CRISPR/Cas9-Mediated Knockout of the Lycopene ε-Cyclase for Efficient Astaxanthin Production in the Green Microalga Chlamydomonas reinhardtii.

Carotenoids are valuable pigments naturally occurring in all photosynthetic plants and microalgae as well as in selected fungi, bacteria, and archaea. Green microalgae developed a complex carotenoid profile suitable for efficient light harvesting and light protection and harbor great capacity for carotenoid production through the substantial power of the endogenous 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. Previous works established successful genome editing and induced significant changes in the cellular carotenoid content in Chlamydomonas reinhardtii. This study employs a tailored carotenoid pathway for engineered bioproduction of the valuable ketocarotenoid astaxanthin. Functional knockout of lycopene ε-cyclase (LCYE) and non-homologous end joining (NHEJ)-based integration of donor DNA at the target site inhibit the accumulation of α-carotene and consequently lutein and loroxanthin, abundant carotenoids in C. reinhardtii without changes in cellular fitness. PCR-based screening indicated that 4 of 96 regenerated candidate lines carried (partial) integrations of donor DNA and increased ß-carotene as well as derived carotenoid contents. Iterative overexpression of CrBKT, PacrtB, and CrCHYB resulted in a 2.3-fold increase in astaxanthin accumulation in mutant ΔLCYE#3 (1.8 mg/L) compared to the parental strain UVM4, which demonstrates the potential of genome editing for the design of a green cell factory for astaxanthin bioproduction.

Outdoor cultivation and metabolomics exploration of Chlamydomonas engineered for bisabolene production.

Demonstrating outdoor cultivation of engineered microalgae at considerable scales is essential for their prospective large-scale deployment. Hence, this study focuses on the outdoor cultivation of an engineered Chlamydomonas reinhardtii strain, 3XAgBs-SQs, for bisabolene production under natural dynamic conditions of light and temperature. Our preliminary outdoor experiments showed improved growth, but frequent culture collapses in conventional Tris-acetate-phosphate medium. In contrast, modified high-salt medium (HSM) supported prolonged cell survival, outdoor. However, their subsequent outdoor scale-up from 250 mL to 5 L in HSM was effective with 10 g/L bicarbonate supplementation. Pulse amplitude modulation fluorometry and metabolomic analysis further validated their improved photosynthesis and uncompromised metabolic fluxes towards the biomass and the products (natural carotenoids and engineered bisabolene). These strains could produce 906 mg/L bisabolene and 54 mg/L carotenoids, demonstrating the first successful outdoor photoautotrophic cultivation of engineeredC. reinhardtii,establishing it as a one-cell two-wells biorefinery.

Bicistronic expression of nuclear transgenes in Chlamydomonas reinhardtii.

In eukaryotic organisms, proteins are typically translated from monocistronic messenger RNAs containing a single coding sequence (CDS). However, recent long transcript sequencing identified 87 nuclear polycistronic mRNAs in Chlamydomonas reinhardtii natively carrying multiple co-expressed CDSs. In this study, we investigated the dynamics of 22 short intergenic sequences derived from these native polycistronic loci by their application in genetic constructs for synthetic transgene expression. A promising candidate sequence was identified based on the quantification of transformation efficiency and expression strength of a fluorescence reporter protein. Subsequently, the expression of independent proteins from one mRNA was verified by cDNA amplification and protein molecular mass characterization. We demonstrated engineered bicistronic expression in vivo to drive successful co-expression of several terpene synthases with the selection marker aphVIII. Bicistronic transgene design resulted in significantly increased (E)-α-bisabolene production of 7.95 mg L-1 from a single open reading frame, 18.1× fold higher than previous reports. Use of this strategy simplifies screening procedures for identification of high-level expressing transformants, does not require the application of additional fluorescence reporters, and reduces the nucleotide footprint compared to classical monocistronic expression cassettes. Although clear advantages for bicistronic transgene expression were observed, this strategy was found to be limited to the aphVIII marker, and further studies are necessary to gain insights into the underlying mechanism that uniquely permits this co-expression from the algal nuclear genome.

Molecular design of microalgae as sustainable cell factories.

