On May 19, 2026, Haolin Han and colleagues from Beijing University of Chemical Technology published a paper in Metabolic Engineering titled "Metabolic rewiring and biosensor-assisted screening for high-level putrescine production from xylose in Escherichia." coli research paper.
Lignocellulosic biomass is a rich renewable raw material for sustainable biomanufacturing, with its main component, xylose, providing an economical and efficient carbon source for producing high value-added chemicals. Putrescine is an important platform compound with broad industrial applications, but its traditional glucose biosynthesis pathway suffers from lengthy paths, low carbon conversion efficiency, and narrow substrate spectrum. To address these challenges in thermodynamics and carbon conservation, an artificial putrescine biosynthetic pathway based on unphosphorylated xylose metabolism was established in E. coli. This streamlined route requires only 12 enzymatic steps to achieve de novo synthesis of putrescine, effectively reducing intermediate side reactions and carbon separation, and providing higher theoretical carbon source conversion efficiency and yield. By systematically modularizing the putrescine biosynthesis network and assisted by biosensors, a microbial platform was developed that achieved a 3.10 g/L putrefine yield in shaken flask culture, a 484.4-fold increase over the initial strains. In batch fermentation of 5-liter feed, putrescine titers reached 34.77 ± 1.06 g/L, yield 0.26 g/g xylose, demonstrating the industrial potential of this synthetic pathway. This work broadens the substrate spectrum of putrescine biosynthesis and provides new directions for the high-value utilization of lignocellulose biomass.
Abstract
Main content
1. Pathway construction: Natural xylA was knocked out, codon-optimized xylose metabolism gene clusters (xylBCXA) and yjhG were expressed, and the first verification was demonstrated that E. coli synthesizes putrescine from xylose (6.64 mg/L).
2. Degradation pathway blockade: sequentially knocking out speDE, puuA, speG, and ygjG, raising putrescine titer to 48.98 mg/L.
3. Modular Optimization:
·Module I (putrescine synthesis): overexpressed speA + speC, titer up to 0.69 g/L.
·Module II (Ornithine Supply): Introduction of the argJBCD gene cluster of Corynebacterium glutamate, with titer increased to 2.15 g/L.
·Module III (glutamate supply): Overexpresses NADPH-dependent CgGDH, titer up to 2.34 g/L.
4. Biosensor-assisted screening: A fluorescent sensor regulated by PuuR-puuO (P115 promoter) was constructed. After two rounds of ARTP mutagenesis screening, strain Put 6-15 (shaken flask 3.10 g/L) was obtained.
5. Fermentation scale-up: Batch fermentation of 5 L bioreactor feed for 96 hours yields 34.77 g/L of putrescine.
Innovation
1. For the first time, a 12-step dephosphorylation and refined pathway for xylose →putrescine was established, avoiding carbon loss and long-path metabolic burden, with theoretical carbon yield superior to traditional PPP pathways.
2. Modular reprogramming systematically optimizes three key modules (synthesis, precursor, cofactor), and introduces C. glutamicum's ArgJBCD pathway enables acetyl cycling, improving carbon/energy efficiency.
3. Develop a novel putrescine responsive biosensor (P115-puuO-egfp), combining ARTP mutagenesis for high-throughput screening, rapidly obtaining high-yield mutant strains, and overcoming rational design bottlenecks.
4. Expand the utilization of lignocellulose carbon sources, providing a universal platform for xylose-based production of other TCA circular derivative chemicals.
Results and discussion
Pathway validation: The non-phosphorylated pathway successfully converted xylose to α-KG, but α-KG accumulation (447.8 mg/L) indicated insufficient downstream conversion efficiency, requiring increased throughput of glutamic acid→ornithine→putrescine flux.
Degradation blocking effect: Five gene knockouts (speDE, puuA, speG, ygjG) effectively eliminate putrescine consumption, and exogenous supplementation experiments confirmed no other significant degradation pathways.
Module optimization differences: SpeC is better than SpeF; ArgA and ArgC are rate-limiting enzymes in the ornithine pathway; CgGDH (NADPH-dependent) is better suited to engineered strains than NADH-dependent exogenous GDH, increasing glutamate supply.
Sensor selection advantages: The P115 promoter sensor has a dynamic range of 2.4 times, and after two mutagenesis rounds, the yield of Put 6-15 is further increased by 32.5% compared to rationally optimized strains. Whole-genome sequencing revealed mutations such as gadA, proB, rpoS, and lacI, which may affect glutamate accumulation, stress response, and expression efficiency.
Fermentation performance and limitations: batch fermentation yield of feed was 0.26 g/g xylose (theoretical value not met), and later glutamate accumulation suggests module I/II rates are lower than module III, requiring differential regulation of expression intensity in each module in the future; The platform demonstrates industrial feasibility but requires further optimization of substrate co-utilization and strain robustness.