Non-canonical d-xylose and l-arabinose metabolism via d-arabitol in the oleaginous yeast Rhodosporidium toruloides

R. toruloides is an oleaginous yeast, with diverse metabolic capacities and high tolerance for inhibitory compounds abundant in plant biomass hydrolysates. While R. toruloides grows on several pentose sugars and alcohols, further engineering of the native pathway is required for efficient conversion of biomass-derived sugars to higher value bioproducts. A previous high-throughput study inferred that R. toruloides possesses a non-canonical l-arabinose and d-xylose metabolism proceeding through d-arabitol and d-ribulose. In this study, we present a combination of genetic and metabolite data that refine and extend that model. Chiral separations definitively illustrate that d-arabitol is the enantiomer that accumulates under pentose metabolism. Deletion of putative d-arabitol-2-dehydrogenase (RTO4_9990) results in > 75% conversion of d-xylose to d-arabitol, and is growth-complemented on pentoses by heterologous xylulose kinase expression. Deletion of putative d-ribulose kinase (RTO4_14368) arrests all growth on any pentose tested. Analysis of several pentose dehydrogenase mutants elucidates a complex pathway with multiple enzymes mediating multiple different reactions in differing combinations, from which we also inferred a putative l-ribulose utilization pathway. Our results suggest that we have identified enzymes responsible for the majority of pathway flux, with additional unknown enzymes providing accessory activity at multiple steps. Further biochemical characterization of the enzymes described here will enable a more complete and quantitative understanding of R. toruloides pentose metabolism. These findings add to a growing understanding of the diversity and complexity of microbial pentose metabolism. Supplementary Information The online version contains supplementary material available at 10.1186/s12934-023-02126-x.


Introduction
Economically viable biorefineries upgrading lignocellulosic biomass to value-added products using microbial platforms will need to efficiently utilize all four major sugar monomers-d-glucose, d-xylose, d-mannose, and l-arabinose [1].While d-glucose is the preferred substrate for most organisms, many have little to no capacity to metabolize d-xylose, the second largest component of biomass [2][3][4].A deeper understanding of pentose catabolism in diverse organisms is required in order to engineer strains with utilization efficiency on par with that of d-glucose.
A recent study of multi-omics analysis and metabolic model curation surmised-with RNAseq, proteomics, and functional genomics-that R. toruloides possesses non-canonical pentose metabolism through d-arabitol, d-ribulose, and d-ribulose-5-phosphate (Ru5P) [13] (Fig. 1).However, R. toruloides has a putative XK (encoded by RTO4_16850), but RNA transcript levels are near the limit of detection and no XK peptides were detected on both pentose and d-glucose [13].Simultaneously, putative genes RTO4_9990 and RTO4_14368 (encoding d-arabitol-2-dehydrogenase (DA2DH) and d-ribulose kinase (RK), respectively) were strongly expressed at the peptide level and mutants in these genes had strong growth defects on nearly all pentose substrates examined, whereas mutants in XK had no growth defects in any condition tested [13].Supporting these data, Jagtap et al. reported that R. toruloides excretes d-arabitol with d-xylose as the sole carbon source; however, they postulated that d-arabitol is a dead-end overflow metabolite to maintain redox homeostasis under high d-xylose utilization conditions alongside traditional flux through XK [14].Under this model, deletion of RTO4_9990 (putative DA2DH) should have a minor effect on d-xylose growth, with eventual complete or near-complete growth recovery, contradicted by Kim et al. [13].An additional study by Jagtap et al. with metabolomics and transcriptomics on several carbon sources recapitulates and reinforces the findings of Kim et al., showing no XK transcription on d-xylose with a simultaneous upregulation of the putative alternative route via d-arabitol [13,15].The authors updated the model in congruence with the findings of Kim et al.; however, no further pathway characterization or verification was performed.
For example, Saha et al. showed that Zygosaccharomyces rouxii is able to produce d-arabitol and/or xylitol from many carbon sources including d-xylose, d-glucose, d-xylulose, d-fructose, and d-mannose, proposing an unverified pentose model accounting for their observations [24,25].A previous study of purified extracts of a different Z. rouxii strain fed [5-14C] d-xylulose demonstrated NADH-linked DA4DH reduction of d-xylulose to d-arabitol [26], with no activity on d-arabitol, but some activity on xylitol (XDH activity), suggesting putative DA4DH is irreversible-either intrinsically so or due to thermodynamic constraints of the tested condition.From other work, it was concluded that DA2DH activity is responsible for production of d-arabitol from d-glucose [27].Unfortunately, d-arabitol production from d-xylose was not assessed, preventing a definitive understanding in Z. rouxii, although production seems likely via simultaneous DA4DH and DA2DH activity.However, there was no verification of their theoretical model of P. anomala pentose metabolism, so details of d-xylose metabolism remain unclear.A study of Candida maltosa grown on d-xylose mother liquor produced mostly xylitol, with a small fraction of d-arabitol [29,30].The authors then tested for growth solely on d-arabitol without observing xylitol production.They surmised an irreversible reaction of d-xylulose reduction (to d-arabitol) and proposed a potential d-xylose catabolic pathway accounting for d-arabitol production that includes both canonical (XR, XDH, XK) and non-canonical routes (XR, XDH, DA4DH, DA2DH).However, no in-depth characterization of the pathway or responsible genes was carried out to confirm this model.Candida arabinofermentans and Pichia guilliermondii fed 13C-labeled l-arabinose produced both labeled d-arabitol and labeled l-arabitol, as detected via NMR.However, the authors inferred that that d-arabitol was produced from d-ribulose via the PPP as opposed to directly from d-xylulose, and hypothesized that this only serves as a means to regenerate NAD + during oxygen-limiting conditions rather than a mainstay for pentose metabolism [31,32].Additional examples and a more comprehensive survey of arabitol and other polyols produced by various yeasts can be found elsewhere [20,[33][34][35][36][37][38][39].
Taken together, data from different fungi suggest a diversity of polyol metabolism linked to pentose catabolism, though in most cases the data are fragmented with uncertainty as to which polyols are intermediates in the main pathway, and which may be side products.Also, while previous work by Kim et al. [13] provides solid evidence that a non-canonical pathway exists in R. toruloides and identifies several enzymes involved, high-throughput fitness studies are limited in their precision, especially for cases of overlapping enzyme function.In order to engineer R. toruloides for optimal pentose conversion, a more complete picture of the catabolic pathway and the enzymes mediating each step is required.To this end, we systematically probed the functions of each of the major putative R. toruloides pentose genes via genomic deletions and selective complementation by heterologous XI and XK.Growth phenotyping on representative pentose substrates, metabolite profiling, and enantiomer determination of arabitol accumulated in the growth medium were employed to piece together a clear picture of this unusual pentose metabolism and further validate previous high-throughput observations.Lastly, we show that the pentose pathway is functionally redundant at nearly every step and explore an unusual substrate-specific bypass to our proposed pentose metabolic model.

