DOI: 10.5061/dryad.ns1rn8q54
title: Harnessing photoenzymatic reactions for unnatural biosynthesis in microorganisms

description: Photoenzymatic catalysis enables new-to-nature transformations, but its
 scalability is limited by high enzyme loading, costly cofactors, and
 radical-induced instability. Here we report the integration of
 light-driven photoenzymatic reactions into the cellular metabolism of
 Escherichia coli, bridging flavin-based photobiocatalysis with
 biosynthesis. Using synthetic biology strategies, we engineered microbial
 cells to continuously produce olefin substrates and ene-reductase
 photoenzyme while regenerating cofactors directly from glucose. By
 externally supplying radical precursors or by introducing synthetic
 pathways for their in situ production, we enabled fermentation-based
 microbial photobiosynthesis, achieving high titers and demonstrating its
 feasibility for scale-up in bioreactor. This approach extends
 photobiocatalysis from in vitro applications to in vivo semi-biosynthesis
 and complete biosynthesis, revealing its full potential for integrating
 light-driven reactions into cellular metabolism.
description: # Harnessing photoenzymatic reactions for unnatural biosynthesis in
 microorganisms Dataset DOI:
 [10.5061/dryad.ns1rn8q54](10.5061/dryad.ns1rn8q54) ## Description of the
 data and file structure This dataset supports the research published in
 “Harnessing Photoenzymatic Reactions for Unnatural Biosynthesis in
 Microorganisms”. It includes all the necessary SnapGene files for the
 plasmids described in the article and the Supplementary Information. All
 files are provided in SnapGene (.dna) format and can be opened and edited
 using SnapGene or SnapGene Viewer. OYE family enzymes (Old Yellow Enzymes
 / ene-reductases) Auxiliary NADPH-regeneration systems Promoter
 comparisons (T7, Trc, lac) 4-vinylphenol and styrene biosynthetic pathway
 construction Photobiocatalysis pathways (using photoenzymes + NADPH
 regeneration) Whole-cell screening of OYE mutants **pACYC_receiver.dna** *
 A p15A-origin backbone is used as a general template for constructing new
 plasmids. **pML43_(pET30a(_)-vector).dna** * Empty pET-30a vector; used as
 a negative control. pET-30a()-_OYE1.dna Expresses OYE1. Mutant or
 promoter-varied OYE plasmids (examples): pML19, pML22, pML23, pML26 →
 OYE3_N194Y under T7 or Trc or lac promoters pML35, pML38, pML39, pML42 →
 same as above but different promoter arrangement pML81–pML86 → OYE1
 variants (WT, H191A, Y82F) for substrate scope pML89, pML90 → GluER_M6
 variants for substrate scope * pACYC_receiver.dna: p15A template *
 pET-30a(_)_-_OYE1.dna: OYE1 expression plasmid *
 pML13_(pET30a(_)-T7-OYE3_N194Y-T7-PntAB).dna: Contains the PntAB gene, for
 NADPH regeneration * pML14_(pET30a(_)-T7-OYE3_N194Y-T7-UdhA).dna: Contains
 the UdhA gene, for NADPH regeneration *
 pML15_(pET30a(_)-T7-OYE3_N194Y-T7-Icd).dna: Contains the Icd gene, for
 NADPH regeneration * pML16_(pET30a(_)-T7-OYE3_N194Y-T7-GapC).dna: Contains
 GapC gene, for NADPH regeneration *
