Biology subjects

Blenner, M.

Publications and source records attributed to Blenner, M..

12 recordsLinked to original sources

bioRxiv2025

Adaptive evolution of Candida maltosa improves the bioconversion of depolymerized plastic feedstock by targeting biosurfactant production

Thermal oxo-degradation (TOD) of plastic can transform plastic waste into fermentable feedstocks. Bioconversion of the TOD products could be used as feedstocks in a biorefinery concept and lead to new avenues for plastic waste upcycling. Previous work demonstrated this concept with high density polyethylene (HDPE), the most abundant type of plastic, and identified the nonconventional yeast Candida maltosa as a promising candidate for this application. Here we describe the evolution of an improved strain of C. maltosa and characterize the uptake mechanisms of TOD products from HDPE (TOD_HDPE). Batch cultures in series passaged at the mid-exponential growth phase applied a selective pressure for faster growth and resulted in a >100% increase in specific growth rate when using TOD_HDPE as a carbon source. The evolved strain was compared to the parent strain to identify the cellular and biochemical changes associated with the improved phenotype and the uptake mechanisms involved in the bioconversion of TOD_HDPE. This comparison found that C. maltosa secretes biosurfactants capable of solubilizing hydrocarbons. The adaptive evolution resulted in changes in biosurfactant production that translated to improved emulsification of alkanes and increased solubilization of fatty alcohols and alkanes. In addition to the changes in metabolites, the study identified increases in membrane permeability associated with a reduction in ergosterol that may also play a role in the improved phenotype. These findings support the development of C. maltosa and other potential microbial cell factories for plastic biorefineries and may inform future design strategies.

microbiology↗

bioRxiv2025

A high throughput assay to detect enzymatic polyethylene oxidation

Biological plastics deconstruction and upcycling have emerged as a sustainable alternative to traditional recycling technologies for plastics waste. The discovery and engineering of efficient thermostable poly(ethylene terephthalate) (PET) hydrolases has made biological PET recycling possible at scale; however, enzymes for non-PET plastics, which account for approximately 70% of all plastics produced, remain largely undiscovered. To accelerate the discovery of such enzymes, a high throughput screening (HTS) platform is needed. Here, we develop a HTS liquid-based assay to detect one of the first committed steps of polyolefin degradation, oxidation of the C-H bond to an aldehyde. We test 4-hydrazino-7-nitro-2,1,3-benxoxadiozole hydrazine (NBD-H), which reacts with generated aldehydes to form a fluorescent hydrazone, on oxidized low-density polyethylene (LDPE) films. Hydrazone generation correlated well with established carbonyl index metrics for polymer oxidation (R2 = 0.97). Moreover, we demonstrate that the probe reliably identifies LDPE-active dye decolorizing peroxidases (DyPs) that generate aldehydes on LDPE films, serving as effective screen as demonstrated by a receiver operating characteristic area under the curve of 0.95. Due to the rapid fluorescent readout and parallelization in microarray plates, this assay enables screening thousands of enzymes in 24 hours compared to time-consuming established approaches, accelerating discovery of enzymes that catalyze the first step of polyolefin biodeconstruction.

biochemistry↗

bioRxiv2025

Biological polyethylene deconstruction initiated by oxidation from DyP peroxidases

Polyethylene (PE) is the most commonly used plastic on Earth due to its favorable material properties such as high ductility, mechanical strength, and bond homogeneity that make the material resistant to deconstruction. However, the lack of robust recycling infrastructure for PE end-of-life management is leading to an estimated 4 million tons of environmental accumulation annually, with implications for human and environmental health. Biological deconstruction and upcycling could potentially aid in PE waste management by allowing for high-yield conversion of waste plastics to high value products, although such processes are not yet possible. In this work, we mined the gut of low-density PE (LDPE) fed mealworms that can reduce LDPE molecular weight by >40% and discovered dye decolorizing peroxidases (DyPs) that oxidized LDPE, initiating biological deconstruction. A plastic-active DyP is characterized by a hydrophobic loop near its active site that helps mediate binding and tunes activity. LDPE oxidation is driven by surface exposed residues proximal to the active site enabling activity on polymeric substrates. Our work provides robust evidence for enzymatic LDPE deconstruction and identifies molecular targets for further development to realize scalable biological LDPE upcycling. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=41 SRC="FIGDIR/small/640435v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@146b8fborg.highwire.dtl.DTLVardef@1a72adorg.highwire.dtl.DTLVardef@5ece5forg.highwire.dtl.DTLVardef@3ee20a_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2024

