De novo production of bioactive sesterterpenoid ophiobolins in Saccharomyces cerevisiae cell factories.

BACKGROUND: Sesterterpenoids are rare species among the terpenoids family. Ophiobolins are sesterterpenes with a 5-8-5 tricyclic skeleton. The oxidized ophiobolins exhibit significant cytotoxic activity and potential medicinal value. There is an urgent need for large amounts of ophiobolins supplication for drug development. The synthetic biology approach has been successfully employed in lots of terpene compound production and inspired us to develop a cell factory for ophiobolin biosynthesis. RESULTS: We developed a systematic metabolic engineering strategy to construct an ophiobolin biosynthesis chassis based on Saccharomyces cerevisiae. The whole-cell biotransformation methods were further combined with metabolic engineering to enhance the expression of key ophiobolin biosynthetic genes and improve the supply of precursors and cofactors. A high yield of 5.1 g/L of ophiobolin F was reached using ethanol and fatty acids as substrates. To accumulate oxidized ophiobolins, we optimized the sources and expression conditions for P450-CPR and alleviated the toxicity of bioactive compounds to cells through PDR engineering. We unexpectedly obtained a novel ophiobolin intermediate with potent cytotoxicity, 5-hydroxy-21-formyl-ophiobolin F, and the known bioactive compound ophiobolin U. Finally, we achieved the ophiobolin U titer of 128.9 mg/L. CONCLUSIONS: We established efficient cell factories based on S. cerevisiae, enabling de novo biosynthesis of the ophiobolin skeleton ophiobolin F and oxidized ophiobolins derivatives. This work has filled the gap in the heterologous biosynthesis of sesterterpenoids in S. cerevisiae and provided valuable solutions for new drug development based on sesterterpenoids.

Production of Terpenoids by Synthetic Biology Approaches.

Terpenoids are a large family of natural products with remarkable diverse biological functions, and have a wide range of applications as pharmaceuticals, flavors, pigments, and biofuels. Synthetic biology is presenting possibilities for sustainable and efficient production of high value-added terpenoids in engineered microbial cell factories, using Escherichia coli and Saccharomyces cerevisiae which are identified as well-known industrial workhorses. They also provide a promising alternative to produce non-native terpenes on account of available genetic tools in metabolic engineering and genome editing. In this review, we summarize the recent development in terpenoids production by synthetic biology approaches.

[Whole-cell biotransformation for simultaneous synthesis of L-2-aminobutyric acid and D-gluconic acid in recombinant Escherichia coli].

A whole-cell catalyst using Escherichia coli BL21(DE3) as a host, expressing L- threonine dehydratase from Escherichia coli, and co-expressing leucine dehydrogenase from Bacillus cereus and glucose dehydrogenase from Bacillus subtilis for cofactor regeneration, was constructed and used for one-pot production of L-2-aminobutyric acid (L-ABA) and D- gluconic acid from L-threonine and D-glucose. We used shake-flask culture to study the whole-cell catalytic condition including temperature, pH, proper permeabilization of cells and optimal wet cells amount. Moreover, the whole-cell catalyst was cultured in 5-L fermentor by fed-batch fermentation, and 164 g/L L-threonine and 248 g/L D-glucose were converted to 141.6 g/L L-ABA and 269.4 g/L D-gluconic acid. The whole-cell catalyst is promising to fulfill industrial requirements for L-ABA and D-gluconic acid.