Time-resolved transcriptome analysis and lipid pathway reconstruction of the oleaginous green microalga Monoraphidium neglectum reveal a model for triacylglycerol and lipid hyperaccumulation.

BACKGROUND: Oleaginous microalgae are promising production hosts for the sustainable generation of lipid-based bioproducts and as bioenergy carriers such as biodiesel. Transcriptomics of the lipid accumulation phase, triggered efficiently by nitrogen starvation, is a valuable approach for the identification of gene targets for metabolic engineering. RESULTS: An explorative analysis of the detailed transcriptional response to different stages of nitrogen availability was performed in the oleaginous green alga Monoraphidium neglectum. Transcript data were correlated with metabolic data for cellular contents of starch and of different lipid fractions. A pronounced transcriptional down-regulation of photosynthesis became apparent in response to nitrogen starvation, whereas glucose catabolism was found to be up-regulated. An in-depth reconstruction and analysis of the pathways for glycerolipid, central carbon, and starch metabolism revealed that distinct transcriptional changes were generally found only for specific steps within a metabolic pathway. In addition to pathway analyses, the transcript data were also used to refine the current genome annotation. The transcriptome data were integrated into a database and complemented with data for other microalgae which were also subjected to nitrogen starvation. It is available at https://tdbmn.cebitec.uni-bielefeld.de. CONCLUSIONS: Based on the transcriptional responses to different stages of nitrogen availability, a model for triacylglycerol and lipid hyperaccumulation is proposed, which involves transcriptional induction of thioesterases, differential regulation of lipases, and a re-routing of the central carbon metabolism. Over-expression of distinct thioesterases was identified to be a potential strategy to increase the oleaginous phenotype of M. neglectum, and furthermore specific lipases were identified as potential targets for future metabolic engineering approaches.

Nuclear transformation and functional gene expression in the oleaginous microalga Monoraphidium neglectum.

Photosynthetic microalgae hold great promise as non-food feedstocks for the sustainable production of a range of bio-products and genetic engineering is an increasingly important strategy to improve the natural cellular features. For the vast majority of microalgal strains, however, genetic engineering is not yet possible and the establishment of efficient genetic tools for a broad range of microalgal species is an important task to reach biotechnological success. Stable DNA transformation is one of the crucial steps for most genetic engineering approaches. In this context, we report the first successful and stable nuclear genetic transformation of the biotechnologically promising oleaginous microalga M. neglectum. Transformation was achieved by electroporation and the efficiency could progressively be improved by implementation of an additional cell pretreatment step, aiming at weakening the rigid natural cell wall. Furthermore, a first reporter transgene was established for M. neglectum. As a result of this work, genetic engineering strategies now become accessible which are required to successfully establish M. neglectum as a novel host for biotechnological applications.

Label-free in vivo analysis of intracellular lipid droplets in the oleaginous microalga Monoraphidium neglectum by coherent Raman scattering microscopy.

Oleaginous photosynthetic microalgae hold great promise as non-food feedstocks for the sustainable production of bio-commodities. The algal lipid quality can be analysed by Raman micro-spectroscopy, and the lipid content can be imaged in vivo in a label-free and non-destructive manner by coherent anti-Stokes Raman scattering (CARS) microscopy. In this study, both techniques were applied to the oleaginous microalga Monoraphidium neglectum, a biotechnologically promising microalga resistant to commonly applied lipid staining techniques. The lipid-specific CARS signal was successfully separated from the interfering two-photon excited fluorescence of chlorophyll and for the first time, lipid droplet formation during nitrogen starvation could directly be analysed. We found that the neutral lipid content deduced from CARS image analysis strongly correlated with the neutral lipid content measured gravimetrically and furthermore, that the relative degree of unsaturation of fatty acids stored in lipid droplets remained similar. Interestingly, the lipid profile during cellular adaption to nitrogen starvation showed a two-phase characteristic with initially fatty acid recycling and subsequent de novo lipid synthesis. This works demonstrates the potential of quantitative CARS microscopy as a label-free lipid analysis technique for any microalgal species, which is highly relevant for future biotechnological applications and to elucidate the process of microalgal lipid accumulation.

