Biology subjects

Decker, E. L.

Publications and source records attributed to Decker, E. L..

At least 19 recordsLinked to original sources

bioRxiv2025

Production of prolyl-hydroxylated human type III collagen in Physcomitrella photobioreactors

Collagens are structural proteins of the extracellular matrix essential for skin elasticity and integrity. They are widely used in dietary supplements and cosmetics. Conventional collagens of animal origin raise concerns regarding ethics, safety, and sustainability. As a vegan alternative, we report on the production of a 30 kDa prolyl-hydroxylated human collagen polypeptide from Physcomitrella moss plants. For secretion-based production and formulation compatibility, a hydrophilic region encompassing 334 amino acids from human type III collagen was selected, which includes four protein domains involved in cell adhesion, collagen binding, integrin recognition and wound healing. Transgenic moss lines were generated via protoplast transformation. Immunodetection identified collagen producing lines, and mass spectrometry validated the product and detected prolyl-hydroxylation on 23 sites. The presence of this important post-translational modification underscores the high biomimetic quality of the product. To enable industrial-scale production, the transformants were quantitatively analysed at the genomic, transcript, and protein levels. The most productive lines were forwarded to process development, where culture conditions including CO2 supplementation, pH, and light intensity were optimized. Upscaling to 5 L photobioreactors established a robust, light- and biomass-dependent production regime that yielded nearly 1 mg/L of secreted collagen polypeptide in the culture supernatant after 11 days of cultivation. Taken together, this study presents the first scalable moss-based production of a post-translationally modified human collagen and offers a sustainable and vegan alternative to conventional collagens for cosmetic formulations. This highlights the versatility of Physcomitrella as a production host for high-quality proteins with industrial applicability that also meet consumer requirements. Key messageScalable moss bioreactors enable the production of high-quality recombinant prolyl-hydroxylated human collagen without heterologous P4H expression, offering a sustainable and vegan alternative to conventional collagens derived from animals.

plant biology↗

bioRxiv2025

Functional Characterization of Target of Rapamycin (TOR) Signalling in Physcomitrella

Target of rapamycin (TOR) is a conserved protein kinase and an important signalling hub in eukaryotes. The moss Physcomitrella (Physcomitrium patens) is a model organism for plant physiology, development, and evolution. However, little is known about TOR signalling in non-vascular plants, including Physcomitrella. Here, we report the effects of inhibiting TOR signalling in Physcomitrella. We identified and characterized Physcomitrella 12-kD FK506-binding protein (FKBP12), which binds TOR in the presence of rapamycin. Whereas the growth of wild-type protonema is unaffected by rapamycin, overexpressing endogenous FKBP12 rendered the plant susceptible to the inhibitor in a dose-dependent manner. Likewise, protonema growth was inhibited when the TOR-specific inhibitor AZD8055 was present in the culture. We show that rapamycin and AZD8055 have pleiotropic effects, as they delay cell cycle progression and development, induce chlorosis, inhibit photosynthesis, and alter total protein content. Additionally, we identified and characterized PpTOR, PpLST8, and PpRAPTOR, key components of TOR complex 1 (TORC1), whereas RICTOR and mSIN1 of TORC2 are not encoded by the P. patens genome. We found that PpLST8 substitutes its homolog in yeast to allow cell growth. Physcomitrella mutants were generated with a conditional downregulation of PpTOR, PpLST8, and PpRAPTOR, respectively. They were impaired in growth. Finally, we show TOR-dependent phosphorylation of a well-known TOR phospho-target, the ribosomal protein RPS6, via LC-MS/MS. Collectively, our results show that Physcomitrella growth and development is positively controlled by a conserved TOR kinase. We suggest to further dissect TOR signalling in Physcomitrella in order to elucidate signalling integration via TORC1 in plants. Key messageThe evolutionary conserved TOR kinase positively controls growth and development of the moss Physcomitrella, development and function of its chloroplasts, its protein synthesis and cell cycle progression.

