Biology subjects

Lauersen, K. J.

Publications and source records attributed to Lauersen, K. J..

At least 19 recordsLinked to original sources

bioRxiv2026

Cultivation and physiological characterization of a desert-derived Halospirulina isolate

Here, we describe a filamentous Halospirulina isolate (Halospirulina saudiensis) obtained from water-clay microhabitat the Empty Quarter desert, (ar-Rub al-Kh[a]l[i]), Saudi Arabia which grows in saline conditions. We present its fully sequenced genome, the first for the genus, and characterize its growth dynamics as well as biochemical composition under a range of cultivation conditions. Protein, carbohydrate, lipid, and phycocyanin content varied with cultivation regime but were largely stable. H. saudiensis reached biomass concentrations of up to 9.83 g L-1 at pH 7, 35 {degrees}C and continuous 325 {micro}mol photons m-2 s-1. Variable climate simulations in lab-scale photobioreactors revealed preference for warmer season cultivation under modeled outdoor conditions. Carotenoid analysis revealed a pigment profile enriched in canthaxanthin and other ketocarotenoids, distinguishing it from industrial Limnospira and positioning its value for neutraceuticals and feed additives. Genome analysis identified a carotene ketolase (crtO) homolog consistent with other cyanobacteria that accumulate ketocarotenoids. Phycocyanin content was heavily dependent on culture health and varied with cultivation pH, irradiance, reaching maximum values of 67.3 {+/-} 0.8 mg gDW-1 (6.73 %). Extracted phycocyanin showed marginal thermal stability compared to that from L. platensis. The findings suggest that H. saudiensis could be a promising source of biomass, ketocarotenoids, and natural pigments, cultivated in saline conditions with elevated temperature and irradiance. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=71 SRC="FIGDIR/small/728284v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@17d6143org.highwire.dtl.DTLVardef@7cb1f7org.highwire.dtl.DTLVardef@880df4org.highwire.dtl.DTLVardef@4c16a6_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIHalospirulina saudiensis sp. nov. isolated from Empty Quarter C_LIO_LIFirst genome-resolved characterization of a Halospirulina strain C_LIO_LIReached 9.83 g L-1 in Red Sea salinity conditions C_LIO_LIAccumulates canthaxanthin as major carotenoid C_LIO_LIPhycocyanin slightly thermotolerant C_LI

microbiology↗

bioRxiv2026

Genome Position Does Not Impact Transgene Expression Efficiency in the Ancient Red Alga Cyanidioschyzon merolae

The thermoacidophilic red alga Cyanidioschyzon merolae represents one of the simplest photosynthetic eukaryotes and an ancient divergent group in the primary endosymbiotic Viridiplantae. Because of its [~]16 Mbp genome, containing few introns, and capacity for transgene integration by homologous recombination, it is an emerging chassis for synthetic biology. However, genomic integration sites and scalable transformation methods have not been established to systematically investigate the effect of genome position on transgene expression. Here, we combined bioinformatic genome analysis, liquid-handling robotics, and assays of heterologous protein and metabolite production to establish a reproducible framework for nuclear genome engineering in C. merolae. We mapped and annotated 40 intergenic loci as candidate neutral sites across 16 out of 20 chromosomes and could validate 38 of them through robotic-assisted transformation. Reporter gene expression analysis revealed highly uniform expression at all integration sites across broad populations of transformants, indicating surprising minimal positional effects and transcriptional neutrality. The functional equivalence of these genomic landing pads was determined by expression of a heterologous isoprene synthase, and coupling algal photobioreactors to headspace analysis to quantify isoprene production driven by transgene expression from different integration sites. Single copy transgene integrants, regardless of genome position, exhibited comparable reporter signals and consequent isoprene production. Together, these results provide the first experimentally validated set of neutral integration sites in C. merolae and establish a high-throughput transformation protocol for its genetic engineering in the context of synthetic genome biology.

synthetic biology↗

bioRxiv2025

Investigating the dynamics of precursor availability for sesquiterpenoid production in the cytoplasm and plastid of Chlamydomonas reinhardtii

