Enhanced protein secretion in reduced genome strains of Streptomyces lividans.

BACKGROUND: S. lividans TK24 is a popular host for the production of small molecules and the secretion of heterologous protein. Within its large genome, twenty-nine non-essential clusters direct the biosynthesis of secondary metabolites. We had previously constructed ten chassis strains, carrying deletions in various combinations of specialized metabolites biosynthetic clusters, such as those of the blue actinorhodin (act), the calcium-dependent antibiotic (cda), the undecylprodigiosin (red), the coelimycin A (cpk) and the melanin (mel) clusters, as well as the genes hrdD, encoding a non-essential sigma factor, and matAB, a locus affecting mycelial aggregation. Genome reduction was aimed at reducing carbon flow toward specialized metabolite biosynthesis to optimize the production of secreted heterologous protein. RESULTS: Two of these S. lividans TK24 derived chassis strains showed ~ 15% reduction in biomass yield, 2-fold increase of their total native secretome mass yield and enhanced abundance of several secreted proteins compared to the parental strain. RNAseq and proteomic analysis of the secretome suggested that genome reduction led to cell wall and oxidative stresses and was accompanied by the up-regulation of secretory chaperones and of secDF, a Sec-pathway component. Interestingly, the amount of the secreted heterologous proteins mRFP and mTNFα, by one of these strains, was 12 and 70% higher, respectively, than that secreted by the parental strain. CONCLUSION: The current study described a strategy to construct chassis strains with enhanced secretory abilities and proposed a model linking the deletion of specialized metabolite biosynthetic clusters to improved production of secreted heterologous proteins.

Extracellular degradation of a polyurethane oligomer involving outer membrane vesicles and further insights on the degradation of 2,4-diaminotoluene in Pseudomonas capeferrum TDA1.

The continuing reports of plastic pollution in various ecosystems highlight the threat posed by the ever-increasing consumption of synthetic polymers. Therefore, Pseudomonas capeferrum TDA1, a strain recently isolated from a plastic dump site, was examined further regarding its ability to degrade polyurethane (PU) compounds. The previously reported degradation pathway for 2,4-toluene diamine, a precursor and degradation intermediate of PU, could be confirmed by RNA-seq in this organism. In addition, different cell fractions of cells grown on a PU oligomer were tested for extracellular hydrolytic activity using a standard assay. Strikingly, purified outer membrane vesicles (OMV) of P. capeferrum TDA1 grown on a PU oligomer showed higher esterase activity than cell pellets. Hydrolases in the OMV fraction possibly involved in extracellular PU degradation were identified by mass spectrometry. On this basis, we propose a model for extracellular degradation of polyester-based PUs by P. capeferrum TDA1 involving the role of OMVs in synthetic polymer degradation.

Whole-Genome Sequence of Streptococcus pyogenes Strain 591, Belonging to the Genotype emm49.

Streptococcus pyogenes strain 591 is a clinical isolate belonging to the genotype emm49. It has been intensively studied for its pathogenicity traits. In this study, the complete genome of strain 591 was sequenced. It consists of a chromosome of 1,762,765 bp with a G+C content of 38.5%.

A targeted transcriptomics approach for the determination of mixture effects of pesticides.

While real-life exposure occurs to complex chemical mixtures, toxicological risk assessment mostly focuses on individual compounds. There is an increasing demand for in vitro tools and strategies for mixture toxicity analysis. Based on a previously established set of hepatotoxicity marker genes, we analyzed mixture effects of non-cytotoxic concentrations of different pesticides in exposure-relevant binary mixtures in human HepaRG hepatocarcinoma cells using targeted transcriptomics. An approach for mixture analysis at the level of a complex endpoint such as a transcript pattern is presented, including mixture design based on relative transcriptomic potencies and similarities. From a mechanistic point of view, goal of the study was to evaluate combinations of chemicals with varying degrees of similarity in order to determine whether differences in mechanisms of action lead to different mixtures effects. Using a model deviation ratio-based approach for assessing mixture effects, it was revealed that most data points are consistent with the assumption of dose addition. A tendency for synergistic effects was only observed at high concentrations of some combinations of the test compounds azoxystrobin, cyproconazole, difenoconazole, propiconazole and thiacloprid, which may not be representative of human real-life exposure. In summary, the findings of our study suggest that, for the pesticide mixtures investigated, risk assessment based on the general assumption of dose addition can be considered sufficiently protective for consumers. The way of data analysis presented in this paper can pave the way for a more comprehensive use of multi-gene expression data in experimental studies related to mixture toxicity.

