Abstract
In Saccharomyces cerevisiae, conventional 2μ-plasmid based plasmid (pC2μ, such as pRS425) have been widely adopted in pathway engineering for multi-copy overexpression of key genes. However, the loss of partition and copy number control elements of yeast endogenous 2μ plasmid (pE2μ) brings the issues concerning plasmid stability and copy number of pC2μ, especially in long-term fermentation. In this study, we developed a method based on CRISPR/Cas9 to edit pE2μ and built the pE2μ multi-copy system by insertion of the target DNA element and elimination of the original pE2μ plasmid. The resulting plasmid pE2μRAF1 and pE2μREP2 demonstrated higher copy number and slower loss rate than a pC2μ control plasmid pRS425RK, when carrying the same target gene. Then, moving the essential gene TPI1 (encoding triose phosphate isomerase) from chromosome to pE2μRAF1 could increase the plasmid viability to nearly 100% and further increase the plasmid copy number by 73.95%. The expression using pE2μ multi-copy system demonstrated much smaller cell-to-cell variation comparing with pC2μ multi-copy system. With auxotrophic complementation of TPI1, the resulting plasmid pE2μRT could undergo cultivation of 90 generations under non-selective conditions without loss. Applying pE2μ multi-copy system for dihydroartemisinic acid (DHAA) biosynthesis, the production of DHAA was increased to 620.9 mg/L at shake-flask level in non-selective rich medium. This titer was 4.73-fold of the strain constructed based on pC2μ due to the more stable pE2μ plasmid system and with higher plasmid copy number. This study provides an improved expression system in yeast, and set a promising platform to construct biosynthesis pathway for valuable products.
Highlights
For the high transformation efficiency and easy manipulation, plasmids have been developed as important tools and were widely applied in many kinds of organisms (Mignon et al, 2015; Lian et al, 2016)
Using multi-copy plasmid such as pRS426 might increase the efficiency of genome or plasmid editing and had been successfully applied for multigenes disruption (Jakounas et al, 2015; Lian et al, 2018), the recombination between pRS426 and endogenous 2μ plasmid through FRT sites by FLP1 was not desired during the plasmid editing process
The foreign gene could be inserted to 2μ plasmid in higher copy number for overexpression
Summary
For the high transformation efficiency and easy manipulation, plasmids have been developed as important tools and were widely applied in many kinds of organisms (Mignon et al, 2015; Lian et al, 2016). In Saccharomyces cerevisiae, conventional 2μ-based plasmid (pC2μ) such as pYES2, Yeast Endogenous 2μ Plasmid Editing pRS426, and pESC were widely used multi-copy plasmids for production of recombinant proteins as well as construction of metabolic pathway. Like all kinds of multi-copy plasmids, the plasmid viability in cells always depends on the selectivity pressure generated by the medium (with antibiotics or auxotroph medium) (Karim et al, 2013; Lian et al, 2016). For strain harboring pC2μ with auxotroph marker, the rich medium such as YPD is not suitable for long-term fermentation because of the loss of the plasmid, the strain grows faster in these kinds of media; And for that with antibiotic marker, the addition of expensive drugs is inevitable and it is not economic for fermentation of industrial scale
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