgcaggtcg gcatgactga gcaggaggcg cgtgaagctg gcctcgacat cactgtgaag 1140 atccagaact actctgatgt ggcttatggc tgggccatgg aagataagga tggattcgtt 1200 aagctcattg ccgataagga caccggcaag ttggtcggcg cgcacatcat tggtgctcaa 1260 gcctcaacac tgatccagca actgatcact gtcatggcat ttggaatcga tgcacgagaa 1320 gctgcaacca agcagtactg gattcaccct gctcttccag aagtcatcga aaacgctctt 1380 ctggggctag agttttag 1398 <210> 39 <211> 465 <212> PRT <213> Artificial Sequence <220> <223> (WT)_mycothione reductase <400> 39 Met Ser Glu Gln Pro Ala Ser Ile Lys His Tyr Asp Leu Ile Ile Ile 1 5 10 15 Gly Thr Gly Ser Gly Asn Ser Ile Pro Gly Pro Glu Phe Asp Asp Lys 20 25 30 Ser Ile Ala Ile Val Glu Lys Gly Ala Phe Gly Gly Thr Cys Leu Asn 35 40 45 Val Gly Cys Ile Pro Thr Lys Met Tyr Val Tyr Ala Ala Asp Ile Ala 50 55 60 Gln Glu Ile Gln Glu Ser Ala Arg Leu Gly Ile Asp Ala Thr Val Asn 65 70 75 80 Ser Val Asp Trp Pro Ser Ile Val Ser Arg Val Phe Asp Lys Arg Ile 85 90 95 Asp Leu Ile Ala Gln Gly Gly Glu Ala Tyr Arg Arg Gly Pro Glu Thr 100 105 110 Pro Asn Ile Asp Val Tyr Asp Met His Ala Ser Phe Val Asp Ser Lys 115 120 125 Thr Ile Ser Thr Gly Ile Ala Gly Gln Glu Gln Leu Ile Ser Gly Thr 130 135 140 Asp Ile Val Ile Ala Thr Gly Ser Arg Pro Tyr Ile Pro Glu Ala Ile 145 150 155 160 Ala Glu Ser Gly Ala Arg Tyr Tyr Thr Asn Glu Asp Ile Met Arg Leu 165 170 175 Ala Gln Gln Pro Glu Ser Leu Val Ile Val Gly Gly Gly Phe Ile Ala 180 185 190 Leu Glu Phe Ala His Val Phe Glu Ala Leu Gly Thr Lys Val Thr Ile 195 200 205 Leu Asn Arg Ser Asp Val Leu Leu Arg Glu Ala Asp Ala Asp Ile Ser 210 215 220 Ala Lys Ile Leu Glu Leu Ser Lys Lys Arg Phe Asp Val Arg Leu Ser 225 230 235 240 Thr Ala Val Thr Ala Val His Asn Lys Ala Asp Gly Gly Val Lys Ile 245 250 255 Ser Thr Asp Thr Gly Asp Asp Ile Glu Ala Asp Ile Leu Leu Val Ala 260 265 270 Thr Gly Arg Thr Pro Asn Gly Asn Gln Met Asn Leu Asp Ala Ala Gly 275 280 285 Ile Glu Met Asn Gly Arg Ser Ile Lys Val Asp Glu Phe Gly Arg Thr 290 295 300 Ser Val Glu Gly Val Trp Ala Leu Gly Asp Val Ser Ser Pro Tyr Lys 305 310 315 320 Leu Lys His Val Ala Asn Ala Glu Met Arg Ala Ile Lys His Asn Leu 325 330 335 Ala Asn Pro Asn Asp Leu Gln Lys Met Pro His Asp Phe Val Pro Ser 340 345 350 Ala Val Phe Thr Asn Pro Gln Ile Ser Gln Val Gly Met Thr Glu Gln 355 360 365 Glu Ala Arg Glu Ala Gly Leu Asp Ile Thr Val Lys Ile Gln Asn Tyr 370 375 380 Ser Asp Val Ala Tyr Gly Trp Ala Met Glu Asp Lys Asp Gly Phe Val 385 390 395 400 Lys Leu Ile Ala Asp Lys Asp Thr Gly Lys Leu Val Gly Ala His Ile 405 410 415 Ile Gly Ala Gln Ala Ser Thr Leu Ile Gln Gln Leu Ile Thr Val Met 420 425 430 Ala Phe Gly Ile Asp Ala Arg Glu Ala Ala Thr Lys Gln Tyr Trp Ile 435 440 445 His Pro Ala Leu Pro Glu Val Ile Glu Asn Ala Leu Leu Gly Leu Glu 450 455 460 Phe 465 <210> 40 <211> 1398 <212> DNA <213> Artificial Sequence <220> <223> (WT)_mtr <400> 40 atgtctgagc agccagcttc cattaagcat tatgacctca tcatcattgg taccggctct 60 ggaaactcca ttcctggacc agagtttgat gataaatcaa ttgccatcgt ggaaaagggt 120 gctttcggcg gaacttgcct caatgtgggc tgcatcccta ccaagatgta cgtttacgct 180 gcggatatcg ctcaagaaat tcaggagtct gctcgcctgg gtatcgatgc gacggtcaac 240 agcgtggatt ggccttccat cgtcagccgc gttttcgaca agcgcatcga cctcatcgcg 300 caaggcggcg aggcttatcg acgtggcccc gaaactccaa acatcgatgt gtatgacatg 360 cacgcatcgt ttgttgattc caagacaatc tccactggta ttgccggcca agaacagctg 420 atcagcggta ctgacattgt aatcgcaacc ggctcccgcc cttacatccc tgaagctatt 480 gcagagtccg gcgcacgcta ctacaccaac gaagacatca tgcgcctggc acagcagcct 540 gaatctttgg tgattgttgg tggcggtttc atcgctttgg aatttgctca cgtttttgaa 600 gcgcttggca ccaaggtcac catcctcaac cgctctgacg tgctgctgcg cgaggcagat 660 gcagacatct ccgcgaaaat cctcgagctt tccaaaaagc gtttcgacgt ccgcctcagc 720 actgcggtca ccgcagtaca caacaaggcc gacggcggcg tgaagatctc caccgacacc 780 ggcgacgaca tcgaggcaga tattttgctc gttgccactg gtcgcacccc taacggcaac 840 caaatgaact tggacgccgc aggcatcgag atgaacggtc gttccatcaa ggttgatgaa 900 ttcggtcgca ccagtgttga aggcgtgtgg gcgcttggcg atgtctcctc cccttacaag 960 ctcaagcacg tagccaatgc ggaaatgcga gcaatcaagc acaaccttgc caaccctaat 1020 gacctgcaga agatgccaca tgatttcgtg ccatcagctg ttttcaccaa ccctcagatc 1080 tcgcaggtcg gcatgactga gcaggaggcg cgtgaagctg gcctcgacat cactgtgaag 1140 atccagaact actctgatgt ggcttatggc tgggccatgg aagataagga tggattcgtt 1200 aagctcattg ccgataagga caccggcaag ttggtcggcg cgcacatcat tggtgctcaa 1260 gcctcaacac tgatccagca actgatcact gtcatggcat ttggaatcga tgcacgagaa 1320 gctgcaacca agcagtactg gattcaccct gctcttccag aagtcatcga aaacgctctt 1380 ctggggctag agttttag 1398 <210> 41 <211> 205 <212> PRT <213> Artificial Sequence <220> <223> (G130D)_Co/Zn/Cd efflux system component <400> 41 Met Ser Glu Ala Phe Asp Ala Thr Lys Val Arg Lys Ala Val Leu Thr 1 5 10 15 Val Ala Leu Leu Asn Phe Ala Tyr Phe Phe Val Glu Phe Phe Ile Ala 20 25 30 Leu Ser Ala Gly Ser Val Ser Leu Leu Ala Asp Ser Val Asp Phe Leu 35 40 45 Glu Asp Thr Ser Ile Asn Leu Leu Ile Phe Ile Ala Leu Gly Trp Pro 50 55 60 Leu Ala Arg Arg Ala Val Met Gly Lys Leu Met Ala Ile Val Ile Leu 65 70 75 80 Ala Pro Ala Ala Phe Ala Ala Trp Ala Ala Ile Gln Arg Phe Ser Ala 85 90 95 Pro Gln Ala Pro Glu Val Phe Pro Ile Ile Val Ala Ser Leu Gly Ala 100 105 110 Val Val Ile Asn Gly Ala Ser Ala Ile Ile Ile Ser Arg Val Arg Gln 115 120 125 His Asp Gly Ser Leu Gly Gln Ala Ala Phe Leu Ser Ala Arg Asn Asp 130 135 140 Val Leu Ile Asn Ile Ala Ile Ile Met Met Ala Leu Ile Thr Ala Trp 145 150 155 160 Thr Thr Ser Gly Trp Pro Asp Leu Ile Leu Gly Cys Phe Ile Ile Leu 165 170 175 Leu Ala Leu His Ala Ala His Glu Val Trp Glu Val Ser Glu Glu Glu 180 185 190 Arg Leu Ala Ser Lys Ala Leu Ala Gly Glu Ala Ile Asp 195 200 205 <210> 42 <211> 618 <212> DNA <213> Artificial Sequence <220> <223> (G130D)_N1992 <400> 42 atgagcgaag cttttgatgc