Coupling the tools of synthetic biology with traditional molecular genetic techniques can enable the rapid
prototyping and optimization of yeast strains. While the era of yeast synthetic biology began in the
well-characterized model organism Saccharomyces cerevisiae, it is swiftly expanding to include nonconventional
yeast production systems such as Hansenula polymorpha, Kluyveromyces lactis, Pichia pastoris,
and Yarrowia lipolytica. These yeasts already have roles in the manufacture of vaccines, therapeutic
proteins, food additives, and biorenewable chemicals, but recent synthetic biology advances have the
potential to greatly expand and diversify their impact on biotechnology. In this review, we summarize
the development of synthetic biological tools (including promoters and terminators) and enabling molecular
genetics approaches that have been applied in these four promising alternative biomanufacturing
platforms. An emphasis is placed on synthetic parts and genome editing tools. Finally, we discuss examples
of synthetic tools developed in other organisms that can be adapted or optimized for these hosts in
the near future.