Biology subjects

Mukhopadhyay, A.

Publications and source records attributed to Mukhopadhyay, A..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

Biosensor-Driven Strain Engineering Reveals Key Cellular Processes for Maximizing Isoprenol Production in Pseudomonas putida

Synthetic biology tools have accelerated the generation of simple mutants, but combinatorial testing remains challenging. High-throughput methods struggle translating from proof-of-principle molecules to advanced bioproducts. We address this challenge with a biosensor-driven strategy for enhanced isoprenol production in Pseudomonas putida, a key precursor for sustainable aviation fuel and platform chemicals. This biosensor leverages P. putidas native response to short-chain alcohols via a previously uncharacterized hybrid histidine kinase signaling cascade. Refactoring the biosensor for a conditional growth-based selection enabled identification of competing cellular processes with a [~]16,500-member CRISPRi-library. An iterative combinatorial strain engineering approach yielded an integrated P. putida strain producing [~]900 mg/L isoprenol in glucose minimal medium, a 36-fold increase. Ensemble -omics analysis revealed metabolic rewiring, including amino acid accumulation as key drivers of enhanced production. Techno-economic analysis elucidated the path to economic viability and confirmed the benefits of adding amino acids outweigh the additional costs. This study establishes a robust biosensor driven approach for optimizing other heterologous pathways, accelerating microbial cell factory development. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=138 SRC="FIGDIR/small/643695v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@533605org.highwire.dtl.DTLVardef@ff98fforg.highwire.dtl.DTLVardef@9592fdorg.highwire.dtl.DTLVardef@1e52993_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2025

Evolution guided tolerance engineering of Pseudomonas putida KT2440 for production of the sustainable aviation fuel precursor isoprenol

Isoprenol (3-methyl-3-buten-1-ol) is a sustainable aviation fuel precursor and commodity chemical which can be microbially synthesized from renewable carbon streams. Its production has been demonstrated in Pseudomonas putida KT2440 but its titers, rates, and yields have yet to reach commercially viable levels, potentially due to its inherent toxicity. We hypothesized that utilization of Tolerization Adaptive Laboratory Evolution (TALE) would generate P. putida hosts more tolerant to isoprenol and suitable for enhanced production phenotypes. Here, we performed a comprehensive TALE campaign using three strains, the wild-type and two strains lacking subsets of known isoprenol catabolism and transport functions in quadruplicate independently evolved lineages. Several evolved clones from each starting strain displayed robust growth (up to 0.2 h-1) at 8 g/L of isoprenol, where starting strains could not grow. Whole genome resequencing of the 12 independent strain lineages identified convergent mutations. Reverse engineering four commonly mutated regions individually (gnuR, ttgB-PP_1394, PP_3024-PP_5558, PP_1695) each resulted in a partial recovery of the tolerance phenotypes observed in the evolved strains. Additionally, a proteomics-guided deletion of the master motility regulator, fleQ, in an evolved clone alleviated the tolerance vs. production trade-off, restoring isoprenol titers and consumption to levels observed in the starting strains. Collectively, this work demonstrated that an integrated strategy of laboratory evolution and rational engineering was effective to develop robust biofuel production hosts with minimized product toxicity.

microbiology↗

bioRxiv2025

Drug repurposing reveals Posaconazole as a CYP11A1 inhibitor enhancing anti-tumour immunity

Steroid hormones regulate cell physiology and immune function, with dysregulated steroidogenesis promoting cancer progression by supporting tumour growth and suppressing anti-tumour immunity. Targeting CYP11A1, the first and rate-limiting enzyme in steroid biosynthesis, has shown promise in cancer therapy, but safe and effective inhibitors remain an unmet need. Undertaking in silico structure-based drug repurposing approach, we found Posaconazole as an inhibitor of CYP11A1. The docking pose analysis showed that Posaconazole can form multiple hydrogen bonds and hydrophobic interactions with the key residues at the binding site and the cofactor, stabilising the protein-ligand complex. We validated its inhibition efficiency in cell-based assays. In a mouse model of lung metastasis, we demonstrated that Posaconazole restricts metastatic cancer growth by stimulating anti-tumour immunity. These findings highlight Posaconazoles potential as a research tool to study steroidogenesis and as a candidate for further preclinical and clinical evaluation in pathological conditions associated with local steroid production, such as steroidogenic tumours.

