A synthetic, light-driven consortium of cyanobacteria and heterotrophic bacteria enables stable polyhydroxybutyrate production.

We previously reported that Synechococcus elongatus PCC 7942, engineered with the sucrose transporter CscB, can export up to 85% of its photosynthetically-fixed carbon as sucrose and shows considerable promise as an alternative carbohydrate source. One approach to effectively utilize this cyanobacterium is to generate synthetic, light-driven consortia in which sucrose-metabolizing heterotrophs catalyze the conversion of the low-value carbohydrate into higher-value compounds in co-culture. Here, we report an improved synthetic photoautotroph/chemoheterotroph consortial design in which sucrose secreted by S. elongatus CscB directly supports the bacterium Halomonas boliviensis, a natural producer of the bioplastic precursor, PHB. We show that alginate encapsulation of S. elongatus CscB enhances sucrose-export rates ~2-fold within 66h, to ~290mg sucrose L-1d-1 OD750-1 and enhances the co-culture stability. Consortial H. boliviensis accumulate up to 31% of their dry-weight as PHB, reaching productivities up to 28.3mg PHB L-1d-1. This light-driven, alginate-partitioned co-culture platform achieves PHB productivities that match or exceed those of traditionally engineered cyanobacterial monocultures. Importantly, S. elongatus CscB/H. boliviensis co-cultures were continuously productive for over 5 months and resisted invasive microbial species without the application of antibiotics or other chemical selection agents.

Ultrastructure and composition of the Nannochloropsis gaditana cell wall.

Marine algae of the genus Nannochloropsis are promising producers of biofuel precursors and nutraceuticals and are also harvested commercially for aquaculture feed. We have used quick-freeze, deep-etch electron microscopy, Fourier transform infrared spectroscopy, and carbohydrate analyses to characterize the architecture of the Nannochloropsis gaditana (strain CCMP 526) cell wall, whose recalcitrance presents a significant barrier to biocommodity extraction. The data indicate a bilayer structure consisting of a cellulosic inner wall (~75% of the mass balance) protected by an outer hydrophobic algaenan layer. Cellulase treatment of walls purified after cell lysis generates highly enriched algaenan preparations without using the harsh chemical treatments typically used in algaenan isolation and characterization. Nannochloropsis algaenan was determined to comprise long, straight-chain, saturated aliphatics with ether cross-links, which closely resembles the cutan of vascular plants. Chemical identification of >85% of the isolated cell wall mass is detailed, and genome analysis is used to identify candidate biosynthetic enzymes.

The path to triacylglyceride obesity in the sta6 strain of Chlamydomonas reinhardtii.

When the sta6 (starch-null) strain of the green microalga Chlamydomonas reinhardtii is nitrogen starved in acetate and then "boosted" after 2 days with additional acetate, the cells become "obese" after 8 days, with triacylglyceride (TAG)-filled lipid bodies filling their cytoplasm and chloroplasts. To assess the transcriptional correlates of this response, the sta6 strain and the starch-forming cw15 strain were subjected to RNA-Seq analysis during the 2 days prior and 2 days after the boost, and the data were compared with published reports using other strains and growth conditions. During the 2 h after the boost, ∼425 genes are upregulated ≥2-fold and ∼875 genes are downregulated ≥2-fold in each strain. Expression of a small subset of "sensitive" genes, encoding enzymes involved in the glyoxylate and Calvin-Benson cycles, gluconeogenesis, and the pentose phosphate pathway, is responsive to culture conditions and genetic background as well as to boosting. Four genes-encoding a diacylglycerol acyltransferase (DGTT2), a glycerol-3-P dehydrogenase (GPD3), and two candidate lipases (Cre03.g155250 and Cre17.g735600)-are selectively upregulated in the sta6 strain. Although the bulk rate of acetate depletion from the medium is not boost enhanced, three candidate acetate permease-encoding genes in the GPR1/FUN34/YaaH superfamily are boost upregulated, and 13 of the "sensitive" genes are strongly responsive to the cell's acetate status. A cohort of 64 autophagy-related genes is downregulated by the boost. Our results indicate that the boost serves both to avert an autophagy program and to prolong the operation of key pathways that shuttle carbon from acetate into storage lipid, the combined outcome being enhanced TAG accumulation, notably in the sta6 strain.

Colony organization in the green alga Botryococcus braunii (Race B) is specified by a complex extracellular matrix.

