# Patent

## Title
Genetically modified microorganisms for producing itaconic acid with high yields

## Abstract

Genetically modified microorganisms that produce itaconic acid at high yields and uses thereof.

## Background
BACKGROUND OF THE INVENTION
Itaconic acid, an essential precursor to various products (e.g., acrylic fibers, rubbers, artificial diamonds, and lens), is in high demand in the chemical industry. Conventionally, itaconic acid is isolated from Aspergillus terreus. However, A. terreus grows slowly and does not produce itaconic acid in its spore-forming stage. There is a need for a method that produces itaconic acid in high yield.
SUMMARY OF THE INVENTION
The present invention is based on the unexpected discovery that certain genetically modified E. coli strains produce itaconic acid at much higher levels relative to wild-type E. coli strains.
In one aspect, this invention features a genetically modified microorganism containing (i) a mutated endogenous icd gene that expresses a lower level of isocitrate dehydrogenase compared with its wild-type counterpart and (ii) an exogenous nucleotide sequence encoding a cis-aconitic acid decarboxylase (CAD) operably linked to a suitable promoter (i.e., capable of initiating gene transcription in the microorganism). The modified microorganism can be Aspergillus niger, Aspergillus terreus, Escherichia coli, Pseudozyma Antarctica, Yarrowia lipotica, or Saccharomyces cerevisiae. It can further contain at least one exogenous nucleotide sequences encoding one of the following three enzymes: (a) an enzyme that converts phosphoenolpyruvate to oxaloacetate (i.e., phosphoenolpyruvate carboxylase; also known as phosphoenolpyruvate carboxykinase), (b) an enzyme that converts oxaloacetate to citrate (i.e., citrate synthase, 2-methylcitrate synthase, and citrate lyase), and (c) an enzyme that converts citrate or isocitrate to cis-aconitic acid (i.e., aconitase and 2-methylcitrate dehydratase). Each of these exogenous nucleotide sequences is operably linked to a suitable promoter.
In another aspect, this invention features a genetically modified microorganism containing (i) a first exogenous nucleotide sequence encoding CAD, (ii) a second exogenous nucleotide sequence encoding enzyme (a) or enzyme (b) mentioned above, and optionally (iii) a third exogenous nucleotide sequence encoding enzyme (c) also mentioned above. Alternatively, the microorganism contains (i) a first exogenous nucleotide sequence encoding CAD, (ii) a second exogenous nucleotide sequence encoding enzyme (a), (iii) a third exogenous nucleotide encoding enzyme (b), and optionally (iv) a fourth exogenous nucleotide sequence encoding enzyme (c). Each of the exogenous nucleotide sequences is linked operatively to a promoter that drives its expression in the microorganism.
Also within the scope of this invention is a method for producing itaconic acid in any of the genetically modified microorganisms described above. This method includes cultivating the genetically modified microorganism in a medium to produce itaconic acid and collecting the medium for isolation of the itaconic acid thus produced. In one example, the medium contains glucose, as the substrate for making itaconic acid, at a concentration ranging from 5-80 g/L (e.g., 10-40 g/L). In another example, the medium contains citrate, as the substrate for making itaconic acid, at a concentration ranging from 5-80 g/L (e.g., 10-40 g/L). When citrate is used as the substrate, the genetically modified microorganism is preferred to be permeabilized.
The details of one or more embodiments of the invention are set forth in the description below. Other features or advantages of the present invention will be apparent from the following detailed description of several embodiments and also from the appended claims.


DETAILED DESCRIPTION OF THE INVENTION
Disclosed herein is a genetically modified microorganism that overly expresses a cis-aconitic acid decarboxylase (CAD), an enzyme that converts cis-aconitic acid to itaconic acid. Examples of the microorganism include, but are not limited to, Aspergillus, Citrobacter, Corynebacterium, Dekkera, Enterobacter, Enterococcus, Escherichia, Erwinia, Klebsiella, Kluyveromyces, Lactobacillus, Lactococcus, Morganella, Pantoea, Pectobacterium, Penicillium, Pichia, Proteus, Pseudomonas, Pseudozyma, Rhodotorula, Salmonella, Serratia, Shigella, Saccharomyces, Ustilago, and Yarrow. 
The term “cis-aconitic acid decarboxylase” or “CAD” used herein refers to any naturally occurring CADs (e.g., the A. terreus CAD described in Dwiarti et al., J. Bioscience and Bioengineering, 94 (1):29-33, 2002 and WO 2009/014437) and functional equivalents thereof. Provided below are the nucleotide sequence (SEQ ID NO:1) and amino acid sequence (SEQ ID NO:2) of an exemplary A. terreus CAD:









A. terreus Cis-aconitic Acid Decarboxylase




atgaccaagcagtctgctgattccaacgcgaagtctggtgtgacctctgagatctgtcac
(SEQ ID NO: 1)


 M  T  K  Q  S  A  D  S  N  A  K  S  G  V  T  S  E  I  C  H
(SEQ ID NO: 2)


 


tgggcgtctaatctcgccactgatgatatcccgagcgacgttctggagcgtgcaaaatac



 W  A  S  N  L  A  T  D  D  I  P  S  D  V  L  E  R  A  K  Y



 


ctgatcctggatggtatcgcgtgcgcgtgggtaggtgctcgtgtcccatggtctgaaaaa



 L  I  L  D  G  I  A  C  A  W  V  G  A  R  V  P  W  S  E  K



 


tacgttcaagcgaccatgtctttcgaacctccgggtgcgtgtcgtgtcatcggttacggc



 Y  V  Q  A  T  M  S  F  E  P  P  G  A  C  R  V  I  G  Y  G



 


cagaaactgggtccggtagcggctgccatgacgaactctgcatttattcaggcgaccgaa



 Q  K  L  G  P  V  A  A  A  M  T  N  S  A  F  I  Q  A  T  E



 


ctcgatgactatcactctgaagcgccgctgcattccgcgtctatcgttctcccggcagtt



 L  D  D  Y  H  S  E  A  P  L  H  S  A  S  I  V  L  P  A  V



 


ttcgcggcgagcgaagtactggccgaacagggtaaaaccatctctggtattgacgtgatt



 F  A  A  S  E  V  L  A  E  Q  G  K  T  I  S  G  I  D  V  I



 


ctggctgcgatcgttggtttcgagagcggtcctcgcatcggcaaagcgatctacggttct



 L  A  A  I  V  G  F  E  S  G  P  R  I  G  K  A  I  Y  G  S



 


gacctcctgaacaacggctggcactgcggtgcggtatatggcgcaccggctggtgcgctc



 D  L  L  N  N  G  W  H  C  G  A  V  Y  G  A  P  A  G  A  L



 


gcaactggtaagctcctgggcctcacgccggacagcatggaagatgcactgggtattgcc



 A  T  G  K  L  L  G  L  T  P  D  S  M  E  D  A  L  G  I  A



 


tgcacgcaagcatgcggcctcatgtccgcgcagtatggtggcatggttaaacgtgttcag



 C  T  Q  A  C  G  L  M  S  A  Q  Y  G  G  M  V  K  R  V  Q



 


cacggtttcgcagcgcgtaatggtctcctcggtggcctcctggctcacggcggctacgag



 H  G  F  A  A  R  N  G  L  L  G  G  L  L  A  H  G  G  Y  E



 


gcgatgaaaggtgttctcgagcgttcttacggtggcttcctgaagatgttcaccaagggc



 A  M  K  G  V  L  E  R  S  Y  G  G  F  L  K  M  F  T  K  G



 


aacggtcgtgaaccgccgtacaaagaagaagaggttgtggctggtctgggtagcttctgg



 N  G  R  E  P  P  Y  K  E  E  E  V  V  A  G  L  G  S  F  W



 


cacaccttcaccattcgtatcaaactgtacgcgtgctgcggtctcgtacacggtcctgtt



 H  T  F  T  I  R  I  K  L  Y  A  C  C  G  L  V  H  G  P  V



 


gaagccattgaaaacctccagggtcgttacccggaactgctcaatcgtgctaacctgtct



 E  A  I  E  N  L  Q  G  R  Y  P  E  L  L  N  R  A  N  L  S



 


aacatccgccacgttcacgtacaactctctaccgcgagcaactcccactgtggttggatc



 N  I  R  H  V  H  V  Q  L  S  T  A  S  N  S  H  C  G  W  I



 