The green alga Chlamydomonas reinhardtii is an interesting organism for production of heterologous isoprenoid natural products through metabolic engineering as it can grow photosynthetically and represents a plant-like cell environment for synthase expression. Here, we investigated expression of the patchoulol synthase from Pogostemon cablin (Benth) (PcPS) to probe the production of sesquiterpenoids from different sub-cellular compartments of the algal cell. We observed that knock-down of squalene synthase (SQS) in the cytoplasm did not affect sesquiterpene production titers in the plastid, suggesting no backward movement of FPP from cytoplasmic pools. Fusion of PcPS with a farnesyl pyrophosphate synthase (FPPS) did not enhance patchoulol production in the cytoplasm but did in the plastid, as also reported elsewhere. Secondary transformations with cytoplasmic and chloroplast-localized PcPS constructs increased synthase titers and improved patchoulol yields from both compartments. In multi-parallel photobioreactor experiments with patchoulol extraction through two-phase solvent contact, different daily patchoulol production behaviors were observed based on subcellular localization of the PcPS and carbon source. Those with plastid localization of PcPS-FPPS exhibited continued production into later stages of cultivation, while cytoplasm-localized PcPS with SQS k.d. had the highest production rates in mid-logarithmic phase with low productivity in later growth stages. Cultivations in high-density photobioreactors with nutrient-enriched medium generated 15 mg patchoulol/L culture from CO2, the highest yield reported from the alga to date. The results indicate that the algal cell has flexible pools of isoprenoid precursors in different subcellular compartments from which the biotechnologist can design an expression strategy tailored to carbon-feeding regimes.

bioengineering↗

bioRxiv2025

Elevated carbon dioxide stimulates highly efficient organic-carbon consumption and confectionary-waste valorization under mixotrophy in the unicellular alga Galdieria

Unicellular algae are appealing for nutritional and biotechnological utility but have wide variation across strains and can be challenging to produce. The thermo-acidophilic algal genus Galdieria use diverse organic-carbon sources for fermentative growth that can include waste-stream feedstocks and have complete amino-acid compositions for human nutrition. Here, we investigated Galdieria metabolic dynamics to catalog organic-carbon conversion to biomass. Tested strains had enhanced growth upon 3% CO2 supplementation, triggering efficient glucose uptake to reach [~]5 {+/-} 0.3 g dry biomass L-{superscript 1}. Stable-isotope analysis revealed that organic-carbon uptake dominates CO2 fixation in darkness under mixotrophy, with CO2 an apparent metabolic trigger. Galdieria sulphuraria 5587.1 can consume up to 8.3 g carbon L-1 day-1 from industrial confectionery waste, with C-phycocyanin reaching 3.8% of dry biomass and remaining thermostable at 72{degrees}C. This framework can optimize Galdieria-based bioprocesses for inexpensive waste conversion into high-value biomass and identifies CO2 as a trigger of organic-carbon assimilation, even in heterotrophic conditions. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/655468v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@e2ef63org.highwire.dtl.DTLVardef@d28574org.highwire.dtl.DTLVardef@137be97org.highwire.dtl.DTLVardef@c1c0a5_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2025

Engineered isoprene production from Chlamydomonas reinhardtii using herbicide selection markers and CO2-fed cultivation optimization through multi-parallel photobioreactor headspace gas analysis

Metabolic engineering requires selection markers for transformant generation. However, use of antibiotic resistance is of concern for potential horizontal gene transfer in the environment. Herbicide resistance markers are an alternative for photosynthetic cell line engineering as these agents are plant-specific with resistance mechanisms that can be generated from mutations of endogenous genes. Here, we developed norflurazon and oxyfluorfen resistance markers for nuclear genome transformant selection in the model green alga Chlamydomonas reinhardtii. These were used to engineer robust isoprene biosynthesis by facilitating overexpression of a yeast isopentenyl-diphosphate delta-isomerase (ScIDI), the algas own beta carotene ketolase (CrBKT), and the sweet potato isoprene synthase (IbIspS). Further UV-C mutagenesis and colony selection were employed to improve yields to [~]350 mg isoprene L-1 culture on organic carbon. It was then possible to optimize CO2-driven cultivation and isoprene biosynthesis in batch and continuous processes using multi-port, real-time, in-line mass spectrometry coupled to parallel photobioreactors. The highest isoprene yields in batch were achieved under 900 {micro}E illumination and 33 {degrees}C and, in turbidostat mode, [~]51 mg isoprene L culture-1 day-1 was achieved for 3 days concomitant with algal biomass production. Cultivation of the engineered alga directly in effluent from an anaerobic membrane bioreactor was also conducted. Isoprene production was concomitant with removal of ammonium and phosphate from the wastewater, and biomass production was similar to that in replete medium. Isoprene yields exhibited gradual reduction after each successive repetitive refresh, which could be mitigated by supplementation of trace elements. The results demonstrate that engineered algae could be used as a secondary wastewater treatment step while generating both biomass and volatile co-products like isoprene. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC="FIGDIR/small/649625v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@139b191org.highwire.dtl.DTLVardef@b8dfa3org.highwire.dtl.DTLVardef@162615forg.highwire.dtl.DTLVardef@1e6cfc4_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2024

