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PreprintCited by 2Microbial Metabolic Engineering and BioproductionPhotosynthetic Processes and MechanismsEnzyme Catalysis and Immobilization

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Harnessing photosynthetic ATP for whole-cell biocatalysis in the cyanobacterium Synechocystis

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Loprete, Giovanni, Traverso, Eleonora, Vascon, Filippo, Botteri, Marco, Robescu, Marina Simona, Ubiali, Daniela, Cendron, Laura, Morosinotto, Tomas, Bergantino, Elisabetta·2025·ACS Sustainable Chemistry & Engineering
DOI 10.1101/2025.05.29.656799PMC PMC12588301

Preprint — published as 10.1021/acssuschemeng.5c07236 · Paper page

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Abstract

Photosynthetic organisms use sunlight to produce ATP and NADPH powering their metabolism. Harnessing these products for driving biocatalytic reactions would enable to develop clean and sustainable alternatives for chemical reactions. In this study, we present the first demonstration that ATP produced from the photosynthetic process, can fuel a biocatalytic transformation in the whole-cell configuration. This result was achieved by expressing in the cyanobacterium Synechocystis sp. PCC 6803 an ATP-dependent enzyme, the γ-Glutamyl-MethylAmide Synthetase from Methylovorus mays No. 9 ( Mm GMAS). The expressed enzyme was able to drive in the transgenic strain the light-driven biosynthesis of L-theanine. Consumption of ATP by the recombinant Mm GMAS was even beneficial under strong illumination, protecting the photosynthetic electron transport from photodamage. These findings demonstrate the possibility of using photosynthetic microorganisms like Synechocystis as potential platform for sunlight driven biotransformations with wide potential biocatalytic applications. In this perspective, we further present the tridimensional structure of Mm GMAS, which explains its promiscuous in vivo activity and provides the basis for its rational evolution.