ccagaagagcgcccaatctcttctatcgcgggtcaaatgtctgtcgcatatatcctcgcc



 P  E  E  R  P  I  S  S  I  A  G  Q  M  S  V  A  Y  I  L  A



 


gttcagctcgttgaccaacagtgtctgctcagccagttctccgagtttgacgataatctg



 V  Q  L  V  D  Q  Q  C  L  L  S  Q  F  S  E  F  D  D  N  L



 


gaacgcccggaagtgtgggacctggcacgtaaggttaccagctctcaatctgaggagttc



 E  R  P  E  V  W  D  L  A  R  K  V  T  S  S  Q  S  E  E  F



 


gaccaggacggtaactgtctctctgccggtcgcgtccgtattgagttcaacgacggctcc



 D  Q  D  G  N  C  L  S  A  G  R  V  R  I  E  F  N  D  G  S



 


tccatcaccgaatccgttgagaagccgctcggtgtaaaggaaccaatgccaaatgaacgc



 S  I  T  E  S  V  E  K  P  L  G  V  K  E  P  M  P  N  E  R



 


atcctgcacaaataccgtaccctggcgggttctgtaacggacgaaagccgtgttaaggag



 I  L  H  K  Y  R  T  L  A  G  S  V  T  D  E  S  R  V  K  E



 


atcgaggatctcgtgctcggcctggaccgtctgaccgatattagcccgctcctcgagctg



 I  E  D  L  V  L  G  L  D  R  L  T  D  I  S  P  L  L  E  L



 


Ctgaattgtccggttaaatccccactggtttaa



 L  N  C  P  V  K  S  P  L  V  -







As used herein, a functional equivalent of a reference enzyme (i.e., the A. terreus CAD or any of the enzymes mentioned below) is a polypeptide having an amino acid sequence at least 60% (e.g., 85%, 90%, or 95%) identical to that of the reference enzyme and possessing the same enzymatic activity as the reference enzyme.
The percent identity of two amino acid sequences is determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, as modified in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such an algorithm is incorporated into the BLASTN and BLASTX programs (version 2.0) of Altschul, et al. J. Mol. Biol. 215:403-10, 1990. BLAST protein searches can be performed with the BLASTX program, score=50, wordlength=3 to obtain amino acid sequences homologous to the protein molecules of the invention. Where gaps exist between two sequences, Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res. 25:3389-3402, 1997. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., BLASTX and BLASTN) can be used.
The genetically modified microorganism described above can have a mutated endogenous icd gene (encoding isocitrate decarboxylase) so that it expresses a lower level of isocitrate decarboxylase as compared with its wild-type counterpart. Isocitrate decarboxylase converts isocitrate to α-ketoglutarate. Icd gene exists in various types of microorganisms, including Aspergillus terreus (GenBank Accession Nos. XM—001210553 and XP—001210553), Citrobacter koseri (GenBank Accession No. YP—001453397), Lactobacillus fermentum (GenBank Accession Nos. NC—009792 and YP—001843755), Saccharomyces cerevisiae (GenBank Accession Nos. NC—001146 and NP—014361), Yarrowia lipolytica (GenBank Accession Nos. XM—503571 and XP—503571), and Escherichia coli (GenBank Accession Nos. NC—000913 and NP—415654) As an example, the coding region of the E. coli icd gene is shown below:








Nucleotide Sequence and Encoded Amino Acid Sequence of



E. coli Icd gene









atggaaagtaaagtagttgttccggcacaaggcaagaagatcaccctgcaaaacggcaaa
(SEQ ID NO: 3)



 M  E  S  K  V  V  V  P  A  Q  G  K  K  I  T  L  Q  N  G  K
(SEQ ID NO: 4)


 


ctcaacgttcctgaaaatccgattatcccttacattgaaggtgatggaatcggtgtagat



 L  N  V  P  E  N  P  I  I  P  Y  I  E  G  D  G  I  G  V  D



 


gtaaccccagccatgctgaaagtggtcgacgctgcagtcgagaaagcctataaaggcgag



 V  T  P  A  M  L  K  V  V  D  A  A  V  E  K  A  Y  K  G  E



 


cgtaaaatctcctggatggaaatttacaccggtgaaaaatccacacaggtttatggtcag



 R  K  I  S  W  M  E  I  Y  T  G  E  K  S  T  Q  V  Y  G  Q



 


gacgtctggctgcctgctgaaactcttgatctgattcgtgaatatcgcgttgccattaaa



 D  V  W  L  P  A  E  T  L  D  L  I  R  E  Y  R  V  A  I  K



 


ggtccgctgaccactccggttggtggcggtattcgctctctgaacgttgccctgcgccag



 G  P  L  T  T  P  V  G  G  G  I  R  S  L  N  V  A  L  R  Q



 


gaactggatctctacatctgcctgcgtccggtacgttactatcagggcactccaagcccg



 E  L  D  L  Y  I  C  L  R  P  V  R  Y  Y  Q  G  T  P  S  P



 


gttaaacaccctgaactgaccgatatggttatcttccgtgaaaactcggaagacatttat



 V  K  H  P  E  L  T  D  M  V  I  F  R  E  N  S  E  D  I  Y



 


gcgggtatcgaatggaaagcagactctgccgacgccgagaaagtgattaaattcctgcgt



 A  G  I  E  W  K  A  D  S  A  D  A  E  K  V  I  K  F  L  R



 


gaagagatgggggtgaagaaaattcgcttcccggaacattgtggtatcggtattaagccg



 E  E  M  G  V  K  K  I  R  F  P  E  H  C  G  I  G  I  K  P



 


tgttcggaagaaggcaccaaacgtctggttcgtgcagcgatcgaatacgcaattgctaac



 C  S  E  E  G  T  K  R  L  V  R  A  A  I  E  Y  A  I  A  N



 


gatcgtgactctgtgactctggtgcacaaaggcaacatcatgaagttcaccgaaggagcg



 D  R  D  S  V  T  L  V  H  K  G  N  I  M  K  F  T  E  G  A



 


tttaaagactggggctaccagctggcgcgtgaagagtttggcggtgaactgatcgacggt



 F  K  D  W  G  Y  Q  L  A  R  E  E  F  G  G  E  L  I  D  G



 


ggcccgtggctgaaagttaaaaacccgaacactggcaaagagatcgtcattaaagacgtg



 G  P  W  L  K  V  K  N  P  N  T  G  K  E  I  V  I  K  D  V



 


attgctgatgcattcctgcaacagatcctgctgcgtccggctgaatatgatgttatcgcc



 I  A  D  A  F  L  Q  Q  I  L  L  R  P  A  E  Y  D  V  I  A



 


tgtatgaacctgaacggtgactacatttctgacgccctggcagcgcaggttggcggtatc



 C  M  N  L  N  G  D  Y  I  S  D  A  L  A  A  Q  V  G  G  I



 


ggtatcgcccctggtgcaaacatcggtgacgaatgcgccctgtttgaagccacccacggt



 G  I  A  P  G  A  N  I  G  D  E  C  A  L  F  E  A  T  H  G



 


actgcgccgaaatatgccggtcaggacaaagtaaatcctggctctattattctctccgct



 T  A  P  K  Y  A  G  Q  D  K  V  N  P  G  S  I  I  L  S  A



 


gagatgatgctgcgccacatgggttggaccgaagcggctgacttaattgttaaaggtatg



 E  M  M  L  R  H  M  G  W  T  E  A  A  D  L  I  V  K  G  M



 


gaaggcgcaatcaacgcgaaaaccgtaacctatgacttcgagcgtctgatggatggcgct



 E  G  A  I  N  A  K  T  V  T  Y  D  F  E  R  L  M  D  G  A



 


 Aaactgctgaaatgttcagagtttggtgacgcgatcatcgaaaacatgtaa



  K  L  L  K  C  S  E  F  G  D  A  I  I  E  N  M  -







Methods for producing a microorganism with a mutated endogenous icd gene are well known in the art. For example, mutations (e.g., insertion, deletion, site mutation) of the icd gene can be introduced by homologous recombination.
Alternatively or in addition, the genetically modified microorganism also overly expresses one or more of the following enzymes: (a) an enzyme that converts phosphoenolpyruvate to oxaloacetate (i.e., phosphoenolpyruvate carboxylase/carboxykinase; including three isoforms EC 4.1.1.32, EC 4.1.1.38, and EC 4.1.1.49), (b) an enzyme that converts oxaloacetate to citrate (i.e., citrate synthase, 2-methylcitrate synthase, and citrate lyase), and (c) an enzyme that converts citrate or isocitrate to cis-aconitic acid (i.e., aconitase and 2-methylcitrate dehydratase).
The terms “phosphoenolpyruvate carboxylase/carboxykinase,” “citrate synthase,” “2-methylcitrate synthase,” “citrate lyase,” “aconitase,” and “2-methylcitrate dehydratase” used herein refers to all enzymes that possess the enzymatic activity described above, including both naturally-occurring enzymes and their functional equivalents. Provided below are nucleotide sequences and amino acid sequences of E. coli phosphoenolpyruvate carboxylase (encoded by ppc gene), citrate synthase (encoded by gltA gene), aconitase A (encoded by acnA gene), and aconitase B (encoded by acnB gene):









