Pseudozyma microorganisms and the production of itaconic acid
AssigneeBASF SE
InventorGerold Barth
Life of this patent
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Abstract
Described herein is the fermentative production of fine chemicals, notably itaconate or itaconic acid, including production microorganisms, fermentation compositions and media, proteins useful in the production of the products, and nucleic acids for expression of such proteins, as well as methods for the production of fine chemicals.
Claims21
An itaconic acid production host microorganism of genus Pseudozyma, wherein the microorganism comprises at least one heterologous expression cassette integrated into the genome of the microorganism outside of an ip locus, wherein the expression cassette comprises:
- a) a heterologous RIA1 gene encoding an RIA1 protein, wherein the RIA1 protein comprises at least 90% amino acid sequence identity to SEQ ID NO:
- 35, under the operable control of a functional promoter,
- and/or
- b) a RIA1 gene encoding an RIA1 protein, wherein the RIA1 protein comprises at least 90% amino acid sequence identity to SEQ ID NO:
- 35, under the operable control of a heterologous functional promoter.
An itaconic acid production host microorganism of genus Pseudozyma, wherein the host is a recombinant microorganism comprising an expression cassette
- for expression of a RIA1 gene encoding an RIA1 protein, wherein the RIA1 protein comprises at least 90% amino acid sequence identity to SEQ ID NO:
- 35, and
- for expression of at least two genes selected from the group consisting of ADI1, MTT1, and TAD1,
- wherein said expression cassette is integrated into the genome of the microorganism at an integration site, wherein the integration site
- a) is located between a left border gene and a right border gene, wherein the respective first nucleotides of the respective translation start codons of the left and right border genes are separated by at most 51600 nucleotides in the corresponding wild type microorganism,
- wherein the left border gene codes for a protein having acetyl-CoA synthetase activity, and
- wherein the right border gene codes for a protein consisting of an amino acid sequence having at least 21% identity to SEQ ID NO:
- 25, and/or
- b) is located up to 51600 nucleotides of the nearest border of an open reading frame translating into an amino acid sequence having at least 30% sequence identity to any of SEQ ID NO:
- 3, SEQ ID NO:
- 5, SEQ ID NO:
- 7, SEQ ID NO:
- 9, SEQ ID NO:
- 11, SEQ ID NO:
- 13, SEQ ID NO:
- 15, SEQ ID NO:
- 17, SEQ ID NO:
- 19, SEQ ID NO:
- 21, SEQ ID NO:
- 23, SEQ ID NO:
- 25, and/or
- c) is located within or replaces an open reading frame translating into an amino acid sequence having at least 30% sequence identity to any of SEQ ID NO:
- 3, SEQ ID NO:
- 5, SEQ ID NO:
- 7, SEQ ID NO:
- 9, SEQ ID NO:
- 11, SEQ ID NO:
- 13, SEQ ID NO:
- 15, SEQ ID NO:
- 17, SEQ ID NO:
- 19, SEQ ID NO:
- 21, SEQ ID NO:
- 23, SEQ ID NO:
- 25.
An itaconic acid production host microorganism of genus Pseudozyma, wherein the host is a recombinant microorganism
- a) comprising an active itaconic acid metabolic pathway for producing itaconic acid, wherein the active itaconic acid pathway comprises at least one RIA1 gene encoding an RIA1 protein, wherein the RIA1 protein comprises at least 90% amino acid sequence identity to SEQ ID NO:
- 35, and
- b) wherein at least one gene coding for a protein having at least 30% sequence identity to any of the protein sequences SEQ ID NO:
- 3, SEQ ID NO:
- 5, SEQ ID NO:
- 7, SEQ ID NO:
- 9, SEQ ID NO:
- 11, SEQ ID NO:
- 13, SEQ ID NO:
- 15, SEQ ID NO:
- 17, SEQ ID NO:
- 19, SEQ ID NO:
- 21, SEQ ID NO:
- 23, or SEQ ID NO:
- 25 is inactivated.
An integration vector comprising a RIA1 gene encoding an RIA1 protein, wherein the RIA1 protein comprises at least 90% amino acid sequence identity to SEQ ID NO:
- 35, operably linked to a strong constitutively active promoter for integration outside of an ip locus.
A method for alteration of an itaconic acid production host microorganism of genus Pseudozyma, the method comprising integrating at least one expression cassette
- for expression of a RIA1 gene encoding an RIA1 protein, wherein the RIA1 protein comprises at least 90% amino acid sequence identity to SEQ ID NO:
- 35, and
- for expression of at least two genes selected from the group consisting of ADI1, MTT1, and TAD1,
- into the genome of a microorganism at an integration site other than an ip locus.
A method for obtaining a recombinant itaconic acid production host microorganism of genus Pseudozyma, the method comprising:
- a) cultivating a parent microorganism,
- b) performing, in any order and/or simultaneously,
- if so required:
- one or more transformations to provide the microorganism with any heterologous ADI1, MTT1, and TAD1 gene to obtain an active itaconic acid pathway in the microorganism,
- at least one integration of a RIA1 gene encoding an RIA1 protein, wherein the RIA1 protein comprises at least 90% amino acid sequence identity to SEQ ID NO:
- 35, under the control of a constitutively active promoter, wherein integration is not in an ip-locus, and
- inactivation of at least one gene coding for a protein having at least 30% sequence identity to any of the protein sequences SEQ ID NO:
- 3, SEQ ID NO:
- 5, SEQ ID NO:
- 7, SEQ ID NO:
- 9, SEQ ID NO:
- 11, SEQ ID NO:
- 13, SEQ ID NO:
- 15, SEQ ID NO:
- 17, SEQ ID NO:
- 19, SEQ ID NO:
- 21, SEQ ID NO:
- 23, or SEQ ID NO:
- 25;
- and
- c) isolating the recombinant itaconic acid production host microorganism resulting from step b).
Dependencies are inferred from the claim text.