Proximate biomass characterization of the high productivity marine microalga Picochlorum celeri TG2.

Microalgae are compelling renewable resources with applications including biofuels, bioplastics, nutrient supplements, and cosmetic products. Picochlorum celeri is an alga with high industrial interest due to exemplary outdoor areal biomass productivities in seawater. Detailed proximate analysis is needed in multiple environmental conditions to understand the dynamic biomass compositions of P. celeri, and how these compositions might be leveraged in biotechnological applications. In this study, biomass characterization of P. celeri was performed under nutrient-replete, nitrogen-restricted, and hyper-saline conditions. Nutrient-replete cultivation of P. celeri resulted in protein-rich biomass (∼50% ash-free dry weight) with smaller carbohydrate (∼12% ash-free dry weight) and lipid (∼11% ash-free dry weight) partitions. Gradual nitrogen depletion elicited a shift from proteins to carbohydrates (∼50% ash-free dry weight, day 3) as cells transitioned into the production of storage metabolites. Importantly, dilutions in nitrogen-restricted 40 parts per million (1.43 mM nitrogen) media generated high-carbohydrate (∼50% ash-free dry weight) biomass without substantially compromising biomass productivity (36 g ash-free dry weight m-2 day-1) despite decreased chlorophyll (∼2% ash-free dry weight) content. This strategy for increasing carbohydrate content allowed for the targeted production of polysaccharides, which could potentially be utilized to produce fuels, oligosaccharides, and bioplastics. Cultivation at 2X sea salts resulted in a shift towards carbohydrates from protein, with significantly increased levels of the amino acid proline, which putatively acts as an osmolyte. A detailed understanding of the biomass composition of P. celeri in nutrient-replete, nitrogen-restricted, and hyper saline conditions informs how this strain can be useful in the production of biotechnological products.

Simultaneous CAS9 editing of cpSRP43, LHCA6, and LHCA7 in Picochlorum celeri lowers chlorophyll levels and improves biomass productivity.

High cellular pigment levels in dense microalgal cultures contribute to excess light absorption. To improve photosynthetic yields in the marine microalga Picochlorum celeri, CAS9 gene editing was used to target the molecular chaperone cpSRP43. Depigmented strains (>50% lower chlorophyll) were generated, with proteomics showing attenuated levels of most light harvesting complex (LHC) proteins. Gene editing generated two types of cpSRP43 transformants with distinct lower pigment phenotypes: (i) a transformant (Δsrp43) with both cpSRP43 diploid alleles modified to encode non-functional polypeptides and (ii) a transformant (STR30309) with a 3 nt in-frame insertion in one allele at the CAS9 cut site (non-functional second allele), leading to expression of a modified cpSRP43. STR30309 has more chlorophyll than Δsrp43 but substantially less than wild type. To further decrease light absorption by photosystem I in STR30309, CAS9 editing was used to stack in disruptions of both LHCA6 and LHCA7 to generate STR30843, which has higher (5-24%) productivities relative to wild type in solar-simulating bioreactors. Maximal productivities required frequent partial harvests throughout the day. For STR30843, exemplary diel bioreactor yields of ~50 g m-2 day-1 were attained. Our results demonstrate diel productivity gains in P. celeri by lowering pigment levels.

Chlamydomonas mutants lacking chloroplast TRIOSE PHOSPHATE TRANSPORTER3 are metabolically compromised and light sensitive.

