Metabolic engineering of lipid pathways in an oleaginous yeast and optimization of fermentation parameters for enhanced lipid production from the organic fraction of municipal solid waste

Abstract:
One of the largest issues for the world population in the near future is the production of fuels that are renewable, biodegradable, non-toxic and have low negative environmental impacts (Alishah Aratboni et al. 2019; Zhu, Li, and Hiltunen 2016). The expected increase in world population to higher than 9.8 billion by 2050, combined with significant economic growth in emerging economies will result in substantially increasing energy consumption (Petrovič 2015). The decreasing amount of fossil fuels available, the consequences for the environment associated with the use of these fossil fuels as well as humanity's increased dependence on fuels in everyday life means that new alternative ways of producing fuels must be developed to reduce the impact of this sector on the environment (Popp et al. 2014). To be able to respond to this growing demand, it is necessary to use natural resources more efficiently and to increase the use of renewable energy, such as biofuels (Khan et al. 2021).
The replacement of fossil fuels with biofuels has been suggested as a promising solution for the transportation sector by several researchers within the field of bioenergy (Ghanavati, Nahvi, and Karimi 2015; Khan et al. 2021). However, a major concern with the production of biofuels for the transportation sector is the actual sustainability of these biofuels with respect to the reduction of green house gasses, issues with the use of large areas of land, and the availability of biomass (Jeswani, Chilvers, and Azapagic 2020; Popp et al. 2014). Furthermore, the cost competitiveness with fossil fuels is a barrier for large-scale production with the currently developed technologies for production of biofuels (Jeswani, Chilvers, and Azapagic 2020; Petrovič 2015).
The environmental and economic sustainability of producing biofuels is largely dependent on the feedstock as well as the technology used for the process (Bombo et al. 2021; Jeswani, Chilvers, and Azapagic 2020). The most common raw materials used to produce biodiesel are vegetable oils derived from edible plants, but the ethical conflicts that arise from the use of food as fuel and the high costs that are associated with its use have encouraged researchers to search for cheaper non-food alternative feedstocks to produce biodiesel (Khan et al. 2021; Popp et al. 2014). This has led to an increased interest in the production of microbial oils, from microalgae, yeasts, fungi or bacteria, and the potential development of a large-scale production process for commercialization.
Oleaginous yeasts produce lipids consisting of long-chain fatty acids comparable to those of conventional vegetable oils (Ghanavati, Nahvi, and Karimi 2015). Thus, microbial lipids produced by oleaginous yeasts have been suggested as a potential feedstock for environmental and economic sustainable biodiesel production due to several advantages, such as accumulation of large amounts of lipids, shorter incubation time compared to plant and animal resources, less need for labor, independent of season, climate, and geography, easier to scale up and no need for agricultural land (Ghanavati, Nahvi, and Karimi 2015; Phukan et al. 2019). Furthermore, oleaginous yeast can be used to convert cheap agro-industrial waste and even municipal waste into high quality lipids (Ghanavati, Nahvi, and Karimi 2015; Phukan et al. 2019). However, the conversion of these lignocellulosic feedstocks to high-quality lipids is dependent on the addition of commercially available enzymes to hydrolyze the carbohydrates to free fermentable sugar molecules (Damayanti et al. 2021). This need for large amounts of commercial enzymes at a large-scale production facility makes the economic aspect of the production process of lipids from oleaginous yeast unfavorable (Damayanti et al. 2021). Thus, the entire production process must be optimized, and new techniques must be developed to make it environmentally as well as economically sustainable. One possibility to enhance the economic viability of the lipid production process is to genetically engineer the oleaginous yeasts (Shi and Zhao 2017). There are several ways in which genetic engineering of oleaginous yeasts could conceivably contribute to a more economically favorable lipid production process. Genes involved in metabolic pathways towards the production of lipids within the yeasts could be altered, overexpressed, or deleted depending on the function of the genes (Görner et al. 2016; Koivuranta et al. 2018). Furthermore, the need for pretreatment of the lignocellulosic materials with commercial enzymes could potentially be reduced by inserting genes responsible for the production of specific enzymes from other microorganisms to the genome of the oleaginous yeasts (Kricka, Fitzpatrick, and Bond 2014).
In the light of the above, this PhD project aims to study the production of lipids in oleaginous yeasts using the organic fraction of solid municipal waste (OFMSW) as the feedstock. Furthermore, it aims to develop a sustainable and efficient extraction method for lipid extraction from the fermentation product. Lastly, it is intended to reduce the need for addition of commercial enzymes in the pretreatment of the OFMSW by genetic engineering of Cutaneotrichosporon oleaginosus and to test the genetically engineered yeast strains as well as the wild-type yeast strain in batch fermentations.

Funding: H2020, EU, GA 10100713