Efficiency and biotechnological aspects of biogas production from microalgal substrates.

Photosynthetic organisms like plants and algae can harvest, convert, and store solar energy and thus represent readily available sources for renewable biofuels production on a domestic or industrial scale. Anaerobic digestion (AD) of the organic biomass yields biogas, containing methane and carbon dioxide as major constituents. Combustion of the biogas or purification of the energy-rich methane fraction can be applied to provide electricity or fuel. AD procedures have been applied for several decades with organic waste, animal products, or higher plants and more recently, utilization of photosynthetic algae as substrates have gained considerable research interest. To provide an overview of recent research efforts made to characterize the AD process of microalgal biomass, we present extended summaries of experimentally determined biochemical methane potentials (BMP), biomass pretreatment options and digestion strategies in this article. We conclude that cultivation options, biomass composition and time of harvesting, application of biomass pretreatment strategies, and parameters of the digestion process are all important factors, which can significantly affect the AD process efficiency. The transition from batch to continuous microalgal biomass digestion trials, accompanied by state-of-the-art analytical techniques, is now in demand to refine the assessments of the overall process feasibility.

Efficient phototrophic production of a high-value sesquiterpenoid from the eukaryotic microalga Chlamydomonas reinhardtii.

The heterologous expression of terpene synthases in microbial hosts has opened numerous possibilities for bioproduction of desirable metabolites. Photosynthetic microbial hosts present a sustainable alternative to traditional fermentative systems, using freely available (sun)light and carbon dioxide as inputs for bio-production. Here, we report the expression of a patchoulol synthase from Pogostemon cablin Benth in the model green microalga Chlamydomonas reinhardtii. The sesquiterpenoid patchoulol was produced from the alga and was used as a marker of sesquiterpenoid production capacity. A novel strategy for gene loading was employed and patchoulol was produced up to 922±242µgg-1 CDW in six days. We additionally investigated the effect of carbon source on sesquiterpenoid productivity from C. reinhardtii in scale-up batch cultivations. It was determined that up to 1.03mgL-1 sesquiterpenoid products could be produced in completely photoautotrophic conditions and that the alga exhibited altered sesquiterpenoid production metabolism related to carbon source.

Genetic tools and techniques for Chlamydomonas reinhardtii.

The development of tools has always been a major driving force for the advancement of science. Optical microscopes were the first instruments that allowed discovery and descriptive studies of the subcellular features of microorganisms. Although optical and electron microscopes remained at the forefront of microbiological research tools since their inventions, the advent of molecular genetics brought about questions which had to be addressed with new "genetic tools". The unicellular green microalgal genus Chlamydomonas, especially the most prominent species C. reinhardtii, has become a frequently used model organism for many diverse fields of research and molecular genetic analyses of C. reinhardtii, as well as the available genetic tools and techniques, have become increasingly sophisticated throughout the last decades. The aim of this review is to provide an overview of the molecular key features of C. reinhardtii and summarize the progress related to the development of tools and techniques for genetic engineering of this organism, from pioneering DNA transformation experiments to state-of-the-art techniques for targeted nuclear genome editing and high-throughput screening approaches.

A novel one-stage cultivation/fermentation strategy for improved biogas production with microalgal biomass.