Hydrophobins from Aspergillus mediate fungal interactions with microplastics

Microplastics present myriad ecological and human health risks including serving as a vector for pathogens in human and animal food chains. However, the specific mechanisms by which pathogenic fungi colonize these microplastics have yet to be explored. In this work, we examine the opportunistic fungal pathogen, Aspergillus fumigatus, and other common soil and marine Aspergilli, which we found bind microplastics tightly. Up to 3.85+/-1.48 g microplastic plastic/g fungi were bound and flocculated for polypropylene (PP), polyethylene (PE), and polyethylene terephthalate (PET) powders and particles ranging in size from 0.05 - 5 mm. Gene knockouts revealed hydrophobins as a key biomolecule driving microplastic-fungi binding. Moreover, purified hydrophobins were still able to flocculate microplastics independent of the fungus. Our work elucidates a role for hydrophobins in fungal colonization of microplastics and highlights a potential target for mitigating the harm of microplastics through engineered fungal-microplastic interactions. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/622132v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@13eb679org.highwire.dtl.DTLVardef@3dc9c0org.highwire.dtl.DTLVardef@888c96org.highwire.dtl.DTLVardef@c5e1aa_HPS_FORMAT_FIGEXP M_FIG C_FIG Significance StatementMicroplastics pose serious ecological and human health effects by introducing pathogens and toxins into animal and human food chains. Many pathogenic microorganisms preferentially form biofilms on microplastic particles that are then ingested. Here, we demonstrate that hydrophobins, highly hydrophobic, cell surface proteins, enable microplastic binding and colonization by the opportunistic pathogen Aspergillus fumigatus and other fungi within the Aspergillus genus. Our work recognizes a novel role for hydrophobin proteins, identifying potential strategies for pathogen control and protein-based microplastics recovery.

molecular biology↗

bioRxiv2024

Genetically-Encoded Fluorescence Barcodes for Single-Cell Analysis

Genetically-encoded, single-cell barcodes are broadly useful for experimental tasks such as lineage tracing or genetic screens. For such applications, a barcode library would ideally have high diversity (many unique barcodes), non-destructive identification (repeated measurements in the same cells or population), and fast, inexpensive readout (many cells and conditions). Current nucleic acid barcoding methods generate high diversity but require destructive and slow/expensive readout, and current fluorescence barcoding methods are non-destructive, fast, and inexpensive to readout but lack high diversity. We recently proposed theory for how fluorescent protein combinations may generate a high-diversity barcode library with non-destructive, fast and inexpensive identification. Here, we present an initial experimental proof-of-concept by generating a library of [~]150 barcodes from two-way combinations of 18 fluorescent proteins, 61 of which are tested experimentally. We use a pooled cloning strategy to generate a barcode library that is validated to contain every possible combination of the 18 fluorescent proteins. Experimental results using single mammalian cells and spectral flow cytometry demonstrate excellent classification performance of individual fluorescent proteins, with the exception of mTFP1, and of most evaluated barcodes, with many true positive rates >99%. The library is compatible with genetic screening for hundreds of genes (or gene pairs) and lineage tracing hundreds of clones. This work lays a foundation for greater diversity libraries (potentially [~]105 and more) generated from hundreds of spectrally-resolvable tandem fluorescent protein probes.

synthetic biology↗

bioRxiv2024

Heritable non-genetic phenotypes are enriched for stress responses as a form of bet hedging

