Production of p-hydroxycinnamic acid from glucose in Saccharomyces cerevisiae and Escherichia coli by expression of heterologous genes from plants and fungi.
Biological production of p-hydroxycinnamic acid (pHCA) from glucose can be achieved via deamination of the aromatic amino acids l-tyrosine or l-phenylalanine. Deamination of l-phenylalanine produces trans-cinnamic acid (CA) which is further hydroxylated in the para position to produce pHCA. However, when tyrosine is used as the substrate, trans-pHCA is produced in one step. This reaction is accomplished by phenylalanine ammonia-lyase (PAL)/tyrosine ammonia-lyase (TAL). Various bacteria and eukaryotic microorganisms were screened for their ability to produce a PAL/TAL enzyme with high TAL activity. Cell-free extracts of the yeast Rhodotorula glutinis possessed the highest level of TAL activity (0.0143U/mg protein) and the lowest PAL/TAL ratio (1.68) amongst species examined. The gene for this enzyme was cloned and expressed in Escherichia coli and the kinetics of the purified PAL/TAL determined. The recombinant PAL/TAL possessed characteristics similar to those of the wild-type enzyme. Functional expression of R. glutinis PAL/TAL enzyme in Saccharomyces cerevisiae cells containing the plant C4H P-450 and P-450 reductase enzymes from Helianthus tuberosus allowed conversion of glucose to pHCA. Addition of l-phenylalanine to these cultures increased pHCA production confirming its production via the PAL route. When R. glutinis PAL/TAL was synthesized in an E. colil-phenylalanine producing strain (ATCC 31882) and grown on glucose, pHCA was formed in the absence of the Cytochrome P-450 and the P-450 reductase enzymes underlining its production via the TAL route without CA intermediacy.
Vannelli
Todd
T
Cornell University/Ludwig Institute for Cancer Research, Ithaca, NY 14853, USA.
Wei Qi
Wei
W
Sweigard
James
J
Gatenby
Anthony A
AA
Sariaslani
F Sima
FS
D003373:Coumaric Acids; D004926:Escherichia coli / Q000502:physiology*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D024861:Genetic Enhancement / Q000379:methods; D005947:Glucose / Q000378:metabolism*; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D011422:Propionates; D015202:Protein Engineering / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D014162:Transfection / Q000379:methods
D003373:Coumaric Acids; D029968:Escherichia coli Proteins; D005656:Fungal Proteins; D010940:Plant Proteins; D011422:Propionates; D029701:Saccharomyces cerevisiae Proteins; C495469:p-coumaric acid; D005947:Glucose