A toolbox to engineer the highly productive cyanobacterium Synechococcus sp. PCC 11901.

Synechococcus sp. PCC 11901 (PCC 11901) is a fast-growing marine cyanobacterial strain that has a capacity for sustained biomass accumulation to very high cell densities, comparable to that achieved by commercially relevant heterotrophic organisms. However, genetic tools to engineer PCC 11901 for biotechnology applications are limited. Here we describe a suite of tools based on the CyanoGate MoClo system to unlock the engineering potential of PCC 11901. First, we characterised neutral sites suitable for stable genomic integration that do not affect growth even at high cell densities. Second, we tested a suite of constitutive promoters, terminators, and inducible promoters including a 2,4-diacetylphloroglucinol (DAPG)-inducible PhlF repressor system, which has not previously been demonstrated in cyanobacteria, and showed tight regulation and a 228-fold dynamic range of induction. Lastly, we developed a DAPG-inducible dCas9-based CRISPR interference (CRISPRi) system and a modular method to generate markerless mutants using CRISPR-Cas12a. Based on our findings, PCC 11901 is highly responsive to CRISPRi-based repression and showed high efficiencies for single insertion (31-81%) and multiplex double insertion (25%) genome editing with Cas12a. We envision that these tools will lay the foundations for the adoption of PCC 11901 as a robust model strain for engineering biology and green biotechnology.

The effect of Alcanivorax borkumensis SK2, a hydrocarbon-metabolising organism, on gas holdup in a 4-phase bubble column bioprocess.

To design bioprocesses utilising hydrocarbon-metabolising organisms (HMO) as biocatalysts, the effect of the organism on the hydrodynamics of bubble column reactor (BCR), such as gas holdup, needs to be investigated. Therefore, this study investigates the first use of an HMO, Alcanivorax borkumensis SK2, as a solid phase in the operation and hydrodynamics of a BCR. The study investigated the gas holdup in 3-phase and 4-phase systems in a BCR under ranges of superficial gas velocities (UG) from 1 to 3 cm/s, hydrocarbon (chain length C13-21) concentrations (HC) of 0, 5, and 10% v/v and microbial concentrations (MC) of 0, 0.35, 0.6 g/l. The results indicated that UG was the most significant parameter, as gas holdup increases linearly with increasing UG from 1 to 3 cm/s. Furthermore, the addition of hydrocarbons into the air-deionized water -SK2 system showed the highest increase in the gas holdup, particularly at high UG (above 2 cm/s). The solids (yeast, cornflour, and SK2) phases had differing effects on gas holdup, potentially due to the difference in surface activity. In this work, SK2 addition caused a reduction in the fluid surface tension in the bioprocess which therefore resulted in an increase in the gas holdup in BCR. This work builds upon previous investigations in optimising the hydrodynamics for bubble column hydrocarbon bioprocesses for the application of alkane bioactivation.

A dual compartment cuvette system for correcting scattering in whole-cell absorbance spectroscopy of photosynthetic microorganisms.

Absorption spectroscopy is widely used to determine absorption and transmission spectra of chromophores in solution, in addition to suspensions of particles, including micro-organisms. Light scattering, caused by photons deflected from part or all of the cells or other particles in suspension, results in distortions to the absorption spectra, lost information and poor resolution. A spectrophotometer with an integrating sphere may be used to alleviate this problem. However, these instruments are not universally available in biology laboratories, for reasons such as cost. Here, we describe a novel, rapid, and inexpensive technique that minimises the effect of light scattering when performing whole-cell spectroscopy. This method involves using a custom made dual compartment cuvette containing titanium dioxide in one chamber as a scattering agent. Measurements were conducted of a range of different photosynthetic micro-organisms of varying cell size and morphology, including cyanobacteria, eukaryotic microalgae and a purple non-sulphur bacterium. A concentration of 1 mg ml-1 titanium dioxide, using a spectrophotometer with a slit width of 5 nm, produced spectra for cyanobacteria and microalgae similar (1-4% difference) to those obtained using an integrating sphere. The spectrum > 520 nm was similar to that with an integrating sphere with the purple non-sulphur bacterium. This system produced superior results to those obtained using a recently reported method, the application of the diffusing agent, Scotch™ Magic tape, to the side of the cuvette. The protocol can be completed in an equivalent period of time to standard whole-cell absorbance spectroscopy techniques, and is, in principle, suitable for any dual-beam spectrophotometer.