Sustainable in situ extraction of microalgae-derived terpenoids using functionalized silica microparticles

Chlamydomonas reinhardtii is a green microalga that has been genetically engineered to produce heterologous terpenoid metabolites. While this green biochemical approach for producing high-value chemicals holds tremendous potential, the current in situ extraction methods, utilizing traditional solvents such as dodecane and perfluorinated chemicals, present separation challenges, unfavorable economics, and risk of cell toxicity. Here, we develop a low-cost solvent-free approach based on silanized silica particles to fill the technology gap. We determine the feasibility and specificity of three differently coated (C11, C16, C18) silica particles to extract nine terpenoid metabolites from C. reinhardtii cultures during cultivation. The results reveal that the extraction efficiencies of functionalized particles, particularly those coated with C18, demonstrated high potential as an alternative to traditional extraction methods. While the extraction efficiencies of coated microparticles are for most compounds lower than those observed for dodecane-based extractions, it holds higher potential for larger-scale cultivations where the use of dodecane is restricted due to its flammability and tendency to form emulsions. In addition, the present study provides insights into the optimization of particle-to-culture volume ratio, particle saturation, and the required number of product elution steps, and proves the feasibility of an upscaled extraction of the sesquiterpenoid patchoulol as a representative metabolite in 5 L hanging bag reactors. This study paves the way for a circular bioprocess where product capture with traditional solvent overlays is impractical due to the formation of emulsions, and at the same time allows for an easy product recovery (solid-liquid separation) and reuse of functionalized particles over numerous cycles.

bioengineering↗

bioRxiv2024

Treatment with Metal-Organic Frameworks (MOFs) elicits secondary metabolite accumulation in Aquilaria crassna (Agarwood) callus culture

Thymelaceaous trees are prized for accumulating fragrant resins composed of hundreds of secondary metabolites in their woody tissues. Slow growth and increasing consumer demand have stretched natural sources of agarwood trees to being endangered and alternative production modes, including silviculture and tissue culture, are currently being investigated. Dedifferentiated tissue culture of agarwood trees provides a means of cell propagation independent of environmental context. However, secondary metabolite accumulation, as found in fragrant resins, occurs largely in response to wounding. Here, we investigated the application of metal-organic frameworks (MOFs) as potential elicitors of secondary metabolite formation in Aquilaria crassna tissue culture samples. Callus cultures were exposed to five commercially available MOFs: UiO-67, MOF-808, HKUST-1, ZIF-67, and MOF-74, and ethanol extracts were used to quantify secondary metabolite accumulation compared to untreated cultures. Samples that were exposed to Zr-based MOFs exhibited similar metabolite production profiles, (trans-2-Carboxy-cyclo-hexyl)-acetic acid was reduced in the presence of all MOFs, the Cu-containing HKUST-1 MOF increased palmitic acid levels, and MOF-808 and ZIF-67 were found to elicit the highest accumulation of secondary metabolites with potential fragrance applications. These results demonstrate the possibility of eliciting secondary metabolites from dedifferentiated agarwood tree cell culture and may provide an alternative means of sourcing fragrant specialty chemicals from these plants.

biochemistry↗

bioRxiv2024

Compartmentalized sesquiterpenoid biosynthesis and functionalization in the Chlamydomonas reinhardtii plastid

