Biology subjects

Moseley, J. L.

Publications and source records attributed to Moseley, J. L..

6 recordsLinked to original sources

bioRxiv2026

A Natural qE- and qZ-Deficient Alga Retains Functional Plant-Like and Chlorophycean Violaxanthin De-Epoxidases

Photosynthetic organisms protect themselves from excess light through non-photochemical quenching (NPQ), a process that safely dissipates excess light energy as heat. Current understanding of NPQ is largely drawn from a few model systems, leaving it unclear how universal NPQ mechanisms are across the green lineage. We characterize the slow, sustained NPQ of the green alga, Auxenochlorella protothecoides x symbiontica (UTEX 250-A). The Auxenochlorella genome lacks Light-Harvesting Complex Stress Related (LHCSR), one of the two proteins required for the fast energy-dependent quenching (qE) component of NPQ. Mutants lacking the Photosystem II Subunit S (PSBS)-like gene (psbsl1) in Auxenochlorella had no effect on NPQ, indicating this alga lacks canonical qE. We identified two candidate enzymes associated with zeaxanthin-dependent quenching (qZ), which has slower kinetics: a plant-type Violaxanthin De-Epoxidase (VDE) and an algal-type Chlorophycean Violaxanthin De-Epoxidase (CVDE). High light did not trigger the expected conversion of violaxanthin to zeaxanthin, a pigment change that is a hallmark of qZ. Mutants lacking VDE (vde1), CVDE (cvde1), or both enzymes (vde1 cvde1) still displayed slow, reversible NPQ. However, when the Auxenochlorella VDE1 and CVDE1 were expressed in a VDE-deficient mutant of Nicotiana benthamiana, both enzymes restored zeaxanthin production and NPQ, confirming they are catalytically functional de-epoxidases. Together, these results reveal an alga that retains two functional, evolutionarily distinct xanthophyll-cycle enzymes yet does not rely on typical xanthophyll pigment dynamics for photoprotection. This points to a biological function that is decoupled from de-epoxidase activity, highlighting unexplored diversity in green algal photoprotection.

plant biology↗

bioRxiv2026

Bioengineered algal lipids enriched in structured medium- and long-chain triacylglycerols, linoleate, and sn-2 palmitate for human milk fat substitutes

Triacylglycerols (TAGs) in human milkfat (HMF) provide over 50% of the calories for infant nutrition. Their unique architecture, featuring palmitic acid predominantly esterified at the sn-2 position, facilitates absorption as 2-palmitoyl monoacylglycerol after hydrolysis of the sn-1 and sn-3 fatty acids by gut lipases. Conventional vegetable oil-based infant formulas mimic the HMF fatty acid profile but lack its distinct regioisomeric structure, abundant medium- and long-chain triglycerides (MLCTs), and oleic-to-linoleic acid ratios that vary with maternal diet and geography. We have engineered the oleaginous green alga Auxenochlorella for biosynthesis of TAGs enriched in MLCTs, sn-2 palmitate, and linoleate via heterologous expression of acyl-acyl-carrier-protein (ACP) thioesterases, palmitate-specific lysophosphatidic acid acyltransferases, and lysophosphatidylcholine acyltransferase. These structured algal lipids replicate both the major fatty acid proportions and regioisomeric composition of authentic HMF, providing single-source bioengineered alternatives to plant-derived HMF substitutes for infant formula.

synthetic biology↗

bioRxiv2025

A defined growth medium for trace metal limitation studies in Auxenochlorella sp. (UTEX 250-A) during mixotrophic growth with glucose.

Auxenochlorella sp. (strain UTEX 250-A) is a fast-growing, oleaginous alga that is an emerging reference organism for basic research, with broad biotechnological applications. Advancing UTEX 250-A as a biotechnological workhorse requires a deeper understanding of its nutritional demands, particularly under different trophic conditions. However, little is known about the specific acclimations that allow UTEX 250-A to thrive when essential nutrients are scarce. Here, we describe the formulation of a defined growth medium that supports the cultivation of UTEX 250-A with controlled trace levels of the essential micronutrients iron, copper, and zinc. Special attention was given to ensure that the medium was compatible with inductively coupled plasma mass spectrometry (ICP-MS) analysis, enabling the detection and minimization of metal contamination in the nanomolar range. The medium was designed to provide sufficient nutrients to support the mixotrophic growth of UTEX 250-A from an initial density of 105 cells/mL to a stationary density of 2.4x108 cells/mL, with additional nutrients supplied to accommodate metabolic and trophic transitions during stationary phase, when photosynthesis is restored due to carbon limitation. This replete medium provides a foundation for robust nutrient limitation studies in Auxenochlorella sp. (UTEX 250-A).

systems biology↗

bioRxiv2025

Allodiploid hybridization, loss-of-heterozygosity and aneuploidy in the green alga Auxenochlorella, an emerging model for discovery research and bioengineering

