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Genome streamlining is an evolutionary strategy used by natural living systems to dispense unnecessary genes from their genome as a mechanism to adapt and evolve. While this strategy has been successfully borrowed to develop synthetic heterotrophic microbial systems with desired phenotype, it has not been extensively explored in photoautotrophs. Genome streamlining strategy incorporates both computational predictions to identify the dispensable regions and experimental validation using genome-editing tool, and in this study, we have employed a modified strategy with the goal to minimize the genome size to an extent that allows optimal cellular fitness under specified conditions. Our strategy has explored a novel genome-editing tool in photoautotrophs, which, unlike other existing tools, enables large, spontaneous optimal deletions from the genome. Our findings demonstrate the effectiveness of this modified strategy in obtaining strains with streamlined genome, exhibiting improved fitness and productivity.

 

Light energy is essential for the existence of life on this planet, and only photosynthetic organisms, equipped with light-harvesting antenna protein complexes, can capture this energy, making it readily accessible to all other life forms. However, these light-harvesting antennae are not designed to function optimally under extreme high light, a condition which can cause photodamage and significantly reduce photosynthetic productivity.

 

ABSTRACT Synechococcus elongatus UTEX 2973, the fastest-growing cyanobacterial strain known, optimally grows under extreme high light (HL) intensities of 1,500–2,500 μmol photons m −2 s −1 , which is lethal to most other photosynthetic microbes. We leveraged the few genetic differences between Synechococcus 2973 and the HL sensitive strain Synechococcus elongatus PCC 7942 to unravel factors essential for the high light tolerance. We identified a novel protein in Synechococcus 2973 that we have termed HltA for H igh l ight t olerance protein A . Using bioinformatic tools, we determined that HltA contains a functional PP2C-type protein phosphatase domain. Phylogenetic analysis showed that the PP2C domain belongs to the bacterial-specific Group II family and is closely related to the environmental stress response phosphatase RsbU. Additionally, we showed that unlike any previously described phosphatases, HltA contains a single N-terminal regulatory GAF domain. We found hltA to be ubiquitous throughout cyanobacteria, indicative of its potentially important role in the photosynthetic lifestyle of these oxygenic phototrophs. Mutations in the hltA gene resulted in severe defects specific to high light growth. These results provide evidence that hltA is a key factor in the tolerance of Synechococcus 2973 to high light and will open new insights into the mechanisms of cyanobacterial light stress response. IMPORTANCE Cyanobacteria are a diverse group of photosynthetic prokaryotes. The cyanobacterium Synechococcus 2973 is a high light tolerant strain with industrial promise due to its fast growth under high light conditions and the availability of genetic modification tools. Currently, little is known about the high light tolerance mechanisms of Synechococcus 2973, and there are many unknowns overall regarding high light tolerance of cyanobacteria. In this study, a comparative genomic analysis of Synechococcus 2973 identified a single nucleotide polymorphism in a locus encoding a serine phosphatase as a key factor for high light tolerance. This novel GAF-containing phosphatase was found to be the sole Group II metal-dependent protein phosphatase that is evolutionarily conserved throughout cyanobacteria. These results shed new light on the light response mechanisms of Synechococcus 2973, improving our understanding of environmental stress response. Additionally, this work will help facilitate the development of Synechococcus 2973 as an industrially useful organism. 

Cyanobacteria are the only oxygenic photosynthetic organisms that can fix nitrogen. In diazotrophic cyanobacteria, the regulation of photosynthesis during the diurnal cycle is hypothesized to be linked with nitrogen fixation and involve the D1 protein isoform PsbA4. The amount of bioavailable nitrogen has a major impact on productivity in aqueous environments. In contrast to low- or nitrogen-fixing (−N) conditions, little data on photosynthetic regulation under nitrogen-replete (+ N) conditions are available. We compared the regulation of photosynthesis under −N and + N conditions during the diurnal cycle in wild type and apsbA4deletion strain of the unicellular diazotrophic cyanobacteriumCyanothecesp. ATCC 51142. We observed common changes to light harvesting and photosynthetic electron transport during the dark in + N and −N conditions and found that these modifications occur in both diazotrophic and non-diazotrophic cyanobacteria. Nitrogen availability increased PSII titer when cells transitioned from dark to light and promoted growth. Under −N conditions, deletion of PsbA4 modified charge recombination in dark and regulation of PSII titer during dark to light transition. We conclude that darkness impacts the acceptor-side modifications to PSII and photosynthetic electron transport in cyanobacteria independently of the nitrogen-fixing status and the presence of PsbA4.

 

Engineered cyanobacteriumSynechococcus elongatuscan use light and CO₂to produce sucrose, making it a promising candidate for use in co-cultures with heterotrophic workhorses. However, this process is challenged by the mutual stresses generated from the multispecies microbial culture. Here we demonstrate an ecosystem whereS. elongatusis freely grown in a photo-bioreactor (PBR) containing an engineered heterotrophic workhorse (either β-carotene-producingYarrowia lipolytica or indigoidine-producingPseudomonas putida) encapsulated in calcium-alginate hydrogel beads. The encapsulation prevents growth interference, allowing the cyanobacterial culture to produce high sucrose concentrations enabling the production of indigoidine and β-carotene in the heterotroph. Our experimental PBRs yielded an indigoidine titer of 7.5 g/L hydrogel and a β-carotene titer of 1.3 g/L hydrogel, amounts 15–22-fold higher than in a comparable co-culture without encapsulation. Moreover,¹³C-metabolite analysis and protein overexpression tests indicated that the hydrogel beads provided a favorable microenvironment where the cell metabolism inside the hydrogel was comparable to that in a free culture. Finally, the heterotroph-containing hydrogels were easily harvested and dissolved by EDTA for product recovery, while the cyanobacterial culture itself could be reused for the next batch of immobilized heterotrophs. This co-cultivation and hydrogel encapsulation system is a successful demonstration of bioprocess optimization under photobioreactor conditions.

 

Marine nitrogen-fixing microorganisms are an important source of fixed nitrogen in oceanic ecosystems. The colonial cyanobacterium Trichodesmium and diatom symbionts were thought to be the primary contributors to oceanic N 2 fixation until the discovery of the unusual uncultivated symbiotic cyanobacterium UCYN-A ( Candidatus Atelocyanobacterium thalassa ). UCYN-A has atypical metabolic characteristics lacking the oxygen-evolving photosystem II, the tricarboxylic acid cycle, the carbon-fixation enzyme RuBisCo and de novo biosynthetic pathways for a number of amino acids and nucleotides. Therefore, it is obligately symbiotic with its single-celled haptophyte algal host. UCYN-A receives fixed carbon from its host and returns fixed nitrogen, but further insights into this symbiosis are precluded by both UCYN-A and its host being uncultured. In order to investigate how this syntrophy is coordinated, we reconstructed bottom-up genome-scale metabolic models of UCYN-A and its algal partner to explore possible trophic scenarios, focusing on nitrogen fixation and biomass synthesis. Since both partners are uncultivated and only the genome sequence of UCYN-A is available, we used the phylogenetically related Chrysochromulina tobin as a proxy for the host. Through the use of flux balance analysis (FBA), we determined the minimal set of metabolites and biochemical functions that must be shared between the two organisms to ensure viability and growth. We quantitatively investigated the metabolic characteristics that facilitate daytime N 2 fixation in UCYN-A and possible oxygen-scavenging mechanisms needed to create an anaerobic environment to allow nitrogenase to function. This is the first application of an FBA framework to examine the tight metabolic coupling between uncultivated microbes in marine symbiotic communities and provides a roadmap for future efforts focusing on such specialized systems.