immunology↗

bioRxiv2025

RB-TnSeq elucidates dicarboxylic acid specific catabolism in β-proteobacteria for improved plastic monomer upcycling

Dicarboxylic acids are key components of many polymers and plastics, making them a target for both engineered microbial degradation and sustainable bioproduction. In this study, we generated a comprehensive dataset of functional evidence for the genetic basis of dicarboxylic and fatty acid metabolism using randomly-barcoded transposon sequencing (RB-TnSeq). We identified four {beta}-proteobacteria that displayed robust growth with dicarboxylic acid sole carbon sources and cultured their mutant libraries with dicarboxylic and fatty acids with carbon chain lengths from C3 to C12. The resulting fitness data suggested that dicarboxylic and fatty acid metabolisms are largely distinct, and different sets of {beta}-oxidation genes are required for catabolizing dicarboxylic versus fatty acids of the same carbon chain lengths. Additionally, we identified transcriptional regulators and transporters with strong fitness phenotypes related to dicarboxylic acid utilization. In Ralstonia sp. UNC404CL21Col (R. CL21), we deleted two transcriptional repressors to improve its utilization of short chain dicarboxylic acids. We exploited the diacid-utilizing catabolism of R. CL21 to upcycle a mock mixture of the dicarboxylic acids produced when polyethylene is oxidized. After introducing a heterologous indigoidine production pathway, this engineered Rastonia produced 0.56 {+/-} 0.02 g/L indigoidine from a mixture of dicarboxylic acids as a carbon source, demonstrating the potential of R. CL21 to upcycle plastics waste to products derived from tricarboxylic acid (TCA) cycle intermediates. ImportanceUpcycling the carbon in plastic wastes to value-added products is a promising approach to address the plastics waste and climate crises, and dicarboxylic acid metabolism is an important facet of several approaches. Improving our understanding of the genetic basis of this metabolism has the potential to uncover new enzymes and genetic parts for engineered pathways involving dicarboxylic acids. Our dataset is the most comprehensive interrogation of dicarboxylic acid catabolism to date, and this work will be of utility to researchers interested in both plastics bioproduction and upcycling applications.

microbiology↗

bioRxiv2025

Pneumolysin nanopores with 20 nm inner diameter to characterize the size and shape of Tau oligomers

Protein nanopores are emerging as versatile tools to fingerprint biomolecules due to their capability to characterize single molecules without the requirement for labeling. A long-standing challenge with biological nanopores is, however, that large biomolecules in their native state are often too large to enter these pores. Here, we report the self-assembly of approximately 35 {+/-} 5 pneumolysin (PLY) toxins to a stable transmembrane pore with a diameter of 20 {+/-} 3 nm, an effective length of 9.5 nm, and excellent low noise characteristics in the context of nanopore-based resistive pulse recordings. The exceptionally large pore diameter enables the characterization of the size and shape of individual proteins and protein complexes ranging in molecular weight from 50 kDa to 0.8 MDa. Moreover, PLY pores make it possible to follow the time course of the formation of oligomers of tau protein in solution by revealing the size, monomer number, approximate shape, and abundance of these oligomers. At least four characteristics make PLY pores well suited for the characterization of heterogeneous amyloid oligomer samples: First, they are not prone to clogging. Second, they provide label-free single particle analysis. Third, their large diameters make it possible to characterize a wide range of amyloid oligomer sizes with high resolution. And fourth, resistive pulse recordings from these pores provide stable open pore current baselines with low electrical noise.

biophysics↗

bioRxiv2024

Implication of TITIN Variations in Dilated Cardiomyopathy: Integrating Whole Exome Sequencing With Molecular Dynamics Simulation Study