Modulation of photoassimilate export from the chloroplast is essential for controlling the distribution of fixed carbon in the cell and maintaining optimum photosynthetic rates. In this study, we identified chloroplast TRIOSE PHOSPHATE/PHOSPHATE TRANSLOCATOR 2 (CreTPT2) and CreTPT3 in the green alga Chlamydomonas (Chlamydomonas reinhardtii), which exhibit similar substrate specificities but whose encoding genes are differentially expressed over the diurnal cycle. We focused mostly on CreTPT3 because of its high level of expression and the severe phenotype exhibited by tpt3 relative to tpt2 mutants. Null mutants for CreTPT3 had a pleiotropic phenotype that affected growth, photosynthetic activities, metabolite profiles, carbon partitioning, and organelle-specific accumulation of H2O2. These analyses demonstrated that CreTPT3 is a dominant conduit on the chloroplast envelope for the transport of photoassimilates. In addition, CreTPT3 can serve as a safety valve that moves excess reductant out of the chloroplast and appears to be essential for preventing cells from experiencing oxidative stress and accumulating reactive oxygen species, even under low/moderate light intensities. Finally, our studies indicate subfunctionalization of the TRIOSE PHOSPHATE/PHOSPHATE TRANSLOCATOR (CreTPT) transporters and suggest that there are differences in managing the export of photoassimilates from the chloroplasts of Chlamydomonas and vascular plants.

Cas9 deletion of lutein biosynthesis in the marine alga Picochlorum celeri reduces photosynthetic pigments while sustaining high biomass productivity.

Domestication of algae for food and renewable biofuels remains limited by the low photosynthetic efficiencies of processes that have evolved to be competitive for optimal light capture, incentivizing the development of large antennas in light-limiting conditions, thus decreasing efficient light utilization in cultivated ponds or photobioreactors. Reducing the pigment content to improve biomass productivity has been a strategy discussed for several decades and the ability to reduce pigment significantly is now fully at hand thanks to the widespread use of genome editing tools. Picochlorum celeri is one of the fastest growing marine algae identified and holds particular promise for outdoor cultivation, especially in saline water and warm climates. We show that while chlorophyll b is essential to sustain high biomass productivities under dense cultivation, removing Picochlorum celeri's main carotenoid, lutein, leads to a decreased total chlorophyll content, higher a/b ratio, reduced functional LHCII cross section and higher maximum quantum efficiencies at lower light intensities, resulting in an incremental increase in biomass productivity and increased PAR-to-biomass conversion efficiency. These findings further strengthen the existing strategies to improve photosynthetic efficiency and biomass production in algae.

Picochlorum celeri as a model system for robust outdoor algal growth in seawater.

With fast growth rates, broad halotolerance and the ability to thrive at high temperatures, algae in the genus Picochlorum are emerging as promising biomass producers. Recently, we isolated a remarkably productive strain, Picochlorum celeri, that attains > 40 g m-2 day-1 productivities using simulated outdoor light. To test outdoor productivities, Picochlorum celeri was cultivated in 820 L raceway ponds at the Arizona Center for Algae Technology and Innovation. Picochlorum celeri demonstrated the highest outdoor biomass productivities reported to date at this testbed averaging ~ 31 g m-2 day-1 over four months with a monthly (August) high of ~ 36 g m-2 day-1. Several single day productivities were > 40 g m-2 day-1. Importantly for sustainability, Picochlorum celeri achieved these productivities in saline water ranging from seawater to 50 parts per thousand sea salts, without any biocides or pond crashes, for over 143 days. Lastly, we report robust genetic engineering tools for future strain improvements.

Biosynthesis of Fatty Alcohols in Engineered Microbial Cell Factories: Advances and Limitations.

Concerns about climate change and environmental destruction have led to interest in technologies that can replace fossil fuels and petrochemicals with compounds derived from sustainable sources that have lower environmental impact. Fatty alcohols produced by chemical synthesis from ethylene or by chemical conversion of plant oils have a large range of industrial applications. These chemicals can be synthesized through biological routes but their free forms are produced in trace amounts naturally. This review focuses on how genetic engineering of endogenous fatty acid metabolism and heterologous expression of fatty alcohol producing enzymes have come together resulting in the current state of the field for production of fatty alcohols by microbial cell factories. We provide an overview of endogenous fatty acid synthesis, enzymatic methods of conversion to fatty alcohols and review the research to date on microbial fatty alcohol production. The primary focus is on work performed in the model microorganisms, Escherichia coli and Saccharomyces cerevisiae but advances made with cyanobacteria and oleaginous yeasts are also considered. The limitations to production of fatty alcohols by microbial cell factories are detailed along with consideration to potential research directions that may aid in achieving viable commercial scale production of fatty alcohols from renewable feedstock.

