Biology subjects

Borodina, I.

Publications and source records attributed to Borodina, I..

9 recordsLinked to original sources

bioRxiv2026

Unlocking substrate specificities of human solute carrier proteins using untargeted metabolomics

The limited understanding of transporter substrate spectra constrains our ability to interpret cell and membrane function, highlighting the need for methods that enable transporter deorphanization and characterization of promiscuous transport activities. Here, we present a Xenopus oocyte-based platform for unbiased transporter substrate discovery. Oocytes expressing heterologous solute carrier proteins (SLC) were incubated in human blood serum, a chemically complex metabolite library containing thousands of endogenous metabolites and xenobiotics, followed by paired untargeted LC-MS/MS profiling of intracellular extracts and surrounding medium to capture metabolite exchange events. Across the five human SLC transporters, viz. SLC10A2, SLC10A6, SLC13A2, SLC16A10, and SLC46A1, metabolite exchange signatures were detected, and automated feature annotation was refined by manual chromatographic peak inspection. The workflow recovered known substrates of SLC10A2 and SLC16A10 and identified additional transported metabolites with MS/MS confirmation. This method provides a scalable framework for transporter substrate profiling and prioritization of candidates for targeted validation.

systems biology↗

bioRxiv2024

Kinetic-model-guided engineering of multiple S. cerevisiae strains improves p-coumaric acid production

The use of kinetic models of metabolism in design-build-learn-test cycles is limited despite their potential to guide and accelerate the optimization of cell factories. This is primarily due to difficulties in constructing kinetic models capable of capturing the complexities of the fermentation conditions. Building on recent advances in kinetic-model-based strain design, we present the rational metabolic engineering of an S. cerevisiae strain designed to overproduce p-coumaric acid (p-CA), an aromatic amino acid with valuable nutritional and therapeutic applications. To this end, we built nine kinetic models of an already engineered p-CA-producing strain by integrating different types of omics data and imposing physiological constraints pertinent to the strain. These nine models contained 268 mass balances involved in 303 reactions across four compartments and could reproduce the dynamic characteristics of the strain in batch fermentation simulations. We used constraint-based metabolic control analysis to generate combinatorial designs of 3 enzyme manipulations that could increase p-CA yield on glucose while ensuring that the resulting engineering strains did not deviate far from the reference phenotype. Among 39 unique designs, 10 proved robust across the phenotypic uncertainty of the models and could reliably increase p-CA yield in nonlinear simulations. We implemented these top 10 designs in a batch fermentation setting using a promoter-swapping strategy for down-regulations and plasmids for up-regulations. Eight out of the ten designs produced higher p-CA titers than the reference strain, with 19 - 32% increases at the end of fermentation. All eight designs also maintained at least 90% of the reference strains growth rate, indicating the critical role of the phenotypic constraint. The high experimental success of our in-silico predictions lays the foundation for accelerated design-build-test-learn cycles enabled by large-scale kinetic modeling.

systems biology↗

bioRxiv2024

Essential sterols from engineered yeast prevent honeybee colony population decline

Honeybees, the worlds most important crop pollinators, are increasingly facing pollen starvation arising from agricultural intensification and climate change. Frequent flowering dearth periods and high-density rearing conditions weaken colonies, often leading to their demise. Beekeepers provide colonies with pollen substitutes, but these feeds cannot sustain brood production because they lack essential sterols found in pollen. Here, we describe a technological breakthrough in honeybee nutrition with wide-reaching impacts on global food security. We first measured the quantity and proportion of sterols found in honeybee tissues. Using this information, we genetically engineered a strain of the oleaginous yeast, Yarrowia lipolytica, to produce a mixture of essential sterols for bees and incorporated it into an otherwise nutritionally complete diet. Colonies fed exclusively with this diet reared brood for significantly longer than those fed diets without suitable sterols. Incorporating sterol supplements into pollen substitutes using this method will enable beekeepers to rear healthier, longer-lived colonies to meet the growing demands for global crop pollination. It could also reduce competition between bee species for access to natural floral resources, stemming the decline of wild bee populations.

