Biology subjects

Kevvai, K.

Publications and source records attributed to Kevvai, K..

3 recordsLinked to original sources

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↗

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↗