Rewiring Yarrowia lipolytica toward triacetic acidlactone for materials generationKelly A. Markhama,1, Claire M. Palmerb,1, Malgorzata Chwatkoa, James M. Wagnera, Clare Murraya, Sofia Vazqueza,Arvind Swaminathana, Ishani Chakravartya, Nathaniel A. Lynda, and Hal S. Alpera,b,2

aMcKetta Department of Chemical Engineering, The University of Texas at Austin, Austin, TX 78712; and bInstitute for Cellular and Molecular Biology, TheUniversity of Texas at Austin, Austin, TX 78712

Edited by Sang Yup Lee, Korea Advanced Institute of Science and Technology, Daejeon, Republic of Korea, and approved January 22, 2018 (received forreview December 6, 2017)

Polyketides represent an extremely diverse class of secondary me-tabolites often explored for their bioactive traits. These molecules arealso attractive building blocks for chemical catalysis and polymeriza-tion. However, the use of polyketides in larger scale chemistryapplications is stymied by limited titers and yields from bothmicrobialand chemical production. Here, we demonstrate that an oleaginousorganism (specifically, Yarrowia lipolytica) can overcome such produc-tion limitations owing to a natural propensity for high flux throughacetyl–CoA. By exploring three distinct metabolic engineering strate-gies for acetyl–CoA precursor formation, we demonstrate that a pre-viously uncharacterized pyruvate bypass pathway supports increasedproduction of the polyketide triacetic acid lactone (TAL). Ultimately,we establish a strain capable of producing over 35%of the theoreticalconversion yield to TAL in an unoptimized tube culture. This strainalso obtained an averaged maximum titer of 35.9 ± 3.9 g/L with anachieved maximum specific productivity of 0.21 ± 0.03 g/L/h in bio-reactor fermentation. Additionally, we illustrate that a β-oxidation-related overexpression (PEX10) can support high TAL production andis capable of achieving over 43% of the theoretical conversion yieldunder nitrogen starvation in a test tube. Next, through use of thisbioproduct, we demonstrate the utility of polyketides like TAL tomodify commodity materials such as poly(epichlorohydrin), resultingin an increased molecular weight and shift in glass transition temper-ature. Collectively, these findings establish an engineering strategyenabling unprecedented production from a type III polyketide syn-thase as well as establish a route through O-functionalization forconverting polyketides into new materials.

triacetic acid lactone | Yarrowia lipolytica | polyketide synthase |biorenewable chemicals | O-functionalization

The growing demand for renewable chemicals and fuels hasspurred great interest in using cells as biochemical factories

(1). Metabolic engineering enables this goal by rewiring cells’metabolism toward desirable chemical compounds (2–4). Amongpossible molecules, polyketides are an interesting class of sec-ondary metabolites produced by microbes and plants with nativeroles in processes such as cellular defense and communication(5–7). While many polyketides can serve as potent antibiotics,this class of molecules also encompasses chemicals with otheruseful properties such as pigments, antioxidants, antifungals, andother bioactive traits (5, 8). However, the use of polyketides inmore unique and nonmedical applications has been partiallylimited due to low natural abundance and difficult cultivation ofnative hosts. Specifically, polyketide-producing organisms aretypically unusual plants and microbial organisms that are notwell-suited for high-level industrial production (6). Syntheticproduction of these molecules in model host organisms has alsoproven quite difficult with titers and yields insufficient for in-dustrial production (10–13). Likewise, traditional chemical syn-thesis of polyketides is limited by low concentrations andchallenging chiral centers (14). While the scale and price-point forpharmaceuticals can tolerate plant-based sourcing of polyketides

or challenging syntheses, this is not an option for any larger-scalechemistry application.Here, we focus on the interesting, yet simple, polyketide, tri-