plant biology↗

bioRxiv2025

Production of HPV-16 L1 virus-like particles in Physcomitrella photobioreactors

Virus-like particles (VLPs) are self-assembling nanoparticles composed of viral structural proteins which mimic native virions but lack viral DNA and infectivity. VLPs are a resourceful class of biopharmaceuticals applied as subunit vaccines or as delivery vehicles for drugs and nucleic acids. Similar to viruses, VLPs are diverse in structure, composition, and assembly, requiring a tailored production platform aligned with the intended application. The moss plant Physcomitrella (Physcomitrium patens) is an emerging expression system offering humanized N-glycosylation, scalability, and adaptability to existing industry settings. Here, we used Physcomitrella to produce human papillomavirus (HPV) 16 VLPs. HPV VLPs are composed of the major structural protein L1 and are used as vaccines against HPV infections which are the main causal agent of cervical and other anogenital cancers. We characterized Physcomitrella chloroplast transit peptides, which we used for targeting of moss-produced L1 to chloroplasts, leading to higher recombinant protein yield compared to nuclear or cytoplasmic localization. We confirmed subcellular localization with confocal laser scanning microscopy and found L1 to accumulate within the chloroplast stroma. Production in 5-liter photobioreactors yielded over 0.3 mg L1 per gram fresh weight. We established a purification protocol for moss-produced L1 using a combination of ammonium sulfate precipitation and cation exchange chromatography. Purified samples were subjected to a controlled dis- and reassembly, yielding fully assembled HPV-16 L1 VLPs. This is the first report of production, purification, and assembly of VLPs in a non-vascular plant.

plant biology↗

bioRxiv2024

Recombinant production of spider silk protein in Physcomitrella photobioreactors

Spider dragline silk stands out as a remarkable biomaterial, representing one of natures toughest fibres. Its strength rivals that of many synthetic fibres used commercially, rendering it applicable across various industrial and medical domains. However, its widespread utilisation requires cost-effective mass production. Biotechnology presents a promising avenue for achieving this goal, particularly through the production of recombinant dragline silk proteins in transgenic plant systems. This study aimed to assess the feasibility of producing one key protein component of dragline silk, MaSp1, from the western black widow spider, Latrodectus hesperus, (LhMaSp1) in the moss Physcomitrella (Physcomitrium patens). Here, we present the successful recombinant production of spider silk protein containing both the N- and C-terminal domains (NTD and CTD) of LhMaSp1 protein in moss cells. NTD and CTD are necessary domains for protein assembly in spider silk. The production of recombinant LhMaSp1 protein in Physcomitrella was performed in shake flasks and in five-litre photobioreactors and the correct synthesis of LhMaSp1 was proven via mass spectrometry. Key messageWe report the first successful plant-produced recombinant spider silk key protein component containing both the N- and the C-terminal domain.

plant biology↗

bioRxiv2024

Thrombospondin-1 inhibits alternative complement pathway activation in vasculitis synergistically to factor H

Complement activation is a relevant driver in the pathomechanisms of vasculitis. The involved proteins in the interaction between endothelia, complement and platelets in these conditions are only partially understood. Thrombospondin-1 (TSP-1), found in platelet -granules and released from activated endothelial cells, interacts with factor H (FH) and von Willebrand factor (vWF). However, direct regulatory interaction with the complement cascade has not yet been described. We could show that TSP-1 is a potent, FH-independent inhibitor of the alternative complement pathway. TSP-1 binds to complement proteins, inhibits cleavage of C3 and C5 and the formation of the membrane attack complex. Complement-regulatory function is validated in blood samples from patients with primary complement defects. Physiological relevance of TSP-1 is demonstrated in ANCA-vasculitis patients by significantly enhanced TSP-1 staining in glomerular lesions and increased complement activity and NETosis following TSP-1 deficiency in an ANCA-vasculitis model. The newly described complement-inhibiting function of TSP-1 represents an important mechanism in the interaction of endothelia, complement and platelets. In particular, the interplay between released TSP-1, vWF and the complement system locally, especially on surfaces, influences the balance between complement activation and inhibition and may be relevant in various vascular diseases.