Dilated cardiomyopathy (DCM) is one of the leading causes of heart failure, characterized by ventricular dilation and impaired systolic function. Variations in the TITIN (TTN) gene, which encodes the giant muscle protein TTN, play a pivotal role in the genetic underpinnings of DCM. We conducted WES on 15 patients (5 familial and 10 sporadic) diagnosed with idiopathic DCM and identified 88 exonic variants including four novel variants. These variants were predominantly located in the A-band region (39 variants) of TTN, a critical region for its mechanical stability and interaction with other sarcomeric proteins, followed by the I-band domain (33 variants), Z-disc domain (7 variants) and M-band region (9 variants). To discern the functional repercussions of these variations, we performed several bioinformatics analyses including pathogenicity prediction, protein stability, and protein-protein docking followed by MD simulations on both wild-type and mutant TTN fragments with their corresponding interacting partners (TCAP, MYH7, LMNA). We revealed that variations in the A-band domain significantly alter the proteins structural dynamics, leading to decreased mechanical stability and altered protein-protein interactions. These changes are likely to disrupt sarcomere function, thereby elucidating their role in the pathogenesis of DCM. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=102 SRC="FIGDIR/small/622829v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@15d3eb1org.highwire.dtl.DTLVardef@bf096dorg.highwire.dtl.DTLVardef@4141dorg.highwire.dtl.DTLVardef@1c4948b_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2024

Biosynth Pipeline: An integrated platform for merging the computational design of chimeric type I polyketide synthases with enzymatic pathways for chemical biosynthesis

Synthetic biology offers the promise of manufacturing chemicals more sustainably than petrochemistry. Yet, both the rate at which biomanufacturing can synthesize these molecules and the net chemical accessible space are limited by existing pathway discovery methods which can often rely on arduous literature searches. Here, we present an automated retrobiosynthesis tool, BioPKS Pipeline, that simultaneously tackles both problems by integrating multifunctional type I polyketide synthases (PKSs) with monofunctional enzymes to propose the synthesis of desired target chemicals via two new tools: DORAnet and RetroTide. While monofunctional enzymes are valuable for carefully decorating a substrates carbon backbone, they typically cannot expand the backbone itself. PKSs can, instead, predictably do this through their unique ability to catalyze carbon-carbon bond formation reactions iteratively. We have evaluated the performance of BioPKS Pipeline against a previously published set of 155 molecules of interest for biomanufacturing, and report that BioPKS Pipeline could produce exact designs for 93 of them, as well as pipelines to a chemically similar product for most of the remaining molecules. Furthermore, BioPKS Pipeline successfully proposes biosynthetic routes for complex therapeutic natural products (cryptofolione and basidalin) for which no known biosynthetic pathway currently exists.

bioinformatics↗

bioRxiv2024

Alternate routes to acetate tolerance lead to varied isoprenol production from mixed carbon sources in Pseudomonas putida

Lignocellulose is a renewable resource for the production of a diverse array of platform chemicals, including the biofuel isoprenol. While this carbon stream provides a rich source of sugars, other organic compounds, such as acetate, can be used by microbial hosts. Here we examined growth and isoprenol production in a Pseudomonas putida strain pre-tolerized ("PT") background where its native isoprenol catabolism pathway is deleted, using glucose and acetate as carbon source. We found that PT displays impaired growth in minimal medium containing acetate, and often fails to grow in glucose-acetate medium. Using a mutant recovery-based approach, we generated tolerized strains that overcame these limitations, achieving fast growth and isoprenol production in the mixed carbon feed. Changes in the glucose and acetate assimilation routes, including an upregulation in PP_0154 (SpcC, succinyl-CoA:acetate CoA-transferase) and differential expression of the gluconate assimilation pathways, were key for higher isoprenol titers in the tolerized strains, while a different set of mechanisms were likely enabling tolerance phenotypes in media containing acetate. Among these, a coproporphyrinogen-III oxidase (HemN) was upregulated across all tolerized strains, and in one isolate required for acetate tolerance. Utilizing a defined glucose and acetate mixture ratio reflective of lignocellulosic feedstocks for isoprenol production in P. putida allowed us to obtain insights into the dynamics and challenges unique to dual carbon source utilization that are obscured when studied separately. Together, this enabled development of a P. putida bioconversion chassis able to use a more complex carbon stream to produce isoprenol. ImportanceAcetate is a relatively abundant component of many lignocellulosic carbon streams and has the potential of being used together with sugars, especially in microbes with versatile catabolism such as P. putida. However, the use of mixed carbon streams necessitates additional optimization. Further, the use of P. putida for the production of the biofuel target, isoprenol, requires the use of engineered strains that have additional growth and production constraints when cultivated in acetate and glucose mixtures. In this study we generate acetate tolerant P. putida strains that overcome these challenges and examine their ability to produce isoprenol. We show that acetate tolerance and isoprenol production, though independent phenotypes can both be optimized in a given P. putida strain. Using proteomics and whole genome sequencing we examine the molecular basis of both phenotypes and show that tolerance to acetate can occur via alternate routes and result in different impacts on isoprenol production.