Cytochrome c M Decreases Photosynthesis under Photomixotrophy in Synechocystis sp. PCC 6803.

Photomixotrophy is a metabolic state that enables photosynthetic microorganisms to simultaneously perform photosynthesis and metabolism of imported organic carbon substrates. This process is complicated in cyanobacteria, since many, including Synechocystis sp. PCC 6803, conduct photosynthesis and respiration in an interlinked thylakoid membrane electron transport chain. Under photomixotrophy, the cell must therefore tightly regulate electron fluxes from photosynthetic and respiratory complexes. In this study, we demonstrate, via characterization of photosynthetic apparatus and the proteome, that photomixotrophic growth results in a gradual inhibition of QA - reoxidation in wild-type Synechocystis, which largely decreases photosynthesis over 3 d of growth. This process is circumvented by deleting the gene encoding cytochrome c M (CytM), a cryptic c-type heme protein widespread in cyanobacteria. The ΔCytM strain maintained active photosynthesis over the 3-d period, demonstrated by high photosynthetic O2 and CO2 fluxes and effective yields of PSI and PSII. Overall, this resulted in a higher growth rate compared to that of the wild type, which was maintained by accumulation of proteins involved in phosphate and metal uptake, and cofactor biosynthetic enzymes. While the exact role of CytM has not been determined, a mutant deficient in the thylakoid-localized respiratory terminal oxidases and CytM (ΔCox/Cyd/CytM) displayed a phenotype similar to that of ΔCytM under photomixotrophy. This, in combination with other physiological data, and in contrast to a previous hypothesis, suggests that CytM does not transfer electrons to these complexes. In summary, our data suggest that CytM may have a regulatory role in photomixotrophy by modulating the photosynthetic capacity of cells.

Proteome Mapping of a Cyanobacterium Reveals Distinct Compartment Organization and Cell-Dispersed Metabolism.

Cyanobacteria are complex prokaryotes, incorporating a Gram-negative cell wall and internal thylakoid membranes (TMs). However, localization of proteins within cyanobacterial cells is poorly understood. Using subcellular fractionation and quantitative proteomics, we produced an extensive subcellular proteome map of an entire cyanobacterial cell, identifying ∼67% of proteins in Synechocystis sp. PCC 6803, ∼1000 more than previous studies. Assigned to six specific subcellular regions were 1,712 proteins. Proteins involved in energy conversion localized to TMs. The majority of transporters, with the exception of a TM-localized copper importer, resided in the plasma membrane (PM). Most metabolic enzymes were soluble, although numerous pathways terminated in the TM (notably those involved in peptidoglycan monomer, NADP+, heme, lipid, and carotenoid biosynthesis) or PM (specifically, those catalyzing lipopolysaccharide, molybdopterin, FAD, and phylloquinol biosynthesis). We also identified the proteins involved in the TM and PM electron transport chains. The majority of ribosomal proteins and enzymes synthesizing the storage compound polyhydroxybuyrate formed distinct clusters within the data, suggesting similar subcellular distributions to one another, as expected for proteins operating within multicomponent structures. Moreover, heterogeneity within membrane regions was observed, indicating further cellular complexity. Cyanobacterial TM protein localization was conserved in Arabidopsis (Arabidopsis thaliana) chloroplasts, suggesting similar proteome organization in more developed photosynthetic organisms. Successful application of this technique in Synechocystis suggests it could be applied to mapping the proteomes of other cyanobacteria and single-celled organisms. The organization of the cyanobacterial cell revealed here substantially aids our understanding of these environmentally and biotechnologically important organisms.