molecular biology↗

bioRxiv2024

Differential prolyl hydroxylation by six Physcomitrella prolyl-4 hydroxylases

The hydroxylation of proline residues to 4-trans-hydroxyproline (Hyp) is a common post-translational protein modification in plants, mediated by prolyl 4-hydroxylases (P4Hs). Hyps predominantly occur in a group of cell wall proteins, the Hydroxyproline-rich glycoproteins (HRGPs), where they are frequently O-glycosylated. While prolyl-hydroxylation and O-glycosylation are important, e.g. for cell wall stability, they are undesired on plant-made pharmaceuticals. Sequence motifs recognized for prolyl-hydroxylation derived from vascular plants were proposed but did not include data from mosses, such as Physcomitrella. Here, a phylogenetic reconstruction of plant P4Hs identified six P4Hs in four subfamilies in mosses. We analysed the amino acid sequences and structural environments around Hyps in Physcomitrella utilizing 73 Hyp sites in 24 secretory proteins from multiple MS/MS datasets, and found that prolines in close proximity to other prolines, alanine, serine, threonine and valine were preferentially hydroxylated. About 95% of the Hyp sites were predictable with a combination of previously defined motifs and methods. In our data, AOV (Ala-Hyp-Val) was the most frequent prolyl-hydroxylation pattern. Additionally, short arabinose chains were attached to Hyps in two cell-wall pectinesterases. A combination of 443 AlphaFold structure models and our MS data of peptides with nearly 3000 proline sites found Hyps predominantly on protein surfaces in disordered regions. Moss-produced human erythropoietin (EPO) exhibited plant-specific O-glycosylation with arabinose chains on two Hyps. This modification was significantly reduced in a P4H1 single knock-out (KO) Physcomitrella mutant. Quantitative proteomics after isotope labelling with different P4H-KO moss mutants revealed specific changes in the amount of proteins, including HRGPs, and modified prolyl-hydroxylation pattern from the mutants, suggesting a differential function of the six Physcomitrella P4Hs. Quantitative RT-PCR proved a differential effect of single P4H KOs on the expression of the other five p4h genes, suggesting a partial compensation of the mutation. AlphaFold-Multimer models for Physcomitrella P4H1 and its target EPO peptide superposed with the crystal structure of Chlamydomonas P4H1 and a peptide substrate suggested significant amino acids in the active centre of the enzyme that form H-bonds with the peptide substrate, and revealed differences between P4H1 and the other five Physcomitrella P4Hs. HighlightsMosses, such as Physcomitrella, encode six prolyl 4-hydroxylases in four subfamilies Computational analysis reveals specific environment for proline hydroxylation AlphaFold structure models and mass spectrometry reveal features of modification Quantitative proteomics and mutant analysis identify relevant modifying enzyme Modelling suggests relevant amino acids in active centre of the modifying enzyme

plant biology↗

bioRxiv2024

Differential GTP-dependent in-vitro polymerization of recombinant Physcomitrella FtsZ proteins