acetic acid lactone (TAL) as it is derived from two commonpolyketide precursors, acetyl–CoA and malonyl–CoA. TAL hasbeen demonstrated as a platform chemical that can be convertedinto a variety of valuable products traditionally derived fromfossil fuels including sorbic acid, a common food preservativewith a global demand of 100,000 t (1, 15–18). However, meetingthis annual demand using the low concentrations of TAL derivedfrom native plants like gerbera daisies (9) would require fourtimes the quantity of global arable land. As a result, utilization ofpolyketides for unique industrial applications including poly-mers, coatings, and even commodity chemical production has notbeen implemented despite the promising chemical nature ofthese molecules. To address these limitations, previous efforts haveexplored microbial production of TAL. However, these efforts havebeen restricted to conventional organisms [like Escherichia coli (10,19) and Saccharomyces cerevisiae (11–13)] and are limited with re-spect to titer only reaching 5.2 g/L with low yields (12).In this work, we explore the unique application of an oleagi-

nous, nonconventional yeast (Yarrowia lipolytica) based on itspotential for high flux through the key polyketide precursors,acetyl–CoA and malonyl–CoA. By investigating three distinctpathways toward CoA precursor formation along with targetshypothesized to enhance β-oxidation, we demonstrate the utilityof a previously uncharacterized pyruvate bypass pathway for

Significance

Polyketides are important molecules for both their bioactivetraits and their potential as chemical building blocks. However,production of these molecules through chemistry and bio-catalysts is restricted in yield and titer. Here, we demonstrate thatthe nonconventional yeast Yarrowia lipolytica can serve as apotent host for such production. This work provides a compre-hensive evaluation of three separate pathways toward acetyl–CoA andmalonyl–CoA in this host, enabling high-titer productionof triacetic acid lactone. Beyond achieving unprecedented titersand appreciable yields, this production capacity allows for bothpurification from fermentation broth and conversion into a ma-terial using simple reaction conditions.

Author contributions: K.A.M., C.M.P., N.A.L., and H.S.A. designed research; K.A.M., C.M.P.,M.C., J.M.W., C.M., S.V., A.S., and I.C. performed research; K.A.M., C.M.P., M.C., N.A.L.,and H.S.A. analyzed data; and K.A.M., C.M.P., and H.S.A. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Published under the PNAS license.1K.A.M. and C.M.P. contributed equally to this work.2To whom correspondence should be addressed. Email: halper@che.utexas.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1721203115/-/DCSupplemental.

Published online February 12, 2018.

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significantly increasing TAL production. After subsequent opti-mization, our final strain achieved over 35% of the theoreti-cal conversion yield to TAL in unoptimized tube culture andachieved a maximum observed titer of 35.9 ± 3.9 g/L in bio-reactor operation. We demonstrate that a higher-yield strain(43% of theoretical conversion yield in a test tube) is possibleby overexpressing a β-oxidation–related target. Finally, we

demonstrate the chemical opportunities gained by high polyketidetiters for novel materials modification by O-functionalization ofbiosourced TAL with commodity poly(epichlorohydrin) to tuneand upgrade thermal properties of the parent material. This workboth establishes a host organism for polyketide overproductionand demonstrates the potential utility of polyketides for materialssynthesis and modification.

Fig. 1. Strain engineering scheme to evaluate native Yarrowia lipolytica pathway potential for the production of TAL. Four overall schemes were tested inthis work. Illustrated here are the three anabolic pathways targeted for overexpression in this work: the citrate route (shown in blue), the pyruvate de-hydrogenase complex (green), and the pyruvate bypass pathway (purple). Additionally, shown in red are two potential β-oxidation up-regulation targets.These color schemes are maintained in future figures to enable continuity and rapid identification.

Fig. 2. Difference of means plots demonstratingthe effect of overexpressing acetyl–CoA productionpathways. TAL titers were measured following 96-htube fermentations in defined media and presentedas the increase in titer over the YT parental strain(the color scheme used in Fig. 1 has been retained).Error bars represent the SE of n ≥ 2. Significance wastested using Dunnett’s test, *P < 0.05, **P < 0.01,***P < 0.001. (A) The effect of sequential over-expression of genes involved in the citrate pathway.(B) The effect of sequential overexpression of pyru-vate dehydrogenase complex genes. (C) The effectof sequential overexpression of pyruvate bypasspathway genes in a full combinatorial fashion; i.e.,every combination of five potential acetylaldehydedehydrogenase genes and two pyruvate decarbox-ylase genes was tested.

