Rewiring Yarrowia Lipolytica Toward Triacetic Acid Lactone for Materials Generation

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Rewiring Yarrowia lipolytica toward triacetic acid lactone for materials generation

Kelly 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, The University 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 for review December 6, 2017) Polyketides represent an extremely diverse class of secondary me- or challenging syntheses, this is not an option for any larger-scale tabolites often explored for their bioactive traits. These molecules are chemistry application. also attractive building blocks for chemical catalysis and polymeriza- Here, we focus on the interesting, yet simple, polyketide, tri- tion. However, the use of polyketides in larger scale chemistry acetic acid lactone (TAL) as it is derived from two common applications is stymied by limited titers and yields from both microbial polyketide precursors, acetyl–CoA and malonyl–CoA. TAL has and chemical production. Here, we demonstrate that an oleaginous been demonstrated as a platform chemical that can be converted organism (specifically, Yarrowia lipolytica) can overcome such produc- into a variety of valuable products traditionally derived from tion limitations owing to a natural propensity for high flux through fossil fuels including sorbic acid, a common food preservative acetyl–CoA. By exploring three distinct metabolic engineering strate- with a global demand of 100,000 t (1, 15–18). However, meeting gies for acetyl–CoA precursor formation, we demonstrate that a pre- this annual demand using the low concentrations of TAL derived viously uncharacterized pyruvate bypass pathway supports increased from native plants like gerbera daisies (9) would require four production of the polyketide triacetic acid lactone (TAL). Ultimately, times the quantity of global arable land. As a result, utilization of we establish a strain capable of producing over 35% of the theoretical polyketides for unique industrial applications including poly- conversion yield to TAL in an unoptimized tube culture. This strain mers, coatings, and even commodity chemical production has not ± also obtained an averaged maximum titer of 35.9 3.9 g/L with an been implemented despite the promising chemical nature of ± achieved maximum specific productivity of 0.21 0.03 g/L/h in bio- these molecules. To address these limitations, previous efforts have β reactor fermentation. Additionally, we illustrate that a -oxidation- explored microbial production of TAL. However, these efforts have PEX10 related overexpression ( ) can support high TAL production and been restricted to conventional organisms [like Escherichia coli (10, is capable of achieving over 43% of the theoretical conversion yield 19) and Saccharomyces cerevisiae (11–13)]andarelimitedwithre- under nitrogen starvation in a test tube. Next, through use of this spect to titer only reaching 5.2 g/L with low yields (12). bioproduct, we demonstrate the utility of polyketides like TAL to In this work, we explore the unique application of an oleagi- modify commodity materials such as poly(epichlorohydrin), resulting nous, nonconventional yeast (Yarrowia lipolytica) based on its in an increased molecular weight and shift in glass transition temper- potential for high flux through the key polyketide precursors, ature. Collectively, these findings establish an engineering strategy acetyl–CoA and malonyl–CoA. By investigating three distinct enabling unprecedented production from a type III polyketide syn- pathways toward CoA precursor formation along with targets thase as well as establish a route through O-functionalization for hypothesized to enhance β-oxidation, we demonstrate the utility converting polyketides into new materials. of a previously uncharacterized pyruvate bypass pathway for triacetic acid lactone | Yarrowia lipolytica | polyketide synthase | biorenewable chemicals | O-functionalization Significance