aaccaaagtg cgcaaagctg tgctcaccgt cgcgctgctt 60 aacttcgctt atttctttgt agaattcttt attgcattaa gcgcaggctc cgtttctcta 120 ctggctgaca gtgtcgattt tcttgaagac acctccatca acctgctcat tttcattgcc 180 ctaggatggc cgttggcgag gcgcgcagtg atgggcaaac ttatggcgat tgtgattctt 240 gcacctgctg cttttgctgc gtgggcagcg attcaacggt tttccgcacc gcaagcgccc 300 gaagtgtttc cgatcatcgt cgcttctctg ggcgccgtcg tgatcaacgg cgcgagtgcc 360 atcattattt ctcgagtgcg acaacatgat ggctcgcttg gccaagctgc cttcctatcc 420 gcccgaaatg acgtcctgat caacattgcc atcatcatga tggccttaat taccgcatgg 480 acgacgtctg gatggccaga tttgatccta ggttgtttca tcattctgct cgcactgcac 540 gccgctcacg aggtgtggga agtcagtgag gaagaacgcc tcgcctccaa agcccttgct 600 ggggaagcca tcgattag 618 <210> 43 <211> 205 <212> PRT <213> Artificial Sequence <220> <223> (WT)_Co/Zn/Cd efflux system component <400> 43 Met Ser Glu Ala Phe Asp Ala Thr Lys Val Arg Lys Ala Val Leu Thr 1 5 10 15 Val Ala Leu Leu Asn Phe Ala Tyr Phe Phe Val Glu Phe Phe Ile Ala 20 25 30 Leu Ser Ala Gly Ser Val Ser Leu Leu Ala Asp Ser Val Asp Phe Leu 35 40 45 Glu Asp Thr Ser Ile Asn Leu Leu Ile Phe Ile Ala Leu Gly Trp Pro 50 55 60 Leu Ala Arg Arg Ala Val Met Gly Lys Leu Met Ala Ile Val Ile Leu 65 70 75 80 Ala Pro Ala Ala Phe Ala Ala Trp Ala Ala Ile Gln Arg Phe Ser Ala 85 90 95 Pro Gln Ala Pro Glu Val Phe Pro Ile Ile Val Ala Ser Leu Gly Ala 100 105 110 Val Val Ile Asn Gly Ala Ser Ala Ile Ile Ile Ser Arg Val Arg Gln 115 120 125 His Gly Gly Ser Leu Gly Gln Ala Ala Phe Leu Ser Ala Arg Asn Asp 130 135 140 Val Leu Ile Asn Ile Ala Ile Ile Met Met Ala Leu Ile Thr Ala Trp 145 150 155 160 Thr Thr Ser Gly Trp Pro Asp Leu Ile Leu Gly Cys Phe Ile Ile Leu 165 170 175 Leu Ala Leu His Ala Ala His Glu Val Trp Glu Val Ser Glu Glu Glu 180 185 190 Arg Leu Ala Ser Lys Ala Leu Ala Gly Glu Ala Ile Asp 195 200 205 <210> 44 <211> 618 <212> DNA <213> Artificial Sequence <220> <223> (WT)_N1992 <400> 44 atgagcgaag cttttgatgc aaccaaagtg cgcaaagctg tgctcaccgt cgcgctgctt 60 aacttcgctt atttctttgt agaattcttt attgcattaa gcgcaggctc cgtttctcta 120 ctggctgaca gtgtcgattt tcttgaagac acctccatca acctgctcat tttcattgcc 180 ctaggatggc cgttggcgag gcgcgcagtg atgggcaaac ttatggcgat tgtgattctt 240 gcacctgctg cttttgctgc gtgggcagcg attcaacggt tttccgcacc gcaagcgccc 300 gaagtgtttc cgatcatcgt cgcttctctg ggcgccgtcg tgatcaacgg cgcgagtgcc 360 atcattattt ctcgagtgcg acaacatggt ggctcgcttg gccaagctgc cttcctatcc 420 gcccgaaatg acgtcctgat caacattgcc atcatcatga tggccttaat taccgcatgg 480 acgacgtctg gatggccaga tttgatccta ggttgtttca tcattctgct cgcactgcac 540 gccgctcacg aggtgtggga agtcagtgag gaagaacgcc tcgcctccaa agcccttgct 600 ggggaagcca tcgattag 618 <210> 45 <211> 466 <212> PRT <213> Artificial Sequence <220> <223> (G382C)_3-deoxy-D-arabinoheptulosonate-7-phosphate synthase <400> 45 Met Asn Arg Gly Val Ser Trp Thr Val Asp Ile Pro Lys Glu Val Leu 1 5 10 15 Pro Asp Leu Pro Pro Leu Pro Glu Gly Met Gln Gln Gln Phe Glu Asp 20 25 30 Thr Ile Ser Arg Asp Ala Lys Gln Gln Pro Thr Trp Asp Arg Ala Gln 35 40 45 Ala Glu Asn Val Arg Lys Ile Leu Glu Ser Val Pro Pro Ile Val Val 50 55 60 Ala Pro Glu Val Leu Glu Leu Lys Gln Lys Leu Ala Asp Val Ala Asn 65 70 75 80 Gly Lys Ala Phe Leu Leu Gln Gly Gly Asp Cys Ala Glu Thr Phe Glu 85 90 95 Ser Asn Thr Glu Pro His Ile Arg Ala Asn Val Lys Thr Leu Leu Gln 100 105 110 Met Ala Val Val Leu Thr Tyr Gly Ala Ser Thr Pro Val Ile Lys Met 115 120 125 Ala Arg Ile Ala Gly Gln Tyr Ala Lys Pro Arg Ser Ser Asp Leu Asp 130 135 140 Gly Asn Gly Leu Pro Asn Tyr Arg Gly Asp Ile Val Asn Gly Val Glu 145 150 155 160 Ala Thr Pro Glu Ala Arg Arg His Asp Pro Ala Arg Met Ile Arg Ala 165 170 175 Tyr Ala Asn Ala Ser Ala Ala Met Asn Leu Val Arg Ala Leu Thr Ser 180 185 190 Ser Gly Thr Ala Asp Leu Tyr Arg Leu Ser Glu Trp Asn Arg Glu Phe 195 200 205 Val Ala Asn Ser Pro Ala Gly Ala Arg Tyr Glu Ala Leu Ala Arg Glu 210 215 220 Ile Asp Ser Gly Leu Arg Phe Met Glu Ala Cys Gly Val Ser Asp Glu 225 230 235 240 Ser Leu Arg Ala Ala Asp Ile Tyr Cys Ser His Glu Ala Leu Leu Val 245 250 255 Asp Tyr Glu Arg Ser Met Leu Arg Leu Ala Thr Asp Glu Glu Gly Asn 260 265 270 Glu Glu Leu Tyr Asp Leu Ser Ala His Gln Leu Trp Ile Gly Glu Arg 275 280 285 Thr Arg Gly Met Asp Asp Phe His Val Asn Phe Ala Ser Met Ile Ser 290 295 300 Asn Pro Ile Gly Ile Lys Ile Gly Pro Gly Ile Thr Pro Glu Glu Ala 305 310 315 320 Val Ala Tyr Ala Asp Lys Leu Asp Pro Asn Phe Glu Pro Gly Arg Leu 325 330 335 Thr Ile Val Ala Arg Met Gly His Asp Lys Val Arg Ser Val Leu Pro 340 345 350 Gly Val Ile Gln Ala Val Glu Ala Ser Gly His Lys Val Ile Trp Gln 355 360 365 Ser Asp Pro Met His Gly Asn Thr Phe Thr Ala Ser Asn Cys Tyr Lys 370 375 380 Thr Arg His Phe Asp Lys Val Ile Asp Glu Val Gln Gly Phe Phe Glu 385 390 395 400 Val His Arg Ala Leu Gly Thr His Pro Gly Gly Ile His Ile Glu Phe 405 410 415 Thr Gly Glu Asp Val Thr Glu Cys Leu Gly Gly Ala Glu Asp Ile Thr 420 425 430 Asp Val Asp Leu Pro Gly Arg Tyr Glu Ser Ala Cys Asp Pro Arg Leu 435 440 445 Asn Thr Gln Gln Ser Leu Glu Leu Ala Phe Leu Val Ala Glu Met Leu 450 455 460 Arg Asn 465 <210> 46 <211> 1401 <212> DNA <213> Artificial Sequence <220> <223> (G382C)_aroG <400> 46 atgaataggg gtgtgagttg gacagttgat atccctaaag aagttctccc tgatttgcca 60 ccattgccag aaggcatgca gcagcagttc gaggacacca tttcccgtga cgctaagcag 120 caacctacgt gggatcgtgc acaggcagaa aacgtgcgca agatccttga gtcggttcct 180 ccaatcgttg ttgcccctga ggtacttgag ctgaagcaga agcttgctga tgttgccaac 240 ggtaaggcct tcctcttgca gggtggtgac tgtgcggaaa ctttcgagtc aaacactgag 300 ccgcacattc gcgccaacgt aaagactctg ctgcagatgg cagttgtttt gacctacggt 360 gcatccactc ctgtgatcaa gatggctcgt attgctggtc agtacgcaaa gcctcgctct 420 tctgatctgg atggaaatgg tctgccaaac taccgtggcg atatcgtcaa cggtgtggag 480 gcaaccccag