Fusicocca-2,10(14)-diene (FCdiene) is a tricyclic diterpene which has many pharmaceutical applications, for example, it is a precursor for different anticancer drugs, including fusicoccin A. Chemical synthesis of this diterpene is not economical as it requires 14 steps with several stereospecific reactions. FCdiene is naturally produced at low titers in phytopathogenic filamentous fungi. However, production of FCdiene can be achieved via expression of fusicoccadiene synthase in yeast. The objective of this study is to increase FCdiene production by optimizing the yeast fermentation process. Our preliminary fermentations showed influences of carbon sources, buffer agents, and oxygen supply on FCdiene production. Buffer agents as well as oxygen supply were investigated in detail at 0.2 and 1.8 L cultivation volumes. Using glucose as the carbon source, FCdiene concentrations were increased to 240 mgFCdiene/L by optimizing pH and oxygen conditions. In situ extraction and adsorption techniques were examined at the 0.2 L scale to determine if these techniques could improve FCdiene yields. Different adsorbents and solvents were tested with in situ product recovery and 4-fold increases in FCdiene productivity could be shown. The results generated in this work provide a proof-of-concept for the fermentative production of FCdiene from S. cerevisiae as a practical alternative to chemical synthesis.

Die fermentative Herstellung von Biotensiden wird meist durch die exzessive Bildung von stabilem Schaum erschwert. Am Beispiel von Monorhamnolipiden werden Vorschläge sowohl zur stabilen Aufrechterhaltung von begasten und gerührten Fermentationen wie auch zur Isolierung dieser Biotenside im größeren Maßstab gegeben, da diese kommerziell nur in sehr kleinen Mengen erhältlich sind. Ein besonderer Fokus liegt dabei auf der Verwendung nachwachsender Rohstoffe.

Fusicocca-2,10(14)-diene (FCdiene) is a diterpene which is interesting as a precursor of the anti-cancer drug fusicoccin A and therefore for pharmaceutical applications. Production of FCdiene using a genetically modified Saccharomyces cerevisiae has been previously demonstrated with batch cultivations with a product concentration up to 10 mg/L. However, it is widely known that fed-batch processes can significantly improve product titer in yeast fermentations. This study focuses on the establishment of fed-batch fermentation for FCdiene production because fed-batch cultivations using FeedBeads® indicated that limiting glucose supply could increase yields of biomass (1.07 gCDW/gGlucose instead of 0.20 gCDW/gGlucose) and FCdiene (21.54 mgFCdiene/gGlucose instead of 9.74 mgFCdiene/gGlucose) in shake flask scale and may have implications for larger scale processes. We implemented a new exponential glucose feed profile in a 1.8 L stirred tank reactor. This reduced overfeeding and the consequent, ethanol production. As a result improvements in cell concentrations up to 246% could be achieved and FCdiene yield increased up to 2.8X in the first 28 h. FCdiene concentration reached 161 mg/L and 320 mg/L at 44 h. Fed-batch and batch mode were combined to examine dynamics of bi-modal cultivation where a fed-batch phase was used for biomass production and a batch phase used for FCdiene production potentially supported by ethanol consumption as reported on production of betulinic acid. The present study highlights the potential of process development improvements which increase high-value heterologous diterpene yields from S. cerevisiae.Electronic supplementary materialThe online version of this article (10.1186/s13568-018-0662-8) contains supplementary material, which is available to authorized users.

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