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Results and DiscussionY. lipolytica Can Support TAL Production. High lipid flux in oleagi-nous organisms like Y. lipolytica suggests a strong potential for theseorganisms to produce alternative acetyl–CoA-derived products likepolyketides. Moreover, Y. lipolytica exhibits sufficient tolerance tomany chemicals (20) including TAL to concentrations approach-ing the soluble limit (SI Appendix, Fig. S1). With these two fea-tures in place, we first established heterologous TAL productionin Y. lipolytica through expression of the codon-optimized Gerberahybrida 2-pyrone synthase gene, g2ps1. While this initial strainproduced TAL (SI Appendix, Fig. S13), further amplifying thegene copy number to four enabled 2.1 g/L production in tubefermentations (defined medium including CSM, YNB, and glu-cose). This strain, named YT, was selected as the starting point forfurther metabolic engineering work. Additional studies of pre-viously characterized mutants of g2ps1 established in E. coli (10)were also tested but produced lower titers than the wild-type allele(SI Appendix, Fig. S2).

A Previously Uncharacterized Y. lipolytica Pyruvate Bypass PathwayImproves TAL Production. To increase metabolic flux throughacetyl–CoA and malonyl–CoA, we investigated three in-dependent metabolic engineering strategies (Fig. 1). First, weexplored the citrate route—a pathway that has been extensivelystudied for its capacity to increase lipid production in Y. lipolytica(21–23). Overexpression of ACC1, which codes for the enzymethat converts acetyl–CoA to malonyl–CoA, has also been shownto promote lipid production (24, 25). When the pathway genes(ACL1, ACL2, and AMPD) were concurrently overexpressed,TAL production was significantly reduced and only marginallyimproved with the addition of ACC1 (Fig. 2A).Second, we explored the pyruvate dehydrogenase (PDH)

complex pathway [located in the mitochondria in S. cerevisiae(26)]. In contrast to the citrate route, this pathway has not beenextensively studied in Y. lipolytica, but would theoretically enablea direct path to convert pyruvate to acetyl–CoA. The relatedalpha-ketoglutarate dehydrogenase complex, which shares onesubunit with the PDH complex, has been previously overex-pressed to promote alpha-ketoglutaric acid production (27),suggesting a similar strategy may be successful here. To test this

approach, we established a coordinated overexpression of thedifferent subunits for this complex (encoded by PDA1, PDE2,PDE3, and PDB1). By combining this pathway with ACC1overexpression, overall TAL production was significantly im-proved by 23%, achieving 2.5 g/L (Fig. 2B).Third, we investigated the pyruvate bypass pathway, which

converts pyruvate to acetaldehyde through pyruvate decarbox-ylase (PDC), then to acetate through acetylaldehyde de-hydrogenase (ALD), and finally to acetyl–CoA via acetyl–CoAsynthetase (ACS) (26, 28). Previous work has targeted thispathway using heterologous enzymes (29); however, the functionand potential of the native Y. lipolytica pyruvate bypass pathwayhas not been previously explored. While a single ACS gene hadbeen previously characterized in Y. lipolytica (27), two PDC ho-mologs (arbitrarily named PDC1 and PDC2) and five potentialALD homologs were identified based on previous yeast homol-ogy studies (30). Next, we established a full combinatorial as-sembly of this pathway in the YT strain background. Unlike theprevious two approaches, ACC1 overexpression did not consis-tently increase production for all combinations tested. Never-theless, four genetic combinations emerged as the top TAL-producing strains including ACS1, ALD5, PDC2, ACC1 (64.7%improvement over YT), ACS1, ALD3, PDC1, ACC1 (61.1%improvement), ACS1, ALD2, PDC2, ACC1 (31.7% improve-ment), and ACS1, ALD3, PDC2, ACC1 (17.9% improvement)(Fig. 2C). The top strain from this effort (YT- ACS1, ALD5,PDC2, ACC1) produced 2.8 g/L of TAL in tube fermentations,equivalent to 30.4% of the theoretical yield.

