title: An enzyme-coupled biosensor enables (S)-reticuline production in yeast from glucose.
abstract: Benzylisoquinoline alkaloids (BIAs) are a diverse family of plant-specialized metabolites that include the pharmaceuticals codeine and morphine and their derivatives. Microbial synthesis of BIAs holds promise as an alternative to traditional crop-based manufacturing. Here we demonstrate the production of the key BIA intermediate (S)-reticuline from glucose in Saccharomyces cerevisiae. To aid in this effort, we developed an enzyme-coupled biosensor for the upstream intermediate L-3,4-dihydroxyphenylalanine (L-DOPA). Using this sensor, we identified an active tyrosine hydroxylase and improved its L-DOPA yields by 2.8-fold via PCR mutagenesis. Coexpression of DOPA decarboxylase enabled what is to our knowledge the first demonstration of dopamine production from glucose in yeast, with a 7.4-fold improvement in titer obtained for our best mutant enzyme. We extended this pathway to fully reconstitute the seven-enzyme pathway from L-tyrosine to (S)-reticuline. Future work to improve titers and connect these steps with downstream pathway branches, already demonstrated in S. cerevisiae, will enable low-cost production of many high-value BIAs.
authors.affiliation: Department of Bioengineering, University of California, Berkeley, Berkeley, California, USA.
authors.forename: William C
authors.initials: WC
authors.lastname: DeLoache
authors.affiliation: Department of Bioengineering, University of California, Berkeley, Berkeley, California, USA.
authors.forename: Zachary N
authors.identifier: 0000000205718252
authors.initials: ZN
authors.lastname: Russ
authors.affiliation: 1] Department of Biology, Concordia University, Montréal, Québec, Canada. [2] Centre for Structural and Functional Genomics, Concordia University, Montréal, Québec, Canada.
authors.forename: Lauren
authors.initials: L
authors.lastname: Narcross
authors.affiliation: Department of Bioengineering, University of California, Berkeley, Berkeley, California, USA.
authors.forename: Andrew M
authors.initials: AM
authors.lastname: Gonzales
authors.affiliation: 1] Department of Biology, Concordia University, Montréal, Québec, Canada. [2] Centre for Structural and Functional Genomics, Concordia University, Montréal, Québec, Canada.
authors.forename: Vincent J J
authors.initials: VJ
authors.lastname: Martin
authors.affiliation: Department of Bioengineering, University of California, Berkeley, Berkeley, California, USA.
authors.forename: John E
authors.initials: JE
authors.lastname: Dueber
mesh_terms: D000470:Alkaloids / Q000096:biosynthesis*; D044182:Benzylisoquinolines / Q000378:metabolism*; D015374:Biosensing Techniques; D004295:Dihydroxyphenylalanine / Q000032:analysis* / Q000378:metabolism; D004296:Dopa Decarboxylase / Q000235:genetics / Q000378:metabolism; D004298:Dopamine / Q000096:biosynthesis; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D005947:Glucose / Q000378:metabolism; D060847:Metabolic Engineering; D016297:Mutagenesis, Site-Directed; D009294:Narcotics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D014446:Tyrosine 3-Monooxygenase / Q000235:genetics / Q000378:metabolism
chemical_list: D000470:Alkaloids; D044182:Benzylisoquinolines; D005656:Fungal Proteins; D009294:Narcotics; D004295:Dihydroxyphenylalanine; D014446:Tyrosine 3-Monooxygenase; D004296:Dopa Decarboxylase; D005947:Glucose; D004298:Dopamine; C003298:reticuline