Rewiring of Cyanobacterial Metabolism for Hydrogen Production: Synthetic Biology Approaches and Challenges.

With the demand for renewable energy growing, hydrogen (H2) is becoming an attractive energy carrier. Developing H2 production technologies with near-net zero carbon emissions is a major challenge for the "H2 economy." Certain cyanobacteria inherently possess enzymes, nitrogenases, and bidirectional hydrogenases that are capable of H2 evolution using sunlight, making them ideal cell factories for photocatalytic conversion of water to H2. With the advances in synthetic biology, cyanobacteria are currently being developed as a "plug and play" chassis to produce H2. This chapter describes the metabolic pathways involved and the theoretical limits to cyanobacterial H2 production and summarizes the metabolic engineering technologies pursued.

Consequences of ccmR deletion on respiration, fermentation and H2 metabolism in cyanobacterium Synechococcus sp. PCC 7002.

CcmR, a LysR-type transcriptional regulator, represses the genes encoding components of the high-affinity carbon concentration mechanism in cyanobacteria. Unexpectedly, deletion of the ccmR gene was found to alter the expression of the terminal oxidase and fermentative genes, especially the hydrogenase operon in the cyanobacterium Synechococcus sp. PCC 7002. Consistent with the transcriptomic data, the deletion strain exhibits flux increases (30-50%) in both aerobic O2 respiration and anaerobic H2 evolution. To understand how CcmR influences anaerobic metabolism, the kinetics of autofermentation were investigated following photoautotrophic growth. The autofermentative H2 yield increased by 50% in the CcmR deletion strain compared to the wild-type strain, and increased to 160% (within 20 h) upon continuous removal of H2 from the medium ("milking") to suppress H2 uptake. Consistent with this greater reductant flux to H2 , the mutant excreted less lactate during autofermentation (NAD(P)H consuming pathway). To enhance the rate of NADH production during anaerobic metabolism, the ccmR mutant was engineered to introduce GAPDH overexpression (more NADH production) and LDH deletion (less NADH consumption). The triple mutant (ccmR deletion + GAPDH overexpression + LDH deletion) showed 6-8-fold greater H2 yield than the WT strain, achieving conversion rates of 17 nmol 10(8) cells(-1) h(-1) and yield of 0.87 H2 per glucose equivalent (8.9% theoretical maximum). Simultaneous monitoring of the intracellular NAD(P)H concentration and H2 production rate by these mutants reveals an inverse correspondence between these variables indicating hydrogenase-dependent H2 production as a major sink for consuming NAD(P)H in preference to excretion of reduced carbon as lactate during fermentation. Biotechnol. Bioeng. 2016;113: 1448-1459. © 2015 Wiley Periodicals, Inc.

Metabolic and photosynthetic consequences of blocking starch biosynthesis in the green alga Chlamydomonas reinhardtii sta6 mutant.

Upon nutrient deprivation, microalgae partition photosynthate into starch and lipids at the expense of protein synthesis and growth. We investigated the role of starch biosynthesis with respect to photosynthetic growth and carbon partitioning in the Chlamydomonas reinhardtii starchless mutant, sta6, which lacks ADP-glucose pyrophosphorylase. This mutant is unable to convert glucose-1-phosphate to ADP-glucose, the precursor of starch biosynthesis. During nutrient-replete culturing, sta6 does not re-direct metabolism to make more proteins or lipids, and accumulates 20% less biomass. The underlying molecular basis for the decreased biomass phenotype was identified using LC-MS metabolomics studies and flux methods. Above a threshold light intensity, photosynthetic electron transport rates (water → CO2) decrease in sta6 due to attenuated rates of NADPH re-oxidation, without affecting photosystems I or II (no change in isolated photosynthetic electron transport). We observed large accumulations of carbon metabolites that are precursors for the biosynthesis of lipids, amino acids and sugars/starch, indicating system-wide consequences of slower NADPH re-oxidation. Attenuated carbon fixation resulted in imbalances in both redox and adenylate energy. The pool sizes of both pyridine and adenylate nucleotides in sta6 increased substantially to compensate for the slower rate of turnover. Mitochondrial respiration partially relieved the reductant stress; however, prolonged high-light exposure caused accelerated photoinhibition. Thus, starch biosynthesis in Chlamydomonas plays a critical role as a principal carbon sink influencing cellular energy balance however, disrupting starch biosynthesis does not redirect resources to other bioproducts (lipids or proteins) during nutrient-replete culturing, resulting in cells that are susceptible to photochemical damage caused by redox stress.

