A process is reported for efficient, enantioselective production of key intermediates for the common chiral side chain of statin-type cholesterol-lowering drugs such as Lipitor (atorvastatin) and Crestor (rosuvastatin). The process features a one-pot tandem aldol reaction catalyzed by a deoxyribose-5-phosphate aldolase (DERA) to form a 6-carbon intermediate with installation of two stereogenic centers from 2-carbon starting materials. An improvement of almost 400-fold in volumetric productivity relative to the published enzymatic reaction conditions has been achieved, resulting in a commercially attractive process that has been run on up to a 100-g scale in a single batch at a rate of 30.6 g͞liter per h. Catalyst load has been improved by 10-fold as well, from 20 to 2.0 wt % DERA. These improvements were achieved by a combination of discovery from environmental DNA of DERAs with improved activity and reaction optimization to overcome substrate inhibition. The two stereogenic centers are set by DERA with enantiomeric excess at >99.9% and diastereomeric excess at 96.6%. In addition, downstream chemical steps have been developed to convert the enzymatic product efficiently to versatile intermediates applicable to preparation of atorvastatin and rosuvastatin. Biocatalysis has received attention recently as a tool of enormous potential for the synthesis of pharmaceutical, industrial, and agricultural chemicals and intermediates (1-5). To capitalize further on this potential, enzymes must demonstrate not only superior performance characteristics such as high enantioselectivity and low catalyst loading but also costeffectiveness and scalability. A successful industrial biocatalytic process requires enzymes that can be produced cheaply in kilogram quantities and are stable and active at very high substrate and product concentrations, frequently in the presence of organic solvents. Although many remarkable biocatalytic transformations have been reported on the milligram-to-gram scale, especially in light of recent advances in directed evolution and high-throughput screening for novel enzymes (6-8), the number of processes that have been applied on an industrial scale remains limited (5, 9). This is especially true for biocatalytic processes involving formation of carbon-carbon bonds.The aldol reaction is an extremely useful transformation that allows the formation of new carbon-carbon bonds and the introduction of up to two new stereocenters into the product. Controlling the stereochemistry of aldol reactions can be challenging and has been an area of intense research. Although several elegant asymmetric methods have been developed (10-12), there remains a need for additional methods, and in recent years enzyme-catalyzed approaches using aldolases have received attention as alternatives to chemical methodologies (13).Several years ago, Wong and coworkers (14, 15) reported unprecedented one-pot tandem aldol reactions catalyzed by a deoxyribose-5-phosphate aldolase (DERA), in which 2 eq of acetaldehyde were added in sequen...

The NAD(+)-dependent protein deacetylase SIRT6 regulates genome stability, cancer, and lifespan. Mice overexpressing SIRT6 (MOSES) have lower low-density lipoprotein cholesterol levels and are protected against the physiological damage of obesity. Here, we examined the role of SIRT6 in cholesterol regulation via the lipogenic transcription factors SREBP1 and SREBP2, and AMP-activated protein kinase (AMPK). We show that SIRT6 represses SREBP1 and SREBP2 by at least three mechanisms. First, SIRT6 represses the transcription levels of SREBP1/SREBP2 and that of their target genes. Second, SIRT6 inhibits the cleavage of SREBP1/SREBP2 into their active forms. Third, SIRT6 activates AMPK by increasing the AMP/ATP ratio, which promotes phosphorylation and inhibition of SREBP1 by AMPK. Reciprocally, the expression of miR33a and miR33b from the introns of SREBP2 and SREBP1, respectively, represses SIRT6 levels. Together, these findings explain the mechanism underlying the improved cholesterol homeostasis in MOSES mice, revealing a relationship between fat metabolism and longevity.

Neurogenesis, the formation of new neurons in the adult brain, is important for memory formation and extinction. One of the most studied external interventions that affect the rate of adult neurogenesis is physical exercise. Physical exercise promotes adult neurogenesis via several factors including brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF). Here, we identified L -lactate, a physical exercise-induced metabolite, as a factor that promotes adult hippocampal neurogenesis. While prolonged exposure to L -lactate promoted neurogenesis, no beneficial effect was exerted on cognitive learning and memory. Systemic pharmacological blocking of monocarboxylate transporter 2 (MCT2), which transports L -lactate to the brain, prevented lactate-induced neurogenesis, while 3,5-dihydroxybenzoic acid (3,5-DHBA), an agonist for the lactate-receptor hydroxycarboxylic acid receptor 1 (HCAR1), did not affect adult neurogenesis. These data suggest that L -lactate partially mediates the effect of physical exercise on adult neurogenesis, but not cognition, in a MCT2-dependent manner.

