Oksik Choi

Artificial biosynthesis of phenylpropanoic acids in a tyrosine overproducing Escherichia coli strain

BackgroundThe phenylpropanoid metabolites are an extremely diverse group of natural products biosynthesized by plants, fungi, and bacteria. Although these compounds are widely used in human health care and nutrition services, their availability is limited by regional variations, and isolation of single compounds from plants is often difficult. Recent advances in synthetic biology and metabolic engineering have enabled artificial production of plant secondary metabolites in microorganisms.ResultsWe develop an Escherichia coli system containing an artificial biosynthetic pathway that yields phenylpropanoic acids, such as 4-coumaric acid, caffeic acid, and ferulic acid, from simple carbon sources. These artificial biosynthetic pathways contained a codon-optimized tal gene that improved the productivity of 4-coumaric acid and ferulic acid, but not caffeic acid in a minimal salt medium. These heterologous pathways extended in E. coli that had biosynthesis machinery overproducing tyrosine. Finally, the titers of 4-coumaric acid, caffeic acid, and ferulic acid reached 974 mg/L, 150 mg/L, and 196 mg/L, respectively, in shake flasks after 36-hour cultivation.ConclusionsWe achieved one gram per liter scale production of 4-coumaric acid. In addition, maximum titers of 150 mg/L of caffeic acid and 196 mg/L of ferulic acid were achieved. Phenylpropanoic acids, such as 4-coumaric acid, caffeic acid, and ferulic acid, have a great potential for pharmaceutical applications and food ingredients. This work forms a basis for further improvement in production and opens the possibility of microbial synthesis of more complex plant secondary metabolites derived from phenylpropanoic acids.

Rational Biosynthetic Engineering for Optimization of Geldanamycin Analogues

Tailor made: We report the rational biosynthesis of C15 hydroxylated non‐quinone geldanamycin analogues by site‐directed mutagenesis of the geldanamycin polyketide synthase (PKS), together with a combination of post‐PKS tailoring genes. Rational biosynthetic engineering allowed the generation of geldanamycin derivatives, such as DHQ3 illustrated in the figure, which had superior pharmacological properties in comparison to the parent compound.

Biosynthesis of methylated resveratrol analogs through the construction of an artificial biosynthetic pathway in E. coli

BackgroundMethylated resveratrol analogs show similar biological activities that are comparable with those of the resveratrol. However, the methylated resveratrol analogs exhibit better bioavailability as they are more easily transported into the cell and more resistant to degradation. Although these compounds are widely used in human health care and in industrial materials, at present they are mainly obtained by extraction from raw plant sources. Accordingly their production can suffer from a variety of economic problems, including low levels of productivity and/or heterogeneous quality. On this backdrop, large-scale production of plant metabolites via microbial approaches is a promising alternative to chemical synthesis and extraction from plant sources.ResultsAn Escherichia coli system containing an artificial biosynthetic pathway that produces methylated resveratrol analogues, such as pinostilbene (3,4’-dihydroxy-5-methoxystilbene), 3,5-dihydroxy-4’-methoxystilbene, 3,4’-dimethoxy-5-hydroxystilbene, and 3,5,4’-trimethoxystilbene, from simple carbon sources is developed. These artificial biosynthetic pathways contain a series of codon-optimized O-methyltransferase genes from sorghum in addition to the resveratrol biosynthetic genes. The E. coli cells that harbor pET-opTLO1S or pET-opTLO3S produce the one-methyl resveratrol analogues of 3,5-dihydroxy-4’-methoxystilbene and pinostilbene, respectively. Furthermore, the E. coli cells that harbor pET-opTLO13S produce 3,5-dihydroxy-4’-methoxystilbene, bis-methyl resveratrol (3,4’-dimethoxy-5-hydroxystilbene), and tri-methyl resveratrol (3,5,4’-trimethoxystilbene).ConclusionsOur strategy demonstrates the first harness microorganisms for de novo synthesis of methylated resveratrol analogs used a single vector system joined with resveratrol biosynthetic genes and sorghum two resveratrol O-methyltransferase genes. Thus, this is also the first report on the production of the methylated resveratrol compounds bis-methyl and tri-methyl resveratrol (3,4’-dimethoxy-5-hydroxystilbene and 3,5,4’-trimethoxystilbene) in the E. coli culture. Thus, the production of the methylated resveratrol compounds was performed on the simple E. coli medium without precursor feeding in the culture.

6-alkylsalicylic acid analogues inhibit in vitro ATPase activity of heat shock protein 90

The molecular chaperone heat shock protein 90 (Hsp90) is responsible for maintaining the correct folding and stability of many signaling proteins. It is a promising target of cancer therapeutics and several other diseases, including neurodegenerative disease, nerve injuries, inflammation, and infection. In an effort to identify new Hsp90 inhibitors from natural sources using an in vitro ATPase inhibition assay, two 6-alkylsalicylic acid analogues, salaceyin A and B were identified from the culture extract of Streptomyces. Salaceyin A and B exhibited moderate ATPase inhibitory activities with IC50 values of 68.3 and 65.2 μM, respectively. Binding of salaceyins to human Hsp90α was examined by competition binding experiments with ATP-Sepharose beads. However, the compounds exhibited no degradation activity of Hsp90 client proteins, Her2, c-Raf, or Akt.