1.Bacterial cell division and plant chloroplast division require self-assembling Filamentous temperature-sensitive Z (FtsZ) proteins. FtsZ proteins are GTPases sharing structural and biochemical similarities with eukaryotic tubulin. In the moss Physcomitrella, the morphology of the FtsZ polymer networks varies between the different FtsZ isoforms. The underlying mechanism and foundation of the distinct networks is unknown. Here, we investigated the interaction of Physcomitrella FtsZ2-1 with FtsZ1 isoforms via co-immunoprecipitation and mass spectrometry, and found protein-protein interaction in vivo. We tagged FtsZ1-2 and FtsZ2-1 with different fluorophores and expressed both in E. coli, which led to the formation of defined structures within the cells and to an influence on bacterial cell division and morphology. Furthermore, we have optimized the purification protocols for FtsZ1-2 and FtsZ2-1 expressed in E. coli and characterized their GTPase activity and polymerization in vitro. Both FtsZ isoforms showed GTPase activity. Stoichiometric mixing of both proteins led to a significantly increased GTPase activity, indicating a synergistic interaction between them. In light scattering assays, we observed GTP-dependent assembly of FtsZ1-2 and of FtsZ2-1 in a protein concentration dependent manner. Stoichiometric mixing of both proteins resulted in significantly faster polymerization, again indicating a synergistic interaction between them. Under the same conditions used for GTPase and light scattering assays both FtsZ isoforms formed filaments in a GTP-dependent manner as visualized by transmission electron microscopy (TEM). Taken together, our results reveal that Physcomitrella FtsZ1-2 and FtsZ2-1 are functionally different, can synergistically interact in vivo and in vitro, and differ in their properties from FtsZ proteins from bacteria, archaea and vascular plants.

plant biology↗

bioRxiv2023

Multifactorial analysis of terminator performance on heterologous gene expression in Physcomitrella

The production of recombinant proteins for health applications accounts for a large share of the biopharmaceutical market. While many drugs are produced in microbial and mammalian systems, plants gain more attention as expression hosts to produce eukaryotic proteins. In particular the GMP-compliant moss Physcomitrella (Physcomitrium patens) has outstanding features such as excellent genetic amenability, reproducible bioreactor cultivation, and humanized protein glycosylation patterns. In this study, we selected and characterized novel terminators for their effects on heterologous gene expression. The Physcomitrella genome contains 53,346 unique 3UTRs (untranslated regions) of which 7,964 transcripts contain at least one intron. Over 91% of 3UTRs exhibit more than one polyadenylation site, indicating the prevalence of alternative polyadenylation in Physcomitrella. Out of all 3UTRs, 14 terminator candidates were selected and characterized via transient Dual Luciferase assays, yielding a collection of endogenous terminators performing equally high as established heterologous terminators CaMV35S, AtHSP90, and NOS. High performing candidates were selected for testing as double terminators which impact reporter levels, dependent on terminator identity and positioning. Testing of 3UTRs among the different promoters NOS, CaMV35S, and PpActin5 showed an increase of more than 1,000-fold between promoters PpActin5 and NOS, whereas terminators increased reporter levels by less than 10-fold, demonstrating the stronger effect promoters play as compared to terminators. The number of polyadenylation sites as well as polyadenylation signals were found to be major determinants of terminator performance. Our results improve the biotechnology platform Physcomitrella and further our understanding of how terminators influence gene expression in plants in general. Key messageCharacterization of Physcomitrella 3UTRs across different promoters yields endogenous single and double terminators for usage in molecular pharming and indicates promoters and terminators to synergistically control gene expression.

plant biology↗

bioRxiv2023

Putative NAD(P)-binding Rossmann fold protein is involved in chitosan-induced peroxidase activity and lipoxygenase expression in Physcomitrella

Oxidative burst, the rapid production of high levels of reactive oxygen species (ROS) in response to external stimuli, is an early defense reaction against pathogens. The fungal elicitor chitosan causes an oxidative burst in the moss Physcomitrella (new species name: Physcomitrium patens) mainly due to the peroxidase enzyme Prx34. To better understand the chitosan responses in Physcomitrella, we conducted a screen of part of a P. patens mutant collection to isolate plants with less peroxidase activity than wild-type plants after chitosan treatment. We isolated a P. patens mutant that affected the gene encoding NAD(P)-binding Rossmann fold protein (hereafter, Rossmann fold protein). Three Rossmann fold protein-knockout (KO) plants (named Rossmann fold KO-lines) were generated and used to assess extracellular peroxidase activity and expression of defense-responsive genes including alternative oxidase (AOX), lipoxygenase (LOX), NADPH-oxidase (NOX) and peroxidase (Prx34) in response to chitosan treatment. Extracellular (apoplastic) peroxidase activity was significantly lower in Rossmann fold KO-lines than in wild-type plants after chitosan treatments. Expression of the LOX gene in Rossmann-fold KO-plants was significantly lower before and after chitosan treatment when compared to WT. Peroxidase activity assays together with gene expression analyses suggest that the Rossmann-fold protein might be an important component of the signaling pathway leading to oxidative burst and basal expression of the LOX gene in Physcomitrella.

