Abstract
BackgroundThe angelic acid moiety represents an essential modification in many biologically active products. These products are commonly known as angelates and several studies have demonstrated their therapeutic benefits, including anti-inflammatory and anti-cancer effects. However, their availability for use in the development of therapeutics is limited due to poor extraction yields. Chemical synthesis has been achieved but its complexity prevents application, therefore microbial production may offer a promising alternative. Here, we engineered the budding yeast Saccharomyces cerevisiae to produce angelyl-CoA, the CoA-activated form of angelic acid.ResultsFor yeast-based production of angelyl-CoA we first expressed genes recently identified in the biosynthetic cluster ssf of Streptomyces sp. SF2575 in S. cerevisiae. Exogenous feeding of propionate and heterologous expression of a propionyl-CoA synthase from Streptomyces sp. were initially employed to increase the intracellular propionyl-CoA level, resulting in production of angelyl-CoA in the order of 5 mg/L. Substituting the Streptomyces sp. propionyl-CoA carboxylase with a carboxylase derived from Streptomyces coelicolor resulted in angelyl-CoA levels up to 6.4 mg/L. In vivo analysis allowed identification of important intermediates in the pathway, including methyl-malonyl-CoA and 3-hydroxyl-2-methyl-butyryl-CoA. Furthermore, methyl-malonate supplementation and expression of matB CoA ligase from S. coelicolor allowed for methyl-malonyl-CoA synthesis and supported, together with parts of the ssf pathway, angelyl-CoA titres of approximately 1.5 mg/L. Finally, feeding of angelic acid to yeasts expressing acyl-CoA ligases from plant species led to angelyl-CoA production rates of approximately 40 mg/L.ConclusionsOur results demonstrate the biosynthesis of angelyl-CoA in yeast from exogenously supplied carboxylic acid precursors. This is the first report on the activity of the ssf genes. We envision that our approach will provide a platform for a more sustainable production of the pharmaceutically important compound class of angelates.
Highlights
The angelic acid moiety represents an essential modification in many biologically active products
Angelyl‐CoA production in yeast starting from propionate Based on the hypotheses of Pickens et al [6] we assembled the pathway to AN-CoA biosynthesis in baker’s yeast
In preliminary experiments we found that two of the CoA-ligases tested showed the ability to accept angelic acid as a substrate for thio-esterification: carboxyl CoA ligase 4 from Humulus lupulus (HlCCL4) and predicted acyl-activating enzyme 6 from Solanum tuberosum (StCCL)
Summary
The angelic acid moiety represents an essential modification in many biologically active products. These products are commonly known as angelates and several studies have demonstrated their therapeutic benefits, including anti-inflammatory and anti-cancer effects. The best-known and most studied example is certainly represented by ingenol-3-angelate ( known as ingenol-mebutate), a topical chemotherapeutic recently approved by the FDA for the treatment of actinic keratosis, a pre-cancerous skin condition. This ester of the diterpenoid ingenol and angelic acid is derived from. Trehangelins are trehalose angelates produced by the endophytic actinomycete Polymorphospora rubra K07-0510, displaying potent inhibitory activity against hemolysis of red blood cells [8]
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