Engineering Pichia pastoris for the efficient production of the high-value steroid intermediate 15α-OH-D-ethylgonendione.

15α-OH-D-ethylgonendione (15α-OH-DE) is a key intermediate for the synthesis of steroid drug gestodene, a major component of a new generation of powerful contraceptives. Synthetic access to 15α-OH-DE by chemical means is limited by low titers and generation of toxic byproducts. To develop a sustainable process for 15α-OH-DE production, a whole-cell catalyst was constructed by engineering Pichia pastoris co-overexpressing the PRH gene from filamentous fungus Penicillium raistrickii, which encodes a steroid 15α-hydroxylase capable of selectively 15α-hydroxylating DE, and the glucose-6-phosphate dehydrogenase gene ZWF1 from the baker's yeast for enhanced NADPH production. Shake-flask cultivation was performed to optimize fermentation parameters and assess the potential of the engineered P. pastoris strains for 15α-OH-DE production. Subsequently, production was scaled up using a fed-batch strategy in a 5-L stirred-tank bioreactor, with pure methanol serving as both the carbon source and inducer. This process achieved a product titer of 5.79 g L⁻¹ with DE feeding of 10 g L