A custom 3D printed paddlewheel improves growth in flat panel photobioreactor.

One of the main challenges with using flat panel photobioreactors for algal growth is uneven mixing and settling of cells in corners, especially when bubbling is the only method used for mixing. In order to improve mixing in our flat panel reactor, we designed a custom paddlewheel. Paddlewheels are frequently used in outdoor algae raceway ponds to improve mixing and we are taking advantage of the same principle for mixing in the reactor.

Investigating the Unique Ability of To Fix Carbon and Nitrogen Simultaneously Using MiMoSA.

The open ocean is an extremely competitive environment, partially due to the dearth of nutrients. Trichodesmium erythraeum, a marine diazotrophic cyanobacterium, is a keystone species in the ocean due to its ability to fix nitrogen and leak 30 to 50% into the surrounding environment, providing a valuable source of a necessary macronutrient to other species. While there are other diazotrophic cyanobacteria that play an important role in the marine nitrogen cycle, is unique in its ability to fix both carbon and nitrogen simultaneously during the day without the use of specialized cells called heterocysts to protect nitrogenase from oxygen.

Rhythm of the Night (and Day): Predictive Metabolic Modeling of Diurnal Growth in .

Economical production of photosynthetic organisms requires the use of natural day/night cycles. These induce strong circadian rhythms that lead to transient changes in the cells, requiring complex modeling to capture. In this study, we coupled times series transcriptomic data from the model green alga Chlamydomonas reinhardtii to a metabolic model of the same organism in order to develop the first transient metabolic model for diurnal growth of algae capable of predicting phenotype from genotype.

Isotopically nonstationary C metabolic flux analysis in resting and activated human platelets.

Platelet metabolism is linked to platelet hyper- and hypoactivity in numerous human diseases. Developing a detailed understanding of the link between metabolic shifts and platelet activation state is integral to improving human health. Here, we show the first application of isotopically nonstationary C metabolic flux analysis to quantitatively measure carbon fluxes in both resting and thrombin activated platelets.

Evaluation of quenching methods for metabolite recovery in photoautotrophic Synechococcus sp. PCC 7002.

The first step of many metabolomics studies is quenching, a technique vital for rapidly halting metabolism and ensuring that the metabolite profile remains unchanging during sample processing. The most widely used approach is to plunge the sample into prechilled cold methanol; however, this led to significant metabolite loss in Synecheococcus sp. PCC 7002.

Multiscale Multiobjective Systems Analysis (MiMoSA): an advanced metabolic modeling framework for complex systems.

In natural environments, cells live in complex communities and experience a high degree of heterogeneity internally and in the environment. Even in 'ideal' laboratory environments, cells can experience a high degree of heterogeneity in their environments. Unfortunately, most of the metabolic modeling approaches that are currently used assume ideal conditions and that each cell is identical, limiting their application to pure cultures in well-mixed vessels.

The challenge and potential of photosynthesis: unique considerations for metabolic flux measurements in photosynthetic microorganisms.

Photosynthetic microorganisms have the potential for sustainable production of chemical feedstocks and products but have had limited success due to a lack of tools and deeper understanding of metabolic pathway regulation. The application of instationary metabolic flux analysis (INST-MFA) to photosynthetic microorganisms has allowed researchers to quantify fluxes and identify bottlenecks and metabolic inefficiencies to improve strain performance or gain insight into cellular physiology. Additionally, flux measurements can also highlight deviations between measured and predicted fluxes, revealing weaknesses in metabolic models and highlighting areas where a lack of understanding still exists.

Metabolic flux analysis of heterotrophic growth in Chlamydomonas reinhardtii.

Despite the wealth of knowledge available for C. reinhardtii, the central metabolic fluxes of growth on acetate have not yet been determined. In this study, 13C-metabolic flux analysis (13C-MFA) was used to determine and quantify the metabolic pathways of primary metabolism in C.

