Biology subjects

Wellman, G. B.

Publications and source records attributed to Wellman, G. B..

8 recordsLinked to original sources

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

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

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

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 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

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↗