microbiology↗

bioRxiv2024

Estrogen receptor alpha (ERα) driven trans-regulation of mitotic checkpoint complex (MCC) components affects the clinical outcome of breast cancer

Hormone receptors (HR), namely estrogen receptor (ER) and progesterone receptor (PR), are prevalent in most malignant tumors. Although previous literature provided clues for ER in regulating mitosis and ploidy status in breast cancer (BC) cells, reports on the mitotic regulators being the targets of HR are sparse. To delve deeper into ERs impact on mitotic execution, our study focuses on examining its transcriptional activity on the core mitotic checkpoint complex (MCC) components, which are involved in ploidy maintenance. The expression of the core MCC components (Bub3, Mad2, and BubR1) was analyzed by quantitative-PCR and immunohistochemistry in breast tumors and adjacent normal tissues from the cancer genome atlas-breast invasive carcinoma collection (TCGA BRCA) dataset and in a prospective cohort of Eastern Indian breast cancer affected individuals. The preliminary data from these cohorts indicated an influence of ER on the two MCC components, namely Mad2 and BubR1. Subsequently, luciferase reporter assays and chromatin immunoprecipitation were performed which revealed that ER promotes transcriptional activation of MAD2 and BUB1B through direct recruitment on these promoters, showing affects in mitotic outcome. Interestingly, the ectopic introduction of ER, in an HR-ve breast cancer line, MDA-MB-231, significantly reduced the percent aneuploidy. Moreover, we found that overexpression of MAD2 and BUB1B is associated with poorer survival in HR-positive (HR+ve) patients in both cohorts. Our findings provide insights into the specific role of ER-mediated transcriptional regulation of mitosis and ploidy outcome. Targeting the deregulated MCC components thus offers translational potential for the therapeutic management of breast cancer.

cancer biology↗

bioRxiv2024

Kinome-wide RNAi screen in Caenorhabditis elegans reveals new modulators of insulin signaling and longevity

The insulin/IGF-I-like signaling (IIS) pathway is a highly conserved signaling cascade that plays a crucial role in regulating longevity across species. Given its significance in aging, identifying novel kinases interacting with the IIS pathway can provide deeper insights into the mechanisms governing longevity. In this study, we performed a targeted RNAi screen of the Caenorhabditis elegans kinome, utilizing dauer formation as a phenotypic readout. We identified several known and novel kinase modulators of the IIS pathway. These hits were enriched with both previously documented as well as undocumented lifespan regulators. Thermotolerance assays revealed mixed trends, with some kinase inhibitions increasing while others decreasing protection. We observed a positive correlation between thermotolerance and lifespan extension, as well as between dauer formation and lifespan extension, with thermotolerance proving to be a better predictor of longevity. Our findings offer a valuable resource for researchers exploring the IIS pathway and highlight novel, unannotated kinases as potential new therapeutic targets for aging interventions.

molecular biology↗

bioRxiv2024

Bottlenecks in the Implementation of Genome Scale Metabolic Model Based Designs for Bioproduction from Aromatic Carbon Sources