Growth phenotyping of putative pentose catabolic enzyme deletions
As in most eukaryotes, the first step in d-xylose metabolism in R. toruloides is likely via an XR as opposed to an XI [40].Protzko et al. found a putative l-glyceraldehyde and general pentose reductase (encoded by RTO4_9774) critical for d-galacturonic acid metabolism.In vitro enzyme activity assays showed that substrates of the reductase include l-glyceraldehyde, l-arabinose, and d-xylose, and NADPH.This broad substrate specificity is not unusual amongst fungi [41,42].Although the preferred substrate of the reductase is not d-xylose, we still assessed its role in d-xylose metabolism.An RTO4_9774 deletion resulted in diminished, but not abolished, growth on d-xylose as the sole carbon source, indicating XR redundancy (Fig. 2).Alignment of characterized fungal XRs from Aspergillus niger and Trichoderma reesei to R. toruloides suggests many additional candidates for this activity.
There is no evidence that R. toruloides has a functional XI, allowing us to employ XI in our investigation of the pentose assimilation pathway.To test its functionality, codon-optimized XI (from Lachnoclostridium phytofermentans) was randomly integrated (via Agrobacterium tumefaciens-mediated transformation (see methods)) into ΔRTO4_9774 (i.e., ΔRTO4_9774 + OE XI), driven by a strong promoter (Rhodotorula graminis Tef1; Fig. 2), recovering growth of the deletion mutant.This indicates that d-xylulose (the product of XI) is likely a native metabolite of pentose metabolism, and that the ratelimiting step of pentose metabolism is probably either downstream of d-xylulose (provided sufficient L. phytofermentans XI expression and activity) or at the point of transport into the cell.
Following reduction of d-xylose, xylitol is likely oxidized to d-xylulose via XDH (Fig. 1).RTO4_8988 mutants have considerable fitness defects on multiple carbon sources (l-arabinose, l-arabitol, l-lyxose, xylitol), and modest defects on d-xylose, d-xylulose [13].Thus, RTO4_8988 likely plays a promiscuous role in l-arabitol and d-xylose metabolism.Orthologous sequences from the filamentous fungi, T. reesei, with empirical data obtained from cell-free extracts and purified enzyme assays include an NADPH-dependent d-mannitol 2-dehydrogenase encoded by lxr1 [43], and an NADPH-dependent LXR encoded by lxr3 [44], the latter showing promiscuous polyol-forming activity on many substrates in addition to l-xylulose-notably d-xylulose [44].Additionally, characterized LXR1 from A. niger shows weak similarity to only a single gene, RTO4_8988 [45,46].Unsurprisingly, ΔRTO4_8988 grown on select pentose substrates shows multiple growth defects in our proposed pentose metabolism model (Fig. 3).Namely, growth defects are observed on d-xylose, xylitol, d-xylulose, l-xylulose, or l-arabitol.The progressively worsening growth defects of RTO4_8988 mutants as the carbon source is moved upstream in the pentose utilization pathway definitively supports RTO4_8988 d-arabitol-4-dehydrogenase (DA4DH) activity and possibly XDH, LXR activities.
Under canonical fungal metabolism, d-xylulose is converted to Xu5P via XK (Fig. 1).Comparative sequence analysis to characterized fungal XKs reveals that R. toruloides RTO4_16850 likely encodes an XK [47,48]; however, under no conditions tested were RTO4_16850 peptides detected or any fitness defects observed upon deletion via RB-TDNAseq fitness profiling [13].Either regulatory mechanisms, improper conditions, coding mutations, or cofactor balancing are suppressing expression and activity.To explore the functionality of RTO4_16850, a plasmid driven by a strong native promoter (P14 Tef1; [49]) expressing native RTO4_16850 sequence was randomly integrated into ΔRTO4_9990 (deletion without growth on d-xylose), and screened for growth on d-xylose as the sole carbon source.No growth was observed in any of the 48 transformants.
With strong evidence of no native XK functionality, XK from A. niger was codon optimized, overexpressed on a plasmid driven by the strong Rhodotorula graminis Tef1 promoter, and randomly integrated in ΔRTO4_8988 (i.e., ΔRTO4_8988 + OE XK).Growth on four of five sugars with observed growth deficits in ΔRTO4_8988 was partially or fully recovered upon XK complementation (Fig. 3).Most notable is the vastly improved growth of ΔRTO4_8988 + OE XK relative to WT on d-xylose (Fig. 3A), implying pentose metabolism is possibly rate limited downstream XDH, and RTO4_8988 may encode minor XDH activity.Interestingly, ΔRTO4_8988 + OE XK has an exacerbated growth deficit on xylitol relative to ΔRTO4_8988, potentially due to unbalanced redox homeostasis in this condition.ΔRTO4_8988 + OE XK grown on l-arabitol and l-xylulose shows partial recovery relative to WT, supporting RTO4_8988 LXR activity (Fig. 3D-E).As expected, the d-xylulose growth deficit from loss of RTO4_8988 DA4DH activity is complemented by ΔRTO4_8988 + OE XK (Fig. 3C).Growth of ΔRTO4_8988 + OE XK on d-arabitol is not impacted by XK expression, possibly due to reaction irreversibility of DA4DH, thermodynamic constraints, or cofactor/ redox imbalance.We further explored the effects of XI and XK overexpression in ΔRTO4_8988 on d-xylose via random integration with the Rhodotorula graminis Tef1 promoter (Fig. 4).ΔRTO4_8988 + OE XI has an identical growth rate to ΔRTO4_8988; however, once XK is overexpressed with ΔRTO4_8988 + OE XI (Fig. 4), growth surpasses WT (similar to Fig. 3A), indicating XR and XDH are not rate limiting.The lack of XI improving growth is similar to Fig. 2
After d-arabitol oxidation, d-ribulose is most likely converted to Ru5P (Fig. 1) via putative RK encoded by RTO4_14368, orthologous to characterized S. cerevisiae RK [66].Like RTO4_9990, RTO4_14368 is highly upregulated on pentose sugars l-arabinose and d-xylose, but unlike ΔRTO4_9990, ΔRTO4_14368 exhibits major fitness defects on d-ribulose in addition to other pentoses [13].We constructed ΔRTO4_14368 and observed growth across 4 pentose substrates (l-ribulose,  8).ΔRTO4_14368 exhibited no growth on any of the 7 substrates tested, implying that all pentose metabolism proceeds through RK.

