Improved E. coli erythromycin A production through the application of metabolic and bioprocess engineering

In this report, small-scale culture and bioreactor experiments were used to compare and improve the heterologous production of the antibiotic erythromycin A across a series of engineered prototype Escherichia coli strains. The original strain, termed BAP1(pBPJW130, pBPJW144, pHZT1, pHZT2, pHZT4, pGro7), was designed to allow full erythromycin A biosynthesis from the exogenous addition of propionate. This strain was then compared against two alternatives hypothesized to increase final product titer. Strain TB3(pBPJW130, pBPJW144, pHZT1, pHZT2, pHZT4, pGro7) is a derivative of BAP1 designed to increase biosynthetic pathway carbon flow as a result of a ygfH deletion; whereas, strain TB3(pBPJW130, pBPJW144, pHZT1, pHZT2, pHZT4-2, pGro7) provided an extra copy of a key deoxysugar glycosyltransferase gene. Production was compared across the three strains with TB3(pBPJW130, pBPJW144, pHZT1, pHZT2, pHZT4, pGro7) showing significant improvement in erythronolide B (EB), 3-mycarosylerythronolide B (MEB), and erythromycin A titers. This strain was further tested in the context of batch bioreactor production experiments with time-course titers leveling at 4 mg/L, representing an approximately sevenfold increase in final erythromycin A titer.     

Annokey: an annotation tool based on key term search of the NCBI Entrez Gene database

Background

The NCBI Entrez Gene and PubMed databases contain a wealth of high-quality information about genes for many different organisms. The NCBI Entrez online web-search interface is convenient for simple manual search for a small number of genes but impractical for the kinds of outputs seen in typical genomics projects.

Results

We have developed an efficient open source tool implemented in Python called Annokey, which annotates gene lists with the results of a keyword search of the NCBI Entrez Gene database and linked Pubmed article information. The user steers the search by specifying a ranked list of keywords (including multi-word phrases and regular expressions) that are correlated with their topic of interest. Rank information of matched terms allows the user to guide further investigation.We applied Annokey to the entire human Entrez Gene database using the key-term “DNA repair” and assessed its performance in identifying the 176 members of a published “gold standard” list of genes established to be involved in this pathway. For this test case we observed a sensitivity and specificity of 97% and 96%, respectively.

Conclusions

Annokey facilitates the identification of genes related to an area of interest, a task which can be onerous if performed manually on a large number of genes. Annokey provides a way to capitalize on the high quality information provided by the Entrez Gene database allowing both scalability and compatibility with automated analysis pipelines, thus offering the potential to significantly enhance research productivity.

     

S/T Phosphorylation of DLL1 Is Required for Full Ligand Activity In Vitro but Dispensable for DLL1 Function In Vivo during Embryonic Patterning and Marginal Zone B Cell Development

Interaction of Notch receptors with Delta- and Serrate-type ligands is an evolutionarily conserved mechanism that mediates direct communication between adjacent cells and thereby regulates multiple developmental processes. Posttranslational modifications of both receptors and ligands are pivotal for normal Notch pathway function. We have identified by mass spectrometric analysis two serine and one threonine phosphorylation sites in the intracellular domain of the mouse Notch ligand DLL1. Phosphorylation requires cell membrane association of DLL1 and occurs sequentially at the two serine residues. Phosphorylation of one serine residue most likely by protein kinase B primes phosphorylation of the other serine. A DLL1 variant, in which all three identified phosphorylated serine/threonine residues are mutated to alanine and valine, was more stable than wild-type DLL1 but had reduced relative levels on the cell surface and was more effectively cleaved in the extracellular domain. In addition, the mutant variant activated Notch1 significantly less efficient than wild-type DLL1 in a coculture assay in vitro. Mice, however, whose endogenous DLL1 was replaced with the phosphorylation-deficient triple mutant developed normally, suggesting compensatory mechanisms under physiological conditions in vivo.     

Wolframin deficiency is accompanied with metabolic inflexibility in rat striated muscles

The protein wolframin is localized in the membrane of the endoplasmic reticulum (ER), influencing Ca2+ metabolism and ER interaction with mitochondria, but the exact role of the protein remains unclear. Mutations in Wfs1 gene cause autosomal recessive disorder Wolfram syndrome (WS). The first symptom of the WS is diabetes mellitus, so accurate diagnosis of the disease as WS is often delayed. In this study we aimed to characterize the role of the Wfs1 deficiency on bioenergetics of muscles. Alterations in the bioenergetic profiles of Wfs1-exon-5-knock-out (Wfs1KO) male rats in comparison with their wild-type male littermates were investigated using high-resolution respirometry, and enzyme activity measurements. The changes were followed in oxidative (cardiac and soleus) and glycolytic (rectus femoris and gastrocnemius) muscles. There were substrate-dependent alterations in the oxygen consumption rate in Wfs1KO rat muscles. In soleus muscle, decrease in respiration rate was significant in all the followed pathways. The relatively small alterations in muscle during development of WS, such as increased mitochondrial content and/or increase in the OxPhos-related enzymatic activity could be an adaptive response to changes in the metabolic environment. The significant decrease in the OxPhos capacity is substrate dependent indicating metabolic inflexibility when multiple substrates are available.     

