Total biosynthesis of hydrocortisone from a simple carbon source in yeast

Abstract

We report on the production of hydrocortisone, the major adrenal glucocorticoid of mammals and an important intermediate of steroidal drug synthesis, from a simple carbon source by recombinant Saccharomyces cerevisiae strains. An artificial and fully self-sufficient biosynthetic pathway involving 13 engineered genes was assembled and expressed in a single yeast strain. Endogenous sterol biosynthesis was rerouted to produce compatible sterols to serve as substrates for the heterologous part of the pathway. Biosynthesis involves eight mammalian proteins (mature forms of CYP11A1, adrenodoxin (ADX), and adrenodoxin reductase (ADR); mitochondrial forms of ADX and CYP11B1; 3beta-HSD, CYP17A1, and CYP21A1). Optimization involved modulating the two mitochondrial systems and disrupting of unwanted side reactions associated with ATF2, GCY1, and YPR1 gene products. Hydrocortisone was the major steroid produced. This work demonstrates the feasibility of transfering a complex biosynthetic pathway from higher eukaryotes into microorganisms.

Increased TCA cycle activity and reduced oxygen consumption during cytochrome P450‐dependent biotransformation in fission yeast

“…The maximal conversion rate of progesterone is attained during the first 24 h ( Figure 1C) and was determined to be 21 ± 1 µM/day [or 1.26 ± 0.06 µmol/g (CWW)/day], while for 17α-hydroxyprogesterone the rate was 259 ± 9 µM/day [or 15.6 ± 0.5 µmol/g (CWW)/d], which is one order of magnitude higher than for the former substrate. Biotransformations with recombinant baker's yeast expressing bovine CYP21 reported previously (Sakaki et al, 1990(Sakaki et al, , 1991Szczebara et al, 2003) might have had significantly lower steroid conversion rates, but the experimental setup was not entirely comparable. Nevertheless, the strong steroid hydroxylation activity of fission yeast strain CAD18 demonstrates its usefulness as a eukaryotic model for P450-driven biocatalysis.…”

“…However, a large number of reactions were developed at the laboratory scale, using a variety of host organisms (e.g. Dragan et al, 2005;Munzer et al, 2005;Nthangeni et al, 2004), with the total synthesis of hydrocortisone from a simple carbon source by a recombinant Saccharomyces cerevisiae strain being a major milestone (Szczebara et al, 2003). The biotechnological prospects of P450-dependent biotransformations are, among other topics (such as enzyme activity and stability), closely related to the questions of co-factor regeneration and oxygen consumption in whole cells, which we address in this study.…”

Reconstitution of a 10-gene pathway for synthesis of the plant alkaloid dihydrosanguinarine in Saccharomyces cerevisiae

“…Cytochrome P450s are required for the synthesis of a wide range of plant natural products and the efficient recombinant expression of this class of enzyme can be difficult. Of all pathways reconstituted in microbes thus far, only those of the mammalian hydrocortisone 49 and plant dihydrosanguinarine pathways require the heterologous expression of four cytochrome P450s. The dihydrosanguinarine pathway described in this work represents the most complex plant alkaloid biosynthetic pathway ever reconstituted in yeast and provides a glimpse into the potential of engineering microbes for the synthesis of ever more complex plant natural products.…”

Chémobiologie à l’happy hour

“…La levure est éga-lement de plus en plus utilisée comme « usine » de production de molécules thérapeutiques, en particulier parce qu'elle appartient à la catégorie des organismes dits GRAS (generally recognized as safe). L'exemple le plus spectaculaire est la construction d'une souche de S. cerevisiae produisant, à partir d'un substrat peu onéreux, des taux élevés d'hydrocortisone, un stéroïde majeur pour l'industrie pharmacologique et dont la synthèse était auparavant à la fois longue, complexe et très coûteuse [7]. En plus de ces situations dans lesquelles elle est employée comme outil, la levure est également très utilisée pour modéliser différentes maladies humaines [8].…”