Microalgae are regarded as sustainable and potent chassis for biotechnology. Their capacity for efficient photosynthesis fuels dynamic growth independent from organic carbon sources and converts atmospheric CO2 directly into various valuable hydrocarbon-based metabolites. However, approaches to gene expression and metabolic regulation have been inferior to those in more established heterotrophs (e.g., prokaryotes or yeast) since the genetic tools and insights in expression regulation have been distinctly less advanced. In recent years, however, these tools and their efficiency have dramatically improved. Various examples have demonstrated new trends in microalgal biotechnology and the potential of microalgae for the transition towards a sustainable bioeconomy.

Microbial Diversity and Community Structure of Wastewater-Driven Microalgal Biofilms.

Dwindling water sources increase the need for efficient wastewater treatment. Solar-driven algal turf scrubber (ATS) system may remediate wastewater by supporting the development and growth of periphytic microbiomes that function and interact in a highly dynamic manner through symbiotic interactions. Using ITS and 16S rRNA gene amplicon sequencing, we profiled the microbial communities of four microbial biofilms from ATS systems operated with municipal wastewater (mWW), diluted cattle and pig manure (CattleM and PigM), and biogas plant effluent supernatant (BGE) in comparison to the initial inocula and the respective wastewater substrates. The wastewater-driven biofilms differed significantly in their biodiversity and structure, exhibiting an inocula-independent but substrate-dependent establishment of the microbial communities. The prokaryotic communities were comparable among themselves and with other microbiomes of aquatic environments and were dominated by metabolically flexible prokaryotes such as nitrifiers, polyphosphate-accumulating and algicide-producing microorganisms, and anoxygenic photoautotrophs. Striking differences occurred in eukaryotic communities: While the mWW biofilm was characterized by high biodiversity and many filamentous (benthic) microalgae, the agricultural wastewater-fed biofilms consisted of less diverse communities with few benthic taxa mainly inhabited by unicellular chlorophytes and saprophytes/parasites. This study advances our understanding of the microbiome structure and function within the ATS-based wastewater treatment process.

Farnesyl pyrophosphate compartmentalization in the green microalga Chlamydomonas reinhardtii during heterologous (E)-α-bisabolene production.

BACKGROUND: Eukaryotic algae have recently emerged as hosts for metabolic engineering efforts to generate heterologous isoprenoids. Isoprenoid metabolic architectures, flux, subcellular localization, and transport dynamics have not yet been fully elucidated in algal hosts. RESULTS: In this study, we investigated the accessibility of different isoprenoid precursor pools for C15 sesquiterpenoid generation in the cytoplasm and chloroplast of Chlamydomonas reinhardtii using the Abies grandis bisabolene synthase (AgBS) as a reporter. The abundance of the C15 sesquiterpene precursor farnesyl pyrophosphate (FPP) was not increased in the cytosol by co-expression and fusion of AgBS with different FPP synthases (FPPSs), indicating limited C5 precursor availability in the cytoplasm. However, FPP was shown to be available in the plastid stroma, where bisabolene titers could be improved several-fold by FPPSs. Sesquiterpene production was greatest when AgBS-FPPS fusions were directed to the plastid and could further be improved by increasing the gene dosage. During scale-up cultivation with different carbon sources and light regimes, specific sesquiterpene productivities from the plastid were highest with CO2 as the only carbon source and light:dark illumination cycles. Potential prenyl unit transporters are proposed based on bioinformatic analyses, which may be in part responsible for our observations. CONCLUSIONS: Our findings indicate that the algal chloroplast can be harnessed in addition to the cytosol to exploit the full potential of algae as green cell factories for non-native sesquiterpenoid generation. Identification of a prenyl transporter may be leveraged for further extending this capacity.

Advanced pathway engineering for phototrophic putrescine production.