Terpenoids play key roles in cellular metabolism, with some organisms having evolved expanded terpenoid profiles for specialized functions such as signaling and defense. Many terpenoids have applications in pharmaceuticals, fragrances, and agriculture, but their harvest from natural sources can be challenging. Heterologous production of specialty terpenoids in microbial hosts offers an alternative, using terpene synthases and further enzymatic decoration to expand chemical complexity and functionality. Here, we explored the heterologous production of 10 different sesquiterpenoids (STPs, C15) and their further biofunctionalization mediated by cytochrome P450s (CYPs) in the green alga Chlamydomonas reinhardtii. STP synthases were expressed from the nuclear genome and localized to the algal plastid, coupled with co-expression of selected CYPs. STP production in the plastid was supported by farnesyl pyrophosphate synthase fusions to STP synthases, and CYPs were modified for soluble localization in the plastid stroma by removing transmembrane domains. Various target CYPs were screened for STP functionalization in the alga, and different product ratios were generated based on trophic modes. Overall STP yields ranged between 250-2500 {micro}g L-1 under screening conditions, with CYP-mediated functionalization reaching up to 80% of accumulated heterologous STP products. Living two-phase terpenoid extractions with different perfluorinated solvents revealed variable performances based on sesquiterpenoid functionalization and solvent type. This work demonstrates the feasibility of generating heterologous functionalized terpenoid products in alga using soluble, plastid-localized CYPs without reductase partners. However, overall improvements in photobioreactor cultivation concepts will be required to facilitate the use of algal chassis for scaled production. Significance StatementThis study demonstrates the feasibility of producing and modifying heterologous terpenoid products in the algal plastid using sesquiterpene synthases (STPS) and soluble cytochrome P450s (CYPs). We show that algal cultivation conditions influence the composition and ratios of functionalized terpenoid products, which can be extracted through solvent-based milking during growth. The reducing equivalents that enable CYP activity in the plastid appear derived from photosynthetic electrons without requiring the co-expression of a cytochrome P450 reductase (CPR) partner, simplifying engineering strategies. As algae can be cultivated with minimal inputs (trace elements, light, and CO2) from sources like wastewater, this approach offers the potential for sustainable production of complex specialty terpenoid chemicals.

bioengineering↗

bioRxiv2024

A rapid CAT transformation protocol and nuclear transgene expression tools for metabolic engineering in Cyanidioschyzon merolae 10D

The eukaryotic red alga Cyanidioschyzon merolae 10D is an emerging algal host for synthetic biology and metabolic engineering. Its small nuclear genome (16.5 Mb; 4775 genes), low intron content (38), stable transgene expression, and capacity for homologous recombination into its nuclear genome make it ideal for genetic and metabolic engineering endeavors. Here, we present an optimized transformation and selection protocol, which yields single chloramphenicol-resistant transformants in under two weeks. Transformation dynamics and a synthetic modular plasmid toolkit are reported, including several new fluorescent reporters. Techniques for fluorescence reporter imaging and analysis at different scales are presented to facilitate high-throughput screening of C. merolae transformants. We use this plasmid toolkit to overexpress the Ipomoea batatas isoprene synthase and demonstrate the dynamics of engineered volatile isoprene production during different light regimes using multi-port headspace analysis coupled to parallel photobioreactors. This work seeks to promote C. merolae as an algal system for metabolic engineering and future sustainable biotechnological production.

bioengineering↗

bioRxiv2023

Comparison of alternative solvents for in situ extraction of hydrocarbons from the colonial green alga Botryococcus braunii race B (Showa)

The colony-forming, green microalga Botryococcus braunii secretes petroleum-like hydrocarbons, which enables the non-destructive continuous in situ extraction, milking, of these extracellular products during culture growth without cell lysis. This work compares the suitability of 15 different solvents, including alkanes, halogenated solvents, and green solvents, for in situ extraction of B. braunii race B (Showa strain) hydrocarbons after acclimation to moderate salinity stress. After 24 h of extraction, bio-based terpene green solvents such as {gamma}-terpinene showed the highest hydrocarbon recovery, around 10-fold greater than with conventional alkane solvents. Brominated alkanes and liquid perfluorocarbons (FCs) formed a lower phase to algal cultures rather than an upper phase as with other solvents, but only bromodecane effectively captured extracellular hydrocarbons similar to conventional alkane solvents. However, bromodecane and all green solvents were too toxic for two-phase continuous culture contact hydrocarbon milking, leading to 33-100% chlorophyll content loss. To overcome the biologically adverse effects of these solvents with suitable hydrocarbon recovery, future research should focus on their application in short-term extraction period milking systems to minimize algal-solvent contact and enable continuous extraction. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=98 SRC="FIGDIR/small/563540v2_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@1072e6borg.highwire.dtl.DTLVardef@787c8forg.highwire.dtl.DTLVardef@39af4dorg.highwire.dtl.DTLVardef@123ed79_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO C_FIG