The use of alga biomass for biogas generation has been studied for over fifty years but until today, several distinct features, like inefficient degradation and low C/N ratios, limit the applicability of algal biomass for biogas production in larger scale. In this work we investigated a novel, one-stage combined cultivation/fermentation strategy including inherently progressing nitrogen starvation conditions to generate improved microalgal biomass substrates. For this strategy, comparable low amounts of nitrogen fertilizers were applied during cultivation and no additional enzymatic, chemical or physical pretreatments had to be performed. The results of this study demonstrate that progressing nitrogen limitation leads to continuously increasing C/N ratios of the biomass up to levels of 24-26 for all three tested alga strains (Chlamydomonas reinhardtii, Parachlorella kessleri and Scenedesmus obliquus). Importantly, the degradation efficiency of the algal cells increased with progressing starvation, leading to strain-specific cell disintegration efficiencies of 35%-100% during the fermentation process. Nitrogen limitation treatment resulted in a 65% increase of biogas yields for C. reinhardtii biomass (max. 698±23mL biogas g(-1) VS) when compared to replete conditions. For P. kessleri and S. obliquus, yields increased by 94% and 106% (max. 706±39mL and 586±36mL biogas g(-1) VS, respectively). From these results we conclude that this novel one-stage cultivation strategy with inherent nitrogen limitation can be used as a pretreatment for microalgal biomass generation, in order to produce accessible substrates with optimized C/N ratios for the subsequent anaerobic fermentation process, thus increasing methane production and avoiding the risk of ammonia inhibition effects within the fermenter.

Investigating the dynamics of recombinant protein secretion from a microalgal host.

Production of recombinant proteins with microalgae represents an alternative platform over plant- or bacterial-based expression systems for certain target proteins. Secretion of recombinant proteins allows accumulation of the target product physically separate from the valuable algal biomass. To date, there has been little investigation into the dynamics of recombinant protein secretion from microalgal hosts-the culture parameters that encourage secreted product accumulation and stability, while encouraging biomass production. In this work, the efficiency of recombinant protein production was optimized by adjusting cultivation parameters for a strain of Chlamydomonas reinhardtii previously engineered to secrete a functional recombinant Lolium perenne ice binding protein (LpIBP), which has applications as a frozen food texturing and cryopreservation additive, into its culture medium. Three media and several cultivation styles were investigated for effects on secreted LpIBP titres and culture growth. A combination of acetate and carbon dioxide feeding with illumination resulted in the highest overall biomass and recombinant protein titres up to 10mgL(-1) in the culture medium. Pure photoautotrophic production was possible using two media types, with recombinant protein accumulation in all cultivations correlating to culture cell density. Two different cultivation systems were used for scale-up to 10L cultivations, one of which produced yields of secreted recombinant protein up to 12mgL(-1) within six cultivation days. Functional ice recrystallization inhibition (IRI) of the LpIBP from total concentrated extracellular protein extracts was demonstrated in a sucrose solution used as a simplified ice cream model. IRI lasted up to 7 days, demonstrating the potential of secreted products from microalgae for use as food additives.

Targeted expression of nuclear transgenes in Chlamydomonas reinhardtii with a versatile, modular vector toolkit.

We present a versatile vector toolkit for nuclear transgene expression in the model green microalga Chlamydomonas reinhardtii. The vector was designed in a modular fashion which allows quick replacement of regulatory elements and genes of interest. The current toolkit comprises two antibiotic resistance markers (paromomycin and hygromycin B), five codon-optimized light emission reporters, including the Gaussia princeps luciferase, as well as bright cyan, green, yellow, and red fluorescent protein variants. The system has demonstrated robust functional flexibility with signal options to target the protein of interest to the cytoplasm, the nucleus, cellular microbodies, the chloroplast, mitochondria, or via the endoplasmic reticulum-Golgi apparatus secretory pathway into the culture medium. Successful fluorescent reporter protein fusion to C. reinhardtii Rubisco small subunit 1 was accomplished with this system. Localization of the fluorescently tagged protein was observed in the chloroplast pyrenoid via live cell fluorescence microscopy, the first report of heterologous protein localization to this cellular structure. The functionalities of the vector toolkit, the individual modular elements, as well as several combinations thereof are demonstrated in this manuscript. Due to its strategic design, this vector system can quickly be adapted to individual tasks and should therefore be of great use to address specific scientific questions requiring nuclear recombinant protein expression in C. reinhardtii.

Light-Harvesting Complex Protein LHCBM9 Is Critical for Photosystem II Activity and Hydrogen Production in Chlamydomonas reinhardtii.