aggctcgtcg ccacgatcct gcccgcatga tccgtgctta cgctaacgct 540 tctgctgcga tgaacttggt gcgcgcgctc accagctctg gcaccgctga tctttaccgt 600 ctcagcgagt ggaaccgcga gttcgttgcg aactccccag ctggtgcacg ctacgaggct 660 cttgctcgtg agatcgactc cggtctgcgc ttcatggaag catgtggcgt gtccgatgag 720 tccctgcgtg ctgcagatat ctactgctcc cacgaggctt tgctggtgga ttacgagcgt 780 tccatgctgc gtcttgcaac cgatgaggaa ggcaacgagg aactttacga tctttcagct 840 caccagctgt ggatcggcga gcgcacccgt ggcatggatg atttccatgt gaacttcgca 900 tccatgatct ctaacccaat cggcatcaag attggtcctg gtatcacccc tgaagaggct 960 gttgcatacg ctgacaagct cgatccgaac ttcgagcctg gccgtttgac catcgttgct 1020 cgcatgggcc acgacaaggt tcgctccgta cttcctggtg ttatccaggc tgttgaggca 1080 tccggacaca aggttatttg gcagtccgat ccgatgcacg gcaacacttt caccgcatcc 1140 aattgctaca agacccgtca cttcgacaag gttatcgatg aggtccaggg cttcttcgag 1200 gtccaccgcg cattgggcac ccacccaggc ggaatccaca ttgagttcac tggtgaagat 1260 gtcaccgagt gcctcggtgg cgctgaagac atcaccgatg ttgatctgcc aggccgctac 1320 gagtccgcat gcgatcctcg cctgaacact cagcagtctt tggagttggc tttcctcgtt 1380 gcagaaatgc tgcgtaacta a 1401 <210> 47 <211> 466 <212> PRT <213> Artificial Sequence <220> <223> (WT)_3-deoxy-D-arabinoheptulosonate-7-phosphate synthase <400> 47 Met Asn Arg Gly Val Ser Trp Thr Val Asp Ile Pro Lys Glu Val Leu 1 5 10 15 Pro Asp Leu Pro Pro Leu Pro Glu Gly Met Gln Gln Gln Phe Glu Asp 20 25 30 Thr Ile Ser Arg Asp Ala Lys Gln Gln Pro Thr Trp Asp Arg Ala Gln 35 40 45 Ala Glu Asn Val Arg Lys Ile Leu Glu Ser Val Pro Pro Ile Val Val 50 55 60 Ala Pro Glu Val Leu Glu Leu Lys Gln Lys Leu Ala Asp Val Ala Asn 65 70 75 80 Gly Lys Ala Phe Leu Leu Gln Gly Gly Asp Cys Ala Glu Thr Phe Glu 85 90 95 Ser Asn Thr Glu Pro His Ile Arg Ala Asn Val Lys Thr Leu Leu Gln 100 105 110 Met Ala Val Val Leu Thr Tyr Gly Ala Ser Thr Pro Val Ile Lys Met 115 120 125 Ala Arg Ile Ala Gly Gln Tyr Ala Lys Pro Arg Ser Ser Asp Leu Asp 130 135 140 Gly Asn Gly Leu Pro Asn Tyr Arg Gly Asp Ile Val Asn Gly Val Glu 145 150 155 160 Ala Thr Pro Glu Ala Arg Arg His Asp Pro Ala Arg Met Ile Arg Ala 165 170 175 Tyr Ala Asn Ala Ser Ala Ala Met Asn Leu Val Arg Ala Leu Thr Ser 180 185 190 Ser Gly Thr Ala Asp Leu Tyr Arg Leu Ser Glu Trp Asn Arg Glu Phe 195 200 205 Val Ala Asn Ser Pro Ala Gly Ala Arg Tyr Glu Ala Leu Ala Arg Glu 210 215 220 Ile Asp Ser Gly Leu Arg Phe Met Glu Ala Cys Gly Val Ser Asp Glu 225 230 235 240 Ser Leu Arg Ala Ala Asp Ile Tyr Cys Ser His Glu Ala Leu Leu Val 245 250 255 Asp Tyr Glu Arg Ser Met Leu Arg Leu Ala Thr Asp Glu Glu Gly Asn 260 265 270 Glu Glu Leu Tyr Asp Leu Ser Ala His Gln Leu Trp Ile Gly Glu Arg 275 280 285 Thr Arg Gly Met Asp Asp Phe His Val Asn Phe Ala Ser Met Ile Ser 290 295 300 Asn Pro Ile Gly Ile Lys Ile Gly Pro Gly Ile Thr Pro Glu Glu Ala 305 310 315 320 Val Ala Tyr Ala Asp Lys Leu Asp Pro Asn Phe Glu Pro Gly Arg Leu 325 330 335 Thr Ile Val Ala Arg Met Gly His Asp Lys Val Arg Ser Val Leu Pro 340 345 350 Gly Val Ile Gln Ala Val Glu Ala Ser Gly His Lys Val Ile Trp Gln 355 360 365 Ser Asp Pro Met His Gly Asn Thr Phe Thr Ala Ser Asn Gly Tyr Lys 370 375 380 Thr Arg His Phe Asp Lys Val Ile Asp Glu Val Gln Gly Phe Phe Glu 385 390 395 400 Val His Arg Ala Leu Gly Thr His Pro Gly Gly Ile His Ile Glu Phe 405 410 415 Thr Gly Glu Asp Val Thr Glu Cys Leu Gly Gly Ala Glu Asp Ile Thr 420 425 430 Asp Val Asp Leu Pro Gly Arg Tyr Glu Ser Ala Cys Asp Pro Arg Leu 435 440 445 Asn Thr Gln Gln Ser Leu Glu Leu Ala Phe Leu Val Ala Glu Met Leu 450 455 460 Arg Asn 465 <210> 48 <211> 1401 <212> DNA <213> Artificial Sequence <220> <223> (WT)_aroG <400> 48 atgaataggg gtgtgagttg gacagttgat atccctaaag aagttctccc tgatttgcca 60 ccattgccag aaggcatgca gcagcagttc gaggacacca tttcccgtga cgctaagcag 120 caacctacgt gggatcgtgc acaggcagaa aacgtgcgca agatccttga gtcggttcct 180 ccaatcgttg ttgcccctga ggtacttgag ctgaagcaga agcttgctga tgttgccaac 240 ggtaaggcct tcctcttgca gggtggtgac tgtgcggaaa ctttcgagtc aaacactgag 300 ccgcacattc gcgccaacgt aaagactctg ctgcagatgg cagttgtttt gacctacggt 360 gcatccactc ctgtgatcaa gatggctcgt attgctggtc agtacgcaaa gcctcgctct 420 tctgatctgg atggaaatgg tctgccaaac taccgtggcg atatcgtcaa cggtgtggag 480 gcaaccccag aggctcgtcg ccacgatcct gcccgcatga tccgtgctta cgctaacgct 540 tctgctgcga tgaacttggt gcgcgcgctc accagctctg gcaccgctga tctttaccgt 600 ctcagcgagt ggaaccgcga gttcgttgcg aactccccag ctggtgcacg ctacgaggct 660 cttgctcgtg agatcgactc cggtctgcgc ttcatggaag catgtggcgt gtccgatgag 720 tccctgcgtg ctgcagatat ctactgctcc cacgaggctt tgctggtgga ttacgagcgt 780 tccatgctgc gtcttgcaac cgatgaggaa ggcaacgagg aactttacga tctttcagct 840 caccagctgt ggatcggcga gcgcacccgt ggcatggatg atttccatgt gaacttcgca 900 tccatgatct ctaacccaat cggcatcaag attggtcctg gtatcacccc tgaagaggct 960 gttgcatacg ctgacaagct cgatccgaac ttcgagcctg gccgtttgac catcgttgct 1020 cgcatgggcc acgacaaggt tcgctccgta cttcctggtg ttatccaggc tgttgaggca 1080 tccggacaca aggttatttg gcagtccgat ccgatgcacg gcaacacttt caccgcatcc 1140 aatggctaca agacccgtca cttcgacaag gttatcgatg aggtccaggg cttcttcgag 1200 gtccaccgcg cattgggcac ccacccaggc ggaatccaca ttgagttcac tggtgaagat 1260 gtcaccgagt gcctcggtgg cgctgaagac atcaccgatg ttgatctgcc aggccgctac 1320 gagtccgcat gcgatcctcg cctgaacact cagcagtctt tggagttggc tttcctcgtt 1380 gcagaaatgc tgcgtaacta a 1401 <210> 49 <211> 367 <212> PRT <213> Artificial Sequence <220> <223> (P209L)_N-succinyldiaminopimelate aminotransferase <400> 49 Met Thr Ser Arg Thr Pro Leu Val Ser Val Leu Pro Asp Phe Pro Trp 1 5 10 15 Asp Ser Leu Ala Ser Ala Lys Ala Lys Ala Ala Ser His Pro Asp Gly 20 25 30 Ile Val Asn Leu Ser Val Gly Thr Pro Val Asp Pro Val Ala Pro Ser 35 40 45 Ile Gln Ile Ala Leu Ala Glu Ala Ala Gly Phe Ser Gly Tyr Pro Gln 50 55 60 Thr Ile Gly Thr Pro Glu Leu Arg Ala