he growing demand for renewable chemicals and fuels has Polyketides are important molecules for both their bioactive Tspurred great interest in using cells as biochemical factories traits and their potential as chemical building blocks. However, (1). Metabolic engineering enables this goal by rewiring cells’ production of these molecules through chemistry and bio- catalysts is restricted in yield and titer. Here, we demonstrate that metabolism toward desirable chemical compounds (2–4). Among the nonconventional yeast Yarrowia lipolytica can serve as a possible molecules, polyketides are an interesting class of sec- potent host for such production. This work provides a compre- ondary metabolites produced by microbes and plants with native hensive evaluation of three separate pathways toward acetyl– roles in processes such as cellular defense and communication CoA and malonyl–CoA in this host, enabling high-titer production (5–7). While many polyketides can serve as potent antibiotics, of triacetic acid lactone. Beyond achieving unprecedented titers this class of molecules also encompasses chemicals with other and appreciable yields, this production capacity allows for both useful properties such as pigments, antioxidants, antifungals, and purification from fermentation broth and conversion into a ma- other bioactive traits (5, 8). However, the use of polyketides in terial using simple reaction conditions. more unique and nonmedical applications has been partially limited due to low natural abundance and difficult cultivation of 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., native hosts. Specifically, polyketide-producing organisms are and H.S.A. analyzed data; and K.A.M., C.M.P., and H.S.A. wrote the paper. typically unusual plants and microbial organisms that are not The authors declare no conflict of interest. well-suited for high-level industrial production (6). Synthetic This article is a PNAS Direct Submission. production of these molecules in model host organisms has also Published under the PNAS license. proven quite difficult with titers and yields insufficient for in- 1K.A.M. and C.M.P. contributed equally to this work. – dustrial production (10 13). Likewise, traditional chemical syn- 2To whom correspondence should be addressed. Email: [email protected]. thesis of polyketides is limited by low concentrations and This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. challenging chiral centers (14). While the scale and price-point for 1073/pnas.1721203115/-/DCSupplemental. pharmaceuticals can tolerate plant-based sourcing of polyketides Published online February 12, 2018.

2096–2101 | PNAS | February 27, 2018 | vol. 115 | no. 9 www.pnas.org/cgi/doi/10.1073/pnas.1721203115 Downloaded by guest on September 30, 2021 Fig. 1. Strain engineering scheme to evaluate native Yarrowia lipolytica pathway potential for the production of TAL. Four overall schemes were tested in this work. Illustrated here are the three anabolic pathways targeted for overexpression in this work: the citrate route (shown in blue), the pyruvate dehydrogenase 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.

significantly increasing TAL production. After subsequent opti- demonstrate the chemical opportunities gained by high polyketide mization, our final strain achieved over 35% of the theoreti- titers for novel materials modification by O-functionalization of cal conversion yield to TAL in unoptimized tube culture and biosourced TAL with commodity poly(epichlorohydrin) to tune achieved a maximum observed titer of 35.9 ± 3.9 g/L in bio- and upgrade thermal properties of the parent material. This work reactor operation. We demonstrate that a higher-yield strain both establishes a host organism for polyketide overproduction (43% of theoretical conversion yield in a test tube) is possible and demonstrates the potential utility of polyketides for materials by overexpressing a β-oxidation–related target. Finally, we synthesis and modification.

Fig. 2. Difference of means plots demonstrating the effect of overexpressing acetyl–CoA production pathways. TAL titers were measured following 96-h tube fermentations in defined media and presented as 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 was tested using Dunnett’s test, *P < 0.05, **P < 0.01, ***P < 0.001. (A) The effect of sequential overexpression of genes involved in the citrate pathway. (B) The effect of sequential overexpression of pyruvate dehydrogenase complex genes. (C) The effect of sequential overexpression of pyruvate bypass pathway genes in a full combinatorial fashion; i.e.,

every combination of five potential acetylaldehyde SCIENCES dehydrogenase genes and two pyruvate decarbox-

