Coenzyme A (CoA) is an essential cofactor present in all domains of life and is involved in numerous metabolic pathways, including fatty acid metabolism, pyruvate oxidation through the TCA cycle, and production of secondary metabolites. This characteristic makes CoA a commercially valuable compound in the pharmaceutical, cosmetic, and clinical industries. However, CoA is difficult to accumulate in living cells at a high level as it is consumed in multiple metabolic pathways, hampering its manufacturing by typical cell cultivation and extraction approaches. The feedback inhibition by CoA to a biosynthetic enzyme, pantothenate kinase (PanK), is also a serious obstacle for high-titer production of CoA. To overcome this challenge, in vitro production of CoA, in which the CoA biosynthetic pathway was reconstructed outside of cells using recombinant thermophilic enzymes, was performed. The in vitro pathway was designed to be insensitive to the feedback inhibition of CoA using a CoA-insensitive type-III PanK from the thermophilic bacterium Thermus thermophilus. Furthermore, a statistical approach using Design of Experiments was employed to rationally determine the enzyme loading ratio to maximize CoA production rate. Consequently, 0.94 mM CoA could be produced from 2 mM d-pantetheine through the designed pathway. We hypothesized that the insufficient conversion yield is attributed to the high Km value of T. thermophilus PanK towards ATP. Based on these observations, possible CoA regulation mechanisms in T. thermophilus and approaches to improve the feasibility of CoA production through the in vitro pathway have been investigated. IMPORTANCE The biosynthesis of coenzyme A (CoA) in bacteria and eukaryotes is regulated by feedback inhibition targeting type-I and type-II pantothenate kinase (PanK). Type-III PanK is only found in bacteria and is generally insensitive to CoA. Previously, type-III PanK from the hyperthermophilic bacterium Thermotoga maritima was shown to defy this typical characteristic, and instead shows inhibition towards CoA. In the present study, phylogenetic analysis combined with functional analysis of type-III PanK from thermophiles revealed that the CoA-sensitive behavior of type-III PanK from T. maritima is uncommon. We cloned type-III PanKs from Thermus thermophilus and Geobacillus sp. 30 and showed that neither enzyme’s activities were inhibited by CoA. Furthermore, we utilized type-III PanK for a one-pot cascade reaction to produce CoA.

Adenosine triphosphate (ATP) is an essential cofactor for energy‐dependent enzymatic reactions that occur during in vitro biochemical conversion. Recently, an enzyme cascade based on non‐oxidative glycolysis, which uses starch and orthophosphate as energy and phosphate sources, respectively, for the regeneration of ATP from adenosine diphosphate, has been developed (Wei et al., ChemCatChem 2018, 10, 5597–5601). However, the 12 enzymes required for this system hampered its practical usability and further testing potential. Here, we addressed this issue by constructing co‐expression vectors for the simultaneous gene expression of the 12 enzymes in a single expression strain. All enzymes were sourced from (hyper)thermophiles, which enabled a one‐step purification via a heat‐treatment process. We showed that the combination of the two enabled the ATP regeneration system to function in a single recombinant Escherichia coli strain. Additionally, this work provides a strategy to rationally design and control proteins expression levels in the co‐expression vectors.

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