Metabolite excretion profiles of key pentose mutants
To complement growth phenotyping data, we collected pentose intermediate time-course data from select strains grown on 40 g/L d-xylose with 40 g/L glycerol (Fig. 9).ΔRTO4_8988 is the only strain that accumulates d-xylulose (Fig. 9E), supporting RTO4_8988 DA4DH activity (Fig. 3C).Glycerol consumption is uninhibited, but d-xylose utilization is heavily impacted; however, at peak titers (48 h), nearly 50% of consumed d-xylose is temporarily converted to xylitol, supporting XDH activity (Fig. 9D).In vitro characterization of P. anomala arabitol dehydrogenase (38% identify, 70% coverage to RTO4_8988) shows reversible DA4DH activity and irreversible XDH activity (xylitol to d-xylulose) [28].After 48 h, though an additional 10 g/L d-xylose are consumed, 4 g/L of xylitol are also consumed.This may reflect ΔRTO4_9990 converted the majority of d-xylose to d-arabitol with a small fraction to xylitol that was eventually consumed (Fig. 9C-D), consistent with RTO4_9990 encoding a DA2DH (Fig. 6).If RTO4_9990 additionally encoded for significant DA4DH activity, we would expect to see an accumulation of d-xylulose (similar to ΔRTO4_8988), but we do not (Fig. 9E).Curiously, ΔRTO4_9990 is able to consume both d-xylose and glycerol more rapidly than WT, hinting that RTO4_9990 itself might be one of the suspected rate-limiting steps in pentose metabolism.Lastly, ΔRTO4_16452 glycerol and d-xylose utilization were similar to WT (Fig. 9A-B); however, there was a modest, but significant temporal decrease and corresponding increase of d-arabitol and xylitol, respectively, relative to WT-supporting RTO4_16452 XDH activity (Fig. 9C-D).No excretion of d-xylulose was observed in ΔRTO4_16452, matching that of WT (Fig. 9E).