molecular biology↗

bioRxiv2022

Structural modelling of human complement FHR1 and two of its synthetic derivatives provides insight into their in-vivo functions

Human complement is the first line of defence against invading pathogens and is involved in tissue homeostasis. Complement-targeted therapies to treat several diseases caused by a dysregulated complement are highly desirable. Despite huge efforts invested in their development, only very few are currently available, and a deeper understanding of the numerous interactions and complement regulation mechanisms is indispensable. Two important complement regulators are human Factor H (FH) and Factor H-related protein 1 (FHR1). MFHR1 and MFHR13, two promising therapeutic candidates based on these regulators, combine the dimerization and C5-regulatory domains of FHR1 with the central C3-regulatory and cell surface-recognition domains of FH. Here, we used AlphaFold2 to model the structure of these two synthetic regulators. Moreover, we used AlphaFold-Multimer (AFM) to study possible interactions of C3 fragments and membrane attack complex (MAC) components C5, C7 and C9 in complex with FHR1, MFHR1, MFHR13 as well as the best-known MAC regulators vitronectin (Vn), clusterin and CD59, whose experimental structures remain undetermined. AFM successfully predicted the binding interfaces of FHR1 and the synthetic regulators with C3 fragments and suggested binding to C3. The models revealed structural differences in binding to these ligands through different interfaces. Additionally, AFM predictions of Vn, clusterin or CD59 with C7 or C9 agreed with previously published experimental results. Because the role of FHR1 as a MAC regulator has been controversial, we analysed possible interactions with C5, C7 and C9. AFM predicted interactions of FHR1 with proteins of the terminal complement complex (TCC) as indicated by experimental observations, and located the interfaces in FHR11-2 and FHR4-5. According to AFM predictions, FHR1 might partially block the C3b binding site in C5, inhibiting C5 activation, and block C5b-7 complex formation and C9 polymerization, with similar mechanisms of action as clusterin and vitronectin. Here, we generate hypotheses and provide the basis for the design of rational approaches to understand the molecular mechanism of MAC inhibition, which will facilitate the development of further complement therapeutics.

immunology↗

bioRxiv2022

A Physcomitrella PIN protein acts in spermatogenesis and sporophyte abortion

O_LIThe auxin efflux PIN-FORMED (PIN) proteins are conserved in all land plants and important players in plant development. In the moss Physcomitrella (Physcomitrium patens) three canonical PINs (PpPINA-C) are expressed in the leafy shoot (gametophore). PpPINA and PpPINB show functional activity in vegetative growth and sporophyte development. Here, we examined the role of PpPINC in the life cycle of Physcomitrella. C_LIO_LIWe established reporter and knockout lines for PpPINC and analysed vegetative and reproductive tissues using microscopy and transcriptomic sequencing of moss gametangia. C_LIO_LIPpPINC is expressed in immature leaves, mature gametangia and during sporophyte development. The sperm cells (spermatozoids) of pinC knockout mutants exhibit increased motility and an altered flagella phenotype. Further, the pinC mutants have a higher portion of differentially expressed genes (DEGs) related to spermatogenesis, increased fertility, and an increased abortion rate of premeiotic sporophytes. C_LIO_LIHere, we show that PpPINC is important for spermatogenesis and sporophyte retention. We propose an evolutionary conserved way of polar growth during early moss embryo development and sporophyte attachment to the gametophore, while suggesting the mechanical function in sporophyte retention of a ring structure, the Lorch ring. C_LI

plant biology↗

bioRxiv2021

Process engineering of biopharmaceutical production in moss bioreactors via model-based description and evaluation of phytohormone impact

