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Cofactor engineering can redistribute carbon fluxes from biomass accumulation toward the synthesis of high-value products, enabling sustainable production of pharmaceuticals and other fine chemicals with enhanced purity and yield.Light-driven and electrochemical cofactor regeneration can transform microbial platforms into autotrophic systems, potentially allowing the continuous bioproduction of chemicals from CO2 with a negative carbon footprint.Biocompatible materials can create robust microenvironments for cofactor recycling and enzyme preservation, potentially advancing the industrial-scale biomanufacturing of bulk chemicals such as biofuels and polymers.Integrated bioprocesses can leverage cofactor dynamics to achieve stable, large-scale production, overcoming metabolic bottlenecks in continuous-flow systems for green manufacturing. Cofactor engineering is revolutionizing green biomanufacturing by overcoming the fundamental bottleneck between the limited supply/regeneration of cellular energy cofactors [e.g., NAD(P)H, ATP] and the high demands of efficient bioproduction. This review highlights advanced strategies, such as orthogonal systems that separate product synthesis pathways from basal metabolism, external energy sources (e.g., light or electricity) for cofactor regeneration, and material-enabled immobilization for scalable processes. These approaches enable high-yield production of diverse compounds, from specialized optically pure pharmaceuticals to bulk chemicals, by addressing critical limitations in yield, purity, and industrial scalability beyond conventional fermentation. Finally, we discuss challenges in process stability and economic viability, underscoring cofactor engineering’s potential as a versatile strategy for sustainable, next-generation biomanufacturing. Cofactor engineering is revolutionizing green biomanufacturing by overcoming the fundamental bottleneck between the limited supply/regeneration of cellular energy cofactors [e.g., NAD(P)H, ATP] and the high demands of efficient bioproduction. This review highlights advanced strategies, such as orthogonal systems that separate product synthesis pathways from basal metabolism, external energy sources (e.g., light or electricity) for cofactor regeneration, and material-enabled immobilization for scalable processes. These approaches enable high-yield production of diverse compounds, from specialized optically pure pharmaceuticals to bulk chemicals, by addressing critical limitations in yield, purity, and industrial scalability beyond conventional fermentation. Finally, we discuss challenges in process stability and economic viability, underscoring cofactor engineering’s potential as a versatile strategy for sustainable, next-generation biomanufacturing.

Abstract NAD and NADP, a pivotal class of cofactors, which function as essential electron donors or acceptors in all biological organisms, drive considerable catabolic and anabolic reactions. Furthermore, they play critical roles in maintaining intracellular redox homeostasis. However, many metabolic engineering efforts in industrial microorganisms towards modification or introduction of metabolic pathways, especially those involving consumption, generation or transformation of NAD/NADP, often induce fluctuations in redox state, which dramatically impede cellular metabolism, resulting in decreased growth performance and biosynthetic capacity. Here, we comprehensively review the cofactor engineering strategies for solving the problematic redox imbalance in metabolism modification, as well as their features, suitabilities and recent applications. Some representative examples of in vitro biocatalysis are also described. In addition, we briefly discuss how tools and methods from the field of synthetic biology can be applied for cofactor engineering. Finally, future directions and challenges for development of cofactor redox engineering are presented.

•Redox balance is achieved through three cofactor systems.•Synthetic balance reshapes the whole-cell response to redox balance.•Future research on redox balance will enable advancements in cofactor engineering. Redox balance plays an important role in the production of enzymes, pharmaceuticals, and chemicals. To meet the demands of industrial production, it is desirable that microbes maintain a maximal carbon flux towards target metabolites with no fluctuations in redox. This requires functional cofactor systems that support dynamic homeostasis between different redox states or functional stability in a given redox state. Redox balance can be achieved by improving the self-balance of a cofactor system, regulating the substrate balance of a cofactor system, and engineering the synthetic balance of a cofactor system. This review summarizes how cofactor systems can be manipulated to improve redox balance in microbes.

Optimizing the overall NADPH turnover is one of the key challenges in various value-added biochemical syntheses. In this work, we first analyzed the NADPH regeneration potentials of common cell factories, including Escherichia coli, Saccharomyces cerevisiae, Bacillus subtilis, and Pichia pastoris across multiple environmental conditions and determined E. coli and glycerol as the best microbial chassis and most suitable carbon source, respectively. In addition, we identified optimal cofactor specificity engineering (CSE) enzyme targets, whose cofactors when switched from NAD(H) to NADP(H) improve the overall NADP(H) turnover. Among several enzyme targets, glyceraldehyde-3-phosphate dehydrogenase was recognized as a global candidate since its CSE improved the NADP(H) regeneration under most of the conditions examined. Finally, by analyzing the protein structures of all CSE enzyme targets via homology modeling, we established that the replacement of conserved glutamate or aspartate with serine in the loop region could change the cofactor dependence from NAD(H) to NADP(H).