Modification of β-oxidation Can Likewise Improve TAL Production.Asan alternative (and potentially complementary) approach toincrease acetyl–CoA pools, we targeted participants in theβ-oxidation pathway for overexpression. Specifically, we evalu-ated the transcription factor Por1 [reported in other hosts toincrease polyketide formation (31)] and the peroxisomal matrixprotein Pex10. When overexpressed in the YT background,POR1 had no effect on TAL production, whereas PEX10 over-expression increased TAL titer by 22% (2.4 g/L) (Fig. 3), sug-gesting β-oxidation up-regulation as a strategy for acetyl–CoArecycling if it cannot be shuttled away from lipid synthesis ef-fectively. This result is intriguing as Pex10p is not directly in-volved in the catalytic conversion of fatty acids to acetyl–CoAand thus provides an area for further biochemical study.

Fig. 3. Difference of means plot demonstrating the effect of over-expressing β-oxidation targets. TAL titers were measured following 96-htube fermentations in defined media and presented as the increase in titerover the YT parental strain. Error bars represent the SE of n ≥ 2. Significancewas tested using Dunnett’s test, **P < 0.01.

Fig. 4. Lipid production as a function of TAL production. Following definedmedia tube fermentation of YT and strains containing the overexpressionsoutlined in Fig. 1, average TAL titer and average total lipids were assessed.An inverse correlation between TAL titer and lipid titer was observed, R2 =0.89. Error bars represent the SD of n = 3.

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Strain Engineering Effectively Diverted Flux from Lipids to TAL. Toevaluate the true efficacy of these four rewiring strategies, bothlipid and TAL production were evaluated from tube fermenta-tions. We observed a clear, inverse correlation between TALtiter and lipid titer with an R2 of 0.89 (Fig. 4). Collectively,these results demonstrate that the PDH complex, pyruvatebypass, and PEX10 overexpressions can divert acetyl–CoA fromlipids into TAL, whereas the citrate pathway is strongly coupledto lipid formation.

Culture Conditions Differentially Alter TAL Production. Y. lipolytica ishighly responsive to environmental factors, and thus we evalu-ated a series of conditions for increased TAL production. First,we evaluated the impact of nitrogen starvation, a strategy com-monly used in Y. lipolytica to induce lipid formation (22). Underthese conditions, the impact was varied across the variousrewiring schemes (SI Appendix, Fig. S3A) with the most signifi-cant improvement observed in the PEX10 overexpression strain

(Fig. 5A). When grown in C20N2 media, this strain achieved agreater than twofold increase over the YT strain under normalconditions (reaching 4.1 g/L in a test tube, 43.4% theoreticalyield). Further nitrogen limitation did not improve TAL pro-duction as a result of the expense to growth.Second, a series of supplements (SI Appendix) and different

carbon sources were tested as spikes or sole carbon sources alongwith glucose as a control (SI Appendix, Fig. S4). The largest gainsin TAL titer were realized through providing an acetate spikewhen the pyruvate bypass pathway was overexpressed. Underthese conditions, the top strain from the pyruvate bypass path-way produced 4.9 g/L TAL in a test tube (representing over 35%of the theoretical conversion yield calculated from both glucoseand acetate fed) (Fig. 5B). Analysis of acetate consumption in-dicates this result is not simply due to acetate acting as a carbonsource for TAL production, as a substantial portion of the fedacetate still remains at the end of the fermentation (SI Appendix,Fig. S5). This result suggests a more regulatory or redox-relatedimpact on metabolism. Further to this point, in S. cerevisiae,acetate feeding has been shown to induce changes to metabo-lism mediated through protein acetylation (32, 33), a possiblemechanism to be explored here. Additionally, acetylaldehydedehydrogenase activity assays suggest pathway engineering (es-pecially with ALD5) resulted in altered redox cofactor usagefavoring NAD+ over NADP+ (SI Appendix, Fig. S6). Thus, anoverall redox and regulatory mechanism may explain the almosttwofold increase in TAL production observed in this study.Intriguingly, this improvement was not seen under nitrogen-limited conditions (SI Appendix, Fig. S3B), suggesting that

Fig. 5. Impact of nitrogen starvation and acetate spike on TAL production.TAL production under different conditions was assessed following tubefermentation in defined media. Error bars represent the SD of n = 3. Sta-tistical significance was determined by a Dunnett’s test; each new conditionwas compared with the relevant control (C20N5 in the case of nitrogenlimitation and glucose in the case of feeding spikes), **P < 0.01, ***P <0.001. (A) Nitrogen limitation enhances the effect of gene overexpressionsrelated to β-oxidation. (B) Feeding assay demonstrating the effect of adding10 g/L carbon molar equivalent of glucose as a feeding spike 24 h intostandard tube fermentation.

