Metabolic engineering of the L-phenylalanine pathway in Escherichia coli for the production of S- or R-mandelic acid

<p>Abstract</p> <p>Background</p> <p>Mandelic acid (MA), an important component in pharmaceutical syntheses, is currently produced exclusively via petrochemical processes. Growing concerns over the environment and fossil energy costs have inspired a quest to develop alternative routes to MA using renewable resources. Herein we report the first direct route to optically pure MA from glucose via genetic modification of the L-phenylalanine pathway in <it>E. coli</it>.</p> <p>Results</p> <p>The introduction of hydroxymandelate synthase (HmaS) from <it>Amycolatopsis orientalis </it>into <it>E. coli </it>led to a yield of 0.092 g/L S-MA. By combined deletion of competing pathways, further optimization of S-MA production was achieved, and the yield reached 0.74 g/L within 24 h. To produce R-MA, hydroxymandelate oxidase (Hmo) from <it>Streptomyces coelicolor </it>and D-mandelate dehydrogenase (DMD) from <it>Rhodotorula graminis </it>were co-expressed in an S-MA-producing strain, and the resulting strain was capable of producing 0.68 g/L R-MA. Finally, phenylpyruvate feeding experiments suggest that HmaS is a potential bottleneck to further improvement in yields.</p> <p>Conclusions</p> <p>We have constructed <it>E. coli </it>strains that successfully accomplished the production of S- and R-MA directly from glucose. Our work provides the first example of the completely fermentative production of S- and R-MA from renewable feedstock.</p

Endoglin Targeting: Lessons Learned and Questions That Remain

Approximately 30 years ago, endoglin was identified as a transforming growth factor (TGF)-&beta; coreceptor with a crucial role in developmental biology and tumor angiogenesis. Its selectively high expression on tumor vessels and its correlation with poor survival in cancer patients led to the exploration of endoglin as a therapeutic target for cancer. The endoglin neutralizing antibody TRC105 (Carotuximab&reg;, Tracon Pharmaceuticals (San Diego, CA, USA) was subsequently tested in a wide variety of preclinical cancer models before being tested in phase I-III clinical studies in cancer patients as both a monotherapy and in combination with other chemotherapeutic and anti-angiogenic therapies. The combined data of these studies have revealed new insights into the role of endoglin in angiogenesis and its expression and functional role on other cells in the tumor microenvironment. In this review, we will summarize the preclinical work, clinical trials and biomarker studies of TRC105 and explore what these studies have enabled us to learn and what questions remain unanswered

An uncapped RNA suggests a model for Caenorhabditis elegans polycistronic pre-mRNA processing

Polycistronic pre-mRNAs from Caenohabditis elegans operons are processed by internal cleavage and polyadenylation to create 3’ ends of mature mRNAs. This is accompanied by trans-splicing with SL2 ∼100 nucleotides downstream of the 3’ end formation sites to create the 5’ ends of downstream mRNAs. SL2 trans-splicing depends on a U-rich element (Ur), located ∼70 nucleotides upstream of the trans-splice site in the intercistronic region (ICR), as well as a functional 3′ end formation signal. Here we report the existence of a novel gene-length RNA, the Ur-RNA, starting just upstream of the Ur element. The expression of Ur-RNA is dependent on 3′ end formation as well as on the presence of the Ur element, but does not require a trans-splice site. The Ur-RNA is not capped, and alteration of the location of the Ur element in either the 5′ or 3′ direction alters the location of the 5′ end of the Ur-RNA. We propose that a 5’ to 3’ exonuclease degrades the precursor RNA following cleavage at the poly(A) site, stopping when it reaches the Ur element, presumably attributable to a bound protein. Part of the function of this protein can be performed by the MS2 coat protein. Recruitment of coat protein to the ICR in the absence of the Ur element results in accumulation of an RNA equivalent to Ur-RNA, and restores trans-splicing. Only SL1, however, is used. Therefore, coat protein is sufficient for blocking the exonuclease and thereby allowing formation of a substrate for trans-splicing, but it lacks the ability to recruit the SL2 snRNP. Our results also demonstrate that MS2 coat protein can be used as an in vivo block to an exonuclease, which should have utility in mRNA stability studies

An air and moisture tolerant iminotrihydroquinoline-ruthenium(ii) catalyst for the transfer hydrogenation of ketones.

Reaction of 8-amino-5,6,7,8-tetrahydroquinoline with RuCl2(PPh3)3 at room temperature affords the ruthenium(ii) chelate (8-NH2-C9H10N)RuCl2(PPh3)2 (E), in which the two triphenylphosphine ligands are disposed mutually cis. By contrast, when the reaction is performed at reflux ligand oxidation/dehydrogenation occurs along with cis-trans reorganization of the triphenylphosphines to form the 8-imino-5,6,7-trihydroquinoline-ruthenium(ii) complex, (8-NH-C9H9N)RuCl2(PPh3)2 (F). Complex F can also be obtained in higher yield by heating a solution of E alone to reflux. Comparison of their molecular structures highlights the superior binding properties of the bidentate imine ligand in F over its amine-containing counterpart in E. Both complexes are highly effective in the transfer hydrogenation of a wide range of alkyl-, aryl- and cycloalkyl-containing ketones affording their corresponding secondary alcohols with loadings of as low as 0.1 mol%. Significantly, F can deliver excellent conversions even in bench quality 2-propanol in reaction vessels open to the air, whereas the catalytic efficiency of E is diminished by the presence of air but only operates efficiently under inert conditions