title: Rational and combinatorial approaches to engineering styrene production by Saccharomyces cerevisiae.
abstract: BACKGROUND
  Styrene is an important building-block petrochemical and monomer used to produce numerous plastics. Whereas styrene bioproduction by Escherichia coli was previously reported, the long-term potential of this approach will ultimately rely on the use of hosts with improved industrial phenotypes, such as the yeast Saccharomyces cerevisiae.
  
  
  RESULTS
  Classical metabolic evolution was first applied to isolate a mutant capable of phenylalanine over-production to 357 mg/L. Transcription analysis revealed up-regulation of several phenylalanine biosynthesis pathway genes including ARO3, encoding the bottleneck enzyme DAHP synthase. To catalyze the first pathway step, phenylalanine ammonia lyase encoded by PAL2 from A. thaliana was constitutively expressed from a high copy plasmid. The final pathway step, phenylacrylate decarboxylase, was catalyzed by the native FDC1. Expression of FDC1 was naturally induced by trans-cinnamate, the pathway intermediate and its substrate, at levels sufficient for ensuring flux through the pathway. Deletion of ARO10 to eliminate the competing Ehrlich pathway and expression of a feedback-resistant DAHP synthase encoded by ARO4K229L preserved and promoted the endogenous availability precursor phenylalanine, leading to improved pathway flux and styrene production. These systematic improvements allowed styrene titers to ultimately reach 29 mg/L at a glucose yield of 1.44 mg/g, a 60% improvement over the initial strain.
  
  
  CONCLUSIONS
  The potential of S. cerevisiae as a host for renewable styrene production has been demonstrated. Significant strain improvements, however, will ultimately be needed to achieve economical production levels.
authors.forename: Rebekah
authors.initials: R
authors.lastname: McKenna
authors.forename: Brian
authors.initials: B
authors.lastname: Thompson
authors.forename: Shawn
authors.initials: S
authors.lastname: Pugh
authors.affiliation: Chemical Engineering, School for Engineering of Matter, Transport, and Energy, Arizona State University, Tempe 85287-6106, AZ, USA. David.R.Nielsen@asu.edu.
authors.forename: David R
authors.initials: DR
authors.lastname: Nielsen
mesh_terms: D001692:Biological Transport / Q000187:drug effects; D002262:Carboxy-Lyases / Q000378:metabolism; D002934:Cinnamates / Q000378:metabolism; D020650:Combinatorial Chemistry Techniques / Q000379:methods*; D005947:Glucose / Q000378:metabolism; D060847:Metabolic Engineering; D009154:Mutation / Q000235:genetics; D010641:Phenotype; D010649:Phenylalanine / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D020058:Styrene / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects
chemical_list: D002934:Cinnamates; C029010:cinnamic acid; D020058:Styrene; D010649:Phenylalanine; D002262:Carboxy-Lyases; C087194:phenylacrylic acid decarboxylase; D005947:Glucose