Metabolism of aromatics by Trichosporon oleaginosus while remaining oleaginous

Valorization of lignocellulosic biomass wastes is critical for the economic viability of the biomass economy [1]. Furthermore, valorization of wastewater and food and agricultural wastes may enhance sustainability and provide additional economic benefits [1, 2]. Waste streams are often heterogeneous in nature and contain additives and by-products, such as phenolics, that are toxic to human health. A by-product or waste that has gained considerable attention recently is lignin. It is the second-most abundant biopolymer on Earth and the only renewable, readily-available biopolymer comprised of aromatics [1]. Lignin is a by-product of biomass used as a feedstock for biofuels and biochemical production, and is a prominent by-product of pulp and paper mills. Kraft lignin is typically burned for its heating value, and only 2% is recovered for nonfuel uses [3]. Rather than burning it, lignin could be utilized in biorefineries as a feedstock for microbial production of higher value products [2, 4]. In addition, the large quantities of aromatics in industrial wastewater effluents makes aromatic compounds a prime target for waste valorization [5].

Bacterial metabolism and growth on various phenolic compounds is well-characterized. Many of these aromatics are representative lignin hydrolysate compounds or common products of lignin depolymerization [6]. Significant work has characterized phenolic metabolism in Rhodococcus opacus PD630 [7–9], Acinetobacter baylyi ADP1 [10, 11], and Pseudomonas putida [4, 12–15]. There are fewer examples of yeast that have been characterized to grow on phenolics, such as Pichia holstii [16], Candida tropicalis [17], and Trichosporon cutaneum [18]. These yeasts have evolved to handle a wide variety of aromatic substrates, utilizing so-called funneling pathways to convert diverse molecular species into a small number of metabolites [19]. There are many pathways for aerobic aromatic metabolism, with the ortho- and meta-cleavage pathways being most common [19]. These pathways generate central metabolites, such as pyruvate and acetyl-CoA [20–23]. Understanding the biochemical pathways of aromatic metabolizing organisms enables downstream engineering for high value products, such as oleochemicals for pharmaceutical, fuel, and specialty chemical applications.

Oleaginous microorganisms are a rational starting point for microbial production of oleochemicals, such as lipids for biofuels and omega-3 fatty acids. These microbes are characterized by their capacity to accumulate at least 20% of their mass as lipids. Significant attention has been given to several oleaginous yeast, including Yarrowia lipolytica [24–27], Lipomyces starkeyi [28], and Rhodosporidium toruloides [29]. Substantial work has been done to expand the genetic engineering tools available for these non-conventional oleaginous yeasts, enabling metabolic engineering of these species [30–36]. Despite being established industrial hosts with significant prior work, these yeast species are not suitable for utilizing certain low-cost feedstocks, such as aromatic-rich lignin and phenolic wastewater streams. Given the 50 million tons of lignin currently produced per year [3], there is a great need for microorganisms that are able to tolerate and even metabolize aromatic feedstocks. Of the oleaginous microorganisms that tolerate aromatic toxicity, bacteria do not achieve a high enough biomass and are prone to phage infection, and white-rot fungi grow too slowly. Similarly, few oleaginous yeasts are known to metabolize aromatics. Furthermore, the oleaginous yeast shown to metabolize aromatics do not maintain high lipid accumulation under aromatic growth conditions.

This study addresses the narrow crossover between efficient aromatic metabolism, rapid growth kinetics, and high endogenous lipid accumulation by investigating Trichosporon oleaginosus, a non-model, non-conventional yeast previously known as Cryptococcus curvatus. A recent review summarizes the ability of T. oleaginosus to metabolize a number of non-conventional feedstocks and maintain oleagincity [37]. In this study, we found T. oleaginosus tolerates several aromatics and metabolizes them when used as a sole carbon source. Simultaneous growth on mixtures of sugars and aromatics appeared diauxic; however, both substrates were completely consumed. Finally, we demonstrate fed-batch growth on aromatics results in over 69% of biomass accumulated as lipids in a simple shake flask. Due to its rapid growth rate on aromatics, and its significant lipid accumulation, we suggest T. oleaginosus has great potential as a model system for aromatic metabolizing oleaginous yeast.

