Towards the Microbial Production of Plant-Derived Anticancer Drugs

Abstract

Many of the plant-derived compounds used in chemotherapies are currently produced by semisynthesis, which results in limited supplies at exorbitant market prices. However, the synthetic biology era, which began ca 15 years ago, has progressively yielded encouraging advances by using engineered microbes for the practical production of cheaper plant anticancer drugs. Many of the plant-derived compounds used in chemotherapies are currently produced by semisynthesis, which results in limited supplies at exorbitant market prices. However, the synthetic biology era, which began ca 15 years ago, has progressively yielded encouraging advances by using engineered microbes for the practical production of cheaper plant anticancer drugs. Plants represent a seemingly inexhaustible source of biologically active compounds. Some of these have been essential components of the anticancer therapeutic arsenal for many years [1.Srivastava V. et al.Plant-based anticancer molecules: a chemical and biological profile of some important leads.Bioorg. Med. Chem. 2005; 13: 5892-5908Crossref PubMed Scopus (305) Google Scholar]. Unfortunately, most of these chemicals naturally accumulate in the plant in only very small quantities and their complex structures render total chemical syntheses highly impractical. As a consequence, although pharmaceutical companies have optimized the extraction of precursors and developed semisynthetic processes for most of the valuable plant drugs currently used in the clinic, these active compounds usually have a high cost of production. In an attempt to circumvent these production limits, alternative strategies based on metabolic engineering have been widely explored over the past 15 years. Taking inspiration from the pioneering success of the antimalarial semisynthetic artemisinin [2.Paddon C.J. Keasling J.D. Semi-synthetic artemisinin: a model for the use of synthetic biology in pharmaceutical development.Nat. Rev. Microbiol. 2014; 12: 355-367Crossref PubMed Scopus (456) Google Scholar], microbial cell factories are increasingly developed for the production of plant-derived drugs. Numerous examples of genetically engineered bacteria and/or yeasts have emerged as suitable production hosts in the past decade. In this context, the plant genes that are heterologously expressed in these microbes allow de novo synthesis of the expected drugs. This can be achieved either through the derivatization of endogenous metabolites from these microbes or by the biotransformation of a cheap and abundant precursor that can be exogenously supplied to the microbe culture medium. In this Forum, we discuss the current status of this field by describing several major advances recently achieved for prominent classes of plant-derived anticancer drugs. Several MIAs found in Apocynaceae, in particular those from the Madagascar periwinkle (Catharanthus roseus), are long known for their antimitotic activity and are still used for treating various types of cancers [1.Srivastava V. et al.Plant-based anticancer molecules: a chemical and biological profile of some important leads.Bioorg. Med. Chem. 2005; 13: 5892-5908Crossref PubMed Scopus (305) Google Scholar]. This series of vinca alkaloids includes the natural compounds vincristine and vinblastine as well as the ‘non-natural’ derivatives vinorelbine, vindesine, and vinflunine. All of these compounds are powerful chemotherapy medications against various forms of leukemia, lymphoma, and solid tumors. After FDA approval in 1963, these anticancer MIAs have been produced by semisynthesis; that is, partial chemical synthesis using compounds isolated from natural sources as starting materials. Specifically, the production of these compounds relies on the coupling of the precursor monomers vindoline and catharanthine to generate the biologically active vinca-type alkaloids, with the first chemical procedure for coupling vindoline and catharanthine to form vinblastine described in 1974. Because both vindoline and catharanthine monomers accumulate in the aerial parts of C. roseus in only small quantities, the current low-yield production of vinca alkaloids leads to exorbitant market prices that can exceed several tens of millions of dollars per kilogram for vincristine [3.Gordon N. et al.Trajectories of injectable cancer drug costs after launch in the United States.J. Clin. Oncol. 2018; 36: 319-325Crossref PubMed Scopus (61) Google Scholar]. Thus, by the end of the 1970s, various research groups began to search for enzymes involved in MIA biosynthetic pathways with the goal of using metabolic engineering to improve the production of vindoline and catharanthine monomers. Forty years later, almost all of the genes involved in MIA synthesis have been characterized in C. roseus [4.Caputi L. et al.Missing enzymes in the biosynthesis of the anticancer drug vinblastine in Madagascar periwinkle.Science. 