Production of abscisic acid in the oleaginous yeast Yarrowia lipolytica.

Abscisic acid (ABA) is a phytohormone with applications in agriculture and human health. ABA can be produced by Botrytis cinerea, a plant pathogenic filamentous fungus. However, the cultivation process is lengthy and strain improvement by genetic engineering is difficult. Therefore, we engineered the oleaginous yeast Yarrowia lipolytica as an alternative host for ABA production. First, we expressed five B. cinerea genes involved in ABA biosynthesis (BcABA1,BcABA2,BcABA3,BcABA4 and BcCPR1) in a Y. lipolytica chassis with optimized mevalonate flux. The strain produced 59.2 mg/L of ABA in small-scale cultivation. Next, we expressed an additional copy of each gene in the strain, but only expression of additional copy of BcABA1 gene increased the ABA titer to 168.5 mg/L. We then integrated additional copies of the mevalonate pathway and ABA biosynthesis encoding genes, and we expressed plant ABA transporters resulting in an improved strain producing 263.5 mg/L and 9.1 mg/g dry cell weight (DCW) ABA. Bioreactor cultivation resulted in a specific yield of 12.8 mg/g DCW ABA; however, surprisingly, the biomass level obtained in bioreactors was only 10.5 g DCW/L, with a lower ABA titer of 133.6 mg/L. While further optimization is needed, this study confirms Y. lipolytica as a potential alternative host for the ABA 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.

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.

Recombinant ß-Carotene Production by Yarrowia lipolytica - Assessing the Potential of Micro-Scale Fermentation Analysis in Cell Factory Design and Bioreaction Optimization.

The production of ß-carotene has become increasingly interesting within the biotechnological industry due to a rising demand for safer and more natural colorants, nutritional supplements, and antioxidants. A recent study has described the potential of Yarrowia lipolytica as a ß-carotene-producing cell factory, reporting the highest titer of recombinant ß-carotene produced to date. Finding the best conditions to maximize production and scaling up the process to full scale, a costly and time-consuming process, it is often a bottleneck in biotechnology. In this work, we explored the benefits of using micro-fermentation equipment to significantly reduce the time spent on design and optimization of bioreaction conditions, especially in the early stages of process development. In this proof-of-concept study, a ß-carotene producing Y. lipolytica strain was tested in micro-fermentations partly to assess the robustness of the cell factory design and partly to perform media optimization. The medium optimization led us to an improvement of up to 50% in the yield of ß-carotene production in the best of the conditions. Overall, the micro-fermentation system had a high degree of reliability in all tests.