CyanoGate: A Modular Cloning Suite for Engineering Cyanobacteria Based on the Plant MoClo Syntax.

Recent advances in synthetic biology research have been underpinned by an exponential increase in available genomic information and a proliferation of advanced DNA assembly tools. The adoption of plasmid vector assembly standards and parts libraries has greatly enhanced the reproducibility of research and the exchange of parts between different labs and biological systems. However, a standardized modular cloning (MoClo) system is not yet available for cyanobacteria, which lag behind other prokaryotes in synthetic biology despite their huge potential regarding biotechnological applications. By building on the assembly library and syntax of the Plant Golden Gate MoClo kit, we have developed a versatile system called CyanoGate that unites cyanobacteria with plant and algal systems. Here, we describe the generation of a suite of parts and acceptor vectors for making (1) marked/unmarked knock-outs or integrations using an integrative acceptor vector, and (2) transient multigene expression and repression systems using known and previously undescribed replicative vectors. We tested and compared the CyanoGate system in the established model cyanobacterium Synechocystis sp. PCC 6803 and the more recently described fast-growing strain Synechococcus elongatus UTEX 2973. The UTEX 2973 fast-growth phenotype was only evident under specific growth conditions; however, UTEX 2973 accumulated high levels of proteins with strong native or synthetic promoters. The system is publicly available and can be readily expanded to accommodate other standardized MoClo parts to accelerate the development of reliable synthetic biology tools for the cyanobacterial community.

Contribution of cyanobacterial alkane production to the ocean hydrocarbon cycle.

Hydrocarbons are ubiquitous in the ocean, where alkanes such as pentadecane and heptadecane can be found even in waters minimally polluted with crude oil. Populations of hydrocarbon-degrading bacteria, which are responsible for the turnover of these compounds, are also found throughout marine systems, including in unpolluted waters. These observations suggest the existence of an unknown and widespread source of hydrocarbons in the oceans. Here, we report that strains of the two most abundant marine cyanobacteria, Prochlorococcus and Synechococcus, produce and accumulate hydrocarbons, predominantly C15 and C17 alkanes, between 0.022 and 0.368% of dry cell weight. Based on global population sizes and turnover rates, we estimate that these species have the capacity to produce 2-540 pg alkanes per mL per day, which translates into a global ocean yield of ∼ 308-771 million tons of hydrocarbons annually. We also demonstrate that both obligate and facultative marine hydrocarbon-degrading bacteria can consume cyanobacterial alkanes, which likely prevents these hydrocarbons from accumulating in the environment. Our findings implicate cyanobacteria and hydrocarbon degraders as key players in a notable internal hydrocarbon cycle within the upper ocean, where alkanes are continually produced and subsequently consumed within days. Furthermore we show that cyanobacterial alkane production is likely sufficient to sustain populations of hydrocarbon-degrading bacteria, whose abundances can rapidly expand upon localized release of crude oil from natural seepage and human activities.

Photosynthetic, respiratory and extracellular electron transport pathways in cyanobacteria.

Cyanobacteria have evolved elaborate electron transport pathways to carry out photosynthesis and respiration, and to dissipate excess energy in order to limit cellular damage. Our understanding of the complexity of these systems and their role in allowing cyanobacteria to cope with varying environmental conditions is rapidly improving, but many questions remain. We summarize current knowledge of cyanobacterial electron transport pathways, including the possible roles of alternative pathways in photoprotection. We describe extracellular electron transport, which is as yet poorly understood. Biological photovoltaic devices, which measure electron output from cells, and which have been proposed as possible means of renewable energy generation, may be valuable tools in understanding cyanobacterial electron transfer pathways, and enhanced understanding of electron transfer may allow improvements in the efficiency of power output. This article is part of a Special Issue entitled Organization and dynamics of bioenergetic systems in bacteria, edited by Conrad Mullineaux.