 pML17_(pET30a(_)-T7-OYE3_N194Y-T7-PTDH_mutant).dna: Contains the PTDH
 gene, for NADPH regeneration *
 pML18_(pET30a(_)-T7-OYE3_N194Y-T7-BstFDH_mutant).dna: Contains the BstFDH
 gene, for NADPH regeneration *
 pML19_(pET30a(_)-T7-OYE3_N194Y-Trc-GDH).dna: Contain T7-OYE3_N194Y
 and Trc-GDH, for promoters evaluation *
 pML22_(pET30a(_)-T7-OYE3_N194Y-Trc-Icd).dna: Contain T7-OYE3_N194Y
 and Trc-Icd, for promoters evaluation *
 pML23_(pET30a(_)-T7-OYE3_N194Y-lac-GDH).dna: Contain T7-OYE3_N194Y
 and lac-GDH, for promoters evaluation *
 pML26_(pET30a(_)-T7-OYE3_N194Y-lac-Icd).dna: Contain T7-OYE3_N194Y
 and lac-Icd, for promoters evaluation *
 pML35_(pET30a(_)-TRC-OYE3_N194Y-T7-GDH).dna: Contain TRC-OYE3_N194Y
 and T7-GDH, for promoters evaluation *
 pML38_(pET30a(_)-TRC-OYE3_N194Y-T7-Icd).dna: Contain TRC-OYE3_N194Y
 and T7-Icd, for promoters evaluation *
 pML39_(pET30a(_)-TRC-OYE3_N194Y-lac-GDH).dna: Contain TRC-OYE3_N194Y
 and lac-GDH, for promoters evaluation *
 pML42_(pET30a(_)-TRC-OYE3_N194Y-lac-Icd).dna: Contain TRC-OYE3_N194Y
 and lac-Icd, for promoters evaluation * pML43_(pET30a(_)-vector).dna:
 empty vector, for control experiments *
 pML7_(pET30a(_)-T7-OYE3_N194Y-T7-GDH).dna: Contains the GDH gene, for
 NADPH regeneration *
 pML70_(pET30a(_)-TRC-OYE3_N194Y-lac-GDH-no_tag).dna: Contain
 TRC-OYE3_N194Y and lac-GDH, for promoters evaluation *
 pML71_(pET30a(_)-TRC-OYE3_N194Y-lac-Icd-no_tag).dna: Contain
 TRC-OYE3_N194Y and lac-Icd, for promoters evaluation *
 pML8_(pET30a(_)-T7-OYE3_N194Y-T7-zwf).dna: Contains zwf gene, for NADPH
 regeneration * pML81_(pET30a(_)-TRC-OYE1-lac-GDH).dna: Contain TRC-OYE1
 and lac-GDH, for substrate scope *
 pML82_(pET30a(_)-TRC-OYE1-lac-Icd).dna: Contain TRC-OYE1 and lac-Icd, for
 substrate scope * pML83_(pET30a(_)-TRC-OYE1_H191A-lac-GDH).dna: Contain
 TRC-OYE1_H191A and lac-GDH, for substrate scope *
 pML84_(pET30a(_)-TRC-OYE1_H191A-lac-Icd).dna: Contain TRC-OYE1_H191A
 and lac-Icd, for substrate scope *
 pML85_(pET30a(_)-TRC-OYE1_Y82F-lac-GDH).dna: Contain TRC-OYE1_Y82F
 and lac-GDH, for substrate scope *
 pML86_(pET30a(_)-TRC-OYE1_Y82F-lac-Icd).dna: Contain TRC-OYE1_Y82F
 and lac-Icd, for substrate scope *
 pML89_(pET30a(_)-TRC-GluER_T36E_W66F_F68Y_P263L_F269Y_Q293T-lac-GDH).dna: Contain TRC-GluER_M6 and lac-GDH, for substrate scope * pML9_(pET30a(_)-T7-OYE3_N194Y-T7-pos5).dna: Contain pos5 gene, for NADPH regeneration * pML90_(pET30a(_)-TRC-GluER_T36E_W66F_F68Y_P263L_F269Y_Q293T-lac-Icd).dna: Contain TRC-GluER_M6 and lac-Icd, for substrate scope * pYJ700_(pET30a(_)-OYE1-GDH).dna:  Contains the n GDH gene, for NADPH regeneration * pYJ702_(pET30a(_)-OYE1-Pos5).dna: Contain Pos5 gene, for NADPH regeneration * pYJ727_(pET30a-Ptrc-PALY_RHOTO-PadA_LACPL-ppsA-tktA).dna: Contains 4-vinylphenol pathway genes, for 4-vinylphenol generation * pYJ728_(pACYC_aroF-tyrA).dna: Contains 4-vinylphenol pathway genes, for 4-vinylphenol generation * pYJ729_(pRSFDuet_Amp__Plac-tnaA).dna: Contain tnaA gene, for indole generation * pYJ749_(4-vinylphenol_biosynthetic_genes_(pACYC-aroF-tyrA__Plac__T7p__PALY_RHOTO__PadA_LACPL__ppsA__tktA)).dna: Contain 4-vinylphenol pathway genes, for 4-vinylphenol generation * pYJ750_(styrene_biosynthetic_genes_(pACYC-aroF-pheA__T7p__Ptrc__PAL2__FDC1__ppsA__tktA)).dna: Contain styrene biosynthetic genes, for styrene generation * pYJ751_(pET30a-aroF-tyrA__T7p__Ptrc__PALY_RHOTO__PadA_LACPL__ppsA__tktA__pBR322_ori).dna: Contain 4-vinylphenol pathway genes, for 4-vinylphenol generation * pYJ753_(pET30a-aroF-pheA__T7p__Ptrc__PAL2__FDC1__ppsA__tktA__pBR322_ori).dna: Contain styrene biosynthetic genes, for styrene generation * pYJ755_(pACYC_vector__Chl__Plac-tnaA).dna: Contain tnaA gene, for indole generation * pYJ761_(pET30a-aroF-tyrA__T7p__Ptrc__PALY_RHOTO__PadA_LACPL__ppsA__tktA__pBR322_ori__Trc-OYE3_N194Y__Lac-GDH).dna: Contain 4-vinylphenol and photoenzyme pathway genes, for photobiosynthesis fermentation * pYJ762_(pET30a-aroF-tyrA__T7p__Ptrc__PALY_RHOTO__PadA_LACPL__ppsA__tktA__pBR322_ori__Trc-OYE3_N194Y__Lac-Icd).dna: Contain 4-vinylphenol and photoenzyme pathway genes, for photobiosynthesis fermentation * pYJ763_(pRSF_ori__Trc-OYE3_N194Y__Lac-GDH).dna: Contain Trc-OYE3_N194Y and Lac-GDH, for photobiosynthesis fermentation * pYJ764_(pRSF_ori__Trc-OYE3_N194Y__Lac-Icd).dna: Contain Trc-OYE3_N194Y and Lac-Icd, for photobiosynthesis fermentation * pYJ767_(pRSF_ori__Trc-OYE1__Lac-GDH).dna: Contain Trc-OYE1 and Lac-GDH, for photobiosynthesis fermentation * pYJ768_(pRSF_ori__Trc-OYE1_H191A__Lac-GDH).dna: Contain Trc-OYE1_H191A and Lac-GDH, for photobiosynthesis fermentation * pYJ769_(pRSF_ori__Trc-OYE1_Y82F__Lac-GDH).dna: Contain Trc-OYE1_Y82F and Lac-GDH, for photobiosynthesis fermentation * pYJ770_(pRSF_ori__Trc-GluER_M6__Lac-GDH).dna: Contain Trc-GluER_M6 and Lac-GDH, for photobiosynthesis fermentation * pYJ771_(pRSF_ori__Trc-OYE1__Lac-Icd).dna: Contain Trc-OYE1 and Lac-Icd, for photobiosynthesis fermentation * pYJ772_(pRSF_ori__Trc-OYE1_H191A__Lac-Icd).dna: Contain Trc-OYE1_H191A and Lac-Icd, for photobiosynthesis fermentation * pYJ773_(pRSF_ori__Trc-OYE1_Y82F__Lac-Icd).dna: Contain Trc-OYE1_Y82F and Lac-Icd, for photobiosynthesis fermentation * pYJ774_(pRSF_ori__Trc-GluER_M6__Lac-Icd).dna: Contain Trc-GluER_M6 and Lac-Icd, for photobiosynthesis fermentation * pYJ775_(pRSF_ori__T7-OYE1-Y82G_T37Q).dna: Contain OYE1-Y82G_T37Q, for whole-cell mutants screening * pYJ776_(pRSF_ori__T7-OYE1-WT).dna: Contain OYE1-WT, for whole-cell mutants screening * pYJ777_(pRSF_ori__T7-OYE1-Y82G).dna: Contain OYE1-Y82G, for whole-cell mutants screening * pYJ779_(pRSF_ori__T7-OYE1-Y82G_T37Q_W116A_H191S).dna: Contain OYE1-Y82G_T37Q_W116A_H191S, for whole-cell mutants screening * pYJ783_(p15A_CmR-araBAD-TnaA).dna: Contains tnaA gene, for indole generation * pYJ784_(pET30a(_)-T7-OYE3_N194Y-T7-GDH-ICD).dna: Contains GDH-ICD genes, for NADPH regeneration * pYJ785_(pET30a(_)-T7-OYE3_N194Y-T7-Icd-GDH).dna:  Contains Icd-GDH genes, for NADPH regeneration * pYJ786_(pRSF_ori__Lac-GDH).dna: Contain Lac-GDH, without photoenzyme, for control experiments * pYJ787_(pRSF_ori__Lac-Icd).dna: Contain Lac-Icd, without photoenzyme, for control experiments