Author Contributions RAL, AHN, and SEB contributed equally to this publication. RAL, SEB, ZC, WRPN, and DB conceived of the idea and initial steps for designing protein switches from de novo designed helical bundles. RAL and DB developed the thermodynamic model and the code upon which it works. RAL, SEB, and WRPN designed and biophysically characterized LOCKR scaffolds and BimLOCKR. RAL performed mutagenesis and Bio-layer interferometry experiments. SB characterized Bim interactions to Bcl2 homologs and aided experimental design. RAL performed design calculations for orthogonal LOCKR designs using code from SEB and VKM. AHN and RAL conceived of caging cODC. RAL performed design calculations to cage cODC and tune degronLOCKR. AHN conceived of and contributed to all experiments with degronLOCKR. THN performed dynamic measurement of degronLOCKR. AMW tested degronLOCKR in HEK293T cells. MJL, SEB, and RAL performed design calculations for asymmetric LOCKR. GD performed experiments with degronLOCKR and dCas9. GD contributed to plasmid and strain construction. RAL, SEB, and MJL conceived of caging sequences to control subcellular location and RAL performed design calculations for nesLOCKR. JAS and AHN performed all experiments for nesLOCKR. RAL, SEB, AHN, HE-S, and DB wrote the manuscript, all authors edited and approved.

No abstract

No abstract

Eukaryotic cells compartmentalize metabolic pathways in organelles to achieve optimal reaction conditions and avoid crosstalk with other factors in the cytosol. Increasingly, engineers are researching ways in which synthetic compartmentalization could be used to address challenges in metabolic engineering. Here, we identified that norcoclaurine synthase (NCS), the enzyme which catalyzes the first committed reaction in benzylisoquinoline alkaloid (BIA) biosynthesis, is toxic when expressed cytosolically in Saccharomyces cerevisiae and, consequently, restricts (S)-reticuline production. We developed a compartmentalization strategy that alleviates NCS toxicity while promoting increased (S)-reticuline titer, achieved through efficient targeting of toxic NCS to the peroxisome while, crucially, taking advantage of the free flow of metabolite substrates and product across the peroxisome membrane. We identified that peroxisome protein capacity in S. cerevisiae becomes a limiting factor for further improvement of BIA production and demonstrate that expression of engineered transcription factors can mimic the oleate response for larger peroxisomes, further increasing BIA titer without the requirement for peroxisome induction with fatty acids. This work specifically addresses the challenges associated with toxic NCS expression and, more broadly, highlights the potential for engineering organelles with desired characteristics for metabolic engineering.

No abstract