Ala Ile Arg Gly Ala Leu Glu 65 70 75 80 Arg Arg Tyr Asn Met Thr Lys Leu Val Asp Ala Ser Leu Leu Pro Val 85 90 95 Val Gly Thr Lys Glu Ala Ile Ala Leu Leu Pro Phe Ala Leu Gly Ile 100 105 110 Ser Gly Thr Val Val Ile Pro Glu Ile Ala Tyr Pro Thr Tyr Glu Val 115 120 125 Ala Val Val Ala Ala Gly Cys Thr Val Leu Arg Ser Asp Ser Leu Phe 130 135 140 Lys Leu Gly Pro Gln Ile Pro Ser Met Met Phe Ile Asn Ser Pro Ser 145 150 155 160 Asn Pro Thr Gly Lys Val Leu Gly Ile Pro His Leu Arg Lys Val Val 165 170 175 Lys Trp Ala Gln Glu Asn Asn Val Ile Leu Ala Ala Asp Glu Cys Tyr 180 185 190 Leu Gly Leu Gly Trp Asp Asp Glu Asn Pro Pro Ile Ser Ile Leu Asp 195 200 205 Leu Arg Val Cys Asp Gly Asp His Thr Asn Leu Ile Ala Ile His Ser 210 215 220 Leu Ser Lys Thr Ser Asn Leu Ala Ser Tyr Arg Ala Gly Tyr Leu Val 225 230 235 240 Gly Asp Pro Ala Leu Ile Gly Glu Leu Thr Glu Val Arg Lys Asn Leu 245 250 255 Gly Leu Met Val Pro Phe Pro Ile Gln Gln Ala Met Ile Ala Ala Leu 260 265 270 Asn Asp Asp Asp Gln Glu Ala Gly Gln Lys Leu Thr Tyr Ala Ile Arg 275 280 285 Arg Ala Lys Leu Met Arg Ala Leu Leu Glu Ser Gly Phe Gln Val Asp 290 295 300 Asn Ser Glu Ala Gly Leu Tyr Leu Trp Ala Thr Arg Glu Glu Pro Cys 305 310 315 320 Arg Asp Thr Val Asp Trp Phe Ala Glu Arg Gly Ile Leu Val Ala Pro 325 330 335 Gly Asp Phe Tyr Gly Pro Arg Gly Ala Gln His Val Arg Val Ala Met 340 345 350 Thr Glu Thr Asp Glu Arg Val Asp Ala Phe Val Ser Arg Leu Ser 355 360 365 <210> 50 <211> 1104 <212> DNA <213> Artificial Sequence <220> <223> (P209L)_NCgl1058 <400> 50 atgacctctc gcaccccgct tgtttctgtt cttcctgatt ttccgtggga ttcgctcgct 60 tccgcaaaag ccaaagctgc gtctcacccg gatgggatcg tgaatctttc tgttggcact 120 ccggttgatc cggtcgcgcc cagcattcag atcgcgttgg cagaagcagc ggggttttcg 180 ggttaccctc aaaccatcgg caccccggaa ctccgcgcag ccatcagggg cgcgcttgag 240 cggcgctaca acatgacaaa gcttgtcgac gcctccctcc tccccgtcgt gggtaccaag 300 gaggcaattg cccttcttcc attcgcgttg ggtatttccg gcaccgttgt catcccagag 360 attgcgtacc caacctacga agtcgctgtc gtggccgcag gatgcaccgt gttgcgttct 420 gattcgctgt ttaagctcgg cccgcagatc ccgtcgatga tgtttatcaa ctcaccatcc 480 aaccccacag gcaaggttct gggcatccca cacttgcgca aggttgtgaa gtgggcgcag 540 gaaaacaacg tgatcctcgc agctgatgaa tgctacttgg gtcttggctg ggacgatgaa 600 aacccaccga tctcaatttt ggatctacgt gtctgcgatg gcgaccacac caacttgatc 660 gccattcact cgctgtctaa aacctcaaac ctcgcttctt accgcgcagg ttacctcgtt 720 ggcgatccag cgctgattgg tgaactcacg gaagtccgta agaacttggg tctcatggtt 780 cctttcccaa tccagcaggc catgatcgca gccctcaacg acgatgacca agaggcaggg 840 cagaagctca cctacgcgat tcgtcgagca aaactcatgc gcgccctgtt ggaatccggc 900 tttcaggtag ataattctga agcgggtctg tacctctggg cgacgcgtga agaaccttgc 960 cgtgacactg tcgattggtt cgctgagcgt ggcattctcg ttgccccagg agacttctat 1020 ggccctcgcg gagcgcagca tgtgcgtgtg gcgatgaccg aaaccgacga gcgcgtcgac 1080 gcctttgttt ctcgcctgag ctaa 1104 <210> 51 <211> 367 <212> PRT <213> Artificial Sequence <220> <223> (WT)_N-succinyldiaminopimelate aminotransferase <400> 51 Met Thr Ser Arg Thr Pro Leu Val Ser Val Leu Pro Asp Phe Pro Trp 1 5 10 15 Asp Ser Leu Ala Ser Ala Lys Ala Lys Ala Ala Ser His Pro Asp Gly 20 25 30 Ile Val Asn Leu Ser Val Gly Thr Pro Val Asp Pro Val Ala Pro Ser 35 40 45 Ile Gln Ile Ala Leu Ala Glu Ala Ala Gly Phe Ser Gly Tyr Pro Gln 50 55 60 Thr Ile Gly Thr Pro Glu Leu Arg Ala Ala Ile Arg Gly Ala Leu Glu 65 70 75 80 Arg Arg Tyr Asn Met Thr Lys Leu Val Asp Ala Ser Leu Leu Pro Val 85 90 95 Val Gly Thr Lys Glu Ala Ile Ala Leu Leu Pro Phe Ala Leu Gly Ile 100 105 110 Ser Gly Thr Val Val Ile Pro Glu Ile Ala Tyr Pro Thr Tyr Glu Val 115 120 125 Ala Val Val Ala Ala Gly Cys Thr Val Leu Arg Ser Asp Ser Leu Phe 130 135 140 Lys Leu Gly Pro Gln Ile Pro Ser Met Met Phe Ile Asn Ser Pro Ser 145 150 155 160 Asn Pro Thr Gly Lys Val Leu Gly Ile Pro His Leu Arg Lys Val Val 165 170 175 Lys Trp Ala Gln Glu Asn Asn Val Ile Leu Ala Ala Asp Glu Cys Tyr 180 185 190 Leu Gly Leu Gly Trp Asp Asp Glu Asn Pro Pro Ile Ser Ile Leu Asp 195 200 205 Pro Arg Val Cys Asp Gly Asp His Thr Asn Leu Ile Ala Ile His Ser 210 215 220 Leu Ser Lys Thr Ser Asn Leu Ala Ser Tyr Arg Ala Gly Tyr Leu Val 225 230 235 240 Gly Asp Pro Ala Leu Ile Gly Glu Leu Thr Glu Val Arg Lys Asn Leu 245 250 255 Gly Leu Met Val Pro Phe Pro Ile Gln Gln Ala Met Ile Ala Ala Leu 260 265 270 Asn Asp Asp Asp Gln Glu Ala Gly Gln Lys Leu Thr Tyr Ala Ile Arg 275 280 285 Arg Ala Lys Leu Met Arg Ala Leu Leu Glu Ser Gly Phe Gln Val Asp 290 295 300 Asn Ser Glu Ala Gly Leu Tyr Leu Trp Ala Thr Arg Glu Glu Pro Cys 305 310 315 320 Arg Asp Thr Val Asp Trp Phe Ala Glu Arg Gly Ile Leu Val Ala Pro 325 330 335 Gly Asp Phe Tyr Gly Pro Arg Gly Ala Gln His Val Arg Val Ala Met 340 345 350 Thr Glu Thr Asp Glu Arg Val Asp Ala Phe Val Ser Arg Leu Ser 355 360 365 <210> 52 <211> 1104 <212> DNA <213> Artificial Sequence <220> <223> (WT)_NCgl1058 <400> 52 atgacctctc gcaccccgct tgtttctgtt cttcctgatt ttccgtggga ttcgctcgct 60 tccgcaaaag ccaaagctgc gtctcacccg gatgggatcg tgaatctttc tgttggcact 120 ccggttgatc cggtcgcgcc cagcattcag atcgcgttgg cagaagcagc ggggttttcg 180 ggttaccctc aaaccatcgg caccccggaa ctccgcgcag ccatcagggg cgcgcttgag 240 cggcgctaca acatgacaaa gcttgtcgac gcctccctcc tccccgtcgt gggtaccaag 300 gaggcaattg cccttcttcc attcgcgttg ggtatttccg gcaccgttgt catcccagag 360 attgcgtacc caacctacga agtcgctgtc gtggccgcag gatgcaccgt gttgcgttct 420 gattcgctgt ttaagctcgg cccgcagatc ccgtcgatga tgtttatcaa ctcaccatcc 480 aaccccacag gcaaggttct gggcatccca cacttgcgca aggttgtgaa gtgggcgcag 540 gaaaacaacg tgatcctcgc agctgatgaa tgctacttgg gtcttggctg ggacgatgaa 600 aacccaccga tctcaatttt ggatccacgt gtctgcgatg gcgaccacac caacttgatc 660 gccattcact cgctgtctaa aacctcaaac ctcgcttctt accgcgcagg ttacctcgtt 720 ggcgatccag cgctgattgg