Centromeric DNA Facilitates Nonconventional Yeast Genetic Engineering

Abstract

Many nonconventional yeast species have highly desirable features that are not possessed by model yeasts, despite that significant technology hurdles to effectively manipulate them lay in front. Scheffersomyces stipitis is one of the most important exemplary nonconventional yeasts in biorenewables industry, which has a high native xylose utilization capacity. Recent study suggested its much better potential than Saccharomyces cerevisiae as a well-suited microbial biomanufacturing platform for producing high-value compounds derived from shikimate pathway, many of which are associated with potent nutraceutical or pharmaceutical properties. However, the broad application of S. stipitis is hampered by the lack of stable episomal expression platforms and precise genome-editing tools. Here we report the success in pinpointing the centromeric DNA as the partitioning element to guarantee stable extra-chromosomal DNA segregation. The identified centromeric sequence not only stabilized episomal plasmid, enabled homogeneous gene expression, increased the titer of a commercially relevant compound by 3-fold, and also dramatically increased gene knockout efficiency from <1% to more than 80% with the expression of CRISPR components on the new stable plasmid. This study elucidated that establishment of a stable minichromosome-like expression platform is key to achieving functional modifications of nonconventional yeast species in order to expand the current collection of microbial factories.

The reported point centromeres of Scheffersomyces stipitis are retrotransposon long terminal repeats

“…They initially identified this sequence as a subcloned 125‐bp region from S . stipitis CEN5 (Cao, Gao, et al, ). In their second study, they found that 125‐bp sequences almost identical to this core sequence were present in all but one of the stability‐conferring 3‐kb fragments from other chromosomes.…”

“…Among the 27 locations, there are 10 different variants of the 125‐bp sequence, which we designated 125a–125j (Figure ; sequences are available at https://doi.org/10.6084/m9.figshare.7364738). Each of these variants has a slightly different sequence, but they all have ≤11 mismatches to the 125‐bp sequence initially isolated from CEN5 (Cao, Gao, et al, ). Variant 125a is actually 127‐bp long; the others are all 125 bp.…”

“…stipitis has point centromeres, which is surprising because it is outside the family Saccharomycetaceae and hence not closely related to known point‐centromere species. They identified the centromeres by using a plasmid stability assay, first for CEN5 (Cao, Gao, et al, ) and subsequently for the other seven centromeres (Cao, Seetharam, Severin, & Shao, ). In their work, they assumed that the centromeres would be located within the large G + C‐poor noncoding regions identified by Lynch et al ().…”

“…stipitis origin of replication. Compared with controls, the plasmids containing fragments with centromere‐like activity were not lost after many generations of mitotic growth and did not preferentially accumulate in mother cells (Cao, Gao, et al, ). Surprisingly, in their first study, Cao, Gao, et al () found that the 17‐kb noncoding region at CEN5 could be reduced to a fragment of only 500 bp without loss of mitotic stability and that it could be further shortened to a 125‐bp “core” sequence with only a small reduction in stability.…”

CRISPR–Mediated Genome Editing and Gene Repression in Scheffersomyces stipitis

“…For engineering nonconventional microbial species, the challenges ahead mainly include low transformation efficiency and the lack of genetic manipulation tools. Recently, we developed a CRISPR‐based gene knockout system in S. stipitis and improved the gene knockout efficiency from <1% to more than 80% based on indel mutations …”