A programmable high-expression yeast platform responsive to user-defined signals

Abstract

Rapidly growing yeasts with appropriate posttranslational modifications are favored hosts for protein production in the biopharmaceutical industry. However, limited production capacity and intricate transcription regulation restrict their application and adaptability. Here, we describe a programmable high-expression yeast platform, SynPic-X, which responds to defined signals and is broadly applicable. We demonstrated that a synthetic improved transcriptional signal amplification device (iTSAD) with a bacterial-yeast transactivator and bacterial-yeast promoter markedly increased expression capacity in Pichia pastoris . CRISPR activation and interference devices were designed to strictly regulate iTSAD in response to defined signals. Engineered switches were then constructed to exemplify the response of SynPic-X to exogenous signals. Expression of α-amylase by SynPic-R, a specific SynPic-X, in a bioreactor proved a methanol-free high-production process of recombinant protein. Our SynPic-X platform provides opportunities for protein production in customizable yeast hosts with high expression and regulatory flexibility.

Yeast transcriptional device libraries enable precise synthesis of value-added chemicals from methanol

“…Using the domain-swapping strategy, we constructed TSAD using an sTF of LacI–Mit1AD ( E. coli lac regulator fused with the activation domain of Mit1) and a hybrid promoter of lacO– cP AOX1 ( E. coli lac operator linked to the core region of P AOX1 ) ( 29 ). The improvement of the functional components further generated an iTSAD with high expression capacity ( 54 ). These preliminary results provide the basic structure of the transcriptional device libraries that we aimed to develop in this study.…”

“…Therefore, the combination of diverse DBP–BS and TFAD–CP may lead to the development of various engineered devices (Figure 1A ). We first selected different bacterial regulators, LacI ( 29 ), AraC ( 54 ), LexA ( 58 ), AcrR and BetI ( 59 ), as DBPs to fuse with Mit1AD using a GGGGS linker ( Supplementary Figure S1 ; Supplementary Table S1 ). An SV40 nuclear localization sequence (NLS) was fused at the N-terminus of DBPs (except LacI) to allow them to enter the nucleus of P. pastoris .…”

“…An improved TSAD (iTSAD) was obtained using 18 combination groups of cis- and trans- acting elements in E. coli and P. pastoris . The reporter protein expressed by the iTSAD with glucose was 4.2-fold higher than that expressed by the strong methanol-inducible promoter P AOX1 with methanol ( 54 ). This represents a useful strategy that far exceeds the amplification of the P AOX1 variant library (up to 1.6-fold) ( 42 , 43 ).…”

“…The plasmids BB1_12, BB1_23, BB1_34, BB2_AB, BB2_BC and BB3eN_14 for GoldenPiCS cloning were purchased from Addgene (#98549). The plasmids pP-P AOX1 G (pP-GFP), pPcAG, pPlacOncAG ( n = 1–9), pParaIcAG, pGP GAP LacIP1AD, pGP GAP LacIX1AD, pGP GAP LacIM1AD, pGP GAP AraCM1AD, pZ_BCGN and pK_sAR were constructed and stored in our laboratory ( 28 , 29 , 32 , 43 , 54 ). Escherichia coli was incubated at 37°C in LLB medium (0.5% yeast extract, 1% tryptone and 0.5% NaCl).…”

“…The plasmids used in this study were constructed by seamless cloning and/or Gibson assembly (ClonExpress™ II one-step cloning kit, VazymeBiotech Co., Ltd, China). Molecular genetic analyses of E. coli and P. pastoris were performed as previously described ( 29 , 54 ). The construction details of the plasmids and strains are described in supplementary Materials and methods.…”