tgaactcacg gaagtccgta agaacttggg tctcatggtt 780 cctttcccaa tccagcaggc catgatcgca gccctcaacg acgatgacca agaggcaggg 840 cagaagctca cctacgcgat tcgtcgagca aaactcatgc gcgccctgtt ggaatccggc 900 tttcaggtag ataattctga agcgggtctg tacctctggg cgacgcgtga agaaccttgc 960 cgtgacactg tcgattggtt cgctgagcgt ggcattctcg ttgccccagg agacttctat 1020 ggccctcgcg gagcgcagca tgtgcgtgtg gcgatgaccg aaaccgacga gcgcgtcgac 1080 gcctttgttt ctcgcctgag ctaa 1104 <210> 53 <211> 892 <212> PRT <213> Artificial Sequence <220> <223> (Y592F)_helicase <400> 53 Met Ala Glu Ser Asn Ala Met Asp Arg Ala Gln Ile Ser Ala Leu Leu 1 5 10 15 Asp Arg Ala Gln His Thr Ile Asn Leu Ala Glu Gln Ala Asn Asn Val 20 25 30 Leu Arg Leu Leu Lys Thr Pro Gly Thr Ala Thr Val Gly Asp Asn Gly 35 40 45 Thr Leu Gly Thr Asp Thr Tyr Leu Ile Pro Ser Arg Asn Ile Thr Trp 50 55 60 Pro Asp Asn Leu Tyr Val Asn Val Phe Leu Asp Gly Met Asn Ala Glu 65 70 75 80 Ala Thr Leu Thr Asp Tyr Val Ala Ser Val Ala Ser Ile Pro Arg Leu 85 90 95 Cys Gln Ile Ile Asn Glu Gly Gln Gly Gly Met Phe Arg Arg Leu Phe 100 105 110 Asn Pro Thr Lys Val Gln Ala Gly Asp Gln Ala Val Phe Asp Leu Met 115 120 125 Val Lys Leu Asp Glu Ile Ser Ser Thr Thr His Glu Val Ser Arg Met 130 135 140 Leu Glu Gly Val His Ala Ala Arg Thr Arg Gln Gln Gln Gly Val Ala 145 150 155 160 Leu Phe Pro Gly Ile His Gly Val Gly Glu Arg Tyr Ile Glu Arg Ala 165 170 175 Gln Gln Val Leu Ala Ser Ala Leu Gly Ile Ala Gly Phe Gly Ala Glu 180 185 190 Pro Trp Asp Gly His Thr Leu Ala Gln Ala Arg Arg Val Val Gln Arg 195 200 205 Tyr Ala Gln Asp Pro Asn Ser Glu Tyr Arg Leu Lys Ser Glu Ala Glu 210 215 220 Lys His Leu Thr Ser Ile Asn Glu Leu Arg Val Gln Ile Leu Leu Glu 225 230 235 240 Gln Leu Pro Val Asp Ala Leu Arg Met Ala Thr Asp His Arg Leu Arg 245 250 255 Phe Gly Ser Leu Asp Ser Ile His Val Ala Thr Val Ala Asp Val Leu 260 265 270 Lys Thr His Thr Ser Ile Leu Thr Thr Val Gln Gly Ile Gly Ala Gln 275 280 285 Thr Ala Gly Arg Met Lys Ala Ala Ala Glu Thr Leu Lys Gln Glu Ala 290 295 300 Leu Arg Arg Gln Asn Thr Ser Ile Gly Asp Glu Pro Thr Gln Pro Ala 305 310 315 320 Met Arg Leu Ile Asn Val Leu Ala Arg Phe Asp Gln Thr Glu Thr Ile 325 330 335 Thr Pro Glu Glu Arg Ala Arg Arg Thr Arg Val Ile Asp Tyr Val Glu 340 345 350 His Ile Pro Pro Ser Leu Asp Pro Tyr Ile Val Ile Asn Pro Ala Thr 355 360 365 Pro Glu Phe Asn Asn Phe Thr Asp Asp Leu Arg Trp Ile Asp Ala Asn 370 375 380 Pro Asn Leu Phe His Pro Gln Thr Ile Thr Thr Pro Pro Ala Asp Ile 385 390 395 400 Trp Asp Asp Tyr Ile Ser Arg Pro Ala His Tyr Gln Gly Leu Leu Ala 405 410 415 Thr Leu Leu Gly Arg Asp Ile Glu Gly Ala Asp Glu Leu Leu Asp Ala 420 425 430 Thr Thr Leu Gln Lys Ile Arg Asp Leu Thr Leu Asp Lys Thr His Leu 435 440 445 Thr Asp Leu His Leu Arg Gly Tyr Gln Ser Phe Gly Ala Arg Phe Ala 450 455 460 Ile Ile Gln Lys Lys Thr Leu Leu Gly Asp Asp Met Gly Leu Gly Lys 465 470 475 480 Thr Val Gln Ala Leu Ser Ala Ala Ala His Leu Ala Ala Thr Glu Lys 485 490 495 Asp Phe Arg Thr Leu Val Val Val Pro Ala Ser Val Ile Val Asn Trp 500 505 510 Thr Arg Glu Cys Lys Arg Phe Leu Asn Leu Pro Val Phe Ile Ala His 515 520 525 Gly Asp Asn Lys Gln Asp Ala Ile Asn Ala Trp Ser Asn Thr Asn Gly 530 535 540 Ile Ala Ile Cys Thr Tyr Asp Gly Val Arg Thr Met Asp Ile Pro Ala 545 550 555 560 Pro Gly Leu Val Ile Ala Asp Glu Ala His Leu Ile Lys Asn Pro Ser 565 570 575 Thr Lys Arg Thr Gln Ala Leu Arg Lys Leu Ile Asp Ala Ala Pro Phe 580 585 590 Thr Leu Leu Met Thr Gly Thr Pro Leu Glu Asn Lys Val Glu Glu Phe 595 600 605 Val Asn Leu Val Arg Tyr Ile Gln Pro Glu Leu Ile Thr Arg Gly Met 610 615 620 Ser Lys Met Gln Ala Glu Asn Phe Arg Glu Arg Ile Ala Pro Ala Tyr 625 630 635 640 Leu Arg Arg Asn Gln Ala Asp Val Leu Asp Glu Leu Pro Glu Arg Thr 645 650 655 Asp Ser Ile Asp Trp Ile Asp Leu Thr Pro Glu Asp Arg Ser Ala Tyr 660 665 670 Asp Asp Gln Val Arg Gln Gly Ser Trp Met Gly Met Arg Arg Ser Ala 675 680 685 Met Leu Ser Pro Thr Pro Arg Leu Thr Ser Ala Lys Met Gln Arg Ile 690 695 700 Leu Glu Leu Phe Glu Glu Ala Glu Glu His Gly Arg Lys Ala Leu Ile 705 710 715 720 Phe Thr Tyr Phe Leu Asp Val Leu Asp Glu Leu Glu Lys His Leu Gly 725 730 735 Glu Arg Val Ile Gly Arg Ile Ser Gly Asp Val Pro Ala Thr Lys Arg 740 745 750 Gln Leu Leu Val Asp Ala Leu Ser His Ser Lys Pro Gly Ser Ala Leu 755 760 765 Ile Ala Gln Ile Thr Ala Gly Gly Val Gly Leu Asn Ile Gln Ser Ala 770 775 780 Ser Leu Cys Ile Ile Cys Glu Pro Gln Val Lys Pro Thr Ile Glu Gln 785 790 795 800 Gln Ala Val Ala Arg Val His Arg Met Gly Gln Thr Ala Thr Val Gln 805 810 815 Val His Arg Leu Ile Gly Asp Glu Thr Ala Asp Glu Arg Met Leu Glu 820 825 830 Ile Leu Ala Gly Lys Thr His Val Phe Asp Val Tyr Ala Arg Leu Ser 835 840 845 Glu Thr Ala Glu Ile Pro Asp Ala Val Asp Ile Thr Glu Ser Gln Leu 850 855 860 Ala Ala Arg Val Ile Asp Glu Glu Arg Ala Arg Leu Gly Leu Thr Glu 865 870 875 880 Ser Thr Gly Pro Lys Asp Glu Glu Thr Ala Leu Ser 885 890 <210> 54 <211> 2679 <212> DNA <213> Artificial Sequence <220> <223> (Y592F)_NCgl1575 <400> 54 atggcagaat caaacgctat ggaccgggca caaatctctg cactgctaga tagagcacag 60 cacacaatca accttgccga acaagcaaac aacgtgctcc gactgttgaa aacacccgga 120 acggccacag taggggacaa cgggacactc ggcaccgata cctatctgat cccatcccgc 180 aacatcacct ggcctgacaa cctgtatgtc aacgtctttc tagacggcat gaatgcagaa 240 gccaccctta ccgattacgt cgcatcagtc gcttcgatcc cacgcctatg ccagatcatc 300 aacgagggcc aaggcggcat gttccgcaga ctattcaacc ccaccaaggt ccaagccggc 360 gaccaagctg tcttcgacct catggtcaaa ctcgacgaga tttcatctac cacccacgaa 420 gtctcccgca tgctcgaggg cgtccacgct gcccgcaccc gccaacaaca aggcgttgca 480 cttttcccag gtattcatgg agtgggagag cgctacatcg aacgcgcaca acaggtactc 540 gcctcagccc tcggtatcgc tggattcggt gccgaaccct gggacggaca tacccttgcc 600 caagcgcgcc gggtagtcca acgctacgcc