Date of Award
1-11-2022
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Applied Science
First Advisor
Qingfang He
Abstract
Caffeic acid and resveratrol are polyphenol compounds produced by plants as secondary metabolites to protect against biotic and abiotic stresses. In the last decades, numerous studies have demonstrated the beneficial effects of these compounds on human health as anticancer, antioxidant, anti-virus and anti-inflammatory agents. In plants, both caffeic acid and resveratrol are synthesized either from phenylalanine or tyrosine. Recently, heterologous production of phenylpropanoids using genetically engineered bacteria is gaining more and more attentions from industry because it can dramatically improve the yield with reduced costs. The major aim of the work is to explore the possibility of using Synechocystis or Escherichia coli for the biosynthesis of caffeic acid and resveratrol. Three plant genes coding for a tyrosine ammonia-lyase (TAL), a coumaroyl-CoA ligase (4CL, codon optimized) and a stilbene synthase (STS, codon optimized), respectively, were introduced into E. coli for de novo biosynthesis of resveratrol. Resveratrol production was detected by HPLC and verified by Mass spectrometry from E.coli culture medium without adding any substrates. The maximum yield of resveratrol reached 115.3 mg/L upon feeding of p-coumaric acid, the immediate precursor of resveratrol. The application of cyanobacteria for phenylpropanoid production offers great potentials. Their photosynthetic capability makes it possible to eliminate carbohydrate feedstock, which further reduces the production costs. A plasmid containing sam8 (coding for TAL) was genetically engineered into Synechocystis sp. PCC 6803 and the strain was found to accumulate no or trace amounts of p-coumaric acid. Upon deletion of slr1573, a putative laccase coding gene, from the genome of the strain, the accumulation of p-coumaric acid was detected using LC/MS and the maximum titer is about 82.6 mg/L. The enzymatic assay of the recombinant Slr1573 protein showed that Slr1573 is indeed a laccase that breaks down polyphenols in Synechocystis cells. The enzyme p-coumarate 3-hydroxylase (C3H) converts p-coumaric acid into caffeic acid. A ref8 gene (coding for C3H) was targeted into the genome of Synechocystis sp. PCC 6803. The successful insertion of ref8 gene was verified by polymerase chain reactions. The protein product of ref8 in Synechocystis sp. PCC 6803 was detected by Western Blot, confirming the expression of the transgene. Upon feeding of p-coumaric acid to the cyanobacterial culture, production of caffeic acid was measured by HPLC and LC/MS. Production improvement was achieved in the Synechocystis strain by optimization of ref8 codons and by deletion of slr1573 encoding the laccase. The caffeic acid production yield was increased from 5.2 to 7.3 mg/L by codon optimization, and the yield was further increased to 72.4 mg/L upon deletion of slr1573. This is the first report so far that has demonstrated the suitability of using cyanobacteria for production of plant phenylpropanoids.
Recommended Citation
Xue, Yong, "Heterologous Production of Plant Secondary Metabolites Resveratrol and Caffeic Acid in E.coli and Synechocystis Sp. PCC 6803" (2022). Theses and Dissertations. 1050.
https://research.ualr.edu/etd/1050
