Complete biosynthesis of noscapine and halogenated alkaloids in yeast Yanran Lia,1, Sijin Lib,1, Kate Thodeyc, Isis Trenchardc, Aaron Cravensb, and Christina D. Smolkeb,d,2 aDepartment of Chemical and Environmental Engineering, University of California, Riverside, CA 92521; bDepartment of Bioengineering, Stanford University, Stanford, CA 94305; cAntheia Inc., Menlo Park, CA 94025; and dChan Zuckerberg Biohub, San Francisco, CA 94158 Edited by James C. Liao, Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan, and approved March 7, 2018 (received for review December 9, 2017) Microbial biosynthesis of plant natural products from simple derivatives have been reported, but none are currently feasible for building blocks is a promising approach toward scalable production large-scale production (5, 9). and modification of high-value compounds. The pathway for bio- A microbial-based manufacturing process for noscapinoids, synthesis of noscapine, a potential anticancer compound, from which are part of a larger class of benzylisoquinoline alkaloids canadine was recently elucidated as a 10-gene cluster from opium (BIAs), has the potential to address many of the challenges as- poppy. Here we demonstrate the de novo production of noscapine in sociated with the current poppy-based supply chain. Industrial Saccharomyces cerevisiae, through the reconstruction of a biosyn- cultivation of microorganisms, such as the baker’s yeast Saccharomyces thetic pathway comprising over 30 enzymes from plants, bacteria, cerevisiae, occurs over days, whereas poppies are annuals. Also, mammals, and yeast itself, including 7 plant endoplasmic reticulum because microbes are grown in closed fermentation vessels, the (ER)-localized enzymes. Optimization directed to tuning expression of production process is not susceptible to external environmental pathway enzymes, host endogenous metabolic pathways, and fer- factors and could provide greater consistency in product com- mentation conditions led to an over 18,000-fold improvement from position and impurity profiles across batches. Finally, by engineering initial noscapine titers to ∼2.2 mg/L. By feeding modified tyrosine a microorganism that does not make the broader profile of nar- derivatives to the optimized noscapine-producing strain we further cotic alkaloids present naturally in the opium poppy, one can remove demonstrated microbial production of halogenated benzylisoquino- the unnecessary hurdles associated with a tightly controlled supply line alkaloids. This work highlights the potential for microbial bio- chain. synthetic platforms to support the synthesis of valuable and novel In recent years, a number of pioneering studies have demon- alkaloid compounds, which can advance alkaloid-based drug discov- strated the reconstruction of complex plant-based pathways in ery and development. yeast, including the complete biosynthesis of strictosidine (10) and opioids (11). A 10-gene cluster encoding enzymes respon- benzylisoquinoline alkaloids | metabolic engineering | plant alkaloids | sible for the biosynthesis of noscapine from (S)-scoulerine in Saccharomyces cerevisiae opium poppy was recently identified (12, 13). We reconstructed the noscapine gene cluster in yeast to achieve microbial biosynthesis oscapine is a natural phthalideisoquinoline alkaloid drug Nthat is extracted from opium poppy (Papaver somniferum). Significance Although present in opium, noscapine is a nonnarcotic drug that has been used worldwide for over 50 years as a safe, nonnarcotic We demonstrate yeast’s capacity for biosynthesis of complex antitussive. Noscapine has also been demonstrated to exhibit an- plant natural products by reconstructing a de novo noscapine ticancer activity (1, 2); however, the dose required for treating biosynthetic pathway and optimizing noscapine production to- cancer is much higher than when it is used as a cough suppressant ward scalable manufacturing. Engineered strain contains 25 het- (1,000–2,250 mg/d for cancer treatment vs. 45–200 mg/d for cough erologous plant, bacteria, and mammalian genes and 6 mutant or treatment). Due to its long-established safety record, noscapine is overexpressed yeast genes. The noscapine biosynthetic path- a compelling candidate anticancer drug and has been used off- way incorporates seven endomembrane-localized plant en- label to treat cancers in some countries (3). In addition, a number zymes, highlighting yeast’s ability to functionally express and of noscapine derivatives have been shown to exhibit higher ther- properly localize large numbers of heterologous enzymes into apeutic potential toward certain cancer cell lines resulting from the endoplasm reticulum. Noscapine titers were improved by enhanced in vitro and in vivo activities or water solubility (4, 5). 