E. coli Phosphoenolpyruvate Carboxylase




atgaacgaacaatattccgcattgcgtagtaatgtcagtatgctcggcaaagtgctggga
(SEQ ID NO: 5)


 M  N  E  Q  Y  S  A  L  R  S  N  V  S  M  L  G  K  V  L  G
(SEQ ID NO: 6)


 


gaaaccatcaaggatgcgttgggagaacacattcttgaacgcgtagaaactatccgtaag



 E  T  I  K  D  A  L  G  E  H  I  L  E  R  V  E  T  I  R  K



 


ttgtcgaaatcttcacgcgctggcaatgatgctaaccgccaggagttgctcaccacctta



 L  S  K  S  S  R  A  G  N  D  A  N  R  Q  E  L  L  T  T  L



 


caaaatttgtcgaacgacgagctgctgcccgttgcgcgtgcgtttagtcagttcctgaac



 Q  N  L  S  N  D  E  L  L  P  V  A  R  A  F  S  Q  F  L  N



 


ctggccaacaccgccgagcaataccacagcatttcgccgaaaggcgaagctgccagcaac



 L  A  N  T  A  E  Q  Y  H  S  I  S  P  K  G  E  A  A  S  N



 


ccggaagtgatcgcccgcaccctgcgtaaactgaaaaaccagccggaactgagcgaagac



 P  E  V  I  A  R  T  L  R  K  L  K  N  Q  P  E  L  S  E  D



 


accatcaaaaaagcagtggaatcgctgtcgctggaactggtcctcacggctcacccaacc



 T  I  K  K  A  V  E  S  L  S  L  E  L  V  L  T  A  H  P  T



 


gaaattacccgtcgtacactgatccacaaaatggtggaagtgaacgcctgtttaaaacag



 E  I  T  R  R  T  L  I  H  K  M  V  E  V  N  A  C  L  K  Q



 


ctcgataacaaagatatcgctgactacgaacacaaccagctgatgcgtcgcctgcgccag



 L  D  N  K  D  I  A  D  Y  E  H  N  Q  L  M  R  R  L  R  Q



 


ttgatcgcccagtcatggcataccgatgaaatccgtaagctgcgtccaagcccggtagat



 L  I  A  Q  S  W  H  T  D  E  I  R  K  L  R  P  S  P  V  D



 


gaagccaaatggggctttgccgtagtggaaaacagcctgtggcaaggcgtaccaaattac



 E  A  K  W  G  F  A  V  V  E  N  S  L  W  Q  G  V  P  N  Y



 


ctgcgcgaactgaacgaacaactggaagagaacctcggctacaaactgcccgtcgaattt



 L  R  E  L  N  E  Q  L  E  E  N  L  G  Y  K  L  P  V  E  F



 


gttccggtccgttttacttcgtggatgggcggcgaccgcgacggcaacccgaacgtcact



 V  P  V  R  F  T  S  W  M  G  G  D  R  D  G  N  P  N  V  T



 


gccgatatcacccgccacgtcctgctactcagccgctggaaagccaccgatttgttcctg



 A  D  I  T  R  H  V  L  L  L  S  R  W  K  A  T  D  L  F  L



 


aaagatattcaggtgctggtttctgaactgtcgatggttgaagcgacccctgaactgctg



 K  D  I  Q  V  L  V  S  E  L  S  M  V  E  A  T  P  E  L  L



 


gcgctggttggcgaagaaggtgccgcagaaccgtatcgctatctgatgaaaaacctgcgt



 A  L  V  G  E  E  G  A  A  E  P  Y  R  Y  L  M  K  N  L  R



 


tctcgcctgatggcgacacaggcatggctggaagcgcgcctgaaaggcgaagaactgcca



 S  R  L  M  A  T  Q  A  W  L  E  A  R  L  K  G  E  E  L  P



 


aaaccagaaggcctgctgacacaaaacgaagaactgtgggaaccgctctacgcttgctac



 K  P  E  G  L  L  T  Q  N  E  E  L  W  E  P  L  Y  A  C  Y



 


cagtcacttcaggcgtgtggcatgggtattatcgccaacggcgatctgctcgacaccctg



 Q  S  L  Q  A  C  G  M  G  I  I  A  N  G  D  L  L  D  T  L



 


cgccgcgtgaaatgtttcggcgtaccgctggtccgtattgatatccgtcaggagagcacg



 R  R  V  K  C  F  G  V  P  L  V  R  I  D  I  R  Q  E  S  T



 


cgtcataccgaagcgctgggcgagctgacccgctacctcggtatcggcgactacgaaagc



 R  H  T  E  A  L  G  E  L  T  R  Y  L  G  I  G  D  Y  E  S



 


tggtcagaggccgacaaacaggcgttcctgatccgcgaactgaactccaaacgtccgctt



 W  S  E  A  D  K  Q  A  F  L  I  R  E  L  N  S  K  R  P  L



 


ctgccgcgcaactggcaaccaagcgccgaaacgcgcgaagtgctcgatacctgccaggtg



 L  P  R  N  W  Q  P  S  A  E  T  R  E  V  L  D  T  C  Q  V



 


attgccgaagcaccgcaaggctccattgccgcctacgtgatctcgatggcgaaaacgccg



 I  A  E  A  P  Q  G  S  I  A  A  Y  V  I  S  M  A  K  T  P



 


tccgacgtactggctgtccacctgctgctgaaagaagcgggtatcgggtttgcgatgccg



 S  D  V  L  A  V  H  L  L  L  K  E  A  G  I  G  F  A  M  P



 


gttgctccgctgtttgaaaccctcgatgatctgaacaacgccaacgatgtcatgacccag



 V  A  P  L  F  E  T  L  D  D  L  N  N  A  N  D  V  M  T  Q



 


ctgctcaatattgactggtatcgtggcctgattcagggcaaacagatggtgatgattggc



 L  L  N  I  D  W  Y  R  G  L  I  Q  G  K  Q  M  V  M  I  G



 


tattccgactcagcaaaagatgcgggagtgatggcagcttcctgggcgcaatatcaggca



 Y  S  D  S  A  K  D  A  G  V  M  A  A  S  W  A  Q  Y  Q  A



 


caggatgcattaatcaaaacctgcgaaaaagcgggtattgagctgacgttgttccacggt



 Q  D  A  L  I  K  T  C  E  K  A  G  I  E  L  T  L  F  H  G



 


cgcggcggttccattggtcgcggcggcgcacctgctcatgcggcgctgctgtcacaaccg



 R  G  G  S  I  G  R  G  G  A  P  A  H  A  A  L  L  S  Q  P



 


ccaggaagcctgaaaggcggcctgcgcgtaaccgaacagggcgagatgatccgctttaaa



 P  G  S  L  K  G  G  L  R  V  T  E  Q  G  E  M  I  R  F  K



 


tatggtctgccagaaatcaccgtcagcagcctgtcgctttataccggggcgattctggaa



 Y  G  L  P  E  I  T  V  S  S  L  S  L  Y  T  G  A  I  L  E



 


gccaacctgctgccaccgccggagccgaaagagagctggcgtcgcattatggatgaactg



 A  N  L  L  P  P  P  E  P  K  E  S  W  R  R  I  M  D  E  L



 


tcagtcatctcctgcgatgtctaccgcggctacgtacgtgaaaacaaagattttgtgcct



 S  V  I  S  C  D  V  Y  R  G  Y  V  R  E  N  K  D  F  V  P



 


tacttccgctccgctacgccggaacaagaactgggcaaactgccgttgggttcacgtccg



 Y  F  R  S  A  T  P  E  Q  E  L  G  K  L  P  L  G  S  R  P



 


gcgaaacgtcgcccaaccggcggcgtcgagtcactacgcgccattccgtggatcttcgcc



 A  K  R  R  P  T  G  G  V  E  S  L  R  A  I  P  W  I  F  A



 


