A Chloroplast-Localised Fluorescent Protein Enhances the Photosynthetic Action Spectrum in Green Algae

Abstract

Green microalgae are important sources of natural products and are attractive cell factories for manufacturing high-value products such as recombinant proteins. Increasing scales of production must address the bottleneck of providing sufficient light energy for photosynthesis. Enhancing the photosynthetic action spectrum of green algae to improve the utilisation of yellow light would provide additional light energy for photosynthesis. Here, we evaluated the Katushka fluorescent protein, which converts yellow photons to red photons, to drive photosynthesis and growth when expressed in Chlamydomonas reinhardtii chloroplasts. Transplastomic algae expressing a codon-optimised Katushka gene accumulated the active Katushka protein, which was detected by excitation with yellow light. Removal of chlorophyll from cells, which captures red photons, led to increased Katushka fluorescence. In yellow light, emission of red photons by fluorescent Katushka increased oxygen evolution and photosynthetic growth. Utilisation of yellow photons increased photosynthetic growth of transplastomic cells expressing Katushka in light deficient in red photons. These results showed that Katushka was a simple and effective yellow light-capturing device that enhanced the photosynthetic action spectrum of C. reinhardtii.

Advances in light system engineering across the phototrophic spectrum

“…This protein lowers the energy of yellow light photons (590 nm) to red light photons (649 nm) through fluorescence, converting the otherwise underused wavelengths into more photoactive radiation. They further showed that diminishing cell chlorophyll content enhanced the transgenic protein expression as the cells used the heterologous protein for light harvesting ( Suarez et al., 2022 ). It should be noted that this study relied mostly on optical density measurements for monitoring growth and grew cells at very low light levels (30 µmol m -2 s -1 ) in mixotrophic conditions.…”

Advances in Genetic Engineering in Improving Photosynthesis and Microalgal Productivity

“…The overexpression of endogenous CBPII (chlorophyll d-binding light-harvesting protein) from Acaryochloris marina suppressed the phycobiliproteins of Synechocystis 6803, resulting in a low ratio of phycobilins to chlorophyll a [29]. Transplastomic algae expressing the Katushka fluorescent protein increased oxygen evolution and photosynthetic growth in yellow light and enhanced the photosynthetic action spectrum of C. reinhardtii [32]. Introducing alternative light-harvesting complexes that absorb more efficiently in areas where chlorophyll is less efficient, like chlorophyll f [31], chlorophyll d-binding light-harvesting proteins [29,30], engineered fluorescence proteins [32], or diatom fucoxanthin components [33] is a possibility.…”

“…Transplastomic algae expressing the Katushka fluorescent protein increased oxygen evolution and photosynthetic growth in yellow light and enhanced the photosynthetic action spectrum of C. reinhardtii [32]. Introducing alternative light-harvesting complexes that absorb more efficiently in areas where chlorophyll is less efficient, like chlorophyll f [31], chlorophyll d-binding light-harvesting proteins [29,30], engineered fluorescence proteins [32], or diatom fucoxanthin components [33] is a possibility. Although this idea is plausible, whether it is feasible is a long-term question.…”

Antisense transcription from a neighboring gene interferes with the expression of mNeonGreen as a functionalin vivofluorescent reporter in the chloroplast ofChlamydomonas reinhardtii

“…Despite substantial progress in the study of the chloroplast's biology, fluorescent proteins have met limited success as reporters in the chloroplast of C. reinhardtii. Previous works have shown the use of several fluorescent proteins including GFP (Franklin et al, 2002;Noor-Mohammadi et al, 2012), mCherry (Kim et al, 2019), the Katushka yellow excitation-red emission protein that showed temporary enhanced fluorescence when chlorophyll was depleted (Suarez et al, 2022), and even the bright Vivid Verde (VFP) (Braun-Galleani et al, 2015) and Venus Q69M (Jackson et al, 2022) fluorescent proteins. However, conflicting evidence has been presented for their poor performance as an in vivo reporter, where presence of gene transcripts and protein has been shown but observation of fluorescence through microscopy has been challenging, suggesting that chlorophyll autofluorescence may impede fluorescent reporter visualization (Franklin et al, 2002;Braun-Galleani et al, 2015).…”