Rhodosporidium toruloides: a new platform organism for conversion of lignocellulose into terpene biofuels and bioproducts

Background Economical conversion of lignocellulosic biomass into biofuels and bioproducts is central to the establishment of a robust bioeconomy. This requires a conversion host that is able to both efficiently assimilate the major lignocellulose-derived carbon sources and divert their metabolites toward specific bioproducts. Results In this study, the carotenogenic yeast Rhodosporidium toruloides was examined for its ability to convert lignocellulose into two non-native sesquiterpenes with biofuel (bisabolene) and pharmaceutical (amorphadiene) applications. We found that R. toruloides can efficiently convert a mixture of glucose and xylose from hydrolyzed lignocellulose into these bioproducts, and unlike many conventional production hosts, its growth and productivity were enhanced in lignocellulosic hydrolysates relative to purified substrates. This organism was demonstrated to have superior growth in corn stover hydrolysates prepared by two different pretreatment methods, one using a novel biocompatible ionic liquid (IL) choline α-ketoglutarate, which produced 261 mg/L of bisabolene at bench scale, and the other using an alkaline pretreatment, which produced 680 mg/L of bisabolene in a high-gravity fed-batch bioreactor. Interestingly, R. toruloides was also observed to assimilate p-coumaric acid liberated from acylated grass lignin in the IL hydrolysate, a finding we verified with purified substrates. R. toruloides was also able to consume several additional compounds with aromatic motifs similar to lignin monomers, suggesting that this organism may have the metabolic potential to convert depolymerized lignin streams alongside lignocellulosic sugars. Conclusions This study highlights the natural compatibility of R. toruloides with bioprocess conditions relevant to lignocellulosic biorefineries and demonstrates its ability to produce non-native terpenes. Electronic supplementary material The online version of this article (doi:10.1186/s13068-017-0927-5) contains supplementary material, which is available to authorized users.


Abstract (Word count 251, Limit 350 words) 22
Background: 23 Economical conversion of lignocellulosic biomass into biofuels and bioproducts is central to the 24 establishment of a robust bioeconomy. This requires a conversion host that is able to both 25 efficiently assimilate the major lignocellulose-derived carbon sources and divert their 26 metabolites toward specific bioproducts.

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Results: 28 In this study, the carotenogenic yeast Rhodosporidium toruloides was examined for its ability to 29 convert lignocellulose into two non-native sesquiterpenes with biofuel (bisabolene) and 30 pharmaceutical (amorphadiene) applications. We found that R. toruloides can efficiently convert 31 a mixture of glucose and xylose from hydrolyzed lignocellulose into these bioproducts, and 32 unlike many conventional production hosts, its growth and productivity were enhanced in 33 lignocellulosic hydrolysates relative to purified substrates. This organism was demonstrated to 34 have superior growth in corn stover hydrolysates prepared by two different pretreatment 35 methods, one using a novel biocompatible ionic liquid (IL) choline α-ketoglutarate, which 36 produced 261 mg/L of bisabolene at bench-scale, and the other using an alkaline pretreatment, 37 which produced 680 mg/L of bisabolene in a high gravity fed-batch bioreactor. Interestingly, R. 38 toruloides was also observed to assimilate p-coumaric acid liberated from acylated grass lignin 39 in the IL hydrolysate, a finding we verified with purified substrates. R. toruloides was also able 40 to consume several additional compounds with aromatic motifs similar to lignin monomers, 41 suggesting that this organism may have the metabolic potential to convert depolymerized lignin have indicated that lignin valorization will be critical for maintaining the economic viability and 68 sustainability of lignocellulosic biorefineries [3]. Therefore, conversion strategies that 69 incorporate lignin will have an economic advantage over those that focus solely on carbohydrate 70 conversion, creating a strong incentive for biorefineries to adopt microbial conversion hosts that 71 can achieve this feat.

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While well-established microbes such as Escherichia coli and Saccharomyces cerevisiae are 73 convenient hosts for bioproduct synthesis from glucose or xylose, they do not readily utilize 74 multiple carbon sources simultaneously, especially not those derived from lignin, making it 75 difficult to efficiently use lignocellulose as a carbon source [4]. Two approaches to circumvent 76 this problem are to 1) engineer commonly used hosts such as E. coli and S. cerevisiae to 77 efficiently utilize cellulose, hemicellulose, and lignin depolymerization products, or 2) find a 78 host that naturally has this ability and engineer it to make bioproducts. Rhodosporidium 79 toruloides, an oleaginous, carotenogenic basidiomycete yeast, has been studied as a model 80 organism for lipid production and has been shown to co-utilize both hexose and pentose sugars 81 [5], suggesting potential advantages of R. toruloides over conventional lignocellulosic 82 conversion hosts. R. toruloides accumulates high concentrations of lipids and carotenoids, both 83 of which are derived from acetyl-CoA [6]. This suggests that it may be a promising host for the 84 production of compounds synthesized from acetyl-CoA, especially terpene and lipid-based 85 bioproducts. Not only does it make these natural bioproducts, it can also grow to very high cell 86 densities (100 g/L dry cell mass) [7], another important industrially-relevant characteristic.