Trichosporon oleaginosus has been studied for its ability to accumulate a significant amount of lipids from a variety of feedstocks. This is the first report showing that T. oleaginosus is able to metabolize model lignin monoaromatic compounds when used as a sole carbon source. It is likewise the first report of T. oleaginosus metabolizing aromatics in mixed sugar and aromatic substrates. While other yeast have been shown to tolerate and metabolize aromatic compounds [41], this is the first demonstration of yeast growing on aromatics and accumulating lipids greater than 20% on a dry weight basis. In fed-batch experiments, T. oleaginosus was able to accumulate over 69% of its dry cell weight as lipids using aromatic compound resorcinol as the sole carbon source, demonstrating its promise as a model organism for aromatic to oleochemical conversion.

To date, the highest reported lipid accumulation from T. oleaginosus is 73.4% on a dry weight basis using acetate as the sole carbon source [42]. This lipid accumulation compares favorably to the lipid accumulation of 69.5% on a dry weight basis from resorcinol demonstrated in this work. We were unable to find any report of lipid accumulation in a microbe grown on resorcinol as the sole carbon source. The literature has focused on bioremediation of xenobiotics and anthropomorphic aromatic substrates rather than lipid accumulation. Amongst oleaginous bacteria, Rhodococcus opacus strains DSM 1069 and PD630, accumulate up to 20% of dry weight as lipids when grown on pHBA and vanillic acid in optimized fed-batch reactors [8]. Phenol toxicity impacted the growth rate when reaching 0.3 g/L [9]. A strain of R. opacus PD630 evolved for higher tolerance, increasing from 0.3 g/L up to 1.5 g/L achieving lipid accumulation of 11.7% on a dry weight basis [9]. The titer was not reported, but could not be higher than 1.5 g/L, the concentration of phenol used in the experiments, and theoretically must be much lower. By comparison, our highest lipid titer was 1.64 ± 0.2 g/L with a lipid accumulation of 69.3% and was achieved using a wild-type strain. Trichosporon oleaginosus is able to accumulate significantly higher titers of lipids when grown on higher concentrations of preferred substrates such as glucose [38, 39, 43]; however, the toxicity of aromatic compounds limits the concentration of aromatics dosed at a single time. The toxicity limitation of aromatic substrates limits the carbon to nitrogen (C:N) ratio when growing initial cell biomass, which benefits significantly from a small amount of yeast extract. In one study, T. oleaginosus was previously shown to achieve the highest percent accumulation and lipid titer at a C:N ratio of 99 [44], whereas another study used a C:N ratio of 360 [40]. These high C:N ratios are consistent with our observations of poor lipid accumulation using a C:N ratio of 36, but drastically greater lipid accumulation using a ratio of 360:1 in fed-batch experiments, emphasizing the role of the C:N ratio on lipid accumulation in T. oleaginosus.

In nature, lignin can be enzymatically depolymerized to a wide variety of aromatics. Similarly, catalytic depolymerization also results in a heterogeneous mixture of aromatic compounds [41]. Funneling pathways overcome the inherent heterogeneity of lignin by converting key aromatic compounds to important intermediates for the TCA cycle [4, 20–23]. The beta-ketoadipate pathway is well-conserved amongst aromatic metabolizing bacteria and yeast. The most common pathways are intra-diol (ortho) ring cleavage and extra-diol (meta) ring cleavage pathways. Based on our demonstration of resorcinol, pHBA, and phenol metabolism, T. oleaginosus appears to have each of the three major funneling pathways. The enzymatic activity of aromatic metabolism was previously studied in Trichosporon cutaneum. Metabolite analysis demonstrated ortho ring cleavage reaction products and the absence of meta ring cleavage products [45, 46]. Given both are from the same genus, it is likely that T. oleaginosus uses the ortho ring cleavage pathway. A more detailed study is needed to definitively identify the pathways for aromatic metabolism.