biochemistry↗

bioRxiv2023

A green alternative to fragrant agarwood sesquiterpenoid production

Certain endangered Thymelaeaceous trees are major sources of the fragrant and highly valued resinous agarwood, comprised of hundreds of oxygenated sesquiterpenoids (STPs). Despite growing pressure on natural agarwood sources, the chemical complexity of STPs severely limits synthetic production. Here, we catalogued the chemical diversity in 58 agarwood samples by two-dimensional gas chromatography-mass spectrometry and partially recreated complex STP mixtures through synthetic biology. We improved STP yields in the unicellular alga Chlamydomonas reinhardtii by combinatorial engineering to biosynthesise nine macrocyclic STP backbones found in agarwood. A bioprocess following green-chemistry principles was developed that exploits milking of STPs without cell lysis, solvent-solvent STP extraction, solvent-STP nanofiltration, and bulk STP oxy-functionalisation to obtain terpene mixtures like those of agarwood. This process occurs with total solvent recycling and enables continuous production. Our synthetic-biology approach offers a sustainable alternative to harvesting agarwood trees to obtain mixtures of complex, fragrant, oxygenated STPs.

synthetic biology↗

bioRxiv2023

Engineered ketocarotenoid biosynthesis in the polyextremophilic red microalga Cyanidioschyzon merolae 10D

The polyextremophilic Cyanidiales are eukaryotic red microalgae with promising biotechnological properties arising from their low pH and elevated temperature requirements which can minimize culture contamination at scale. Cyanidioschyzon merolae 10D is a cell wall deficient species with a fully sequenced genome that is amenable to nuclear transgene integration by targeted homologous recombination. C. merolae maintains a minimal carotenoid profile and here, we sought to determine its capacity for ketocarotenoid accumulation mediated by heterologous expression of a green algal {beta}-carotene ketolase (BKT) and hydroxylase (CHYB). To achieve this, a synthetic transgene expression cassette system was built to integrate and express Chlamydomonas reinhardtii (Cr) sourced enzymes by fusing native C. merolae transcription, translation and chloroplast targeting signals to codon-optimized coding sequences. Chloramphenicol resistance was used to select for the integration of synthetic linear DNAs into a neutral site within the host genome. CrBKT expression caused accumulation of canthaxanthin and adonirubin as major carotenoids while co-expression of CrBKT with CrCHYB generated astaxanthin as the major carotenoid in C. merolae. Unlike green algae and plants, ketocarotenoid accumulation in C. merolae did not reduce total carotenoid contents, but chlorophyll a reduction was observed. Light intensity affected global ratios of all pigments but not individual pigment compositions and phycocyanin contents were not markedly different between parental strain and transformants. Continuous illumination was found to encourage biomass accumulation and all strains could be cultivated in simulated summer conditions from two different extreme desert environments. Our findings present the first example of carotenoid metabolic engineering in a red eukaryotic microalga and open the possibility for use of C. merolae 10D for simultaneous production of phycocyanin and ketocarotenoid pigments. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=71 SRC="FIGDIR/small/530181v2_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@dd010dorg.highwire.dtl.DTLVardef@1700e5corg.highwire.dtl.DTLVardef@1bee8eaorg.highwire.dtl.DTLVardef@ad67da_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2023

Cultivation of the polyextremophile Cyanidioschyzon merolae 10D during summer conditions on the coast of the Red Sea and its adaptation to hypersaline sea water

