Enzymes and methods for production of malonic acid and derivatives thereof

An engineered microorganism comprising:

• a heterologous gene, which encodes a heterologous malonate-semialdehyde dehydrogenase that comprises at least 90% sequence identity to SEQ ID NO:
• 6, wherein the engineered microorganism is capable of producing 3 g/L to 250 g/L of malonic acid, malonate, esters of malonic acid, or mixtures thereof at a pH between 2 and 7, wherein at least one of:
• an amino acid residue of the malonate-semialdehyde dehydrogenase that aligns with amino acid residue 160 of SEQ ID NO:
• 6 is tryptophan;
• an amino acid residue of the malonate-semialdehyde dehydrogenase that aligns with amino acid residue 290 of SEQ ID NO:
• 6 is serine;
• an amino acid residue of the malonate-semialdehyde dehydrogenase that aligns with amino acid residue 89 of SEQ ID NO:
• 6 is serine, arginine, or phenylalanine;
• an amino acid residue of the malonate-semialdehyde dehydrogenase that aligns with amino acid residue 200 of SEQ ID NO:
• 6 is lysine;
• an amino acid residue of the malonate-semialdehyde dehydrogenase that aligns with amino acid residue 227 of SEQ ID NO:
• 6 is glutamine, methionine, or cysteine;
• an amino acid residue of the malonate-semialdehyde dehydrogenase that aligns with amino acid residue 332 of SEQ ID NO:
• 6 is lysine or arginine;
• an amino acid residue of the malonate-semialdehyde dehydrogenase that aligns with amino acid residue 217 of SEQ ID NO:
• 6 is cysteine;
• an amino acid residue of the malonate-semialdehyde dehydrogenase that aligns with amino acid residue 368 of SEQ ID NO:
• 6 is histidine;
• an amino acid residue of the malonate-semialdehyde dehydrogenase that aligns with amino acid residue 310 of SEQ ID NO:
• 6 is leucine;
• an amino acid residue of the malonate-semialdehyde dehydrogenase that aligns with amino acid residue 233 of SEQ ID NO:
• 6 is alanine, threonine, or valine;
• an amino acid residue of the malonate-semialdehyde dehydrogenase that aligns with amino acid residue 80 of SEQ ID NO:
• 6 is histidine;
• an amino acid residue of the malonate-semialdehyde dehydrogenase that aligns with amino acid residue 175 of SEQ ID NO:
• 6 is threonine or serine;
• an amino acid residue of the malonate-semialdehyde dehydrogenase that aligns with amino acid residue 246 of SEQ ID NO:
• 6 is phenylalanine;
• an amino acid residue of the malonate-semialdehyde dehydrogenase that aligns with amino acid residue 319 of SEQ ID NO:
• 6 is aspartic acid;
• an amino acid residue of the malonate-semialdehyde dehydrogenase that aligns with amino acid residue 192 of SEQ ID NO:
• 6 is threonine;
• an amino acid residue of the malonate-semialdehyde dehydrogenase that aligns with amino acid residue 137 of SEQ ID NO:
• 6 is arginine;
• an amino acid residue of the malonate-semialdehyde dehydrogenase that aligns with amino acid residue 158 of SEQ ID NO:
• 6 is tyrosine;
• an amino acid residue of the malonate-semialdehyde dehydrogenase that aligns with amino acid residue 452 of SEQ ID NO:
• 6 is threonine;
• an amino acid residue of the malonate-semialdehyde dehydrogenase that aligns with amino acid residue 195 of SEQ ID NO:
• 6 is isoleucine;
• an amino acid residue of the malonate-semialdehyde dehydrogenase that aligns with amino acid residue 77 of SEQ ID NO:
• 6 is alanine;
• an amino acid residue of the malonate-semialdehyde dehydrogenase that aligns with amino acid residue 85 of SEQ ID NO:
• 6 is valine;
• an amino acid residue of the malonate-semialdehyde dehydrogenase that aligns with amino acid residue 33 of SEQ ID NO:
• 6 is asparagine;
• an amino acid residue of the malonate-semialdehyde dehydrogenase that aligns with amino acid residue 221 of SEQ ID NO:
• 6 is valine;
• an amino acid residue of the malonate-semialdehyde dehydrogenase that aligns with amino acid residue 218 of SEQ ID NO:
• 6 is glycine;
• an amino acid residue of the malonate-semialdehyde dehydrogenase that aligns with amino acid residue 50 of SEQ ID NO:
• 6 is threonine;
• an amino acid residue of the malonate-semialdehyde dehydrogenase that aligns with amino acid residue 106 of SEQ ID NO:
• 6 is glutamine;
• an amino acid residue of the malonate-semialdehyde dehydrogenase that aligns with amino acid residue 305 of SEQ ID NO:
• 6 is aspartic acid;
• an amino acid residue of the malonate-semialdehyde dehydrogenase that aligns with amino acid residue 415 of SEQ ID NO:
• 6 is asparagine;
• an amino acid residue of the malonate semialdehyde dehydrogenase that aligns with amino acid residue 68 of SEQ ID NO:
• 6 is valine;
• an amino acid residue of the malonate semialdehyde dehydrogenase that aligns with amino acid residue 22 of SEQ ID NO:
• 6 is isoleucine;
• or
• combinations thereof.