ylase genes was tested. APPLIED BIOLOGICAL

Markham et al. PNAS | February 27, 2018 | vol. 115 | no. 9 | 2097 Downloaded by guest on September 30, 2021 approach, we established a coordinated overexpression of the different subunits for this complex (encoded by PDA1, PDE2, PDE3, and PDB1). By combining this pathway with ACC1 overexpression, overall TAL production was significantly improved 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),thentoacetatethroughacetylaldehydede- hydrogenase (ALD), and finally to acetyl–CoA via acetyl–CoA synthetase (ACS) (26, 28). Previous work has targeted this pathway using heterologous enzymes (29); however, the function and potential of the native Y. lipolytica pyruvate bypass pathway has not been previously explored. While a single ACS gene had been previously characterized in Y. lipolytica (27), two PDC homologs (arbitrarily named PDC1 and PDC2) and five potential ALD homologs were identified based on previous yeast homol- ogy studies (30). Next, we established a full combinatorial assembly of this pathway in the YT strain background. Unlike the previous two approaches, ACC1 overexpression did not consis- tently increase production for all combinations tested. Never- Fig. 3. Difference of means plot demonstrating the effect of over- theless, four genetic combinations emerged as the top TAL- expressing β-oxidation targets. TAL titers were measured following 96-h producing strains including ACS1, ALD5, PDC2, ACC1 (64.7% tube fermentations in defined media and presented as the increase in titer improvement over YT), ACS1, ALD3, PDC1, ACC1 (61.1% over the YT parental strain. Error bars represent the SE of n ≥ 2. Significance was tested using Dunnett’s test, **P < 0.01. improvement), ACS1, ALD2, PDC2, ACC1 (31.7% improvement), and ACS1, ALD3, PDC2, ACC1 (17.9% improvement) (Fig. 2C). The top strain from this effort (YT- ACS1, ALD5, Results and Discussion PDC2, ACC1) produced 2.8 g/L of TAL in tube fermentations, Y. lipolytica Can Support TAL Production. High lipid flux in oleagi- equivalent to 30.4% of the theoretical yield. nous organisms like Y. lipolytica suggests a strong potential for these organisms to produce alternative acetyl–CoA-derived products like Modification of β-oxidation Can Likewise Improve TAL Production. As polyketides. Moreover, Y. lipolytica exhibits sufficient tolerance to an alternative (and potentially complementary) approach to many chemicals (20) including TAL to concentrations approach- increase acetyl–CoA pools, we targeted participants in the ing the soluble limit (SI Appendix,Fig.S1). With these two fea- β-oxidation pathway for overexpression. Specifically, we evalu- tures in place, we first established heterologous TAL production ated the transcription factor Por1 [reported in other hosts to in Y. lipolytica through expression of the codon-optimized Gerbera increase polyketide formation (31)] and the peroxisomal matrix hybrida 2-pyrone synthase gene, g2ps1. While this initial strain protein Pex10. When overexpressed in the YT background, produced TAL (SI Appendix,Fig.S13), further amplifying the POR1 had no effect on TAL production, whereas PEX10 over- gene copy number to four enabled 2.1 g/L production in tube expression increased TAL titer by 22% (2.4 g/L) (Fig. 3), sug- fermentations (defined medium including CSM, YNB, and glu- gesting β-oxidation up-regulation as a strategy for acetyl–CoA cose). This strain, named YT, was selected as the starting point for recycling if it cannot be shuttled away from lipid synthesis ef- further metabolic engineering work. Additional studies of pre- fectively. This result is intriguing as Pex10p is not directly in- viously characterized mutants of g2ps1 established in E. coli (10) volved in the catalytic conversion of fatty acids to acetyl–CoA were also tested but produced lower titers than the wild-type allele and thus provides an area for further biochemical study. (SI Appendix,Fig.S2).

A Previously Uncharacterized Y. lipolytica Pyruvate Bypass Pathway Improves TAL Production. To increase metabolic flux through acetyl–CoA and malonyl–CoA, we investigated three independent metabolic engineering strategies (Fig. 1). First, we explored the citrate route—a pathway that has been extensively studied for its capacity to increase lipid production in Y. lipolytica (21–23). Overexpression of ACC1, which codes for the enzyme that converts acetyl–CoA to malonyl–CoA, has also been shown to promote lipid production (24, 25). When the pathway genes (ACL1, ACL2, and AMPD) were concurrently overexpressed, TAL production was significantly reduced and only marginally improved 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 been extensively studied in Y. lipolytica, but would theoretically enable a direct path to convert pyruvate to acetyl–CoA. The related Fig. 4. Lipid production as a function of TAL production. Following defined alpha-ketoglutarate dehydrogenase complex, which shares one media tube fermentation of YT and strains containing the overexpressions subunit with the PDH complex, has been previously overex- outlined in Fig. 1, average TAL titer and average total lipids were assessed. pressed to promote alpha-ketoglutaric acid production (27), An inverse correlation between TAL titer and lipid titer was observed, R2 = suggesting a similar strategy may be successful here. To test this 0.89. Error bars represent the SD of n = 3.

