Along with the increased concern about the intensive usage of fossil fuels and global warming, a growing need has been motivated to find sustainable and renewable resources. Lignocellulosic biomass, one of the most abundant natural resources, is a promising alternative for replacing fossil carbon resources (Han et al., 2019). In recent years, numerous studies have been conducted to investigate the unitization of lignocellulosic biomass for the production of biofuels and other value-added chemicals, such as bioethanol, lipid, and lactic acid (Qiu et al., 2020, Wang et al., 2022, Yu et al., 2020). This bioconversion process has enormous potential for mitigating the dependence on fossil resources and fostering the sustainable development of global economies.

Lignocellulosic biomass is composed of cellulose, hemicellulose, and lignin through the ester and ether linkages, which confer a recalcitrant structure to resist microbial or enzymatic degradation. Pretreatment is an indispensable step to disrupt the complex rigid structure to release the fermentable sugars (mainly glucose and xylose) from lignocellulosic biomass (Mankar et al., 2021). However, a complex mix of toxic by-products, such as phenolic compounds, organic acids, and furans derivatives, are also generated during the pretreatment step, which poses a significant threat to microbial fermentation. Among these toxic by-products, phenolic compounds were commonly known as the most toxic compounds to most microorganisms (Adeboye et al., 2014). Aromatic acids, including vanillic acid and ferulic acid, existed as the common components of phenolic compounds (Xu et al., 2019) and showed particular toxicity to cell growth even in minute quantities. For example, vanillic acid with a concentration of 0.8 g/L could cause significant growth inhibition on Rhodosporidium toruloides, and similar growth inhibition on Saccharomyces cerevisiae was seen when 0.2 g/L ferulic acid was present (Larsson et al., 2000, Liu et al., 2021). Furthermore, the concentration of aromatic acids in lignocellulosic hydrolysate is primarily determined by the characteristics of the biomass and the pretreatment methods (Chen et al., 2020). For instance, the wet oxidation-treated rice straw generated 0.36 g/L vanillic acid (van der Pol et al., 2014). Similar concentrations were seen for ferulic acid, with 0.4 g/L in acid-treated corn stover (Lopez et al., 2004) and 0.2 g/L in alkali-treated rice straw (Hou et al., 2017). Given the inhibitory effects and the concentration of these aromatic acid compounds, efficient strategies to limit the effects of aromatic acids are required for promoting lignocellulosic hydrolysate fermentation.

In recent years, multiple strategies have been developed to reduce the inhibition of phenolic compounds on microbial fermentation. One approach is to use physical, chemical, and biological methods to remove the inhibitors (Lin et al., 2020, Liu et al., 2019). However, this detoxification process would lead to increased production costs, loss of fermentable sugars, and complex operations. Another potential alternative strategy is to enhance the phenolic compounds tolerance of microbes through adaptive laboratory evolution (ALE) (Wang et al., 2018, Wu et al., 2022). Adaptive laboratory evolution was considered an effective method for obtaining strains with enhanced inhibitor tolerance without detailed information about inhibitor action and responsible genes. Currently, adaptive evolution has been used successfully in improving the tolerance of strain to various inhibitors and environmental stresses. For instance, S. cerevisiae was conferred with different types of tolerance through adaptive evolution, including the tolerance to lignocellulose hydrolysates, individual inhibitors (e.g. acetic acid, furfural, and vanillin) (Ko et al., 2020, Stovicek et al., 2022), high temperature (Garcia-Rios et al., 2021), and low pH (Salas-Navarrete et al., 2022). In addition, the emergence of advanced RNA-seq technology can provide much information for uncovering the correlation between the tolerance phenotypes and genetic basis, further promoting ALE as a powerful tool to reveal the underlying molecular mechanisms of stress tolerance.

Yarrowia lipolytica is a typical oleaginous yeast that has been genetically modified to produce biofuels, biochemical, and enzymes (Blazeck et al., 2014, Liu et al., 2015). Recently, Y. lipolytica has shown great potential in the exploitation of lignocellulosic biomass for biofuels production due to its superior lipid accumulation capacity (Xu et al., 2016) and robustness in harsh environments including high salt and broad-range pH (2-11) conditions (Andreishcheva et al., 1999, Epova et al., 2012). Nevertheless, the fermentation performance of Y. lipolytica is still poor in lignocellulosic hydrolysate owing to the existence of inhibitors. As described above, the inhibitory effects of the phenolic compounds (mainly aromatic aldehydes and aromatic acids) were considered the main obstacle in the bioconversion of lignocellulosic biomass. Previous studies have studied the tolerance mechanism of Y. lipolytica to aromatic aldehyde inhibitors (Zhou et al., 2021). The main tolerance mechanism in Y. lipolytica is the conversion of aromatic aldehydes to corresponding aromatic acids. However, further information about the underlying mechanism and engineering efforts for improved aromatic acid tolerance was less studied in Y. lipolytica.

In this study, we performed an ALE experiment to improve the tolerance of Y. lipolytica towards a representative aromatic acid inhibitor: vanillic acid. Transcriptome analysis was subsequently carried out to identify the essential genes responsible for enhanced aromatic acid tolerance after evolution. The candidate genes were further identified and studied for their physiological functions in resistance to different aromatic acid inhibitors to reveal the underlying mechanisms. This work provides valuable guidance for tolerance engineering in constructing robust strains for lignocellulosic biorefinery.