Abstract
Background21-deoxycortisol (21-DF) is the key intermediate to manufacture pharmaceutical glucocorticoids. Recently, a Japan patent has realized 21-DF production via biotransformation of 17-hydroxyprogesterone (17-OHP) by purified steroid 11β-hydroxylase CYP11B1. Due to the less costs on enzyme isolation, purification and stabilization as well as cofactors supply, whole-cell should be preferentially employed as the biocatalyst over purified enzymes. No reports as so far have demonstrated a whole-cell system to produce 21-DF. Therefore, this study aimed to establish a whole-cell biocatalyst to achieve 21-DF transformation with high catalytic activity and product specificity.ResultsIn this study, Escherichia coli MG1655(DE3), which exhibited the highest substrate transportation rate among other tested chassises, was employed as the host cell to construct our biocatalyst by co-expressing heterologous CYP11B1 together with bovine adrenodoxin and adrenodoxin reductase. Through screening CYP11B1s (with mutagenesis at N-terminus) from nine sources, Homo sapiens CYP11B1 mutant (G25R/G46R/L52 M) achieved the highest 21-DF transformation rate at 10.6 mg/L/h. Furthermore, an optimal substrate concentration of 2.4 g/L and a corresponding transformation rate of 16.2 mg/L/h were obtained by screening substrate concentrations. To be noted, based on structural analysis of the enzyme-substrate complex, two types of site-directed mutations were designed to adjust the relative position between the catalytic active site heme and the substrate. Accordingly, 1.96-fold enhancement on 21-DF transformation rate (to 47.9 mg/L/h) and 2.78-fold improvement on product/by-product ratio (from 0.36 to 1.36) were achieved by the combined mutagenesis of F381A/L382S/I488L. Eventually, after 38-h biotransformation in shake-flask, the production of 21-DF reached to 1.42 g/L with a yield of 52.7%, which is the highest 21-DF production as known.ConclusionsHeterologous CYP11B1 was manipulated to construct E. coli biocatalyst converting 17-OHP to 21-DF. Through the strategies in terms of (1) screening enzymes (with N-terminal mutagenesis) sources, (2) optimizing substrate concentration, and most importantly (3) rational design novel mutants aided by structural analysis, the 21-DF transformation rate was stepwise improved by 19.5-fold along with 4.67-fold increase on the product/byproduct ratio. Eventually, the highest 21-DF reported production was achieved in shake-flask after 38-h biotransformation. This study highlighted above described methods to obtain a high efficient and specific biocatalyst for the desired biotransformation.
Highlights
Manufacturing steroid compounds with therapeutic usage and commercial value is a successful example to apply biocatalysts in large-scale industrial processes [1,2,3,4]
Through measuring the extracellular 17-OHP concentration at each time point, it was demonstrated that 17-OHP concentration was decreased significantly only in the media culturing Y. lipolytica ATCC201249 or E. coli MG1655(DE3) (Fig. 2a)
As previous study demonstrated that endogenous protein GCY1 could convert 17-OHP into 17α,20α-dihydroxypregn-4ene-3-one in S. cerevisiae [45], and E. coli protein Akr is the isoenzyme of GCY1
Summary
Manufacturing steroid compounds with therapeutic usage and commercial value is a successful example to apply biocatalysts in large-scale industrial processes [1,2,3,4]. 21-deoxycortisol (21-DF) is the key intermediate for pharmaceutical glucocorticoids with anti-inflammatory effect. It can be used as the substrate to chemosynthesize cortisol via a matured and environmental friendly process [7]. The accessibility of 21-DF in bulks remains a great challenge due to catalytic selectivity for 11β-hydroxylation, which has restricted the large-scale application of 21-DF in associated steroid industry. This study aims to establish a whole-cell biocatalyst of CYP11B1 to achieve the desired conversion (Fig. 1) with high catalytic activity and product specificity
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