Genome scale metabolic models (GSMM) are commonly used to identify gene deletion sets that result in growth coupling, pairing product formation with substrate utilization. While such approaches can improve strain performance beyond levels typically accessible using targeted strain engineering approaches, sustainable feedstocks often pose a challenge for GSMM-based methods due to incomplete underlying metabolic data. Specifically, we address a four-gene deletion design for the lignin-derived non-sugar carbon source, para-coumarate, that proved challenging to implement. We examine the performance of the fully implemented design for p-coumarate to glutamine, a useful biomanufacturing intermediate. In this study glutamine is then converted to indigoidine, an alternative sustainable pigment and a model heterologous product. Through omics, promoter-variation and growth characterization of a fully implemented gene deletion design, we provide evidence that aromatic catabolism in the completed design is rate-limited by fumarate hydratase activity in the citrate cycle and required careful optimization of the final fumarate hydratase protein (PP_0897) expression to achieve growth and production. A metabolic cross-feeding experiment with the completed design strain also revealed an unanticipated nutrient requirement suggesting additional functions for the fumarate hydratase protein. A double sensitivity analysis confirmed a strict requirement for fumarate hydratase activity in the strain where all genes in the growth coupling design have been implemented. While a complete implementation of the design was achieved, this study highlights the challenge of precisely inactivating metabolic reactions encoded by under-characterized proteins especially in the context of multi-gene edits.

microbiology↗

bioRxiv2024

Assessing horizontal gene transfer in the rhizosphere of Brachypodium distachyon using fabricated ecosystems (EcoFABs)

Horizontal gene transfer (HGT) is a major process by which genes are transferred within microbes in the rhizosphere. However, examining HGT remains challenging due to the complexity of mimicking conditions within the rhizosphere. Fabricated ecosystems (EcoFABs) have been used to investigate several complex processes in plant associated environments. Here we show that EcoFABs are efficient tools to examine and measure HGT frequency in the rhizosphere. We provided the first demonstration of gene transfer via triparental conjugation system in the Brachypodium distachyon rhizosphere in the EcoFABs using Pseudomonas putida KT2440 as both donor and recipient bacterial strain with the donor having the mobilizable and non-self-transmissible plasmid. We also observed that the frequency of conjugal plasmid transfer in the rhizosphere is potentially dependent on the plant developmental stage, and composition and amount of root exudates. The frequency of conjugation also increased with higher numbers of donor cells. We have also shown the transfer of plasmid from P. putida to another B. distachyon root colonizer, Burkholderia sp. showing the possibility of HGT within a rhizosphere microbial community. Environmental stresses also influence the rate of HGT in the rhizosphere between species and genera. Additionally, we observed transfer of a non-self transmissible donor plasmid without the helper strain on agar plates when supplemented with environmental stressors, indicating reduced dependency on the helper plasmid under certain conditions. This study provides a robust workflow to evaluate conjugal transfer of engineered plasmids in the rhizosphere when such plasmids are introduced in a field or plant associated environment. ImportanceWe report the use of EcoFABs to investigate the HGT process in a rhizosphere environment. It highlights the potential of EcoFABs in recapitulating the dynamic rhizosphere conditions as well as their versatility in studying plant-microbial interactions. This study also emphasizes the importance of studying the parameters impacting the HGT frequency. Several factors such as plant developmental stages, nutrient conditions, number of donor cells and environmental stresses influence gene transfer within the rhizosphere microbial community. This study paves the way for future investigations into understanding the fate and movement of engineered plasmids in a field environment.

microbiology↗

bioRxiv2024

Methionine cycle in a pair of serotonergic neurons regulates diet-dependent behavior and longevity through a neuron-gut signaling

The folate-methionine cycle (Met-C) is a central metabolic pathway that is regulated by vitamin B12 (B12), a micronutrient obtained exclusively from diet and microbiota. This metabolic hub supports amino acid, nucleotide and lipid biosynthesis apart from its central role of providing one carbon (-CH3) moiety for methylation reactions. While deficiency of B12 as well as polymorphism in enzymes of the Met-C has been clinically attributed to neurological and metabolic disorders, how this pathway cell non-autonomously regulates systemic physiological processes is less understood. Using a B12-sensitive mutant of Caenorhabditis elegans, we show that the neuronal Met-C responds to differential B12 content in diet to regulate p38-MAPK activation in intestinal cells, thereby modulating cytoprotective gene expression, stress tolerance and longevity. Mechanistically, B12-driven changes in the metabolic flux through the Met-C in the serotonergic ADF neurons of the mutant lead to the release of serotonin (5-hydroxytryptamine, 5-HT). 5-HT activates its receptor, MOD-1, in the post-synaptic interneurons that then secretes the neuropeptide FLR-2. FLR-2 binds to FSHR-1, its cognate receptor in the intestine, and induces the phase transition of the SARM domain protein TIR-1, thereby activating the p38-MAPK pathway. Importantly, this cascade influences the foraging behaviour of the mutant worms such that they prefer a B12-rich diet. Together, our study reveals a dynamic neuron-gut signaling axis that helps an organism modulate behaviour and life history traits based on the neuronal Met-C metabolic flux determined by B12 availability in its diet. Understandably, disruption of the optimum functioning of this axis may have debilitating effects on the health of an organism and the survival of the species.

