Microbial Synthesis of the Forskolin Precursor Manoyl Oxide in an Enantiomerically Pure Form

Abstract

bForskolin is a promising medicinal compound belonging to a plethora of specialized plant metabolites that constitute a rich source of bioactive high-value compounds. A major obstacle for exploitation of plant metabolites is that they often are produced in small amounts and in plants difficult to cultivate. This may result in insufficient and unreliable supply leading to fluctuating and high sales prices. Hence, substantial efforts and resources have been invested in developing sustainable and reliable supply routes based on microbial cell factories. Here, we report microbial synthesis of (13R)-manoyl oxide, a proposed intermediate in the biosynthesis of forskolin and other medically important labdane-type terpenoids. Process optimization enabled synthesis of enantiomerically pure (13R)-manoyl oxide as the sole metabolite, providing a pure compound in just two steps with a yield of 10 mg/liter. The work presented here demonstrates the value of a standardized bioengineering pipeline and the large potential of microbial cell factories as sources for sustainable synthesis of complex biochemicals.

Reconstructing the chemical diversity of labdane-type diterpene biosynthesis in yeast

“…The titer of manoyl oxide (4) production was 36 mg/L of culture (Table 2), suggesting that in our in vivo system SpMilS can utilize alternative substrates to synthesize different diterpene compounds with comparable efficiency. Manoyl oxide is a central molecule in the biosynthesis of forskolin (Pateraki et al, 2014), a potent stimulator of intracellular cAMP generation (Shoback and Brown, 1984), and the ability to produce it in yeast with SpMilS1 as a surrogate synthase provides an efficient alternative to existing production systems (Nielsen et al, 2014).…”

Uracil Excision for Assembly of Complex Pathways

“…This was recently demonstrated for the uracil excision assembly and engineering of a six-gene biosynthetic pathway for porphyrin production [12] and a seven-gene heterologous pathway for production of a diterpene in Escherichia coli [13]. This type of standardization perfectly fits large collaborative efforts, much like BioBricks in the global iGEM project [14], and reuse of parts also enables better comparison of data.…”

“…The setup will allow the buildup of a library of benchmarked oligonucleotides where differently designed linkers have been validated for correct assembly. Examples of validated linkers are 12,13]. Examples of control elements are constitutive promoters such as P trc followed by randomized ShineDalgarno sequences (for details, see 12] and the phage promoter P T7 followed by the lac operator and a consensus Shine-Dalgarno sequence (for details, see 13].…”

Stable heterologous expression of biologically active terpenoids in green plant cells

“…This level of expression is significantly higher than in Artemisia annua , the natural producer of artemisinin (Ro et al, 2006 ). The commercialization of yeast-based pharmaceuticals such as artemisinin (Paddon et al, 2013 ), the forskolin precursor manoyl oxide (Nielsen et al, 2014 ; Pateraki et al, 2014 ), and some fragrance compounds is imminent, or already available.…”

“…For many bioactive terpenoids of interest to the pharmaceutical, food, or fragrance industries, heterologous expression in engineered organisms is the most commercially sustainable method of production, due to the limited availability from natural sources or the structural complexity of the target compound (Simonsen et al, 2009 ; Daviet and Schalk, 2010 ; Nielsen et al, 2014 ). This however requires extensive knowledge about the native in planta biosynthesis of terpenoids in what are often complex metabolic networks, as opposed to linear biosynthetic pathways leading to a single product.…”