Process to prepare elongated 2-ketoacids and C-5-C10 compounds therefrom via genetic modifications to microbial metabolic pathways

A LeuCD′ enzyme complex selected from the group consisting of:

• (i) a combination of a genetically modified LeuC′ subunit comprising an amino acid sequence with at least 95% identity to SEQ ID NO:
• 1 and at least one modification wherein the amino acid at the position corresponding to position 35 of SEQ ID NO:
• 1 is substituted with alanine and a native LeuD subunit comprising an amino acid sequence with at least 94% identity to SEQ ID NO:
• 2;
• (ii) a combination of a native LeuC subunit comprising an amino acid sequence with at least 95% identity to SEQ ID NO:
• 1 and a genetically modified LeuD′ subunit comprising an amino acid sequence with at least 94% identity to SEQ ID NO:
• 2 and at least one modification wherein the amino acid at the position corresponding to position 31 of SEQ ID NO:
• 2 is substituted with alanine;
• (iii) a combination of a native LeuC subunit comprising an amino acid sequence with at least 95% identity to SEQ ID NO:
• 1 and a genetically modified LeuD′ subunit comprising an amino acid sequence with at least 94% identity to SEQ ID NO:
• 2 and at least one modification wherein the amino acid at the position corresponding to position 31 of SEQ ID NO:
• 2 is substituted with glycine;
• (iv) a combination of a genetically modified LeuC′ subunit comprising an amino acid sequence with at least 95% identity to SEQ ID NO:
• 1 and at least one modification wherein the amino acid at the position corresponding to position 35 of SEQ ID NO:
• 1 is substituted with alanine and a genetically modified LeuD′ subunit comprising an amino acid sequence with at least 94% identity to SEQ ID NO:
• 2 and at least one modification wherein the amino acid at the position corresponding to position 31 of SEQ ID NO:
• 2 is substituted with glycine;
• (v) a combination of a genetically modified LeuC′ subunit comprising an amino acid sequence with at least 95% identity to SEQ ID NO:
• 1 and at least one modification wherein the amino acid at the position corresponding to position 35 of SEQ ID NO:
• 1 is substituted with glycine and a genetically modified LeuD′ subunit comprising an amino acid sequence with at least 94% identity to SEQ ID NO:
• 2 and at least one modification wherein the amino acid at the position corresponding to position 31 of SEQ ID NO:
• 2 is substituted with valine;
• (vi) a combination of a genetically modified LeuC′ subunit comprising an amino acid sequence with at least 95% identity to SEQ ID NO:
• 1 and at least one modification wherein the amino acid at the position corresponding to position 411 of SEQ ID NO:
• 1 is substituted with valine and a genetically modified LeuD′ subunit comprising an amino acid sequence with at least 94% identity to SEQ ID NO:
• 2 and at least one modification wherein the amino acid at the position corresponding to position 31 of SEQ ID NO:
• 2 is substituted with glycine;
• (vii) a combination of a genetically modified LeuC′ subunit comprising an amino acid sequence with at least 95% identity to SEQ ID NO:
• 1 and at least the modifications wherein the amino acid at the position corresponding to position 35 of SEQ ID NO:
• 1 is substituted with alanine and the amino acid at the position corresponding to position 411 of SEQ ID NO:
• 1 is substituted with valine and a genetically modified LeuD′ subunit comprising an amino acid sequence with at least 94% identity to SEQ ID NO:
• 2 and at least one modification wherein the amino acid at the position corresponding to position 31 of SEQ ID NO:
• 2 is substituted with valine;
• (viii) a combination of a genetically modified LeuC′ subunit comprising an amino acid sequence with at least 95% identity to SEQ ID NO:
• 1 and at least the modifications wherein the amino acid at the position corresponding to position 35 of SEQ ID NO:
• 1 is substituted with alanine and the amino acid at the position corresponding to position 411 of SEQ ID NO:
• 1 is substituted with valine and a genetically modified LeuD′ subunit comprising an amino acid sequence with at least 94% identity to SEQ ID NO:
• 2 and at least one modification wherein the amino acid at the position corresponding to position 31 of SEQ ID NO:
• 2 is substituted with alanine;
• (ix) a combination of a genetically modified LeuC′ subunit comprising an amino acid sequence with at least 95% identity to SEQ ID NO:
• 1 and at least the modifications wherein the amino acid at the position corresponding to position 35 of SEQ ID NO:
• 1 is substituted with alanine and the amino acid at the position corresponding to position 411 of SEQ ID NO:
• 1 is substituted with glycine and a genetically modified LeuD′ subunit comprising an amino acid sequence with at least 94% identity to SEQ ID NO:
• 2 and at least one modification wherein the amino acid at the position corresponding to position 31 of SEQ ID NO:
• 2 is substituted with alanine;
• (x) a combination of a genetically modified LeuC′ subunit comprising an amino acid sequence with at least 95% identity to SEQ ID NO:
• 1 and at least the modifications wherein the amino acid at the position corresponding to position 35 of SEQ ID NO:
• 1 is substituted with alanine and the amino acid at the position corresponding to position 411 of SEQ ID NO:
• 1 is substituted with glycine and a genetically modified LeuD′ subunit comprising an amino acid sequence with at least 94% identity to SEQ ID NO:
• 2 and at least one modification wherein the amino acid at the position corresponding to position 31 of SEQ ID NO:
• 2 is substituted with glycine;
• (xi) a combination of a native LeuC subunit comprising an amino acid sequence with at least 95% identity to SEQ ID NO:
• 1 and a genetically modified LeuD′ subunit comprising an amino acid sequence with at least 94% identity to SEQ ID NO:
• 2 and at least the modifications wherein the amino acid at the position corresponding to position 31 of SEQ ID NO:
• 2 is substituted with valine and the amino acid at the position corresponding to position 88 of SEQ ID NO:
• 2 is substituted with alanine;
• (xii) a combination of a native LeuC subunit comprising an amino acid sequence with at least 95% identity to SEQ ID NO:
• 1 and a genetically modified LeuD′ subunit comprising an amino acid sequence with at least 94% identity to SEQ ID NO:
• 2 and at least the modifications wherein the amino acid at the position corresponding to position 31 of SEQ ID NO:
• 2 is substituted with glycine and the amino acid at the position corresponding to position 88 of SEQ ID NO:
• 2 is substituted with alanine;
• (xiii) a combination of a native LeuC subunit comprising an amino acid sequence with at least 95% identity to SEQ ID NO:
• 1 and a genetically modified LeuD′ subunit comprising an amino acid sequence with at least 94% identity to SEQ ID NO:
• 2 and at least the modifications wherein the amino acid at the position corresponding to position 31 of SEQ ID NO:
• 2 is substituted with glycine and the amino acid at the position corresponding to position 88 of SEQ ID NO:
• 2 is substituted with serine;
• and
• (xiv) a combination of a genetically modified LeuC′ subunit comprising an amino acid sequence with at least 95% identity to SEQ ID NO:
• 1 and at least the modifications wherein the amino acid at the position corresponding to position 35 of SEQ ID NO:
• 1 is substituted with glycine and the amino acid at the position corresponding to position 411 of SEQ ID NO:
• 1 is substituted with valine and a genetically modified LeuD′ subunit comprising an amino acid sequence with at least 94% identity to SEQ ID NO:
• 2 and at least the modifications wherein the amino acid at the position corresponding to position 31 of SEQ ID NO:
• 2 is substituted with alanine and the amino acid at the position corresponding to position 88 of SEQ ID NO:
• 2 is substituted with serine;
• wherein the genetically modified LeuCD′ enzyme complex has isopropylmalate isomerase activity.

A protein complex having isopropylmalate isomerase activity, where said complex comprises:

• (i) a polypeptide that comprises all of SEQ ID NO:
• 1 except for one or more amino acid substitutions at positions corresponding to positions of SEQ ID NO:
• 1 selected from positions 35 and 411, wherein the amino acid at the position corresponding to position 35 of the polypeptide of SEQ ID NO:1 is replaced with glycine or alanine, and the amino acid at the position corresponding to position 411 of the polypeptide of SEQ ID NO:1 is replaced with valine or glycine, and
• (ii) a polypeptide that comprises all of SEQ ID NO:
• 2 except for one or more amino acid substitutions at positions corresponding to positions of SEQ ID NO:
• 2 selected from positions 31 and 88, wherein the amino acid at the position corresponding to position 31 of the polypeptide of SEQ ID NO:
• 2 is replaced with alanine, valine, or glycine, and wherein the amino acid at the positon corresponding to position 88 of the polypeptide of SEQ ID NO:
• 2 is replaced with alanine gr serine.