Distinguishing the Roles of Thylakoid Respiratory Terminal Oxidases in the Cyanobacterium Synechocystis sp. PCC 6803.

Various oxygen-utilizing electron sinks, including the soluble flavodiiron proteins (Flv1/3), and the membrane-localized respiratory terminal oxidases (RTOs), cytochrome c oxidase (Cox) and cytochrome bd quinol oxidase (Cyd), are present in the photosynthetic electron transfer chain of Synechocystis sp. PCC 6803. However, the role of individual RTOs and their relative importance compared with other electron sinks are poorly understood, particularly under light. Via membrane inlet mass spectrometry gas exchange, chlorophyll a fluorescence, P700 analysis, and inhibitor treatment of the wild type and various mutants deficient in RTOs, Flv1/3, and photosystem I, we investigated the contribution of these complexes to the alleviation of excess electrons in the photosynthetic chain. To our knowledge, for the first time, we demonstrated the activity of Cyd in oxygen uptake under light, although it was detected only upon inhibition of electron transfer at the cytochrome b6f site and in ∆flv1/3 under fluctuating light conditions, where linear electron transfer was drastically inhibited due to impaired photosystem I activity. Cox is mostly responsible for dark respiration and competes with P700 for electrons under high light. Only the ∆cox/cyd double mutant, but not single mutants, demonstrated a highly reduced plastoquinone pool in darkness and impaired gross oxygen evolution under light, indicating that thylakoid-based RTOs are able to compensate partially for each other. Thus, both electron sinks contribute to the alleviation of excess electrons under illumination: RTOs continue to function under light, operating on slower time ranges and on a limited scale, whereas Flv1/3 responds rapidly as a light-induced component and has greater capacity.

Hydrocarbons Are Essential for Optimal Cell Size, Division, and Growth of Cyanobacteria.

Cyanobacteria are intricately organized, incorporating an array of internal thylakoid membranes, the site of photosynthesis, into cells no larger than other bacteria. They also synthesize C15-C19 alkanes and alkenes, which results in substantial production of hydrocarbons in the environment. All sequenced cyanobacteria encode hydrocarbon biosynthesis pathways, suggesting an important, undefined physiological role for these compounds. Here, we demonstrate that hydrocarbon-deficient mutants of Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803 exhibit significant phenotypic differences from wild type, including enlarged cell size, reduced growth, and increased division defects. Photosynthetic rates were similar between strains, although a minor reduction in energy transfer between the soluble light harvesting phycobilisome complex and membrane-bound photosystems was observed. Hydrocarbons were shown to accumulate in thylakoid and cytoplasmic membranes. Modeling of membranes suggests these compounds aggregate in the center of the lipid bilayer, potentially promoting membrane flexibility and facilitating curvature. In vivo measurements confirmed that Synechococcus sp. PCC 7002 mutants lacking hydrocarbons exhibit reduced thylakoid membrane curvature compared to wild type. We propose that hydrocarbons may have a role in inducing the flexibility in membranes required for optimal cell division, size, and growth, and efficient association of soluble and membrane bound proteins. The recent identification of C15-C17 alkanes and alkenes in microalgal species suggests hydrocarbons may serve a similar function in a broad range of photosynthetic organisms.

Phycobilisome-Deficient Strains of Synechocystis sp. PCC 6803 Have Reduced Size and Require Carbon-Limiting Conditions to Exhibit Enhanced Productivity.