Botryococcus braunii is a colonial green alga whose cells associate via a complex extracellular matrix (ECM) and produce prodigious amounts of liquid hydrocarbons that can be readily converted into conventional combustion engine fuels. We used quick-freeze deep-etch electron microscopy and biochemical/histochemical analysis to elucidate many new features of B. braunii cell/colony organization and composition. Intracellular lipid bodies associate with the chloroplast and endoplasmic reticulum (ER) but show no evidence of being secreted. The ER displays striking fenestrations and forms a continuous subcortical system in direct contact with the cell membrane. The ECM has three distinct components. (i) Each cell is surrounded by a fibrous ß-1, 4- and/or ß-1, 3-glucan-containing cell wall. (ii) The intracolonial ECM space is filled with a cross-linked hydrocarbon network permeated with liquid hydrocarbons. (iii) Colonies are enclosed in a retaining wall festooned with a fibrillar sheath dominated by arabinose-galactose polysaccharides, which sequesters ECM liquid hydrocarbons. Each cell apex associates with the retaining wall and contributes to its synthesis. Retaining-wall domains also form "drapes" between cells, with some folding in on themselves and penetrating the hydrocarbon interior of a mother colony, partitioning it into daughter colonies. We propose that retaining-wall components are synthesized in the apical Golgi apparatus, delivered to apical ER fenestrations, and assembled on the surfaces of apical cell walls, where a proteinaceous granular layer apparently participates in fibril morphogenesis. We further propose that hydrocarbons are produced by the nonapical ER, directly delivered to the contiguous cell membrane, and pass across the nonapical cell wall into the hydrocarbon-based ECM.

Bio-crude transcriptomics: gene discovery and metabolic network reconstruction for the biosynthesis of the terpenome of the hydrocarbon oil-producing green alga, Botryococcus braunii race B (Showa).

BACKGROUND: Microalgae hold promise for yielding a biofuel feedstock that is sustainable, carbon-neutral, distributed, and only minimally disruptive for the production of food and feed by traditional agriculture. Amongst oleaginous eukaryotic algae, the B race of Botryococcus braunii is unique in that it produces large amounts of liquid hydrocarbons of terpenoid origin. These are comparable to fossil crude oil, and are sequestered outside the cells in a communal extracellular polymeric matrix material. Biosynthetic engineering of terpenoid bio-crude production requires identification of genes and reconstruction of metabolic pathways responsible for production of both hydrocarbons and other metabolites of the alga that compete for photosynthetic carbon and energy. RESULTS: A de novo assembly of 1,334,609 next-generation pyrosequencing reads form the Showa strain of the B race of B. braunii yielded a transcriptomic database of 46,422 contigs with an average length of 756 bp. Contigs were annotated with pathway, ontology, and protein domain identifiers. Manual curation allowed the reconstruction of pathways that produce terpenoid liquid hydrocarbons from primary metabolites, and pathways that divert photosynthetic carbon into tetraterpenoid carotenoids, diterpenoids, and the prenyl chains of meroterpenoid quinones and chlorophyll. Inventories of machine-assembled contigs are also presented for reconstructed pathways for the biosynthesis of competing storage compounds including triacylglycerol and starch. Regeneration of S-adenosylmethionine, and the extracellular localization of the hydrocarbon oils by active transport and possibly autophagy are also investigated. CONCLUSIONS: The construction of an annotated transcriptomic database, publicly available in a web-based data depository and annotation tool, provides a foundation for metabolic pathway and network reconstruction, and facilitates further omics studies in the absence of a genome sequence for the Showa strain of B. braunii, race B. Further, the transcriptome database empowers future biosynthetic engineering approaches for strain improvement and the transfer of desirable traits to heterologous hosts.

Raman spectroscopy analysis of botryococcene hydrocarbons from the green microalga Botryococcus braunii.

Botryococcus braunii, B race is a unique green microalga that produces large amounts of liquid hydrocarbons known as botryococcenes that can be used as a fuel for internal combustion engines. The simplest botryococcene (C(30)) is metabolized by methylation to give intermediates of C(31), C(32), C(33), and C(34), with C(34) being the predominant botryococcene in some strains. In the present work we have used Raman spectroscopy to characterize the structure of botryococcenes in an attempt to identify and localize botryococcenes within B. braunii cells. The spectral region from 1600-1700 cm(-1) showed ν(C=C) stretching bands specific for botryococcenes. Distinct botryococcene Raman bands at 1640 and 1647 cm(-1) were assigned to the stretching of the C=C bond in the botryococcene branch and the exomethylene C=C bonds produced by the methylations, respectively. A Raman band at 1670 cm(-1) was assigned to the backbone C=C bond stretching. Density function theory calculations were used to determine the Raman spectra of all botryococcenes to compare computed theoretical values with those observed. The analysis showed that the ν(C=C) stretching bands at 1647 and 1670 cm(-1) are actually composed of several closely spaced bands arising from the six individual C=C bonds in the molecule. We also used confocal Raman microspectroscopy to map the presence and location of methylated botryococcenes within a colony of B. braunii cells based on the methylation-specific 1647 cm(-1) botryococcene Raman shift.

Draft Nuclear Genome Sequence of the Liquid Hydrocarbon-Accumulating Green Microalga Botryococcus braunii Race B (Showa).

Botryococcus braunii has long been known as a prodigious producer of liquid hydrocarbon oils that can be converted into combustion engine fuels. This draft genome for the B race of B. braunii will allow researchers to unravel important hydrocarbon biosynthetic pathways and identify possible regulatory networks controlling this unusual metabolism.

A microfluidic photobioreactor array demonstrating high-throughput screening for microalgal oil production.

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.