Verification of arabitol enantiomer production
In fungi, few studies have definitively verified the actual production pathway of arabitol or distinguished the enantiomer produced [31], which is important for downstream applications [33].Neither of these two inquiries have been satisfactorily investigated in R. toruloides or related Rhodotorula species; therefore, we performed chiral separations of d-arabitol, l-arabitol standards, and the supernatant from ΔRTO4_9990 grown on glycerol plus d-xylose (Fig. 9C) via gas chromatography-mass  9).Solid lines are the average of 3 (biological) replicates; shaded regions indicate 100% percentile intervals spectrometry (GC-MS; see methods).Indeed, Fig. 10 shows that the arabitol isomer produced from d-xylose is d-arabitol.

Enzymatic d-arabitol-2-dehydrogenase redundancy
In Fig. 6, ΔRTO4_9990 did not grow on any substrate except for l-arabitol, with slow growth between 40 and 100 h (Fig. 6D), suggesting latent DA2DH redundancy in addition to activity encoded via RTO4_9990.In Fig. 9, a carbon balance between d-xylose, d-xylulose, d-arabitol, and xylitol was approximately closed until mid-run, then approximately 10% of carbon was unaccounted for by the end of the experiment.The onset of this carbon balance gap coincided with the onset of slow growth of ∆RTO4_9990 (Fig. 6D).To test if latent expression of RTO4_16850 was responsible for ∆RTO4_9990's slow growth on l-arabitol, we constructed a double-deletion strain (i.e., ΔRTO4_9990 ΔRTO4_16850); however, mild growth was still observed on l-arabitol.ΔRTO4_9990 ΔRTO4_16850 was then adapted on 40 g/L l-arabitol (see methods) to generate ΔRTO4_9990 ΔRTO4_16850*.The evolved strain and controls were then cultured on 6 pentose substrates, including l-arabinose (Fig. 11).The evolved strain (ΔRTO4_9990 ΔRTO4_16850*) grew faster than the parent strain on both l-arabitol and l-arabinose, surpassing WT biomass yields with a similar growth rate.However, ΔRTO4_9990 ΔRTO4_16850* is still not able to grow on any other pentose substrate.Further, the evolved strain and controls were grown on 40 g/L l-arabinose, monitoring pentose intermediates (Fig. 12).Over 10 g/L of d-arabitol accumulated in cultures of the evolved strain.This, coupled with xylitol excretion, provides strong evidence of unknown redundant DA2DH activity alongside activity encoded via RTO4_9990.One potential homology-based candidate is RTO4_8988, the only gene that shows any similarity to RTO4_9990 (93% coverage and 40% identity); however, no growth defects were observed for RTO4_8988 mutants on d-arabitol in Fig. 3F.If the late growth on l-arabitol and l-arabinose is occurring through alternative DA2DH function, we would expect to see growth halted if RTO4_14368 is deleted in a ΔRTO4_9990 ΔRTO4_16850 background.Indeed, after 21 days of culturing on 40 g/L l-arabitol, no growth could be measured for the triple-deletion strain (ΔRTO4_9990 ΔRTO4_16850 ΔRTO4_14368), whereas significant growth was measured for ΔRTO4_9990 ΔRTO4_16850 (Fig. 13).