Photosynthetic organisms developed multiple strategies for balancing light-harvesting versus intracellular energy utilization to survive ever-changing environmental conditions. The light-harvesting complex (LHC) protein family is of paramount importance for this function and can form light-harvesting pigment protein complexes. In this work, we describe detailed analyses of the photosystem II (PSII) LHC protein LHCBM9 of the microalga Chlamydomonas reinhardtii in terms of expression kinetics, localization, and function. In contrast to most LHC members described before, LHCBM9 expression was determined to be very low during standard cell cultivation but strongly increased as a response to specific stress conditions, e.g., when nutrient availability was limited. LHCBM9 was localized as part of PSII supercomplexes but was not found in association with photosystem I complexes. Knockdown cell lines with 50 to 70% reduced amounts of LHCBM9 showed reduced photosynthetic activity upon illumination and severe perturbation of hydrogen production activity. Functional analysis, performed on isolated PSII supercomplexes and recombinant LHCBM9 proteins, demonstrated that presence of LHCBM9 resulted in faster chlorophyll fluorescence decay and reduced production of singlet oxygen, indicating upgraded photoprotection. We conclude that LHCBM9 has a special role within the family of LHCII proteins and serves an important protective function during stress conditions by promoting efficient light energy dissipation and stabilizing PSII supercomplexes.

Reconstruction of the lipid metabolism for the microalga Monoraphidium neglectum from its genome sequence reveals characteristics suitable for biofuel production.

BACKGROUND: Microalgae are gaining importance as sustainable production hosts in the fields of biotechnology and bioenergy. A robust biomass accumulating strain of the genus Monoraphidium (SAG 48.87) was investigated in this work as a potential feedstock for biofuel production. The genome was sequenced, annotated, and key enzymes for triacylglycerol formation were elucidated. RESULTS: Monoraphidium neglectum was identified as an oleaginous species with favourable growth characteristics as well as a high potential for crude oil production, based on neutral lipid contents of approximately 21% (dry weight) under nitrogen starvation, composed of predominantly C18:1 and C16:0 fatty acids. Further characterization revealed growth in a relatively wide pH range and salt concentrations of up to 1.0% NaCl, in which the cells exhibited larger structures. This first full genome sequencing of a member of the Selenastraceae revealed a diploid, approximately 68 Mbp genome with a G + C content of 64.7%. The circular chloroplast genome was assembled to a 135,362 bp single contig, containing 67 protein-coding genes. The assembly of the mitochondrial genome resulted in two contigs with an approximate total size of 94 kb, the largest known mitochondrial genome within algae. 16,761 protein-coding genes were assigned to the nuclear genome. Comparison of gene sets with respect to functional categories revealed a higher gene number assigned to the category "carbohydrate metabolic process" and in "fatty acid biosynthetic process" in M. neglectum when compared to Chlamydomonas reinhardtii and Nannochloropsis gaditana, indicating a higher metabolic diversity for applications in carbohydrate conversions of biotechnological relevance. CONCLUSIONS: The genome of M. neglectum, as well as the metabolic reconstruction of crucial lipid pathways, provides new insights into the diversity of the lipid metabolism in microalgae. The results of this work provide a platform to encourage the development of this strain for biotechnological applications and production concepts.

Synthesis of transparent aminosilane-derived silica based networks for entrapment of sensitive materials.

A novel sol-gel synthesis route is reported which results in the formation of optically transparent silica based hydro- and xerogels from an aminosilane precursor in aqueous solutions. These materials can be used for entrapment of microalgae and light-harvesting complex (LHC) samples.

Ice recrystallization inhibition mediated by a nuclear-expressed and -secreted recombinant ice-binding protein in the microalga Chlamydomonas reinhardtii.

