Dipicolinic acid (DPA), a cyclic diacid, has garnered significant interest due to its potential applications in antimicrobial agents, antioxidants, chelating reagents, and polymer precursors. However, its natural bioproduction is limited since DPA is only accumulated in Bacillus and Clostridium species during sporulation. Thus, heterologous production by engineered strains is of paramount importance for developing a sustainable biological route for DPA production. Pseudomonas putida KT2440 has emerged as a promising host for the production of various chemicals thanks to its robustness, metabolic versatility, and genetic tractability. The dominant Entner–Doudoroff (ED) pathway for glucose metabolism in this strain offers an ideal route for DPA production due to the advantage of NADPH generation and the naturally balanced flux between glyceraldehyde-3-phosphate and pyruvate, which are both precursors for DPA synthesis. In this study, DPA production via the ED pathway was in silico designed in P. putida KT2440. The systematically engineered strain produced dipicolinate with a titer of 11.72 g/L from glucose in a 5 L fermentor. This approach not only provides a sustainable green route for DPA production but also expands our understanding of the metabolic potential of the ED pathway in P. putida KT2440.
Zi-Qian Geng,Ding-Kang Qian,Zhi-Yi Hu,Shuai Wang,Yang Yan,Mark C. M. van Loosdrecht,Raymond Jianxiong Zeng,Fang Zhang
Uronic acid in extracellular polymeric substances is a primary but often ignored factor related to the difficult hydrolysis of waste-activated sludge (WAS), with alginate as a typical polymer. Previously, we enriched alginate-degrading consortia (ADC) in batch reactors that can enhance methane production from WAS, but the enzymes and metabolic pathway are not well documented. In this work, two chemostats in series were operated to enrich ADC, in which 10 g/L alginate was wholly consumed. Based on it, the extracellular alginate lyase (∼130 kD, EC 4.2.2.3) in the cultures was identified by metaproteomic analysis. This enzyme offers a high specificity to convert alginate to disaccharides over other mentioned hydrolases. Genus Bacteroides (>60%) was revealed as the key bacterium for alginate conversion. A new Entner–Doudoroff pathway of alginate via 5-dehydro-4-deoxy-d-glucuronate (DDG) and 3-deoxy-d-glycerol-2,5-hexdiulosonate (DGH) as the intermediates to 2-keto-3-deoxy-gluconate (KDG) was constructed based on the metagenomic and metaproteomic analysis. In summary, this work documented the core enzymes and metabolic pathway for alginate degradation, which provides a good paradigm when analyzing the degrading mechanism of unacquainted substrates. The outcome will further contribute to the application of Bacteroides-dominated ADC on WAS methanogenesis in the future.
Boyu Yang,Chao Li,Yongshuo Ren,Weichen Wang,Xiangxiang Zhang,Xiaojun Han
The bottom-up construction of artificial cells is beneficial for understanding cell working mechanisms. The glycolysis metabolism mimicry inside artificial cells is challenging. Herein, the glycolytic pathway (Entner–Doudoroff pathway in archaea) is reconstituted inside artificial cells. The glycolytic pathway comprising glucose dehydrogenase (GDH), gluconate dehydratase (GAD), and 2-keto-3-deoxygluconate aldolase (KDGA) converts glucose molecules to pyruvate molecules. Inside artificial cells, pyruvate molecules are further converted into alanine with the help of alanine dehydrogenase (AlaDH) to build a metabolic pathway for synthesizing amino acid. On the other hand, the pyruvate molecules from glycolysis stimulate the living mitochondria to produce ATP inside artificial cells, which further trigger actin monomers to polymerize to form actin filaments. With the addition of methylcellulose inside the artificial cell, the actin filaments form adjacent to the inner lipid bilayer, deforming the artificial cell from a spherical shape to a spindle shape. The spindle-shaped artificial cell reverses to a spherical shape by depolymerizing the actin filament upon laser irradiation. The glycolytic pathway and its further extension to produce amino acids (or ATP) inside artificial cells pave the path to build functional artificial cells with more complicated metabolic pathways.
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