Understanding the structural basis for protein thermostability is of considerable biological and biotechnological importance as exemplified by the industrial use of xylanases at elevated temperatures in the paper pulp and animal feed sectors. Here we have used directed protein evolution to generate hyperthermostable variants of a thermophilic GH11 xylanase, EvXyn11. The Gene Site Saturation Mutagenesis TM (GSSM) methodology employed assesses the influence on thermostability of all possible amino acid substitutions at each position in the primary structure of the target protein. The 15 most thermostable mutants, which generally clustered in the N-terminal region of the enzyme, had melting temperatures (T m ) 1-8°C higher than the parent protein. Screening of a combinatorial library of the single mutants identified a hyperthermostable variant, EvXyn11TS , containing seven mutations. EvXyn11 TS had a T m ϳ 25°C higher than the parent enzyme while displaying catalytic properties that were similar to EvXyn11. The crystal structures of EvXyn11 and EvXyn11 TS revealed an absence of substantial changes to identifiable intramolecular interactions. The only explicable mutations are T13F, which increases hydrophobic interactions, and S9P that apparently locks the conformation of a surface loop. This report shows that the molecular basis for the increased thermostability is extraordinarily subtle and points to the requirement for new tools to interrogate protein folding at non-ambient temperatures.

Mice overexpressing the longevity protein SIRT6 or deficient for the liver's most prevalent microRNA miR-122 display a similar set of phenotypes, including improved lipid profile and protection against damage linked to obesity. Here, we show that miR-122 and SIRT6 negatively regulate each other's expression. SIRT6 downregulates miR-122 by deacetylating H3K56 in the promoter region. MiR-122 binds to three sites on the SIRT6 3' UTR and reduces its levels. The interplay between SIRT6 and miR-122 is manifested in two physiologically relevant ways in the liver. First, they oppositely regulate a similar set of metabolic genes and fatty acid β-oxidation. Second, in hepatocellular carcinoma patients, loss of a negative correlation between SIRT6 and miR-122 expression is significantly associated with better prognosis. These findings show that SIRT6 and miR-122 negatively regulate each other to control various aspects of liver physiology and SIRT6-miR-122 correlation may serve as a biomarker for hepatocarcinoma prognosis.

The rapid emergence of drug resistant bacteria is occurring worldwide, outpacing the development of new antibiotics. It is known that some of the main sources of antibiotics are the bacteria themselves, many of which are secondary metabolites of Gram positive bacteria. Siderophores, which are secondary metabolites, function as natural chelators (e.g., iron). They are produced and secreted by many bacteria and have been experimented on as “carriers” of several types of antibiotics that pass the cell membrane of challenging Gram negative bacteria. Delftibactin A is a non-ribosomal peptide (NRP), which is known to detoxify gold in Delftia spp. and form gold nuggets, and is considered to be a siderophore. In this study we demonstrate that the supernatant from novel environmental isolates of Delftia spp. have antimicrobial activity. We characterized the active fraction and identified delftibactin A as a compound with antimicrobial activity. Delftibactin A exhibits potent antimicrobial activity against Gram positive multi drug resistant (MDR) bacteria like Methicillin-resistant Staphylococcus aureus (MRSA), and Vancomycin resistant Enterococcus (VRE), and also against the Gram negative pathogens Acinetobacter baumannii and Klebsiella pneumoniae. We discovered that the production of delftibactin A is greatly influenced by temperature. Furthermore, we have demonstrated the possibility of utilizing delftibactin A as a siderophore carrier of toxic metals such as gallium into Gram negative bacteria. These findings expose new opportunities of yet unexploited natural products such as delftibactin A, which have been known for other bacterial uses, as potent factors in the battle against MDR bacteria.

Celiac disease (CD) is an autoimmune disorder in individuals that carry DQ2 or DQ8 MHC class II haplotypes, triggered by the ingestion of gluten. There is no current treatment other than a gluten-free diet (GFD). We have previously shown that the BL-7010 copolymer poly(hydroxyethyl methacrylate-co-styrene sulfonate) (P(HEMA-co-SS)) binds with higher efficiency to gliadin than to other proteins present in the small intestine, ameliorating gliadin-induced pathology in the HLA-HCD4/DQ8 model of gluten sensitivity. The aim of this study was to investigate the efficiency of two batches of BL-7010 to interact with gliadin, essential vitamins and digestive enzymes not previously tested, and to assess the ability of the copolymer to reduce gluten-associated pathology using the NOD-DQ8 mouse model, which exhibits more significant small intestinal damage when challenged with gluten than HCD4/DQ8 mice. In addition, the safety and systemic exposure of BL-7010 was evaluated in vivo (in rats) and in vitro (genetic toxicity studies). In vitro binding data showed that BL-7010 interacted with high affinity with gliadin and that BL-7010 had no interaction with the tested vitamins and digestive enzymes. BL-7010 was effective at preventing gluten-induced decreases in villus-to-crypt ratios, intraepithelial lymphocytosis and alterations in paracellular permeability and putative anion transporter-1 mRNA expression in the small intestine. In rats, BL-7010 was well-tolerated and safe following 14 days of daily repeated administration of 3000 mg/kg. BL-7010 did not exhibit any mutagenic effect in the genetic toxicity studies. Using complementary animal models and chronic gluten exposure the results demonstrate that administration of BL-7010 is effective and safe and that it is able to decrease pathology associated with gliadin sensitization warranting the progression to Phase I trials in humans.

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