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Taking advantage of the recently developed genetic tools for R. toruloides [8][9][10][11], we explored 88 its utility as a new platform for production of non-native terpenes from lignocellulose. We 89 demonstrate that R. toruloides has the unique ability to simultaneously utilize glucose and xylose 90 derived from depolymerized cellulose and hemicellulose in addition to compounds associated 91 with lignin, such as p-coumaric acid. This compound is associated with grass lignins (like corn, 92 sorghum, switchgrass, etc.) where it is conjugated to lignin through an ester linkage that is easily 93 cleavable using existing alkaline lignocellulosic pretreatment technologies [12]. Unlike this 94 lignin conjugate, the lignin polymer itself is very heterogeneous and its depolymerization has the 95 potential to yield a wide variety of compounds derived from the lignin p-hydroxyphenyl (H), 96 guaiacyl (G), and syringyl (S) phenylpropanoid units. Therefore, R. toruloides was also 97 examined for its ability to consume several compounds containing aromatic motifs similar to the 98 H, G and S lignin subunits. Demonstration of the conversion of these types of compounds along 99 with lignocellulosic sugars opens the possibility of including a lignin as a carbon source in 100 lignocellulose conversion schemes, a process that would increase the efficiency and commercial 101 viability of a biorefinery. Finally, we demonstrate that R. toruloides is compatible with a single-102 unit or one-pot lignocellulose pretreatment, saccharification, and fermentation process ( Fig. 1) 103 that potentially reduce biorefinery capital and operating expenses (CAPEX,and OPEX,104 respectively) and wastewater treatment [13,14]. Together, these abilities suggest that R. 105 toruloides has the potential to be a platform organism for the conversion of the majority of the 106 carbon present in lignocellulose into advanced biofuels and bioproducts. YPD agar plate containing antibiotics at the following concentrations: nourseothricin, 100 120 µg/mL, and cefotaxime, 300 µg/mL. The seed cultures were used to inoculate 5 mL SD media 121 with a starting optical density at 600 nm (OD 600 ) of 0.1. The same inoculation strategy was used 122 for cultivations of lignin-related monoaromatics, with the following compounds being added 123 separately to SD medium: p-coumaric acid, ferulic acid, p-hydroxybenzoic acid, vanillic acid, 124 sinapic acid, benzoic acid and vanillin, at a final concentration of 2 g/L for each compound.

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Cultures of terpene-producing strains were overlaid with 20% (v/v) dodecane. All cultures were 126 grown at 30°C with shaking at 200 rpm. Growth was monitored by measuring OD 600 . Samples in 127 which the OD measurements were significantly different from others in the sample set were 128 excluded from the analysis. Strains and plasmids used in this study can be found in Table 1 dodecane was sampled to quantify bisabolene production. This process was repeated 2 additional 157 times, spanning four rounds of culture over 24 days.

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Measurement of lipid and carotenoid content 160 Total lipid content was quantified gravimetrically following extraction with Folch reagent (2:1 161 chloroform/methanol) as described previously [18]. Carotenoids were extracted with acetone and 162 quantified by high performance liquid chromatography (HPLC) as described previously [19].

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Analysis of sugars and α-ketoglutarate 167 The concentrations of sugars and α-ketoglutarate were quantified on an Agilent Technologies 168 1200 series HPLC equipped with an Aminex HPX-87H column (BioRad, Hercules, CA) as 169 described previously [20]. Sugars were monitored by a refractive index detector, and α-

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The raw and pretreated corn stover were dried and characterized with powder X-ray diffraction 207 (XRD). The XRD analyses were performed on a PANalytical Empyrean X-ray diffractometer 208 equipped with a PIXcel3D detector and operated at 45 kV and 40 kA using Cu Kα radiation 209 (λ=1.5418Å). The patterns were collected in the 2θ range from 5 to 60° with a step size of 0.039° 210 and an exposure time of 300 seconds. A reflection-transmission spinner was used as a sample 211 holder and the spinning rate was set at 8 rpm throughout the experiment. Crystallinity index (CrI) 212 was determined by Segal's method [21].

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X-ray diffraction (XRD) studies were conducted to determine the changes in the crystalline vs.