18,000-fold (to low mg/L levels) via a combination of enzyme Noscapine and its analogs (collectively referred to as noscapi- engineering, pathway and strain engineering, and fermentation noids) serve as an emerging class of microtubule-modulating an- optimization. We demonstrated that microbial fermentation can ticancer agents that exhibit fewer side effects than traditional be used to produce halogenated alkaloid derivatives, which can chemotherapy drugs (6). ultimately serve as potential drug leads, through feeding amino All noscapinoids are currently derived from noscapine isolated acid derivatives to strains. from the opium poppy. Tens of tons of noscapine are extracted from thousands of tons of raw narcotic poppy straw annually to Author contributions: Y.L., S.L., K.T., I.T., and C.D.S. designed research; Y.L., S.L., K.T., I.T., and A.C. performed research; Y.L., S.L., K.T., I.T., and C.D.S. analyzed data; and Y.L., S.L., meet medical and scientific demand (7). Industrial poppy farming and C.D.S. wrote the paper. is susceptible to environmental factors such as pests, disease, and Conflict of interest statement: C.D.S. and Y.L. report a patent application published on climate, resulting in instability and variability in this geographically May 26, 2016, “Noscapinoid-Producing Microbes and Methods of Making and Using the concentrated supply chain (8). Relying on a narcotic supply chain Same,” application WO 2016/081371. (i.e., opium poppy) to produce a nonnarcotic drug introduces This article is a PNAS Direct Submission. additional unnecessary hurdles and costs to procurement, which Published under the PNAS license. are amplified due to the small size of the noscapinoid market 1Y.L. and S.L. contributed equally to this work. relative to the opioid market. Despite these challenges, poppy 2To whom correspondence should be addressed. Email: [email protected]. farming remains the sole source of noscapinoids, in part because This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. chemical synthesis of these complex molecules is not commercially 1073/pnas.1721469115/-/DCSupplemental. competitive. A number of chemical syntheses of noscapine and Published online April 2, 2018. E3922–E3931 | PNAS | vol. 115 | no. 17 www.pnas.org/cgi/doi/10.1073/pnas.1721469115 Downloaded by guest on September 24, 2021 of noscapine from (S)-canadine and norlaudanosoline (14). Sepa- more than 25 heterologous enzymes required for complete bio- PNAS PLUS rately, we engineered the first part of the biosynthetic pathway from synthesis of these complex molecules is nontrivial because of the glucose to (S)-reticuline (15) and (S)-reticuline to (S)-canadine (16) decreases in titer observed with each additional enzymatic step. In into yeast (Fig. 1A). These earlier studies suggested that yeast might addition, microbial expression of multiple endomembrane-localized be capable of synthesizing noscapinoids via fermentation of simple plant enzymes, such as cytochrome P450s, has remained challeng- carbon and nitrogen sources. However, functionally expressing the ing (17), and full reconstruction of the noscapine biosynthetic AB+ESI MRM 414220 Increased pentose phosphate 5 BH Biosynthetic, Sugar Tkl1p 4 pathway flux to E4P * recycling and salvage enzymes Redox partner for CPR cytochrome P450 enzymes E4P CO2H Aro8p Q166K DAHP Aro1p Aro2p T226I Tyr1p Aro4p Aro7p 4-HPP Aro9p + NH2 PEP HO Aro10p L-Tyrosine O MeO HO TyrHWR* 6OMT NCS OH NMe NH 4-HPAA HO CNMT HO HO + NMCH HO NH2 HO CO2H 2 3 4 5 4’OMT DODC MeO HO NH2 HO HO Time (minute) (S)-Reticuline (S)-Norcoclaurine Dopamine L-DOPA BBE MeO MeO O O O N S9OMT N CAS N TNMT N CYP82Y1 N HO HO O O O H H H H H OH OMe OMe OMe OH OMe OMe OMe OMe OMe OMe (S)-Scoulerine (S)-Tetrahydrocolumbamine (S)-Canadine (S)-N-methylcanadine (S)-1-Hydoxy-N-methylcanadine CYP82X2 O O O O NMe NMe O O O H O O H O OH OH SDR1 HO CXE1 NMe CYP82X1 NMe AT1 NMe O CHO O O H H HO HO H OMe OMe OMe OMe HO OMe AcO AcO HO OMe OMe OMe OMe OMe 4'-O-Desmethyl- 1-Hydroxy-13-O-acetyl- 1,13-Dihydroxy- Narcotoline Narcotolinehemiacetal 3-O-acetylpapaveroxine N-methylcanadine N-methylcanadine N4’OMT N4’OMT O O NMe NMe O O H O O H O OH SDR1 OMe OMe OMe OMe OMe OMe Noscapine Narcotinehemiacetal Fig. 1. De novo noscapine biosynthesis in S. cerevisiae.(A) The proposed biosynthetic pathway of noscapine in yeast. Block arrows indicate enzyme-catalyzed steps. Light gray arrows, unmodified yeast enzymes; dark gray arrows, overexpressed and modified yeast enzymes; purple arrows, mammalian (Rattus norvegicus) enzymes; orange arrows, bacterial (Pseudomonas putida) enzymes; green arrows, plant (Papaver somniferum, Coptis japonica) enzymes. Aro10p, phenylpyruvate decarboxylase; Aro1p, pentafunctional arom enzyme;