Bujdoš Dalimil, Popelářová Barbora, Volke Daniel C, Nikel Pablo I, Sonnenschein Nikolaus, Dvořák Pavel
Department of Experimental Biology (Section of Microbiology, Microbial Bioengineering Laboratory), Faculty of Science, Masaryk University, Kamenice 753/5, 62500, Brno, Czech Republic.
The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kemitorvet 220, 2800 Kgs, Lyngby, Denmark.
Metab Eng. 2023 Jan;75:29-46. doi: 10.1016/j.ymben.2022.10.011. Epub 2022 Nov 4.
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.
恶臭假单胞菌KT2440是一种颇具吸引力的细菌宿主，可用于从可再生木质纤维素原料中生物技术生产有价值的化学品，因为它能够利用木质素衍生的芳烃或从纤维素中获取的葡萄糖。恶臭假单胞菌EM42是KT2440菌株的一种基因组精简变体，具有有利的生理特性，最近经过工程改造以利用纤维二糖生长，纤维二糖是纤维素酶促水解的主要低聚纤维素产物。在缺乏周质葡萄糖脱氢酶Gcd（PP_1444）的突变体中实现了纤维二糖和葡萄糖的共利用。然而，共利用表型的原因仍有待了解，且Δgcd菌株存在显著的生长缺陷。在本研究中，我们研究了两种糖同时摄取的基础，并加速了恶臭假单胞菌EM42 Δgcd突变体的生长，以便从葡萄糖和纤维二糖生物生产有价值的化合物。我们表明，gcd缺失解除了周质葡萄糖氧化途径中间体对来自嗜热栖热菌的外源β-葡萄糖苷酶BglC的抑制。编码上糖酵解基因阻遏物的hexR基因的额外缺失未能恢复在葡萄糖上的快速生长。通过分别植入来自运动发酵单胞菌的异源葡萄糖转运蛋白（Glf）和来自大肠杆菌的乳糖转运蛋白（LacY），部分补偿了Δgcd突变体降低的生长速率。值得注意的是，这种干预导致需氧恶臭假单胞菌培养物中丙酮酸的积累。我们证明，这种关键代谢中间体的过量可重新导向乙醇和乳酸的增强生物合成。然后，通过用蛋白质组学和动力学数据约束的升级基因组规模代谢模型揭示了丙酮酸过量生产表型。该模型指出由于底物摄取不受调控导致葡萄糖分解代谢酶饱和，并预测工程菌株可改善丙酮酸衍生化学品的生物生产。这项工作揭示了一种有吸引力的生物技术宿主中纤维素糖的共代谢，并引入了一种在需氧条件下细菌培养物中丙酮酸过量生产的新策略。