2098 | www.pnas.org/cgi/doi/10.1073/pnas.1721203115 Markham et al. Downloaded by guest on September 30, 2021 (Fig. 5A). When grown in C20N2 media, this strain achieved a greater than twofold increase over the YT strain under normal conditions (reaching 4.1 g/L in a test tube, 43.4% theoretical yield). Further nitrogen limitation did not improve TAL production 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 along with glucose as a control (SI Appendix, Fig. S4). The largest gains in TAL titer were realized through providing an acetate spike when the pyruvate bypass pathway was overexpressed. Under these conditions, the top strain from the pyruvate bypass pathway produced 4.9 g/L TAL in a test tube (representing over 35% of the theoretical conversion yield calculated from both glucose and acetate fed) (Fig. 5B). Analysis of acetate consumption in- dicates this result is not simply due to acetate acting as a carbon source for TAL production, as a substantial portion of the fed acetate still remains at the end of the fermentation (SI Appendix, Fig. S5). This result suggests a more regulatory or redox-related impact on metabolism. Further to this point, in S. cerevisiae, acetate feeding has been shown to induce changes to metabolism mediated through protein acetylation (32, 33), a possible mechanism to be explored here. Additionally, acetylaldehyde dehydrogenase activity assays suggest pathway engineering (es- pecially with ALD5) resulted in altered redox cofactor usage favoring NAD+ over NADP+ (SI Appendix, Fig. S6). Thus, an overall redox and regulatory mechanism may explain the almost twofold 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 tube fermentation in defined media. Error bars represent the SD of n = 3. Sta- tistical significance was determined by a Dunnett’s test; each new condition was compared with the relevant control (C20N5 in the case of nitrogen limitation and glucose in the case of feeding spikes), **P < 0.01, ***P < 0.001. (A) Nitrogen limitation enhances the effect of gene overexpressions related to β-oxidation. (B) Feeding assay demonstrating the effect of adding 10 g/L carbon molar equivalent of glucose as a feeding spike 24 h into standard tube fermentation.

Strain Engineering Effectively Diverted Flux from Lipids to TAL. To evaluate the true efficacy of these four rewiring strategies, both lipid and TAL production were evaluated from tube fermentations. We observed a clear, inverse correlation between TAL titer and lipid titer with an R2 of 0.89 (Fig. 4). Collectively, these results demonstrate that the PDH complex, pyruvate bypass, and PEX10 overexpressions can divert acetyl–CoA from lipids into TAL, whereas the citrate pathway is strongly coupled to lipid formation.

Culture Conditions Differentially Alter TAL Production. Y. lipolytica is highly responsive to environmental factors, and thus we evalu- Fig. 6. Bioreactor cultivation of pyruvate bypass overexpression strain. YT- ated a series of conditions for increased TAL production. First, ACS1, ALD5, PDC2, ACC1 was fermented in a 3-L bioreactor with YP media, we evaluated the impact of nitrogen starvation, a strategy com- 180 g/L glucose, and a 13.7 g/L sodium acetate spike at 36 h. This figure demonstrates a representative run with a duplicate presented in SI Appen- monly used in Y. lipolytica to induce lipid formation (22). Under dix, Fig. S7.(A) Concentrations of TAL, citrate, and glucose were determined

these conditions, the impact was varied across the various from three independent samples taken at each time point; error bars rep- SCIENCES rewiring schemes (SI Appendix, Fig. S3A) with the most signifi- resent SD of n = 3. (B) Viable cell count was determined by plating different

cant improvement observed in the PEX10 overexpression strain dilutions of sample; error bars represent SD of n ≥ 2. APPLIED BIOLOGICAL

Markham et al. PNAS | February 27, 2018 | vol. 115 | no. 9 | 2099 Downloaded by guest on September 30, 2021 Fig. 7. Production of poly[(epichlorohydrin)-co(epoxy triacetic acid lactone)] or PETAL. (A) Reaction scheme to create PETAL. (B) H NMR characterization of PETAL which shows distinct new peaks and shifts from the start molecules. (C) Photo of PETAL pressed into a film.

