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α-Dioxygenases (α-DOXs) are known as plant enzymes involved in the α-oxidation of fatty acids through which fatty aldehydes, with a high commercial value as flavor and fragrance compounds, are synthesized as products. Currently, little is known about α-DOXs from non-plant organisms. The phylogenic analysis reported here identified a substantial number of α-DOX enzymes across various taxa. Here, we report the functional characterization and Escherichia coli whole-cell application of two novel α-DOXs identified from cyanobacteria: CalDOX from Calothrix parietina and LepDOX from Leptolyngbya sp. The catalytic behavior of the recombinantly expressed CalDOX and LepDOX revealed that they are heme-dependent like plant α-DOXs but exhibit activities toward medium carbon fatty acids ranging from C10 to C14 unlike plant α-DOXs. The in-depth molecular investigation of cyanobacterial α-DOXs and their application in an E. coli whole system employed in this study is useful not only for the understanding of the molecular function of α-DOXs, but also for their industrial utilization in fatty aldehyde biosynthesis.Key points• Two novel α-dioxygenases from Cyanobacteria are reported• Both enzymes prefer medium-chain fatty acids• Both enzymes are useful for fatty aldehyde biosynthesis Graphical abstract

p-Coumaric acid (p-CA) is a key precursor for the biosynthesis of flavonoids. Tyrosine ammonia lyases (TALs) specifically catalyze the synthesis of p-CA from l-tyrosine, which is a convenient enzymatic pathway. To explore novel and highly active TALs, a phylogenetic tree-building approach was conducted including 875 putative TALs and 46 putative phenylalanine/tyrosine ammonia lyases (PTALs). Among them, 5 TALs and 3 PTALs were successfully characterized and found to exhibit the proposed enzymatic activity. The TAL from Chryseobacterium luteum sp. nov (TAL

TfCa, a promiscuous carboxylesterase from Thermobifida fusca, was found to hydrolyze polyethylene terephthalate (PET) degradation intermediates such as bis(2-hydroxyethyl) terephthalate (BHET) and mono-(2-hydroxyethyl)-terephthalate (MHET). In this study, we elucidated the structures of TfCa in its apo form, as well as in complex with a PET monomer analogue and with BHET. The structure–function relationship of TfCa was investigated by comparing its hydrolytic activity on various ortho- and para-phthalate esters of different lengths. Structure-guided rational engineering of amino acid residues in the substrate-binding pocket resulted in the TfCa variant I69W/V376A (WA), which showed 2.6-fold and 3.3-fold higher hydrolytic activity on MHET and BHET, respectively, than the wild-type enzyme. TfCa or its WA variant was mixed with a mesophilic PET depolymerizing enzyme variant [Ideonella sakaiensis PETase (IsPETase) PM] to degrade PET substrates of various crystallinity. The dual enzyme system with the wild-type TfCa or its WA variant produced up to 11-fold and 14-fold more terephthalate (TPA) than the single IsPETase PM, respectively. In comparison to the recently published chimeric fusion protein of IsPETase and MHETase, our system requires 10% IsPETase and one-fourth of the reaction time to yield the same amount of TPA under similar PET degradation conditions. Our simple dual enzyme system reveals further advantages in terms of cost-effectiveness and catalytic efficiency since it does not require time-consuming and expensive cross-linking and immobilization approaches.