The west coast of Saudi Arabia borders the Red Sea, which maintains high average temperatures and increased salinity compared to other seas or oceans. Summer conditions in the Arabian Peninsula may exceed the temperature tolerance of most currently cultivated microalgae. The Cyanidiales are polyextremophilic red algae whose native habitats are at the edges of acidic hot springs. Cyanidioschyzon merolae 10D has recently emerged as an interesting model organism capable of high-cell density cultivation on pure CO2 with optimal growth at 42 {degrees}C and low pH between 0.5-2. C. merolae biomass has an interesting macromolecular composition, is protein rich, and contains valuable bio-products like heat-stable phycocyanin, carotenoids, {beta}-glucan, and starch. Here, photobioreactors were used to model C. merolae 10D growth performance in simulated environmental conditions of the mid-Red Sea coast across four seasons, it was then grown at various scales outdoors in Thuwal, Saudi Arabia during the Summer of 2022. We show that C. merolae 10D is amenable to cultivation with industrial-grade nutrient and CO2 inputs outdoors in this location and that its biomass is relatively constant in biochemical composition across culture conditions. We also show the adaptation of C. merolae 10D to high salinity levels of those found in Red Sea waters and conducted further modeled cultivations in nutrient enriched local sea water. It was determined that salt-water adapted C. merolae 10D could be cultivated with reduced nutrient inputs in local conditions. The results presented here indicate this may be a promising alternative species for algal bioprocesses in outdoor conditions in extreme desert summer environments.

bioengineering↗

bioRxiv2023

Engineered production of isoprene from the model green microalga Chlamydomonas reinhardtii

1.Isoprene is a clear, colorless, volatile 5-carbon hydrocarbon that is one monomer of all cellular isoprenoids and a platform chemical with multiple applications in industry. Many plants have evolved isoprene synthases (IspSs) with the capacity to liberate isoprene from dimethylallyl pyrophosphate (DMAPP) as part of cellular protection mechanisms. Isoprene is hydrophobic and volatile, rapidly leaves plant tissues and is one of the main carbon emission sources from vegetation globally. The universality of isoprenoid metabolism allows volatile isoprene production from microbes expressing heterologous IspSs. Here, we compared heterologous overexpression from the nuclear genome and localization into the plastid of four plant terpene synthases (TPs) in the green microalga Chlamydomonas reinhardtii. Using sealed vial mixotrophic cultivation, direct quantification of isoprene production was achieved from the headspace of living cultures, with the highest isoprene production observed in algae expressing the Ipomoea batatas IspS. Perturbations of the downstream carotenoid pathway through keto carotenoid biosynthesis enhanced isoprene titers, which could be further enhanced by increasing flux towards DMAPP through heterologous co-expression of a yeast isopentenyl-PP delta isomerase. Multiplexed controlled-environment testing revealed that cultivation temperature, rather than illumination intensity, was the main factor affecting isoprene yield from the engineered alga. This is the first report of heterologous isoprene production from a eukaryotic alga and sets a foundation for further exploration of carbon conversion to this commodity chemical. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=72 SRC="FIGDIR/small/523746v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@95085aorg.highwire.dtl.DTLVardef@575e72org.highwire.dtl.DTLVardef@1f9c2dborg.highwire.dtl.DTLVardef@67f252_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2022

Biomass generation and heterologous isoprenoid milking from engineered microalgae grown in anaerobic membrane bioreactor effluent

1.Wastewater (WW) treatment in anaerobic membrane bioreactors (AnMBR) is considered more sustainable than in their aerobic counterparts. However, outputs from AnMBR are mixed methane and carbon dioxide gas streams as well as ammonium- (N) and phosphate- (P) containing waters. Using AnMBR outputs as inputs for photoautotrophic algal cultivation can strip the CO2 and remove N and P from effluent which feed algal biomass generation. Recent advances in algal engineering have generated strains for concomitant high-value side product generation in addition to biomass, although only shown in heavily domesticated, lab-adapted strains. Here, investigated whether such a strain of Chlamydomonas reinhardtii could be grown directly in AnMBR effluent with CO2 at concentrations found in its off-gas. The domesticated strain was found to proliferate over bacteria in the non-sterile effluent, consume N and P to levels that meet general discharge or reuse limits, and tolerate cultivation in modelled (extreme) outdoor environmental conditions prevalent along the central Red Sea coast. High-value co-product milking was then demonstrated, up to 837 g L-1 culture in 96 h, in addition to algal biomass production, [~]2.4 g CDW L-1 in 96 h, directly in effluents. This is the first demonstration of a combined bio-process that employs a heavily engineered algal strain to enhance the product generation potentials from AnMBR effluent treatment. This study shows it is possible to convert waste into value through use of engineered algae while also improve wastewater treatment economics through co-product generation. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=124 SRC="FIGDIR/small/510234v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@a7e325org.highwire.dtl.DTLVardef@31c0a2org.highwire.dtl.DTLVardef@5deea8org.highwire.dtl.DTLVardef@df672b_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2022

