Described are methods for the production of isobutene comprising the enzymatic conversion of 3-methylcrotonic acid into isobutene wherein said 3-methylcrotonic acid is obtained by the enzymatic conversion of 3-methylcrotonyl-CoA into 3-methylcrotonic acid or wherein said 3-methylcrotonic acid is obtained by the enzymatic conversion of 3-hydroxyisovalerate (HIV) into 3-methylcrotonic acid. It is described that the enzymatic conversion of 3-methylcrotonic acid into isobutene can, e.g., be achieved by making use of a 3-methylcrotonic acid decarboxylase, preferably an FMN-dependent decarboxylase associated with an FMN prenyl transferase, an aconitate decarboxylase (EC 4.1.1.6), a methylcrotonyl-CoA carboxylase (EC 6.4.1.4), or a geranoyl-CoA carboxylase (EC 6.4.1.5).

The invention relates to the use of a cytosolic citric acid synthase for the heterologous production of citrate outside the mitochondrion of a micro-organism or algae, wherein the protein is selected from A. niger An08g10920, An01g09940, An09g03570 ,or an ortholog of these genes. Such production is achieved by introducing the nucleic acid encoding such a protein into a suitable host cell. Preferably the protein is A. niger An08g10920, An01g09940, An09g03570 or an ortholog thereof, more particularly, wherein such an ortholog is chosen from the group of proteins listed in FIG. 9 and proteins having a percentage identity of 70%, more preferably 75%, more preferably 80%, more preferably 85%, more preferably 90%, more preferably 95%, more preferably 98%, more preferably 99% with An08g10920, An01g09940 or An09g03570.

The invention relates to the use of a cytosolic citric acid synthase for the heterologous production of citrate outside the mitochondrion of a micro-organism or algae, wherein the protein is selected from A. niger An08g10920, An01g09940, An09g03570, or an ortholog of these genes. Such production is achieved by introducing the nucleic acid encoding such a protein into a suitable host cell. Preferably the protein is A. niger An08g10920, An01g09940, An09g03570 or an ortholog thereof, more particularly, wherein such an ortholog is chosen from the group of proteins listed in FIG. 9 and proteins having a percentage identity of 70%, more preferably 75%, more preferably 80%, more preferably 85%, more preferably 90%, more preferably 95%, more preferably 98%, more preferably 99% with An08g10920, An01g09940 or An09g03570.

The present invention relates to a method for the production of itaconic acid, which method comprises fermenting a recombinant cell capable of producing itaconic acid in a suitable fermentation medium, thereby to produce itaconic acid, wherein: (a) the recombinant cell: (i) overexpresses cis-aconitate decarboxylase; and (ii) overexpresses a part of the citric acid cycle and/or has reduced activity of a native metabolic route to acetate and/or lactate; and, optionally, (b) the fermentation is carried out under anaerobic conditions.

The present invention relates to a recombinant yeast cell which is capable of producing one or more of 4-methyl itaconate, 1-methyl itaconate or 1,4-dimethyl itaconate. The invention also relates to a recombinant yeast cell which is capable of producing itaconic acid and which overexpresses: a nucleic acid encoding a polypeptide having cis-aconitate decarboxylase activity; and a nucleic acid encoding a polypeptide which catalyzes a reaction towards acetyl CoA. These recombinant yeast cells may be used in processes for the production of itaconic acid, 4-methyl itaconate, 1-methyl itaconate or 1,4-dimethyl itaconate.

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.

Provided is a novel cis-aconitate synthesis enzyme, more particularly, a recombinant microorganism for producing itaconate including a cis-aconitate synthesis enzyme variant. According to the present disclosure, it was confirmed that the production and yield of itaconate were significantly increased in the recombinant microorganism for producing itaconate into which the novel cis-aconitate synthesis enzyme was introduced. In addition, it was confirmed that in the recombinant microorganism for producing itaconate of the present disclosure, a new carbon flow to itaconate was separated from the existing TCA cycle based on the activity of the corresponding enzyme. Accordingly, the novel aconitate synthesis enzyme of the present disclosure and the recombinant microorganism introduced with the aconitate synthesis enzyme can increase the economic feasibility of itaconate, and thus can be used in various industrial fields, such as synthetic resins, latexes, and food additives in which itaconate is used.

Described is a recombinant organism or microorganism which is capable of enzymatically converting acetyl-CoA into isobutene, (A) wherein in said organism or microorganism: (i) acetyl-CoA is enzymatically converted into acetoacetyl-CoA, (ii) acetoacetyl-CoA is enzymatically converted into 3-hydroxy-3-methylglutaryl-CoA, (iii) 3-hydroxy-3-methylglutaryl-CoA is enzymatically converted into 3-methylglutaconyl-CoA, (iv) 3-methylglutaconyl-CoA is enzymatically converted into 3-methylcrotonyl-CoA, and (v) wherein said 3-methylcrotonyl-CoA is converted into isobutene by: (a) enzymatically converting 3-methylcrotonyl-CoA into 3-methylcrotonic acid which is then further enzymatically converted into said isobutene; or (b) enzymatically converting 3-methylcrotonyl-CoA into 3-hydroxy-3-methylbutyryl-CoA which is then further enzymatically converted into 3-hydroxy-3-methylbutyric acid which is then further enzymatically converted into 3-phosphonoxy-3-methylbutyric acid which is then further enzymatically converted into said isobutene; (B) wherein said recombinant organism or microorganism has an increased pool of coenzyme A (CoA) over the organism or microorganism from which it is derived due to: (i) an increased uptake of pantothenate; and/or (ii) an increased conversion of pantothenate into CoA. Moreover, described is the use of such a recombinant organism or microorganism for the production of isobutene. Further, described is a method for the production of isobutene by culturing such a recombinant organism or microorganism in a suitable culture medium under suitable conditions.