Discussion
Recent studies of R. toruloides are concentrated on metabolic engineering, especially lipid production from biomass hydrolysates [67][68][69], with less attention to aspects of non-lipid metabolism [13,15,40,49,70,71].More of these studies are needed to identify rate-limiting steps, RNAseq, proteomics, and functional genomics [13], together with d-arabitol accumulation in two R. toruloides strains [14,72], as well as documented d-arabitol production from non-l-arabinose sources in other yeasts [33], strongly suggest R. toruloides l-arabinose and d-xylose metabolism does not occur via canonical fungal XR, XDH, and XK.Our growth complementation data (Fig. 6) and d-arabitol accumulation data (Figs.9 and 12) strongly support an alternative pathway through d-arabitol.Moreover, it is an absolute requirement for DA2DH activity encoded via RTO4_9990 to robustly metabolize any pentose substrates.Similarly, expressing a functional RK (encoded via RTO4_14368) is an absolute requirement to metabolize any pentose substrate studied (Figs. 7  and 8)-the only studied enzyme with no known functional redundancy.Lastly, the coordinated transcriptional upregulation of RTO4_14368 and RTO4_9990 strongly suggests simultaneous flux occurs through these steps during d-xylose and l-arabinose metabolism [13].   in this study, accompanied by Table 1, a tabular summary of supporting data for all gene-protein-reaction rules.
Under d-xylose conditions, no evidence exists for expression of a functional R. toruloides XK (RTO4_16850) [13,15].This is corroborated by our inability to complement growth of ΔRTO4_9990 with RTO4_16850 expression under a strong endogenous promoter Tef1 [49].Unexpectedly, XK is highly transcriptionally upregulated in the presence of acetate (whereas the rest of pentose metabolism is downregulated), but the corresponding protein abundance, function, and role XK plays regarding acetate metabolism is unknown [15].Further investigation into RTO4_16850 functionality (e.g., in vitro kinetics or growth complementation in XK-deficient systems) is warranted.Nonfunctional or dormant pathways are not uncommon.In Y. lipolytica, WT is incapable of utilizing both d-xylose [73], but a study complementing mutant E. coli with genes encoding putative xylose-catabolizing proteins from Y. lipolytica demonstrated functional XR, XDH, and XK [74].WT overexpression of endogenous XK and XDH resulted in robust growth on d-xylose, without the need for adaptation.Similarly, Y. lipolytica does not consume l-arabinose despite transcriptomics and enzyme activities showing a potential pathway may be active [75].Culturing an engineered strain (that utilizes d-xylose) on a mixture of d-xylose and l-arabinose, a dramatic improvement in l-arabinose consumption was observed, suggesting l-arabinose catabolism can exist but is dormant due to inhibitory regulation, in addition to rate-limiting LA4DH activity.
l-arabinose and d-xylose metabolism are alternating series of reduction and oxidation steps, presenting a difficult task of cofactor balancing and redox homeostasis, a possible reason R. toruloides, A. niger [83], and other fungi have functional pentose enzyme redundancy [31].Typically, different cofactor preference patterns exist for each step amongst molds and yeast [65], with exceptions such as XRs from 3 different yeasts (S. cerevisiae, P. stipitis, Candida parapsilosis) displaying unique cofactor preferences-solely NADPH, both, or mostly NADH, respectively [84][85][86].Figure 14 shows the predominant cofactors participating in each step of the R. toruloides pathway as predicted by homology to characterized enzymes, but these predictions require experimental validation.Furthermore, in vitro characterization of enzyme kinetics, substrate preference, and cofactor usage might elucidate interesting selective growth of certain substrate-strain combinations tested such as ∆RTO4_8988 + OE XK, ∆RTO4_9990 + OE XK, or ΔRTO4_9990 ΔRTO4_16850* (Figs. 3B, 6B, 11C-F, respectively).l-ribulose is readily metabolized by WT (Fig. 7) through a pathway yet to be elucidated.This may occur by isomerization to l-arabinose (via l-arabinose isomerase) or conversion to l-ribulose-5-phosphate (via l-ribulose kinase) (Fig. 1).Alternatively, l-ribulose could be converted to l-arabitol via an l-arabitol-2-dehydrogenase (LA2DH; Fig. 1), as demonstrated in purified extracts of the fungus Penicillium chrysogenum Table 1 Summary of data supporting pentose gene-protein-reaction rules reflected in Fig. 14 Gene IDs are synonymous with their 'RTO4_' counterparts; 'Functions' column denotes the purported function   Yes KO has slight growth defect on l-arabitol;cannot rule out XDH activity without metabolite profiling [87] and in the bacterium Pantoea ananatis expressing xytf [88,89], with no clear orthologs in R. toruloides.Regardless of mechanism, ΔRTO4_9990 exhibits slow growth only on l-ribulose (not on d-ribulose), notably with the same final OD 600 on l-arabitol (Fig. 7).In contrast, ΔRTO4_14368 neither grows on d-ribulose nor l-ribulose.Together, these data indicate that l-ribulose metabolism is upstream of RTO4_9990, likely part of l-arabinose metabolism (and possibly catalyzed via promiscuous LA2DH activity of unknown origin; Fig. 14).
Secondly, this necessitates that d-ribulose is the product of DA2DH, with d-arabitol as the substrate (as there is no known reaction converting l-arabitol to d-ribulose).

