A nucleosome-free dG-dC-rich sequence element promotes constitutive transcription of the essential yeast RIO1 gene

RIO1 is an essential gene that encodes a protein serine kinase and is transcribed constitutively at a very low level. Transcriptional activation of RIO1 dispenses with a canonical TATA box as well as with classical transactivators or specific DNAbinding factors. Instead, a dGdCrich sequence element, that is located 40 to 48 bp upstream the single site of mRNA initiation, is essential and presumably constitutes the basal promoter. In addition, we demonstrate here that this promoter element comprises a nucleosomefree gap which is centered at the dGdC tract and flanked by two positioned nucleosomes. This element is both, necessary and sufficient, for basal transcription initiation at the RIO1 promoter and, thus, constitutes a novel type of core promoter element

Synthetic biology of cyanobacterial cell factories

In the field of microbial biotechnology rational design approaches are employed for the generation of microbial cells with desired functions, such as the ability to produce precursor molecules for biofuels or bioplastics. In essence, that is the introduction of a (new) biosynthetic pathway into a microbial cell to create a ‘microbial catalyst’. Utilizing cyanobacteria as production hosts has the potential to contribute to a bio-based economy. Cyanobacteria are prokaryotes that perform oxygenic photosynthesis. By ‘re-programming’ their metabolic network their photosynthetic metabolism can be used to synthesize valuable products from CO2, sunlight and water, O2 being the only by-product. Compared to traditional biofuel production this direct production would bypass the need to initially synthesize complex ‘biomass’ which has to be broken down again in later steps. Key to the work presented in this thesis is the optimization of synthetic pathways, which are introduced into the cyanobacterium Synechocystis sp. PCC 6803 by means of genetic engineering. The carbon metabolism, originating from the fixation of CO2, is ‘tapped’ for the production of carbon-based compounds such as lactic acid, butanediol, and ethanol. The introduced biosynthetic pathways, which are originating from chemotrophic organisms, result in a trade-off between production and growth due to the ‘re-channeling’ of the carbon flux of the metabolism. Undoubtedly, different factors contribute to the success of a well-engineered cyanobacterial production strain. General knowledge about regulatory networks and also experience with metabolic engineering of cyanobacteria is still at the beginning

Ethylene production with engineered<i> Synechocystis</i> sp PCC 6803 strains

BACKGROUND: Metabolic engineering and synthetic biology of cyanobacteria offer a promising sustainable alternative approach for fossil-based ethylene production, by using sunlight via oxygenic photosynthesis, to convert carbon dioxide directly into ethylene. Towards this, both well-studied cyanobacteria, i.e., Synechocystis sp PCC 6803 and Synechococcus elongatus PCC 7942, have been engineered to produce ethylene by introducing the ethylene-forming enzyme (Efe) from Pseudomonas syringae pv. phaseolicola PK2 (the Kudzu strain), which catalyzes the conversion of the ubiquitous tricarboxylic acid cycle intermediate 2-oxoglutarate into ethylene. RESULTS: This study focuses on Synechocystis sp PCC 6803 and shows stable ethylene production through the integration of a codon-optimized version of the efe gene under control of the Ptrc promoter and the core Shine–Dalgarno sequence (5′-AGGAGG-3′) as the ribosome-binding site (RBS), at the slr0168 neutral site. We have increased ethylene production twofold by RBS screening and further investigated improving ethylene production from a single gene copy of efe, using multiple tandem promoters and by putting our best construct on an RSF1010-based broad-host-self-replicating plasmid, which has a higher copy number than the genome. Moreover, to raise the intracellular amounts of the key Efe substrate, 2-oxoglutarate, from which ethylene is formed, we constructed a glycogen-synthesis knockout mutant (ΔglgC) and introduced the ethylene biosynthetic pathway in it. Under nitrogen limiting conditions, the glycogen knockout strain has increased intracellular 2-oxoglutarate levels; however, surprisingly, ethylene production was lower in this strain than in the wild-type background. CONCLUSION: Making use of different RBS sequences, production of ethylene ranging over a 20-fold difference has been achieved. However, a further increase of production through multiple tandem promoters and a broad-host plasmid was not achieved speculating that the transcription strength and the gene copy number are not the limiting factors in our system

Ventricular assist devices for heart failure

Background: Heart failure is a clinical syndrome of major epidemiological and economical importance. In Germany as in other industrialised countries it is one of the leading causes of mortality, morbidity and disability. For patients not responding to medical therapies ventricular assist devices are used as bridge to transplant, bridge to recovery or as destination therapy alternative to transplantation. Research questions: This report aims to present the actual evidence on clinical effectiveness, psychological and social aspects and economical aspects of left ventricular assist devices.MethodsWe conducted a systematic research of the literature in different databases (EMBASE, MEDLINE, Cochrane Library). The included studies were assessed by two reviewers and were presented in tables and in a narrative form. We also conducted a survey among hospitals using the technology. Results and discussion: We included 40 references, six of them were HTA-reports. LVAD patients as bridge to transplant had better survival rates and a better quality of life as compared to medical therapy. Mechanical assistance was associated with frequent and often serious adverse events which were often the cause of death. There were numerous psychological and psychiatric problems. Conclusions/Recommendations: LVAD showed to be clinically effective, however with high complications rate and seriousness of them. The use of technology is very costly. Cost-effective should be analysed for Germany. There is a need to further develop the technology before it can contribute seriously to a reduction of the need for heart transplantation

