Despite all the progresses made by metabolic engineering, still only a few biotechnological processes are running at an industrial level. In order to boost the biotechnological sector, integration strategies as well as long-term views are needed. The aim of the present review is to identify the main drawbacks in biotechnological processes, and to propose possible solutions to overcome the issues in question. Novel cell factories and bioreactor design are discussed as possible solutions. In particular, the following microorganisms: Yarrowia lipolytica, Trichosporon oleaginosus, Ustilago cynodontis, Debaryomyces hansenii along with sequential bioreactor configurations are presented as possible cell factories and bioreactor design solutions, respectively.

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The production of β-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 β-carotene-producing cell factory, reporting the highest titer of recombinant β-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 β-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 β-carotene production in the best of the conditions. Overall, the micro-fermentation system had a high degree of reliability in all tests.

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 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 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 that Y. lipolytica as a potential alternative host for the abscisic acid production.

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.