Cong Gao

Microbial CO₂ assimilation provides a strategy for sustainable biomanufacturing. However, CO₂ reduction requires substantial energy input, while native energy systems are tightly regulated and extensively consumed by endogenous metabolism, limiting assimilation efficiency. Here, we design and construct an orthogonal energy system in Escherichia coli, achieving an intracellular NUDH concentration exceeding 7.0 mM and an NUDH/NUD⁺ ratio above 55. Subsequently, we design a CO₂-formate-acetyl-CoA-pyruvate-malate pathway and reprogram key enzymes for NUDH-dependent operation. Integration of the OES-driven CAM* pathway with a rationally rewired native electron transport chain supports growth of the engineered E. coli on CO₂ and methanol, with a doubling time of 8.6 ± 0.3 h and a maximum OD₆₀₀ of 37.5 ± 5.8. Moreover, the platform extends CO₂ assimilation to biosynthesis of multiple chemicals from distinct CAM* pathway nodes, including mevalonate, lactate, 2,3-butanediol, and succinate. Together, these results establish a framework for reprogramming microbial energy metabolism and expanding one-carbon biotransformation.

Microbial production of phenolic acids offers a sustainable alternative to plant extraction, reducing carbon loss and environmental impact. Here, we present a multi-level engineering strategy in Escherichia coli integrating carbon-efficient pathways, enzyme optimization, and subcellular compartmentalization. A synthetic TktA–Rpe–tyrosinase (TRT) pathway increased the theoretical carbon yield from 0.5 to 0.6 mol CaA per mol of glucose. Structure-guided engineering of tyrosinase enhanced catalytic efficiency toward p-coumaric acid by 5.07-fold. Programmable alkaline condensates created intracellular microenvironments optimized for alkaliphilic enzymes. This approach enabled the highest reported space–time yield of 211 mg L–1 h–1 for CaA. This work demonstrates a sustainable, high-efficiency microbial production strategy that maximizes carbon utilization, enhances enzyme performance, and precisely controls intracellular reactions.

Glutaric acid is an important monomer for the synthesis of polymeric materials such as polyesters and polyamides, whereas its bio-based production efficiency has long been limited. In this study, we focused on the key metabolic bottlenecks in glutaric acid biosynthesis and constructed a high-producing Corynebacterium glutamicum cell factory. First, the glutaric acid biosynthetic pathway was integrated into the genome of a high-lysine-producing C. glutamicum strain to establish a basal production strain. Subsequently, modular metabolic engineering strategies were employed to strengthen the biosynthetic modules and eliminate byproduct formation pathways, resulting in a glutaric acid titer of 78.67 g/L. To alleviate α-ketoglutarate depletion, a cofactor regeneration cycle composed of l-glutamate oxidase and catalase was introduced, further increasing the glutaric acid titer to 84.43 g/L. Finally, pH and temperature were optimized in a 5 L bioreactor, leading to a final glutaric acid titer of 97.63 g/L with a yield of 0.36 g/g. This study lays a foundation for the construction of high-producing glutaric acid cell factories based on C. glutamicum.

1,4-Butanediol (1,4-BDO) is an important non-natural platform chemical for biodegradable plastics, yet its microbial production is constrained by inadequate NADPH availability. In this study, five heterologous enzymes were introduced into Escherichia coli for the biosynthesis of 1,4-BDO. Through coordinated regulation of pathway enzyme expression and enhancement of key rate-limiting steps, a 1,4-BDO titer of 0.93 g/L was achieved from glucose. Precursor engineering to increase succinyl-CoA availability and pathway intermediate accumulation further improved the titer to 1.87 g/L. Strengthening endogenous NADPH-generating pathways, combined with the development of an intracellular biohybrid system, increased intracellular NADPH levels by 73%, resulting in a 1,4-BDO titer of 2.54 g/L (35.8% improvement). After optimization of fermentation conditions, the optimal strain BDO-25 produced 3.1 g/L and 7.5 g/L of 1,4-BDO in shake-flask and 5-L bioreactor fermentations, respectively, without antibiotics or inducers. This study offers a highly efficient and scale-up feasible strategy for the biosynthesis of NADPH-dependent diols.

As an essential precursor for numerous high-value chemicals, l-tyrosine is in high demand across the food, medical, and chemical sectors. While microbial cell factories offer a green and sustainable route for its production, their efficiency is often constrained by the inherent complexity of metabolic pathways and the carbon flux competition between cell growth and product synthesis. Building upon a previously established l-tryptophan-producing chassis, we fine-tuned the metabolic flux upstream and downstream of the shikimate node and implemented a cofactor recycling-driven strategy, thereby increasing the l-tyrosine titer from 2.5 to 8.6 g/L in shake-flask fermentation (a 3.4-fold improvement). To address the growth-production trade-off, a quorum-sensing-based dynamic control strategy was employed to regulate the expression of the TCA cycle gene sucA. Further integration of screening and reinforcement of glutamate dehydrogenase enabled the effective spatiotemporal decoupling of cell growth from l-tyrosine biosynthesis. Consequently, the engineered strain achieved an l-tyrosine titer of 81.3 g/L in a 5 L bioreactor, with a record-breaking cell-specific productivity of 0.048 g/g of DCW/h, representing a significant enhancement in the spatiotemporal productivity of microbial l-tyrosine biosynthesis. This study provides a robust strategy for the efficient and sustainable biomanufacturing of aromatic amino acids and their derivatives.