Means and Methods for Itaconic Acid Production
AssigneesRheinisch-Westfaelische Technische Hochschule (Rwth) Aachen, Philipps-Universitaet Marburg
InventorsMichael Boelker, Wolfgang Buckel, Sandra Przybilla, Lars M. Blank, Nick Wierckx, Elena Geiser
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Abstract
The present invention relates to a method of producing itaconic acid. Further the present invention relates to nucleic acids encoding an aconitate-delta-isomerase (ADI) and trans-aconitate decarboxylase (TAD) and uses of such nucleic acids. Provided is additionally a recombinant host cell engineered to overexpress nucleic acids of the present invention. Furthermore an expression cassette and a vector are provided which include the respective nucleic acid.
Claims28
A method of producing itaconic acid, comprising
- (a) culturing a recombinant host cell which is genetically engineered to overexpress
- (i) a polynucleotide sequence having at least 30% sequence identity with the sequence of SEQ ID NO:
- 1, said polynucleotide sequence encoding a protein or fragment thereof having aconitate-delta-isomerase (ADI) activity;
- and
- (ii) a polynucleotide sequence having at least 50% sequence identity with the sequence of SEQ ID NO:
- 6, said polynucleotide sequence encoding a protein or fragment thereof having trans-aconitate decarboxylase (TAD) activity,
- in comparison to a comparable host cell which has not been genetically altered under conditions to allow said host cell to overexpress said polynucleotide sequence (i) and (ii), thereby said host cell is capable of converting cis-aconitate via trans-aconitate to itaconic acid;
- and
- (b) obtaining itaconic acid.
3-4. (canceled)
(canceled)
9-10. (canceled)
(canceled)
23-25. (canceled)
A host cell genetically engineered to overexpress at least one of the nucleic acid molecules having a polynucleotide sequence selected from
- (a) a polynucleotide sequence having at least 30% sequence identity with the sequence of SEQ ID NO:
- 1, said polynucleotide sequence encoding a protein or fragment thereof having aconitate-delta-isomerase (ADI) activity;
- (b) a polynucleotide sequence having at least 50% sequence identity with the sequence of SEQ ID NO:
- 3, said polynucleotide sequence encoding a protein or fragment thereof having the activity of a multidrug transporter of the major facilitator superfamily, preferably of a transporter which exports itaconic acid out of the host cell;
- (c) a polynucleotide sequence having at least 50% sequence identity with the sequence of SEQ ID NO:
- 4, said polynucleotide sequence encoding a protein or fragment thereof having the activity of a mitochondrial citrate transporter, preferably of a mitochondrial cis-aconitate transporter,
- (d) a polynucleotide sequence having at least 50% sequence identity with the sequence of SEQ ID NO:
- 6, said polynucleotide sequence encoding a protein or fragment thereof having trans-aconitate decarboxylase (TAD) activity;
- (e) a polynucleotide sequence having at least 50% sequence identity with the sequence of SEQ ID NO:
- 8, said polynucleotide sequence encoding a protein or fragment thereof having the activity of a transcription factor for at least one polynucleotide sequence selected from the group consisting of SEQ ID NOs:
- 1, 3, 4, and 6
- in comparison to a comparable host cell which has not been genetically altered.
(canceled)
30-31. (canceled)
A method for producing a host cell which is capable of producing itaconic acid, comprising genetically engineering a host cell to overexpress a nucleic acid having a polynucleotide sequence selected from
- (a) a polynucleotide sequence having at least 30% sequence identity with the sequence of SEQ ID NO:
- 1, said polynucleotide sequence encoding a protein or fragment thereof having aconitate-delta-isomerase (ADI) activity;
- (b) a polynucleotide sequence having at least 50% sequence identity with the sequence of SEQ ID NO:
- 3, said polynucleotide sequence encoding a protein or fragment thereof having the activity of a multidrug transporter of the major facilitator superfamily, preferably of a transporter which exports itaconic acid out of the host cell;
- (c) a polynucleotide sequence having at least 50% sequence identity with the sequence of SEQ ID NO:
- 4, said polynucleotide sequence encoding a protein or fragment thereof having the activity of a mitochondrial citrate transporter, preferably of a mitochondrial cis-aconitate transporter,
- (d) a polynucleotide sequence having at least 50% sequence identity with the sequence of SEQ ID NO:
- 6, said polynucleotide sequence encoding a protein or fragment thereof having trans-aconitate decarboxylase (TAD) activity;
- and
- (e) a polynucleotide sequence having at least 50% sequence identity with the sequence of SEQ ID NO:
- 8, said polynucleotide sequence encoding a protein or fragment thereof having the activity of a transcription factor for at least one polynucleotide sequence selected from the group consisting of SEQ ID NOs:
- 1, 3, 4, and 6.
(canceled)
Dependencies are inferred from the claim text.