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 SCIENCES ± also obtained an averaged maximum titer of 35.9 3.9 g/L with an been implemented despite the promising chemical nature of ± APPLIED BIOLOGICAL achieved maximum specific productivity of 0.21 0.03 g/L/h in bio- these molecules. To address these limitations, previous efforts reactor fermentation. Additionally, we illustrate that a β-oxidation- PEX10 have explored microbial production of TAL. However, these related overexpression ( ) can support high TAL production and efforts have been restricted to conventional organisms [like is capable of achieving over 43% of the theoretical conversion yield Escherichia coli (10, 19) and Saccharomyces cerevisiae (11–13)] under nitrogen starvation in a test tube. Next, through use of this and are limited with respect to titer only reaching 5.2 g/L with low bioproduct, we demonstrate the utility of polyketides like TAL to yields (12). modify commodity materials such as poly(epichlorohydrin), resulting In this work, we explore the unique application of an oleagi- in an increased molecular weight and shift in glass transition temper- nous, nonconventional yeast (Yarrowia lipolytica) based on its ature. Collectively, these findings establish an engineering strategy potential for high flux through the key polyketide precursors, enabling unprecedented production from a type III polyketide syn- – – thase as well as establish a route through O-functionalization for acetyl CoA and malonyl CoA. By investigating three distinct converting polyketides into new materials. pathways toward CoA precursor formation along with targets hypothesized to enhance β-oxidation, we demonstrate the utility triacetic acid lactone | Yarrowia lipolytica | polyketide synthase | of a previously uncharacterized pyruvate bypass pathway for biorenewable chemicals | O-functionalization Significance he growing demand for renewable chemicals and fuels has Tspurred great interest in using cells as biochemical factories Polyketides are important molecules for both their bioactive (1). Metabolic engineering enables this goal by rewiring cells’ traits and their potential as chemical building blocks. However, metabolism toward desirable chemical compounds (2–4). Among production of these molecules through chemistry and bio- catalysts is restricted in yield and titer. Here, we demonstrate that possible molecules, polyketides are an interesting class of sec- the nonconventional yeast Yarrowia lipolytica can serve as a ondary metabolites produced by microbes and plants with native potent host for such production. This work provides a compre- roles in processes such as cellular defense and communication hensive evaluation of three separate pathways toward acetyl– – (5 7). While many polyketides can serve as potent antibiotics, CoA and malonyl–CoA in this host, enabling high-titer production this class of molecules also encompasses chemicals with other of triacetic acid lactone. Beyond achieving unprecedented titers useful properties such as pigments, antioxidants, antifungals, and and appreciable yields, this production capacity allows for both other bioactive traits (5, 8). However, the use of polyketides in purification from fermentation broth and conversion into a ma- more unique and nonmedical applications has been partially terial using simple reaction conditions. limited due to low natural abundance and difficult cultivation of native hosts. Specifically, polyketide-producing organisms are 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., typically unusual plants and microbial organisms that are not and H.S.A. analyzed data; and K.A.M., C.M.P., and H.S.A. wrote the paper. well-suited for high-level industrial production (6). Synthetic The authors declare no conflict of interest. production of these molecules in model host organisms has also This article is a PNAS Direct Submission. proven quite difficult with titers and yields insufficient for in- Published under the PNAS license. – dustrial production (10 13). Likewise, traditional chemical syn- 1K.A.M. and C.M.P. contributed equally to this work. thesis of polyketides is limited by low concentrations and 2To whom correspondence should be addressed. Email: [email protected]. challenging chiral centers (14). While the scale and price-point for This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. pharmaceuticals can tolerate plant-based sourcing of polyketides 1073/pnas.1721203115/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1721203115 PNAS Latest Articles | 1of6 Downloaded by guest on October 1, 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 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. 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 over- expression of genes involved in the citrate pathway. (B) The effect of sequential overexpression of pyru- vate 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 dehydrogenase genes and two pyruvate decarbox- ylase genes was tested. 2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1721203115 Markham et al. Downloaded by guest on October 1, 2021 approach, we established a coordinated overexpression of the different