Development of Yeast Cell Factories for Efficient and Affordable Production of Blood Substitutes by Microbial Fermentation

Traditional transfusions using donor blood are life-saving and routine treatments. Especially rich countries have effective blood donor corps where the collected blood is both in abundance, and safe to use. However, blood transfusions in general are associated with several risks, including but not limited to; infectious diseases, insufficient screening, donor incompatibility, and strict storage requirements. These issues call for a donor-independent blood supply, either for the production of fully synthetic whole blood, or as a supplement for use during emergencies.The oxygen-carrying molecule of blood is called haemoglobin (Hb); a tetrameric metalloprotein situated on the red blood cells. Hb can be subdivided into heme; a prosthetic group containing ferrous iron, and globin chains. The model organism and industrial workhorse, Saccharomyces cerevisiae is a potential candidate for the production of recombinant human haemoglobin due to its native production of heme and well-understood metabolism.Our engineering philosophy focuses on upregulating, and de-repressing of heme metabolism, recombinant production of human haemoglobin, better understanding of iron regulation, and growth media optimization through minimal engineering using native genes, promotors, and terminators. The target of the overall project is to reach 25% active rHb of total cell protein. Currently, our best candidates for a production strain are producing 15x total heme compared to the wildtype, and 10.67±0.67% active human haemoglobin of the total cell protein, respectively. Free heme as well as 22.52±1.09nM/mgCell accumulated intracellular iron allow room for improvement in rHb titers, making it realistic to reach 25% active rHb at lab-scale within years.With the ongoing task of further genetic engineering and media optimization we do believe that recombinant human haemoglobin can serve as artificial oxygen carriers in humans in the future to allow for a safer and more readily available treatment for anemia

Engineering ergothioneine production in Yarrowia lipolytica

Ergothioneine is a naturally occurring antioxidant that has shown potential in ameliorating neurodegenerative and cardiovascular diseases. In this study, we investigated the potential of the Crabtree‐negative, oleaginous yeast Yarrowia lipolytica as an alternative host for ergothioneine production. We expressed the biosynthetic enzymes EGT1 from Neurospora crassa and EGT2 from Claviceps purpurea to obtain 158 mg·L(−1) of ergothioneine in small‐scale cultivation, with an additional copy of each gene improving the titer to 205 mg·L(−1). The effect of phosphate limitation on ergothioneine production was studied, and finally, a phosphate‐limited fed‐batch fermentation in 1 L bioreactors yielded 1.63 ± 0.04 g·L(−1) ergothioneine in 220 h, corresponding to an overall volumetric productivity of 7.41 mg·L(−1)·h(−1), showing that Y. lipolytica is a promising host for ergothioneine production

Engineering precursor supply for the high-level production of ergothioneine in Saccharomyces cerevisiae.

Ergothioneine (ERG) is an unusual sulfur-containing amino acid. It is a potent antioxidant, which shows great potential for ameliorating neurodegenerative and cardiovascular diseases. L-ergothioneine is rare in nature, with mushrooms being the primary dietary source. The chemical synthesis process is complex and expensive. Alternatively, ERG can be produced by fermentation of recombinant microorganisms engineered for ERG overproduction. Here, we describe the engineering of S. cerevisiae for high-level ergothioneine production on minimal medium with glucose as the only carbon source. To this end, metabolic engineering targets in different layers of the amino acid metabolism were selected based on literature and tested. Out of 28 targets, nine were found to improve ERG production significantly by 10%-51%. These targets were then sequentially implemented to generate an ergothioneine-overproducing yeast strain capable of producing 106.2 ± 2.6 mg/L ERG in small-scale cultivations. Transporter engineering identified that the native Aqr1 transporter was capable of increasing the ERG production in a yeast strain with two copies of the ERG biosynthesis pathway, but not in the strain that was further engineered for improved precursor supply. Medium optimization indicated that additional supplementation of pantothenate improved the strain's productivity further and that no supplementation of amino acid precursors was necessary. Finally, the engineered strain produced 2.39 ± 0.08 g/L ERG in 160 h (productivity of 14.95 ± 0.49 mg/L/h) in a controlled fed-batch fermentation without supplementation of amino acids. This study paves the way for the low-cost fermentation-based production of ergothioneine