title: Yeast artificial chromosomes employed for random assembly of biosynthetic pathways and production of diverse compounds in Saccharomyces cerevisiae.
abstract: BACKGROUND
  Natural products are an important source of drugs and other commercially interesting compounds, however their isolation and production is often difficult. Metabolic engineering, mainly in bacteria and yeast, has sought to circumvent some of the associated problems but also this approach is impeded by technical limitations. Here we describe a novel strategy for production of diverse natural products, comprising the expression of an unprecedented large number of biosynthetic genes in a heterologous host.
  
  
  RESULTS
  As an example, genes from different sources, representing enzymes of a seven step flavonoid pathway, were individually cloned into yeast expression cassettes, which were then randomly combined on Yeast Artificial Chromosomes and used, in a single transformation of yeast, to create a variety of flavonoid producing pathways. Randomly picked clones were analysed, and approximately half of them showed production of the flavanone naringenin, and a third of them produced the flavonol kaempferol in various amounts. This reflected the assembly of 5-7 step multi-species pathways converting the yeast metabolites phenylalanine and/or tyrosine into flavonoids, normally only produced by plants. Other flavonoids were also produced that were either direct intermediates or derivatives thereof. Feeding natural and unnatural, halogenated precursors to these recombinant clones demonstrated the potential to further diversify the type of molecules that can be produced with this technology.
  
  
  CONCLUSIONS
  The technology has many potential uses but is particularly suited for generating high numbers of structurally diverse compounds, some of which may not be amenable to chemical synthesis, thus greatly facilitating access to a huge chemical space in the search for new commercially interesting compounds.
authors.affiliation: Evolva A/S, Bülowsvej 25, 1870 Frederiksberg C, Denmark. tanjat@evolva.com.
authors.forename: Michael
authors.initials: M
authors.lastname: Naesby
authors.forename: Søren Vs
authors.initials: SV
authors.lastname: Nielsen
authors.forename: Curt Af
authors.initials: CA
authors.lastname: Nielsen
authors.forename: Trine
authors.initials: T
authors.lastname: Green
authors.forename: Thomas O
authors.initials: TO
authors.lastname: Tange
authors.forename: Ernesto
authors.initials: E
authors.lastname: Simón
authors.forename: Philipp
authors.initials: P
authors.lastname: Knechtle
authors.forename: Anders
authors.initials: A
authors.lastname: Hansson
authors.forename: Markus S
authors.initials: MS
authors.lastname: Schwab
authors.forename: Olca
authors.initials: O
authors.lastname: Titiz
authors.forename: Christophe
authors.initials: C
authors.lastname: Folly
authors.forename: Roberto E
authors.initials: RE
authors.lastname: Archila
authors.forename: Milena
authors.initials: M
authors.lastname: Maver
authors.forename: Stephan
authors.initials: S
authors.lastname: van Sint Fiet
authors.forename: Thiamo
authors.initials: T
authors.lastname: Boussemghoune
authors.forename: Michael
authors.initials: M
authors.lastname: Janes
authors.forename: A S Sathish
authors.initials: AS
authors.lastname: Kumar
authors.forename: Shailendra P
authors.initials: SP
authors.lastname: Sonkar
authors.forename: Partha P
authors.initials: PP
authors.lastname: Mitra
authors.forename: V Ajai Kumar
authors.initials: VA
authors.lastname: Benjamin
authors.forename: Nimitha
authors.initials: N
authors.lastname: Korrapati
authors.forename: Inala
authors.initials: I
authors.lastname: Suman
authors.forename: Esben H
authors.initials: EH
authors.lastname: Hansen
authors.forename: Tanja
authors.initials: T
authors.lastname: Thybo
authors.forename: Neil
authors.initials: N
authors.lastname: Goldsmith
authors.forename: Alexandra Santana
authors.initials: AS
authors.lastname: Sorensen
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