cell biology↗

bioRxiv2023

Prognostic relevance of correlated co-expression of coding and noncoding RNAs in cervical cancers

Human papillomavirus (HPV) drives cervical cancer (CaCx) pathogenesis and viral oncoproteins jeopardize global gene expression in such cancers. We aimed to identify differentially expressed coding (DEcGs) and long noncoding (sense intronic and Natural Antisense Transcripts) RNA genes (DElncGs) in HPV16-positive CaCx patients (N=44) compared to HPV-negative normal individuals (N=34). Thereby, employing strand-specific RNA-seq, we determined the relationships between DEcGs and DElncGs and their clinical implications. Gene set enrichment and protein-protein interaction analyses of DEcGs revealed enrichment of processes crucial for abortive virus life cycle and cancer progression. The DEcGs formed 16 gene clusters, portraying cancer-related functions. We recorded significantly correlated co-expression of 79 DElncGs with DEcGs at proximal genomic loci, and 24 such pairs portrayed significantly altered correlation coefficients among patients, compared to normal individuals. Of these, 6 DEcGs belonged to 5 gene clusters, one of which was survival-associated. Out of the 24 correlated DEcG: DElncG pairs, 3 pairs were identified, where expression of both members was significantly associated with patient overall survival. Besides being prognostically relevant, disruption of the significant correlative relationships of such gene pairs in CaCx bears immense potential for patient-targeted therapy. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/573593v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@19091c8org.highwire.dtl.DTLVardef@52fbeaorg.highwire.dtl.DTLVardef@18ecf3org.highwire.dtl.DTLVardef@6c5e30_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical Abstract:C_FLOATNO A schematic representation of the analyses undertaken to draw insights on the biological relevance of joint analysis of co-expressed and significantly correlated DElncGs and DEcGs considering the ncNATs and sense intronic DElncGs, in HPV16-positive CaCx patients compared with HPV-negative normal individuals. C_FIG

cancer biology↗

bioRxiv2023

A non-canonical role of somatic CYCLIN D/CYD-1 in oogenesis and reproductive aging, dependent on the FOXO/DAF-16 activation state

For the optimal survival of a species, an organism coordinates its reproductive decisions with the nutrient availability of its niche. Thus, nutrient-sensing pathways like insulin-IGF-1 signaling (IIS) play an important role in modulating cell division, oogenesis, and reproductive aging. Lowering of the IIS leads to the activation of the downstream FOXO transcription factor (TF) DAF-16 in Caenorhabditis elegans which promotes oocyte quality and delays reproductive aging. However, less is known about how the IIS axis responds to changes in cell cycle proteins, particularly in the somatic tissues. Here, we show a new aspect of the regulation of the germline by this nutrient- sensing axis. First, we show that the canonical G1-S cyclin, cyclin D/cyd-1, regulates reproductive aging from the uterine tissue of wild-type worms. Then, we show that knocking down cyd-1 in the uterine tissue of an IIS receptor mutant arrests oogenesis at the pachytene stage of meiosis-1 in a FOXO/DAF-16-dependent manner. We find that activated FOXO/DAF-16 destroys the somatic gonad tissues like the sheath cells, and transcriptionally prevents the spermatogenesis-to- oogenesis switch to orchestrate this arrest. Deleting FOXO/DAF-16 releases the arrest and restores the somatic gonad but leads to the production of poor-quality oocytes. Together, our study reveals the unrecognized cell non-autonomous interaction of CYD-1 and FOXO/DAF-16 in reproductive aging and the regulation of oogenesis.