The polyamine putrescine (1,4-diaminobutane) contributes to cellular fitness in most organisms, where it is derived from the amino acids ornithine or arginine. In the chemical industry, putrescine serves as a versatile building block for polyamide synthesis. The green microalga Chlamydomonas reinhardtii accumulates relatively high putrescine amounts, which, together with recent advances in genetic engineering, enables the generation of a powerful green cell factory to promote sustainable biotechnology for base chemical production. Here, we report a systematic investigation of the native putrescine metabolism in C. reinhardtii, leading to the first CO2 -based bio-production of putrescine, by employing modern synthetic biology and metabolic engineering strategies. A CRISPR/Cas9-based knockout of key enzymes of the polyamine biosynthesis pathway identified ornithine decarboxylase 1 (ODC1) as a gatekeeper for putrescine accumulation and demonstrated that the arginine decarboxylase (ADC) route is likely inactive and that amine oxidase 2 (AMX2) is mainly responsible for putrescine degradation in C. reinhardtii. A 4.5-fold increase in cellular putrescine levels was achieved by engineered overexpression of potent candidate ornithine decarboxylases (ODCs). We identified unexpected substrate promiscuity in two bacterial ODCs, which exhibited co-production of cadaverine and 4-aminobutanol. Final pathway engineering included overexpression of recombinant arginases for improved substrate availability as well as functional knockout of putrescine degradation, which resulted in a 10-fold increase in cellular putrescine titres and yielded 200 mg/L in phototrophic high cell density cultivations after 10 days.

Engineering a powerful green cell factory for robust photoautotrophic diterpenoid production.

Diterpenoids display a large and structurally diverse class of natural compounds mainly found as specialized plant metabolites. Due to their diverse biological functions they represent an essential source for various industrially relevant applications as biopharmaceuticals, nutraceuticals, and fragrances. However, commercial production utilizing their native hosts is inhibited by low abundances, limited cultivability, and challenging extraction, while the precise stereochemistry displays a particular challenge for chemical synthesis. Due to a high carbon flux through their native 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway towards photosynthetically active pigments, green microalgae hold great potential as efficient and sustainable heterologous chassis for sustainable biosynthesis of plant-derived diterpenoids. In this study, innovative synthetic biology and efficient metabolic engineering strategies were systematically combined to re-direct the metabolic flux through the MEP pathway for efficient heterologous diterpenoid synthesis in C. reinhardtii. Engineering of the 1-Deoxy-D-xylulose 5-phosphate synthase (DXS) as the main rate-limiting enzyme of the MEP pathway and overexpression of diterpene synthase fusion proteins increased the production of high-value diterpenoids. Applying fully photoautotrophic high cell density cultivations demonstrate potent and sustainable production of the high-value diterpenoid sclareol up to 656 mg L-1 with a maximal productivity of 78 mg L-1 day-1 in a 2.5 L scale photobioreactor, which is comparable to sclareol titers reached by highly engineered yeast. Consequently, this work represents a breakthrough in establishing a powerful phototrophic green cell factory for the competetive use in industrial biotechnology.

The Spermidine Synthase Gene SPD1: A Novel Auxotrophic Marker for Chlamydomonas reinhardtii Designed by Enhanced CRISPR/Cas9 Gene Editing.

Biotechnological application of the green microalga Chlamydomonas reinhardtii hinges on the availability of selectable markers for effective expression of multiple transgenes. However, biological safety concerns limit the establishment of new antibiotic resistance genes and until today, only a few auxotrophic markers exist for C. reinhardtii. The recent improvements in gene editing via CRISPR/Cas allow directed exploration of new endogenous selectable markers. Since editing frequencies remain comparably low, a Cas9-sgRNA ribonucleoprotein (RNP) delivery protocol was strategically optimized by applying nitrogen starvation to the pre-culture, which improved successful gene edits from 10% to 66% after pre-selection. Probing the essential polyamine biosynthesis pathway, the spermidine synthase gene (SPD1) is shown to be a potent selectable marker with versatile biotechnological applicability. Very low levels of spermidine (0.75 mg/L) were required to maintain normal mixotrophic and phototrophic growth in newly designed spermidine auxotrophic strains. Complementation of these strains with a synthetic SPD1 gene was achieved when the mature protein was expressed in the cytosol or targeted to the chloroplast. This work highlights the potential of new selectable markers for biotechnology as well as basic research and proposes an effective pipeline for the identification of new auxotrophies in C. reinhardtii.

