Introduction of the early pathway to taxol biosynthesis in yeast by means of biosynthetic gene cluster construction using SOE-PCR and homologous recombination.
Metabolic engineering of plant natural product pathways in heterologous systems requires the highly concerted action of several biosynthetic genes. Besides the functional heterologous expression of the genes encoding the natural product biosynthetic pathway, often additional extensive modifications in the host primary metabolism are also needed, in order to obtain efficient supply of the required biosynthetic building blocks to support the engineered natural product biosynthesis. Selection markers in heterologous expression systems, like baker's yeast (Saccharomyces cerevisiae), are often limited and the chromosomal insertion prevents later modifications of engineered pathway, e.g. exchange of gene promoters, or the introduction of additional genetic regulatory elements in a timely manner. Thus the construction of biosynthetic gene clusters on episomal expression vectors seems a logical solution for this dilemma. Although manipulation of long DNA fragments still represents a challenge, by using PCR and in vitro homologous recombination, we assembled a biosynthetic gene cluster for the concerted heterologous expression of three important genes for the metabolic engineering of taxoid biosynthesis in yeast.
Dahm
Pia
P
Institute of Organic Chemistry and Biochemistry, Technical University of Darmstadt, Darmstadt, Germany.
Jennewein
Stefan
S
D019677:Artificial Gene Fusion; D017931:DNA Primers / Q000235:genetics; D051231:Farnesyltranstransferase / Q000235:genetics; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D006903:Hydroxymethylglutaryl CoA Reductases / Q000235:genetics; D007535:Isomerases / Q000235:genetics; D005810:Multigene Family / Q000235:genetics*; D017239:Paclitaxel / Q000096:biosynthesis*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D016945:Sulfolobus acidocaldarius / Q000235:genetics
D017931:DNA Primers; D006903:Hydroxymethylglutaryl CoA Reductases; D051231:Farnesyltranstransferase; D007535:Isomerases; C095926:taxa-4(5),11(12)-diene synthase; D017239:Paclitaxel