Pesticide mixture effects on liver protein abundance in HepaRG cells.

Toxicological effects of chemicals are mostly tested individually. However, consumers encounter exposure to complex mixtures, for example multiple pesticide residues, by consuming food such as crops, fruits or vegetables. Currently, more than 450 active substances are approved in the European Union, and there is little data on effects after combined exposure to several pesticides. Toxicological animal studies would increase enormously, if pesticide combinations had to be analyzed in vivo. Therefore, in vitro methods addressing this issue are needed. We have developed 32 immunoaffinity-based mass spectrometry assays to investigate the impact of hepatotoxic active substances on liver proteins in human HepaRG cells. Five compounds were selected based on their (dis)similar capability to modulate protein levels, and on their combined use in commercially available formulations. Four binary mixtures were prepared from these five substances and tested in different concentrations over three time points. We applied a novel statistical method to describe deviations from additivity and to detect antagonistic and synergistic effects. The results regarding the abundance of hepatotoxicity-related proteins showed additive behavior for 1323 out of 1427 endpoints tested, while 104 combinatorial effects deviating from additivity, such as antagonism or synergism were observed.

Transcript and protein marker patterns for the identification of steatotic compounds in human HepaRG cells.

Non-alcoholic fatty liver disease is a major health concern especially in Western countries. Animal studies suggest that certain chemicals may contribute to hepatocellular triglyceride accumulation, among them a number of hepatotoxic pesticidal active compounds. In order to improve the identification of potential liver steatosis inducers in vitro in a human cell culture system, HepaRG cells were treated with a selection of 30 steatotic or non-steatotic pesticides. Induction of triglyceride accumulation was monitored, and changes in the expression of hepatotoxicity marker genes were measured at the mRNA and protein levels. Based on these data, transcript and protein marker signatures predictive of triglyceride accumulation in HepaRG cells were derived. The predictive transcript set consisted of POR, ANXA10, ARG1, CCL20, FASN, INSIG1, SREBF1, CD36, CYP2D6, and SLCO1B1. The predictive protein set consisted of NCPR (POR), CYP2E1, CYP1A1, ALDH3A1, UGT2B7, UGT2B15, S100P, LMNA, and PRKDC. In conclusion, the present study presents for the first time transcript and protein marker patterns to separate steatotic from non-steatotic compounds in a human liver cell line.

Heterologous AdpA transcription factors enhance landomycin production in Streptomyces cyanogenus S136 under a broad range of growth conditions.

Streptomyces cyanogenus S136 is the only known producer of landomycin A (LaA), one of the largest glycosylated angucycline antibiotics possessing strong antiproliferative properties. There is rising interest in elucidation of mechanisms of action of landomycins, which, in turn, requires access to large quantities of the pure compounds. Overproduction of LaA has been achieved in the past through manipulation of cluster-situated regulatory genes. However, other components of the LaA biosynthetic regulatory network remain unknown. To fill this gap, we elucidated the contribution of AdpA family pleiotropic regulators in landomycin production via expression of adpA genes of different origins in S. cyanogenus S136. Overexpression of the native S. cyanogenus S136 adpA ortholog had no effect on landomycin titers. In the same time, expression of several heterologous adpA genes led to significantly increased landomycin production under different cultivation conditions. Hence, heterologous adpA genes are a useful tool to enhance or activate landomycin production by S. cyanogenus. Our ongoing research effort is focused on identification of mutations that render S. cyanogenus AdpA nonfunctional.

Fast and reliable strain characterization of Streptomyces lividans through micro-scale cultivation.

