TY  - JOUR
A1  - Harth, Simon
A1  - Wagner, Jacqueline
A1  - Sens, Tamina
A1  - Choe, Jun-yong
A1  - Benz, Philipp
A1  - Weuster-Botz, Dirk
A1  - Oreb, Igor-Mislav
T1  - Engineering cofactor supply and NADH-dependent D-galacturonic acid reductases for redox-balanced production of l-galactonate in Saccharomyces cerevisiae
T2  - Scientific reports
N2  - D-Galacturonic acid (GalA) is the major constituent of pectin-rich biomass, an abundant and underutilized agricultural byproduct. By one reductive step catalyzed by GalA reductases, GalA is converted to the polyhydroxy acid l-galactonate (GalOA), the first intermediate of the fungal GalA catabolic pathway, which also has interesting properties for potential applications as an additive to nutrients and cosmetics. Previous attempts to establish the production of GalOA or the full GalA catabolic pathway in Saccharomyces cerevisiae proved challenging, presumably due to the inefficient supply of NADPH, the preferred cofactor of GalA reductases. Here, we tested this hypothesis by coupling the reduction of GalA to the oxidation of the sugar alcohol sorbitol that has a higher reduction state compared to glucose and thereby yields the necessary redox cofactors. By choosing a suitable sorbitol dehydrogenase, we designed yeast strains in which the sorbitol metabolism yields a “surplus” of either NADPH or NADH. By biotransformation experiments in controlled bioreactors, we demonstrate a nearly complete conversion of consumed GalA into GalOA and a highly efficient utilization of the co-substrate sorbitol in providing NADPH. Furthermore, we performed structure-guided mutagenesis of GalA reductases to change their cofactor preference from NADPH towards NADH and demonstrated their functionality by the production of GalOA in combination with the NADH-yielding sorbitol metabolism. Moreover, the engineered enzymes enabled a doubling of GalOA yields when glucose was used as a co-substrate. This significantly expands the possibilities for metabolic engineering of GalOA production and valorization of pectin-rich biomass in general.
KW  - Metabolic engineering
KW  - Oxidoreductases
Y1  - 2020
UR  - http://publikationen.ub.uni-frankfurt.de/frontdoor/index/index/docId/63718
UR  - https://nbn-resolving.org/urn:nbn:de:hebis:30:3-637186
SN  - 2045-2322
N1  - This work was supported by the German Federal Ministry of Education and Research, grant numbers 031B0342B (to M.O.) and 031B0342C (to D.W.B.).
N1  - Open Access funding enabled and organized by Projekt DEAL.
VL  - 10
IS  - art. 19021
SP  - 1
EP  - 12
PB  - Macmillan Publishers Limited, part of Springer Nature
CY  - [London]
ER  -