synthetic biology↗

bioRxiv2022

TransporterPAL: An integrative database Transporter Prediction ALgorithm

MotivationNatural products are used as drugs, cosmetic ingredients, pigments, flavors, and agricultural products. The compounds are retrievable as extracts from natural sources, but the yields are often low, and the final product may contain various impurities. These challenges can be solved by expressing the biosynthetic pathway in microbial cell factories and ensuring product secretion from the cell by using an appropriate transporter. However, insufficient knowledge of transporters for specific compounds often obstructs efficient secretion of the natural product. Therefore, our goal was to develop an algorithm that predicts transporters for a given compound using available public data. ResultsThe web application TransporterPAL predicts suitable transporters for compounds by interconnecting data for biosynthetic genes and their interactions with transporters. The web application queries the STITCH, STRING, and UniProtKB databases via their respective APIs and returns a set of potential transporters based on a compound and, optionally, the organism as input. For a test set of 61 transporter systems, each containing one or more transporters, a total of 90 unique transporters with a known substrate, we could retrieve 45% of the transporters. To our knowledge, this is the first bioinformatics tool for predicting transporter candidates for a given molecule. Availabilityhttps://transporterpal.com Contactirbo@biosustain.dtu.dk Supplementary informationSupplementary data are available at Bioinformatics online.

bioinformatics↗

bioRxiv2020

Identification and characterization of phlorizin transporter from Arabidopsis thaliana and its application for phlorizin production in Saccharomyces cerevisiae

Bioengineering aimed at producing complex and valuable plant specialized metabolites in microbial hosts requires efficient uptake of precursor molecules and export of final products to alleviate toxicity and feedback inhibition. Plant genomes encode a vast repository of transporters of specialized metabolites that-- due to lack of molecular knowledge--remains largely unexplored in bioengineering. Using phlorizin as a case study--an anti-diabetic and anti-cancerous flavonoid from apple--we demonstrate that brute-force functional screening of plant transporter libraries in Xenopus oocytes is a viable approach to identify transporters for bioengineering. By screening 600 Arabidopsis transporters, we identified and characterized purine permease 8 (AtPUP8) as a bidirectional phlorizin transporter. Functional expression in the plasma membrane of a phlorizin-producing yeast strain increased phlorizin titer by more than 80 %. This study provides a generic approach for identifying plant exporters of specialized metabolites and demonstrates the potential of transport-engineering for improving yield in bioengineering approaches.

molecular biology↗

bioRxiv2020

Production of moth sex pheromones for pest control by yeast fermentation

The use of insect sex pheromones is an alternative technology for pest control in agriculture and forestry, which, in contrast to insecticides, does not have adverse effects on human health or environment and is efficient also against insecticide-resistant insect populations.1,2 Due to the high cost of chemically synthesized pheromones, mating disruption applications are currently primarily targeting higher value crops, such as fruits.3 Here we demonstrate a biotechnological method for the production of pheromones of economically important moth pests using engineered yeast cell factories. Biosynthetic pathways towards several pheromones or their precursors were reconstructed in the oleaginous yeast Yarrowia lipolytica, which was further metabolically engineered for improved pheromone biosynthesis by decreasing fatty alcohol degradation and downregulating storage lipid accumulation. The sex pheromone of the cotton bollworm Helicoverpa armigera was produced by oxidation of fermented fatty alcohols into corresponding aldehydes. The resulting pheromone was just as efficient and specific for trapping of H. armigera male moths in cotton fields in Greece as a synthetic pheromone mixture. We further demonstrated the production of the main pheromone component of the fall armyworm Spodoptera frugiperda. Our work describes a biotech platform for the production of commercially relevant titres of moth pheromones for pest control by yeast fermentation. Significance statementAgriculture largely relies on insecticides and genetically modified crops for pest control, however alternative solutions are required due to emerging resistance, toxicity and regulatory issues, and consumer preferences. Mating disruption with sex pheromones that act by preventing insect reproduction is considered the most promising and scalable alternative to insecticides. This method is highly efficient and safe for human health and environment. The likelihood of insect resistance development is very low and can be handled by adjusting the pheromone composition. The high cost of chemically synthesized pheromones is the major barrier for the wider adoption of pheromones. A novel method based on yeast fermentation enables the production of insect sex pheromones as a lower cost from renewable feedstocks.

