Search bioRxiv⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/166678

Screening and directed evolution of transporters to improve xylodextrin utilization in the yeast Saccharomyces cerevisiae

Zhang, C.·Acosta-Sampson, L.·Yu, V. Y.·Cate, J. H. D.
View original source ↗Export metadata ↓

Abstract

The economic production of cellulosic biofuel requires efficient and full utilization of all abundant carbohydrates naturally released from plant biomass by enzyme cocktails. Recently, we reconstituted the Neurospora crassa xylodextrin transport and consumption system in Saccharomyces cerevisiae, enabling growth of yeast on xylodextrins aerobically. However, the consumption rate of xylodextrin requires improvement for industrial applications, including consumption in anaerobic conditions. As a first step in this improvement, we report analysis of orthologues of the N. crassa transporters CDT-1 and CDT-2. Transporter ST16 from Trichoderma virens enables faster aerobic growth of S. cerevisiae on xylodextrins compared to CDT-2. ST16 is a xylodextrin-specific transporter, and the xylobiose transport activity of ST16 is not inhibited by cellobiose. Other transporters identified in the screen also enable growth on xylodextrins including xylotriose. Taken together, these results indicate that multiple transporters might prove useful to improve xylodextrin utilization in S. cerevisiae. Efforts to use directed evolution to improve ST16 from a chromosomally-integrated copy were not successful, due to background growth of yeast on other carbon sources present in the selection medium. Future experiments will require increasing the baseline growth rate of the yeast population on xylodextrins, to ensure that the selective pressure exerted on xylodextrin transport can lead to isolation of improved xylodextrin transporters.

Source connections

Explore related subjects

synthetic biologyPreprint

Keep this discovery

BibTeXRIS

Zhang, C., Acosta-Sampson, L., Yu, V. Y., Cate, J. H. D.. 2017-07-21. Screening and directed evolution of transporters to improve xylodextrin utilization in the yeast Saccharomyces cerevisiae. https://doi.org/10.1101/166678

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Living electronic transistors with tunable conductivity

Electroactive bacteria, like Shewanella oneidensis, can couple the oxidation of organic electron donors to the reduction of external conductive surfaces, such as minerals and electrodes, by utilizing multiheme cytochromes to carry charge from within the cell to external surfaces. Additionally, multiheme cytochromes facilitate gateable, long-distance (micrometer-scale) redox conduction along the outer membrane and across multiple cells bridging electrodes. While electroactive microbes are being used to develop bioelectrochemical devices, there have been limited efforts to use synthetic biology to exert additional control over microbes serving as device components. Thus, this work implements an optogenetic biofilm patterning gene circuit and a small molecule sensor in S. oneidensis to simultaneously control cell deposition and cytochrome expression. This allows for photolithographic patterning of biofilms possessing tunable electrical properties controlled with small molecules. This system demonstrates tunable electrochemical activity, redox conduction, intrinsic biofilm conductivity, and negative differential transconductance as a function of cytochrome expression. Additionally, temperature-dependent measurements of this tunable biofilm conduction reveal changes in activation energy as a function of cytochrome expression. Through this combination of synthetic biology and electrochemistry, simultaneous control over biofilm geometry and conductivity sheds light on fundamental microbial electron transport processes, and it enables the construction of living electronic devices.

synthetic biology↗
bioRxiv2026

Evolutionary stabilisation of stressful metabolism via integrated biocomputing and essential-gene metabolic locking circuits

Synthetic genetic circuits enable microbial differentiation from growth to production, yet metabolic burden, imbalance and toxicity frequently drive strain degeneration. Yeast strains engineered to produce different terpene products exhibited divergent genetic responses to metabolic stresses, but commonly underwent progressive loss of induction of synthetic GAL regulatory circuits, either across the entire population or within subpopulations. Using di- and tri-input biocomputing circuits, the essential glutamine synthetase gene GLN1 was coupled to GAL induction, thereby enabling stabilisation and evolutionary adaptation of the synthetic genetic circuits and stressful heterologous terpene synthetic pathways. The integrated biocomputing and metabolic coupling circuit systems not only prevent strain degeneration but also enable interrogation of non-degenerative evolutionary shifts, providing a platform for metabolic engineering optimisation.

synthetic biology↗
bioRxiv2026

Unbiased and scalable reduction of diverse bacterial genomes

The genome is a complex, integrated system where the functions and regulatory interactions of its many components remain poorly understood. Genome minimization aims to reduce genomic complexity by removing non-essential elements to reveal the fundamental building blocks of cellular life. However, current minimization strategies are often slow and species-specific due to a reliance on prior information, and limited to producing single, isolated strains, which obscures the diverse ways a genome can adapt to large-scale DNA removal. Here we show the development and application of Stochastic Lineage-based Iterative Minimization (SLIM) a modular, high-throughput platform for unbiased genome reduction across phylogenetically diverse bacteria. We apply SLIM to generate a library of genome-reduced Escherichia coli lineages. We then interrogate the lineages, identifying both universal and lineage-specific transcriptional and translational reprogramming in response to deletions. We demonstrate that these expression dynamics drive environment-dependent fitness, allowing us to pinpoint a single gene deletion in one genome-reduced lineage as the driver of a measurable environmental growth defect. Beyond E. coli, we successfully deploy SLIM in phylogenetically distinct bacterial taxa to rapidly reduce the genomes of Shigella flexneri and Pseudomonas putida, distinct genus and order respectively from E. coli, without species-specific optimization. Our results establish a scalable, generalizable framework for navigating the vast landscape of minimized genomes, providing a powerful new tool for functional discovery and the rational design of synthetic genomic chassis.

synthetic biology↗