BackgroundBiofuel production from plant cell walls offers the potential for sustainable and economically attractive alternatives to petroleum-based products. In particular, Clostridium thermocellum is a promising host for consolidated bioprocessing (CBP) because of its strong native ability to ferment cellulose.ResultsWe tested 12 different enzyme combinations to identify an n-butanol pathway with high titer and thermostability in C. thermocellum. The best producing strain contained the thiolase–hydroxybutyryl-CoA dehydrogenase–crotonase (Thl-Hbd-Crt) module from Thermoanaerobacter thermosaccharolyticum, the trans-enoyl-CoA reductase (Ter) enzyme from Spirochaeta thermophila and the butyraldehyde dehydrogenase and alcohol dehydrogenase (Bad-Bdh) module from Thermoanaerobacter sp. X514 and was able to produce 88 mg/L n-butanol. The key enzymes from this combination were further optimized by protein engineering. The Thl enzyme was engineered by introducing homologous mutations previously identified in Clostridium acetobutylicum. The Hbd and Ter enzymes were engineered for changes in cofactor specificity using the CSR-SALAD algorithm to guide the selection of mutations. The cofactor engineering of Hbd had the unexpected side effect of also increasing activity by 50-fold.ConclusionsHere we report engineering C. thermocellum to produce n-butanol. Our initial pathway designs resulted in low levels (88 mg/L) of n-butanol production. By engineering the protein sequence of key enzymes in the pathway, we increased the n-butanol titer by 2.2-fold. We further increased n-butanol production by adding ethanol to the growth media. By combining all these improvements, the engineered strain was able to produce 357 mg/L of n-butanol from cellulose within 120 h.

Hydroxytyrosol (HT) is a potent polyphenolic antioxidant widely utilized in the biomedical and food industries. However, its high-level microbial biosynthesis is primarily hindered by the metabolic flux imbalances and severe cellular toxicity. In this study, an artificial synthetic pathway from 4-hydroxyphenylpyruvate was constructed in an engineered l-phenylalanine producing E. coli chassis. Building on this, the endogenous precursor supply was strengthened via targeted promoter engineering of aroK, aroC, and tyrA, and the heterologous HT biosynthetic pathway was enhanced by overexpressing ARO10. To mitigate intermediate l-DOPA accumulation, co-expression of l-DOPA decarboxylase (DODC) and tyramine oxidase (TYO) reduced l-DOPA by 63.7%, while expression of l-amino acid deaminase (LAAD) reduced l-DOPA by 76.1%. Additionally, precise cofactor engineering was implemented; overexpressing the riboflavin metabolic genes ribH, ribC, and ribF, alongside introducing pntAB, increased HT production by 30.9% and 12.7%, respectively. Furthermore, transcriptomic analysis under HT stress revealed significant upregulation of genes related to transport and stress responses. Among these targets, overexpressing marR substantially improved cellular tolerance and HT production. Finally, during a 5-L bioreactor fermentation supplemented with Fe2+ and ascorbic acid, the engineered strain achieved an HT titer of 9.25 g/L, a yield of 0.102 g/g glucose, and a productivity of 0.193 g/L/h. This study reports the highest HT titer to date in E. coli using glucose as the carbon source, providing a robust biomanufacturing platform.

α-Farnesene, an acyclic volatile sesquiterpene, plays important roles in aircraft fuel, food flavoring, agriculture, pharmaceutical and chemical industries. Here, by re-creating the NADPH and ATP biosynthetic pathways in Pichia pastoris, we increased the production of α-farnesene. First, the native oxiPPP was recreated by overexpressing its essential enzymes or by inactivating glucose-6-phosphate isomerase (PGI). This revealed that the combined over-expression of ZWF1 and SOL3 increases α-farnesene production by improving NADPH supply, whereas inactivating PGI did not do so because it caused a reduction in cell growth. The next step was to introduce heterologous cPOS5 at various expression levels into P. pastoris. It was discovered that a low intensity expression of cPOS5 aided in the production of α-farnesene. Finally, ATP was increased by the overexpression of APRT and inactivation of GPD1. The resultant strain P. pastoris X33-38 produced 3.09 ± 0.37 g/L of α-farnesene in shake flask fermentation, which was 41.7% higher than that of the parent strain. These findings open a new avenue for the development of an industrial-strength α-farnesene producer by rationally modifying the NADPH and ATP regeneration pathways in P. pastoris.