During manufacturing batches, Chinese hamster ovary (CHO) cells encounter critical levels of environmental stressors such as ammonia, lactate, and osmolality accumulation that can significantly reduce cell health and productivity. It is therefore crucial that stress adaptation and resistance be factored into cell line development (CLD). In this study, we employee population-based transcriptomic and differential gene expression analysis on stress-induced CHO cells to identify biomarkers displaying both heritable and stress-responsive properties. Using this workflow, 199 genes displayed transcriptional variability characteristic of a bistable system that formed four network communities of co-fluctuating genes. These communities were enriched in genes related to the regulation of apoptotic processes and gene expression/metabolic pathways. Seven genes were identified as promising biomarkers for engineering a stress-resistant phenotype. Genetic engineering methods may be employed in the future to bias clonal populations for higher stress tolerance to manufacturing stress, therefore increasing cell health and productivity in at-scale bioreactors.

genomics↗

bioRxiv2024

Revisiting the activity of two poly(vinyl chloride)- and polyethylene-degrading enzymes

Biocatalytic degradation of non-hydrolyzable plastics is a rapidly growing field of research, driven by the global accumulation of waste. Enzymes capable of cleaving the carbon-carbon bonds in synthetic polymers are highly sought-after as they may provide tools for environmentally friendly plastic recycling. Despite some reports of oxidative enzymes acting on non-hydrolyzable plastics, including polyethylene or poly(vinyl chloride), the notion that these materials are susceptible to efficient enzymatic degradation remains controversial, partly driven by a general lack of studies independently reproducing previous observations. We attempted to replicate two recent studies reporting that deconstruction of polyethylene and poly(vinyl chloride) can be achieved using an insect hexamerin from Galleria mellonella (so-called "Ceres") or a bacterial catalase-peroxidase from Klebsiella sp., respectively. Reproducing previously described experiments with the recombinant proteins, we did not observe any activity on plastics using multiple reaction conditions and multiple substrate types. Digging deeper into the discrepancies between the previous data and our observations, we show how and why the original experimental results may have been misinterpreted, leading to the erroneous claim that enzymatic deconstruction of polyethylene and poly(vinyl chloride) had occurred. Our results should lead to caution when interpreting the growing amount of literature claiming enzymatic degradation of non-hydrolyzable plastics.

biochemistry↗

bioRxiv2023

Engineering Y. lipolytica for the biosynthesis of geraniol

Geraniol is a monoterpene with wide applications in the food, cosmetics, and pharmaceutical industries. Microbial production has largely used model organisms lacking favorable properties for monoterpene production. In this work, we produced geraniol in metabolically engineered Yarrowia lipolytica. First, two plant-derived geraniol synthases (GES) from Catharanthus roseus (Cr) and Valeriana officinalis (Vo) were tested based on previous reports of activity. Both wild type and truncated mutants of GES (without signal peptide targeting chloroplast) were examined by co-expressing with MVA pathway enzymes tHMG1 and IDI1. Truncated CrGES (tCrGES) produced the most geraniol and thus was used for further experimentation. The initial strain was obtained by overexpression of the truncated HMG1, IDI and tCrGES. The acetyl-CoA precursor pool was enhanced by overexpressing mevalonate pathway genes such as ERG10, HMGS or MVK, PMK. The final strain overexpressing 3 copies of tCrGES and single copies of ERG10, HMGS, tHMG1, IDI produced approximately 1 g/L in shake-flask fermentation. This is the first demonstration of geraniol production in Yarrowia lipolytica and the highest de novo titer reported to date in yeast.

bioengineering↗

bioRxiv2022

A Theory for High-Throughput Genetic Interaction Screening

Systematic, genome-scale genetic screens have been instrumental for elucidating genotype-phenotype relationships, but approaches for probing genetic interactions have been limited to at most [~]100 pre-selected gene combinations in mammalian cells. Here, we introduce a theory for high-throughput genetic interaction screens. The theory extends our recently developed Multiplexing using Spectral Imaging and Combinatorics (MuSIC) approach to propose [~]105 spectrally unique, genetically-encoded MuSIC barcodes from 18 currently available fluorescent proteins. Simulation studies based on constraints imposed by spectral flow cytometry equipment suggest that genetic interaction screens at the human genome-scale may be possible if MuSIC barcodes can be paired to guide RNAs. While experimental testing of this theory awaits, it offers transformative potential for genetic perturbation technology and knowledge of genetic function. More broadly, the availability of a genome-scale spectral barcode library for non-destructive identification of single-cells could find more widespread applications such as traditional genetic screening and high-dimensional lineage tracing.

