# Patent

## Title
Cis-aconitate decarboxylase mutants having improved enzymatic activity

## Abstract

Cis-aconitate decarboxylase mutants having one or more mutations in a C-terminal region as compared with a wild-type cis-aconitate decarboxylase of Aspergillus terreus.

## Background
RELATED APPLICATION
This application is a continuation-in-part application of U.S. application Ser. No. 12/494,487, filed on Jun. 30, 2009, now abandoned, the content of which is hereby incorporated by reference in its entirety.


BACKGROUND OF THE INVENTION
Itaconic acid (IA), an essential compound used in manufacture of various products (e.g., acrylic fibers, rubbers, artificial diamonds, and lens), is highly demanded in the chemical industry. Certain microorganisms, such as Aspergillus terreus, produce this compound. It has been found that cis-aconitate decarboxylase (CAD) plays the key role in the biosynthesis of this compound.
SUMMARY OF THE INVENTION
The present invention is based on an unexpected discovery that genetically modified Aspergillus stains containing CAD mutants that carry one or more mutations in a C-terminal region produce higher levels of IA as compared with wild-type Aspergillus. 
Accordingly, one aspect of this invention features an isolated polypeptide having the amino acid sequence of a mutated CAD that has a mutation in the region corresponding to 441-490 (e.g., 461-490 or 481-490) of the amino acid sequence of a wild-type CAD (SEQ ID NO:1). The mutation can be located at the position corresponding to position 490 in SEQ ID NO:1. In one example, this mutation is substitution of a peptide fragment (e.g., GI or GIK) for V at position 490 in SEQ ID NO:1. The mutated CAD described above can further include a mutation at the position corresponding to position 489 in SEQ ID NO:1 (e.g., L at position 489 in SEQ ID NO:1 being replaced with F). In another example, the mutation is substitution of the 441-490 fragment in SEQ ID NO:1 with another fragment, e.g., 441-492 in SEQ ID NO:9. Examples of the CAD mutants described herein include, but are not limited to, a polypeptide having the amino acid sequence of SEQ ID NO:3, 5, 7 or 9.
Another aspect of this invention features a nucleic acid encoding any of the polypeptides disclosed above and host cells containing the nucleic acid, which is in operative linkage with a promoter functional in the host cell. A promoter sequence is a nucleotide sequence containing an element(s) necessary for initiating transcription of an operably linked nucleic acid sequence. At a minimum, a promoter contains an RNA polymerase binding site. It can further contain one or more enhancer elements that enhance transcription, or contain one or more regulatory elements that control the on/off status of the promoter.
Also within the scope of this invention is a method for producing IA by culturing the host cell disclosed above in a suitable medium to allow production of IA. The IA thus produced can be isolated from the culture medium.
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 examples, and also from the appended claims.


DETAILED DESCRIPTION OF THE INVENTION
Described herein is a mutated CAD that displays higher enzymatic activity relative to its wild-type counterpart.
CAD, which converts cis-aconitic acid to itaconic acid, is the key enzyme involved in the biosynthesis of IA. The term “CAD” used herein refers to any naturally occurring CADs (i.e., wild-type CAD). One example is the A. terreus CAD described in Dwiarti et al., J. Bioscience and Bioengineering, 94 (1):29-33, 2002 and WO 2009/014437). Provided below are the amino acid sequence (SEQ ID NO:1) of this A. terreus CAD and an example of its encoding nucleotide sequence (SEQ ID NO:2):




Other natural CADs are polypeptides found in non-A. terreus species that possess the same enzymatic activity. These polypeptides are highly homologous to the A. terreus CAD described above, i.e., having an amino acid sequence at least 75% (85%, 90%, or 95%) identical to SEQ ID NO:1. The amino acid sequences and their encoding gene sequences can be retrieved from gene/protein databases, e.g., GenBank, using SEQ ID NOs:1 and 2 as queries.
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 CAD mutant of this invention contains one or more mutations in the region of a naturally-occurring CAD that corresponds to 441-490 in SEQ ID NO:1. The one or more mutations can be deletion, insertion, or amino acid residue substitution (e.g., a fragment being replaced with a single amino acid residue or with another fragment, or a single residue being replaced with a fragment). Listed below are a number of exemplary CAD mutants, including their amino acid sequences (mutated positions bold-faced) and encoding nucleotide sequences:










Any of the above-mentioned CAD mutants can be prepared by conventional recombinant technology. For example, one or more mutations can be introduced into a nucleotide sequence encoding a wild-type CAD and the mutated encoding sequence can be expressed in a suitable host cell to produce the mutated CAD polypeptide. If desired, the coding sequences are subjected to codon optimization based on the optimal codon usage in the host microorganism. Below is an example of an A. terreus CAD coding sequence, in which certain codons were replaced with optimal E. coli codons:







(SEQ ID NO: 11)


atgaccaagcagtctgctgattccaacgcgaagtctggtgtgacctct


 


gagatctgtcactgggcgtctaatctcgccactgatgatatcccgagc


 


gacgttctggagcgtgcaaaatacctgatcctggatggtatcgcgtgc


 


gcgtgggtaggtgctcgtgtcccatggtctgaaaaatacgttcaagcg


 


accatgtctttcgaacctccgggtgcgtgtcgtgtcatcggttacggc


 


cagaaactgggtccggtagcggctgccatgacgaactctgcatttatt


 


caggcgaccgaactcgatgactatcactctgaagcgccgctgcattcc


 


gcgtctatcgttctcccggcagttttcgcggcgagcgaagtactggcc


 


gaacagggtaaaaccatctctggtattgacgtgattctggctgcgatc


 


gttggtttcgagagcggtcctcgcatcggcaaagcgatctacggttct


 


gacctcctgaacaacggctggcactgcggtgcggtatatggcgcaccg


 


gctggtgcgctcgcaactggtaagctcctgggcctcacgccggacagc


 


atggaagatgcactgggtattgcctgcacgcaagcatgcggcctcatg


 


tccgcgcagtatggtggcatggttaaacgtgttcagcacggtttcgca


 


gcgcgtaatggtctcctcggtggcctcctggctcacggcggctacgag


 


gcgatgaaaggtgttctcgagcgttcttacggtggcttcctgaagatg


 


ttcaccaagggcaacggtcgtgaaccgccgtacaaagaagaagaggtt


 


gtggctggtctgggtagcttctggcacaccttcaccattcgtatcaaa


 


ctgtacgcgtgctgcggtctcgtacacggtcctgttgaagccattgaa


 


aacctccagggtcgttacccggaactgctcaatcgtgctaacctgtct


 


aacatccgccacgttcacgtacaactctctaccgcgagcaactcccac


 


tgtggttggatcccagaagagcgcccaatctcttctatcgcgggtcaa


 


atgtctgtcgcatatatcctcgccgttcagctcgttgaccaacagtgt


 


ctgctcagccagttctccgagtttgacgataatctggaacgcccggaa


 


gtgtgggacctggcacgtaaggttaccagctctcaatctgaggagttc


 


gaccaggacggtaactgtctctctgccggtcgcgtccgtattgagttc


 


aacgacggctcctccatcaccgaatccgttgagaagccgctcggtgta


 


aaggaaccaatgccaaatgaacgcatcctgcacaaataccgtaccctg


 


gcgggttctgtaacggacgaaagccgtgttaaggagatcgaggatctc


 


gtgctcggcctggaccgtctgaccgatattagcccgctcctcgagctg


 


ctgaattgtccggttaaatccccactggtttaa






The level of protein expression can be measured by conventional methods. In one example, the levels of a wild-type and mutated CAD are determined by Western analysis. The increased enzymatic activity of the polypeptide can be confirmed by conventional methods. In one example, the IA level produced in the host cells can be determined by HPLC and an elevated level of IA production relative to the same type of host cells expressing a wild-type CAD indicates that the mutant CAD possesses improved enzymatic activity as compared with the wild-type CAD.
After its elevated enzymatic activity has been confirmed, the CAD mutant can be used for producing IA. More specifically, its coding sequence can be cloned into a suitable expression plasmid and introduced into a suitable host microorganism. The microorganism is 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 example is, 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
Itaconic Acid Production in A. terreus Strains Containing Mutated CADs
Generating A. terreus Mutants/Fusants Containing Mutated CADs
Mutants of A. terreus were generated by random mutagenesis as follows. A. terreus ATCC10020 cells at a concentration of 108 cell/ml was incubated overnight in an IA producing medium (pH 2.8) that contains, in one liter distilled water, 100 g glucose, 6 g (NH4)2SO4, 0.2 g KH2PO4, 1 g MgSO4.7H2O, 0.4 g CaSO4, 0.5×10−3 g CuSO4, 0.5×10−3 g ZnSO4.4H2O, 3×10−3 g FeCl3. The cells were collected, washed, and then incubated with 10-15 g/l Lysosome (contains beta-glucanase, cellulose, protease and chitinase) and a mutagen, i.e., 1-methyl-3-nitro-nitrosoguanidine (NTG, 1-5 g/l) or ethylmethanesulfonate (EMS, 2-200 mg/l), to induce mutations. The cells were again collected and re-suspended in a medium, placed on a 2-deoxy-glucose (2-DG)-potato agar plate (2-DG concentration 0.25-5.0 g/l), and cultured under suitable conditions to allow colony formation. Around 12.5-99.3% (3×107-9×108 CFU/ml) of the cells died in the presence of either mutagen. Each of the surviving colonies were cultured in 25 ml of the above-described IA production medium at a concentration of 1010 cell/ml and then plated on an IA-potato dextrose agar plate containing a pH indicator (pH 3.0-5.4), which was selected from bromocresol green, bromophenol blue, or congo red (30-130 mg/L). Colonies producing high levels of IA were selected based on their color shown on the plate.
Mutants M1-M5 were selected in the just-described screening process as producing high levels of IA. These mutants were then cultured in the IA-production medium for 3.5 days and the levels of IA in the culture media, filtered through a membrane having a pore size of 0.22 μm, were analyzed by HPLC, using the LiChroCART column (5-mm particle size, 125-mm length, 4-mm diameter, E. Merck, Germany). IA was eluted at 30° C. with a buffer containing 20 mM ortho-phosphoric acid at a flow rate of 1 ml/min. The eluted IA was detected at 230 nm with a Shimadzu SD-20A Absorbance Detector (Shimadzu, Japan). As shown in Table 1 below, mutants M1-M5 produced higher levels of IA than wild-type A. terreus.







