Towards the Microbial Production of Plant-Derived Anticancer Drugs

CD11bF4/80 macrophages and CD11c MHC-II DCs in the TME and periphery. To study myeloid cell-expressed PD1 in myelopoiesis and tumor growth, the authors generated myeloid cell-specific PD1deficient mice. These mice were equally resistant to tumor growth as global PD1deficient mice. Similar to global PD1deficient mice, myeloid cell PD1-deficient mice presented with fewer MDSCs and more macrophages and DCs in the TME. This immunogenic skew in myeloid lineage commitment was associated with increased IRF8, a major transcription factor implicated in myelopoiesis. The work of Strauss et al. is consistent with previous studies suggesting that downregulation of IRF8 by MDSCinducing factors, such as G-CSF, shifts myelopoiesis towards MDSCs in the TME [7]. G-CSF stimulation of PD1-deficient MPCs leads to enhanced activation of the ERK1/2, mTORC1, and STAT1 pathways, which are known to promote immunogenic myeloid differentiation. However, more effort is needed to elucidate how PD1-deficient MPCsmay be transcriptionally programmed via IRF8.

deficient mice. Similar to global PD1deficient mice, myeloid cell PD1-deficient mice presented with fewer MDSCs and more macrophages and DCs in the TME. This immunogenic skew in myeloid lineage commitment was associated with increased IRF8, a major transcription factor implicated in myelopoiesis. The work of Strauss et al. is consistent with previous studies suggesting that downregulation of IRF8 by MDSCinducing factors, such as G-CSF, shifts myelopoiesis towards MDSCs in the TME [7]. G-CSF stimulation of PD1-deficient MPCs leads to enhanced activation of the ERK1/2, mTORC1, and STAT1 pathways, which are known to promote immunogenic myeloid differentiation. However, more effort is needed to elucidate how PD1-deficient MPCs may be transcriptionally programmed via IRF8.
Strauss et al. also defined metabolic pathways involved in the differentiation of PD1deficient MPCs towards a monocyte-macrophage myeloid lineage. Consistent with their in vivo data, PD1-deficient MPCs effectively differentiated into Ly6C + monocytic cells (macrophage and/or DC precursors) when cultured with cytokines that skew myelopoiesis in the TME [e.g., G-CSF, GM-CSF, and/or interleukin-6 (IL- 6)]. This push towards a monocytic lineage was associated with an increased bioenergetic profile and higher cholesterol activity in PD1-deficient MPCs. Indeed, others have shown that cholesterol promotes the expansion of myeloid cells and the differentiation of macrophages and DCs [8].
Strauss et al. emphasize the differential importance of PD1 within myeloid cells versus T cells in antitumor immunity. Myeloid cellintrinsic PD1 signaling appears critical to alter myelopoiesis during cancer, because the skew in myeloid cell fate commitment from MDSCs to antitumor macrophages and DCs was not observed in T cellspecific PD1-deficient mice. The authors also linked this enhanced myelopoiesis in myeloid cell PD1-deficient mice with improved antitumor T cell function, including increased frequency of interferon (IFN)-γ/IL-17-producing effector memory CD8 + T cells. Despite some tumor-promoting effects of IL-17 [9], IL-17-producing CD4 + Th17 and CD8 + T cells have antitumor functions due to their polyfunctional cytokine profiles (including IFN-γ and stem-like features) [10]. In fact, Strauss et al. directly showed that PD1-deficient DCs from tumor-bearing mice better induced IFN-γsecreting CD4 + and CD8 + T cells. Overall, their finding that tumor growth is better controlled in myeloid cell-specific PD1 deficient mice versus T cell-specific PD1-deficient mice underscores the crucial role of myeloid cell PD1 in antitumor immunity. This is striking given that PD1 cancer studies have thus far predominantly focused on T cell PD1 [2]. Interestingly, the data from Strauss et al. also suggest that improved T cell responses due to PD1 blockade are possibly mediated indirectly via PD1 signaling within myeloid cells.
Consistent with animal models of global and myeloid cell-specific PD1 deletion, anti-PD1 treatment decreases MDSCs while increasing monocytes and DCs in the TME, suggesting that blocking PD1 could work partially by correcting a tumor-promoting shift in myelopoiesis during cancer. Overall, this exciting work adds a new perspective on the key contribution of myeloid-intrinsic PD1 signaling to immune evasion in cancer. Immune phenotyping of TME myeloid cell populations, including their PD1 expression, may be useful as a prognostic biomarker for anti-PD1 therapies. Beyond direct effects on T cells, approaches for PD1 blockade combination therapies should consider synergy with myeloid cell biology, such as agents that promote myeloid cell immunogenicity and recruitment. Given that PD1 is expressed on many types of myeloid cell, human PD1 function on specific myeloid cell subsets in cancer and other contexts awaits further discoveries. 1 Ann Romney Center for Neurologic Diseases, Department of 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.

Plant-Derived Anticancer Drugs: Low Supply, High Cost
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]. 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], 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.

Monoterpene Indole Alkaloids (MIAs) from Apocynaceae
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]. 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]. Thus, by the end of the 1970s, various research groups began to search for enzymes involved in MIA biosynthetic pathways with th 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]. Moreover, some proof-ofconcept 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]. 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].

Taxane Derivatives from the Pacific Yew
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]. 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]. While this type of production has been a commercial success, additional effort implementing the production by engineered microbes of monoterpene indole alkaloids of the Madagascar periwinkle, yew taxane-type terpenoids, mayapple lignans, poppy isoquinoline alkaloids, and hemp cannabinoids. The development process relies on a combination of systems biology, synthetic biology, and metabolic engineering strategies. Usually, this begins with the search for biosynthetic genes in the medicinal plant. Once identified and characterized, these genes are transferred into a microbial host by genetic transformation. Improvement of the metabolic flux and optimization of the fermentation strategy must be rationally achieved to improve the production yields of these engineered microbes, whether as a precursor or a final product.

Trends Trends in in Cancer Cancer
has been made to engineer taxane biosynthesis, especially by creating bacterial or yeast strains and bacterial/yeast consortia that produce upstream precursors [8,9]. 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], the identification of these enzymes could be achieved in the foreseeable future.

Lignan Derivatives from the Mayapple Podophyllotoxin
Etoposide and teniposide are lignan derivatives widely used in chemotherapies for testicular cancer, lung cancer, lymphoma, leukemia, neuroblastoma, and ovarian cancer [1]. 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]. 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.

Benzylisoquinoline Alkaloids from Opium Poppy
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] 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.

Cannabinoids from Hemp
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]. 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] 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.

Concluding Remarks
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 drugsthat is, opioids, cannabinoids, and alkaloidsand 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]. 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].