A roadmap to establish a comprehensive platform for sustainable manufacturing of natural products in yeast

Abstract

Secondary natural products (NPs) are a rich source for drug discovery. However, the low abundance of NPs makes their extraction from nature inefficient, while chemical synthesis is challenging and unsustainable. Saccharomyces cerevisiae and Pichia pastoris are excellent manufacturing systems for the production of NPs. This Perspective discusses a comprehensive platform for sustainable production of NPs in the two yeasts through system-associated optimization at four levels: genetics, temporal controllers, productivity screening, and scalability. Additionally, it is pointed out critical metabolic building blocks in NP bioengineering can be identified through connecting multilevel data of the optimized system using deep learning.

Efficient PCR-based gene targeting in isolates of the non-conventional yeastDebaryomyces hansenii

“…As a test we generated a universal peroxisomal marker by appending the red fluorescent protein mCherry with a peroxisomal targeting signal type 1 (PTS1). Peroxisomes are small organelles that posttranslationally import proteins into their lumen and that are gaining interest in synthetic biology circles to house new biosynthetic pathways for metabolic engineering purposes (see for instance Cross et al, 2017;DeLoache, Russ, & Dueber, 2016;Dusseaux, Wajn, Liu, Ignea, & Kampranis, 2020;Naseri, 2023). Using the KanR cassette flanked by long ARG1 homology arms (Fig.…”

Efficient PCR‐based gene targeting in isolates of the nonconventional yeast Debaryomyces hansenii

“…As a test, we generated a universal peroxisomal marker by appending the red fluorescent protein mCherry with a PTS1. Peroxisomes are small organelles that posttranslationally import proteins into their lumen and that are gaining interest in synthetic biology circles to house new biosynthetic pathways for metabolic engineering purposes (see for instance Cross et al, 2017; DeLoache et al, 2016; Dusséaux et al, 2020; Naseri, 2023). Using the KanR cassette flanked by long ARG1 homology arms (Figure 4a) as a starting point, we subsequently inserted mCherry‐PTS1 under control of the S. stipitis GPD1 promoter into this cassette.…”

“…We conclude that gene targeting occurs efficiently at both the ARG1 and the ADE2 locus using short regions of homology. DeLoache et al, 2016;Dusséaux et al, 2020;Naseri, 2023). Using the KanR cassette flanked by long ARG1 homology arms (Figure 4a) as a starting point, we subsequently inserted mCherry-PTS1 under control of the S. stipitis GPD1…”

Combinatorial optimization of gene expression through recombinase-mediated promoter and terminator shuffling in yeast

“…Metabolic engineering allows rewiring of cellular metabolism to increase product titers by enhancing precursor supply 3 , 4 , tackling co-factor limitations 5 – 7 , interrupting competitive pathways 8 , 9 , preventing buildup of toxic intermediates 10 and improving heterologous pathway flux 11 . Over the past years, several tools and strategies have been developed to facilitate yield optimization, including chassis strain improvement 12 – 14 , directed mutagenesis 15 – 17 , synthetic compartmentalization 18 , 19 and pathway gene expression optimization 20 .…”