The moss Physcomitrella is an interesting production host for recombinant biopharmaceuticals. Here we produced MFHR1, a synthetic complement regulator which has been proposed for the treatment of diseases associated to the complement system as part of human innate immunity. We studied the impact of different operation modes for the production process in 5 L stirred-tank photobioreactors. The total amount of recombinant protein was doubled by using fed-batch or batch compared to semi-continuous operation, although the maximum specific productivity (mg MFHR1/g FW) increased just by 35%. We proposed an unstructured kinetic model which fits accurately with the experimental data in batch and semi-continuous operation under autotrophic conditions with 2% CO2 enrichment. The model is able to predict recombinant protein production, nitrate uptake and biomass growth, which is useful for process control and optimization. We investigated strategies to further increase MFHR1 production. While mixotrophic and heterotrophic conditions decreased the MFHR1-specific productivity compared to autotrophic conditions, addition of the phytohormone auxin (NAA, 10 {micro}M) to the medium enhanced it by 470% in shaken flasks and up to 230% and 260%, in batch and fed-batch bioreactors, respectively. Supporting this finding, the auxin-synthesis inhibitor L-Kynurenine (100 {micro}M) decreased MFHR1 production significantly by 110% and 580% at day 7 and 18, respectively. Expression analysis revealed that the MFHR1 transgene, driven by the Physcomitrella actin5 (PpAct5) promoter, was upregulated 16 hours after NAA addition and remained enhanced over the whole process, whereas the auxin-responsive gene PpIAA1A was upregulated within the first two hours, indicating that the effect of auxin on PpAct5 promoter-driven expression is indirect. Furthermore, the day of NAA supplementation was crucial, leading to an up to 8-fold increase of MFHR1-specific productivity (0.82 mg MFHR1/ g fresh weight, 150 mg accumulated over 7 days) compared to the productivity reported previously. Our findings are likely to be applicable to other plant-based expression systems to increase biopharmaceutical production and yields.

bioengineering↗

bioRxiv2021

Unexpected arabinosylation after humanization of plant protein N-glycosylation

As biopharmaceuticals, recombinant proteins have become indispensable tools in medicine. An increasing demand, not only in quantity but also in diversity, drives the constant development and improvement of production platforms. The N-glycosylation pattern on biopharmaceuticals plays an important role in activity, serum half-life and immunogenicity. Therefore, production platforms with tailored protein N-glycosylation are of great interest. Plant-based systems have already demonstrated their potential to produce pharmaceutically relevant recombinant proteins, although their N-glycan patterns differ from those in humans. Plants have shown great plasticity towards the manipulation of their glycosylation machinery, and some have already been glyco-engineered in order to avoid the attachment of plant-typical, putatively immunogenic sugar residues. This resulted in complex-type N-glycans with a core structure identical to the human one. Compared to humans, plants lack the ability to elongate these N-glycans with {beta}1,4-linked galactoses and terminal sialic acids. However, these modifications, which require the activity of several mammalian enzymes, have already been achieved for Nicotiana benthamiana and the moss Physcomitrella. Here, we present the first step towards sialylation of recombinant glycoproteins in Physcomitrella, human {beta}1,4-linked terminal N-glycan galactosylation, which was achieved by the introduction of a chimeric {beta}1,4-galactosyltransferase (FTGT). This chimeric enzyme consists of the moss 1,4-fucosyltransferase transmembrane domain, fused to the catalytic domain of the human {beta}1,4-galactosyltransferase. Stable FTGT expression led to the desired {beta}1,4-galactosylation. However, additional pentoses of unknown identity were also observed. The nature of these pentoses was subsequently determined by Western blot and enzymatic digestion followed by mass spectrometric analysis and resulted in their identification as -linked arabinoses. Since a pentosylation of {beta}1,4-galactosylated N-glycans was reported earlier, e.g. on recombinant human erythropoietin produced in glyco-engineered Nicotiana tabacum, this phenomenon is of a more general importance for plant-based production platforms. Arabinoses, which are absent in humans, may prevent the full humanization of plant-derived products. Therefore, the identification of these pentoses as arabinoses is important as it creates the basis for their abolishment to ensure the production of safe biopharmaceuticals in plant-based systems.