TABLE 1







Itaconic Acid Production by M1-M5












IA concentration
IA concentration
Yield
Yield


Strains
(g/l)
(folds)
(%)
(folds)














WT
10.87
1
23.38
1


M1
17.43
1.60
28.30
1.21


M2
17.12
1.57
29.06
1.24


M3
21.68
1.99
33.55
1.44


M4
28.04
2.58
36.85
1.58


M5
23.51
2.16
39.32
1.68









A. terreus mutants were also generated by genome shuffling with A. niger as follows. Aspergillus terreus ATCC10020 cells (108 cell/ml) and Aspergillus niger NRRRL330 cells (108 cell/ml) were cultured separately overnight at 35° C. in a medium (pH 3) containing, in one liter distilled water, 150 g glucose, 2.5 g (NH4)2SO4, 2 g KH2PO4, 0.5 g MgSO4, 0.06×10−3 g CuSO4.5H2O, 0.1×10−3 g ZnSO4.7H2O, 0.1×103 g FeSO4.24H2O. All cells were collected, washed with a phosphate buffer (pH 6) containing 0.7 M KCl, and incubated in Lysosome for 2 hours with gentle shaking. The protoplasts thus released were subjected to osmosis using 1.4 M sorbitol and then collected by centrifugation at 1800 g. The protoplasts were re-suspended in distilled water to form crude protoplast suspensions. Equal volumes of A. terreus and A. niger crude protoplast suspensions were mixed and centrifuged at 1000 g, 4° C. for 10 min. The protoplast pellet thus formed was resuspended in a 0.05 M glycin-NaOH buffer (pH7.5) containing 30% (w/v) polyethylene glycol (PEG) 6000, 100 mM CaCl2, and 0.7 M KCl. The suspension was incubated at 30° C. for 20 min, plated onto the surface of a potato dextrose agar plate, and incubated at 30° C. for 5-7 days to allow formation of colonies, which were fusants. The fusion frequency in this study was found to be  ̃200 CFU/ml. Each of the fusants was then cultured in 25 ml of the IA production medium described above at 35° C. for 5.5 days in a rotary shaker (150 rev/min). Fusants G1-G6 were found to produce higher levels of IA than wild-type A. terreus.







TABLE 2







Itaconic Acid Production by G1-G6












IA concentration
IA concentration
Yield
Yield


Strains
(g/l)
(folds)
(%)
(folds)














WT
21.83
1
27.20
1


G1
35.97
1.65
49.69
1.83


G2
35.63
1.63
50.26
1.85


G3
29.33
1.34
67.12
2.47


G4
31.90
1.46
50.46
1.86


G5
31.62
1.45
55.29
2.03


G6
30.98
1.42
53.69
1.97









Determining Nucleotide Sequences Encoding CAD Mutants

The CAD genomic sequences of certain mutants/fusants were determined by conventional methods. Briefly, genomic DNAs and total RNAs were isolated from the mutants/fusants of interest using Wizard® Genomic DNA Purification kit (Promega, USA) and Epicentre® MasterPureTM RNA Purification kit (Biotechnologies, USA), respectively. A DNA fragment containing the CAD gene was amplified from the genomic DNAs by polymerase chain reaction (PCR) with tag polymerase (InvitrogenTM, US) using the primers listed below:










CAD1 (forward):




5′-CAGCCATGACCAATTCCGCTTTCA-3′
(SEQ ID NO: 12)



 



CAD1 (reverse):




5′-AAGACCTCACTTGCTGCAAAGACC-3′
(SEQ ID NO: 13)



 



Cad-f-2 (504-523):




5′-TTGTGGAGCTGTGTATGGCG-3′
(SEQ ID NO: 14)



 



Cad-(700-716)-F:




5′-GTTGGCCCATGGTGGG-3′
(SEQ ID NO: 15)



 



Cad-(251-270)-R:




5′-CATGGCTGCTGCAACAGGCC-3′
(SEQ ID NO: 16)






The PCR conditions were: 94° C. for 5 s, 30 cycles of 94° C. for 20 s, 56° C. for 30 s, 72° C. for 2 min, and a final extension at 72° C. for 10 s.
The CAD coding sequences were also amplified by reverse transcription polymerase chain reaction (RT-PCR) using the total RNAs mentioned above as the template and the primers listed above with the Verso 1-step RT-PCR kit (AB gene, US) under the following conditions: 47° C. for 30 s, 94° C. for 2 min, 30 cycles of 94° C. for 20 s, 55° C. for 30 s, 72° C. for 2 min, and a final extension at 72° C. for 5 min.
Results obtained from this study indicate that M1 and M4 contain mCAD1, G2 and G4 contain mCAD2, and G3 contains mCAD3. The amino acid sequences and nucleotide sequences of these CAD mutants are shown in pages 4-7 above.
Determining Enzymatic Activity of CAD Mutants
The CAD activity of these mutated CAD were examined following the method described in Dwiarti et al., J. Bioscience and Bioengineering 94:29-33 (2002) with slight modifications. Briefly, A. terreus strains containing these CAD mutants were cultured in a suitable medium (1010 cells/ml) for 3 days at 35° C. in a rotary shaker (150 rev/min). The cultured cells were harvested, suspended in a sodium phosphate buffer (0.2 M, pH 6.2) at a volume ratio of 1:10, and lyzed by pulsed sonication for 10 min to form crude lysates. The lysates were centrifuged and the resultant supernatants were collected. 4.5 ml of each supernatant were incubated with 0.5 ml of a solution containing cis-aconitic acid at a final concentration of 5.0 mM for 10 min at 40° C. The enzyme reaction was terminated by addition of 0.1 ml 12 M HCl into the reaction mixture. The amount of itaconic acid, the product of the enzymatic reaction, was measured by HPLC (column: LiChroCART, detector: SPD-20AD, 20 mM ortho-phosphoric acid, at 30° C., at 230 nm).







TABLE 3







CAD Activity in A. terreus Strains Containing Mutated CADs













CAD activity
CAD activity




Strains
(umol/mg/min)
(folds)
CAD
















WT
0.007
1
WT



M1
0.056
8
mCAD1



M4
0.064
9.1
mCAD1



G2
0.009
1.3
mCAD2



G3
0.008
1.1
mCAD3



G4
0.018
2.6
mCAD2










As shown in Table 3 above, A. terreus strains containing the mutated CADs all exhibited increased CAD activity as compared with wild-type A. terreus. 

EXAMPLE 2
Preparation of CAD Mutants in E. Coli 
mCAD1, mCAD2, and mCAD3 were amplified by PCR using primers list below:








CAD (forward):



actcatatgATGACCAAACAATCTGCGGA
(SEQ ID NO: 17)


 


mCAD1 (reverse):



tacggatcc TATACCAAGGGGCGATTTCAC
(SEQ ID NO: 18)


 


mCAD2 (reverse):



tacggatcc TTTTATACCCAGGGGCGATTTC
(SEQ ID NO: 19)


 


mCAD3 (reverse):



tacggatccTATACCAAAGGGCGATTTCACG
(SEQ ID NO: 20)






The PCR condition were as follows: 94° C. for 5 s, 25 cycles of 94° C. for 20 s, 56° C. for 30 s, 72° C. for 2 min, and a final extension at 72° C. for 10 s.
The PCR products were cloned into pET-24a DNA 69749-3 (Novagen), which carries a His-tag, via restriction sites BamHI and NdeI. The resultant plasmids were introduced into DH5α (GeneMark, Cat. No. DH01-10). Positive clones were determined by colony PCR, using the primers 5′-ctcagcttcctttcgggctt-3′ (SEQ ID NO:21) and 5′-cgcacctgtggcgcc-3′ (SEQ ID NO:22) and further confirmed by DNA sequencing. Plasmid DNAs carrying coding sequences for mCAD1, mCAD2, and mCAD3 were purified using Plasmid Miniprep Purification Kit (GeneMark, Cat. No: DP01-300).
mCAD1, mCAD2, and mCAD3, as well as wild-type (wt) CAD, were overexpressed in DH5α and purified using His Binding® Kits (Novagen, Cat. No. 70239-3). As confirmed by Westernblot, the expression levels of these mutants were similar to that of the wt CAD, indicating that the mutations had no impact on protein expression.
The CAD activities of the purified CAD mutants were examined following the method described in Example 1 above. As shown Table 4 below, the mutated CADs all exhibited increased CAD activity as compared with wt CAD.