bioengineering↗

bioRxiv2020

A Novel Efficient L-Lysine Exporter Identified by Functional Metagenomics

Lack of active export system often limits the industrial bio-based production processes accumulating the intracellular product and hence complexing the purification steps. L-lysine, an essential amino acid, is produced biologically in quantities exceeding two million tons per year; yet, L-lysine production is challenged by efficient export system at high titres during fermentation. To address this issue, new exporter candidates for efficient efflux of L-lysine are needed. Using metagenomic functional selection, we identified 58 genes encoded on 28 unique metagenomic fragments from cow gut microbiome library that improved L-lysine tolerance. These genes include a novel putative L-lysine transporter, belonging to a previously uncharacterized EamA superfamily. Characterization using Xenopus oocyte expression system as well as an Escherichia coli host demonstrates activity as a L-lysine transporter. This novel exporter improved L-lysine tolerance in E. coli by 40% and enhanced the specific productivity of L-lysine in an industrial Corynebacterium glutamicum strain by 12%. Our approach allows the sequence-independent discovery of novel exporters and can be deployed to increase titres and productivity of toxicity-limited bioprocesses.

molecular biology↗

bioRxiv2019

Engineering the yeast Saccharomyces cerevisiae for the production of L-(+)-ergothioneine

L-(+)-Ergothioneine is an unusual, naturally occurring antioxidant nutraceutical that has been shown to help reduce cellular oxidative damage. Humans do not biosynthesise it, so can acquire it only from their diet; it exploits a specific transporter (SLC22A4) for its uptake. ERG is considered to be a nutraceutical and possible vitamin that is involved in the maintenance of health, and seems to be at too low a concentration in several diseases in vivo. Ergothioneine is thus a potentially useful dietary supplement. Present methods of commercial production rely on extraction from natural sources or on chemical synthesis. Here we describe the engineering of the bakers yeast Saccharomyces cerevisiae to produce ergothioneine by fermentation in defined media. After integrating combinations of ERG biosynthetic pathways from different organisms, we screened yeast strains for their production of ERG. The highest-producing strain was also engineered with known ergothioneine transporters. The effect of amino acid supplementation of the medium was investigated and the nitrogen metabolism of S. cerevisiae was altered by knock-out of TOR1 or YIH1. We also optimized the media composition using fractional factorial methods. Our optimal strategy led to a titer of 598 {+/-} 18 mg/L ergothioneine in fed-batch culture in bioreactors. Because S. cerevisiae is a GRAS ( generally recognised as safe) organism that is widely used for nutraceutical production, this work provides a promising process for the biosynthetic production of ERG.

bioengineering↗

bioRxiv2017

Energetic Evolution of Cellular Transportomes

Transporter proteins mediate the translocation of substances across the membranes of living cells. We performed a genome-wide analysis of the compositional reshaping of cellular transporters (the transportome) across the kingdoms of bacteria, archaea, and eukarya. We show that the transportomes of eukaryotes evolved strongly towards a higher energetic efficiency, as ATP-dependent transporters diminished and secondary transporters and ion channels proliferated. This change has likely been important in the development of tissues performing energetically costly cellular functions. The transportome analysis also indicated seven bacterial species, including Neorickettsia risticii and Neorickettsia sennetsu, as likely origins of the mitochondrion in eukaryotes, due to the restricted presence therein of clear homologues of modern mitochondrial solute carriers.

evolutionary biology↗