New promoters for strain engineering of Penicillium chrysogenum.

Filamentous fungi such as Aspergillus and Penicillium are widely used as hosts for the industrial products such as proteins and secondary metabolites. Although filamentous fungi are versatile in recognizing transcriptional and translational elements present in genes from other filamentous fungal species, only few promoters have been applied and compared in performance so far in Penicillium chrysogenum. Therefore, a set of homologous and heterologous promoters were tested in a reporter system to obtain a set of potential different strengths. Through in vivo homologous recombination in Saccharomyces cerevisiae, twelve Aspergillus niger and P. chrysogenum promoter-reporter pathways were constructed that drive the expression of green fluorescent protein while concurrent expression of the red fluorescent protein was used as an internal standard and placed under control of the PcPAF promoter. The pathways were integrated into the genome of P. chrysogenum and tested using the BioLector system for fermentation. Reporter gene expression was monitored during growth and classified according to promoter strength and expression profile. A set of novel promoters was obtained that can be used to tune the expression of target genes in future strain engineering programs.

Single-step fermentative production of the cholesterol-lowering drug pravastatin via reprogramming of Penicillium chrysogenum.

The cholesterol-lowering blockbuster drug pravastatin can be produced by stereoselective hydroxylation of the natural product compactin. We report here the metabolic reprogramming of the antibiotics producer Penicillium chrysogenum toward an industrial pravastatin production process. Following the successful introduction of the compactin pathway into the ß-lactam-negative P. chrysogenum DS50662, a new cytochrome P450 (P450 or CYP) from Amycolatopsis orientalis (CYP105AS1) was isolated to catalyze the final compactin hydroxylation step. Structural and biochemical characterization of the WT CYP105AS1 reveals that this CYP is an efficient compactin hydroxylase, but that predominant compactin binding modes lead mainly to the ineffective epimer 6-epi-pravastatin. To avoid costly fractionation of the epimer, the enzyme was evolved to invert stereoselectivity, producing the pharmacologically active pravastatin form. Crystal structures of the optimized mutant P450(Prava) bound to compactin demonstrate how the selected combination of mutations enhance compactin binding and enable positioning of the substrate for stereo-specific oxidation. Expression of P450(Prava) fused to a redox partner in compactin-producing P. chrysogenum yielded more than 6 g/L pravastatin at a pilot production scale, providing an effective new route to industrial scale production of an important drug.