Reducing excessive light harvesting in photosynthetic organisms may increase biomass yields by limiting photoinhibition and increasing light penetration in dense cultures. The cyanobacterium Synechocystis sp. PCC 6803 harvests light via the phycobilisome, which consists of an allophycocyanin core and six radiating rods, each with three phycocyanin (PC) discs. Via targeted gene disruption and alterations to the promoter region, three mutants with two (pcpcT→C) and one (ΔCpcC1C2:pcpcT→C) PC discs per rod or lacking PC (olive) were generated. Photoinhibition and chlorophyll levels decreased upon phycobilisome reduction, although greater penetration of white light was observed only in the PC-deficient mutant. In all strains cultured at high cell densities, most light was absorbed by the first 2 cm of the culture. Photosynthesis and respiration rates were also reduced in the ΔCpcC1C2:pcpcT→C and olive mutants. Cell size was smaller in the pcpcT→C and olive strains. Growth and biomass accumulation were similar between the wild-type and pcpcT→C under a variety of conditions. Growth and biomass accumulation of the olive mutant were poorer in carbon-saturated cultures but improved in carbon-limited cultures at higher light intensities, as they did in the ΔCpcC1C2:pcpcT→C mutant. This study shows that one PC disc per rod is sufficient for maximal light harvesting and biomass accumulation, except under conditions of high light and carbon limitation, and two or more are sufficient for maximal oxygen evolution. To our knowledge, this study is the first to measure light penetration in bulk cultures of cyanobacteria and offers important insights into photobioreactor design.

Thylakoid terminal oxidases are essential for the cyanobacterium Synechocystis sp. PCC 6803 to survive rapidly changing light intensities.

Cyanobacteria perform photosynthesis and respiration in the thylakoid membrane, suggesting that the two processes are interlinked. However, the role of the respiratory electron transfer chain under natural environmental conditions has not been established. Through targeted gene disruption, mutants of Synechocystis sp. PCC 6803 were generated that lacked combinations of the three terminal oxidases: the thylakoid membrane-localized cytochrome c oxidase (COX) and quinol oxidase (Cyd) and the cytoplasmic membrane-localized alternative respiratory terminal oxidase. All strains demonstrated similar growth under continuous moderate or high light or 12-h moderate-light/dark square-wave cycles. However, under 12-h high-light/dark square-wave cycles, the COX/Cyd mutant displayed impaired growth and was completely photobleached after approximately 2 d. In contrast, use of sinusoidal light/dark cycles to simulate natural diurnal conditions resulted in little photobleaching, although growth was slower. Under high-light/dark square-wave cycles, the COX/Cyd mutant suffered a significant loss of photosynthetic efficiency during dark periods, a greater level of oxidative stress, and reduced glycogen degradation compared with the wild type. The mutant was susceptible to photoinhibition under pulsing but not constant light. These findings confirm a role for thylakoid-localized terminal oxidases in efficient dark respiration, reduction of oxidative stress, and accommodation of sudden light changes, demonstrating the strong selective pressure to maintain linked photosynthetic and respiratory electron chains within the thylakoid membrane. To our knowledge, this study is the first to report a phenotypic difference in growth between terminal oxidase mutants and wild-type cells and highlights the need to examine mutant phenotypes under a range of conditions.

CyanoCyc cyanobacterial web portal.

CyanoCyc is a web portal that integrates an exceptionally rich database collection of information about cyanobacterial genomes with an extensive suite of bioinformatics tools. It was developed to address the needs of the cyanobacterial research and biotechnology communities. The 277 annotated cyanobacterial genomes currently in CyanoCyc are supplemented with computational inferences including predicted metabolic pathways, operons, protein complexes, and orthologs; and with data imported from external databases, such as protein features and Gene Ontology (GO) terms imported from UniProt. Five of the genome databases have undergone manual curation with input from more than a dozen cyanobacteria experts to correct errors and integrate information from more than 1,765 published articles. CyanoCyc has bioinformatics tools that encompass genome, metabolic pathway and regulatory informatics; omics data analysis; and comparative analyses, including visualizations of multiple genomes aligned at orthologous genes, and comparisons of metabolic networks for multiple organisms. CyanoCyc is a high-quality, reliable knowledgebase that accelerates scientists' work by enabling users to quickly find accurate information using its powerful set of search tools, to understand gene function through expert mini-reviews with citations, to acquire information quickly using its interactive visualization tools, and to inform better decision-making for fundamental and applied research.

