Functionalized PHB granules provide the basis for the efficient side-chain cleavage of cholesterol and analogs in recombinant Bacillus megaterium

Abstract

BackgroundCholesterol, the precursor of all steroid hormones, is the most abundant steroid in vertebrates and exhibits highly hydrophobic properties, rendering it a difficult substrate for aqueous microbial biotransformations. In the present study, we developed a Bacillus megaterium based whole-cell system that allows the side-chain cleavage of this sterol and investigated the underlying physiological basis of the biocatalysis.ResultsCYP11A1, the side-chain cleaving cytochrome P450, was recombinantly expressed in the Gram-positive soil bacterium B. megaterium combined with the required electron transfer proteins. By applying a mixture of 2-hydroxypropyl-β-cyclodextrin and Quillaja saponin as solubilizing agents, the zoosterols cholesterol and 7-dehydrocholesterol, as well as the phytosterol β-sitosterol could be efficiently converted to pregnenolone or 7-dehydropregnenolone. Fluorescence-microscopic analysis revealed that cholesterol accumulates in the carbon and energy storage-serving poly(3-hydroxybutyrate) (PHB) bodies and that the membrane proteins CYP11A1 and its redox partner adrenodoxin reductase (AdR) are likewise localized to their surrounding phospholipid/protein monolayer. The capacity to store cholesterol was absent in a mutant strain devoid of the PHB-producing polymerase subunit PhaC, resulting in a drastically decreased cholesterol conversion rate, while no effect on the expression of the recombinant proteins could be observed.ConclusionWe established a whole-cell system based on B. megaterium, which enables the conversion of the steroid hormone precursor cholesterol to pregnenolone in substantial quantities. We demonstrate that the microorganism’s PHB granules, aggregates of bioplastic coated with a protein/phospholipid monolayer, are crucial for the high conversion rate by serving as substrate storage. This microbial system opens the way for an industrial conversion of the abundantly available cholesterol to any type of steroid hormones, which represent one of the biggest groups of drugs for the treatment of a wide variety of diseases.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0300-y) contains supplementary material, which is available to authorized users.

Biochemical and structural characterization of CYP109A2, a vitamin D3 25‐hydroxylase from Bacillus megaterium

“…Its use in the pharmaceutical and food industries is preferred due to the ability of this bacterium to grow on a variety of carbon sources and simple media as well as due to its high protein production capacity and plasmid stability . Importantly, as shown by research results considering various P450 enzymes, the B. megaterium offers certain advantages, of being not only a source of new, uncharacterized P450s but also a very good host for P450s and/or their homologous or heterologous redox partners . Use of the whole‐cell expressed P450(s) in a biotransformation process allows for regeneration of the expensive NADPH cofactor that is necessary for P450 activity.…”

CYP109E1 from Bacillus megaterium Acts as a 24‐ and 25‐Hydroxylase for Cholesterol

“…megaterium membrane, Quillaja saponin, with a final concentration of 0.2 %, was used. This surface‐active glycoside was previously demonstrated to strongly increase the cholesterol conversion rate of another P450‐based whole‐cell system in B. megaterium . Interestingly, the CYP109E1 whole‐cell system showed limited activity towards cholesterol, with a conversion ratio of about 20 %, yielding (13.6±2.4) mg L −1 of products after 48 h of reaction (Figure B).…”

“…The reasons for the observed low activity of CYP109E1 towards cholesterol in B. megaterium cells are not yet clear, but might be related to limited cholesterol accessibility for P450 during whole‐cell conversion. It was previously shown that cholesterol tends to accumulate in the poly(3‐hydroxybutyrate) (PHB) bodies in B. megaterium , whereas CYP109E1 is a cytosolic P450 located in the cytoplasm. Moreover, the strain encodes other P450s with unknown natural substrates, which might also bind cholesterol or consume electrons for other reactions.…”

Microbial Biotransformation for the Production of Steroid Medicament

“…The steroid pharmaceuticals are of great importance for their role in the management of human fertility, osteoporosis, menopause and blood pressure regulation [2,3]. Commercially, steroid production represents one of the largest sectors of medical products manufactured by the pharmaceutical industry [4]. Production of AD and androstadienedione (ADD) exceeds 1000 tons per year in the world.…”