Application of rational enzyme engineering in a new route to etonogestrel and levonorgestrel: carbonyl reductase bioreduction of ethyl secodione

Abstract

Women in developing countries still face enormous challenges when accessing reproductive health care. Access to voluntary family planning empowers women allowing them to complete their education and join the paid workforce. This effectively helps to end poverty, hunger and promotes good health for all. According to the United Nations (UN) organization, in 2022, an estimated 257 million women still lacked access to safe and effective family planning methods globally. One of the main barriers is the associated cost of modern contraceptive methods. Funded by the Bill & Melinda Gates Foundation, Almac Group worked on the development of a novel biocatalytic route to etonogestrel and levonorgestrel, two modern contraceptive APIs, with the goal of substantially decreasing the cost of production and so enabling their use in developing nations. This present work combines the selection and engineering of a carbonyl reductase (CRED) enzyme from Almac’s selectAZyme™ panel, with process development, to enable efficient and economically viable bioreduction of ethyl secodione to (13R,17S)-secol, the key chirality introducing intermediate en route to etonogestrel and levonorgestrel API. CRED library screening returned a good hit with an Almac CRED from Bacillus weidmannii, which allowed for highly stereoselective bioreduction at low enzyme loading of less than 1% w/w under screening assay conditions. However, the only co-solvent tolerated was DMSO up to ∼30% v/v, and it was impossible to achieve reaction completion with any enzyme loading at substrate titres of 20 g L⁻¹ and above, due to the insolubility of the secodione. This triggered a rapid enzyme engineering program fully based on computational mutant selection. A small panel of 93 CRED mutants was rationally designed to increase the catalytic activity as well as thermal and solvent stability. The best mutant, Mutant-75, enabled a reaction at 45 °C to go to completion at 90 g L⁻¹ substrate titre in a buffer/DMSO/heptane reaction medium fed over 6 h with substrate DMSO stock solution, with a low enzyme loading of 3.5% w/w wrt substrate. In screening assay conditions, Mutant-75 also showed a 2.2-fold activity increase. Our paper shows which computations and rational decisions enabled this outcome.