Auxenochlorella spp. are diploid oleaginous green algae whose streamlined genomes can be readily manipulated by homologous recombination, making them highly amenable to discovery research and bioengineering. Vegetatively diploid organisms experience specific evolutionary phenomena, including allodiploid hybridization, mitotic recombination, loss-of-heterozygosity and aneuploidy; however, studies of these forces have largely focused on yeasts. Here, we present a telomere-to-telomere phased diploid genome assembly of Auxenochlorella UTEX 250-A (haploid length 22 Mb) and introduce a genetic toolkit for site-specific manipulation of the nuclear genome in multiple strains, featuring several selectable markers, inducible promoters, and fluorescent reporters for protein localization. UTEX 250-A is an allodiploid hybrid of Auxenochlorella protothecoides and Auxenochlorella symbiontica, two species differentiated by extensive chromosomal rearrangements. UTEX 250-A haplotypes are a mosaic of each parental species following mitotic recombination, and two chromosomes are trisomic. Loss-of-heterozygosity events are pervasive across Auxenochlorella and can evolve rapidly in the laboratory. High-quality structural annotation yielded [~]7,500 genes per haplotype. Auxenochlorella have experienced gene family loss and reduction, including core photosynthesis genes, and exhibit periodic adenine and cytosine methylation at promoters and gene bodies, respectively. Approximately 10% of genes, especially those involved in DNA repair and sex, overlap antisense long noncoding RNAs, which may participate in a regulatory mechanism. We demonstrate the utility of Auxenochlorella for fundamental research by knockout of a chlorophyll biosynthesis enzyme, and confirm one trisomy by allele-specific transformation. These results demonstrate the generality of several evolutionary forces associated with vegetative diploidy and provide a foundation for use of Auxenochlorella as a reference organism. One-sentence summaryAuxenochlorella, green algae shaped by evolutionary forces acting on vegetative diploids, are amenable to discovery research and bioengineering via efficient site-specific homologous recombination

plant biology↗

bioRxiv2024

Leaky ribosomal scanning enables tunable translation of bicistronic ORFs in green algae.

Advances in sequencing technology have unveiled examples of nucleus-encoded polycistronic genes, once considered rare. Exclusively polycistronic transcripts are prevalent in green algae, although the mechanism by which multiple polypeptides are translated from a single transcript is unknown. Here, we used bioinformatic and in vivo mutational analyses to evaluate competing mechanistic models for polycistronic expression in green algae. High-confidence manually curated datasets of bicistronic loci from two divergent green algae, Chlamydomonas reinhardtii and Auxenochlorella protothecoides, revealed 1) a preference for weak Kozak-like sequences for ORF 1 and 2) an underrepresentation of potential initiation codons before ORF 2, which are suitable conditions for leaky scanning to allow ORF 2 translation. We used mutational analysis in Auxenochlorella protothecoides to test the mechanism. In vivo manipulation of the ORF 1 Kozak-like sequence and start codon altered reporter expression at ORF 2, with a weaker Kozak-like sequence enhancing expression and a stronger one diminishing it. A synthetic bicistronic dual reporter demonstrated inversely adjustable activity of green fluorescent protein expressed from ORF 1 and luciferase from ORF 2, depending on the strength of the ORF 1 Kozak-like sequence. Our findings demonstrate that translation of multiple ORFs in green algal bicistronic transcripts is consistent with episodic leaky ribosome scanning of ORF 1 to allow translation at ORF 2. This work has implications for the potential functionality of upstream open reading frames found across eukaryotic genomes and for transgene expression in synthetic biology applications. Significance StatementTextbook dogma states that nucleus-encoded genes are monocistronic, producing transcripts with a single translated open reading frame. However, highly conserved bicistronic loci are pervasive in green algae that are separated by several hundred million years of evolution, speaking to their ancestral origins and functions within the Chlorophyte lineage. A combination of bioinformatic analysis and in vivo gene manipulation supports leaky ribosomal scanning as the primary mechanism for translation of multiple ORFs from bicistronic transcripts. We have successfully tuned synthesis levels of two proteins encoded on one mRNA by modifying the ORF 1 Kozak-like sequence. These findings may have broad applications in synthetic biology.

plant biology↗

bioRxiv2023

Distinct function of Chlamydomonas CTRA-CTR transporters in Cu assimilation and intracellular mobilization

Successful acclimation to copper (Cu) deficiency involves a fine balance between Cu import and export. In the unicellular green alga Chlamydomonas reinhardtii, Cu import is dependent on Copper Response Regulator 1 (CRR1), the master regulator of Cu homeostasis. Among CRR1 target genes are two Cu transporters belonging to the CTR/COPT gene family (CTR1 and CTR2) and a related soluble cysteine-rich protein (CTR3). The ancestor of these green algal proteins was likely acquired from an ancient chytrid and contained conserved cysteine-rich domains (named the CTR-associated domains, CTRA) that are predicted to be involved in Cu acquisition. We show by reverse genetics that Chlamydomonas CTR1 and CTR2 are canonical Cu importers albeit with distinct affinities, while loss of CTR3 did not result in an observable phenotype under the conditions tested. Mutation of CTR1, but not CTR2, recapitulate the poor growth of crr1 in Cu-deficient medium, consistent with a dominant role for CTR1 in high affinity Cu(I) uptake. Notably, the over-accumulation of Cu(I) in Zinc (Zn)-deficiency (20 times the quota) depends on CRR1 and both CTR1 and CTR2. CRR1-dependent activation of CTR gene expression needed for Cu over-accumulation can be bypassed by the provision of excess Cu in the growth medium. Over-accumulated Cu is sequestered into the acidocalcisome but can become remobilized by restoring Zn nutrition. This mobilization is also CRR1-dependent, and requires activation of CTR2 expression, again distinguishing CTR2 from CTR1 and is consistent with the lower substrate affinity of CTR2.

biochemistry↗