Genome of the red alga Porphyridium purpureum.

The limited knowledge we have about red algal genomes comes from the highly specialized extremophiles, Cyanidiophyceae. Here, we describe the first genome sequence from a mesophilic, unicellular red alga, Porphyridium purpureum. The 8,355 predicted genes in P. purpureum, hundreds of which are likely to be implicated in a history of horizontal gene transfer, reside in a genome of 19.7 Mbp with 235 spliceosomal introns. Analysis of light-harvesting complex proteins reveals a nuclear-encoded phycobiliprotein in the alga. We uncover a complex set of carbohydrate-active enzymes, identify the genes required for the methylerythritol phosphate pathway of isoprenoid biosynthesis, and find evidence of sexual reproduction. Analysis of the compact, function-rich genome of P. purpureum suggests that ancestral lineages of red algae acted as mediators of horizontal gene transfer between prokaryotes and photosynthetic eukaryotes, thereby significantly enriching genomes across the tree of photosynthetic life.

Metabolic pathways for photobiological hydrogen production by nitrogenase- and hydrogenase-containing unicellular cyanobacteria Cyanothece.

Current biotechnological interest in nitrogen-fixing cyanobacteria stems from their robust respiration and capacity to produce hydrogen. Here we quantify both dark- and light-induced H(2) effluxes by Cyanothece sp. Miami BG 043511 and establish their respective origins. Dark, anoxic H(2) production occurs via hydrogenase utilizing reductant from glycolytic catabolism of carbohydrates (autofermentation). Photo-H(2) is shown to occur via nitrogenase and requires illumination of PSI, whereas production of O(2) by co-illumination of PSII is inhibitory to nitrogenase above a threshold pO(2). Carbohydrate also serves as the major source of reductant for the PSI pathway mediated via nonphotochemical reduction of the plastoquinone pool by NADH dehydrogenases type-1 and type-2 (NDH-1 and NDH-2). Redirection of this reductant flux exclusively through the proton-coupled NDH-1 by inhibition of NDH-2 with flavone increases the photo-H(2) production rate by 2-fold (at the expense of the dark-H(2) rate), due to production of additional ATP (via the proton gradient). Comparison of photobiological hydrogen rates, yields, and energy conversion efficiencies reveals opportunities for improvement.

Gene expression: protein interaction systems network modeling identifies transformation-associated molecules and pathways in ovarian cancer.

Multiple, dissimilar genetic defects in cancers of the same origin contribute to heterogeneity in tumor phenotypes and therapeutic responses of patients, yet the associated molecular mechanisms remain elusive. Here, we show at the systems level that serous ovarian carcinoma is marked by the activation of interconnected modules associated with a specific gene set that was derived from three independent tumor-specific gene expression data sets. Network prediction algorithms combined with preestablished protein interaction networks and known functionalities affirmed the importance of genes associated with ovarian cancer as predictive biomarkers, besides "discovering" novel ones purely on the basis of interconnectivity, whose precise involvement remains to be investigated. Copy number alterations and aberrant epigenetic regulation were identified and validated as significant influences on gene expression. More importantly, three functional modules centering on c-Myc activation, altered retinoblastoma signaling, and p53/cell cycle/DNA damage repair pathways have been identified for their involvement in transformation-associated events. Further studies will assign significance to and aid the design of a panel of specific markers predictive of individual- and tumor-specific pathways. In the parlance of this emerging field, such networks of gene-hub interactions may define personalized therapeutic decisions.