developmental biology↗

bioRxiv2023

Desiccated cyanobacteria serve as efficient plasmid DNA carrier in space flight

Effective transport of biological systems as cargo during space travel is a critical requirement to use synthetic biology and biomanufacturing in outer space. Bioproduction using microbes will drive the extent to which many human needs can be met in environments with limited resources. Vast repositories of biological parts and strains are available to meet this need, but their on-site availability requires effective transport. Here, we explore an approach that allows DNA plasmids, a ubiquitous synthetic biology part, to be safely transported to the International Space Station and back to the Kennedy Space center, without low-temperature or cryogenic stowage. Our approach relied on the cyanobacterium Nostoc punctiforme PC73102, that is naturally tolerant to prolonged desiccation. Desiccated N. punctiforme was able to carry the non-native pSCR119 plasmid as intracellular cargo safely to space and back. Upon return to the laboratory, the extracted plasmid showed no DNA damage or additional mutations and could be used as intended to transform the model Synbio host Escherichia coli to bestow Kanamycin resistance. This proof-of-concept study provides the foundation for a ruggedized transport host for DNA to environments where there is a need to reduce equipment and infrastructure for biological parts stowage and storage.

microbiology↗

bioRxiv2023

Sustainable production of 2,3,5,6-Tetramethylpyrazine at high titer in engineered Corynebacterium glutamicum using statistical design of experiments

BackgroundThe industrial amino acid production workhorse, Corynebacterium glutamicum naturally produces low levels of 2,3,5,6-tetramethylpyrazine (TMP), a valuable flavor, fragrance and commodity chemical. We have previously reported serendipitous production of TMP in C. glutamicum strains. In the present study, we demonstrate TMP production in C. glutamicum type strain ATCC13032 via the expression of a heterologous TMP pathway in a defined medium followed by statistical design of experiments to understand the effect of the media composition on TMP production. ResultsThe C. glutamicum strain engineered to overexpress acetolactate synthase and alpha-acetolactate decarboxylase from Lactococcus lactis produced [~]0.8 g/L TMP in CGXII minimal medium supplemented with 40 g/L glucose in 24-deep well plates. This engineered strain also demonstrated growth and TMP production when the minimal medium was supplemented with up to 40% (v/v) hydrolysates derived from ionic liquid pretreated sorghum biomass. A screen for improvements in media composition on TMP titer was conducted using fractional factorial design that identified glucose and urea as significant components affecting TMP production. These two components were further optimized using response surface methodology. In the optimized CGXII medium, the engineered strain could produce up to 3.56 g/L TMP (4-fold enhancement in titers and 2-fold enhancement in yield, mol/mol) from 80 g/L glucose and 11.9 g/L urea in shake flask batch cultivation. ConclusionsWe engineered the industrially relevant host, C. glutamicum for targeted production of TMP by heterologous expression of pathway proteins. We demonstrated the capability of the engineered strain for growth and TMP production utilizing real world carbon streams such as hydrolysates. We further identified glucose and urea as the key minimal media components significantly affecting TMP production using statistical media optimization.

synthetic biology↗

bioRxiv2023

ZymePackNet: rotamer-sampling free graph neural network method for protein sidechain prediction

Protein sidechain conformation prediction, or packing, is a key step in many in silico protein modeling and design tasks. Popular protein packing methods typically rely on approximated energy functions and complex algorithms to search dense rotamer libraries. Inspired by the recent success of deep learning in protein modeling tasks, we present ZymePackNet, a graph neural network based protein packing tool that does not require a rotamer library, scoring functions or a search algorithm. We train regression models using protein crystal structures represented as graphs, which are employed sequentially to "germinate" the sidechain starting from atoms anchoring the protein backbone to the sidechains termini, followed by an iterative refinement stage. ZymePackNet is fast and accurate compared to state-of-the-art protein packing methods. We validate our model on three native backbone datasets achieving a mean average error of 16.6{degrees}, 24.1{degrees}, 42.1{degrees}, and 53.0{degrees} for sidechain dihedral angles ({chi}1 to{chi} 4). ZymePackNet captures complex physical interactions such as{pi} stacking without explicitly accounting for it in the model; such effects are currently lacking in the energy terms used in traditional packing tools. Contactabmukho@vt.edu Supplementary informationSupplementary data are available at Bioinformatics online.

biochemistry↗