Conclusion
We have strongly improved upon and verified results from Kim et al. [13], positing that the primary route of For strains constructed by homologous recombination (e.g., full-deletion mutants), the parental strain was wild type.Homologous recombination and non-homologous end-joining (i.e., for generating randomly integrated mutants) was achieved by transforming R. toruloides via TDNA insertion with 1 kbp homology arms to the targeted locus by Agrobacterium tumefaciens-mediated transformation as described in [92].Strain construction methods are listed for each strain in Additional file 1.For all deletion mutants, successful deletion was confirmed by diagnostic PCR at the altered locus.Plasmids with heterologous gene expression were codon optimized via the high-CAI method (i.e., the most used codons in R. toruloides).For construction of overexpression strains by random insertion, ~ 48 randomly selected transformants were screened for growth in liquid culture with 100 µg/ mL of the appropriate selective agent, comparing growth to WT, and selecting the best-performing strain for further analysis.

Media and growth conditions
All chemicals used in this study were from Sigma Aldrich unless otherwise stated.l-xylulose, d-xylulose, l-ribulose, and d-ribulose (XYU-009, XYU-001, RBU-005, and RBU-004, respectively) were purchased from Omicron Biochemicals.For regular strain maintenance and transformation, cells were grown in 10 g/L yeast extract, 20 g/L peptone, 20 g/L glucose (YPD).All strains were first grown on YPD agar plates (15 g/L agar), followed by picking of individual colonies to obtain biological replicates.

Adaptive evolution
Generation of the adapted strain ΔRTO4_9990 ΔRTO4_16850* began with a colony of ΔRTO4_9990 ΔRTO4_16850 isolated from plating on YPD agar.A single colony was picked and serially passaged every 3-5 days by transferring 5 µL of cells from the previous culture to a new well of 800 µL fresh media containing 1.7 g/L yeast nitrogen base without ammonium sulfate and amino acids (BD 233520), 5 g/L ammonium sulfate, 75 mM KH 2 PO 4 , 25 mM K 2 HPO 4 , 40 g/L l-arabitol, pH 6.2.Cells were grown in a microtiter plate format: 48-well M2P Labs Flower Plate (MTP-48-B) at 1000 RPM agitation, 30 °C, and 85% relative humidity in a 3 mm throw shaking incubator.Serial transfers occurred a total of 8 times.The final generation was stopped and plated onto YPD agar.Afterwards, to isolate a pure strain and test for stability of the phenotype, serial plating was completed on YPD agar by choosing a single colony each time, for a total of 5 times.8 colonies were chosen from the final plating and tested in biological triplicate relative to WT on 40 g/L l-arabitol for improved growth.The most consistently reproducible isolate was chosen for further analysis and named ΔRTO4_9990 ΔRTO4_16850*.

