Advances in genetic tools for metabolic engineering of non-conventional yeasts

Journal Article · Tue Jan 06 00:00:00 UTC 2026 · Frontiers in Synthetic Biology

DOI: https://doi.org/10.3389/fsybi.2025.1729790 · OSTI ID:3029034

Frousnoon, Thasneem Banu ^[1]; Mondo, Stephen ^[1]; Yoshikuni, Yasuo ^[2]

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1. Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)
2. Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States); Univ. of California, Berkeley, CA (United States); Hokkaido Univ., Sapporo (Japan)

Non-conventional yeasts are emerging as powerful alternatives to Saccharomyces cerevisiae for metabolic engineering, owing to their innate stress tolerance, broad substrate utilization, and distinctive metabolic capabilities. These attributes position them as promising chassis for producing biofuels, pharmaceuticals, and specialty chemicals. This review synthesizes recent advances in genetic toolkits for four such species—Pichia kudriavzevii (Issatchenkia orientalis), Starmerella bombicola, Debaryomyces hansenii, and Pachysolen tannophilus—highlighting progress across plasmid architectures (episomal and integrative), identification of autonomously replicating sequences and centromeric elements, and the development of safe-harbor genomic loci. We summarize promoter and terminator libraries enabling tunable expression, the expansion of auxotrophic and antifungal selection markers with recycling strategies, and the rapid adaptation of CRISPR-based systems (Cas9 and Cas12a) with optimized guide RNA expression, multiplex editing, and approaches that enhance homologous recombination (e.g., KU70/80 disruption). We also review landing-pad platforms for modular, repeated integrations and transposon-based tools (e.g., piggyBac) that facilitate multigene pathway assembly. Collectively, these innovations are accelerating design-build-test-learn cycles and enabling precise, scalable engineering of non-conventional yeasts. Remaining challenges—including limited species-specific episomal systems, variable transformation efficiencies, genome-stability concerns, and alternative codon usage—define clear priorities for future toolkit development. Together, these advances and open needs chart a path toward robust, sustainable biomanufacturing using diverse non-conventional yeast chassis.

• Research Organization: Univ. of Illinois at Urbana-Champaign, IL (United States)
• Sponsoring Organization: USDOE Office of Science (SC), Biological and Environmental Research (BER)
• Grant/Contract Number: AC02-05CH11231; SC0018420
• OSTI ID: 3029034
• Journal Information: Frontiers in Synthetic Biology, Journal Name: Frontiers in Synthetic Biology Vol. 3; ISSN 2813-818X
• Publisher: Frontiers Media S.A.Copyright Statement
• Country of Publication: United States
• Language: English