title: Optimization of yeast-based production of medicinal protoberberine alkaloids.
abstract: BACKGROUND
  Protoberberine alkaloids are bioactive molecules abundant in plant preparations for traditional medicines. Yeast engineered to express biosynthetic pathways for fermentative production of these compounds will further enable investigation of the medicinal properties of these molecules and development of alkaloid-based drugs with improved efficacy and safety. Here, we describe the optimization of a biosynthetic pathway in Saccharomyces cerevisiae for conversion of rac-norlaudanosoline to the protoberberine alkaloid (S)-canadine.
  
  
  RESULTS
  This yeast strain is engineered to express seven heterologous enzymes, resulting in protoberberine alkaloid production from a simple benzylisoquinoline alkaloid precursor. The seven enzymes include three membrane-bound enzymes: the flavin-dependent oxidase berberine bridge enzyme, the cytochrome P450 canadine synthase, and a cytochrome P450 reductase. A number of strategies were implemented to improve flux through the pathway, including enzyme variant screening, genetic copy number variation, and culture optimization, that led to an over 70-fold increase in canadine titer up to 1.8 mg/L. Increased canadine titers enable extension of the pathway to produce berberine, a major constituent of several traditional medicines, for the first time in a microbial host. We also demonstrate that this strain is viable at pilot scale.
  
  
  CONCLUSIONS
  By applying metabolic engineering and synthetic biology strategies for increased conversion of simple benzylisoquinoline alkaloids to complex protoberberine alkaloids, this work will facilitate chemoenzymatic synthesis or de novo biosynthesis of these and other high-value compounds using a microbial cell factory.
authors.affiliation: Department of Chemistry, Stanford University, 443 Via Ortega, MC 4245, Stanford, CA, 94305, USA. sgalanie@stanford.edu.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Galanie
authors.affiliation: Department of Bioengineering, Stanford University, 443 Via Ortega, MC 4245, Stanford, CA, 94305, USA. csmolke@stanford.edu.
authors.forename: Christina D
authors.initials: CD
authors.lastname: Smolke
mesh_terms: D061249:Batch Cell Culture Techniques; D001599:Berberine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D001600:Berberine Alkaloids / Q000737:chemistry / Q000378:metabolism*; D019149:Bioreactors; D053898:Biosynthetic Pathways; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D005285:Fermentation; D018628:Gene Dosage; D060847:Metabolic Engineering / Q000379:methods*; D010865:Pilot Projects; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D013765:Tetrahydropapaveroline / Q000737:chemistry / Q000378:metabolism
chemical_list: D001600:Berberine Alkaloids; D001599:Berberine; C009090:protoberberine; D013765:Tetrahydropapaveroline; D003577:Cytochrome P-450 Enzyme System; C004645:canadine