caagatccta actccgaata ccggctgaaa 660 agcgaagccg agaaacacct cacatccatc aacgagctcc gcgtacagat actcctcgaa 720 caactccccg ttgatgccct acgcatggct accgaccacc gcctgcgctt tggatccctc 780 gattccatcc acgtcgcaac cgtcgccgac gtcctaaaaa cacacacctc catcctcacc 840 accgtgcaag gtatcggcgc ccaaaccgcg gggcggatga aagccgcagc agaaacactc 900 aaacaagaag cactacgccg ccaaaacacc tccatcggcg acgaacctac ccaacccgcc 960 atgcgtctaa tcaacgtgct ggcccgcttc gaccaaaccg aaaccatcac gcccgaagaa 1020 cgcgcccgcc gcacccgcgt catcgactac gtagaacaca tacccccaag cctcgacccc 1080 tacatcgtca tcaacccagc aacgcctgag ttcaacaact tcaccgacga cctccgctgg 1140 atcgacgcaa accccaacct cttccaccca caaacaatca ccaccccacc cgccgacatc 1200 tgggacgact acatctcccg tcccgctcac taccaaggcc tgctagccac gctgctcggc 1260 cgcgacatcg aaggcgcaga cgaactcctc gacgccacca ccctccaaaa aatcagagac 1320 ctcaccctcg acaaaactca tctcaccgac ctccacctcc gcggatacca atcattcggc 1380 gcccgcttcg ccatcatcca aaagaaaacc ctcctcggcg acgacatggg actcggcaaa 1440 acagtccaag ccctctccgc agctgcacac cttgccgcca ccgaaaaaga cttccgcacc 1500 ctcgtcgtcg tacccgcatc cgtcattgtt aactggaccc gcgaatgcaa acgcttcctc 1560 aacctccccg tattcatcgc ccacggagac aacaaacaag acgccatcaa cgcctggtct 1620 aacaccaacg gaatcgcaat ctgcacctac gacggcgtcc gcaccatgga catccccgcg 1680 ccgggtctgg tcattgccga tgaagcccac ctgatcaaaa acccctccac caaacgcacc 1740 caagcactgc gcaaacttat cgacgccgcc ccattcaccc ttctgatgac cggcacacca 1800 ctagaaaaca aagtggaaga gtttgtaaat ctcgtgcgct acatccaacc ggagctgatc 1860 acccgtggca tgtccaaaat gcaggccgag aatttccgcg agcgcatcgc accagcctat 1920 ctgcgcagaa atcaagctga tgtgcttgac gaactcccag agcgcaccga ctccatcgac 1980 tggatcgacc tcaccccaga agaccgcagc gcctacgacg accaagtccg ccaaggcagc 2040 tggatgggca tgcgccgctc cgccatgctc tcaccaacac cacgcctaac ttccgcaaaa 2100 atgcaacgca tcctagaact cttcgaagaa gcagaagaac acggccgcaa agccctcatc 2160 ttcacctact tcctcgacgt cctcgacgaa ctggaaaagc atctaggcga gcgcgtcatc 2220 ggccgcattt ccggcgacgt gccagccacc aagcgccaat tgcttgtcga cgccctgtcc 2280 cactccaaac ccggatccgc cctcattgcc caaatcaccg ccgggggagt aggcctaaac 2340 atccaatccg cgagcctatg cattatttgt gaacctcaag taaagccaac catcgaacag 2400 caggccgtcg cccgagtcca ccgcatgggc caaaccgcca ccgtccaagt ccaccgactc 2460 atcggcgacg aaaccgcaga cgaacgcatg ctagaaatcc tggcaggcaa aactcacgtc 2520 ttcgacgtct acgcccggct atctgaaacc gcagagattc cagatgctgt ggatatcact 2580 gaatcacagc tggcagcacg ggttattgat gaggagcgtg cacggttagg gcttactgaa 2640 tccactggcc ctaaagatga agaaacggcc ttaagctag 2679 <210> 55 <211> 892 <212> PRT <213> Artificial Sequence <220> <223> (WT)_helicase <400> 55 Met Ala Glu Ser Asn Ala Met Asp Arg Ala Gln Ile Ser Ala Leu Leu 1 5 10 15 Asp Arg Ala Gln His Thr Ile Asn Leu Ala Glu Gln Ala Asn Asn Val 20 25 30 Leu Arg Leu Leu Lys Thr Pro Gly Thr Ala Thr Val Gly Asp Asn Gly 35 40 45 Thr Leu Gly Thr Asp Thr Tyr Leu Ile Pro Ser Arg Asn Ile Thr Trp 50 55 60 Pro Asp Asn Leu Tyr Val Asn Val Phe Leu Asp Gly Met Asn Ala Glu 65 70 75 80 Ala Thr Leu Thr Asp Tyr Val Ala Ser Val Ala Ser Ile Pro Arg Leu 85 90 95 Cys Gln Ile Ile Asn Glu Gly Gln Gly Gly Met Phe Arg Arg Leu Phe 100 105 110 Asn Pro Thr Lys Val Gln Ala Gly Asp Gln Ala Val Phe Asp Leu Met 115 120 125 Val Lys Leu Asp Glu Ile Ser Ser Thr Thr His Glu Val Ser Arg Met 130 135 140 Leu Glu Gly Val His Ala Ala Arg Thr Arg Gln Gln Gln Gly Val Ala 145 150 155 160 Leu Phe Pro Gly Ile His Gly Val Gly Glu Arg Tyr Ile Glu Arg Ala 165 170 175 Gln Gln Val Leu Ala Ser Ala Leu Gly Ile Ala Gly Phe Gly Ala Glu 180 185 190 Pro Trp Asp Gly His Thr Leu Ala Gln Ala Arg Arg Val Val Gln Arg 195 200 205 Tyr Ala Gln Asp Pro Asn Ser Glu Tyr Arg Leu Lys Ser Glu Ala Glu 210 215 220 Lys His Leu Thr Ser Ile Asn Glu Leu Arg Val Gln Ile Leu Leu Glu 225 230 235 240 Gln Leu Pro Val Asp Ala Leu Arg Met Ala Thr Asp His Arg Leu Arg 245 250 255 Phe Gly Ser Leu Asp Ser Ile His Val Ala Thr Val Ala Asp Val Leu 260 265 270 Lys Thr His Thr Ser Ile Leu Thr Thr Val Gln Gly Ile Gly Ala Gln 275 280 285 Thr Ala Gly Arg Met Lys Ala Ala Ala Glu Thr Leu Lys Gln Glu Ala 290 295 300 Leu Arg Arg Gln Asn Thr Ser Ile Gly Asp Glu Pro Thr Gln Pro Ala 305 310 315 320 Met Arg Leu Ile Asn Val Leu Ala Arg Phe Asp Gln Thr Glu Thr Ile 325 330 335 Thr Pro Glu Glu Arg Ala Arg Arg Thr Arg Val Ile Asp Tyr Val Glu 340 345 350 His Ile Pro Pro Ser Leu Asp Pro Tyr Ile Val Ile Asn Pro Ala Thr 355 360 365 Pro Glu Phe Asn Asn Phe Thr Asp Asp Leu Arg Trp Ile Asp Ala Asn 370 375 380 Pro Asn Leu Phe His Pro Gln Thr Ile Thr Thr Pro Pro Ala Asp Ile 385 390 395 400 Trp Asp Asp Tyr Ile Ser Arg Pro Ala His Tyr Gln Gly Leu Leu Ala 405 410 415 Thr Leu Leu Gly Arg Asp Ile Glu Gly Ala Asp Glu Leu Leu Asp Ala 420 425 430 Thr Thr Leu Gln Lys Ile Arg Asp Leu Thr Leu Asp Lys Thr His Leu 435 440 445 Thr Asp Leu His Leu Arg Gly Tyr Gln Ser Phe Gly Ala Arg Phe Ala 450 455 460 Ile Ile Gln Lys Lys Thr Leu Leu Gly Asp Asp Met Gly Leu Gly Lys 465 470 475 480 Thr Val Gln Ala Leu Ser Ala Ala Ala His Leu Ala Ala Thr Glu Lys 485 490 495 Asp Phe Arg Thr Leu Val Val Val Pro Ala Ser Val Ile Val Asn Trp 500 505 510 Thr Arg Glu Cys Lys Arg Phe Leu Asn Leu Pro Val Phe Ile Ala His 515 520 525 Gly Asp Asn Lys Gln Asp Ala Ile Asn Ala Trp Ser Asn Thr Asn Gly 530 535 540 Ile Ala Ile Cys Thr Tyr Asp Gly Val Arg Thr Met Asp Ile Pro Ala 545 550 555 560 Pro Gly Leu Val Ile Ala Asp Glu Ala His Leu Ile Lys Asn Pro Ser 565 570 575 Thr Lys Arg Thr Gln Ala Leu Arg Lys Leu Ile Asp Ala Ala Pro Tyr 580 585 590 Thr Leu Leu Met Thr Gly Thr Pro Leu Glu Asn Lys Val Glu Glu Phe 595 600 605 Val Asn Leu Val Arg Tyr Ile Gln Pro Glu Leu Ile Thr Arg Gly Met 610 615 620 Ser Lys Met Gln Ala Glu Asn Phe Arg Glu Arg Ile Ala Pro Ala Tyr 625 630 635 640 Leu Arg Arg Asn Gln Ala Asp Val Leu Asp