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

Abstract

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^{- 1} after 170 h of methanol feeding (196 h fermentation), representing the highest reported titer of 15α-OH-DE to date. The above results highlight the potential of developing P. pastoris-based biotransformation systems for the efficient production of key intermediates of steroid pharmaceuticals and other high-value fine chemicals.

Keywords: Pichia pastoris; D-ethylgonendione; Fed-batch fermentation; Glucose-6-phosphate dehydrogenase; Intermediate; Steroid 15α-hydroxylase.

Fig. 1

Strategy for construction of the recombinant P. pastoris strain 15,134 with genomic integration of multiple copies of PRH. Generating and characterizing the recombinant strain 15,134 includes five steps. Construction of the recombinant plasmid (Step 1), plasmid linearization using restriction enzyme Sal I (Step 2), yeast cell transformation (Step 3), recombinant strain identification (Step 4), and shaking flask cultivation (Step 5)

Fig. 2

Transcript levels of steroid 15α-hydroxylase gene PRH and steroid transformation of recombinant strain 15,134. (A) RT-qPCR was conducted to analyze the expression level of the 15α-hydroxylase gene PRH compared to the control of β-actin. (B) Chromatography analysis of transformation product in 96 h fermentation broth of strain 15,134. Each experiment was conducted in triplicate

Fig. 3

Schematic view of the methanol metabolism pathway in recombinant strain Z15134 and enhanced NADPH regeneration (F6P, fructose 6-phosphate; DHA, dihydroxyacetone; DHAP, dihydroxyacetone phosphate; G3P, glyceraldehyde-3-phosphate; FBP, fructose 1,6-phosphate; PGI, phosphoglucose isomerase; ZWF1, encoding glucose-6-phosphate dehydrogenase). Highlighted in red depicts production of NADPH through the engineered reaction. Dashed line: multi-step reactions

Fig. 4

Strategy for construction of recombinant strain Z15134 with genomic integration of multiple copies of PRH and ZWF1. The entire process includes: (1) recombinant plasmid construction, (2) plasmid linearization using restriction enzyme Sac I, (3) yeast cell transformation, (4) recombinant strain identification, (5) shake flask cultivation, (6) optimization strategy, and (7) high-cell-density cultivation

Fig. 5

Improvement of DE transformation by regenerating NADPH in recombinant strain Z15134. (A) RT-qPCR was conducted to analyze the transcript levels of steroid 15α-hydroxylase gene PRH and G6PDH gene ZWF1 compared to the control of β-actin. (B) Growth curve of recombinant strain Z15134 and 15,134 during fermentation. (C) The time course of 15α-hydroxylation of DE catalyzed by recombinant strain Z15134 and 15,134 by TLC analysis. (D) The time course of DE transformation by recombinant strain Z15134 and 15,134 as measured HPLC. The data represents the mean ± SD from three data points. Different letters indicate significant differences at P < 0.05 for conversion. Each experiment was conducted in triplicate

Fig. 6

Effect of optimized fermentation parameters on recombinant strain Z15134 growth. (A) Growth curve of strain Z15134 in BMGY broth. (B) Effect of different amounts of methanol addition on strain Z15134 growth during fermentation. (C) Effect of different amounts of DMF addition on strain Z15134 growth during fermentation. The data was derived from the results of three repeated experiments

Fig. 7

Effect of BMMY medium volume on strain Z15134 growth (A), 15α-OH-DE titers and DE conversion (B). The data shown represents the mean ± SD from three data points. Different letters indicate significant differences at P < 0.05 for 15α-OH-DE titers and conversion, respectively

Fig. 8

Optimization of 15α-OH-DE production. Effect of different methanol pre-induction time (A), pH value (B), methanol content (C), substrate concentrations (D), Tween 80 content (E), on 15α-OH-DE titers and DE conversion in fermentation. (F) Effects of 0.06% (v/v) Tween 80 (with or without) on 15α-OH-DE titers and DE conversion during fermentation. The data shown represents the mean ± SD from three data points. Different letters indicate significant differences at P < 0.05 for 15α-OH-DE titers and conversion, respectively

Fig. 9

High-cell-density cultivation of recombinant strain Z15134 in 5-L fermenter. The recombinant strain Z15134 was further evaluated in 5-L high-cell-density cultivation experiments. Every 4 h after cultivation, samples were taken from the fermenter for quantifying cell density. Every 8 h after methanol induction, samples were taken from the fermenter, followed by HPLC assay. (A) Optimized procedure of high-cell-density cultivation. (B) Time courses showing changes in cell growth (OD₆₀₀) and 15α-OH-DE titers during fed-batch fermentation. Mean values and standard errors from triplicates