Uncovering Key Metabolic Determinants of the Drug Interactions Between Trimethoprim and Erythromycin in Escherichia coli

Understanding interactions between antibiotics used in combination is an important theme in microbiology. Using the interactions between the antifolate drug trimethoprim and the ribosome-targeting antibiotic erythromycin in Escherichia coli as a model, we applied a transcriptomic approach for dissecting interactions between two antibiotics with different modes of action. When trimethoprim and erythromycin were combined, the transcriptional response of genes from the sulfate reduction pathway deviated from the dominant effect of trimethoprim on the transcriptome. We successfully altered the drug interaction from additivity to suppression by increasing the sulfate level in the growth environment and identified sulfate reduction as an important metabolic determinant that shapes the interaction between the two drugs. Our work highlights the potential of using prioritization of gene expression patterns as a tool for identifying key metabolic determinants that shape drug-drug interactions. We further demonstrated that the sigma factor-binding protein gene crl shapes the interactions between the two antibiotics, which provides a rare example of how naturally occurring variations between strains of the same bacterial species can sometimes generate very different drug interactions

Exploring metabolic engineering design principles for the photosynthetic production of lactic acid by Synechocystis sp. PCC6803

Background: Molecular engineering of the intermediary physiology of cyanobacteria has become important for the sustainable production of biofuels and commodity compounds from CO2 and sunlight by "designer microbes." The chemical commodity product L-lactic acid can be synthesized in one step from a key intermediary metabolite of these organisms, pyruvate, catalyzed by a lactate dehydrogenase. Synthetic biology engineering to make "designer microbes" includes the introduction and overexpression of the product-forming biochemical pathway. For further optimization of product formation, modifications in the surrounding biochemical network of intermediary metabolism have to be made. Results: To improve light-driven L-lactic acid production from CO2, we explored several metabolic engineering design principles, using a previously engineered L-lactic acid producing mutant strain of Synechocystis sp. PCC6803 as the benchmark. These strategies included: (i) increasing the expression level of the relevant product-forming enzyme, lactate dehydrogenase (LDH), for example, via expression from a replicative plasmid; (ii) co-expression of a heterologous pyruvate kinase to increase the flux towards pyruvate; and (iii) knockdown of phosphoenolpyruvate carboxylase to decrease the flux through a competing pathway (from phosphoenolpyruvate to oxaloacetate). In addition, we tested selected lactate dehydrogenases, some of which were further optimized through site-directed mutagenesis to improve the enzyme’s affinity for the co-factor nicotinamide adenine dinucleotide phosphate (NADPH). The carbon partitioning between biomass and lactic acid was increased from about 5% to over 50% by strain optimization. Conclusion: An efficient photosynthetic microbial cell factory will display a high rate and extent of conversion of substrate (CO2) into product (here: L-lactic acid). In the existing CO2-based cyanobacterial cell factories that have been described in the literature, by far most of the control over product formation resides in the genetically introduced fermentative pathway. Here we show that a strong promoter, in combination with increased gene expression, can take away a significant part of the control of this step in lactic acid production from CO2. Under these premises, modulation of the intracellular precursor, pyruvate, can significantly increase productivity. Additionally, production enhancement is achieved by protein engineering to increase co-factor specificity of the heterologously expressed LDH

Single-cell screening of photosynthetic growth and lactate production by cyanobacteria

Background Photosynthetic cyanobacteria are attractive for a range of biotechnological applications including biofuel production. However, due to slow growth, screening of mutant libraries using microtiter plates is not feasible. Results We present a method for high-throughput, single-cell analysis and sorting of genetically engineered l-lactate-producing strains of Synechocystis sp. PCC6803. A microfluidic device is used to encapsulate single cells in picoliter droplets, assay the droplets for l-lactate production, and sort strains with high productivity. We demonstrate the separation of low- and high-producing reference strains, as well as enrichment of a more productive l-lactate-synthesizing population after UV-induced mutagenesis. The droplet platform also revealed population heterogeneity in photosynthetic growth and lactate production, as well as the presence of metabolically stalled cells. Conclusions The workflow will facilitate metabolic engineering and directed evolution studies and will be useful in studies of cyanobacteria biochemistry and physiology

Rio1 mediates ATP-dependent final maturation of 40S ribosomal subunits

During the last step in 40S ribosome subunit biogen-esis, the PIN-domain endonuclease Nob1 cleaves the 20S pre-rRNA at site D, to form the mature 18S rRNAs. Here we report that cleavage occurs in particles that have largely been stripped of previously character-ized pre-40S components, but retain the endonu-clease Nob1, its binding partner Pno1 (Dim2) and the atypical ATPase Rio1. Within the Rio1-associated pre-40S particles, in vitro pre-rRNA cleavage was strongly stimulated by ATP and required nucleotide binding by Rio1. In vivo binding sites for Rio1, Pno1 and Nob1 were mapped by UV cross-linking in ac-tively growing cells. Nob1 and Pno1 bind overlap-ping regions within the internal transcribed spacer 1, and both bind directly over cleavage site D. Bind-ing sites for Rio1 were within the core of the 18S rRNA, overlapping tRNA interaction sites and distinct from the related kinase Rio2. Site D cleavage occurs within pre-40S-60S complexes and Rio1-associated particles efficiently assemble into these complexes, whereas Pno1 appeared to be depleted relative to Nob1. We speculate that Rio1-mediated dissociation of Pno1 from cleavage site D is the trigger for final 18S rRNA maturation