Glu Leu Pro Glu Arg Thr 645 650 655 Asp Ser Ile Asp Trp Ile Asp Leu Thr Pro Glu Asp Arg Ser Ala Tyr 660 665 670 Asp Asp Gln Val Arg Gln Gly Ser Trp Met Gly Met Arg Arg Ser Ala 675 680 685 Met Leu Ser Pro Thr Pro Arg Leu Thr Ser Ala Lys Met Gln Arg Ile 690 695 700 Leu Glu Leu Phe Glu Glu Ala Glu Glu His Gly Arg Lys Ala Leu Ile 705 710 715 720 Phe Thr Tyr Phe Leu Asp Val Leu Asp Glu Leu Glu Lys His Leu Gly 725 730 735 Glu Arg Val Ile Gly Arg Ile Ser Gly Asp Val Pro Ala Thr Lys Arg 740 745 750 Gln Leu Leu Val Asp Ala Leu Ser His Ser Lys Pro Gly Ser Ala Leu 755 760 765 Ile Ala Gln Ile Thr Ala Gly Gly Val Gly Leu Asn Ile Gln Ser Ala 770 775 780 Ser Leu Cys Ile Ile Cys Glu Pro Gln Val Lys Pro Thr Ile Glu Gln 785 790 795 800 Gln Ala Val Ala Arg Val His Arg Met Gly Gln Thr Ala Thr Val Gln 805 810 815 Val His Arg Leu Ile Gly Asp Glu Thr Ala Asp Glu Arg Met Leu Glu 820 825 830 Ile Leu Ala Gly Lys Thr His Val Phe Asp Val Tyr Ala Arg Leu Ser 835 840 845 Glu Thr Ala Glu Ile Pro Asp Ala Val Asp Ile Thr Glu Ser Gln Leu 850 855 860 Ala Ala Arg Val Ile Asp Glu Glu Arg Ala Arg Leu Gly Leu Thr Glu 865 870 875 880 Ser Thr Gly Pro Lys Asp Glu Glu Thr Ala Leu Ser 885 890 <210> 56 <211> 2679 <212> DNA <213> Artificial Sequence <220> <223> (WT)_NCgl1575 <400> 56 atggcagaat caaacgctat ggaccgggca caaatctctg cactgctaga tagagcacag 60 cacacaatca accttgccga acaagcaaac aacgtgctcc gactgttgaa aacacccgga 120 acggccacag taggggacaa cgggacactc ggcaccgata cctatctgat cccatcccgc 180 aacatcacct ggcctgacaa cctgtatgtc aacgtctttc tagacggcat gaatgcagaa 240 gccaccctta ccgattacgt cgcatcagtc gcttcgatcc cacgcctatg ccagatcatc 300 aacgagggcc aaggcggcat gttccgcaga ctattcaacc ccaccaaggt ccaagccggc 360 gaccaagctg tcttcgacct catggtcaaa ctcgacgaga tttcatctac cacccacgaa 420 gtctcccgca tgctcgaggg cgtccacgct gcccgcaccc gccaacaaca aggcgttgca 480 cttttcccag gtattcatgg agtgggagag cgctacatcg aacgcgcaca acaggtactc 540 gcctcagccc tcggtatcgc tggattcggt gccgaaccct gggacggaca tacccttgcc 600 caagcgcgcc gggtagtcca acgctacgcc caagatccta actccgaata ccggctgaaa 660 agcgaagccg agaaacacct cacatccatc aacgagctcc gcgtacagat actcctcgaa 720 caactccccg ttgatgccct acgcatggct accgaccacc gcctgcgctt tggatccctc 780 gattccatcc acgtcgcaac cgtcgccgac gtcctaaaaa cacacacctc catcctcacc 840 accgtgcaag gtatcggcgc ccaaaccgcg gggcggatga aagccgcagc agaaacactc 900 aaacaagaag cactacgccg ccaaaacacc tccatcggcg acgaacctac ccaacccgcc 960 atgcgtctaa tcaacgtgct ggcccgcttc gaccaaaccg aaaccatcac gcccgaagaa 1020 cgcgcccgcc gcacccgcgt catcgactac gtagaacaca tacccccaag cctcgacccc 1080 tacatcgtca tcaacccagc aacgcctgag ttcaacaact tcaccgacga cctccgctgg 1140 atcgacgcaa accccaacct cttccaccca caaacaatca ccaccccacc cgccgacatc 1200 tgggacgact acatctcccg tcccgctcac taccaaggcc tgctagccac gctgctcggc 1260 cgcgacatcg aaggcgcaga cgaactcctc gacgccacca ccctccaaaa aatcagagac 1320 ctcaccctcg acaaaactca tctcaccgac ctccacctcc gcggatacca atcattcggc 1380 gcccgcttcg ccatcatcca aaagaaaacc ctcctcggcg acgacatggg actcggcaaa 1440 acagtccaag ccctctccgc agctgcacac cttgccgcca ccgaaaaaga cttccgcacc 1500 ctcgtcgtcg tacccgcatc cgtcattgtt aactggaccc gcgaatgcaa acgcttcctc 1560 aacctccccg tattcatcgc ccacggagac aacaaacaag acgccatcaa cgcctggtct 1620 aacaccaacg gaatcgcaat ctgcacctac gacggcgtcc gcaccatgga catccccgcg 1680 ccgggtctgg tcattgccga tgaagcccac ctgatcaaaa acccctccac caaacgcacc 1740 caagcactgc gcaaacttat cgacgccgcc ccatacaccc ttctgatgac cggcacacca 1800 ctagaaaaca aagtggaaga gtttgtaaat ctcgtgcgct acatccaacc ggagctgatc 1860 acccgtggca tgtccaaaat gcaggccgag aatttccgcg agcgcatcgc accagcctat 1920 ctgcgcagaa atcaagctga tgtgcttgac gaactcccag agcgcaccga ctccatcgac 1980 tggatcgacc tcaccccaga agaccgcagc gcctacgacg accaagtccg ccaaggcagc 2040 tggatgggca tgcgccgctc cgccatgctc tcaccaacac cacgcctaac ttccgcaaaa 2100 atgcaacgca tcctagaact cttcgaagaa gcagaagaac acggccgcaa agccctcatc 2160 ttcacctact tcctcgacgt cctcgacgaa ctggaaaagc atctaggcga gcgcgtcatc 2220 ggccgcattt ccggcgacgt gccagccacc aagcgccaat tgcttgtcga cgccctgtcc 2280 cactccaaac ccggatccgc cctcattgcc caaatcaccg ccgggggagt aggcctaaac 2340 atccaatccg cgagcctatg cattatttgt gaacctcaag taaagccaac catcgaacag 2400 caggccgtcg cccgagtcca ccgcatgggc caaaccgcca ccgtccaagt ccaccgactc 2460 atcggcgacg aaaccgcaga cgaacgcatg ctagaaatcc tggcaggcaa aactcacgtc 2520 ttcgacgtct acgcccggct atctgaaacc gcagagattc cagatgctgt ggatatcact 2580 gaatcacagc tggcagcacg ggttattgat gaggagcgtg cacggttagg gcttactgaa 2640 tccactggcc ctaaagatga agaaacggcc ttaagctag 2679

Claims (5)

Protein variants selected from the group consisting of the following (1) to (14):
(1) an endogenous membrane transport protein variant consisting of the amino acid sequence set forth in SEQ ID NO: 1 in which phenylalanine, an amino acid corresponding to position 220 of SEQ ID NO: 3, is substituted with cysteine;
(2) DNA polymerase III gamma and tau subunit variants consisting of the amino acid sequence set forth in SEQ ID NO: 5 in which proline, an amino acid corresponding to position 43 of SEQ ID NO: 7, is substituted with leucine;
(3) a protein variant consisting of the amino acid sequence shown in SEQ ID NO: 9, in which cysteine, an amino acid corresponding to position 33 of SEQ ID NO: 11, is substituted with serine;
(4) a transcriptional anti-termination protein variant consisting of the amino acid sequence shown in SEQ ID NO: 13, in which asparagine, an amino acid corresponding to position 210 of SEQ ID NO: 15, is substituted with aspartic acid;
(5) an ABC transporter ATP-binding protein variant consisting of the amino acid sequence shown in SEQ ID NO: 17 in which valine, an amino acid corresponding to position 199 of SEQ ID NO: 19, is substituted with methionine;
