4-hydroxy-6-methyl-2-pyrone and 2-pyrone

An environmentally sustainable world can be realized by using microorganisms to produce value-added materials from renewable biomass. Triacetic acid lactone (TAL) is a high-value-added compound that is used as a precursor of various organic compounds such as food additives and pharmaceuticals. In this study, we used metabolic engineering to produce TAL from glucose using an oleaginous yeast Yarrowia lipolytica. We first introduced TAL-producing gene 2-pyrone synthase into Y. lipolytica, which enabled TAL production. Next, we increased TAL production by engineering acetyl-CoA and malonyl-CoA biosynthesis pathways by redirecting carbon flux to glycolysis. Finally, we optimized the carbon and nitrogen ratios in the medium, culminating in the production of 4078 mg/L TAL. The strategy presented in this study had the potential to improve the titer and yield of polyketide biosynthesis.

Topics: Gene Deletion; Metabolic Engineering; Yarrowia

Triacetic acid lactone (TAL) (4-hydroxy-6-methyl-2-pyrone) can be upgraded into a variety of higher-value products, and has potential to be developed into a renewable platform chemical through metabolic engineering. We previously developed an endogenous TAL sensor based on the regulatory protein AraC, and applied it to screen 2-pyrone synthase (2-PS) variant libraries in E. coli, resulting in the identification of variants conferring up to 20-fold improved TAL production in liquid culture. In this study, the sensor-reporter system was further optimized and used to further improve TAL production from recombinant E. coli, this time by screening a genomic overexpression library. We identified new and unpredictable gene targets (betT, ompN, and pykA), whose plasmid-based expression improved TAL yield (mg/L/OD

Topics: Biosensing Techniques; Catalysis; Escherichia coli; Gene Deletion; Gene Library; Genes, Reporter; Genome, Bacterial; Industrial Microbiology; Metabolic Engineering; Plasmids; Pyrones; Recombinant Proteins

Type III polyketide synthases (PKSs) contribute to the synthesis of many economically important natural products, which are typically produced by direct extraction from plants or synthesized chemically. For example, humulone and lupulone (Fig. 1a) in hops (Humulus lupulus) account for the characteristic bitter taste of beer and display multiple pharmacological effects. 4-Hydroxy-6-methyl-2-pyrone is a precursor of parasorboside contributing to insect and disease resistance of plant Gerbera hybrida, and was recently demonstrated to be a potential platform chemical. Fig. 1 Examples of phloroglucinols (a) and 2-pyrones (b) synthesized by type III PKS. PIBP phlorisobutyrophenone; PIVP phlorisovalerophenone; TAL 4-hydroxy-6-methyl-2-pyrone (triacetic acid lactone); HIPP 4-hydroxy-6-isopropyl-2-pyrone; HIBP 4-hydroxy-6-isobutyl-2-pyrone. In this study, we achieved simultaneous biosynthesis of phlorisovalerophenone, a key intermediate of humulone biosynthesis and 4-hydroxy-6-isobutyl-2-pyrone in Escherichia coli from glucose. First, we constructed a biosynthetic pathway of isovaleryl-CoA via hydroxy-3-methylglutaryl CoA followed by dehydration, decarboxylation and reduction in E. coli. Subsequently, the type III PKSs valerophenone synthase or chalcone synthase from plants were introduced into the above E. coli strain, to produce phlorisovalerophenone and 4-hydroxy-6-isobutyl-2-pyrone at the highest titers of 6.4 or 66.5 mg/L, respectively.. The report of biosynthesis of phlorisovalerophenone and 4-hydroxy-6-isobutyl-2-pyrone in E. coli adds a new example to the list of valuable compounds synthesized in E. coli from renewable carbon resources by type III PKSs.

Topics: Acyltransferases; Amino Acid Sequence; Biosynthetic Pathways; Cyclohexenes; Escherichia coli; Glucose; Phloroglucinol; Polyketide Synthases; Pyrones; Terpenes

Triacetic acid lactone (TAL) is a potential platform chemical that can be produced in yeast. To evaluate the potential for industrial yeast strains to produce TAL, the g2ps1 gene encoding 2-pyrone synthase was transformed into 13 industrial yeast strains of varied genetic background. TAL production varied 63-fold between strains when compared in batch culture with glucose. Ethanol, acetate, and glycerol were also tested as potential carbon sources. Batch cultures with ethanol medium produced the highest titers. Therefore, fed-batch cultivation with ethanol feed was assayed for TAL production in bioreactors, producing our highest TAL titer, 5.2 g/L. Higher feed rates resulted in a loss of TAL and subsequent production of additional TAL side products. Finally, TAL efflux was measured and TAL is actively exported from S. cerevisiae cells. Percent yield for all strains was low, indicating that further metabolic engineering of the strains is required.

Topics: Acetic Acid; Batch Cell Culture Techniques; Bioreactors; Ethanol; Glucose; Glycerol; Metabolic Engineering; Pyrones; Saccharomyces cerevisiae