Fig. 6. Bioreactor cultivation of pyruvate bypass overexpression strain. YT-ACS1, ALD5, PDC2, ACC1 was fermented in a 3-L bioreactor with YP media,180 g/L glucose, and a 13.7 g/L sodium acetate spike at 36 h. This figuredemonstrates a representative run with a duplicate presented in SI Appen-dix, Fig. S7. (A) Concentrations of TAL, citrate, and glucose were determinedfrom three independent samples taken at each time point; error bars rep-resent SD of n = 3. (B) Viable cell count was determined by plating differentdilutions of sample; error bars represent SD of n ≥ 2.

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these two strategies (acetate feed and nitrogen starvation) arenot compatible.

Bioreactor Cultivation Boosts Overall TAL Titer. Fermentation of thepyruvate bypass overexpression strain (YT- ACS1, ALD5, PDC2,ACC1) with acetate spiking was scaled up to the 3-L bioreactorscale (YP, 18% glucose, 13.7 g/L acetate spike). After optimi-zation, fermentation resulted in production of 35.9 ± 3.9 g/L ofTAL (Fig. 6A). In this scale-up, we achieve a substantially im-proved productivity over the previous batch cultures, upward of afourfold increase to a glucose-phase maximum specific pro-ductivity of 0.21 ± 0.03 g/L/h (SI Appendix, Table S4). Although along fermentation time was necessary, this timescale is compa-rable to a previously published TAL study (12). The overallproductivity reported here [0.12 g/L/h (SI Appendix, Table S4)]improves upon the previous report in S. cerevisiae by greater thansixfold (12). As this production level is well outside the solublerange of TAL, substantial in situ precipitation occurred, an at-tractive feature for industrial production, but a unique source ofsampling error. Likewise, we observed a diauxic shift from glu-cose to (produced) citrate utilization (Fig. 6A) that leads to anincreased production as cell viability stagnates and even de-creases throughout the process (Fig. 6B).

Biosourced TAL Can Be Incorporated into a Polymer ThroughO-Functionalization. Finally, we leveraged the newfound bulk-availability of polyketides such as TAL to demonstrate theirutility in the modification of polymer properties. TAL servesa dual role as both a polymer modifier but also a func-tional adduct for later chemical derivatization owing to its lactoneand unsaturated functionalities (34). TAL was rapidly extractedand purified from fermentation broth and ultimately used forthe modification of structure and properties of commoditypoly(epichlorohydrin). This was achieved through heat and anactivating organic base 1,8-Diazabicyclo(5.4.0)undec-7-ene(DBU) (Fig. 7A) (34, 35). In this process, the displacement ofchloride through the O-functionalization of TAL was evidentspectroscopically, chromatographically, and thermally throughthe use of NMR spectroscopy (Fig. 7B and SI Appendix, Fig. S8),size exclusion chromatography (SEC), and differential scanningcalorimetry (DSC) (Table 1), respectively. The amount of TALincorporated along the poly(epichlorohydrin) backbone wastuned stoichiometrically from 16 to 83% by mole resulting inpoly[(epichlorohydrin)-co-(epoxy triacetic acid lactone)] orPETAL. We measured the compositionally dependent changesin the glassy amorphous solid based on molecular weight andglass transition temperature of PETAL. Molecular weight was

Fig. 7. Production of poly[(epichlorohydrin)-co(epoxytriacetic acid lactone)] or PETAL. (A) Reaction schemeto create PETAL. (B) H NMR characterization of PETALwhich shows distinct new peaks and shifts from thestart molecules. (C) Photo of PETAL pressed into a film.