TABLE 4







CAD Activity in E. Coli Containing Mutated CADs










CAD activity
CAD activity


Strains
(%)
(folds)












WT
100
1


mCAD1
118.24
1.2


mCAD2
153.88
1.5


mCAD3
104.16
1.04









Moreover, enzyme kinetics was examined following the method described in Dwiarti et al., J. Bioscience and Bioengineering 94:29-33; 2002 with slight modifications. Briefly, an E. coli strain containing one of the CAD mutants was cultured in LB medium for a suitable period at 35° C. in a rotary shaker (250 rev/min). When the optical density of the culture reached 0.4, the bacterial cells were cultured in M9 medium (1 g/l yeast extract and 20 g/l glucose) supplemented with IPTG for 5-6 hours at 30° C. in a rotary shaker (250 rev/min) to allow expression of the CAD mutant. The cells were harvested, suspended in a sodium phosphate buffer (0.2 M, pH 6.5) at a volume ratio of 1:10, and lyzed by a bead beater for 1 min to form a crude lysate. The lysate was centrifuged and the resultant supernatant was collected. To determine the enzymatic activity of the CAD mutant, 0.5 ml of the supernatant was incubated with 0.5 ml of a solution containing cis-aconitic acid at a final concentration of 12 mM at 37° C. for various periods and then mixed with 1 μl 12 M HCl to terminate the enzymatic reaction. The amount of itaconic acid, converted from cis-aconitic acid by the CAD mutant, was measured by HPLC (column: LiChroCART, detector: SPD-20AD, 20 mM ortho-phosphoric acid, at 30° C., at 230 nm).








TABLE 5







Enzyme Kinetics of CAD Mutants












Km (mM)
Vm (mM/min)
Kcat (1/s)
kcat/Km (mM-1s-1)















WT
5
0.0024
60.61
12.12


mCAD3
3.7
0.002
81.82
22.11


mCAD2
5
0.0027
72.73
14.55


mCAD1
5.55
0.0025
75.76
13.65









In sum, the results from this study indicate that mCAD1, mCAD2, and mCAD3 exhibited higher enzymatic activity than wt CAD.
EXAMPLE 3
Itaconic Acid Production in E. Coli Expressing mCAD4
mCAD4 was generated by random mutagenesis as follows. Error-Prone PCR was performed using SEQ ID NO:11 as a template and the GeneMorph® II Random Mutagenesis Kit (Stratagene, Cat. No. 200550). A mutated sequence encoding mCAD4 was identified. Compared to wt CAD, mCAD4 contains a substituted fragment (52 amino acids) at positions 441-490 in wt CAD (SEQ ID NO:1).
IA production in E. coli strains expressing either mCAD4 or wt CAD was determined following the method described in Example 1 and the results are shown in Table 6 below:







TABLE 6







Itaconic Acid Production in E. Coli


Strains Expressing mCAD4 and wt CAD










IA concentration
IA concentration


Strains
(g/l)
(folds)












WT
0.020121
1


mCAD4
0.024648
1.22









The above results indicate that an E. coli strain expressing mCAD4 produced a higher level of IA as compared to an E. coli strain expressing wt CAD. This indicates that mCAD4 possesses higher enzymatic activity than wt CAD.
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. An isolated polypeptide, comprising the amino acid sequence of a mutated cis-aconitate decarboxylase (CAD), wherein the mutated CAD has a mutation in the region corresponding to positions 441-490 in the wild-type CAD sequence of SEQ ID NO:1, the amino acid sequence being that of SEQ ID NO: 3, 5, 7, or 9.

2. The isolated polypeptide of claim 1, wherein the amino acid sequence is that of SEQ ID NO:3.

3. The isolated polypeptide of claim 1, wherein the amino acid sequence is that of SEQ ID NO:5.

4. The isolated polypeptide of claim 1, wherein the amino acid sequence is that of SEQ ID NO:7.

5. The isolated polypeptide of claim 1, wherein the amino acid sequence is that of SEQ ID NO:9.