Yeast artificial chromosomes employed for random assembly of biosynthetic pathways and production of diverse compounds in Saccharomyces cerevisiae.
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.
Naesby
Michael
M
Evolva A/S, Bülowsvej 25, 1870 Frederiksberg C, Denmark. tanjat@evolva.com.
Nielsen
Søren Vs
SV
Nielsen
Curt Af
CA
Green
Trine
T
Tange
Thomas O
TO
Simón
Ernesto
E
Knechtle
Philipp
P
Hansson
Anders
A
Schwab
Markus S
MS
Titiz
Olca
O
Folly
Christophe
C
Archila
Roberto E
RE
Maver
Milena
M
van Sint Fiet
Stephan
S
Boussemghoune
Thiamo
T
Janes
Michael
M
Kumar
A S Sathish
AS
Sonkar
Shailendra P
SP
Mitra
Partha P
PP
Benjamin
V Ajai Kumar
VA
Korrapati
Nimitha
N
Suman
Inala
I
Hansen
Esben H
EH
Thybo
Tanja
T
Goldsmith
Neil
N
Sorensen
Alexandra Santana
AS
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