Background Menaquinone-7 (MK-7), which is associated with complex and tightly regulated pathways and redox imbalances, is produced at low titres in Bacillus subtilis . Synthetic biology provides a rational engineering principle for the transcriptional optimisation of key enzymes and the artificial creation of cofactor regeneration systems without regulatory interference. This holds great promise for alleviating pathway bottlenecks and improving the efficiency of carbon and energy utilisation. Results We used a bottom-up synthetic biology approach for the synthetic redesign of central carbon and to improve the adaptability between material and energy metabolism in MK-7 synthesis pathways. First, the rate-limiting enzymes, 1-deoxyxylulose-5-phosphate synthase (DXS), isopentenyl-diphosphate delta-isomerase (Fni), 1-deoxyxylulose-5-phosphate reductase (DXR), isochorismate synthase (MenF), and 3-deoxy-7-phosphoheptulonate synthase (AroA) in the MK-7 pathway were sequentially overexpressed. Promoter engineering and fusion tags were used to overexpress the key enzyme MenA, and the titre of MK-7 was 39.01 mg/L. Finally, after stoichiometric calculation and optimisation of the cofactor regeneration pathway, we constructed two NADPH regeneration systems, enhanced the endogenous cofactor regeneration pathway, and introduced a heterologous NADH kinase (Pos5P) to increase the availability of NADPH for MK-7 biosynthesis. The strain expressing pos5P was more efficient in converting NADH to NADPH and had excellent MK-7 synthesis ability. Following three Design-Build-Test-Learn cycles, the titre of MK-7 after flask fermentation reached 53.07 mg/L, which was 4.52 times that of B. subtilis 168. Additionally, the artificially constructed cofactor regeneration system reduced the amount of NADH-dependent by-product lactate in the fermentation broth by 9.15%. This resulted in decreased energy loss and improved carbon conversion. Conclusions In summary, a \"high-efficiency, low-carbon, cofactor-recycling\" MK-7 synthetic strain was constructed, and the strategy used in this study can be generally applied for constructing high-efficiency synthesis platforms for other terpenoids, laying the foundation for the large-scale production of high-value MK-7 as well as terpenoids.

α-Alkenes (terminal alkenes) are important fuel and platform chemicals that are mainly produced from petroleum. Microbial synthesis might provide a sustainable approach for α-alkenes. In this work, we engineered the methylotrophic yeast Pichia pastoris to produce long-chain (C15:1, C17:1 and C17:2) α-alkenes via a decarboxylation of fatty acids. Combinatorial engineering, including enzyme selection, expression optimization and peroxisomal compartmentalization, enabled the production of 1.6 mg/L α-alkenes from sole methanol. This study represents the first case of α-alkene biosynthesis from methanol and also provides a reference for the construction of methanol microbial cell factories of other high-value chemicals.

Vitamin A is a micronutrient critical for versatile biological functions and has been widely used in the food, cosmetics, pharmaceutical, and nutraceutical industries. Synthetic biology and metabolic engineering enable microbes, especially the model organism Saccharomyces cerevisiae (generally recognised as safe) to possess great potential for the production of vitamin A. Herein, we first generated a vitamin A-producing strain by mining β-carotene 15,15′-mono(di)oxygenase from different sources and identified two isoenzymes Mbblh and Ssbco with comparable catalytic properties but different catalytic mechanisms. Combinational expression of isoenzymes increased the flux from β-carotene to vitamin A metabolism. To modulate the vitamin A components, retinol dehydrogenase 12 from Homo sapiens was introduced to achieve more than 90 % retinol purity using shake flask fermentation. Overexpressing POS5Δ17 enhanced the reduced nicotinamide adenine dinucleotide phosphate pool, and the titer of vitamin A was elevated by almost 46 %. Multi-copy integration of the key rate-limiting step gene Mbblh further improved the synthesis of vitamin A. Consequently, the titer of vitamin A in the strain harbouring the Ura3 marker was increased to 588 mg/L at the shake-flask level. Eventually, the highest reported titer of 5.21 g/L vitamin A in S. cerevisiae was achieved in a 1-L bioreactor. This study unlocked the potential of S. cerevisiae for synthesising vitamin A in a sustainable and economical way, laying the foundation for the commercial-scale production of bio-based vitamin A. [Display omitted]