Covering: 2000 to 2020
Natural products and their derivatives are commercially important medicines, agrochemicals, flavors, fragrances, and food ingredients. Industrial strategies to produce these ...structurally complex molecules encompass varied combinations of chemical synthesis, biocatalysis, and extraction from natural sources. Interest in engineering natural product biosynthesis began with the advent of genetic tools for pathway discovery. Genes and strains can now readily be synthesized, mutated, recombined, and sequenced. Enzyme engineering has succeeded commercially due to the development of genetic methods, analytical technologies, and machine learning algorithms. Today, engineered biosynthetic enzymes from organisms spanning the tree of life are used industrially to produce diverse molecules. These biocatalytic processes include single enzymatic steps, multienzyme cascades, and engineered native and heterologous microbial strains. This review will describe how biosynthetic enzymes have been engineered to enable commercial and near-commercial syntheses of natural products and their analogs.
This review describes examples of the broadening industrial relevance of engineered secondary metabolism enzymes, natural products and analogs being made with these enzymes, and technology improvements that have enabled their development since 1999.
Opiates and related molecules are medically essential, but their production via field cultivation of opium poppy Papaver somniferum leads to supply inefficiencies and insecurity. As an alternative ...production strategy, we developed baker's yeast Saccharomyces cerevisiae as a microbial host for the transformation of opiates. Yeast strains engineered to express heterologous genes from P. somniferum and bacterium Pseudomonas putida M10 convert thebaine to codeine, morphine, hydromorphone, hydrocodone and oxycodone. We discovered a new biosynthetic branch to neopine and neomorphine, which diverted pathway flux from morphine and other target products. We optimized strain titer and specificity by titrating gene copy number, enhancing cosubstrate supply, applying a spatial engineering strategy and performing high-density fermentation, which resulted in total opioid titers up to 131 mg/l. This work is an important step toward total biosynthesis of valuable benzylisoquinoline alkaloid drug molecules and demonstrates the potential for developing a sustainable and secure yeast biomanufacturing platform for opioids.
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.
Protoberberine alkaloids are bioactive molecules abundant in plant preparations for traditional medicines. Yeast engineered to express biosynthetic pathways for fermentative production of these ...compounds will further enable investigation of the medicinal properties of these molecules and development of alkaloid-based drugs with improved efficacy and safety. Here, we describe the optimization of a biosynthetic pathway in Saccharomyces cerevisiae for conversion of rac-norlaudanosoline to the protoberberine alkaloid (S)-canadine.
This yeast strain is engineered to express seven heterologous enzymes, resulting in protoberberine alkaloid production from a simple benzylisoquinoline alkaloid precursor. The seven enzymes include three membrane-bound enzymes: the flavin-dependent oxidase berberine bridge enzyme, the cytochrome P450 canadine synthase, and a cytochrome P450 reductase. A number of strategies were implemented to improve flux through the pathway, including enzyme variant screening, genetic copy number variation, and culture optimization, that led to an over 70-fold increase in canadine titer up to 1.8 mg/L. Increased canadine titers enable extension of the pathway to produce berberine, a major constituent of several traditional medicines, for the first time in a microbial host. We also demonstrate that this strain is viable at pilot scale.
By applying metabolic engineering and synthetic biology strategies for increased conversion of simple benzylisoquinoline alkaloids to complex protoberberine alkaloids, this work will facilitate chemoenzymatic synthesis or de novo biosynthesis of these and other high-value compounds using a microbial cell factory.
is an economically and ecologically important fungal pathogen that causes Septoria stem canker and leaf spot disease of
species. To bridge the gap between genetic markers and structural barriers ...previously found to be linked to Septoria canker disease resistance in poplar, we used hydrophilic interaction liquid chromatography and tandem mass spectrometry to identify and quantify metabolites involved with signaling and cell wall remodeling. Fluctuations in signaling molecules, organic acids, amino acids, sterols, phenolics, and saccharides in resistant and susceptible
inoculated with
were observed. The patterns of 222 metabolites in the resistant host implicate systemic acquired resistance (SAR), cell wall apposition, and lignin deposition as modes of resistance to this hemibiotrophic pathogen. This pattern is consistent with the expected response to the biotrophic phase of
colonization during the first 24 h postinoculation. The fungal pathogen metabolized key regulatory signals of SAR, other phenolics, and precursors of lignin biosynthesis that were depleted in the susceptible host. This is the first study to characterize metabolites associated with the response to initial colonization by
between resistant and susceptible hosts.