The use of genome-scale metabolic network reconstruction to predict fluxes and equilibrium composition of N-fixing versus C-fixing cells in a diazotrophic cyanobacterium, Trichodesmium erythraeum.

Background: Computational, genome based predictions of organism phenotypes has enhanced the ability to investigate the biological phenomena that help organisms survive and respond to their environments. In this study, we have created the first genome-scale metabolic network reconstruction of the nitrogen fixing cyanobacterium T. erythraeum and used genome-scale modeling approaches to investigate carbon and nitrogen fluxes as well as growth and equilibrium population composition.

Omics in Chlamydomonas for Biofuel Production.

In response to demands for sustainable domestic fuel sources, research into biofuels has become increasingly important. Many challenges face biofuels in their effort to replace petroleum fuels, but rational strain engineering of algae and photosynthetic organisms offers a great deal of promise. For decades, mutations and stress responses in photosynthetic microbiota were seen to result in production of exciting high-energy fuel molecules, giving hope but minor capability for design.

Genome Engineering in Cyanobacteria: Where We Are and Where We Need To Go.

Genome engineering of cyanobacteria is a promising area of development in order to produce fuels, feedstocks, and value-added chemicals in a sustainable way. Unfortunately, the current state of genome engineering tools for cyanobacteria lags far behind those of model organisms such as Escherichia coli and Saccharomyces cerevisiae. In this review, we present the current state of synthetic biology tools for genome engineering efforts in the most widely used cyanobacteria strains and areas that need concerted research efforts to improve tool development.

Multiplexed tracking of combinatorial genomic mutations in engineered cell populations.

Multiplexed genome engineering approaches can be used to generate targeted genetic diversity in cell populations on laboratory timescales, but methods to track mutations and link them to phenotypes have been lacking. We present an approach for tracking combinatorial engineered libraries (TRACE) through the simultaneous mapping of millions of combinatorially engineered genomes at single-cell resolution. Distal genomic sites are assembled into individual DNA constructs that are compatible with next-generation sequencing strategies.

Nitrogen-Sparing Mechanisms in Chlamydomonas Affect the Transcriptome, the Proteome, and Photosynthetic Metabolism.

Nitrogen (N) is a key nutrient that limits global primary productivity; hence, N-use efficiency is of compelling interest in agriculture and aquaculture. We used Chlamydomonas reinhardtii as a reference organism for a multicomponent analysis of the N starvation response. In the presence of acetate, respiratory metabolism is prioritized over photosynthesis; consequently, the N-sparing response targets proteins, pigments, and RNAs involved in photosynthesis and chloroplast function over those involved in respiration.

Systems-level analysis of nitrogen starvation-induced modifications of carbon metabolism in a Chlamydomonas reinhardtii starchless mutant.

To understand the molecular basis underlying increased triacylglycerol (TAG) accumulation in starchless (sta) Chlamydomonas reinhardtii mutants, we undertook comparative time-course transcriptomics of strains CC-4348 (sta6 mutant), CC-4349, a cell wall-deficient (cw) strain purported to represent the parental STA6 strain, and three independent STA6 strains generated by complementation of sta6 (CC-4565/STA6-C2, CC-4566/STA6-C4, and CC-4567/STA6-C6) in the context of N deprivation. Despite N starvation-induced dramatic remodeling of the transcriptome, there were relatively few differences (5 × 10(2)) observed between sta6 and STA6, the most dramatic of which were increased abundance of transcripts encoding key regulated or rate-limiting steps in central carbon metabolism, specifically isocitrate lyase, malate synthase, transaldolase, fructose bisphosphatase and phosphoenolpyruvate carboxykinase (encoded by ICL1, MAS1, TAL1, FBP1, and PCK1 respectively), suggestive of increased carbon movement toward hexose-phosphate in sta6 by upregulation of the glyoxylate pathway and gluconeogenesis. Enzyme assays validated the increase in isocitrate lyase and malate synthase activities.

Recombineering to homogeneity: extension of multiplex recombineering to large-scale genome editing.