A unique subseafloor microbiosphere in the Mariana Trench driven by episodic sedimentation.

Hadal trenches are characterized by enhanced and infrequent high-rate episodic sedimentation events that likely introduce not only labile organic carbon and key nutrients but also new microbes that significantly alter the subseafloor microbiosphere. Currently, the role of high-rate episodic sedimentation in controlling the composition of the hadal subseafloor microbiosphere is unknown. Here, analyses of carbon isotope composition in a ~ 750 cm long sediment core from the Challenger Deep revealed noncontinuous deposition, with anomalous 14C ages likely caused by seismically driven mass transport and the funneling effect of trench geomorphology. Microbial community composition and diverse enzyme activities in the upper ~ 27 cm differed from those at lower depths, probably due to sudden sediment deposition and differences in redox condition and organic matter availability. At lower depths, microbial population numbers, and composition remained relatively constant, except at some discrete depths with altered enzyme activity and microbial phyla abundance, possibly due to additional sudden sedimentation events of different magnitude. Evidence is provided of a unique role for high-rate episodic sedimentation events in controlling the subsurface microbiosphere in Earth's deepest ocean floor and highlight the need to perform thorough analysis over a large depth range to characterize hadal benthic populations. Such depositional processes are likely crucial in shaping deep-water geochemical environments and thereby the deep subseafloor biosphere. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-023-00212-y.

Alternative dimethylsulfoniopropionate biosynthesis enzymes in diverse and abundant microorganisms.

Dimethylsulfoniopropionate (DMSP) is an abundant marine organosulfur compound with roles in stress protection, chemotaxis, nutrient and sulfur cycling and climate regulation. Here we report the discovery of a bifunctional DMSP biosynthesis enzyme, DsyGD, in the transamination pathway of the rhizobacterium Gynuella sunshinyii and some filamentous cyanobacteria not previously known to produce DMSP. DsyGD produces DMSP through its N-terminal DsyG methylthiohydroxybutyrate S-methyltransferase and C-terminal DsyD dimethylsulfoniohydroxybutyrate decarboxylase domains. Phylogenetically distinct DsyG-like proteins, termed DSYE, with methylthiohydroxybutyrate S-methyltransferase activity were found in diverse and environmentally abundant algae, comprising a mix of low, high and previously unknown DMSP producers. Algae containing DSYE, particularly bloom-forming Pelagophyceae species, were globally more abundant DMSP producers than those with previously described DMSP synthesis genes. This work greatly increases the number and diversity of predicted DMSP-producing organisms and highlights the importance of Pelagophyceae and other DSYE-containing algae in global DMSP production and sulfur cycling.

Terminal oxidase mutants of the cyanobacterium Synechocystis sp. PCC 6803 show increased electrogenic activity in biological photo-voltaic systems.

Biological photo-voltaic systems are a type of microbial fuel cell employing photosynthetic microbes at the anode, enabling the direct transduction of light energy to electrical power. Unlike the anaerobic bacteria found in conventional microbial fuel cells that use metals in the environment as terminal electron acceptors, oxygenic photosynthetic organisms are poorly adapted for electron transfer out of the cell. Mutant strains of the cyanobacterium Synechocystis sp. PCC 6803 were created in which all combinations of the three respiratory terminal oxidase complexes had been inactivated. These strains were screened for the ability to reduce the membrane-impermeable soluble electron acceptor ferricyanide, and the results were compared to the performance of the mutants in a biological photo-voltaic system. Deletion of the two thylakoid-localised terminal oxidases, the bd-quinol oxidase and cytochrome c oxidase resulted in a 16-fold increase in ferricyanide reduction rate in the dark compared to the wild-type. A further improvement to a 24-fold increase was seen upon deletion of the remaining "alternative respiratory terminal oxidase". These increases were reflected in the peak power generated in the biological photo-voltaic systems. Inactivation of all three terminal oxidase complexes resulted in a substantial redirection of reducing power; in the dark the equivalent of 10% of the respiratory electron flux was channelled to ferricyanide, compared to less than 0.2% in the wild-type. Only minor improvements in ferricyanide reduction rates over the wild-type were seen in illuminated conditions, where carbon dioxide is preferentially used as an electron sink. This study demonstrates the potential for optimising photosynthetic microbes for direct electrical current production.