Filamentous organisms of the genus Streptomyces play an important role in industrial production processes, due to their extensive secondary metabolism variability, as well as their ability to secrete efficiently large amounts of (heterologous) proteins. While genetic engineering tools are available to rapidly build up large strain libraries, the subsequent strain screening and bioprocess development still constitutes a bottleneck. This is due to the lack of reliable parallelized and accelerated cultivation techniques for morphologically challenging organisms. To address this challenge, we developed an integrated cultivation workflow for Streptomyces lividans based on a parallelized shaken 48-well microtiter-plate (MTP) cultivation device. In a first step, a feasible pre-culture method was identified and validated, revealing high comparability in subsequent main cultivations (coefficient of variation of 1.1% for in-plate replicates and 3.2% between different pre-cultures). When validating the growth performance in 1 mL MTP cultivation against an established 1,000 mL lab-scale cultivation system, highly comparable cultivation patterns were found for online (pH, dissolved oxygen), as well as for offline derived parameters (glucose uptake, cell-dry-weight, and pellet size). Additionally, the two cultivation regimes were compared with respect to transcriptional and protein secretion activity of Streptomyces, showing overall good comparability with minor, but well explainable discrepancies, most probably caused by different energy dissipation (shaking vs. stirring) and adaption effects due to different illumination conditions. Embedded within the presented cultivation workflow, the 1 mL MTP-based parallelized cultivation system seems to be a suitable screening tool for filamentous and industrial relevant organisms like Streptomyces. This can contribute to widen the field of application for these organisms and facilitate screening and early-stage bioprocess development. Biotechnol. Bioeng. 2017;114: 2011-2022. © 2017 Wiley Periodicals, Inc.

High-throughput sequencing and graph-based cluster analysis facilitate microsatellite development from a highly complex genome.

Despite recent advances in high-throughput sequencing, difficulties are often encountered when developing microsatellites for species with large and complex genomes. This probably reflects the close association in many species of microsatellites with cryptic repetitive elements. We therefore developed a novel approach for isolating polymorphic microsatellites from the club-legged grasshopper (Gomphocerus sibiricus), an emerging quantitative genetic and behavioral model system. Whole genome shotgun Illumina MiSeq sequencing was used to generate over three million 300 bp paired-end reads, of which 67.75% were grouped into 40,548 clusters within RepeatExplorer. Annotations of the top 468 clusters, which represent 60.5% of the reads, revealed homology to satellite DNA and a variety of transposable elements. Evaluating 96 primer pairs in eight wild-caught individuals, we found that primers mined from singleton reads were six times more likely to amplify a single polymorphic microsatellite locus than primers mined from clusters. Our study provides experimental evidence in support of the notion that microsatellites associated with repetitive elements are less likely to successfully amplify. It also reveals how advances in high-throughput sequencing and graph-based repetitive DNA analysis can be leveraged to isolate polymorphic microsatellites from complex genomes.

Complete mitochondrial genome of the scleractinian coral Porites rus.

Scleractinian corals of the genus Porites are found across a wide geographical range along the tropical seas. Some species of the genus such as Porites rus are important reef builders in coral reef ecosystems and display a remarkable stress tolerance. Despite their physiological particularities and ecological importance, there is a scarcity of molecular data for members of this genus. Here, we report the first complete mitochondrial genome of Porites rus (Genbank accession number LN864762) which is 18 647 bp in size. It has the typical coral mitochondrial gene arrangement, consisting of 14 protein-coding genes, with a GC content of 36.2%, 12 tRNAs and two rRNAs. The P. rus mitochondrial genome displays two groups: 1 intron in cox1 and nad5 genes. Phylogenetic analyses support the monophyly of the genus Porites. The complete mitochondrial genome will be helpful for addressing questions regarding mitochondrial gene evolution.

Morphological changes and proteome response of Corynebacterium glutamicum to a partial depletion of FtsI.

In Corynebacterium glutamicum, as in many Gram-positive bacteria, the cell division gene ftsI is located at the beginning of the dcw cluster, which comprises cell division- and cell wall-related genes. Transcriptional analysis of the cluster revealed that ftsI is transcribed as part of a polycistronic mRNA, which includes at least mraZ, mraW, ftsL, ftsI and murE, from a promoter that is located upstream of mraZ. ftsI appears also to be expressed from a minor promoter that is located in the intergenic ftsL-ftsI region. It is an essential gene in C. glutamicum, and a reduced expression of ftsI leads to the formation of larger and filamentous cells. A translational GFP-FtsI fusion protein was found to be functional and localized to the mid-cell of a growing bacterium, providing evidence of its role in cell division in C. glutamicum. This study involving proteomic analysis (using 2D SDS-PAGE) of a C. glutamicum strain that has partially depleted levels of FtsI reveals that at least 20 different proteins were overexpressed in the organism. Eight of these overexpressed proteins, which include DivIVA, were identified by MALDI-TOF. Overexpression of DivIVA was confirmed by Western blotting using anti-DivIVA antibodies, and also by fluorescence microscopy analysis of a C. glutamicum RESF1 strain expressing a chromosomal copy of a divIVA-gfp transcriptional fusion. Overexpression of DivIVA was not observed when FtsI was inhibited by cephalexin treatment or by partial depletion of FtsZ.