Abstract
In addition to its essential role in viral polyprotein processing, the SARS-CoV-2 3C-like protease (3CLpro) can cleave human immune signaling proteins, like NF-κB Essential Modulator (NEMO) ...and deregulate the host immune response. Here, in vitro assays show that SARS-CoV-2 3CLpro cleaves NEMO with fine-tuned efficiency. Analysis of the 2.50 Å resolution crystal structure of 3CLpro C145S bound to NEMO
226–234
reveals subsites that tolerate a range of viral and host substrates through main chain hydrogen bonds while also enforcing specificity using side chain hydrogen bonds and hydrophobic contacts. Machine learning- and physics-based computational methods predict that variation in key binding residues of 3CLpro-NEMO helps explain the high fitness of SARS-CoV-2 in humans. We posit that cleavage of NEMO is an important piece of information to be accounted for, in the pathology of COVID-19.
Metabolite genome-wide association studies (mGWASs) are increasingly used to discover the genetic basis of target phenotypes in plants such as
, a biofuel feedstock and model woody plant species. ...Despite their growing importance in plant genetics and metabolomics, few mGWASs are experimentally validated. Here, we present a functional genomics workflow for validating mGWAS-predicted enzyme-substrate relationships. We focus on uridine diphosphate-glycosyltransferases (UGTs), a large family of enzymes that catalyze sugar transfer to a variety of plant secondary metabolites involved in defense, signaling, and lignification. Glycosylation influences physiological roles, localization within cells and tissues, and metabolic fates of these metabolites. UGTs have substantially expanded in
, presenting a challenge for large-scale characterization. Using a high-throughput assay, we produced substrate acceptance profiles for 40 previously uncharacterized candidate enzymes. Assays confirmed 10 of 13 leaf mGWAS associations, and a focused metabolite screen demonstrated varying levels of substrate specificity among UGTs. A substrate binding model case study of UGT-23 rationalized observed enzyme activities and mGWAS associations, including glycosylation of trichocarpinene to produce trichocarpin, a major higher-order salicylate in
We identified UGTs putatively involved in lignan, flavonoid, salicylate, and phytohormone metabolism, with potential implications for cell wall biosynthesis, nitrogen uptake, and biotic and abiotic stress response that determine sustainable biomass crop production. Our results provide new support for
analyses and evidence-based guidance for
functional characterization.
The COVID-19 pandemic caused by SARS-CoV-2 requires rapid development of specific therapeutics and vaccines. The main protease of SARS-CoV-2, 3CL M
, is an established drug target for the design of ...inhibitors to stop the virus replication. Repurposing existing clinical drugs can offer a faster route to treatments. Here, we report on the binding mode and inhibition properties of several inhibitors using room temperature X-ray crystallography and in vitro enzyme kinetics. The enzyme active-site cavity reveals a high degree of malleability, allowing aldehyde leupeptin and hepatitis C clinical protease inhibitors (telaprevir, narlaprevir, and boceprevir) to bind and inhibit SARS-CoV-2 3CL M
. Narlaprevir, boceprevir, and telaprevir are low-micromolar inhibitors, whereas the binding affinity of leupeptin is substantially weaker. Repurposing hepatitis C clinical drugs as COVID-19 treatments may be a useful option to pursue. The observed malleability of the enzyme active-site cavity should be considered for the successful design of specific protease inhibitors.
This review describes examples of the broadening industrial relevance of engineered secondary metabolism enzymes, natural products and analogs being made with these enzymes, and technology ...improvements that have enabled their development since 1999.
Covering: 2000 to 2020 Natural products and their derivatives are commercially important medicines, agrochemicals, flavors, fragrances, and food ingredients. Industrial strategies to produce these ...structurally complex molecules encompass varied combinations of chemical synthesis, biocatalysis, and extraction from natural sources. Interest in engineering natural product biosynthesis began with the advent of genetic tools for pathway discovery. Genes and strains can now readily be synthesized, mutated, recombined, and sequenced. Enzyme engineering has succeeded commercially due to the development of genetic methods, analytical technologies, and machine learning algorithms. Today, engineered biosynthetic enzymes from organisms spanning the tree of life are used industrially to produce diverse molecules. These biocatalytic processes include single enzymatic steps, multienzyme cascades, and engineered native and heterologous microbial strains. This review will describe how biosynthetic enzymes have been engineered to enable commercial and near-commercial syntheses of natural products and their analogs.
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