Continuous extraction and concentration of secreted metabolites from engineered microbes using membrane technology

Microalgal cultivation in photobioreactors and membrane separations are both considered sustainable processes. Here we explore their synergistic combination to extract and concentrate a heterologous sesquiterpenoid produced by engineered green algal cells. A hydrophobic hollow-fiber membrane contactor was used to allow interaction of culture broth and cells with a dodecane solvent phase to accumulate algal produced patchoulol. Subsequent continuous membrane extraction of patchoulol from dodecane enabled product concentration in a methanol stream as well as dodecane recovery for its reuse. A structure-based prediction using machine learning was used to model a process whereby 100% patchoulol recovery from dodecane could be achieved with solvent-resistant nanofiltration membranes. Solvent consumption, E-factor, and economic sustainability were assessed and compared with existing patchoulol production processes. Our extraction and product purification process offers six- and two-orders of magnitude lower solvent consumption compared to synthetic production and thermal-based separation, respectively. Our proposed methodology is transferable to other microbial systems for the isolation of high-value isoprenoid and hydrocarbon products.

bioengineering↗

bioRxiv2022

Combinatorial engineering for photoautotrophic production of recombinant products from the green microalga Chlamydomonas reinhardtii

Chlamydomonas reinhardtii has emerged as a powerful green cell factory for metabolic engineering of sustainable products created from the photosynthetic lifestyle of this microalga. Advances in nuclear genome and transgene expression engineering are allowing robust engineering strategies to be demonstrated in this host. However, commonly used lab strains are not equipped with features to enable their broader implementation in non-sterile conditions and high-cell density concepts. Here, we use combinatorial chloroplast and nuclear genome engineering to complement the C. reinhardtii strain UVM4 with publicly available genetic tools to enable the use of inorganic phosphite and nitrate as a sole the source phosphorous and nitrogen, respectively. We present recipes to create phosphite-buffered media solutions that enable high cell density algal cultivation. We then combine previously reported engineering strategies to produce the heterologous sesquiterpenoid patchoulol to high titers from our engineered green cell factories and show these products are possible to produce in under non-sterile conditions. Our work presents a straightforward means to generate C. reinhardtii strains for broader application in bio-processes for the sustainable generation of products.

bioengineering↗

bioRxiv2022

Biocompatible fluorocarbon liquid underlays for in situ extraction of isoprenoids from microbial cultures

Microbial production of heterologous metabolites is now a mature technology in many host organisms, opening new avenues for green production processes for specialty chemicals. At lab scale, petroleum-based hydrophobic bio-compatible solvents like dodecane can be used as a second phase on top of microbial cultures to act as a physical sink for heterologous hydrocarbon products like isoprenoids. However, this approach has significant drawbacks at scale due to the difficulty of handling solvents and their potential contamination with unwanted byproducts of their manufacture. We discovered that synthetic perfluorocarbon liquids (FCs), commonly used for heat transfer, can also act as physical sinks for microbially produced isoprenoid compounds. FCs are stable, inert, and are amenable to direct liquid-liquid extraction with alcohols for rapid product isolation. These liquids are more dense than water and form a lower phase to microbial cultures rather than an upper phase as with other solvents. Their ability to form an under-layer or underlay also enables the cultivation of microbes directly at the FC-culture medium interface via gravity settling, which could open their application for filamentous or mat-forming organisms. We present comparisons of the isoprenoid extraction potential of three commercial FCs: FC-3283, FC-40, and FC-770 with engineered green microalga cultures producing patchoulol, taxadiene, casbene, or 13R(+) manoyl oxide. We demonstrate that FCs are promising alternatives to traditional solvents and open new avenues in bio-process design for microbial heterologous metabolite milking.

synthetic biology↗