Biology subjects

Pyne, M. E.

Publications and source records attributed to Pyne, M. E..

5 recordsLinked to original sources

bioRxiv2025

An Ehrlich-inspired retrobiosynthesis of pharmaceutical scaffolds

Brewers yeast (Saccharomyces cerevisiae) acquires nitrogen from branched-chain and aromatic amino acids via the Ehrlich pathway, generating flavor (fusel) byproducts. Recently, diverting 4-hydroxyphenylacetaldehyde from Ehrlich catabolism of -tyrosine has enabled microbial production of opioids and other plant benzylisoquinolines. Yet, fusel metabolism is versatile in substrate scope, offering an untapped entry point for synthesizing structurally diverse aldehydes. Here, we repurpose the yeast Ehrlich pathway into a modular biocatalytic conduit for manufacturing privileged pharmaceutical alkaloids. We utilize retrobiosynthetic analysis and enzyme screening to derive scaffolds representative of solifenacin, colchicine, and ephedrine pharmaceuticals from simple amino acids. We survey wild yeasts for catabolism of -phenylglycine and demonstrate Ehrlich conversion to benzyl alcohol or (R)-phenylacetylcarbinol by 29 strains across nine genera. Implementing an {omega}-transaminase enables production of norephedrine from a simple amino acid input. This work unveils a generalizable biocatalytic route to clinically important alkaloids by exploiting metabolic logic from a yeast flavor pathway.

synthetic biology↗

bioRxiv2023

Benzylisoquinoline alkaloid production in yeast via norlaudanosoline improves selectivity and yield

The benzylisoquinoline alkaloid (BIA) family of tetrahydroisoquinolines (THIQs) comprises over 2,500 members, including the pharmaceuticals morphine, codeine, and papaverine as well as the antibiotics sanguinarine and chelerythrine used in animal husbandry. Agricultural cultivation can currently supply the demand for the BIAs that accumulate in plants, but broader access to the entire BIA family would open new avenues of research and commercialization. Microbial synthesis presents an attractive option due to cheap feedstock, genetic tractability, and ease of scale-up. Previously we reported titers of the key branch-point BIA (S)-reticuline of 4.6 g/L in yeast, which was achieved through leveraging the Ehrlich pathway 2-oxoacid decarboxylase Aro10 to generate the intermediate 4-hydroxyphenylacetaldehyde (4-HPAA). Here, we establish a superior route to (S)-reticuline by switching the pathway intermediate from 4-HPAA to 3,4-dihydroxyphenylacetaldehyde (3,4-dHPAA) using human monoamine oxidase A (MAO). The resulting (S)-norlaudanosoline route to (S)-reticuline synthesis is more selective, resolving prior issues with off-pathway THIQs synthesized due to concerted enzyme promiscuity. The new pathway is also more efficient, enabling titers of 4.8 g/L (S)-reticuline while improving yields over 40%, from 17 mg/g sucrose to 24 mg/g sucrose in fed-batch fermentations. Finally, we extend de novo (S)-reticuline synthesis to dihydrosanguinarine, achieving 635 mg/L dihydrosanguinarine and sanguinarine in fed-batch fermentation the highest reported titer of these BIAs by a factor of 40. HighlightsO_LIMonoamine oxidase A (MAO) supports high-titer (S)-reticuline synthesis in yeast C_LIO_LIMAO route to (S)-reticuline improves specificity compared to Aro10 route C_LIO_LIThis work represents a 40% increase in highest reported (S)-reticuline yield C_LIO_LI653 mg/L (dihydro-) sanguinarine was produced by extending the pathway C_LI

synthetic biology↗

bioRxiv2023

Screening non-conventional yeasts for organic acid tolerance and engineering Pichia occidentalis for production of cis,cis-muconic acid

Saccharomyces cerevisiae is a workhorse of industrial biotechnology owing to the organisms prominence in alcohol fermentation and the suite of sophisticated genetic tools available to manipulate its metabolism. However, S. cerevisiae is not suited to overproduce many bulk bioproducts, as toxicity constrains production at high titers. Here we employ a high-throughput assay to screen 108 publicly accessible yeast strains for tolerance to 20 g L-1 adipic acid (AA), a nylon precursor. We identify 15 tolerant yeasts and select Pichia occidentalis for production of cis,cis-muconic acid (CCM), a precursor to AA. By developing a genome editing toolkit for P. occidentalis, we demonstrate fed-batch production of CCM with a maximum titer (38.8 g L-1), yield (0.134 g g-1 glucose) and productivity (0.511 g L-1 h-1) that surpasses all metrics achieved using S. cerevisiae. This work brings us closer to the industrial bioproduction of AA and underscores the importance of host selection in bioprocessing.

synthetic biology↗

bioRxiv2023

CRAPS: Chromosomal-Repair-Assisted Pathway Shuffling in yeast

A fundamental challenge of metabolic engineering involves assembling and screening vast combinations of orthologous enzymes across a multi-step biochemical pathway. Current pathway assembly workflows involve combining genetic parts ex vivo and assembling one pathway configuration per tube or well. Here we present CRAPS, Chromosomal-Repair-Assisted Pathway Shuffling, an in vivo pathway engineering technique that enables the self-assembly of one pathway configuration per cell. CRAPS leverages the yeast chromosomal repair pathway and utilizes a pool of inactive, chromosomally integrated orthologous gene variants corresponding to a target multi-step pathway. Supplying gRNAs to the CRAPS host activates the expression of one gene variant per pathway step, resulting in a unique pathway configuration in each cell. We deployed CRAPS to build more than 1,000 combinations of a four-step carotenoid biosynthesis network. Sampling the CRAPS pathway space yielded strains with distinct color phenotypes and carotenoid product profiles. We anticipate that CRAPS will expedite strain engineering campaigns by enabling the generation and sampling of vast biochemical spaces.

synthetic biology↗

bioRxiv2019

A yeast platform for high-level synthesis of natural and unnatural tetrahydroisoquinoline alkaloids

The tetrahydroisoquinoline (THIQ) moiety is a privileged substructure of many bioactive natural products and semi-synthetic analogues. The plant kingdom manufactures more than 3,000 THIQ alkaloids, including the opioids morphine and codeine. While microbial species have been engineered to synthesize a few compounds from the benzylisoquinoline alkaloid (BIA) family of THIQs, low product titers impede industrial viability and limit access to the full chemical space. Here we report a THIQ platform by increasing yeast production of the central BIA intermediate (S)-reticuline to more than 3 g L-1, a 38,000-fold improvement over our first-generation strain. Gains in BIA output coincided with the formation of several substituted THIQs derived from host amino acid catabolism. Enabled by this activity, we repurposed the yeast Ehrlich pathway and demonstrate the synthesis of an array of unnatural THIQ scaffolds. This work provides a blueprint for synthesizing new privileged structures and will enable the targeted overproduction of thousands of THIQ products, including natural and semi-synthetic opioids.

synthetic biology↗