biochemistry↗

bioRxiv2021

Rational engineering of low temperature activity in thermoalkalophilic Geobacillus thermocatenulatus lipase

While thermophilic enzymes have thermostability desired for broad industrial applications, they can lose activity at ambient temperatures far from their optimal. Engineering cold activity into thermophilic enzymes has the potential to broaden the range of temperatures resulting in significant activity (i.e., decreasing the temperature dependence of kcat). Even though it has been widely suggested that cold temperature enzyme activity results from active flexibility that is at odds with the rigidity necessary for thermostable enzymes; however, directed evolution experiments have shown us these properties are not mutually exclusive. In this study, rational protein engineering was used to introduce flexibility inducing mutations around the active sites of Geobacillus thermocatenulatus lipase (GTL). Two mutants were found to have enhanced specific activity compared to wild-type at temperatures between 283 K to 363 K with p-nitrophenol butyrate but not with larger substrates. Kinetics assay revealed both mutations resulted in psychrophilic traits, such as lower activation enthalpy and more negative entropy values compared to wild type in all substrates. Furthermore, the mutants had significantly improved thermostability compared to wild type enzyme, which proves that it is feasible to improve the cold activity without trade-off. Our study provides insight into the enzyme cold adaptation mechanism and design principles for engineering cold activity into thermostable enzymes.

biochemistry↗

bioRxiv2020

The eccDNA Replicon: A heritable, extra-nuclear vehicle that enables gene amplification and glyphosate resistance in Amaranthus palmeri

Gene copy number variation is a predominant mechanism by which organisms respond to selective pressures in nature, which often results in unbalanced structural variations that perpetuate as adaptations to sustain life. However, the underlying mechanisms that give rise to gene proliferation are poorly understood. Here, we show a unique result of genomic plasticity in Amaranthus palmeri, a massive, [~]400kb extrachromosomal circular DNA (eccDNA), that harbors the 5-enoylpyruvylshikimate-3-phosphate synthase (EPSPS) gene and 58 other encoded genes whose functions traverse detoxification, replication, recombination, transposition, tethering, and transport. Gene expression analysis under glyphosate stress showed transcription of 41 of the 59 genes, with high expression of EPSPS, aminotransferase, zinc-finger, and several uncharacterized proteins. The genomic architecture of the eccDNA replicon is comprised of a complex arrangement of repeat sequences and mobile genetic elements interspersed among arrays of clustered palindromes that may be crucial for stability, DNA duplication and tethering, and/or a means of nuclear integration of the adjacent and intervening sequences. Comparative analysis of orthologous genes in grain amaranth (Amaranthus hypochondriacus) and water-hemp (Amaranthus tuberculatus) suggest higher order chromatin interactions contribute to the genomic origins of the Amaranthus palmeri eccDNA replicon structure. One-sentence summaryThe eccDNA replicon is a large extra-nuclear circular DNA that is composed of a sophisticated repetitive structure, harbors the EPSPS and several other genes that are transcribed during glyphosate stress.

genomics↗

bioRxiv2018

Validating genome-wide CRISPR-Cas9 function in the non-conventional yeast Yarrowia lipolytica

Genome-wide mutational screens are central to understanding the genetic underpinnings of evolved and engineered phenotypes. The widespread adoption of CRISPR-Cas9 genome editing has enabled such screens in many organisms, but identifying functional sgRNAs still remains a challenge. To address this limitation, we developed a methodology to quantify the cutting efficiency of each sgRNA in a genome-scale library in the biotechnologically important yeast Yarrowia lipolytica. Screening in the presence and absence of native DNA repair enabled high-throughput quantification of sgRNA function leading to the identification of high efficiency sgRNAs that cover 94% of genes. Library validation enhanced the classification of essential genes by identifying inactive guides that create false negatives and mask the effects of successful disruptions. Quantification of guide effectiveness also creates a dataset from which functional determinants of CRISPR-Cas9 can be identified. Finally, application of the library identified mutations that led to high lipid accumulation and eliminated pseudohyphal morphology.

synthetic biology↗