Deep-sea Bacteroidetes from the Mariana Trench specialize in hemicellulose and pectin degradation typically associated with terrestrial systems.

BACKGROUND: Hadal trenches (>6000 m) are the deepest oceanic regions on Earth and depocenters for organic materials. However, how these enigmatic microbial ecosystems are fueled is largely unknown, particularly the proportional importance of complex polysaccharides introduced through deposition from the photic surface waters above. In surface waters, Bacteroidetes are keystone taxa for the cycling of various algal-derived polysaccharides and the flux of carbon through the photic zone. However, their role in the hadal microbial loop is almost unknown. RESULTS: Here, culture-dependent and culture-independent methods were used to study the potential of Bacteroidetes to catabolize diverse polysaccharides in Mariana Trench waters. Compared to surface waters, the bathypelagic (1000-4000 m) and hadal (6000-10,500 m) waters harbored distinct Bacteroidetes communities, with Mesoflavibacter being enriched at ≥ 4000 m and Bacteroides and Provotella being enriched at 10,400-10,500 m. Moreover, these deep-sea communities possessed distinct gene pools encoding for carbohydrate active enzymes (CAZymes), suggesting different polysaccharide sources are utilised in these two zones. Compared to surface counterparts, deep-sea Bacteroidetes showed significant enrichment of CAZyme genes frequently organized into polysaccharide utilization loci (PULs) targeting algal/plant cell wall polysaccharides (i.e., hemicellulose and pectin), that were previously considered an ecological trait associated with terrestrial Bacteroidetes only. Using a hadal Mesoflavibacter isolate (MTRN7), functional validation of this unique genetic potential was demonstrated. MTRN7 could utilize pectic arabinans, typically associated with land plants and phototrophic algae, as the carbon source under simulated deep-sea conditions. Interestingly, a PUL we demonstrate is likely horizontally acquired from coastal/land Bacteroidetes was activated during growth on arabinan and experimentally shown to encode enzymes that hydrolyze arabinan at depth. CONCLUSIONS: Our study implies that hadal Bacteroidetes exploit polysaccharides poorly utilized by surface populations via an expanded CAZyme gene pool. We propose that sinking cell wall debris produced in the photic zone can serve as an important carbon source for hadal heterotrophs and play a role in shaping their communities and metabolism. Video Abstract.

Development of a Biotechnology Platform for the Fast-Growing Cyanobacterium Synechococcus sp. PCC 11901.

Synechococcus sp. PCC 11901 reportedly demonstrates the highest, most sustained growth of any known cyanobacterium under optimized conditions. Due to its recent discovery, our knowledge of its biology, including the factors underlying sustained, fast growth, is limited. Furthermore, tools specific for genetic manipulation of PCC 11901 are not established. Here, we demonstrate that PCC 11901 shows faster growth than other model cyanobacteria, including the fast-growing species Synechococcuselongatus UTEX 2973, under optimal growth conditions for UTEX 2973. Comparative genomics between PCC 11901 and Synechocystis sp. PCC 6803 reveal conservation of most metabolic pathways but PCC 11901 has a simplified electron transport chain and reduced light harvesting complex. This may underlie its superior light use, reduced photoinhibition, and higher photosynthetic and respiratory rates. To aid biotechnology applications, we developed a vitamin B12 auxotrophic mutant but were unable to generate unmarked knockouts using two negative selectable markers, suggesting that recombinase- or CRISPR-based approaches may be required for repeated genetic manipulation. Overall, this study establishes PCC 11901 as one of the most promising species currently available for cyanobacterial biotechnology and provides a useful set of bioinformatics tools and strains for advancing this field, in addition to insights into the factors underlying its fast growth phenotype.