these two strategies (acetate feed and nitrogen starvation) are Biosourced TAL Can Be Incorporated into a Polymer Through not compatible. O-Functionalization. Finally, we leveraged the newfound bulk- availability of polyketides such as TAL to demonstrate their Bioreactor Cultivation Boosts Overall TAL Titer. Fermentation of the utility in the modification of polymer properties. TAL serves pyruvate bypass overexpression strain (YT- ACS1, ALD5, PDC2, a dual role as both a polymer modifier but also a func- ACC1) with acetate spiking was scaled up to the 3-L bioreactor tional adduct for later chemical derivatization owing to its lactone scale (YP, 18% glucose, 13.7 g/L acetate spike). After optimi- and unsaturated functionalities (34). TAL was rapidly extracted zation, fermentation resulted in production of 35.9 ± 3.9 g/L of and purified from fermentation broth and ultimately used for TAL (Fig. 6A). In this scale-up, we achieve a substantially im- the modification of structure and properties of commodity proved productivity over the previous batch cultures, upward of a poly(epichlorohydrin). This was achieved through heat and an fourfold increase to a glucose-phase maximum specific pro- activating organic base 1,8-Diazabicyclo(5.4.0)undec-7-ene ductivity of 0.21 ± 0.03 g/L/h (SI Appendix, Table S4). Although a (DBU) (Fig. 7A) (34, 35). In this process, the displacement of long fermentation time was necessary, this timescale is compa- chloride through the O-functionalization of TAL was evident rable to a previously published TAL study (12). The overall spectroscopically, chromatographically, and thermally through productivity reported here [0.12 g/L/h (SI Appendix, Table S4)] the use of NMR spectroscopy (Fig. 7B and SI Appendix, Fig. S8), improves upon the previous report in S. cerevisiae by greater than size exclusion chromatography (SEC), and differential scanning sixfold (12). As this production level is well outside the soluble calorimetry (DSC) (Table 1), respectively. The amount of TAL range of TAL, substantial in situ precipitation occurred, an at- incorporated along the poly(epichlorohydrin) backbone was tractive feature for industrial production, but a unique source of tuned stoichiometrically from 16 to 83% by mole resulting in sampling error. Likewise, we observed a diauxic shift from glu- poly[(epichlorohydrin)-co-(epoxy triacetic acid lactone)] or cose to (produced) citrate utilization (Fig. 6A) that leads to an PETAL. We measured the compositionally dependent changes increased production as cell viability stagnates and even de- in the glassy amorphous solid based on molecular weight and creases throughout the process (Fig. 6B). glass transition temperature of PETAL. Molecular weight was

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 −30

P(ECH-TALcom) 1: 1.5: 0.75 1: 1 23,700 30 PECH 1:0 7,200 −30

P(ECH-TALcom) 1: 1.5: 1 1: 5 9,400 70

P(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.

2100 | www.pnas.org/cgi/doi/10.1073/pnas.1721203115 Markham et al. Downloaded by guest on September 30, 2021 seen to increase proportionately to the amount of TAL in- Materials Generation. Biosourced TAL was purified from fermentation broth corporated as measured by size exclusion chromatography with through an ethyl acetate/acetic acid extraction. Commodity poly(epichloro- multiangle light-scattering detection to yield absolute number- hydrin) was functionalized with TAL using the activating organic base 1,8- average molecular weights (Mn) (Table 1). While poly(epichlo- Diazabicyclo(5.4.0)undec-7-ene and the resulting material characterized by rohydrin) exhibits a native glass transition temperature (Tg)of NMR, differential scanning calorimetry, UV-Vis spectroscopy, and size −30 °C, this value was markedly increased and strongly de- exclusion chromatography. pendent on TAL incorporation with the highest TAL composition (83% by mole) exhibiting a Tg of 70 °C. The final product Conclusions can be formed into a film and is seen to exhibit a unique hue and In summary, this work demonstrates the use of an oleaginous relative transparency (Fig. 7C). In particular, the orange color of organism for high-level production of an acetyl–CoA and the polymer is due to the native color of covalently bound TAL, malonyl–CoA-derived polyketide. Moreover, we establish a as no other characteristic spectroscopic differences were ob- previously uncharacterized pyruvate bypass pathway as superior served via UV-Vis spectroscopy (SI Appendix, Fig. S9). It should for rewiring CoA flux from lipid biosynthesis and into high-level be noted that the reaction rate obtained with this purified, bio- ± sourced TAL was comparable to a reaction conducted with TAL production reaching a titer of 35.9 3.85 g/L in a bio- commercially sourced TAL. Moreover, the biosourced PETAL reactor and overall yield of 0.164 g/g in a tube. Additional en- β structure and properties fall exactly along the trend observed gineering efforts related to the -oxidation pathway increased using commercially sourced TAL, again supporting that there yields to 0.203 g/g. The achieved titer far exceeds previous efforts is no difference (SI Appendix, Fig. S10). Finally, the residual in the field with conventional organisms (a summary of the lactone and unsaturated functionality from TAL repeat units achieved titers and yields in this work is provided in SI Appendix, offer a unique future strategy toward further modification of Table S4). This high-level production enabled rapid purification material properties. and conversion into a unique polymer with favorable molecular Methods weight and glass transition temperature. This work and resulting strain provides a path forward for microbial production of other AfullMaterials and Methods section is provided in the SI Appendix. acetyl–CoA and malonyl–CoA-derived polyketides for novel Strain Engineering and Analysis. Strains used in this study were constructed in applications such as polymers and chemical conversion. the wild-type Y. lipolytica strain, PO1f, through random genomic integration employing a sequential overexpression strategy. TAL concentrations were ACKNOWLEDGMENTS. We thank Yuki Naito for updating CRISPRdirect to assessed via reverse-phase HPLC following 96-h tube fermentations at 28 °C include a specificity check to Y. lipolytica. We would also like to thank Cory in defined media containing 2% glucose. Bioreactor fermentations were Schwartz and Ian Wheeldon for providing the pCRISPRyl plasmid. This work performed at the 3-L scale at pH 6.5 in YP media with 18% glucose and a was funded through the Camille and Henry Dreyfus Foundation. N.A.L. ac- 13.7 g/L sodium acetate spike at 36 h. knowledges support through Welch Foundation Grant F-1904.

