Microalgae are envisioned as a future source of renewable oil. The feasibility of producing high-value biomolecules from microalgae is strongly dependent on developing strains with increased productivity and environmental tolerance, understanding algal gene regulation, and optimizing growth conditions for higher production of target molecules. We present a high-throughput microfluidic microalgal photobioreactor array capable of applying 64 different light conditions to arrays of microscale algal photobioreactors and apply this device to investigate how light conditions influence algal growth and oil production. Using the green colony-forming microalga Botryococcus braunii, the light intensity and light-dark cycle conditions were identified that induced 1.8-fold higher oil accumulation over the typically used culture conditions. Additionally, the studies revealed that the condition under which maximum oil production occurs is significantly different from that of maximum growth. This screening test was accomplished using the developed photobioreactor array at 250 times higher throughput compared to conventional flask-scale photobioreactors.

Biofuels derived from microalgal lipids have demonstrated a promising potential as future renewable bioenergy. However, the production costs for microalgae-based biofuels are not economically competitive, and one strategy to overcome this limitation is to develop better-performing microalgal strains that have faster growth and higher lipid content through genetic screening and metabolic engineering. In this work, we present

Microalgae have emerged as a promising source for producing future renewable biofuels. Developing better microalgal strains with faster growth and higher oil production rates is one of the major routes towards economically viable microalgal biofuel production. In this work, we present a droplet microfluidics-based microalgae analysis platform capable of measuring growth and oil content of various microalgal strains with single-cell resolution in a high-throughput manner. The platform allows for encapsulating a single microalgal cell into a water-in-oil emulsion droplet and tracking the growth and division of the encapsulated cell over time, followed by on-chip oil quantification. The key feature of the developed platform is its capability to fluorescently stain microalgae within microdroplets for oil content quantification. The performance of the developed platform was characterized using the unicellular microalga Chlamydomonas reinhardtii and the colonial microalga Botryococcus braunii. The application of the platform in quantifying growth and oil accumulation was successfully confirmed using C. reinhardtii under different culture conditions, namely nitrogen-replete and nitrogen-limited conditions. These results demonstrate the capability of this platform as a rapid screening tool that can be applied to a wide range of microalgal strains for analyzing growth and oil accumulation characteristics relevant to biofuel strain selection and development. Biotechnol. Bioeng. 2016;113: 1691-1701. © 2016 Wiley Periodicals, Inc.

The green microalga Botryococcus braunii is considered a promising biofuel feedstock producer due to its prodigious accumulation of hydrocarbon oils that can be converted into fuels. B. braunii Race L produces the C40 tetraterpenoid hydrocarbon lycopadiene via an uncharacterized biosynthetic pathway. Structural similarities suggest this pathway follows a biosynthetic mechanism analogous to that of C30 squalene. Confirming this hypothesis, the current study identifies C20 geranylgeranyl diphosphate (GGPP) as a precursor for lycopaoctaene biosynthesis, the first committed intermediate in the production of lycopadiene. Two squalene synthase (SS)-like complementary DNAs are identified in race L with one encoding a true SS and the other encoding an enzyme with lycopaoctaene synthase (LOS) activity. Interestingly, LOS uses alternative C15 and C20 prenyl diphosphate substrates to produce combinatorial hybrid hydrocarbons, but almost exclusively uses GGPP in vivo. This discovery highlights how SS enzyme diversification results in the production of specialized tetraterpenoid oils in race L of B. braunii.

Marine macroalgae, seaweeds, are exceptionally prolific producers of halogenated natural products. Biosynthesis of halogenated molecules in seaweeds is inextricably linked to reactive oxygen species (ROS) signaling as hydrogen peroxide serves as a substrate for haloperoxidase enzymes that participate in the construction these halogenated molecules. Here, using red macroalga Asparagopsis taxiformis, a prolific producer of the ozone depleting molecule bromoform, we provide the discovery and biochemical characterization of a ROS-producing NAD­(P)H oxidase from seaweeds. This discovery was enabled by our sequencing of Asparagopsis genomes, in which we find the gene encoding the ROS-producing enzyme to be clustered with genes encoding bromoform-producing haloperoxidases. Biochemical reconstitution of haloperoxidase activities establishes that fatty acid biosynthesis can provide viable hydrocarbon substrates for bromoform production. The ROS production haloperoxidase enzymology that we describe here advances seaweed biology and biochemistry by providing the molecular basis for decades worth of physiological observations in ROS and halogenated natural product biosyntheses.

Sequential enzymatic reactions on substrates tethered to carrier proteins (CPs) generate thiotemplated building blocks that are then delivered to nonribosomal peptide synthetases (NRPSs) to generate peptidic natural products. The underlying diversity of these thiotemplated building blocks is the principal driver of the chemical diversity of NRPS-derived natural products. Structural insights into recognition of CPs by tailoring enzymes that generate these building blocks are sparse. Here we present the crystal structure of a flavin-dependent prolyl oxidase that furnishes thiotemplated pyrrole as the product, in complex with its cognate CP in the holo and product-bound states. The thiotemplated pyrrole is an intermediate that is well-represented in natural product biosynthetic pathways. Our results delineate the interactions between the CP and the oxidase while also providing insights into the stereospecificity of the enzymatic oxidation of the prolyl heterocycle to the aromatic pyrrole. Biochemical validation of the interaction between the CP and the oxidase demonstrates that NRPSs recognize and bind to their CPs using interactions quite different from those of fatty acid and polyketide biosynthetic enzymes. Our results posit that structural diversity in natural product biosynthesis can be, and is, derived from subtle modifications of primary metabolic enzymes.

Widespread testing for the presence of the novel coronavirus SARS-CoV-2 in individuals remains vital for controlling the COVID-19 pandemic prior to the advent of an effective treatment. Challenges in testing can be traced to an initial shortage of supplies, expertise and/or instrumentation necessary to detect the virus by quantitative reverse transcription polymerase chain reaction (RT-qPCR), the most robust, sensitive, and specific assay currently available. Here we show that academic biochemistry and molecular biology laboratories equipped with appropriate expertise and infrastructure can replicate commercially available SARS-CoV-2 RT-qPCR test kits and backfill pipeline shortages. The Georgia Tech COVID-19 Test Kit Support Group, composed of faculty, staff, and trainees across the biotechnology quad at Georgia Institute of Technology, synthesized multiplexed primers and probes and formulated a master mix composed of enzymes and proteins produced in-house. Our in-house kit compares favorably to a commercial product used for diagnostic testing. We also developed an environmental testing protocol to readily monitor surfaces across various campus laboratories for the presence of SARS-CoV-2. Our blueprint should be readily reproducible by research teams at other institutions, and our protocols may be modified and adapted to enable SARS-CoV-2 detection in more resource-limited settings.

Widespread testing for the presence novel coronavirus SARS-CoV-2 in patients remains vital for controlling the COVID-19 pandemic prior to the advent of an effective treatment. The early testing shortfall in some parts of the US can be traced to an initial shortage of supplies, expertise and/or instrumentation necessary to detect the virus by quantitative reverse transcription polymerase chain reaction (RT-qPCR). Here we show that academic biochemistry and molecular biology laboratories equipped with appropriate expertise and infrastructure can produce the RT-qPCR assay and backfill pipeline shortages. The Georgia Tech COVID-19 Test Kit Support Group synthesized multiplexed primers and probes and formulated a master mix composed of enzymes and proteins produced in-house. We compare the performance of our in-house kit to a commercial product used for diagnostic testing and describe implementation of environmental testing to monitor surfaces across various campus laboratories for the presence of SARS-CoV-2.