GC-MS analysis
End-point culture samples of ΔRTO4_9990 from Fig. 9 were processed and analyzed as described previously [31].Briefly, 50 μl of each sample were evaporated to dryness under a stream of nitrogen, dissolved in dichloromethane (200 μl) and trifluoroacetic anhydride (400 μl), heated at 80 °C for 30 min, followed by nitrogen stream drying, and then redissolved in dichloromethane.GC-MS analysis was performed using a Chiraldex G-TA glass capillary column (ASTEC, Sigma 73035AST) and a single quadrupole Agilent GC-MS set at 70 eV.The helium carrier gas flow rate was set at 1 mL/min, and a four-step program was followed: 90 °C for 13 min, 0.8 °C/ min up to 110 °C, 4 °C/min up to 180 °C, and 10 min at 180 °C.The injection port and ion source temperatures were maintained at 180 °C.Identification of the sample enantiomer was completed by retention time comparison to pure l-arabitol and d-arabitol standards.

Sugar and sugar alcohol quantification
Sugars were quantified on a Dionex Ultimate 3000 system UHPLC (Agilent Technologies) using an Aminex HPX-87C column (Bio-Rad 1250095) and Thermo Scientific RefractoMax 520 Refractive Index Detector (RID) held at 35 °C.Prior to analysis, samples were diluted to 1:10 and filtered through a 0.45 µM polypropylene membrane microplate filter (Agilent 200983-100) by centrifugation at 3000 RCF for 3 min.Samples were run for 26 min using an isocratic HPLC-grade water mobile phase at 0.6 mL/min and 85 °C.Quantification was completed via peak area measurements compared to standard curves of pure compounds within their linear range of detection.Sandia National Laboratories is a multi-mission laboratory managed and operated by National Technology and Engineering Solutions of Sandia LLC., a wholly-owned subsidiary of Honeywell International Inc., for the U.S. Department of Energy's National Nuclear Security Administration under contract DE-NA0003525.The views and opinions of the authors expressed herein do not necessarily state or reflect those of the United States Government or any agency thereof.Neither the United States Government nor any agency thereof, nor any of their employees, makes any warranty, expressed or implied, or assumes any legal liability or responsibility for the accuracy, completeness, or usefulness of any information, apparatus, product, or process disclosed or represents that its use would not infringe privately owned rights.The

Fig. 2
Fig. 2 Growth of WT vs putative d-xylose reductase deletion complemented with XI on 40 g/L d-xylose medium.Solid lines are the average of 3 biological replicates; shaded regions indicate 100% percentile intervals; OE: overexpression; A.U.: Arbitrary Units

Fig. 3
Fig. 3 Growth curves of WT, ΔRTO4_8988, ΔRTO4_8988 + OE XK on 5 g/L per sugar.Solid lines are the average of 3 biological replicates; shaded regions indicate 100% percentile intervals

Fig. 6
Fig. 6 Growth curves of WT, ΔRTO4_9990, ΔRTO4_9990 + OE XK on 5 g/L per sugar.Solid lines are the average of 3 biological replicates; shaded regions indicate 100% percentile intervals

Fig. 9
Fig. 9 WT, ΔRTO4_9990, ΔRTO4_16452, ΔRTO4_8988 time-series measurements of supernatants grown on 40 g/L d-xylose + 40 g/L glycerol.WT did not produce any detectable d-arabitol or xylitol with glycerol as the sole carbon source.ΔRTO4_9990 is unable to grow on d-xylose (Fig. 6A); hence, all strains were supplemented with glycerol to aid biomass production.Solid lines are the average of 3 biological replicates; shaded regions indicate 100% percentile intervals

Fig. 11
Fig. 11 WT, ΔRTO4_9990, ΔRTO4_9990 ΔRTO4_16850, ΔRTO4_9990 ΔRTO4_16850* biomass growth curves on 10 g/L per sugar.Solid lines are the average of 3 biological replicates; shaded regions indicate 100% percentile intervals Figure 14 is an updated pathway reflecting all data collected