Table 1. Characteristics of copolymers in comparison with PECH

Monomer pairs Monomer feed* ECH:TAL:DBU Polymer composition† ECH:TAL Mn‡ (g/mol) Tg

§ (°C)

PECH 1: 0 19,700 −30P(ECH-TALcom) 1: 1.5: 0.75 1: 1 23,700 30PECH 1:0 7,200 −30P(ECH-TALcom) 1: 1.5: 1 1: 5 9,400 70P(ECH-TALbio) 1: 0.5: 1 6: 1 7,900 −11

Polymer characteristics were measured for PETAL.*Determined by gravimetry.†Determined by 1H NMR spectroscopy.‡Number-average molecular weight determined by size exclusion chromatography in chloroform using light-scattering and differential refractometer detectors.§Thermal properties determined by differential scanning calorimetry.

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seen to increase proportionately to the amount of TAL in-corporated as measured by size exclusion chromatography withmultiangle light-scattering detection to yield absolute number-average molecular weights (Mn) (Table 1). While poly(epichlo-rohydrin) exhibits a native glass transition temperature (Tg) of−30 °C, this value was markedly increased and strongly de-pendent on TAL incorporation with the highest TAL composi-tion (83% by mole) exhibiting a Tg of 70 °C. The final productcan be formed into a film and is seen to exhibit a unique hue andrelative transparency (Fig. 7C). In particular, the orange color ofthe polymer is due to the native color of covalently bound TAL,as no other characteristic spectroscopic differences were ob-served via UV-Vis spectroscopy (SI Appendix, Fig. S9). It shouldbe noted that the reaction rate obtained with this purified, bio-sourced TAL was comparable to a reaction conducted withcommercially sourced TAL. Moreover, the biosourced PETALstructure and properties fall exactly along the trend observedusing commercially sourced TAL, again supporting that thereis no difference (SI Appendix, Fig. S10). Finally, the residuallactone and unsaturated functionality from TAL repeat unitsoffer a unique future strategy toward further modification ofmaterial properties.

MethodsA full Materials and Methods section is provided in the SI Appendix.

Strain Engineering and Analysis. Strains used in this study were constructed inthe wild-type Y. lipolytica strain, PO1f, through random genomic integrationemploying a sequential overexpression strategy. TAL concentrations wereassessed via reverse-phase HPLC following 96-h tube fermentations at 28 °Cin defined media containing 2% glucose. Bioreactor fermentations wereperformed at the 3-L scale at pH 6.5 in YP media with 18% glucose and a13.7 g/L sodium acetate spike at 36 h.

Materials Generation. Biosourced TAL was purified from fermentation broththrough an ethyl acetate/acetic acid extraction. Commodity poly(epichloro-hydrin) was functionalized with TAL using the activating organic base 1,8-Diazabicyclo(5.4.0)undec-7-ene and the resulting material characterized byNMR, differential scanning calorimetry, UV-Vis spectroscopy, and sizeexclusion chromatography.

ConclusionsIn summary, this work demonstrates the use of an oleaginousorganism for high-level production of an acetyl–CoA andmalonyl–CoA-derived polyketide. Moreover, we establish apreviously uncharacterized pyruvate bypass pathway as superiorfor rewiring CoA flux from lipid biosynthesis and into high-levelTAL production reaching a titer of 35.9 ± 3.85 g/L in a bio-reactor and overall yield of 0.164 g/g in a tube. Additional en-gineering efforts related to the β-oxidation pathway increasedyields to 0.203 g/g. The achieved titer far exceeds previous effortsin the field with conventional organisms (a summary of theachieved titers and yields in this work is provided in SI Appendix,Table S4). This high-level production enabled rapid purificationand conversion into a unique polymer with favorable molecularweight and glass transition temperature. This work and resultingstrain provides a path forward for microbial production of otheracetyl–CoA and malonyl–CoA-derived polyketides for novelapplications such as polymers and chemical conversion.

ACKNOWLEDGMENTS. We thank Yuki Naito for updating CRISPRdirect toinclude a specificity check to Y. lipolytica. We would also like to thank CorySchwartz and Ian Wheeldon for providing the pCRISPRyl plasmid. This workwas funded through the Camille and Henry Dreyfus Foundation. N.A.L. ac-knowledges support through Welch Foundation Grant F-1904.

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