Expression of Alternative Nitrogenases in Rhodopseudomonas palustris Is Enhanced Using an Optimized Genetic Toolset for Rapid, Markerless Modifications.

The phototrophic bacterium Rhodopseudomonas palustris is emerging as a promising biotechnological chassis organism, due to its resilience to a range of harsh conditions, a wide metabolic repertoire, and the ability to quickly regenerate ATP using light. However, realization of this promise is impeded by a lack of efficient, rapid methods for genetic modification. Here, we present optimized tools for generating chromosomal insertions and deletions employing electroporation as a means of transformation. Generation of markerless strains can be completed in 12 days, approximately half the time for previous conjugation-based methods. This system was used for overexpression of alternative nitrogenase isozymes with the aim of improving biohydrogen productivity. Insertion of the pucBa promoter upstream of vnf and anf nitrogenase operons drove robust overexpression up to 4000-fold higher than wild-type. Transcript quantification was facilitated by an optimized high-quality RNA extraction protocol employing lysis using detergent and heat. Overexpression resulted in increased nitrogenase protein levels, extending to superior hydrogen productivity in bioreactor studies under nongrowing conditions, where promoter-modified strains better utilized the favorable energy state created by reduced competition from cell division. Robust heterologous expression driven by the pucBa promoter is thus attractive for energy-intensive biosyntheses suited to the capabilities of R. palustris. Development of this genetic modification toolset will accelerate the advancement of R. palustris as a biotechnological chassis organism, and insights into the effects of nitrogenase overexpression will guide future efforts in engineering strains for improved hydrogen production.

Current knowledge and recent advances in understanding metabolism of the model cyanobacterium Synechocystis sp. PCC 6803.

Cyanobacteria are key organisms in the global ecosystem, useful models for studying metabolic and physiological processes conserved in photosynthetic organisms, and potential renewable platforms for production of chemicals. Characterizing cyanobacterial metabolism and physiology is key to understanding their role in the environment and unlocking their potential for biotechnology applications. Many aspects of cyanobacterial biology differ from heterotrophic bacteria. For example, most cyanobacteria incorporate a series of internal thylakoid membranes where both oxygenic photosynthesis and respiration occur, while CO2 fixation takes place in specialized compartments termed carboxysomes. In this review, we provide a comprehensive summary of our knowledge on cyanobacterial physiology and the pathways in Synechocystis sp. PCC 6803 (Synechocystis) involved in biosynthesis of sugar-based metabolites, amino acids, nucleotides, lipids, cofactors, vitamins, isoprenoids, pigments and cell wall components, in addition to the proteins involved in metabolite transport. While some pathways are conserved between model cyanobacteria, such as Synechocystis, and model heterotrophic bacteria like Escherichia coli, many enzymes and/or pathways involved in the biosynthesis of key metabolites in cyanobacteria have not been completely characterized. These include pathways required for biosynthesis of chorismate and membrane lipids, nucleotides, several amino acids, vitamins and cofactors, and isoprenoids such as plastoquinone, carotenoids, and tocopherols. Moreover, our understanding of photorespiration, lipopolysaccharide assembly and transport, and degradation of lipids, sucrose, most vitamins and amino acids, and haem, is incomplete. We discuss tools that may aid our understanding of cyanobacterial metabolism, notably CyanoSource, a barcoded library of targeted Synechocystis mutants, which will significantly accelerate characterization of individual proteins.