A Lolium perenne ice-binding protein (LpIBP) demonstrates superior ice recrystallization inhibition (IRI) activity and has proposed applications in cryopreservation, food texturing, as well as in being a "green" gas hydrate inhibitor. Recombinant production of LpIBP has been previously conducted in bacterial and yeast systems for studies of protein characterization, but large-scale applications have been hitherto limited due to high production costs. In this work, a codon-optimized LpIBP was recombinantly expressed and secreted in a novel one-step vector system from the nuclear genome of the green microalga Chlamydomonas reinhardtii. Both mixotrophic and photoautotrophic growth regimes supported LpIBP expression, indicating the feasibility of low-cost production using minimal medium, carbon dioxide, and light energy as input. In addition, multiple growth and bioproduct extraction cycles were performed by repetitive batch cultivation trials, demonstrating the potential for semi-continuous production and biomass harvesting. Concentrations of recombinant protein reached in this proof of concept approach were sufficient to demonstrate IRI activity in culture media without additional purification or concentration, with activity further verified by thermal hysteresis and morphology assays. The incorporation of the recombinant LpIBP into a model gas hydrate offers the promise that algal production may eventually find application as a "green" hydrate inhibitor.

Identification of Monoraphidium contortum as a promising species for liquid biofuel production.

In this work, 30 microalgae strains from 17 genera were investigated in regard to biomass productivity in photoautotrophic growth conditions, lipid amount, lipid quality and biomass degradability. Six strains could be identified with robust phototrophic growth properties and high biomass productivities equal or above 300 mg l(-1) day(-1). Anaerobic fermentation of the algal biomass was most efficient for the marine members of the genera Dunaliella and Navicula, while biogas production with the freshwater strains generally resulted in lower methane yields. Monoraphidium contortum was identified as promising candidate for liquid biofuel production, characterized by high biomass productivity during maximum growth (maximum increase of 896 mg dry biomass weight (DW) l(-1) day(-1)) and a promising lipid profile. Neutral lipid production was strongly induced in M. contortum by nitrogen deficient conditions and accumulated to up to 20.4±2.2% of DW.

Efficient recombinant protein production and secretion from nuclear transgenes in Chlamydomonas reinhardtii.

Microalgae are diverse photosynthetic microbes which offer the potential for production of a number of high value products (HVP) such as pigments, oils, and bio-active compounds. Fast growth rates, ease of photo-autotrophic cultivation, unique metabolic properties and continuing progress in algal transgenics have raised interest in the use of microalgae systems for recombinant protein (RP) production. This work demonstrates the development of an advanced RP production and secretion system for the green unicellular model alga Chlamydomonas reinhardtii. We generated a versatile expression vector that employs the secretion signal of the native extracellular C. reinhardtii carbonic anhydrase for efficient RP secretion into the culture medium. Unique restriction sites were placed between the regulatory elements to allow fast and easy sub-cloning of sequences of interest. Positive transformants can rapidly be identified by high-throughput plate-level screens via a coupled Gaussia luciferase marker. The vector was tested in Chlamydomonas wild type CC-1883 (WT) and in the transgene expression transformant UVM4. Compared to the native secretion signal of the Gaussia luciferase, up to 84% higher RP production could be achieved. With this new expression system we could generate transformants that express up to 10 mg RP per liter culture without further optimization. The target RP is found exclusively in culture medium and can therefore easily be isolated and purified. We conclude that this new expression system will be a valuable tool for many heterologous protein expression applications from C. reinhardtii in the future.

Future prospects of microalgal biofuel production systems.

Climate change mitigation, economic growth and stability, and the ongoing depletion of oil reserves are all major drivers for the development of economically rational, renewable energy technology platforms. Microalgae have re-emerged as a popular feedstock for the production of biofuels and other more valuable products. Even though integrated microalgal production systems have some clear advantages and present a promising alternative to highly controversial first generation biofuel systems, the associated hype has often exceeded the boundaries of reality. With a growing number of recent analyses demonstrating that despite the hype, these systems are conceptually sound and potentially sustainable given the available inputs, we review the research areas that are key to attaining economic reality and the future development of the industry.

The interplay of proton, electron, and metabolite supply for photosynthetic H2 production in Chlamydomonas reinhardtii.

