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.

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BACKGROUND:Treatments now available for mucopolysaccharidosis I require early detection for optimum therapy. Therefore, we have developed an assay appropriate for newborn screening of the activity of the relevant enzyme, ␣-L-iduronidase.

AT2433, an indolocarbazole antitumor antibiotic, is structurally distinguished by its aminodideoxypentose-containing disaccharide and asymmetrically halogenated N-methylated aglycon. Cloning and sequence analysis of AT2433 gene cluster and comparison of this locus with that encoding for rebeccamycin and the gene cluster encoding calicheamicin present an opportunity to study the aminodideoxypentose biosynthesis via comparative genomics. The locus was confirmed via in vitro biochemical characterization of two methyltransferases--one common to AT2433 and rebeccamycin, the other unique to AT2433--as well as via heterologous expression and in vivo bioconversion experiments using the AT2433 N-glycosyltransferase. Preliminary studies of substrate tolerance for these three enzymes reveal the potential to expand upon the enzymatic diversification of indolocarbazoles. Moreover, this work sets the stage for future studies regarding the origins of the indolocarbazole maleimide nitrogen and indolocarbazole asymmetry.

To compare body mass index (BMI) and daily energy intake (DEI) after subthalamic versus pallidal deep brain stimulation (DBS). Weight gain following DBS in Parkinson's disease patients remains largely unexplained and no comparison of subthalamic and pallidal (GPi) stimulation has yet been performed. BMI and DEI, dopaminergic drug administration and motor scores were recorded in 46 patients with PD before STN (n = 32) or GPi (n = 14) DBS and 3 and 6 months after. At M6, BMI had increased by an average of 8.4% in the STN group and 3.2% in the GPi group. BMI increased in 28 STN and 9 GPi patients. This increase was significantly higher in the STN group (P < 0.048) and the difference remained significant after adjustment for reduced dopaminergic medication; 28.6% of GPi patients were overweight at 6 months (14.3% preoperatively) versus 37.5% of STN patients (21.9% preoperatively). Changes in BMI were negatively correlated with changes in dyskinesia in the GPi-DBS group. Food intake did not change in the two groups, either quantitatively or qualitatively. Frequent weight gain, inadequately explained by motor improvement or reduced dopaminergic drug dosage, occurred in subthalamic DBS patients. The difference between groups suggests additional factors in the STN group, such as homeostatic control center involvement.