1. Shanks BH, Keeling PL (2017) Bioprivileged molecules: Creating value from biomass. 19. Xie D, et al. (2006) Microbial synthesis of triacetic acid lactone. Biotechnol Bioeng 93: Green Chem 19:3177–3185. 727–736. 2. Liu L, Redden H, Alper HS (2013) Frontiers of yeast metabolic engineering: Di- 20. Madzak C (2015) Yarrowia lipolytica: Recent achievements in heterologous protein versifying beyond ethanol and Saccharomyces. Curr Opin Biotechnol 24:1023–1030. expression and pathway engineering. Appl Microbiol Biotechnol 99:4559–4577. 3. Markham KA, Cordova L, Hill A, Alper HS (2017) Yarrowia lipolytica as a cell factory for 21. Holdsworth JE, Veenhuis M, Ratledge C (1988) Enzyme activities in oleaginous yeasts oleochemical biotechnology. Consequences of Microbial Interactions with Hydrocarbons, accumulating and utilizing exogenous or endogenous lipids. J Gen Microbiol 134: Oils, and Lipids: Production of Fuels and Chemicals, ed Lee SY (Springer Int Publ, Cham, 2907–2915. Switzerland), pp 1–18. 22. Beopoulos A, et al. (2009) Yarrowia lipolytica as a model for bio-oil production. Prog 4. Markham KA, Alper HS (2015) Synthetic biology for specialty chemicals. Annu Rev Lipid Res 48:375–387. Chem Biomol Eng 6:35–52. 23. Nicaud J-M (2012) Yarrowia lipolytica. Yeast 29:409–418. 5. Austin MB, Noel JP (2003) The chalcone synthase superfamily of type III polyketide 24. Tai M, Stephanopoulos G (2013) Engineering the push and pull of lipid biosynthesis in – synthases. Nat Prod Rep 20:79 110. oleaginous yeast Yarrowia lipolytica for biofuel production. Metab Eng 15:1–9. 6. Robinson JA (1991) Polyketide synthase complexes: Their structure and function in 25. Yuzbasheva EY, et al. (2017) A metabolic engineering strategy for producing free – antibiotic biosynthesis. Philos Trans R Soc Lond B Biol Sci 332:107 114. fatty acids by the Yarrowia lipolytica yeast based on impairment of glycerol metab- 7. Lim YP, Go MK, Yew WS (2016) Exploiting the biosynthetic potential of type III pol- olism. Biotechnol Bioeng 115:433–443. – yketide synthases. Molecules 21:1 37. 26. Flores CL, Rodríguez C, Petit T, Gancedo C (2000) Carbohydrate and energy-yielding – 8. Eckermann S, et al. (1998) New pathway to polyketides in plants. Nature 396:387 390. metabolism in non-conventional yeasts. FEMS Microbiol Rev 24:507–529. 9. Yrjönen T, et al. (2002) Application of centrifugal force to the extraction and separation 27. Holz M, et al. (2011) Overexpression of alpha-ketoglutarate dehydrogenase in Yar- of parasorboside and gerberin from Gerbera hybrida. Phytochem Anal 13:349–353. rowia lipolytica and its effect on production of organic acids. Appl Microbiol 10. Tang S-Y, et al. (2013) Screening for enhanced triacetic acid lactone (TAL) production Biotechnol 89:1519–1526. by recombinant Escherichia coli expressing a designed TAL reporter. J Am Chem Soc 28. Kujau M, Weber H, Barth G (1992) Characterization of mutants of the yeast Yarrowia 135:10099–10103. lipolytica defective in acetyl-coenzyme A synthetase. Yeast 8:193–203. 