To obtain a detailed picture of sulfur deprivation-induced H(2) production in microalgae, metabolome analyses were performed during key time points of the anaerobic H(2) production process of Chlamydomonas reinhardtii. Analyses were performed using gas chromatography coupled to mass spectrometry (GC/MS), two-dimensional gas chromatography combined with time-of-flight mass spectrometry (GCxGC-TOFMS), lipid and starch analysis, and enzymatic determination of fermentative products. The studies were designed to provide a detailed metabolite profile of the solar Bio-H(2) production process. This work reports on the differential analysis of metabolic profiles of the high H(2)-producing strain Stm6Glc4 and the wild-type cc406 (WT) before and during the H(2) production phase. Using GCxGC-TOFMS analysis the number of detected peaks increased from 128 peaks, previously detected by GC/MS techniques, to ∼1168. More detailed analysis of the anaerobic H(2) production phase revealed remarkable differences between wild-type and mutant cells in a number of metabolic pathways. Under these physiological conditions the WT produced up to 2.6 times more fatty acids, 2.2 times more neutral lipids, and up to 4 times more fermentation products compared with Stm6Glc4. Based on these results, specific metabolic pathways involving the synthesis of fatty acids, neutral lipids, and fermentation products during anaerobiosis in C. reinhardtii have been identified as potential targets for metabolic engineering to further enhance substrate supply for the hydrogenase(s) in the chloroplast.

Cysteine modification of a specific repressor protein controls the translational status of nucleus-encoded LHCII mRNAs in Chlamydomonas.

The cytosolic RNA-binding protein NAB1 represses translation of LHCII (light-harvesting complex of photosystem II) encoding mRNAs by sequestration into translationally silent mRNP complexes in the green alga Chlamydomonas reinhardtii. NAB1 contains 2 cysteine residues, Cys-181 and Cys-226, within its C-terminal RRM motif. Modification of these cysteines either by oxidation or by alkylation in vitro was accompanied by a decrease in RNA-binding affinity for the target mRNA sequence. To confirm the relevance of reversible NAB1 cysteine oxidation for the regulation of its activity in vivo, we replaced both cysteines with serines. All examined cysteine single and double mutants exhibited a reduced antenna at PSII caused by a perturbed NAB1 deactivation mechanism, with double mutations and Cys-226 single mutations causing a stronger and more distinctive phenotype compared with the Cys-181 mutation. Our data indicated that the responsible redox control mechanism is mediated by modification of single cysteines. Polysome analyses and RNA co-immunoprecipitation experiments demonstrated the interconnection of the NAB1 thiol state and its activity as a translation repressor in vivo. NAB1 is fully active in its dithiol state and is reversibly deactivated by modification of its cysteines. In summary, this work is an example that cytosolic translation of nucleus encoded photosynthetic genes is regulated via a reversible cysteine-based redox switch in a RNA-binding translation repressor protein.

Transcriptome for photobiological hydrogen production induced by sulfur deprivation in the green alga Chlamydomonas reinhardtii.

Photobiological hydrogen production using microalgae is being developed into a promising clean fuel stream for the future. In this study, microarray analyses were used to obtain global expression profiles of mRNA abundance in the green alga Chlamydomonas reinhardtii at different time points before the onset and during the course of sulfur-depleted hydrogen production. These studies were followed by real-time quantitative reverse transcription-PCR and protein analyses. The present work provides new insights into photosynthesis, sulfur acquisition strategies, and carbon metabolism-related gene expression during sulfur-induced hydrogen production. A general trend toward repression of transcripts encoding photosynthetic genes was observed. In contrast to all other LHCBM genes, the abundance of the LHCBM9 transcript (encoding a major light-harvesting polypeptide) and its protein was strongly elevated throughout the experiment. This suggests a major remodeling of the photosystem II light-harvesting complex as well as an important function of LHCBM9 under sulfur starvation and photobiological hydrogen production. This paper presents the first global transcriptional analysis of C. reinhardtii before, during, and after photobiological hydrogen production under sulfur deprivation.