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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.

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).

•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.

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.

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.

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.

Background 2,3-Butanediol (2,3-BD) with low toxicity to microbes, could be a promising alternative for biofuel production. However, most of the 2,3-BD producers are opportunistic pathogens that are not suitable for industrial-scale fermentation. In our previous study, wild-type Bacillus subtilis 168, as a class I microorganism, was first found to generate only d-(-)-2,3-BD (purity >99 %) under low oxygen conditions. Results In this work, B. subtilis was engineered to produce chiral pure meso-2,3-BD. First, d-(-)-2,3-BD production was abolished by deleting d-(-)-2,3-BD dehydrogenase coding gene bdhA, and acoA gene was knocked out to prevent the degradation of acetoin (AC), the immediate precursor of 2,3-BD. Next, both pta and ldh gene were deleted to decrease the accumulation of the byproducts, acetate and l-lactate. We further introduced the meso-2,3-BD dehydrogenase coding gene budC from Klebsiella pneumoniae CICC10011, as well as overexpressed alsSD in the tetra-mutant (ΔacoAΔbdhAΔptaΔldh) to achieve the efficient production of chiral meso-2,3-BD. Finally, the pool of NADH availability was further increased to facilitate the conversion of meso-2,3-BD from AC by overexpressing udhA gene (coding a soluble transhydrogenase) and low dissolved oxygen control during the cultivation. Under microaerobic oxygen conditions, the best strain BSF9 produced 103.7 g/L meso-2,3-BD with a yield of 0.487 g/g glucose in the 5-L batch fermenter, and the titer of the main byproduct AC was no more than 1.1 g/L. Conclusion This work offered a novel strategy for the production of chiral pure meso-2,3-BD in B. subtilis. To our knowledge, this is the first report indicating that metabolic engineered B. subtilis could produce chiral meso-2,3-BD with high purity under limited oxygen conditions. These results further demonstrated that B. subtilis as a class I microorganism is a competitive industrial-level meso-2,3-BD producer.

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]

5-Hydroxytryptophan (5-HTP) is widely used as a natural remedy for sleep disorders. In terms of biosafety, bio-derived 5-HTP is preferred over chemically synthesized 5-HTP. However, the low titer of 5-HTP in the reported microbiological methods (< 10 g/L) limits the industrialization of 5-HTP biosynthesis. In the present study, a Trp-accumulating E. coli strain TRP1 was constructed by blocking the degradation path (Δ tnaA ), branching paths (Δ pheA , Δ tyrA ) and repression system (Δ trpR , Δ trpL ). Next, the hydroxylation module employing a phenylalanine hydroxylase mutant XcPAH W179F (XC2) coupled with an MH4 regenerating system (CvPCD-EcFolM system) was screened to convert L-Trp into 5-HTP. Protein engineering was performed on hydroxylase XC2 based on the molecular dynamics simulation of the enzyme-substrate complex, and the strain TRP1-XC4 harboring the triple-mutant XcPAH L98I/A129K/W179F (XC4) was able to produce 319.4 mg/L 5-HTP. Genome editing was carried out focused on accelerating product efflux (strengthening YddG) and increasing MH4 supply (strengthening FolM, FolE and FolX), resulting in a strain TRP5-XC4 to produce 13.9 g/L 5-HTP in 5 L fed-batch fermentation with a space-time yield of 0.29 g/L/h, which is the highest production and productivity record for 5-HTP biosynthesis. This study successfully provided an engineered strain and an efficient green method for the industrial synthesis of 5-HTP. Graphical Abstract

D-Pantothenic acid (DPA), also known as vitamin B 5 , is a water-soluble organic acid, widely applied in foods, feeds, cosmetics, and medicines. Although numerous and rapidly developing cell factories have been established for DPA biosynthesis, there has been no report of any attempts to engineer Yarrowia lipolytica to synthesize DPA. To explore further possibilities in DPA biosynthesis, we tried to employ systematic metabolic engineering strategies to identify and break the potential bottlenecks in DPA biosynthesis by Y. lipolytica . By improving the rate-limiting steps of the DPA biosynthesis pathway, weakening the strongly competitive pathways, and enhancing the multiple cofactor supplies, a robust Y. lipolytica cell factory for DPA biosynthesis was successfully constructed. Consequently, the resulting strain DPA34 produced 2.18 g/L DPA in a 5-L bioreactor, representing the first report of DPA production to date in Y. lipolytica. This work is believed to facilitate the development of Y. lipolytica for sustainable manufacturing of vitamin B 5 and its derivatives. Graphical abstract