bioengineering↗

bioRxiv2021

O-methylated N-glycans Distinguish Mosses from Vascular Plants

In the animal kingdom, a stunning variety of N-glycan structures has emerged with phylogenetic specificities of various kinds. In the plant kingdom, however, N-glycosylation appears as strictly conservative and uniform. From mosses to all kinds of gymno- and angiosperms, land plants mainly express structures with the common pentasaccharide core substituted with xylose, core 1,3-fucose, maybe terminal GlcNAc residues and Lewis A determinants. In contrast, green algae biosynthesize unique and unusual N-glycan structures with uncommon monosaccharides, a plethora of different structures and various kinds of O-methylation. Mosses, a group of plants that are separated by at least 400 million years of evolution from vascular plants, were hitherto seen as harbouring an N-glycosylation machinery identical to that of vascular plants. To challenge this view, we have analysed the N-glycomes of several moss species using MALDI-TOF/TOF, PGC-MS/MS and GC-MS. While all species contained the plant-typical heptasaccharide with no, one or two terminal GlcNAc residues (MMXF, MGnXF and GnGnXF, respectively), many species exhibited MS signals with 14.02 Da increments as characteristic for O-methylation. Throughout all analysed moss N-glycans the level of methylation differed strongly even in the same family. In some species, methylated glycans dominated, while others had no methylation at all. GC-MS revealed the main glycan from Funaria hygrometrica to contain 2,6-O-methylated terminal mannose. Some mosses additionally presented very large, likewise methylated complex-type N-glycans. This first finding of methylation of N-glycans in land plants mirrors the presumable phylogenetic relation of mosses to green algae, where O-methylation of mannose and many other monosaccharides is a common trait.

biochemistry↗

bioRxiv2021

Autopolyploidization affects transcript patterns and gene targeting frequencies in Physcomitrella

Qualitative changes in gene expression after an autopolyploidization event, a pure duplication of the whole genome, might be relevant for a different regulation of molecular mechanisms between angiosperms growing in a life cycle with a dominant diploid sporophytic stage and the haploid-dominant bryophytes. Whereas angiosperms repair DNA double strand breaks (DSB) preferentially via non-homologous end joining (NHEJ), in bryophytes homologous recombination (HR) is the main DNA-DSB repair pathway facilitating the precise integration of foreign DNA into the genome via gene targeting (GT). Here, we studied the influence of ploidy on gene expression patterns and GT efficiency in the moss Physcomitrella using haploid plants and autodiploid plants, generated via an artificial duplication of the whole genome. Single cells (protoplasts) were transfected with a GT construct and material from different time-points after transfection was analysed by microarrays and SuperSAGE sequencing. In the SuperSAGE data, we detected 3.7% of the Physcomitrella genes as differentially expressed in response to the whole genome duplication event. Among the differentially expressed genes involved in DNA-DSB repair was an upregulated gene encoding the X-ray repair cross-complementing protein 4 (XRCC4), a key player in NHEJ. Analysing the GT efficiency, we observed that autodiploid plants were significantly GT suppressed (p<0.001) attaining only one third of the expected GT rates. Hence, an alteration of global transcript patterns, including genes related to DNA repair, in autodiploid Physcomitrella plants correlated with a drastic suppression of HR.

plant biology↗

bioRxiv2021

The synthetic protein MFHR13 as efficient multitarget regulator against complement over-activation