Differences in binding behavior of (−)‐epigallocatechin gallate to β‐lactoglobulin heterodimers (AB) compared to homodimers (A) and (B)

The lipocalin β‐lactoglobulin (β‐LG) exists in different natural genetic variants—of which β‐LG A and B are predominant in bovine milk. At physiological conditions the protein dimerizes—building homodimers of β‐LG A and β‐LG B and heterodimers of β‐LG AB. Although β‐LG is one of the most intensely characterized lipocalins, the interaction behavior of ligands with hetero‐ and homodimers of β‐LG is largely unknown. The present findings revealed significant differences for hetero‐ and homodimers regarding ligand binding capacity as tested with a model ligand (i.e. surface binding (?)‐epigallocatechin gallate (EGCG)). These findings were confirmed using FT‐IR, where the addition of EGCG influenced the β‐sheet backbone of homodimer A and B with significantly higher intensity compared to heterodimer AB. Further, shape analysis by SAXS revealed oligomerization of both types of dimers upon addition of EGCG; however, homodimer A and B produced significantly larger aggregates compared to the heterodimer AB. In summary, the present study revealed that EGCG showed significantly different interaction reactivity (binding sites, aggregation size and conformational changes) to the hetero and homodimers of β‐LG in the order β‐LG A > B > AB. The results suggest that conformational differences between homodimers and heterodimers strongly influence the EGCG binding ability. This may also occur with other polyphenols and ligands of β‐LG and gives not only important information for β‐LG binding studies, but may also apply for polymorphisms of other self‐aggregating lipocalins. Copyright © 2015 John Wiley & Sons, Ltd.     

Detection of endocrine disrupters: evaluation of a Fish Sexual Development Test (FSDT)

Managed by the Organisation for Economic Co-operation and Development (OECD), a comprehensive work is carried out in numerous laboratories to develop test guidelines for the detection of endocrine disrupting chemicals in humans, and various animal species. Development of tests to detect chemicals with endocrine disrupting properties in fish is a part of that work. A Fish Sexual Development Test (FSDT) (an extension of the existing OECD TG 210, fish early life stage toxicity test), proposed as an international test guideline for the detection of endocrine disrupting chemicals, was evaluated by water exposure of juvenile zebrafish to the three natural estrogens: estrone, 17beta-estradiol, and estriol and the synthetic androgen trenbolone (trenbolone acetate). As endpoints, vitellogenin induction and histological changes including changes in sex ratios were investigated. The sex ratio was significantly altered towards females from 49 ng/l estrone, 54 ng/l 17beta-estradiol and 22 microg/l estriol, respectively. An all male population was observed from exposure to 9.7 ng/l trenbolone and above. Significant vitellogenin induction in whole body homogenate was measured after exposure to 14 ng/l estrone, 54 ng/l 17beta-estradiol and 0.6 mug/l estriol, respectively. Significant vitellogenin reduction was measured after exposure to 193 ng/l trenbolone or higher. The present results provide strong evidence that the FSDT is a sensitive test toward estrogenic and especially androgenic exposure and the validation of the FSDT as an OECD test guideline should continue.     

The VASP-Spred-Sprouty domain puzzle

Sprouty-related proteins with an EVH1 domain (Spreds) belong to a new protein family harboring a conserved N-terminal EVH1 domain, which is related to the VASP (vasodilator-stimulated phosphoprotein) EVH1 domain (Enabled/VASP homology 1 domain) and a C-terminal Sprouty-related domain, typical for Sprouty proteins. Spreds were, like Sproutys, initially discovered as inhibitors of the Ras/MAPK pathway, and the SPR (Sprouty-related) domains of both protein families seem to be very important for many protein interactions and cellular processes. VASP was initially characterized as a proline-rich substrate of protein kinases A and G in human platelets and later shown to be a scaffold protein, regulating both signal transduction pathways and the actin filament system. The VASP-EVH1 domain is known to bind specifically to a FP(4) binding motif, which is, for example, present in the focal adhesion proteins vinculin and zyxin. In this review we give a structural and functional overview on these three protein families and ask whether nature plays a modular protein domain puzzle with stable exchangeable elements or if these closely related domains have various functions when pasted in a different protein context.