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Sci Rep. 2017 Sep 12;7(1):11284. doi: 10.1038/s41598-017-11624-z.

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

Author information

1
Department of Chemical Engineering, National Tsing Hua University, Hsinchu, Taiwan.
2
Department of Chemical Engineering, National Tsing Hua University, Hsinchu, Taiwan. crshen@mx.nthu.edu.tw.

Abstract

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.

PMID:
28900255
PMCID:
PMC5595793
DOI:
10.1038/s41598-017-11624-z
[Indexed for MEDLINE]
Free PMC Article

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