tggacgcaaaaccgtctgatgctccccgcctggctgggtgcaggtacggcgctgcaaaaa



 W  T  Q  N  R  L  M  L  P  A  W  L  G  A  G  T  A  L  Q  K



 


gtggtcgaagacggcaaacagagcgagctggaggctatgtgccgcgattggccattcttc



 V  V  E  D  G  K  Q  S  E  L  E  A  M  C  R  D  W  P  F  F



 


tcgacgcgtctcggcatgctggagatggtcttcgccaaagcagacctgtggctggcggaa



 S  T  R  L  G  M  L  E  M  V  F  A  K  A  D  L  W  L  A  E



 


tactatgaccaacgcctggtagacaaagcactgtggccgttaggtaaagagttacgcaac



 Y  Y  D  Q  R  L  V  D  K  A  L  W  P  L  G  K  E  L  R  N



 


ctgcaagaagaagacatcaaagtggtgctggcgattgccaacgattcccatctgatggcc



 L  Q  E  E  D  I  K  V  V  L  A  I  A  N  D  S  H  L  M  A



 


gatctgccgtggattgcagagtctattcagctacggaatatttacaccgacccgctgaac



 D  L  P  W  I  A  E  S  I  Q  L  R  N  I  Y  T  D  P  L  N



 


gtattgcaggccgagttgctgcaccgctcccgccaggcagaaaaagaaggccaggaaccg



 V  L  Q  A  E  L  L  H  R  S  R  Q  A  E  K  E  G  Q  E  P



 


gatcctcgcgtcgaacaagcgttaatggtcactattgccgggattgcggcaggtatgcgt



 D  P  R  V  E  Q  A  L  M  V  T  I  A  G  I  A  A  G  M  R



 


aataccggctaa



 N  T  G  -



 


E. coli Citrate Synthase



atggctgatacaaaagcaaaactcaccctcaacggggatacagctgttgaactggatgtg
(SEQ ID NO: 7)


 M  A  D  T  K  A  K  L  T  L  N  G  D  T  A  V  E  L  D  V
(SEQ ID NO: 8)


 


ctgaaaggcacgctgggtcaagatgttattgatatccgtactctcggttcaaaaggtgtg



 L  K  G  T  L  G  Q  D  V  I  D  I  R  T  L  G  S  K  G  V



 


ttcacctttgacccaggcttcacttcaaccgcatcctgcgaatctaaaattacttttatt



 F  T  F  D  P  G  F  T  S  T  A  S  C  E  S  K  I  T  F  I



 


gatggtgatgaaggtattttgctgcaccgcggtttcccgatcgatcagctggcgaccgat



 D  G  D  E  G  I  L  L  H  R  G  F  P  I  D  Q  L  A  T  D



 


tctaactacctggaagtttgttacatcctgctgaatggtgaaaaaccgactcaggaacag



 S  N  Y  L  E  V  C  Y  I  L  L  N  G  E  K  P  T  Q  E  Q



 


tatgacgaatttaaaactacggtgacccgtcataccatgatccacgagcagattacccgt



 Y  D  E  F  K  T  T  V  T  R  H  T  M  I  H  E  Q  I  T  R



 


ctgttccatgctttccgtcgcgactcgcatccaatggcagtcatgtgtggtattaccggc



 L  F  H  A  F  R  R  D  S  H  P  M  A  V  M  C  G  I  T  G



 


gcgctggcggcgttctatcacgactcgctggatgttaacaatcctcgtcaccgtgaaatt



 A  L  A  A  F  Y  H  D  S  L  D  V  N  N  P  R  H  R  E  I



 


gccgcgttccgcctgctgtcgaaaatgccgaccatggccgcgatgtgttacaagtattcc



 A  A  F  R  L  L  S  K  M  P  T  M  A  A  M  C  Y  K  Y  S



 


attggtcagccatttgtttacccgcgcaacgatctctcctacgccggtaacttcctgaat



 I  G  Q  P  F  V  Y  P  R  N  D  L  S  Y  A  G  N  F  L  N



 


atgatgttctccacgccgtgcgaaccgtatgaagttaatccgattctggaacgtgctatg



 M  M  F  S  T  P  C  E  P  Y  E  V  N  P  I  L  E  R  A  M



 


gaccgtattctgatcctgcacgctgaccatgaacagaacgcctctacctccaccgtgcgt



 D  R  I  L  I  L  H  A  D  H  E  Q  N  A  S  T  S  T  V  R



 


accgctggctcttcgggtgcgaacccgtttgcctgtatcgcagcaggtattgcttcactg



 T  A  G  S  S  G  A  N  P  F  A  C  I  A  A  G  I  A  S  L



 


tggggacctgcgcacggcggtgctaacgaagcggcgctgaaaatgctggaagaaatcagc



 W  G  P  A  H  G  G  A  N  E  A  A  L  K  M  L  E  E  I  S



 


tccgttaaacacattccggaatttgttcgtcgtgcgaaagacaaaaatgattctttccgc



 S  V  K  H  I  P  E  F  V  R  R  A  K  D  K  N  D  S  F  R



 


ctgatgggcttcggtcaccgcgtgtacaaaaattacgacccgcgcgccaccgtaatgcgt



 L  M  G  F  G  H  R  V  Y  K  N  Y  D  P  R  A  T  V  M  R



 


gaaacctgccatgaagtgctgaaagagctgggcacgaaggatgacctgctggaagtggct



 E  T  C  H  E  V  L  K  E  L  G  T  K  D  D  L  L  E  V  A



 


atggagctggaaaacatcgcgctgaacgacccgtactttatcgagaagaaactgtacccg



 M  E  L  E  N  I  A  L  N  D  P  Y  F  I  E  K  K  L  Y  P



 


aacgtcgatttctactctggtatcatcctgaaagcgatgggtattccgtcttccatgttc



 N  V  D  F  Y  S  G  I  I  L  K  A  M  G  I  P  S  S  M  F



 


accgtcattttcgcaatggcacgtaccgttggctggatcgcccactggagcgaaatgcac



 T  V  I  F  A  M  A  R  T  V  G  W  I  A  H  W  S  E  M  H



 


agtgacggtatgaagattgcccgtccgcgtcagctgtatacaggatatgaaaaacgcgac



 S  D  G  M  K  I  A  R  P  R  Q  L  Y  T  G  Y  E  K  R  D



 


Tttaaaagcgatatcaagcgttaa



 F  K  S  D  I  K  R  -



 


E. coli Aconitase A



atgtcgtcaaccctacgagaagccagtaaggacacgttgcaggccaaagataaaacttac
(SEQ ID NO: 9)


 M  S  S  T  L  R  E  A  S  K  D  T  L  Q  A  K  D  K  T  Y
(SEQ ID NO: 10)


 


cactactacagcctgccgcttgctgctaaatcactgggcgatatcacccgtctacccaag



 H  Y  Y  S  L  P  L  A  A  K  S  L  G  D  I  T  R  L  P  K



 


tcactcaaagttttgctcgaaaacctgctgcgctggcaggatggtaactcggttaccgaa



 S  L  K  V  L  L  E  N  L  L  R  W  Q  D  G  N  S  V  T  E



 


gaggatatccacgcgctggcaggatggctgaaaaatgcccatgctgaccgtgaaattgcc



 E  D  I  H  A  L  A  G  W  L  K  N  A  H  A  D  R  E  I  A



 


taccgcccggcaagggtgctgatgcaggactttaccggcgtacctgccgttgttgatctg



 Y  R  P  A  R  V  L  M  Q  D  F  T  G  V  P  A  V  V  D  L



 


gcggcaatgcgcgaagcggttaaacgcctcggcggcgatactgcaaaggttaacccgctc



 A  A  M  R  E  A  V  K  R  L  G  G  D  T  A  K  V  N  P  L



 


tcaccggtcgacctggtcattgaccactcggtgaccgtcgatcgttttggtgatgatgag



 S  P  V  D  L  V  I  D  H  S  V  T  V  D  R  F  G  D  D  E



 


gcatttgaagaaaacgtacgcctggaaatggagcgcaaccacgaacgttatgtgttcctg



 A  F  E  E  N  V  R  L  E  M  E  R  N  H  E  R  Y  V  F  L



 


aaatggggaaagcaagcgttcagtcggtttagcgtcgtgccgccaggcacaggcatttgc



 K  W  G  K  Q  A  F  S  R  F  S  V  V  P  P  G  T  G  I  C



 