(6) a malate dehydrogenase variant consisting of the amino acid sequence set forth in SEQ ID NO: 21 in which threonine, an amino acid corresponding to position 272 of SEQ ID NO: 23, is substituted with isoleucine;
(7) a primosome assembly protein variant consisting of the amino acid sequence shown in SEQ ID NO: 25, in which arginine, an amino acid corresponding to position 304 of SEQ ID NO: 27, is substituted with glutamine;
(8) a type II citrate synthase variant consisting of the amino acid sequence shown in SEQ ID NO: 29, in which alanine, an amino acid corresponding to position 169 of SEQ ID NO: 31, is substituted with valine;
(9) a membrane protein variant consisting of the amino acid sequence set forth in SEQ ID NO: 33 in which glutamic acid, an amino acid corresponding to position 294 of SEQ ID NO: 35, is substituted with lysine;
(10) a mycothione reductase variant consisting of the amino acid sequence set forth in SEQ ID NO: 37 in which proline, which is the amino acid corresponding to position 358 of SEQ ID NO: 39, is substituted with serine;
(11) a Co/Zn/Cd efflux system component variant consisting of the amino acid sequence set forth in SEQ ID NO: 41 in which glycine, an amino acid corresponding to position 130 of SEQ ID NO: 43, is substituted with aspartic acid;
(12) a DAHP synthase variant consisting of the amino acid sequence set forth in SEQ ID NO: 45 in which glycine, an amino acid corresponding to position 382 of SEQ ID NO: 47, is substituted with cysteine;
(13) an N-succinyldiaminopimelate aminotransferase variant consisting of the amino acid sequence set forth in SEQ ID NO: 49, wherein proline, which is the amino acid corresponding to position 209 of SEQ ID NO: 51, is substituted with leucine; and
(14) A helicase variant consisting of the amino acid sequence shown in SEQ ID NO: 53, wherein tyrosine, which is an amino acid corresponding to position 592 of SEQ ID NO: 55, is substituted with phenylalanine.
A polynucleotide encoding the variant of claim 1 .
A Corynebacterium glutamicum strain comprising the following (a) and (b) protein variants or polynucleotides encoding the variants:
(a) a helicase variant consisting of the amino acid sequence set forth in SEQ ID NO: 53 in which tyrosine, an amino acid corresponding to position 592 of SEQ ID NO: 55, is substituted with phenylalanine, and
(b) at least one protein variant selected from the group consisting of the following (1) to (13):
(1) an endogenous membrane transport protein variant consisting of the amino acid sequence set forth in SEQ ID NO: 1 in which phenylalanine, an amino acid corresponding to position 220 of SEQ ID NO: 3, is substituted with cysteine;
(2) DNA polymerase III gamma and tau subunit variants consisting of the amino acid sequence set forth in SEQ ID NO: 5 in which proline, an amino acid corresponding to position 43 of SEQ ID NO: 7, is substituted with leucine;
(3) a protein variant consisting of the amino acid sequence shown in SEQ ID NO: 9, in which cysteine, an amino acid corresponding to position 33 of SEQ ID NO: 11, is substituted with serine;
(4) a transcriptional anti-termination protein variant consisting of the amino acid sequence shown in SEQ ID NO: 13, in which asparagine, an amino acid corresponding to position 210 of SEQ ID NO: 15, is substituted with aspartic acid;
(5) an ABC transporter ATP-binding protein variant consisting of the amino acid sequence shown in SEQ ID NO: 17 in which valine, an amino acid corresponding to position 199 of SEQ ID NO: 19, is substituted with methionine;
(6) a malate dehydrogenase variant consisting of the amino acid sequence set forth in SEQ ID NO: 21 in which threonine, an amino acid corresponding to position 272 of SEQ ID NO: 23, is substituted with isoleucine;
(7) a primosome assembly protein variant consisting of the amino acid sequence shown in SEQ ID NO: 25, in which arginine, an amino acid corresponding to position 304 of SEQ ID NO: 27, is substituted with glutamine;
(8) a type II citrate synthase variant consisting of the amino acid sequence shown in SEQ ID NO: 29, in which alanine, an amino acid corresponding to position 169 of SEQ ID NO: 31, is substituted with valine;
(9) a membrane protein variant consisting of the amino acid sequence set forth in SEQ ID NO: 33 in which glutamic acid, an amino acid corresponding to position 294 of SEQ ID NO: 35, is substituted with lysine;
(10) a mycothione reductase mutant consisting of the amino acid sequence set forth in SEQ ID NO: 37 in which proline, an amino acid corresponding to position 358 of SEQ ID NO: 39, is substituted with serine;
(11) a Co/Zn/Cd efflux system component variant consisting of the amino acid sequence set forth in SEQ ID NO: 41 in which glycine, an amino acid corresponding to position 130 of SEQ ID NO: 43, is substituted with aspartic acid;
(12) a DAHP synthase variant consisting of the amino acid sequence shown in SEQ ID NO: 45 in which glycine, an amino acid corresponding to position 382 of SEQ ID NO: 47, is substituted with cysteine; and
(13) An N-succinyldiaminopimelate aminotransferase mutant consisting of the amino acid sequence shown in SEQ ID NO: 49, wherein proline, which is the amino acid corresponding to position 209 of SEQ ID NO: 51, is substituted with leucine.
The strain of claim 3, wherein the L-lysine-producing ability is increased compared to Corynebacterium glutamicum that does not include the protein variant or the polynucleotide encoding the variant.
A method for producing L-lysine, comprising the step of culturing the Corynebacterium glutamicum strain of claim 3 or 4 in a medium.