2018; 360: 1235-1239Crossref PubMed Scopus (192) Google Scholar]. Moreover, some proof-of-concept studies have emerged recently through the development of yeast strains hosting numerous periwinkle biosynthetic genes for the engineered production of anticancer MIA precursors (Figure 1) [5.Brown S. et al.De novo production of the plant-derived alkaloid strictosidine in yeast.Proc. Natl. Acad. Sci. U. S. A. 2015; 112: 3205-3210Crossref PubMed Scopus (304) Google Scholar]. The MIA intermediates strictosidine and nepetalactone have been thus successfully produced de novo in engineered yeast strains, and vindoline has been produced by feeding the intermediate tabersonine to engineered yeasts as well. However, the titers of these engineered yeast strains for de novo production remain at submilligram levels, indicating that substantial optimization is required to commercialize these production processes. Considerable effort is being made in this area, and given the successes with the production of other plant-derived compounds in yeast, we are optimistic that microbial cell factories will be soon available for participation in lower-cost production processes of MIA-based anticancer formulations. Undoubtedly, such advances on vinca alkaloids will also pave the way for implementing microorganisms producing precursors of biosynthetically related topotecan and irinotecan, the highly potent antitumor MIAs, derived from the happy tree (Camptotheca acuminata) camptothecin (Figure 1) [6.Sadre R. et al.Metabolite diversity in alkaloid biosynthesis: a multilane (diastereomer) highway for camptothecin synthesis in Camptotheca acuminata.Plant Cell. 2016; 28: 1926-1944Crossref PubMed Scopus (78) Google Scholar]. Paclitaxel (Taxol), docetaxel (Taxotere), and cabazitaxel represent another important class of plant-derived compounds extensively used in chemotherapy, notably for AIDS-related Kaposi sarcoma and various solid tumors [1.Srivastava V. et al.Plant-based anticancer molecules: a chemical and biological profile of some important leads.Bioorg. Med. Chem. 2005; 13: 5892-5908Crossref PubMed Scopus (305) Google Scholar]. These compounds belong to the taxane family, which are diterpenoids produced by yews (Taxus genus) (Figure 1). Like MIAs, taxanes display complex structures that make bulk chemical synthesis impossible in a cost-effective manner. Notably, the biotechnological production of taxane derivatives has been successful in plant cell cultures since the 2000s [7.Yukimune Y. et al.Methyl jasmonate-induced overproduction of paclitaxel and baccatin III in Taxus cell suspension cultures.Nat. Biotechnol. 1996; 14: 1129-1132Crossref PubMed Scopus (403) Google Scholar]. While this type of production has been a commercial success, additional effort has been made to engineer taxane biosynthesis, especially by creating bacterial or yeast strains and bacterial/yeast consortia that produce upstream precursors [8.Zhou K. et al.Distributing a metabolic pathway among a microbial consortium enhances production of natural products.Nat. Biotechnol. 2015; 33: 377-383Crossref PubMed Scopus (428) Google Scholar,9.Biggs B.W. et al.Overcoming heterologous protein interdependency to optimize P450-mediated Taxol precursor synthesis in Escherichia coli.Proc. Natl. Acad. Sci. U. S. A. 2016; 113: 3209-3214Crossref PubMed Scopus (148) Google Scholar]. However, many hurdles remain in implementing a sustainable supply of taxane using microorganisms. Most importantly, many biosynthetic enzymes from the yew taxane biosynthetic pathway have not been discovered and this clearly prevents the heterologous reconstitution of the complete pathway. Nevertheless, using the same omics-based global strategies as utilized for the MIA pathway [4.Caputi L. et al.Missing enzymes in the biosynthesis of the anticancer drug vinblastine in Madagascar periwinkle.Science. 2018; 360: 1235-1239Crossref PubMed Scopus (192) Google Scholar], the identification of these enzymes could be achieved in the foreseeable future. Etoposide and teniposide are lignan derivatives widely used in chemotherapies for testicular cancer, lung cancer, lymphoma, leukemia, neuroblastoma, and ovarian cancer [1.Srivastava V. et al.Plant-based anticancer molecules: a chemical and biological profile of some important leads.Bioorg. Med. Chem. 2005; 13: 5892-5908Crossref PubMed Scopus (305) Google Scholar]. Both compounds are currently supplied by chemical modifications of the podophyllotoxin skeleton, a lignan that accumulates at low levels in the roots of the mayapple (Podophyllum peltatum) (Figure 1). The restricted access to this endangered medicinal plant, which has been overharvested, has recently caused supply disruption. A few years ago, a huge leap forwards was accomplished with the identification and characterization of the key missing enzymes for podophyllotoxin synthesis in planta [10.Lau W. Sattely E.S. Six enzymes from mayapple that complete the biosynthetic pathway to the etoposide aglycone.Science. 