Characterisation of four hotdog-fold thioesterases for their implementation in a novel organic acid production system.

With increasing interest in the diverse properties of organic acids and their application in synthetic pathways, developing biological tools for producing known and novel organic acids would be very valuable. In such a system, organic acids may be activated as coenzyme A (CoA) esters, then modified by CoA-dependent enzymes, followed by CoA liberation by a broad-acting thioesterase. This study has focused on the identification of suitable thioesterases (TE) for utilisation in such a pathway. Four recombinant hotdog-fold TEs were screened with a range of CoA esters in order to identify a highly active, broad spectrum TE. The TesB-like TE, RpaL, from Rhodopseudomonas palustris was found to be able to use aromatic, alicyclic and both long and short aliphatic CoA esters. Size exclusion chromatography, revealed RpaL to be a monomer of fused hotdog domains, in contrast to the complex quaternary structures found with similar TesB-like TEs. Nonetheless, sequence alignments showed a conserved catalytic triad despite the variation in quaternary arrangement. Kinetic analysis revealed a preference towards short-branched chain CoA esters with the highest specificity towards DL-ß-hydroxybutyryl CoA (1.6 × 104 M-1 s-1), which was found to decrease as the acyl chain became longer and more functionalised. Substrate inhibition was observed with the fatty acyl n-heptadecanoyl CoA at concentrations exceeding 0.3 mM; however, this was attributed to its micellar aggregation properties. As a result of the broad activity observed with RpaL, it is a strong candidate for implementation in CoA ester pathways to generate modified or novel organic acids.

Detecting intratumoral heterogeneity of EGFR activity by liposome-based in vivo transfection of a fluorescent biosensor.

Despite decades of research in the epidermal growth factor receptor (EGFR) signalling field, and many targeted anti-cancer drugs that have been tested clinically, the success rate for these agents in the clinic is low, particularly in terms of the improvement of overall survival. Intratumoral heterogeneity is proposed as a major mechanism underlying treatment failure of these molecule-targeted agents. Here we highlight the application of fluorescence lifetime microscopy (FLIM)-based biosensing to demonstrate intratumoral heterogeneity of EGFR activity. For sensing EGFR activity in cells, we used a genetically encoded CrkII-based biosensor which undergoes conformational changes upon tyrosine-221 phosphorylation by EGFR. We transfected this biosensor into EGFR-positive tumour cells using targeted lipopolyplexes bearing EGFR-binding peptides at their surfaces. In a murine model of basal-like breast cancer, we demonstrated a significant degree of intratumoral heterogeneity in EGFR activity, as well as the pharmacodynamic effect of a radionuclide-labeled EGFR inhibitor in situ. Furthermore, a significant correlation between high EGFR activity in tumour cells and macrophage-tumour cell proximity was found to in part account for the intratumoral heterogeneity in EGFR activity observed. The same effect of macrophage infiltrate on EGFR activation was also seen in a colorectal cancer xenograft. In contrast, a non-small cell lung cancer xenograft expressing a constitutively active EGFR conformational mutant exhibited macrophage proximity-independent EGFR activity. Our study validates the use of this methodology to monitor therapeutic response in terms of EGFR activity. In addition, we found iNOS gene induction in macrophages that are cultured in tumour cell-conditioned media as well as an iNOS activity-dependent increase in EGFR activity in tumour cells. These findings point towards an immune microenvironment-mediated regulation that gives rise to the observed intratumoral heterogeneity of EGFR signalling activity in tumour cells in vivo.

A rapid, sensitive colorimetric assay for the high-throughput screening of transaminases in liquid or solid-phase.

A new colorimetric method has been developed to screen transaminases using an inexpensive amine donor. The assay is sensitive, has a low level of background coloration, and can be used to identify and profile transaminase activities against aldehyde and ketone substrates in a high-throughput format. Significantly it is also amendable to solid phase colony screening.

ω-Transaminases for the amination of functionalised cyclic ketones.

The potential of a number of enantiocomplementary ω-transaminases (ω-TAms) in the amination of cyclic ketones has been investigated. After a preliminary screening of several compounds with increasing complexity, different approaches to shift the equilibrium of the reaction to the amine products were studied, and reaction conditions (temperature and pH) optimised. Interestingly, 2-propylamine as an amine donor was tolerated by all five selected ω-TAms, and therefore used in further experiments. Due to the higher conversions observed and interest in chiral amines studies then focused on the amination of α-tetralone and 2-methylcyclohexanone. Both ketones were aminated to give the corresponding amine with at least one of the employed enzymes. Moreover, the amination of 2-methylcyclohexanone was investigated in more detail due to the different stereoselectivities observed with TAms used. The highest yields and stereoselectivities were obtained using the ω-TAm from Chromobacterium violaceum (CV-TAm), producing 2-methylcyclohexylamine with complete stereoselectivity at the (1S)-amine position and up to 24 : 1 selectivity for the cis : trans [(1S,2R) : (1S,2S)] isomer.

One-pot synthesis of amino-alcohols using a de-novo transketolase and beta-alanine: pyruvate transaminase pathway in Escherichia coli.

Biocatalysis continues to emerge as a powerful technique for the efficient synthesis of optically pure pharmaceuticals that are difficult to access via conventional chemistry. The power of biocatalysis can be enhanced if two or more reactions can be achieved by a single whole cell biocatalyst containing a pathway designed de-novo to facilitate a required synthetic sequence. The enzymes transketolase (TK) and transaminase (TAm) respectively catalyze asymmetric carbon--carbon bond formation and amine group addition to suitable substrate molecules. The ability of a transaminase to accept the product of the transketolase reaction can allow the two catalysts to be employed in series to create chiral amino-alcohols from achiral substrates. As proof of principle, the beta-alanine: pyruvate aminotransferase (beta-A:P TAm) from Pseudomonas aeruginosa has been cloned, to create plasmid pQR428, for overexpression in E.coli strain BL21gold(DE3). Production of the beta-A:P TAm alongside the native transketolase (overexpressed from plasmid pQR411), in a single E.coli host, has created a novel biocatalyst capable of the synthesis of chiral amino alcohols via a synthetic two-step pathway. The feasibility of using the biocatalyst has been demonstrated by the formation of a single diastereoisomer of 2-amino-1,3,4-butanetriol (ABT) product, in up to 21% mol/mol yield, by the beta-A:P TAm, via transamination of L-erythrulose synthesized by TK, from achiral substrates glycolaldehyde (GA) and beta-hydroxypyruvate (beta-HPA). ABT synthesis was achieved in a one-pot process, using either whole cells of the dual plasmid strain or cell lysate, while the dual alcohol-amine functionality of ABT makes it an excellent synthon for many pharmaceutical syntheses.

Royal Society of Chemistry and The Biochemical Society's international symposium on functional genomics: from gene to commercial reality? September 19-22, 1999, Ambleside, UK.

All aspects of genomics were covered during the meeting from gene identification and characterization to exploitation of genomic targets. Speakers highlighted the strategies used together with recent examples, indicating the advantages together with the disadvantages of the methodologies used.