A novel, robust and mating-competent Chlamydomonas reinhardtii strain with an enhanced transgene expression capacity for algal biotechnology.

In the future, algae biotechnology could play an important role in sustainable development, especially with regard to the production of valuable chemicals. Among the established laboratory strains with efficient transgene expression, there are none that have demonstrated the required robustness for industrial applications, which generally require growth at larger scale. Here, we created a robust and mating-competent cell line of the green microalga Chlamydomonas reinhardtii, which also possesses a high transgene expression capacity. This strain shows a comparably high resistance to shear stress by accumulating increased amounts of biomass under these conditions. As a proof-of-concept, a high phototrophic productivity of cadaverine from CO2 and nitrate was demonstrated by efficiently expressing a bacterial l-lysine decarboxylase. In contrast to other established strains, this novel chassis strain for phototrophic production schemes is equipped with the traits required for industrial applications, by combining mating-competence, cell wall-mediated robustness and high level transgene expression.

A gene regulatory network for antenna size control in carbon dioxide-deprived Chlamydomonas reinhardtii cells.

In green microalgae, prolonged exposure to inorganic carbon depletion requires long-term acclimation responses, involving modulated gene expression and the adjustment of photosynthetic activity to the prevailing supply of carbon dioxide. Here, we describe a microalgal regulatory cycle that adjusts the light-harvesting capacity at photosystem II (PSII) to the prevailing supply of carbon dioxide in Chlamydomonas (Chlamydomonas reinhardtii). It engages low carbon dioxide response factor (LCRF), a member of the squamosa promoter-binding protein (SBP) family of transcription factors, and the previously characterized cytosolic translation repressor nucleic acid-binding protein 1 (NAB1). LCRF combines a DNA-binding SBP domain with a conserved domain for protein-protein interaction. LCRF transcription is rapidly induced by carbon dioxide depletion. LCRF activates NAB1 transcription by specifically binding to tetranucleotide motifs present in its promoter. Accumulation of the NAB1 protein enhances translational repression of its prime target mRNA, encoding the PSII-associated major light-harvesting protein LHCBM6. The resulting truncation of the PSII antenna size helps maintaining a low excitation during carbon dioxide limitation. Analyses of low carbon dioxide acclimation in nuclear insertion mutants devoid of a functional LCRF gene confirm the essentiality of this novel transcription factor for the regulatory circuit.

Rational Promoter Engineering Enables Robust Terpene Production in Microalgae.

Microalgal biotechnology promises sustainable light-driven production of valuable bioproducts and addresses urgent demands to attain a sustainable economy. However, to unfold its full potential as a platform for biotechnology, new and powerful tools for nuclear engineering need to be established. Chlamydomonas reinhardtii, the model for microalgal synthetic biology and genetic engineering has already been used to produce various bioproducts. Nevertheless, low transgene titers, the lack of potent expression elements, and sparse comparative evaluation prevents further development of C. reinhardtii as a biotechnological host. By systematically evaluating existing expression elements combined with rational promoter engineering, we established novel synthetic expression elements, improved the standardized application of synthetic biology tools, and unveiled an existing synergism between the PSAD 5' UTR and its corresponding chloroplast targeting peptide. Promoter engineering strategies, implemented in a newly designed synthetic algal promoter, increased the production of the sesquiterpene (E)-α-bisabolene by 18-fold compared to its native version and 4-fold to commonly used expression elements. Our results improve the application of synthetic biology in microalgae and display a significant step toward establishing C. reinhardtii as a sustainable green cell-factory.

Engineering Biocatalytic Solar Fuel Production: The PHOTOFUEL Consortium.

The EU Horizon2020 consortium PHOTOFUEL joined academic and industrial partners from biology, chemistry, engineering, engine design, and lifecycle assessment, making tremendous progress towards engine-ready fuels from CO2 via engineered photosynthetic microbes. Technical, environmental, economic, and societal opportunities and challenges were explored to frame future technology realization at scale.

Phytoplankton consortia as a blueprint for mutually beneficial eukaryote-bacteria ecosystems based on the biocoenosis of Botryococcus consortia.