2015; 349: 1224-1228Crossref PubMed Scopus (254) Google Scholar]. The proof of concept of heterologous production of this precursor was provided by transferring the whole set of mayapple biosynthetic genes into tobacco plants allowing, in turn, the conversion of a common plant natural precursor into podophyllotoxin analogs. Since the biosynthetic pathway of these lignans is shorter than that of vinblastine and taxanes described above, we anticipate that the development of practical microbial cell factories for these important anticancerous lignans can be rapidly achieved. Opium poppy (Papaver somniferum) is a source of potent pharmaceutical compounds showing a wide variety of activities. Among these complex structures related to the family of benzylisoquinoline alkaloids, which include the opioids, noscapine exhibits exciting anticancer potential but is accumulated only in low quantities in planta. In this context, Li et al. [11.Li Y. et al.Complete biosynthesis of noscapine and halogenated alkaloids in yeast.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: E3922-E3931Crossref PubMed Scopus (175) Google Scholar] have recently achieved outstanding de novo production of noscapine in yeast by recreating a biosynthetic pathway comprising over 30 enzymes from plants, bacteria, mammals, and yeast (Figure 1). Although noscapine production titers remain low (2.2 mg/l of yeast culture), this work opens unprecedented perspectives for an upcoming development of production platforms for anticancer opioids and illustrates that sourcing enzymes from distinct organisms is often necessary for efficient microbial production. Although displaying some in vitro antiproliferative and proapoptotic effects, cannabinoids from hemp (Cannabis sativa), mainly tetrahydrocannabinol and cannabidiol, are not clinically used as anticancer medicines. However, the use of these compounds by cancer patients is currently booming due to their capacity to reduce pain caused by cancer and/or chemotherapy. In addition, these cannabinoids have been approved as antiemetics, anticachexics, analgesics, or antispastic medicines by several regulatory agencies [12.Byars T. et al.Using cannabis to treat cancer-related pain.Semin. Oncol. Nurs. 2019; 35: 300-309Crossref PubMed Scopus (7) Google Scholar]. However, cannabinoid synthesis in hemp can be highly variable in planta and the complex structures limit chemical synthesis approaches, thus requiring the development of complementary processes of supply to meet the demand. In this regard, Luo et al. [13.Luo X. et al.Complete biosynthesis of cannabinoids and their unnatural analogues in yeast.Nature. 2019; 567: 123-126Crossref PubMed Scopus (334) Google Scholar] recently performed the complete biosynthesis of several cannabinoids in yeast (Figure 1). Although some strain improvements are required to reach production at an industrial scale, this work provided the proof of concept that engineered microbes can efficiently supply cannabinoids for pain management during cancer. Furthermore, by exploiting enzymes that have the capacity to metabolize different substrates, new-to-nature cannabinoid analogs were also produced in this study, thereby opening further possibilities for the production of new, related structures with novel pharmaceutical properties. Synthetic biology has driven major advances in the development of microbial cell factories that produce natural products since the beginning of the 2000s. A few recent works have demonstrated the feasibility of production platforms for several families of plant-derived anticancer drugs – that is, opioids, cannabinoids, and alkaloids – and these successes suggest that we can anticipate the same for taxanes and lignans (Figure 1). However, although the development of yeast strains at the laboratory scale represents a major step we can celebrate, much work remains to be performed before we reach industrial-scale production of strains that can be used for commercialization. These bottlenecks include: (i) low gene expression, which could be solved by increasing gene copy number using CRISPR/Cas9 technology; (ii) low metabolic fluxes that could be optimized by increasing the production of enzyme co-factors in the microbial host; (iii) improving host tolerance to the produced drug by the timely induction of transgene expression and/or product capture during yeast culture; and (iv) compensating for slow growth of the engineered microbe by optimization of the fermentation conditions, for example (Figure 1) [14.Choi K.R. et al.Systems metabolic engineering strategies: integrating systems and synthetic biology with metabolic engineering.Trends Biotechnol. 2019; 37: 817-837Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar]. Importantly, addressing these drawbacks will benefit all of the aforementioned metabolic engineering approaches. These advances will also pave the way for the future production of new cytotoxic natural products from other biological sources, in particular those identified in marine organisms [15.Blunt J.W. et al.Marine natural products.Nat. Prod. Rep. 2018; 35: 8-53Crossref PubMed Google Scholar]. We acknowledge funding from the EU Horizon 2020 research and innovation program (MIAMi project-grant agreement N°814645), the ARD2020 Biopharmaceutical Program of the Région Centre Val de Loire (BioPROPHARM and CatharSIS projects), La Ligue contre le Cancer and Le Studium (Consortium fellowship), and ERC 788301. The authors apologize for other excellent studies which have not been cited here due to space limitations.

Topics & Concepts