A process for preparing a C7-C11 2-ketoacid, the process comprising providing at least one of a C4-C10 2-ketoacid substrate with:

• (A) at least one isopropylmalate synthase enzyme having isopropylmalate synthase activity;
• (B) at least one isopropylmalate dehydrogenase enzyme having isopropylmalate dehydrogenase activity;
• and
• (C) at least one genetically modified LeuCD′ enzyme complex selected from the group consisting of:
• (i) a combination of a genetically modified LeuC′ subunit comprising an amino acid sequence with at least 95% identity to SEQ ID NO:
• 1 and at least one modification wherein the amino acid at the position corresponding to position 35 of SEQ ID NO:
• 1 is substituted with alanine and a native LeuD subunit comprising an amino acid sequence with at least 94% identity to SEQ ID NO:
• 2;
• (ii) a combination of a native LeuC subunit comprising an amino acid sequence with at least 95% identity to SEQ ID NO:
• 1 and a genetically modified LeuD′ subunit comprising an amino acid sequence with at least 94% identity to SEQ ID NO:
• 2 and at least one modification wherein the amino acid at the position corresponding to position 31 of SEQ ID NO:
• 2 is substituted with alanine;
• (iii) a combination of a native LeuC subunit comprising an amino acid sequence with at least 95% identity to SEQ ID NO:
• 1 and a genetically modified LeuD′ subunit comprising an amino acid sequence with at least 94% identity to SEQ ID NO:
• 2 and at least one modification wherein the amino acid at the position corresponding to position 31 of SEQ ID NO:
• 2 is substituted with glycine;
• (iv) a combination of a genetically modified LeuC′ subunit comprising an amino acid sequence with at least 95% identity to SEQ ID NO:
• 1 and at least one modification wherein the amino acid at the position corresponding to position 35 of SEQ ID NO:
• 1 is substituted with alanine and a genetically modified LeuD′ subunit comprising an amino acid sequence with at least 94% identity to SEQ ID NO:
• 2 and at least one modification wherein the amino acid at the position corresponding to position 31 of SEQ ID NO:
• 2 is substituted with glycine;
• (v) a combination of a genetically modified LeuC′ subunit comprising an amino acid sequence with at least 95% identity to SEQ ID NO:
• 1 and at least one modification wherein the amino acid at the position corresponding to position 35 of SEQ ID NO:
• 1 is substituted with glycine and a genetically modified LeuD′ subunit comprising an amino acid sequence with at least 94% identity to SEQ ID NO:
• 2 and at least one modification wherein the amino acid at the position corresponding to position 31 of SEQ ID NO:
• 2 is substituted with valine;
• (vi) a combination of a genetically modified LeuC′ subunit comprising an amino acid sequence with at least 95% identity to SEQ ID NO:
• 1 and at least one modification wherein the amino acid at the position corresponding to position 411 of SEQ ID NO:
• 1 is substituted with valine and a genetically modified LeuD′ subunit comprising an amino acid sequence with at least 94% identity to SEQ ID NO:
• 2 and at least one modification wherein the amino acid at the position corresponding to position 31 of SEQ ID NO:
• 2 is substituted with glycine;
• (vii) a combination of a genetically modified LeuC′ subunit comprising an amino acid sequence with at least 95% identity to SEQ ID NO:
• 1 and at least the modifications wherein the amino acid at the position corresponding to position 35 of SEQ ID NO:
• 1 is substituted with alanine and the amino acid at the position corresponding to position 411 of SEQ ID NO:
• 1 is substituted with valine and a genetically modified LeuD′ subunit comprising an amino acid sequence with at least 94% identity to SEQ ID NO:
• 2 and at least one modification wherein the amino acid at the position corresponding to position 31 of SEQ ID NO:
• 2 is substituted with valine;
• (viii) a combination of a genetically modified LeuC′ subunit comprising an amino acid sequence with at least 95% identity to SEQ ID NO:
• 1 and at least the modifications wherein the amino acid at the position corresponding to position 35 of SEQ ID NO:
• 1 is substituted with alanine and the amino acid at the position corresponding to position 411 of SEQ ID NO:
• 1 is substituted with valine and a genetically modified LeuD′ subunit comprising an amino acid sequence with at least 94% identity to SEQ ID NO:
• 2 and at least one modification wherein the amino acid at the position corresponding to position 31 of SEQ ID NO:
• 2 is substituted with alanine;
• (ix) a combination of a genetically modified LeuC′ subunit comprising an amino acid sequence with at least 95% identity to SEQ ID NO:
• 1 and at least the modifications wherein the amino acid at the position corresponding to position 35 of SEQ ID NO:
• 1 is substituted with alanine and the amino acid at the position corresponding to position 411 of SEQ ID NO:
• 1 is substituted with glycine and a genetically modified LeuD′ subunit comprising an amino acid sequence with at least 94% identity to SEQ ID NO:
• 2 and at least one modification wherein the amino acid at the position corresponding to position 31 of SEQ ID NO:
• 2 is substituted with alanine;
• (x) a combination of a genetically modified LeuC′ subunit comprising an amino acid sequence with at least 95% identity to SEQ ID NO:
• 1 and at least the modifications wherein the amino acid at the position corresponding to position 35 of SEQ ID NO:
• 1 is substituted with alanine and the amino acid at the position corresponding to position 411 of SEQ ID NO:
• 1 is substituted with glycine and a genetically modified LeuD′ subunit comprising an amino acid sequence with at least 94% identity to SEQ ID NO:
• 2 and at least one modification wherein the amino acid at the position corresponding to position 31 of SEQ ID NO:
• 2 is substituted with glycine;
• (xi) a combination of a native LeuC subunit comprising an amino acid sequence with at least 95% identity to SEQ ID NO:
• 1 and a genetically modified LeuD′ subunit comprising an amino acid sequence with at least 94% identity to SEQ ID NO:
• 2 and at least the modifications wherein the amino acid at the position corresponding to position 31 of SEQ ID NO:
• 2 is substituted with valine and the amino acid at the position corresponding to position 88 of SEQ ID NO:
• 2 is substituted with alanine;
• (xii) a combination of a native LeuC subunit comprising an amino acid sequence with at least 95% identity to SEQ ID NO:
• 1 and a genetically modified LeuD′ subunit comprising an amino acid sequence with at least 94% identity to SEQ ID NO:
• 2 and at least the modifications wherein the amino acid at the position corresponding to position 31 of SEQ ID NO:
• 2 is substituted with glycine and the amino acid at the position corresponding to position 88 of SEQ ID NO:
• 2 is substituted with alanine;
• (xiii) a combination of a native LeuC subunit comprising an amino acid sequence with at least 95% identity to SEQ ID NO:
• 1 and a genetically modified LeuD′ subunit comprising an amino acid sequence with at least 94% identity to SEQ ID NO:
• 2 and at least the modifications wherein the amino acid at the position corresponding to position 31 of SEQ ID NO:
• 2 is substituted with glycine and the amino acid at the position corresponding to position 88 of SEQ ID NO:
• 2 is substituted with serine;
• and
• (xiv) a combination of a genetically modified LeuC′ subunit comprising an amino acid sequence with at least 95% identity to SEQ ID NO:
• 1 and at least the modifications wherein the amino acid at the position corresponding to position 35 of SEQ ID NO:
• 1 is substituted with glycine and the amino acid at the position corresponding to position 411 of SEQ ID NO:
• 1 is substituted with valine and a genetically modified LeuD′ subunit comprising an amino acid sequence with at least 94% identity to SEQ ID NO:
• 2 and at least the modifications wherein the amino acid at the position corresponding to position 31 of SEQ ID NO:
• 2 is substituted with alanine and the amino acid at the position corresponding to position 88 of SEQ ID NO:
• 2 is substituted with serine;
• under conditions that the at least one of the C4-C10 2-ketoacid substrate is converted to the C7-C11 2-ketoacid;
• wherein the at least one genetically modified LeuCD enzyme complex has isopropylmalate isomerase activity;
• and wherein the conversion of the at least one of the C4-C10 2-ketoacid substrate to the C7-C11 2-ketoacid occurs via one or more biochemical reactions.