Biology subjects

Hedin, K. A.

Publications and source records attributed to Hedin, K. A..

3 recordsLinked to original sources

bioRxiv2023

Biosensor-Assisted Laboratory Evolution of Malonyl-CoA production in Saccharomyces cerevisiae

The production of bio-based chemicals and fuels through microbial engineering offers a promising and sustainable alternative to petroleum-based fuels and chemicals, with the potential for scalability. However, engineering microbes and continuously evolving them to enhance the production of industrially relevant products is a complex and challenging task, requiring precise selection of genetic traits to achieve desired outcomes. In this study, we report the development of a novel counter-selectable growth-sensitive malonyl-CoA platform strain by coupling the malonyl-CoA repressor FapR from Bacillus subtilis to essential gene promoters involved in glucose growth and the plasma membrane arginine permease. This platform strain was then coupled with a CRISPR-dCas9 guide-RNA (gRNA) library, which after multiple rounds of dilutions and library sequencing, resulted in the enrichment for gRNAs that increased fluxes towards malonyl-CoA. The enriched gRNAs were validated for their effects on growth enhancement, gene regulation, and the production of an industrially relevant malonyl-CoA product, namely 3-hydroxypropionic acid. This study highlights an innovative approach to microbial engineering and opens up avenues for further exploration in the field of laboratory continuous evolution.

bioengineering↗

bioRxiv2022

Biocontainment strategies for in vivo applications of Saccharomyces boulardii

The human gastrointestinal tract is a complex and dynamic environment, playing a crucial role in human health. Microorganisms engineered to express a therapeutic activity have emerged as a novel modality to manage numerous diseases. Such advanced microbiome therapeutics (AMTs) must be contained within the treated individual. Hence safe and robust biocontainment strategies are required to prevent the proliferation of microbes outside the treated individual. Here we present the first biocontainment strategy for a probiotic yeast, demonstrating a multilayered strategy combining an auxotrophic and environmental-sensitive strategy. We knocked out the genes THI6 and BTS1, causing thiamine auxotrophy and increased sensitivity to cold, respectively. The biocontained Saccharomyces boulardii was unable to grow in the absence of thiamine above 1 ng/mL and exhibited a severe growth defect at temperatures below 20{degrees}C. The biocontained strain was well tolerated and viable in mice and demonstrated equal efficiency in peptide production as the ancestral non-biocontained strain. In combination, the data support that thi6{Delta} and bts1{Delta} enable biocontainment of S. boulardii, which could be a relevant chassis for future yeast-based AMTs.

synthetic biology↗

bioRxiv2022

Oral delivery of GLP-1R agonist by an engineered probiotic yeast strain has anti-obesity effects in mice

Obesity is rapidly increasing within the global population and is one of the leading causes of chronic diseases, including type 2 diabetes (T2D), non-alcoholic fatty liver disease, and cardiovascular diseases. Glucagon-like peptide-1 receptor (GLP-1R) agonists have emerged as promising therapeutic agents for treating T2D and obesity. However, the route of administration of the GLP-1R agonists is currently by injection or high oral dosages of the therapeutic combined with absorption enhancers. Oral delivery of GLP-1R agonists remains the preferred administration route due to convenience and high patient compliance. Thus, strategies to improve the oral delivery of this therapeutic are needed. In this study, we engineered the probiotic yeast Saccharomyces boulardii strain to produce Exendin-4, a GLP-1R agonist, in the gastrointestinal tract to reduce the adverse effects of diet-induced obesity in male C57BL/6 mice. The biological efficiency of the secreted Exendin-4 from S. boulardii was characterised ex vivo on isolated pancreatic islets, demonstrating induced insulin secretion. Furthermore, in vivo characterisation of the engineered strain identified a synergistic effect of cold exposure and Sb-Exe4 by successfully inhibiting appetite and promoting body weight loss under cold exposure (8{degrees}C). In addition, the combination of cold and Sb-Exe4 improved the glucose and lipid homeostasis in the mice by increasing the circulating glucagon level and reducing the inflammatory marker TNF-. Our results demonstrate that S. boulardii can be genetically modified to secrete and deliver active therapeutic GLP-1R agonists in the gastrointestinal tract improving the metabolism of the host.

synthetic biology↗