Complete biosynthesis of noscapine and halogenated alkaloids in yeast.

Microbial biosynthesis of plant natural products from simple building blocks is a promising approach toward scalable production and modification of high-value compounds. The pathway for biosynthesis of noscapine, a potential anticancer compound, from canadine was recently elucidated as a 10-gene cluster from opium poppy. Here we demonstrate the de novo production of noscapine in Saccharomyces cerevisiae, through the reconstruction of a biosynthetic pathway comprising over 30 enzymes from plants, bacteria, mammals, and yeast itself, including 7 plant endoplasmic reticulum (ER)-localized enzymes. Optimization directed to tuning expression of pathway enzymes, host endogenous metabolic pathways, and fermentation conditions led to an over 18,000-fold improvement from initial noscapine titers to ∼2.2 mg/L. By feeding modified tyrosine derivatives to the optimized noscapine-producing strain we further demonstrated microbial production of halogenated benzylisoquinoline alkaloids. This work highlights the potential for microbial biosynthetic platforms to support the synthesis of valuable and novel alkaloid compounds, which can advance alkaloid-based drug discovery and development.

Engineering strategies for the fermentative production of plant alkaloids in yeast.

Microbial hosts engineered for the biosynthesis of plant natural products offer enormous potential as powerful discovery and production platforms. However, the reconstruction of these complex biosynthetic schemes faces numerous challenges due to the number of enzymatic steps and challenging enzyme classes associated with these pathways, which can lead to issues in metabolic load, pathway specificity, and maintaining flux to desired products. Cytochrome P450 enzymes are prevalent in plant specialized metabolism and are particularly difficult to express heterologously. Here, we describe the reconstruction of the sanguinarine branch of the benzylisoquinoline alkaloid pathway in Saccharomyces cerevisiae, resulting in microbial biosynthesis of protoberberine, protopine, and benzophenanthridine alkaloids through to the end-product sanguinarine, which we demonstrate can be efficiently produced in yeast in the absence of the associated biosynthetic enzyme. We achieved titers of 676 µg/L stylopine, 548 µg/L cis-N-methylstylopine, 252 µg/L protopine, and 80 µg/L sanguinarine from the engineered yeast strains. Through our optimization efforts, we describe genetic and culture strategies supporting the functional expression of multiple plant cytochrome P450 enzymes in the context of a large multi-step pathway. Our results also provided insight into relationships between cytochrome P450 activity and yeast ER physiology. We were able to improve the production of critical intermediates by 32-fold through genetic techniques and an additional 45-fold through culture optimization.

De novo production of the key branch point benzylisoquinoline alkaloid reticuline in yeast.

Microbial biosynthesis for plant-based natural products, such as the benzylisoquinoline alkaloids (BIAs), has the potential to address limitations in plant-based supply of established drugs and make new molecules available for drug discovery. While yeast strains have been engineered to produce a variety of downstream BIAs including the opioids, these strains have relied on feeding an early BIA substrate. We describe the de novo synthesis of the major BIA branch point intermediate reticuline via norcoclaurine in Saccharomyces cerevisiae. Modifications were introduced into yeast central metabolism to increase supply of the BIA precursor tyrosine, allowing us to achieve a 60-fold increase in production of the early benzylisoquinoline scaffold from fed dopamine with no supply of exogenous tyrosine. Yeast strains further engineered to express a mammalian tyrosine hydroxylase, four mammalian tetrahydrobiopterin biosynthesis and recycling enzymes, and a bacterial DOPA decarboxylase produced norcoclaurine de novo. We further increased production of early benzylisoquinoline scaffolds by 160-fold through introducing mutant tyrosine hydroxylase enzymes, an optimized plant norcoclaurine synthase variant, and optimizing culture conditions. Finally, we incorporated five additional plant enzymes--three methyltransferases, a cytochrome P450, and its reductase partner--to achieve de novo production of the key branch point molecule reticuline with a titer of 19.2 µg/L. These strains and reconstructed pathways will serve as a platform for the biosynthesis of diverse natural and novel BIAs.

Advancing secondary metabolite biosynthesis in yeast with synthetic biology tools.

Secondary metabolites are an important source of high-value chemicals, many of which exhibit important pharmacological properties. These valuable natural products are often difficult to synthesize chemically and are commonly isolated through inefficient extractions from natural biological sources. As such, they are increasingly targeted for production by biosynthesis from engineered microorganisms. The budding yeast species Saccharomyces cerevisiae has proven to be a powerful microorganism for heterologous expression of biosynthetic pathways. S. cerevisiae's usefulness as a host organism is owed in large part to the wealth of knowledge accumulated over more than a century of intense scientific study. Yet many challenges are currently faced in engineering yeast strains for the biosynthesis of complex secondary metabolite production. However, synthetic biology is advancing the development of new tools for constructing, controlling, and optimizing complex metabolic pathways in yeast. Here, we review how the coupling between yeast biology and synthetic biology is advancing the use of S. cerevisiae as a microbial host for the construction of secondary metabolic pathways.

Complete biosynthesis of opioids in yeast.

Opioids are the primary drugs used in Western medicine for pain management and palliative care. Farming of opium poppies remains the sole source of these essential medicines, despite diverse market demands and uncertainty in crop yields due to weather, climate change, and pests. We engineered yeast to produce the selected opioid compounds thebaine and hydrocodone starting from sugar. All work was conducted in a laboratory that is permitted and secured for work with controlled substances. We combined enzyme discovery, enzyme engineering, and pathway and strain optimization to realize full opiate biosynthesis in yeast. The resulting opioid biosynthesis strains required the expression of 21 (thebaine) and 23 (hydrocodone) enzyme activities from plants, mammals, bacteria, and yeast itself. This is a proof of principle, and major hurdles remain before optimization and scale-up could be achieved. Open discussions of options for governing this technology are also needed in order to responsibly realize alternative supplies for these medically relevant compounds.

Analyzing polyaniline-poly(2-acrylamido-2-methylpropane sulfonic acid) biocompatibility with 3T3 fibroblasts.

Conductive polymers can be utilized as unique functional elements in future intelligent biomaterials required for tissue engineering, cell stimulation and drug delivery. Research in these areas could be limited by established methods of conductive polymer synthesis which do not provide an easy route towards large scale processing of these materials. Typically, conductive polymers are highly insoluble in biocompatible solvents, and can be mechanically brittle, making their integration with biomaterials challenging. As an alternative, the use of a water-soluble conductive polymer for integration with biomaterials, a polyaniline template synthesized with poly(2-acrylamido-2-methylpropanesulfonic acid) (PANI-PAAMPSA), is proposed. To address key fundamental questions about the biocompatibility of this conductive polymer, cell cytotoxicity and proliferation assays have been performed on NIH 3T3 fibroblasts cultured on films of PANI-PAAMPSA. It was determined that the cell cultures maintained growth habits similar to those cultured on the control surfaces. Additionally, conductivity of the PANI-PAAMPSA films subsequent to exposure to the cell culture was demonstrated, indicating the materials retain functionality after cell growth. These results indicate that this type of template synthesized PANI could be successfully implemented as a functional, conductive biomaterial.