11. Cardenas J, Da Silva NA (2014) Metabolic engineering of Saccharomyces cerevisiae for 29. Xu P, Qiao K, Ahn WS, Stephanopoulos G (2016) Engineering Yarrowia lipolytica as a the production of triacetic acid lactone. Metab Eng 25:194–203. platform for synthesis of drop-in transportation fuels and oleochemicals. Proc Natl 12. Saunders LP, Bowman MJ, Mertens JA, Da Silva NA, Hector RE (2015) Triacetic acid Acad Sci USA 113:10848–10853. lactone production in industrial Saccharomyces yeast strains. J Ind Microbiol 30. Sherman D, Durrens P, Beyne E, Nikolski M, Souciet JL; Génolevures Consortium (2004) Biotechnol 42:711–721. Génolevures: Comparative genomics and molecular evolution of hemiascomycetous 13. Cardenas J, Da Silva NA (2016) Engineering cofactor and transport mechanisms in – Saccharomyces cerevisiae for enhanced acetyl-CoA and polyketide biosynthesis. yeasts. Nucleic Acids Res 32:D315 D318. Metab Eng 36:80–89. 31. Luo X, Affeldt KJ, Keller NP (2016) Characterization of the far transcription factor – 14. Keatinge-Clay AT (2016) Stereocontrol within polyketide assembly lines. Nat Prod Rep family in Aspergillus flavus. G3 (Bethesda) 6:3269 3281. 33:141–149. 32. Cai L, Sutter BM, Li B, Tu BP (2011) Acetyl-CoA induces cell growth and proliferation – 15. Sorbic International Plc (2011) Product FAQS, sorbic acid. Available at www. by promoting the acetylation of histones at growth genes. Mol Cell 42:426 437. sorbicinternational.com/media/product-faqs.php. Accessed March 1, 2017. 33. Shi L, Tu BP (2013) Acetyl-CoA induces transcription of the key G1 cyclin CLN3 to 16. Schwartz TJ, et al. (2014) Engineering catalyst microenvironments for metal-catalyzed promote entry into the cell division cycle in Saccharomyces cerevisiae. Proc Natl Acad hydrogenation of biologically derived platform chemicals. Angew Chem Int Ed Engl Sci USA 110:7318–7323. 53:12718–12722. 34. Burns MJ, Ronson TO, Taylor RJ, Fairlamb IJ (2014) 4-Hydroxy-6-alkyl-2-pyrones as 17. Kraus GA, Basemann K, Guney T (2015) Selective pyrone functionalization: Reductive nucleophilic coupling partners in Mitsunobu reactions and oxa-Michael additions. alkylation of triacetic acid lactone. Tetrahedron Lett 56:3494–3496. Beilstein J Org Chem 10:1159–1165. 18. Kraus GA, Wanninayake UK, Bottoms J (2016) Triacetic acid lactone as a common 35. Hansen CA, Frost JW (2002) Deoxygenation of polyhydroxybenzenes: An alternative SCIENCES intermediate for the synthesis of 4-hydroxy-2-pyridones and 4-amino-2-pyrones. strategy for the benzene-free synthesis of aromatic chemicals. J Am Chem Soc 124: Tetrahedron Lett 57:1293–1295. 5926–5927. APPLIED BIOLOGICAL

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