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@article{2235161,
author = {Bujdoš, Dalimil and Popelářová, Barbora and Volke, Daniel C. and Nikel, Pablo I. and Sonnenschein, Nikolaus and Dvořák, Pavel},
article_number = {January},
doi = {http://dx.doi.org/10.1016/j.ymben.2022.10.011},
keywords = {Pseudomonas putida; Metabolic engineering; Glucose; Cellobiose; Co-utilization of sugars; Pyruvate; Metabolic model},
language = {eng},
issn = {1096-7176},
journal = {Metabolic Engineering},
title = {Engineering of Pseudomonas putida for accelerated co-utilization of glucose and cellobiose yields aerobic overproduction of pyruvate explained by an upgraded metabolic model},
url = {https://www.sciencedirect.com/science/article/pii/S1096717622001306},
volume = {75},
year = {2023}
}
TY - JOUR
ID - 2235161
AU - Bujdoš, Dalimil - Popelářová, Barbora - Volke, Daniel C. - Nikel, Pablo I. - Sonnenschein, Nikolaus - Dvořák, Pavel
PY - 2023
TI - Engineering of Pseudomonas putida for accelerated co-utilization of glucose and cellobiose yields aerobic overproduction of pyruvate explained by an upgraded metabolic model
JF - Metabolic Engineering
VL - 75
IS - January
SP - 29-46
EP - 29-46
PB - Elsevier Inc
SN - 10967176
KW - Pseudomonas putida
KW - Metabolic engineering
KW - Glucose
KW - Cellobiose
KW - Co-utilization of sugars
KW - Pyruvate
KW - Metabolic model
UR - https://www.sciencedirect.com/science/article/pii/S1096717622001306
N2 - Pseudomonas putida KT2440 is an attractive bacterial host for biotechnological production of valuable chemicals from renewable lignocellulosic feedstocks as it can valorize lignin-derived aromatics or glucose obtainable from cellulose. P. putida EM42, a genome-reduced variant of strain KT2440 endowed with advantageous physiological properties, was recently engineered for growth on cellobiose, a major cellooligosaccharide product of enzymatic cellulose hydrolysis. Co-utilization of cellobiose and glucose was achieved in a mutant lacking periplasmic glucose dehydrogenase Gcd (PP_1444). However, the cause of the co-utilization phenotype remained to be understood and the Δgcd strain had a significant growth defect. In this study, we investigated the basis of the simultaneous uptake of the two sugars and accelerated the growth of P. putida EM42 Δgcd mutant for the bioproduction of valuable compounds from glucose and cellobiose. We show that the gcd deletion lifted the inhibition of the exogenous β-glucosidase BglC from Thermobifida fusca exerted by the intermediates of the periplasmic glucose oxidation pathway. The additional deletion of hexR gene, which encodes a repressor of the upper glycolysis genes, failed to restore rapid growth on glucose. The reduced growth rate of the Δgcd mutant was partially compensated by the implantation of heterologous glucose and cellobiose transporters (Glf from Zymomonas mobilis and LacY from Escherichia coli, respectively). Remarkably, this intervention resulted in the accumulation of pyruvate in aerobic P. putida cultures. We demonstrated that the excess of this key metabolic intermediate can be redirected to the enhanced biosynthesis of ethanol and lactate. The pyruvate overproduction phenotype was then unveiled by an upgraded genome-scale metabolic model constrained with proteomic and kinetic data. The model pointed to the saturation of glucose catabolism enzymes due to unregulated substrate uptake and it predicted improved bioproduction of pyruvate-derived chemicals by the engineered strain. This work sheds light on the co-metabolism of cellulosic sugars in an attractive biotechnological host and introduces a novel strategy for pyruvate overproduction in bacterial cultures under aerobic conditions.
ER -
BUJDOŠ, Dalimil, Barbora POPELÁŘOVÁ, Daniel C. VOLKE, Pablo I. NIKEL, Nikolaus SONNENSCHEIN a Pavel DVOŘÁK. Engineering of Pseudomonas putida for accelerated co-utilization of glucose and cellobiose yields aerobic overproduction of pyruvate explained by an upgraded metabolic model. \textit{Metabolic Engineering}. Elsevier Inc, 2023, roč.~75, January, s.~29-46. ISSN~1096-7176. Dostupné z: https://dx.doi.org/10.1016/j.ymben.2022.10.011.
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