The complement system constitutes the innate defense against pathogens. Its dysregulation leads to diseases and is a critical determinant in many viral infections, e.g.COVID-19. Factor H (FH) is the main regulator of the alternative pathway of complement activation and could be a therapy to restore homeostasis. However, recombinant FH is not available. Engineered FH versions may present alternative therapeutics. Here, we designed a synthetic protein, MFHR13, as a multitarget complement regulator. It combines the dimerization and C5-regulatory domains of human FH-related protein 1 (FHR1) with the C3-regulatory and cell surface recognition domains of human FH. MFHR13 includes the FH variant I62, which we characterized to induce improved C3b binding and cofactor activity compared to the variant V62. After comparative protein structure modelling, we introduced the SCR FH13, which includes an N-glycosylation site for higher protein stability. In summary, the fusion protein MFHR13 comprises SCRs FHR11-2:FH1-4:FH13:FH19-20. It shows an enhanced heparin binding and protects sheep erythrocytes from complement attack exhibiting 26 and 4-fold the regulatory activity of eculizumab and human FH, respectively. Furthermore, it also blocks the terminal pathway of complement activation and we demonstrate that MFHR13 and FHR1 bind to all proteins forming the membrane attack complex, which contributes to the mechanistic understanding of FHR1. We consider MFHR13 a promising candidate as a therapeutic for complement-associated diseases.

immunology↗

bioRxiv2021

Medium optimization for biomass production of three peat moss (Sphagnum L.) species using fractional factorial design and response surface methodology

Peat moss (Sphagnum) biomass is a promising bioresource to substitute peat in growing media with a renewable material. For sustainable production on a large scale, the productivity of Sphagnum mosses has to be increased by optimizing culture conditions. Optimization was achieved using fractional factorial design and response surface methodology based on central composite design to determine concentrations of eight factors leading to highest biomass yield. We improved a standard Sphagnum medium by reducing the concentrations of NH4NO3, KH2PO4, KCl, MgSO4, Ca(NO3)2, FeSO4 and a microelement solution up to 50 %. Together with a reduced sucrose concentration for Sphagnum fuscum, while it remained unchanged for Sphagnum palustre and Sphagnum squarrosum, moss productivities were enhanced for all tested species in shake flasks. Further upscaling to 5 L photobioreactors increased the biomass yield up to nearly 50-fold for S. fuscum, 40-fold for S. palustre and 25-fold for S. squarrosum in 24 days.

plant biology↗

bioRxiv2020

Stable Protein Sialylation in Physcomitrella

Recombinantly produced proteins are indispensable tools for medical applications. Since the majority of them are glycoproteins, their N-glycosylation profiles are major determinants for their activity, structural properties and safety. For therapeutical applications, a glycosylation pattern adapted to product and treatment requirements is advantageous. Physcomitrella (Physcomitrium patens, moss) is able to perform highly homogeneous complex-type N-glycosylation. Additionally, it has been glyco-engineered to eliminate plant-specific sugar residues by knock-out of the {beta}1,2-xylosyltransferase and 1,3-fucosyltransferase genes ({Delta}xt/ft). Furthermore, P. patens meets wide-ranging biopharmaceutical requirements such as GMP compliance, product safety, scalability and outstanding possibilities for precise genome engineering. However, all plants, in contrast to mammals, lack the capability to perform N-glycan sialylation. Since sialic acids are a common terminal modification on human N-glycans, the property to perform N-glycan sialylation is highly desired within the plant-based biopharmaceutical sector. In this study, we present the successful achievement of protein N-glycan sialylation in stably transformed P. patens. The sialylation ability was achieved in a {Delta}xt/ft moss line by stable expression of six mammalian coding sequences combined with targeted organelle-specific localization of the encoded enzymes responsible for synthesis, activation, transport and transfer of sialic acid. Production of free and (CMP)-activated sialic acid was proven. The glycosidic anchor for the attachment of terminal sialic acid was generated by the introduction of a chimeric human {beta}1,4-galactosyltransferase gene under the simultaneous knock-out of the gene encoding the endogenous {beta}1,3-galactosyltransferase. Functional complex-type N-glycan sialylation was confirmed via mass spectrometric analysis of a stably co-expressed recombinant human protein.

bioengineering↗