Integration of Yeast Episomal/Integrative Plasmid Causes Genotypic and Phenotypic Diversity and Improved Sesquiterpene Production in Metabolically Engineered Saccharomyces cerevisiae

Abstract

The variability in phenotypic outcomes among biological replicates in engineered microbial factories presents a captivating mystery. Establishing the association between phenotypic variability and genetic drivers is important to solve this intricate puzzle. We applied a previously developed auxin-inducible depletion of hexokinase 2 as a metabolic engineering strategy for improved nerolidol production in Saccharomyces cerevisiae, and biological replicates exhibit a dichotomy in nerolidol production of either 3.5 or 2.5 g L −1 nerolidol. Harnessing Oxford Nanopore's long-read genomic sequencing, we reveal a potential genetic cause�the chromosome integration of a 2μ sequence-based yeast episomal plasmid, encoding the expression cassettes for nerolidol synthetic enzymes. This finding was reinforced through chromosome integration revalidation, engineering nerolidol and valencene production strains, and generating a diverse pool of yeast clones, each uniquely fingerprinted by gene copy numbers, plasmid integrations, other genomic rearrangements, protein expression levels, growth rate, and target product productivities. Τhe best clone in two strains produced 3.5 g L −1 nerolidol and ∼0.96 g L −1 valencene. Comparable genotypic and phenotypic variations were also generated through the integration of a yeast integrative plasmid lacking 2μ sequences. Our work shows that multiple factors, including plasmid integration status, subchromosomal location, gene copy number, sesquiterpene synthase expression level, and genome rearrangement, together play a complicated determinant role on the productivities of sesquiterpene product. Integration of yeast episomal/integrative plasmids may be used as a versatile method for increasing the diversity and optimizing the efficiency of yeast cell factories, thereby uncovering metabolic control mechanisms.

The integration of tandem gene repeatsviaa bacterial type-II toxin-antitoxin-mediated gene amplification (ToxAmp) system and stability visualisation inSaccharomyces cerevisiae

“…46 Recently, we showed that the typical yeast integrative plasmid (YIp) could be used to deliver multi-copy genome integration in the pattern of tandem gene repeats. 21 We note that YIp delivering multicopy genome integration has been previously reported in 1981. 47 The current work further confirms that the multi-copy genome integration feature happens during the transformation, and does not depends on a specific selection pressure (Figure 1 & Figure 4).…”

“…Oxford Nanopore whole genome sequencing was performed using the methods described previously. 8, 21 The whole genome DNA was extracted using MagAttract HMW DNA Kit (Qiangen) with a modified protocol. A Rapid Barcoding Kit (SQK-RBK114.24, Oxford Nanopore), a R10.4.1 flow cell (FLO-MIN114, Oxford Nanopore), and a MinION sequencing Device (Oxford Nanopore) were used in Nanopore DNA sequencing.…”

“…Oxford Nanopore whole genome sequencing was performed using the methods described previously. 8,21 The whole genome DNA was extracted using MagAttract HMW DNA Kit (Qiangen) with a modified protocol. A Rapid Barcoding Kit (SQK-RBK114.24, Oxford Nanopore), a R10.…”

“…In these sesquiterpene-producing yeasts, the variance of copy number likely occurred randomly rather than being driven by a strongly linked selection pressure. 21 These show that gene amplification may happen as either a selection-force-driven process or a random process.…”

“…pressure. 21 These show that gene amplification may happen as either a selection-force-driven process or a random process.…”