Design and construction of short synthetic terminators for β-amyrin production in Saccharomyces cerevisiae

Exchange of endogenous and heterogeneous yeast terminators in Pichia pastoris to tune mRNA stability and gene expression

“…As part of this study, we characterized for the first time the activity in P. pastoris of synthetic terminators originally developed for use in S. cerevisiae . Although the synthetic terminators resulted in high expression in S. cerevisiae ( 18 , 19 ), we did not observe this effect for P. pastoris . This suggests that different mechanisms of regulation for protein expression are at play in S. cerevisiae versus P. pastoris .…”

“…To identify terminator sequences with different activities in P. pastoris , we constructed yeast strains carrying various terminator sequences downstream of a GFP-encoding ORF under the control of a constitutive GAPDH promoter derived from P. pastoris (Figure 1 ), then profiled a terminator catalog based on GFP FI to define terminator activity. To create a ‘terminator catalog’ for P. pastoris , we chose 72 terminator sequences representing the following five categories (see also Table 1 ): (i) S. cerevisiae terminator sequences with the top 21-ranked and bottom 18-ranked activities in S. cerevisiae ( 13 ); (ii) P. pastoris terminator sequences with high amino acid sequence similarity to the ORFs upstream of the 30 top- and bottom-ranked S. cerevisiae terminators ( Supplementary Table S3 ) ( 13 ); (iii) previously reported P. pastoris terminator sequences with high activities ( 10 ); (iv) artificial short terminators originally designed for use in S. cerevisiae ( 18 , 19 ); and (v) terminator sequences that served in our study as standards. In total, 72 terminator sequences were inserted into the GFP expression plasmid (Figure 1 ), and each resulting plasmid was linearized and transformed into P. pastoris .…”

“…The weakest terminator among the 72 tested in this study, Sc_GIC1 t, was also the weakest in S. cerevisiae ( 13 ). In this study, we also characterized the P. pastoris strains with each of three different synthetic short terminators, T Guo , T synth27 ( 18 ) and Syn Ter10 ( 19 ), which had 2.3-, 3.5- and 5.5-fold higher terminator activity, respectively, than Sc_CYC1 t in S. cerevisiae . In P. pastoris , the activities of these synthetic terminators were the same or a little higher (1.0- to 1.2-fold) as compared with Sc_CYC1 t, such that the synthetic terminators are in the middle range of activities observed for the full terminator catalog (Figure 2 and Supplementary Table S4 ).…”

“…The trans -acting factors Nab6p and Pap1p, which were identified in a genome library screen, appear to be responsible for the strong activity of the endogenous DIT1 terminator in S. cerevisiae ( 17 ). Artificial terminators have been developed in S. cerevisiae using either of two strategies: connection of three functional parts of S. cerevisiae terminators ( 18 , 19 ) or insertion of additional trans -acting factor binding sites of the DIT1 terminator into an endogenous terminator sequence ( 17 ). Notably, terminator technology based on use of native or artificial terminators has been successfully applied in metabolic engineering ( 15 , 19 ).…”

Self-Redirection of Metabolic Flux toward Squalene and Ethanol Pathways by Engineered Yeast

“…Squalene is a polyunsaturated triterpenoid that acts as a precursor for the biosynthesis of steroids and cholesterol in animals and plants [1,2]. Several terpenoid synthases use squalene as a substrate to synthesize various types of triterpenoids including α-amyrin, β-amyrin, and lupeol [3,4]. Squalene is used in many cosmetic products because it acts as an emollient, antioxidant, and moisturizing agent [5,6].…”