catcaggttaacctcgaatatctcggcaaagcagtgtggagtgaattgcaggacggtgaa



 H  Q  V  N  L  E  Y  L  G  K  A  V  W  S  E  L  Q  D  G  E



 


tggattgcttatccggatacactcgttggtactgactcgcacaccaccatgatcaacggc



 W  I  A  Y  P  D  T  L  V  G  T  D  S  H  T  T  M  I  N  G



 


cttggcgtgctggggtggggcgttggtgggatcgaagcagaagccgcaatgttaggccag



 L  G  V  L  G  W  G  V  G  G  I  E  A  E  A  A  M  L  G  Q



 


ccggtttccatgcttatcccggatgtagtgggcttcaaacttaccggaaaattacgtgaa



 P  V  S  M  L  I  P  D  V  V  G  F  K  L  T  G  K  L  R  E



 


ggtattaccgccacagacctggttctcactgttacccaaatgctgcgcaaacatggcgtg



 G  I  T  A  T  D  L  V  L  T  V  T  Q  M  L  R  K  H  G  V



 


gtggggaaattcgtcgaattttatggtgatggtctggattcactaccgttggcggatcgc



 V  G  K  F  V  E  F  Y  G  D  G  L  D  S  L  P  L  A  D  R



 


gccaccattgccaatatgtcgccagaatatggtgccacctgtggcttcttcccaatcgat



 A  T  I  A  N  M  S  P  E  Y  G  A  T  C  G  F  F  P  I  D



 


gctgtaaccctcgattacatgcgtttaagcgggcgcagcgaagatcaggtcgagttggtc



 A  V  T  L  D  Y  M  R  L  S  G  R  S  E  D  Q  V  E  L  V



 


gaaaaatatgccaaagcgcagggcatgtggcgtaacccgggcgatgaaccaatttttacc



 E  K  Y  A  K  A  Q  G  M  W  R  N  P  G  D  E  P  I  F  T



 


agtacgttagaactggatatgaatgacgttgaagcgagcctggcagggcctaaacgccca



 S  T  L  E  L  D  M  N  D  V  E  A  S  L  A  G  P  K  R  P



 


caggatcgcgttgcactgcccgatgtaccaaaagcatttgccgccagtaacgaactggaa



 Q  D  R  V  A  L  P  D  V  P  K  A  F  A  A  S  N  E  L  E



 


gtgaatgccacgcataaagatcgccagccggtcgattatgttatgaacggacatcagtat



 V  N  A  T  H  K  D  R  Q  P  V  D  Y  V  M  N  G  H  Q  Y



 


cagttacctgatggcgctgtggtcattgctgcgataacctcgtgcaccaacacctctaac



 Q  L  P  D  G  A  V  V  I  A  A  I  T  S  C  T  N  T  S  N



 


ccaagtgtgctgatggccgcaggcttgctggcgaaaaaagccgtaactctgggcctcaag



 P  S  V  L  M  A  A  G  L  L  A  K  K  A  V  T  L  G  L  K



 


cggcaaccatgggtcaaagcgtcgctggcaccgggttcgaaagtcgtttctgattatctg



 R  Q  P  W  V  K  A  S  L  A  P  G  S  K  V  V  S  D  Y  L



 


gcaaaagcgaaactgacaccgtatctcgacgaactggggtttaaccttgtgggatacggt



 A  K  A  K  L  T  P  Y  L  D  E  L  G  F  N  L  V  G  Y  G



 


tgtaccacctgtattggtaactctgggccgctgcccgatcctatcgaaacggcaatcaaa



 C  T  T  C  I  G  N  S  G  P  L  P  D  P  I  E  T  A  I  K



 


aaaagcgatttaaccgtcggtgcggtgctgtccggcaaccgtaactttgaaggccgtatc



 K  S  D  L  T  V  G  A  V  L  S  G  N  R  N  F  E  G  R  I



 


catccgctggttaaaactaactggctggcctcgccgccgctggtggttgcctatgcgctg



 H  P  L  V  K  T  N  W  L  A  S  P  P  L  V  V  A  Y  A  L



 


gcgggaaatatgaatatcaacctggcttctgagcctatcggccatgatcgcaaaggcgat



 A  G  N  M  N  I  N  L  A  S  E  P  I  G  H  D  R  K  G  D



 


ccggtttatctgaaagatatctggccatcggcacaagaaattgcccgtgcggtagaacaa



 P  V  Y  L  K  D  I  W  P  S  A  Q  E  I  A  R  A  V  E  Q



 


gtctccacagaaatgttccgcaaagagtacgcagaagtttttgaaggcacagcagagtgg



 V  S  T  E  M  F  R  K  E  Y  A  E  V  F  E  G  T  A  E  W



 


aagggaattaacgtcacacgatccgatacctacggttggcaggaggactcaacctatatt



 K  G  I  N  V  T  R  S  D  T  Y  G  W  Q  E  D  S  T  Y  I



 


cgcttatcgcctttctttgatgaaatgcaggcaacaccagcaccagtggaagatattcac



 R  L  S  P  F  F  D  E  M  Q  A  T  P  A  P  V  E  D  I  H



 


ggtgcgcggatcctcgcaatgctgggggattcagtcaccactgaccatatctctccggcg



 G  A  R  I  L  A  M  L  G  D  S  V  T  T  D  H  I  S  P  A



 


ggcagtattaagcccgacagcccagcgggtcgatatctacaaggtcggggtgttgagcga



 G  S  I  K  P  D  S  P  A  G  R  Y  L  Q  G  R  G  V  E  R



 


aaagactttaactcctacggttcgcggcgtggtaaccatgaagtgatgatgcgcggcacc



 K  D  F  N  S  Y  G  S  R  R  G  N  H  E  V  M  M  R  G  T



 


ttcgccaatattcgcatccgtaatgaaatggtgcctggcgttgaaggggggatgacgcgg



 F  A  N  I  R  I  R  N  E  M  V  P  G  V  E  G  G  M  T  R



 


catttacctgacagcgacgtagtctctatttatgatgctgcgatgcgctataagcaggag



 H  L  P  D  S  D  V  V  S  I  Y  D  A  A  M  R  Y  K  Q  E



 


caaacgccgctggcggtgattgccgggaaagagtatggatcaggctccagtcgtgactgg



 Q  T  P  L  A  V  I  A  G  K  E  Y  G  S  G  S  S  R  D  W



 


gcggcaaaaggtccgcgtctgcttggtattcgtgtggtgattgccgaatcgtttgaacga



 A  A  K  G  P  R  L  L  G  I  R  V  V  I  A  E  S  F  E  R



 


attcaccgttcgaatttaattggcatgggcatcctgccgctggaatttccgcaaggcgta



 I  H  R  S  N  L  I  G  M  G  I  L  P  L  E  F  P  Q  G  V



 


acgcgtaaaacgttagggctaaccggggaagagaagattgatattggcgatctgcaaaac



 T  R  K  T  L  G  L  T  G  E  E  K  I  D  I  G  D  L  Q  N



 


ctacaacccggcgcgacggttccggtgacgcttacgcgcgcggatggtagccaggaagtc



 L  Q  P  G  A  T  V  P  V  T  L  T  R  A  D  G  S  Q  E  V



 


gtaccctgccgttgtcgtatcgacaccgcgacggagttgacctactaccagaacgacggc



 V  P  C  R  C  R  I  D  T  A  T  E  L  T  Y  Y  Q  N  D  G



Attttgcattatgtcattcgtaatatgttgaagtaa



 I  L  H  Y  V  I  R  N  M  L  K  -



 


E. coli Aconitase B



atgctagaagaataccgtaagcacgtagctgagcgtgccgctgaggggattgcgcccaaa
(SEQ ID NO: 11)


 M  L  E  E  Y  R  K  H  V  A  E  R  A  A  E  G  I  A  P  K
(SEQ ID NO: 12)