Bacteria occupy all major ecosystems and maintain an intensive relationship to the eukaryotes, developing together into complex biomes (i.e., phycosphere and rhizosphere). Interactions between eukaryotes and bacteria range from cooperative to competitive, with the associated microorganisms affecting their host`s development, growth and health. Since the advent of non-culture dependent analytical techniques such as metagenome sequencing, consortia have been described at the phylogenetic level but rarely functionally. Multifaceted analysis of the microbial consortium of the ancient phytoplankton Botryococcus as an attractive model food web revealed that its all abundant bacterial members belong to a niche of biotin auxotrophs, essentially depending on the microalga. In addition, hydrocarbonoclastic bacteria without vitamin auxotrophies seem adversely to affect the algal cell morphology. Synthetic rearrangement of a minimal community consisting of an alga, a mutualistic and a parasitic bacteria underpins the model of a eukaryote that maintains its own mutualistic microbial community to control its surrounding biosphere. This model of coexistence, potentially useful for defense against invaders by a eukaryotic host could represent ecologically relevant interactions that cross species boundaries. Metabolic and system reconstruction is an opportunity to unravel the relationships within the consortia and provide a blueprint for the construction of mutually beneficial synthetic ecosystems.

High cell density cultivation enables efficient and sustainable recombinant polyamine production in the microalga Chlamydomonas reinhardtii.

Modern chemical industry calls for new resource-efficient and sustainable value chains for production of key base chemicals such as polyamines. The green microalga Chlamydomonas reinhardtii offers great potential as an innovative green-cell factory by combining fast and inexpensive, phototrophic growth with mature genetic engineering. Here, overexpression of recombinant lysine decarboxylases in C. reinhardtii enabled the robust accumulation of the non-native polyamine cadaverine, which serves as building block for bio-polyamides. The issue of low cell densities, limiting most microalgal cultivation processes was resolved by systematically optimizing cultivation parameters. A new, easy-to-apply and fully phototrophic medium enables high cell density cultivations of C. reinhardtii with a 6-fold increase in biomass and cell count (20 g/L biomass dry weight, ~2·108 cells/mL). Application of high cell density cultivations in established photobioreactors resulted in a 10-fold increase of cadaverine yields, with up to 0.24 g/L after 9 days and maximal productivity of 0.1 g/L/d.

Wastewater-borne microalga Chlamydomonas sp.: A robust chassis for efficient biomass and biomethane production applying low-N cultivation strategy.

Biogas/biomethane generation from microalgae biomass via anaerobic fermentation is increasingly gaining attention as CO2-neutral energy source. Intensive research has shown, however, that microalgae represent a rather challenging substrate for anaerobic digestion (AD) due to their high cell wall recalcitrance and unfavourable protein content. Previously, the utilization of nitrogen-limited (low-N) microalgal biomass for continuous AD-processes was demonstrated (as proof-of-concept) with remarkable biomethane productivity. The present study shows the efficient portability of the low-N cultivation/fermentation strategy on a robust, wastewater-borne microalga isolate that tolerates high temperature and light conditions and can perfectly cope with microbial contaminations. Continuous long-term anaerobic digestion was characterized by stable and efficient specific biogas and biomethane productivity (765 ± 20 and 478 ± 15 mLNg-1 volatile solids (VS) d-1, respectively), equivalent to volumetric methane productivity of 1912 mLN L-1d-1. The present work underlines the applicability of low-N-biomass of wastewater-borne, robust microalgae as mono-substrate for highly efficient continuous methane generation.

Introns mediate post-transcriptional enhancement of nuclear gene expression in the green microalga Chlamydomonas reinhardtii.

Efficient nuclear transgene expression in the green microalga Chlamydomonas reinhardtii is generally hindered by low transcription rates. Introns can increase transcript abundance by a process called Intron-Mediated Enhancement (IME) in this alga and has been broadly observed in other eukaryotes. However, the mechanisms of IME in microalgae are poorly understood. Here, we identified 33 native introns from highly expressed genes in C. reinhardtii selected from transcriptome studies as well as 13 non-native introns. We investigated their IME capacities and probed the mechanism of action by modification of splice sites, internal sequence motifs, and position within transgenes. Several introns were found to elicit strong IME and found to be broadly applicable in different expression constructs. We determined that IME in C. reinhardtii exclusively occurs from introns within transcribed ORFs regardless of the promoter and is not induced by traditional enhancers of transcription. Our results elucidate some mechanistic details of IME in C. reinhardtii, which are similar to those observed in higher plants yet underly distinctly different induction processes. Our findings narrow the focus of targets responsible for algal IME and provides evidence that introns are underestimated regulators of C. reinhardtii nuclear gene expression.