Lignocellulosic hydrolysates contain sugars derived from the cellulose and hemicellulose content and aromatics from the lignin content; therefore, our demonstration that T. oleaginosus co-utilizes resorcinol and sugars (glucose or xylose) establishes the potential to utilize multiple components of lignocellulosic biomass. Given that T. oleaginosus can co-utilize glucose and xylose (Additional file 1: Figure S1), we expect that this yeast will co-utilize glucose, xylose, and aromatics. In addition, this finding suggests it may be unnecessary to remove phenolic content in recalcitrant feedstocks, as is typically required due aromatic toxicity and lack of metabolism [47]. Related organisms show similar co-utilization. In fact, wild type T. cutaneum was shown to simultaneously uptake glucose and phenol, with the rate of glucose utilization being much higher than that of phenol utilization [48], consistent with our findings. The presence of glucose decreased phenol utilization and catechol-1,2 dioxygenase (an enzyme belonging to the catechol funneling pathway) activity by 60 and 75%, respectively.

We show T. oleaginosus is able to tolerate 15 different compounds to various degrees, with MICs as high as 15.5 g/L for protocatechuate and as low as 0.2 g/L for vanillin. Improving tolerance will be important for further development of industrially relevant strains. Several mechanisms have been reported to avoid aromatic toxicity including dynamic lipid distribution, efficient transporters, and highly active aromatic conversion genes. The mechanism for aromatic tolerance in bacteria is mostly attributed to redistribution of the highly dynamic lipid composition and the activation of aromatic efflux transporters [49–53]. Pseudomonas putida strains isolated from toluene-polluted sites responded to aromatic-induced membrane stress by increasing the ratio of trans and cis fatty acid isomers, making the cell membrane more rigid. Pseudomonas putida DOT-T1E has been shown to tolerate and metabolize 17 g/L pHBA, which the authors attribute to the cell membrane rigidity [54]. A more recent study showed the fatty acid composition of R. opacus strains grown on phenol and benzene also exhibited increased trans-fatty acids and increased 10-methyl branched fatty acid content in the presence of aromatics [51]. While these specific membrane adaptations are unique to prokaryotes, eukaryotes can likewise alter membrane fluidity through alterations in composition and sterols [55]. Aside from altering membrane fluidity, strains of P. putida have been shown to utilize efflux pumps to remove toluene from the cell membrane [50, 53]. However, efflux pumps would be counter to the desired aromatic metabolism. Recently, an evolved strain of R. opacus PD630 showed increased importer activity led to faster growth on phenol, and higher tolerance to phenol compared to the wild type strain [9]. Lastly, a recent study in Saccharomyces cerevisiae shows that enabling efficient conversion genes also improves tolerance by removing the toxic compounds and converting them to a more benign intermediate [56].

We hypothesize that T. oleaginosus uses a combination of these mechanisms. The fatty acid distribution changes in the presence of aromatic compounds as compared to glucose, with glucose resulting in C18:3, but aromatic grown cells have a higher content of C18:1 and C18:2. The identical growth rates of cells using glucose, pHBA, or resorcinol suggest the potential for efficient metabolism of aromatics. Enhancing import or metabolism of aromatics could lead to improved tolerance. The recent demonstration of genetic engineering tools for T. oleaginosus [38] makes it possible to engineer increased tolerance through control of membrane fluidity and overexpression of rate limiting aromatic metabolism genes. Improving aromatic tolerance in this, and any microbe, is necessary to economically utilize higher concentrations of substrate for industrial applications, further motivating future studies understanding the mechanism for aromatic metabolism and tolerance and engineering improvements.

Trichosporon oleaginosus is the first yeast shown to metabolize aromatics and remain oleaginous, accumulating nearly 70% of its biomass as lipids when grown on resorcinol in fed-batch. This yeast shows promise for utilizing aromatic-containing feedstocks, such as lignin and wastewater effluent, for microbial production of oleochemicals or aromatic-derived compounds due to its natural ability to tolerate and metabolize relatively high concentrations of aromatics. A complete understanding coupled to additional metabolic engineering tools will enable pathway engineering for improving tolerance to and conversion of aromatics for industrial applications. Nevertheless, this yeast is well-positioned to become a model system for aromatics metabolism to lipids and oleochemicals.