Recombineering has been an essential tool for genetic engineering in microbes for many years and has enabled faster, more efficient engineering than previous techniques. There have been numerous studies that focus on improving recombineering efficiency, which can be divided into three main areas: (i) optimizing the oligo used for recombineering to enhance replication fork annealing and limit proofreading; (ii) mechanisms to modify the replisome itself, enabling an increased rate of annealing; and (iii) multiplexing recombineering targets and automation. These efforts have increased the efficiency of recombineering several hundred-fold.

Genome-wide identification of genes conferring energy related resistance to a synthetic antimicrobial peptide (Bac8c).

A fundamental issue in the design and development of antimicrobials is the lack of understanding of complex modes of action and how this complexity affects potential pathways for resistance evolution. Bac8c (RIWVIWRR-NH(2)) is an 8 amino acid antimicrobial peptide (AMP) that has been shown to have enhanced activity against a range of pathogenic Gram-positive and Gram-negative bacteria, as well as yeast. We have previously demonstrated that Bac8c appears to interfere with multiple targets, at least in part through the disruption of cytoplasmic membrane related functions, and that resistance to this peptide does not easily develop using standard laboratory methods.

Engineering improved ethanol production in Escherichia coli with a genome-wide approach.

A key challenge to the commercial production of commodity chemical and fuels is the toxicity of such molecules to the microbial host. While a number of studies have attempted to engineer improved tolerance for such compounds, the majority of these studies have been performed in wild-type strains and culturing conditions that differ considerably from production conditions. Here we applied the multiscalar analysis of library enrichments (SCALEs) method and performed a growth selection in an ethanol production system to quantitatively map in parallel all genes in the genome onto ethanol tolerance and production.

Three acyltransferases and nitrogen-responsive regulator are implicated in nitrogen starvation-induced triacylglycerol accumulation in Chlamydomonas.

Algae have recently gained attention as a potential source for biodiesel; however, much is still unknown about the biological triggers that cause the production of triacylglycerols. We used RNA-Seq as a tool for discovering genes responsible for triacylglycerol (TAG) production in Chlamydomonas and for the regulatory components that activate the pathway. Three genes encoding acyltransferases, DGAT1, DGTT1, and PDAT1, are induced by nitrogen starvation and are likely to have a role in TAG accumulation based on their patterns of expression.

Tools for genome-wide strain design and construction.

Advances in DNA sequencing and synthesis technologies concurrent with the development of new recombinant DNA approaches have enabled the extension of directed evolution algorithms to the genome-scale. It is now possible to simultaneously map the effect of mutation(s) in each and every gene in the genome onto almost any screenable or selectable phenotype in less than a week. Such maps can be used to direct the design and construction of libraries containing billions of rationally designed combinatorial mutations.

Computation of metabolic fluxes and efficiencies for biological carbon dioxide fixation.

With rising energy prices and concern over the environmental impact of fossil fuel consumption, the push to develop biomass derived fuels has increased significantly. Although most global carbon fixation occurs via the Calvin Benson Bassham cycle, there are currently five other known pathways for carbon fixation; the goal of this study was to determine the thermodynamic efficiencies of all six carbon fixation pathways for the production of biomass using flux balance analysis. The three chemotrophic pathways, the reductive acetyl-CoA pathway, the 3-hydroxypropionate/4-hydroxybutyrate cycle and the dicarboxylate/4-hydroxybutyrate cycle, were found to be more efficient than photoautotrophic carbon fixation pathways.

Flux balance analysis of primary metabolism in Chlamydomonas reinhardtii.

Background: Photosynthetic organisms convert atmospheric carbon dioxide into numerous metabolites along the pathways to make new biomass. Aquatic photosynthetic organisms, which fix almost half of global inorganic carbon, have great potential: as a carbon dioxide fixation method, for the economical production of chemicals, or as a source for lipids and starch which can then be converted to biofuels. To harness this potential through metabolic engineering and to maximize production, a more thorough understanding of photosynthetic metabolism must first be achieved.