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non-crystalline components found in the untreated corn stover, and to monitor the structural 215 changes in these polymers that occur during the pretreatment process. Additional file 1 shows the 216 X-ray diffractograms of the untreated and pretreated corn stover after processing at 120°C  Bioreactor cultivation using alkaline hydrolysate 245 The seed cultures were prepared by transferring a single colony from a YPD agar plate to a 500 246 mL baffled flask containing 250 mL of seed medium. The seed medium consisted of 10 g/L 247 yeast extract, 20 g/L peptone, and 20 g/L glucose. The seed was grown at 30°C, shaking at 250 248 rpm overnight to reach exponential growth phase. When the seed reached the exponential growth 249 phase, 5.5% (v/v) inoculum was transferred to each bioreactor to reach an initial OD 600 of 0.6.

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Germany) with an extractive fermentation. Fermentation process parameters were controlled 252 with temperature at 30°C, dissolved oxygen at 40% air saturation, and pH 5, respectively. toruloides BIS strain is that bisabolene titers show remarkable stability over extended periods of 298 serial cultivation, varying by less than 16% over the course of four cultures spanning 24 days 299 (Fig. 3). It should be noted that this reproducibility was also achieved without the need for a 300 heterologous inducer or antibiotic selection, since the BIS gene is stably integrated into the 301 genome and its expression is under control of a constitutive GAPDH promoter [8][9][10]. Both of 302 these features reduce OPEX in a biorefinery. In comparison, the bisabolene titer from an 303 engineered strain of S. cerevisiae grown under similar conditions was found to decline by more 304 than 75% over 14 days [29]. The strain stability we observed in engineered R. toruloides is an 305 important industrial phenotype and a critical factor for large-scale economical production of any 306 bioproduct.

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We found that the pH of the growth medium is an important factor for efficient sugar utilization 308 by R. toruloides. After examining a range of starting pH values in unbuffered medium (3 to 8) in 309 batch cultures, a starting pH of 7.4 was determined to be optimal to achieve complete glucose 310 utilization (Table 2) and the highest bisabolene titer (Fig. 4). Interestingly, R. toruloides grew 311 and produced bisabolene at a pH as low as 3.4, suggesting the host may be amenable to 312 production of organic acids or other bioproducts that require low pH. One potential explanation 313 for the decline in pH is that R. toruloides is producing native organic acids of potential value, a 314 topic that merits further investigation. However, once the pH declines to 2.5 (in unbuffered 315 medium starting at pH 7 or below), sugar utilization is strongly inhibited, suggesting that the pH 316 must remain above this level to enable efficient carbon conversion. Therefore, all subsequent 317 experiments in unbuffered media were performed with a starting pH of 7.4. Cultures were carried out as described above, the aqueous layer was sampled for glucose 320 analysis (n=3, data shown as average ±s.d, from a single experiment).

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To demonstrate the capability of engineered R. toruloides to utilize different carbon sources to 324 produce non-native terpenes, we cultivated the bisabolene-producing strain BIS3 with the most 325 abundant sugars present in lignocellulosic hydrolysates: glucose and xylose. In addition, we 326 observed the liberation and consumption of p-coumaric acid in the IL-pretreated cornstover 327 hydrolysate described in detail below (the hydrolysate in Fig. 7), and therefore we examined this the pretreatments used to generate hydrolysates in this study . Initially, these three carbon 332 sources were provided individually and growth, carbon utilization, and bisabolene production 333 were monitored (Fig. 5A-C). Glucose was completely consumed at the fastest rate, followed by 334 p-coumaric acid, then xylose (in 1, 3, and 4 days, respectively). The highest bisabolene titers 335 were observed in the p-coumaric acid cultures, likely due to its higher percentage of carbon 336 relative to the sugars (Fig. 5A-C). Remarkably, when combined, all three carbon sources were 337 completely utilized within four days (Fig. 5D) the level tested (Fig. 6). We also tested a compound with the same aromatic motif as the 4-360 hydroxy-3,5-dimethoxypheny S units (sinapic acid), but the results were inconclusive due to 361 apparent oxidation and precipitation of the substrate during the cultivation. Overall, these results 362 indicate that R. toruloides has the metabolic potential to consume lignin-degradation products 363 derived from depolymerization processes that produce compounds similar to those tested. This 364 capability highlights the potential of R. toruloides to be used as a conversion host of 365 monoaromatic lignin degradation products, a characteristic that will become more important as 366 biomass deconstruction technologies advance to provide more extensive lignin depolymerization.  xylose, and p-coumaric acid, and produce 261 mg/L of bisabolene (Fig. 7A). In fact, it produced 397 higher titers of bisabolene in the hydrolysate than it did in a control medium with matching 398 concentrations of the IL, sugars, and p-coumaric acid (127 mg/L) (Fig. 7B).

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In this study, we demonstrate that this organism is a versatile production host that possesses 437 many features critical to reducing CAPEX and OPEX in a biorefinery: 1) it can be used to make