A Novel Semi-biosynthetic Route for Artemisinin Production Using Engineered Substrate-Promiscuous P450_BM3

Abstract

Production of fine heterologus pathways in microbial hosts is frequently hindered by insufficient knowledge of the native metabolic pathway and its cognate enzymes; often the pathway is unresolved and enzymes lack detailed characterization. An alternative paradigm to using native pathways is de novo pathway design using well-characterized, substrate-promiscuous enzymes. We demonstrate this concept using P450 BM3 from Bacillus megaterium. Using a computer model, we illustrate how key P450 BM3 activ site mutations enable binding of non-native substrate amorphadiene, incorporating these mutations into P450 BM3 enabled the selective oxidation of amorphadiene arteminsinic-11s,12-epoxide, at titers of 250 mg L "1 in E. coli. We also demonstrate high-yeilding, selective transformations to dihydroartemisinic acid, the immediate precursor to the high value anti-malarial drug artemisinin.

Genome Sequences of the Biotechnologically Important Bacillus megaterium Strains QM B1551 and DSM319

“…In addition, a cytochrome P450 gene (BMQ_pBM50008) is plasmidborne. The P450 enzymes of B. megaterium have long been used as a model system (15,20,21). There is a gene cluster that appears to be responsible for the biosynthesis of a fatty acid compound (BMQ_pBM50048 to BMQ_pBM50053).…”

Designing de novo: interdisciplinary debates in synthetic biology

“…Nevertheless, it was questionable as to whether the construct was genuinely new, not only because the synthesized genome was functioning in a naturally existing cell, but also because the sequence of the synthetic genome was for the most part a copy of an existing one (Giuliani et al 2011). To mention just one more remarkable achievement of synthetic biology: metabolic pathways have been re-wired in astounding ways, to the extent that the artemisinin project (Dietrich et al 2009) achieved the synthesis of a precursor of the antimalaria drug artemisinin in yeast and Escherichia coli. Acknowledged as a milestone within the field of synthetic biology, this successful project relied to a large extent on tinkering and trial and error rather than rational ''design''.…”

Metabolic Engineering of Microorganisms for the Production of Natural Compounds

“…In another study, the novel artemisinin pathway through artemisinic-11S,12-epoxide and dihydroartemisinic acid was developed (Figure 4). [45] The bifunctional cytochrome P450/NADPH-P450 reductase (P450 BM3 ) from Bacillus megaterium was evolved by mutagenesis to make it selectively oxidize amorphadiene. The potential mutation sites were screened using in silico ROSETTA-based energy minimization.…”