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Figures

1 (A) In vitro biocatalytic reaction producing l-Thea, catalyzed by recombinant MmGMAS. (B) Schematic diagram of the whole-cell biotransformation investigated in this study and performed by the MmGMAS-expressing transgenic strain Syn_GMAS. Fluxes of electrons, NADPH and ATP cofactors, and protons (H+) are represented by dotted black, gray, green, and red arrows, respectively.
1 (A) In vitro biocatalytic reaction producing l-Thea, catalyzed by recombinant MmGMAS. (B) Schematic diagram of the whole-cell biotransformation investigated in this study and performed by the MmGMAS-expressing transgenic strain Syn_GMAS. Fluxes of electrons, NADPH and ATP cofactors, and protons (H+) are represented by dotted black, gray, green, and red arrows, respectively.CC BY · PubMed Central
2 (A) Results of RP-HPLC analysis of Syn_GMAS whole-cell biotransformations performed at OD730 = 5, supplemented with different concentrations of l-glutamate and ethylamine (2.5, 5, and 10 mM) at different times. Concentration of l-Glu and ethylamine over time are represented by the continuous and dashed red lines, respectively. Control strain (Syn_UV) biotransformation was performed in the presence of 10 mM substrates. (B) Results of RP-HPLC analysis of Syn_GMAS whole-cell biotransformations performed at OD730 = 5, supplemented with different concentrations of ethylamine (1 and 2.5 mM) at different times. Concentration of ethylamine over time is represented by the dashed red line.
2 (A) Results of RP-HPLC analysis of Syn_GMAS whole-cell biotransformations performed at OD730 = 5, supplemented with different concentrations of l-glutamate and ethylamine (2.5, 5, and 10 mM) at different times. Concentration of l-Glu and ethylamine over time are represented by the continuous and dashed red lines, respectively. Control strain (Syn_UV) biotransformation was performed in the presence of 10 mM substrates. (B) Results of RP-HPLC analysis of Syn_GMAS whole-cell biotransformations performed at OD730 = 5, supplemented with different concentrations of ethylamine (1 and 2.5 mM) at different times. Concentration of ethylamine over time is represented by the dashed red line.CC BY · PubMed Central
3 Effects of substrate addition in recombinant Synechocystis growth. (A) Curves comparing growth rates of Syn_GMAS and Syn_UV strains in the absence and presence of 1 mM l-glutamate and ethylamine. Statistical significance (two-sample t test) is represented by ns (not significant) and asterisks (significant, *p < 0.05, **p < 0.01, ***p < 0.001) and refers to each time-point measured in the absence of substrates. (B) Curves comparing growth of strain Syn_GMAS and Syn_UV in the presence of 1 mM l-glutamate, l-theanine, and ethylamine separately. Cultures (three independent biological replicates for each strain and each condition) were cultivated in flasks under continuous shaking and under standard cultivation conditions.
3 Effects of substrate addition in recombinant Synechocystis growth. (A) Curves comparing growth rates of Syn_GMAS and Syn_UV strains in the absence and presence of 1 mM l-glutamate and ethylamine. Statistical significance (two-sample t test) is represented by ns (not significant) and asterisks (significant, *p < 0.05, **p < 0.01, ***p < 0.001) and refers to each time-point measured in the absence of substrates. (B) Curves comparing growth of strain Syn_GMAS and Syn_UV in the presence of 1 mM l-glutamate, l-theanine, and ethylamine separately. Cultures (three independent biological replicates for each strain and each condition) were cultivated in flasks under continuous shaking and under standard cultivation conditions.CC BY · PubMed Central
4 ATP content and electron transport rates of PSII and PSI (rETR). (A) ATP content of Syn_GMAS and Syn_UV grown in standard conditions (up to exponential phase, in BG11 and under 50 μmol photons·m–2·s–1) in the absence of exogenous substrates. (B) ATP quantification of Syn_GMAS whole-cell biotransformations after 2 h of cultivation in the presence or absence of 1 mM substrates, in BG11 and under 150 μmol photons·m–2·s–1 constant illumination. (C, D) Measurements of relative electron transport rate of PSI and PSII, (C) rETR­(I) and (D) rETR­(II), respectively, in Syn_GMAS and control Syn_UV strains. Statistical significance (two-sample t test) is represented by ns (not significant) and asterisks (significant, *p < 0.05, **p < 0.01, ***p < 0.001 and **** p < 0.0001).
4 ATP content and electron transport rates of PSII and PSI (rETR). (A) ATP content of Syn_GMAS and Syn_UV grown in standard conditions (up to exponential phase, in BG11 and under 50 μmol photons·m–2·s–1) in the absence of exogenous substrates. (B) ATP quantification of Syn_GMAS whole-cell biotransformations after 2 h of cultivation in the presence or absence of 1 mM substrates, in BG11 and under 150 μmol photons·m–2·s–1 constant illumination. (C, D) Measurements of relative electron transport rate of PSI and PSII, (C) rETR­(I) and (D) rETR­(II), respectively, in Syn_GMAS and control Syn_UV strains. Statistical significance (two-sample t test) is represented by ns (not significant) and asterisks (significant, *p < 0.05, **p < 0.01, ***p < 0.001 and **** p < 0.0001).CC BY · PubMed Central
5 Crystal structure and computational modeling of MmGMAS. (A) Front and top view of the MmGMAS homo-dodecamer (PDB 9QUR). (B) Interactions between ATPγS, Mg2+, and MmGMAS residues. Omit map of ATPγS and Mg2+ cations at 2.5σ is displayed. H-bonds and metal coordination bonds are displayed as black dashed lines; salt bridges, π-stacking, and cation−π interactions are shown as gray dashed lines; hydrophobic interactions are depicted as gray dotted lines. (C) In silico generated model of the quaternary complex MmGMAS/ADP/P-γ-Glu/MMA. Color gradient depicts the surface electrostatic potential of the MmGMAS active site. The dashed line represents the nucleophilic attack of the MMA amine group on P-γ-Glu Cδ. (D) Interactions between MmGMAS residues, MMA, and P-γ-Glu. Hydrophobic interactions are depicted as gray dotted lines, while electrostatic contacts are shown as black dashed lines.
5 Crystal structure and computational modeling of MmGMAS. (A) Front and top view of the MmGMAS homo-dodecamer (PDB 9QUR). (B) Interactions between ATPγS, Mg2+, and MmGMAS residues. Omit map of ATPγS and Mg2+ cations at 2.5σ is displayed. H-bonds and metal coordination bonds are displayed as black dashed lines; salt bridges, π-stacking, and cation−π interactions are shown as gray dashed lines; hydrophobic interactions are depicted as gray dotted lines. (C) In silico generated model of the quaternary complex MmGMAS/ADP/P-γ-Glu/MMA. Color gradient depicts the surface electrostatic potential of the MmGMAS active site. The dashed line represents the nucleophilic attack of the MMA amine group on P-γ-Glu Cδ. (D) Interactions between MmGMAS residues, MMA, and P-γ-Glu. Hydrophobic interactions are depicted as gray dotted lines, while electrostatic contacts are shown as black dashed lines.CC BY · PubMed Central

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Citations

Cites

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Metadata

Authors
Loprete, G., Traverso, E., Vascon, F., Botteri, M., Robescu, M. S., Ubiali, D., Cendron, L., Morosinotto, T., & Bergantino, E.
Affiliations
  • University of Padua (IT)
  • University of Pavia (IT)
Corresponding author
Bergantino, Elisabetta
ORCID
Venue
ACS Sustainable Chemistry & Engineering
Publisher
openRxiv
Published
2025-05-30
Volume
13
Issue
43
Pages
18667-18677
ISSN
2168-0485 (ISSN-L)
DOI
10.1101/2025.05.29.656799
PMC
PMC12588301
OpenAlex
W4415468576
Cited by
2
References
2
Citations by year
2026: 2
FWCI
0.60
Type
journal article
Language
en
Open access
Licence
Locations
  • ACS Sustainable Chemistry & Engineering (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Padua Research Archive (University of Padova) (submittedVersion, cc-by)
Funders
  • European Commission (ECS_00000036)
  • European Synchrotron Radiation Facility (ID30A-3)
  • Università degli Studi di Padova
  • Dipartimento di Biologia, Università degli Studi di Padova (BIRD221528/22, U-GOV CEND_BIRD24_01)
  • NextGenerationEU (ECS00000036)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Microbial Metabolic Engineering and Bioproduction
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Photosynthetic Processes and Mechanisms
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Enzyme Catalysis and Immobilization
Keywords
whole-cell biocatalysis, Synechocystis sp. PCC 6803, ATP-dependent enzyme, enzyme structure, cyanobacteria

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