 


cccctggatgcaaaccaaatggccgcacttgtagagctgctgaaaaacccgcccgcgggc



 P  L  D  A  N  Q  M  A  A  L  V  E  L  L  K  N  P  P  A  G



 


gaagaagaattcctgttagatctgttaaccaaccgtgttcccccaggcgtcgatgaagcc



 E  E  E  F  L  L  D  L  L  T  N  R  V  P  P  G  V  D  E  A



 


gcctatgtcaaagcaggcttcctggctgctatcgcgaaaggcgaagccaaatcccctctg



 A  Y  V  K  A  G  F  L  A  A  I  A  K  G  E  A  K  S  P  L



 


ctgactccggaaaaagccatcgaactgctgggcaccatgcagggtggttacaacattcat



 L  T  P  E  K  A  I  E  L  L  G  T  M  Q  G  G  Y  N  I  H



 


ccgctgatcgacgcgctggatgatgccaaactggcacctattgctgccaaagcactttct



 P  L  I  D  A  L  D  D  A  K  L  A  P  I  A  A  K  A  L  S



 


cacacgctgctgatgttcgataacttctatgacgtagaagagaaagcgaaagcaggcaac



 H  T  L  L  M  F  D  N  F  Y  D  V  E  E  K  A  K  A  G  N



 


gaatatgcgaagcaggttatgcagtcctgggcggatgccgaatggttcctgaatcgcccg



 E  Y  A  K  Q  V  M  Q  S  W  A  D  A  E  W  F  L  N  R  P



 


gcgctggctgaaaaactgaccgttactgtcttcaaagtcactggcgaaactaacaccgat



 A  L  A  E  K  L  T  V  T  V  F  K  V  T  G  E  T  N  T  D



 


gacctttctccggcaccggatgcgtggtcacgcccggatatcccactgcacgcgctggcg



 D  L  S  P  A  P  D  A  W  S  R  P  D  I  P  L  H  A  L  A



 


atgctgaaaaacgcccgtgaaggtattgagccagaccagcctggtgttgttggtccgatc



 M  L  K  N  A  R  E  G  I  E  P  D  Q  P  G  V  V  G  P  I



 


aagcaaatcgaagctctgcaacagaaaggtttcccgctggcgtacgtcggtgacgttgtg



 K  Q  I  E  A  L  Q  Q  K  G  F  P  L  A  Y  V  G  D  V  V



 


ggtacgggttcttcgcgtaaatccgccactaactccgttctgtggtttatgggcgatgat



 G  T  G  S  S  R  K  S  A  T  N  S  V  L  W  F  M  G  D  D



 


attccacatgtgccgaacaaacgcggcggtggtttgtgcctcggcggtaaaattgcaccc



 I  P  H  V  P  N  K  R  G  G  G  L  C  L  G  G  K  I  A  P



 


atcttctttaacacgatggaagacgcgggtgcactgccaatcgaagtcgacgtctctaac



 I  F  F  N  T  M  E  D  A  G  A  L  P  I  E  V  D  V  S  N



 


ctgaacatgggcgacgtgattgacgtttacccgtacaaaggtgaagtgcgtaaccacgaa



 L  N  M  G  D  V  I  D  V  Y  P  Y  K  G  E  V  R  N  H  E



 


accggcgaactgctggcgaccttcgaactgaaaaccgacgtgctgattgatgaagtgcgt



 T  G  E  L  L  A  T  F  E  L  K  T  D  V  L  I  D  E  V  R



 


gctggtggccgtattccgctgattatcgggcgtggcctgaccaccaaagcgcgtgaagca



 A  G  G  R  I  P  L  I  I  G  R  G  L  T  T  K  A  R  E  A



 


cttggtctgccgcacagtgatgtgttccgtcaggcgaaagatgtcgctgagagcgatcgc



 L  G  L  P  H  S  D  V  F  R  Q  A  K  D  V  A  E  S  D  R



 


ggcttctcgctggcgcaaaaaatggtaggccgtgcctgtggcgtgaaaggcattcgtccg



 G  F  S  L  A  Q  K  M  V  G  R  A  C  G  V  K  G  I  R  P



 


ggcgcgtactgtgaaccgaaaatgacttctgtaggttcccaggacaccaccggcccgatg



 G  A  Y  C  E  P  K  M  T  S  V  G  S  Q  D  T  T  G  P  M



 


acccgtgatgaactgaaagacctggcgtgcctgggcttctcggctgacctggtgatgcag



 T  R  D  E  L  K  D  L  A  C  L  G  F  S  A  D  L  V  M  Q



 


tctttctgccacaccgcggcgtatccgaagccagttgacgtgaacacgcaccacacgctg



 S  F  C  H  T  A  A  Y  P  K  P  V  D  V  N  T  H  H  T  L



 


ccggacttcattatgaaccgtggcggtgtgtcgctgcgtccgggtgacggcgtcattcac



 P  D  F  I  M  N  R  G  G  V  S  L  R  P  G  D  G  V  I  H



 


tcctggctgaaccgtatgctgctgccggataccgtcggtaccggtggtgactcccatacc



 S  W  L  N  R  M  L  L  P  D  T  V  G  T  G  G  D  S  H  T



 


cgtttcccgatcggtatctctttcccggcgggttctggtctggtggcgtttgctgccgca



 R  F  P  I  G  I  S  F  P  A  G  S  G  L  V  A  F  A  A  A



 


actggcgtaatgccgcttgatatgccggaatccgttctggtgcgcttcaaaggcaaaatg



 T  G  V  M  P  L  D  M  P  E  S  V  L  V  R  F  K  G  K  M



 


cagccgggcatcaccctgcgcgatctggtacacgctattccgctgtatgcgatcaaacaa



 Q  P  G  I  T  L  R  D  L  V  H  A  I  P  L  Y  A  I  K  Q



 


ggtctgctgaccgttgagaagaaaggcaagaaaaacatcttctctggccgcatcctggaa



 G  L  L  T  V  E  K  K  G  K  K  N  I  F  S  G  R  I  L  E



 


attgaaggtctgccggatctgaaagttgagcaggcctttgagctaaccgatgcgtccgcc



 I  E  G  L  P  D  L  K  V  E  Q  A  F  E  L  T  D  A  S  A



 


gagcgttctgccgctggttgtaccatcaagctgaacaaagaaccgatcatcgaatacctg



 E  R  S  A  A  G  C  T  I  K  L  N  K  E  P  I  I  E  Y  L



 


aactctaacatcgtcctgctgaagtggatgatcgcggaaggttacggcgatcgtcgtacc



 N  S  N  I  V  L  L  K  W  M  I  A  E  G  Y  G  D  R  R  T



 


ctggaacgtcgtattcagggcatggaaaaatggctggcgaatcctgagctgctggaagcc



 L  E  R  R  I  Q  G  M  E  K  W  L  A  N  P  E  L  L  E  A



 


gatgcagatgcggaatacgcggcagtgatcgacatcgatctggcggatattaaagagcca



 D  A  D  A  E  Y  A  A  V  I  D  I  D  L  A  D  I  K  E  P



 


atcctgtgtgctccgaacgacccggatgacgcgcgtccgctgtctgcggtacagggtgag



 I  L  C  A  P  N  D  P  D  D  A  R  P  L  S  A  V  Q  G  E



 


aagatcgacgaagtgtttatcggttcctgcatgaccaacatcggtcacttccgtgctgcg



 K  I  D  E  V  F  I  G  S  C  M  T  N  I  G  H  F  R  A  A



 


ggtaaactgctggatgcgcataaaggtcagttgccgacccgcctgtgggtggcaccgcca



 G  K  L  L  D  A  H  K  G  Q  L  P  T  R  L  W  V  A  P  P



 


acccgtatggacgccgcacagttgaccgaagaaggctactacagcgtcttcggtaagagt



 T  R  M  D  A  A  Q  L  T  E  E  G  Y  Y  S  V  F  G  K  S



 


ggtgcgcgtatcgagatccctggctgttccctgtgtatgggtaaccaggcgcgtgtggcg



 G  A  R  I  E  I  P  G  C  S  L  C  M  G  N  Q  A  R  V  A



 


gacggtgcaacggtggtttccacctctacccgtaacttcccgaaccgtctgggtactggc



 D  G  A  T  V  V  S  T  S  T  R  N  F  P  N  R  L  G  T  G



 


gcgaatgtcttcctggcttctgcggaactggcggctgttgcggcgctgattggcaaactg



 A  N  V  F  L  A  S  A  E  L  A  A  V  A  A  L  I  G  K  L



 


ccgacgccggaagagtaccagacctacgtggcgcaggtagataaaacagccgttgatact



 P  T  P  E  E  Y  Q  T  Y  V  A  Q  V  D  K  T  A  V  D  T



 


taccgttatctgaacttcaaccagctttctcagtacaccgagaaagccgatggggtgatt



 Y  R  Y  L  N  F  N  Q  L  S  Q  Y  T  E  K  A  D  G  V  I



 


ttccagactgcggtttaa



 F  Q  T  A  V  -







Table 1 below lists additional examples of phosphoenolpyruvate carboxylases/carboxykinase, citrate synthases, and aconitases, as well as exemplary 2-methylcitrate synthases, citrate lyases, and 2-methylcitrate dehydratase:







TABLE 1







Enzymes Involved in Itaconic Acid Synthesis








Enzymes
GenBank Accession Numbers





Phosphoenolpyruvate
NP_417862 (E. coli, EC4.1.1.49); AAB07805 


carboxykinase/carboxylase
(S. aureus, EC4.1.1.32); CAC32156 (M. 



leprae), XP_645396 (D. discoideum); 



EDN6000 (S. cerevisiae); and XP_001210573



(A. terreus); PC2168 (E. coli, EC4.1.1.38), 



NP_850372 (A. thaliana); CAA35251 (S. 



bicolor); CAB95920 (S. coelicolor); 



XP_001391222 (A. niger)


Citrate synthase
AAC73814 (E. coli); NP_001080194 (X. 



laevis); CAB66275 (S. coelicolor); 



NP_080720 (M. musculus); ABP36423 (C. 



phaeovibrioides); XP_001827205 (A. oryzae); 



and EDN 61138 (S. cerevisiae)


2-methylcitrate synthase
ABN63514 (S. baltica); ABI57944 (A. 



ehrlichei); AP_000985 (E. coli); 



XP_001209805 (A. terreus); P45858 (B. 



subtilis); Q56063 (S. typhimurium)


Citrate lyase
YP_662283 (P. atlantica); ABH11558 (L. 



helveticus); AAL50820 (R. erythropolis); 



AP_001263 (E. coli); XP_750953 (A. 



fumigatus); and NP_669690 (Y. pestis)


Aconitase
CAA90177 (B. taurus); CAQ017353 (C. 



michiganesis); CAC37548 (S. coelicolor); 



AAC46192 (M. avium); 1L5JB (E. coli); 



EDN59216 (S. cerevisiae); AAC61778 (A. 



terreus); YP_910600 (C. phaeobacteroides)


2-methylcitrate
ZP_03698315 (L. nitroferrum); 


dehydratase
YP_002029406 (S. maltophilia); AP_000986 



(E. coli); EDV11211 (S. cerevisiae);



XP_001209777 (A. terreus); YP_001860514



(B. phymatum)









The above-described genetically modified microorganisms can be constructed by conventional recombinant technology (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press.)
More specifically, a microorganism strain that overly expresses one or more of the enzymes mentioned above can be obtained as follows. A DNA fragment(s) encoding the one or more of the enzymes mentioned above can be obtained by polymerase chain reaction from its natural source(s) based on its coding sequence(s), which can be retrieved from GenBank. The DNA fragment(s) is then operably linked to a suitable promoter to produce an expression cassette. In one example, one expression cassette includes one coding sequence operably linked to a promoter. In another example, one expression cassette includes multiple coding sequences, all of which are in operative linkage with a promoter. In that case, it is preferred that a ribosomal binding site is incorporated 5′ to each of the coding sequences. If desired, the coding sequences are subjected to codon optimization based on the optimal codon usage in the host microorganism.
As used herein, the term “promoter” refers to a nucleotide sequence containing elements that initiate the transcription of an operably linked nucleic acid sequence in a desired host microorganism. At a minimum, a promoter contains an RNA polymerase binding site. It can further contain one or more enhancer elements which, by definition, enhance transcription, or one or more regulatory elements that control the on/off status of the promoter. When E. coli is used as the host microorganism, representative E. coli promoters include, but are not limited to, the β-lactamase and lactose promoter systems (see Chang et al., Nature 275:615-624, 1978), the SP6, T3, T5, and T7 RNA polymerase promoters (Studier et al., Meth. Enzymol. 185:60-89, 1990), the lambda promoter (Elvin et al., Gene 87:123-126, 1990), the trp promoter (Nichols and Yanofsky, Meth. in Enzymology 101:155-164, 1983), and the Tac and Trc promoters (Russell et al., Gene 20:231-243, 1982). When yeast is used as the host microorganism, exemplary yeast promoters include 3-phosphoglycerate kinase promoter, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter, galactokinase (GAL1) promoter, galactoepimerase promoter, and alcohol dehydrogenase (ADH) promoter. Promoters suitable for driving gene expression in other types of microorganisms are also well known in the art.
The expression cassette(s) described above is then introduced into a suitable microorganism to produce the genetically modified microorganisms disclosed herein. Positive transformants are selected and the over-expression of one or more of the enzymes mentioned above are confirmed by methods known in the art, e.g., immune-blotting or enzymatic activity analysis. The modified microorganisms are then cultured in a suitable medium for itaconic acid production. Preferably, the medium contains glucose or citrate as the precursor for making itaconic acid. After a sufficient culturing period, the medium is collected and the secreted itaconic acid is isolated.
Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present invention to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference.
EXAMPLE 1
Plasmids and Genetically Modified E. coli Strains
E. coli strains BW25113 (rrnBT14 ΔlacZWJ16 hsdR514 ΔaraBADAH33 ΔrhaBADLD78) and BL21 (dcm ompT hsdS(rB-mB-) gal), used as the parent strains (see Datsehko et al., Proc. Natl. Acad. Sci. USA, 97:6640-6645, 2000), were genetically modified by recombinant technology. Briefly, the endogenous icd gene in BW25113 was disrupted by FLP-FRT recombination to produce a BW25113 mutant (i.e., PCI400), which expressed a lower level of isocitrate dehydrogenase as compared to BW25113.
A number of expression plasmids to be introduced into the parent strains and PCI400, shown in Table 2 below, were constructed by recombinant technology.







TABLE 2







Expression Plasmids










Plasmid Name
Genotype






pZE12-luc
ColE1 ori; AmpR; PLlacO1::luc(VF)



pPC1
ColE1 ori; KanR; PLlacO1::cad(AT)



pPC2
from pZE12, PLlacO1::acnA(EC)



pPC3
from pZE12, PLlacO1::acnB(EC)



pPC4
ColE1 ori; SpeR; PLlacO1::gltA(EC)



pPC5
ColE1 ori; SpeR; PLlacO1::ppc(EC)



pPC6
ColE1 ori; SpeR; PLlacO1::ppc(EC)-gltA(EC)





* luc (VF): V. fischeri luciferase


* Cad (AT): A. terreus CAD gene


* acnA(EC): E. coli aconitase A gene


* acnB(EC): E. coli aconitase B gene


* gltA (EC): E. coli citrate synthase gene


* ppc (EC): E. coli phosphoenolpyruvate carboxylase gene






One or more of the plasmids listed in Table 2 above were introduced into BW25113, PCI400, or BL21 to produce various modified E. coli strains, which are listed in Table 3 below:







TABLE 3







Genetically Modified E. coli Strains











Names
Parent strain
Plasmid(s) introduced






PCI 010
BW25133
pPC1



PCI 011
BW25133
pPC1 and pPC5



PCI 012
BW25133
pPC1 and pPC4



PCI 013
BW25133
pPC1 and pPC3



PCI 014
BW25133
pPC1, pPC3, and pPC5



PCI 015
BW25133
pPC1, pPC3, and pPC4



PCI 016
BW25133
pPC1, pPC3, and pPC6



PCI 017
BW25133
pPC1 and pPC6



PCI 019
BW25133
pPC1, pPC2, and pPC6



PCI 213
BL21
pPC1



PCI 510
PCI400
pPC1



PCI 511
PCI400
pPC1 and pPC5



PCI 512
PCI400
pPC1 and pPC4



PCI 513
PCI400
pPC1 and pPC3



PCI 514
PCI400
pPC1, pPC3, and pPC5



PCI 515
PCI400
pPC1, pPC3, and pPC4



PCI 516
PCI400
pPC1, pPC3, and pPC6



PCI 517
PCI400
pPC1 and pPC6



PCI 519
PCI400
pPC1, pPC2, and pPC6









EXAMPLE 2
Producing Itaconic Acid in PCI213
Strain PCI 213 (BL21 over-expressing A. terreus CAD) was cultured overnight at 37° C. in Luria-Bertani (LB) medium. The overnight culture was inoculated (1%) into a minimal medium containing 80 g/L glucose, sodium chloride, and sodium phosphate buffer and cultured at 30° C. for a suitable period until the optical density at 600 nm (OD600) reached 0.2-0.6. Isopropyl β-D-1-thiogalactopyranoside (IPTG) was then added to the culture (0.5 mM) to induce expression of CAD. 24 hours later, the culture medium was collected. The itaconic acid concentration in the medium was about 100 mg/L.
EXAMPLE 3
Producing Itaconic Acid in PCI010
Strain PCI 010 (BW25133 over-expressing A. terreus CAD) was cultured overnight at 37° C. in a minimal medium containing 40 g/L glucose, sodium chloride, and sodium phosphate buffer. The overnight culture was inoculated into M9 medium containing 20 or 40 g/L glucose, sodium chloride, and sodium phosphate buffer, supplemented with or without 1 mM glutamate, and cultured at 30° C. 0.5 mM IPTG was added to the culture when its OD600 reached 0.2 ̃0.4 to induce expression of A. terreus CAD. The culture medium was collected 24 hours later and the amount of itaconic acid was determined. The results obtained from this study were shown in Table 4 below.







