Saturated mutagenesis of ketoisovalerate decarboxylase V461 enabled specific synthesis of 1-pentanol via the ketoacid elongation cycle.

Iterative ketoacid elongation has been an essential tool in engineering artificial metabolism, in particular the synthetic alcohols. However, precise control of product specificity is still greatly challenged by the substrate promiscuity of the ketoacid decarboxylase, which unselectively hijacks ketoacid intermediates from the elongation cycle along with the target ketoacid. In this work, preferential tuning of the Lactococcus lactis ketoisovalerate decarboxylase (Kivd) specificity toward 1-pentanol synthesis was achieved via saturated mutagenesis of the key residue V461 followed by screening of the resulting alcohol spectrum. Substitution of V461 with the small and polar amino acid glycine or serine significantly improved the Kivd selectivity toward the 1-pentanol precursor 2-ketocaproate by lowering its catalytic efficiency for the upstream ketoacid 2-ketobutyrate and 2-ketovalerate. Conversely, replacing V461 with bulky or charged side chains displayed severely adverse effect. Increasing supply of the iterative addition unit acetyl-CoA by acetate feeding further drove 2-ketoacid flux into the elongation cycle and enhanced 1-pentanol productivity. The Kivd V461G variant enabled a 1-pentanol production specificity around 90% of the total alcohol content with or without oleyl alcohol extraction. This work adds insight to the selectivity of Kivd active site.

Engineering efficient production of itaconic acid from diverse substrates in Escherichia coli.

Itaconic acid is an excellent polymeric precursor with wide range of industrial applications. Here, efficient production of itaconate from various renewable substrates was demonstrated by engineered Escherichia coli. Limitation in the itaconate precursor supply was revealed by feeding of the key intermediate citrate to the culture medium. Efforts of enhancing the cis-aconitate flux and preserving the isocitrate pool increased itaconate productivity by nearly 100-fold. Elimination of the isocitrate lyase lowered the itaconate production by 10-30%, suggesting the potential positive role of glyoxylate shunt. High aeration was essential for efficient synthesis of itaconate due to its inability to serve as a fermentation product. Using the best strain, we achieved by far the highest itaconate titer from xylose and glycerol individually, reaching 20-22g/L in 72h with an average yield of 0.5g/g using bench-scale flasks. Compare to the use of phosphoenolpyruvate (PEP) carboxylase, overexpression of pyruvate carboxylase consistently led to higher production of itaconate from substrates such as glucose and glycerol whose dissimilation involves PEP-dependent phosphorylation. With high density fermentation in the fed-batch bioreactor, the titer of itaconate was further pushed to 43g/L in 32h with a final yield around 0.6g/g of glycerol.