Rational engineering of photosynthetic electron flux enhances light-powered cytochrome P450 activity

Light-driven biocatalysis in recombinant cyanobacteria provides highly atom-efficient cofactor regeneration via photosynthesis, thereby remediating constraints associated with sacrificial cosubstrates. However, despite the remarkable specific activities of photobiocatalysts, self-shading at moderate-high cell densities limits efficient space-time-yields of heterologous enzymatic reactions. Moreover, efficient integration of an artificial electron sink into the tightly regulated network of cyanobacterial electron pathways can be highly challenging. Here, we used C=C bond reduction of 2-methylmaleimide by the NADPH-dependent ene-reductase YqjM as a model reaction for light-dependent biotransformations. Time-resolved NADPH fluorescence spectroscopy allowed direct monitoring of in-cell YqjM activity and revealed differences in NADPH steady-state levels and oxidation kinetics between different genetic constructs. This effect correlates with specific activities of whole-cells, which demonstrated conversions of >99%. Further channelling of electrons toward heterologous YqjM by inactivation of the flavodiiron proteins (Flv1/Flv3) led to a 2-fold improvement in specific activity at moderate cell densities, thereby elucidating the possibility of accelerating light-driven biotransformations by the removal of natural competing electron sinks. In the best case, an initial product formation rate of 18.3 mmol h –1 L –1 was reached, allowing the complete conversion of a 60 mM substrate solution within 4 h.

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“…PCC 7002. 38 Similar methods have also been reported for other purposes including the production of sucrose and photohydrogen. 39 , 40 …”

mentioning

confidence: 72%

Engineering of NADPH Supply Boosts Photosynthesis-Driven Biotransformations

et al. 2020

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“…Photosynthetic Enhancements. We validated the effect of expressing a carbon sink (sucrose production) (16,17) or an electron sink (cytochrome P450) (21,22) separately in S. elongatus ( Fig. 1).…”

Section: Expression Of a Single Heterologous Sink Leads To Distinctmentioning

confidence: 99%

Improved photosynthetic capacity and photosystem I oxidation via heterologous metabolism engineering in cyanobacteria

Santos-Merino

Torrado

Davis

et al. 2021

Proc. Natl. Acad. Sci. U.S.A.

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Cyanobacteria must prevent imbalances between absorbed light energy (source) and the metabolic capacity (sink) to utilize it to protect their photosynthetic apparatus against damage. A number of photoprotective mechanisms assist in dissipating excess absorbed energy, including respiratory terminal oxidases and flavodiiron proteins, but inherently reduce photosynthetic efficiency. Recently, it has been hypothesized that some engineered metabolic pathways may improve photosynthetic performance by correcting source/sink imbalances. In the context of this subject, we explored the interconnectivity between endogenous electron valves, and the activation of one or more heterologous metabolic sinks. We coexpressed two heterologous metabolic pathways that have been previously shown to positively impact photosynthetic activity in cyanobacteria, a sucrose production pathway (consuming ATP and reductant) and a reductant-only consuming cytochrome P450. Sucrose export was associated with improved quantum yield of phtotosystem II (PSII) and enhanced electron transport chain flux, especially at lower illumination levels, while cytochrome P450 activity led to photosynthetic enhancements primarily observed under high light. Moreover, coexpression of these two heterologous sinks showed additive impacts on photosynthesis, indicating that neither sink alone was capable of utilizing the full “overcapacity” of the electron transport chain. We find that heterologous sinks may partially compensate for the loss of photosystem I (PSI) oxidizing mechanisms even under rapid illumination changes, although this compensation is incomplete. Our results provide support for the theory that heterologous metabolism can act as a photosynthetic sink and exhibit some overlapping functionality with photoprotective mechanisms, while potentially conserving energy within useful metabolic products that might otherwise be “lost.”

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“…Though there were no QTLs associated with photosynthesis or carbon fixation previously reported for chromosome 15 (Grant et al, 2010), there is a reasonable background to hypothesize the relationship between photosynthesis and cytochrome. In cyanobacterium, for instance, an improved performance in A via increased electron transport rate and ATP production was promoted as consequence of doubling the activity of the cytochrome protein CYP1A1 (Berepiki et al, 2018). Induction of genes associated with these proteins are also reported when atrazine and bentazon, herbicides inhibitor of photosynthesis, are applied in soybean (Zhu et al, 2009).…”

Section: Discussionmentioning

confidence: 99%

Phenotypic Variation and Genetic Architecture for Photosynthesis and Water Use Efficiency in Soybean (Glycine max L. Merr)

2019

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Photosynthesis ( A ) and intrinsic water use efficiency ( WUE ) are physiological traits directly influencing biomass production, conversion efficiency, and grain yield. Though the influence of physiological process on yield is widely known, studies assessing improvement strategies are rare due to laborious phenotyping and specialized equipment needs. This is one of the first studies to assess the genetic architecture underlying A and intrinsic WUE , as well as to evaluate the feasibility of implementing genomic prediction. A panel of 383 soybean recombinant inbred lines were evaluated in a multi-environment yield trial that included measurements of A and intrinsic WUE , using an infrared gas analyzer during R4–R5 growth stages. Genetic variability was found to support the possibility of genetic improvement through breeding. High genetic correlation between grain yield ( GY ) and A (0.80) was observed, suggesting increases in GY can be achieved through the improvement of A . Genome-wide association analysis revealed quantitative trait loci (QTLs) for these physiological traits. Cross-validation studies indicated high predictive ability (>0.65) for the implementation of genomic prediction as a viable strategy to improve physiological efficiency while reducing field phenotyping. This work provides core knowledge to develop new soybean cultivars with enhanced photosynthesis and water use efficiency through conventional breeding and genomic techniques.

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Rational engineering of photosynthetic electron flux enhances light-powered cytochrome P450 activity

Cited by 30 publications

References 53 publications

Engineering of NADPH Supply Boosts Photosynthesis-Driven Biotransformations

Engineering of NADPH Supply Boosts Photosynthesis-Driven Biotransformations

Improved photosynthetic capacity and photosystem I oxidation via heterologous metabolism engineering in cyanobacteria

Phenotypic Variation and Genetic Architecture for Photosynthesis and Water Use Efficiency in Soybean (Glycine max L. Merr)

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