TABLE 4







Itaconic Acid Production in PCI010










Ingredients in minimal medium
Itaconic Acid









glucose (g/L)
glutamate (1 mM)
(mg/L)












20
−
168.86


40
−
163.27


20
+
57.05


40
+
88.85









EXAMPLE 4
Production of Itaconic Acid in Various Modified E. coli Strains Using Glucose as Substrate
The modified E. coli strains listed in Table 5 below were cultured in LB medium overnight at 37° C. in a rotary shaker (250 rpm). The overnight cultures were inoculated into M9 medium containing 20 g/L glucose, 1 g/L yeast extract, sodium chloride, and sodium phosphate buffer, and cultured at 37° C. 0.5 mM IPTG was added to each of the cultures when their OD600 reached 0.2 ̃0.4 to induce exogenous gene expression. The culture media were collected at different time points shown in Table 5 below and the amounts of itaconic acid contained therein were determined.







TABLE 5







Production of Itaconic Acid in Genetically Modified E. coli Strains












Itaconate production
Culture Time



E. coli Strain
(g/L)
(h)














PCI 014
0.005
48



PCI 013
0.021
48



PCI 015
0.032
48



PCI 016
0.034
48



PCI 010
0.054
48



PCI 010
0.057
70



PCI 514
0.074
49



PCI 510
0.280
49



PCI 511
0.288
49



PCI 513
0.346
49



PCI 512
0.404
49



PCI 515
0.598
49



PCI 516
2.106
49



PCI 516
4.02
72



PCI 519
4.158
73









As shown in Table 5 above, both strains PCI 519 (BW25133; Δicd, gltA, ppc, cad and acnA) and PCI 516 (BW25133; Δicd, gltA, ppc, cad and acnB) produced more than 4 g/L itaconic acid in the medium after being cultured for 72-hours. The glucose-to-itaconic acid conversion rates of PCI 519 and PCI 516 were about 0.52 g itaconic acid per gram of glucose and about 0.68 g itaconic acid per gram of glucose.
EXAMPLE 5
Producing Itaconic Acid in Genetically Modified E. coli Strains Using Citrate as Substrate
Strains PCI 513 (BW25133; Δicd, cad, and acnB), PCI 516, and PCI 519 were cultured in LB medium at 37° C. for about 16 hours. The E. coli cells were then cultured in fresh LB medium supplemented with 0.5 mM IPTG for 16 hours. The cells were then permeabilized by Triton X-100 and re-suspended in a phosphate buffer containing citrate (50 g/ml). After 48 or 72 hours, the phosphate buffer was examined for the amount of itaconic acid in it.
PCI 513 produced 1.27 g/L of itaconic acid after being cultured for 48 hours. In addition, this strain converted citrate to itaconic acid at a rate of 0.24 g itaconic acid per gram of citrate.
PCI 519 and PCI 516 produced more than 6 g/L and more than 5 g/L itaconic acid after being cultured for 70 hours. The citrate-to-itaconic acid conversion rates were 0.61 itaconic acid per gram of citrate and 0.34 g itaconic acid per gram citrate, respectively.
Other Embodiments
All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.
From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, other embodiments are also within the claims.

## Claims

1. A genetically modified microorganism, comprising
a first exogenous nucleotide sequence encoding an A. terreus cis-aconitic acid decarboxylase (CAD) capable of converting cis-aconitic acid to itaconic acid, the first exogenous nucleotide sequence being operably linked to an E. coli promoter;
a second exogenous nucleotide sequence encoding a phosphoenolpyruvate carboxylase capable of converting phosphoenolpyruvate to oxaloacetate, the second exogenous nucleotide sequence being operably linked to an E. coli promoter;
a third exogenous nucleotide sequence encoding a citrate synthase capable of converting oxaloacetate to citrate, the third exogenous nucleotide sequence being operably linked to an E. coli promoter;
a fourth exogenous nucleotide sequence encoding an aconitase capable of converting citrate or isocitrate to cis-aconitic acid, the fourth exogenous nucleotide sequence being operably linked to an E. coli promoter; and
a mutated endogenous icd gene disrupted by a deletion or insertion, the mutated endogenous icd gene expressing a lower level of isocitrate dehydrogenase relative to its wild-type counterpart;

wherein the genetically modified microorganism is an E. coli hat over-expresses the CAD, the phosphoenolpyruvate carboxylase, the citrate synthase and the aconitase as compared to its wild-type counterpart, and is capable of producing itaconic acid from glucose or citrate as a substrate.

2. A method for producing itaconic acid in a microorganism, comprising
providing the genetically modified microorganism of claim 1,
cultivating the genetically modified microorganism in a medium to produce itaconic acid, and
collecting the medium for isolation of the itaconic acid thus produced.

3. The method of claim 2, wherein the medium contains glucose at a concentration of 5-80 g/L.

4. The method of claim 3, wherein the glucose concentration is 10-40 g/L.

5. The method of claim 2, wherein the genetically modified microorganism is permeablized and the medium contains citrate at a concentration of 5-80 g/L.

6. The method of claim 5, wherein the citrate concentration is 10-40 g/L.

7. The genetically modified microorganism of claim 1, wherein the first exogenous nucleotide sequence encodes the amino acid sequence of SEQ ID NO:2.

8. The genetically modified microorganism of claim 7, wherein the first exogenous nucleotide sequence comprises SEQ ID NO:1.

9. The genetically modified microorganism of claim 1, wherein the second exogenous nucleotide sequence encodes an E. coli phosphoenolpyruvate carboxylase.

10. The genetically modified microorganism of claim 9, wherein the second exogenous nucleotide sequence encodes the amino acid sequence of SEQ ID NO:6.

11. The genetically modified microorganism of claim 10, wherein the second exogenous nucleotide sequence comprises SEQ ID NO:5.

12. The genetically modified microorganism of claim 1, wherein the third exogenous nucleotide sequence encodes an E. coli citrate synthase.

13. The genetically modified microorganism of claim 12, wherein the third exogenous nucleotide sequence encodes the amino acid sequence of SEQ ID NO:8.

14. The genetically modified microorganism of claim 13, wherein the third exogenous nucleotide sequence comprises SEQ ID NO:7.

15. The genetically modified microorganism of claim 1, wherein the fourth exogenous nucleotide sequence encodes an aconitase A.

16. The genetically modified microorganism of claim 1, wherein the fourth exogenous nucleotide sequence encodes an aconitase B.

17. The genetically modified microorganism of claim 1, wherein the fourth exogenous nucleotide sequence encodes an E. coli aconitase A or an E. coli aconitase B.

18. The genetically modified microorganism of claim 17, wherein the fourth exogenous nucleotide sequence encodes the amino acid sequence of SEQ ID NO:12.

19. The genetically modified microorganism of claim 18, wherein the fourth exogenous nucleotide sequence comprises SEQ ID NO:11.

20. The genetically modified microorganism of claim 1, wherein the CAD is a naturally-occurring enzyme from A. terreus, and wherein the phosphoenolpyruvate carboxylase, the citrate synthase, and the aconitase are naturally-occurring enzymes from E. coli.

21. The genetically modified microorganism of claim 20, wherein the first exogenous nucleotide sequence encodes the amino acid sequence of SEQ ID NO:2, the second exogenous nucleotide sequence encodes the amino acid sequence of SEQ ID NO:6, the third exogenous nucleotide sequence encodes the amino acid sequence of SEQ ID NO:8, and the fourth exogenous nucleotide sequence encodes the amino acid sequence of SEQ ID NO:12.

22. The genetically modified microorganism of claim 21, the E. coli promoter being the PLlacO1 promoter.