Green algal hydrocarbon metabolism is an exceptional source of sustainable chemicals.

Microalgae are rapidly growing, low-input requiring, sun light-utilizing microorganisms capable of converting carbon dioxide into various natural products, a major portion of which are hydrocarbons. Their cellular compartmentalization and photosynthetic apparatus depend on robust turnover of two hydrocarbon classes, isoprenoids and acyl-lipids. This review summarizes the current understanding of algal hydrocarbon metabolism, including carbon partitioning capacities, the localization and size of precursor pools, environmental effects on flux distribution, and limiting factors towards efficient (heterologous) hydrocarbon production. Questions and challenges regarding our knowledge of algal hydrocarbon metabolism as well as guidelines for systematic engineering are presented. Recent engineering achievements indicate fundamental plasticity in the (heterologous) hydrocarbon metabolism of green algae while highlighting their potential as renewable sources of these products.

Ocean acidification has little effect on the biochemical composition of the coccolithophore Emiliania huxleyi.

Owing to the hierarchical organization of biology, from genomes over transcriptomes and proteomes down to metabolomes, there is continuous debate about the extent to which data and interpretations derived from one level, e.g. the transcriptome, are in agreement with other levels, e.g. the metabolome. Here, we tested the effect of ocean acidification (OA; 400 vs. 1000 µatm CO2) and its modulation by light intensity (50 vs. 300 µmol photons m-2 s-1) on the biomass composition (represented by 75 key metabolites) of diploid and haploid life-cycle stages of the coccolithophore Emiliania huxleyi (RCC1216 and RCC1217) and compared these data with interpretations from previous physiological and gene expression screenings. The metabolite patterns showed minor responses to OA in both life-cycle stages. Whereas previous gene expression analyses suggested that the observed increased biomass buildup derived from lipid and carbohydrate storage, this dataset suggests that OA slightly increases overall biomass of cells, but does not significantly alter their metabolite composition. Generally, light was shown to be a more dominant driver of metabolite composition than OA, increasing the relative abundances of amino acids, mannitol and storage lipids, and shifting pigment contents to accommodate increased irradiance levels. The diploid stage was shown to contain vastly more osmolytes and mannitol than the haploid stage, which in turn had a higher relative content of amino acids, especially aromatic ones. Besides the differences between the investigated cell types and the general effects on biomass buildup, our analyses indicate that OA imposes only negligible effects on E. huxleyi´s biomass composition.

Growth and photosynthetic activity of Chlamydomonas reinhardtii entrapped in lens-shaped silica hydrogels.

Entrapment of microalgae in silica hydrogels enables the application as biocatalysts in continuous production of secreted products. Despite a mitigation of substrate and product diffusion limitations by lens-shaped particles, there are no reports on light supply and limitation. This study investigated the impact of hydrogel structure, particle size and biomass loading on the behaviour of the microalga Chlamydomonas reinhardtii entrapped in lens-shaped silica particles. Entrapment in tetraethyl orthosilicate and tetra(n-propylamino)silane based hydrogels reduced the growth rate by 30% and 23%, respectively. In contrast, cells entrapped in sodium silicate based hydrogels displayed a growth rate similar to free cells and cells entrapped in calcium alginate (1.13 d-1), indicating a suitable biocompatibility. Reduction of lens height by 26% maintained the growth rate in silica hydrogel. A fourfold increase in biomass loading reduced the growth rate by 20% and elevated the yield coefficient by 211%, indicating the impact of biomass loading on light and nutrient supply on photosynthetic growth. Finally, hydrogen production was observed by entrapped cells. The results of this work will pave the way for robust biocatalytic processes where photosynthetically active cells are protected against harmful mechanical and biological influences.