Metabolic Fingerprint Analysis of Cytochrome b5-producing E. coli N4830-1 Using FT-IR Spectroscopy

Optimization of recombinant protein expression in bacteria is an important task in order to increase protein yield while maintaining the structural fidelity of the product. In this study, we employ Fourier transform infrared (FT-IR) spectroscopy as a high throughput metabolic fingerprinting approach to optimize and monitor cytochrome b 5 (CYT b 5) production in Escherichia coli N4830-1, as the heterologous host. Cyt b5 was introduced as a plasmid with between 0 and 6 copies under a strong promoter. The FT-IR spectroscopy results combined with multivariate chemometric analysis illustrated discriminations among culture conditions as well as revealing features that correlated to the different cytb 5 gene copy numbers. The second derivative of the FT-IR spectral data allowed for the quantitative detection of Cyt b5 directly inside the intact cells without the need for extraction, and highlighted changes in protein secondary structure that was directly correlated to the cytb 5 gene copy number and protein content, and was in complete agreement with quantitative findings of standard traditional techniques such as SDS-PAGE and western blot analysis

Functional genomics via metabolic footprinting: monitoring metabolite secretion by Escherichia coli tryptophan metabolism mutants using FT-IR and direct injection electrospray mass spectrometry

We sought to test the hypothesis that mutant bacterial strains could be discriminated from each other on the basis of the metabolites they secrete into the medium (their ‘metabolic footprint’), using two methods of ‘global’ metabolite analysis (FT–IR and direct injection electrospray mass spectrometry). The biological system used was based on a published study of Escherichia coli tryptophan mutants that had been analysed and discriminated by Yanofsky and colleagues using transcriptome analysis. Wild-type strains supplemented with tryptophan or analogues could be discriminated from controls using FT–IR of 24 h broths, as could each of the mutant strains in both minimal and supplemented media. Direct injection electrospray mass spectrometry with unit mass resolution could also be used to discriminate the strains from each other, and had the advantage that the discrimination required the use of just two or three masses in each case. These were determined via a genetic algorithm. Both methods are rapid, reagentless, reproducible and cheap, and might beneficially be extended to the analysis of gene knockout libraries

Identification and Characterization of Three Novel Lipases Belonging to Families II and V from Anaerovibrio lipolyticus 5ST

Following the isolation, cultivation and characterization of the rumen bacterium Anaerovibrio lipolyticus in the 1960s, it has been recognized as one of the major species involved in lipid hydrolysis in ruminant animals. However, there has been limited characterization of the lipases from the bacterium, despite the importance of understanding lipolysis and its impact on subsequent biohydrogenation of polyunsaturated fatty acids by rumen microbes. This study describes the draft genome of Anaerovibrio lipolytica 5ST, and the characterization of three lipolytic genes and their translated protein. The uncompleted draft genome was 2.83 Mbp and comprised of 2,673 coding sequences with a G+C content of 43.3%. Three putative lipase genes, alipA, alipB and alipC, encoding 492-, 438- and 248- amino acid peptides respectively, were identified using RAST. Phylogenetic analysis indicated that alipA and alipB clustered with the GDSL/SGNH family II, and alipC clustered with lipolytic enzymes from family V. Subsequent expression and purification of the enzymes showed that they were thermally unstable and had higher activities at neutral to alkaline pH. Substrate specificity assays indicated that the enzymes had higher hydrolytic activity against caprylate (C8), laurate (C12) and myristate (C14)

Isolation and characterization of novel lipases/esterases from a bovine rumen metagenome

Improving the health beneficial fatty acid content of meat and milk is a major challenge requiring an increased understanding of rumen lipid metabolism. In this study, we isolated and characterized rumen bacterial lipases/esterases using functional metagenomics. Metagenomic libraries were constructed from DNA extracted from strained rumen fluid (SRF), solid-attached bacteria (SAB) and liquid-associated rumen bacteria (LAB), ligated into a fosmid vector and subsequently transformed into an Escherichia coli host. Fosmid libraries consisted of 7,744; 8,448; and 7,680 clones with an average insert size of 30 to 35 kbp for SRF, SAB and LAB, respectively. Transformants were screened on spirit blue agar plates containing tributyrin for lipase/esterase activity. Five SAB and four LAB clones exhibited lipolytic activity, and no positive clones were found in the SRF library. Fosmids from positive clones were pyrosequenced and twelve putative lipase/esterase genes and two phospholipase genes retrieved. Although the derived proteins clustered into diverse esterase and lipase families, a degree of novelty was seen, with homology ranging from 40 to 78 % following BlastP searches. Isolated lipases/esterases exhibited activity against mostly short- to medium-chain substrates across a range of temperatures and pH. The function of these novel enzymes recovered in ruminal metabolism needs further investigation, alongside their potential industrial uses. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00253-014-6355-6) contains supplementary material, which is available to authorized users