Use of Genome-scale Models to get new insights into the MarineActinomycete genus Salinispora: Microbial engineering and its application in secondary metabolite production

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Successful examples of the application of novel iterative trainable algorithms to guide rational mutation strategies for enzyme engineering: From prediction to lab testing to algorithm retraining

Both natural mutations occurring in a homologous enzyme family and mutations engineered in a given protein can have a tremendous impact in the activity and binding behavior of the enzyme towards substrates or other molecules. Binding and catalytic properties can be modified by rationally mutating selected amino acids in a protein. For instance, new specificity properties can be engineered into existing enzymes, which can be applied to the rational design of mutations to alter its catalysis. Although this approach has been largely used, the modifications introduced in the target protein have not been exempt of deleterious effects on protein function, binding or physicochemical properties. Much finer tuned modifications should be designed in order to alter the desired catalytic or binding properties of a protein and simultaneously not affecting other protein properties or functions. These engineered mutations usually require a thorough knowledge of the relevant structure-function relationships in the protein molecule. If no precise structure-function information is available for a protein, the amount of possible amino acid mutations to be tested precludes a direct search. Furthermore, in many cases a directed evolution strategy cannot be successfully used to achieve the desired results due to the unavailability of suitable screening tests. In the last years, we have developed new and powerful in silico methodologies to automatically propose, test and redesign mutagenesis strategies for a target protein, based only on evolutionarily conserved physicochemical properties of amino acids in a protein family where the target protein belongs, and on structural properties, including calculation of vibrational entropies, if available, with no need of explicit structure-function relationships. This methodology identifies amino acid positions that are putatively responsible for function, specificity, stability or binding interactions in a family of proteins and calculates amino acid propensity and distributions at each position. Not only conserved amino acid positions in a protein family can be labelled as functionally relevant, but also non-conserved amino acid positions can be identified to have a meaningful functional effect, and even amino acid substitutions that are unobserved in nature. These results can be used to predict if a given mutation can have a functional implication and which mutation is most likely to be functionally silent for a protein. Through several rounds of mutation suggestions, laboratory testing of the mutants and feedback of results to retrain the algorithms, our methodology can be used to rapidly and automatically discard any irrelevant mutation and guide the research focus toward functionally significant mutations. In this work, we will show how we have successfully used our publicly available methods to guide mutant design in enzyme engineering applied to xylanases (producing an improved octuple mutant in a single mutagenesis round), proteases, glucanases, ubiquitin ligases and other enzymes, to alter protein function, stability or thermodynamic properties independently of their catalytic properties in vitro and in vivo. We will also show how the predictions of these methods have been employed to shift chromatographic elution profiles of xylanases and ferritin nanocages for better purification without affecting their activity and to obtain ferritin variants with better properties to be used in nanotechnological applications, including modifications to the external and internal surface of the protein to change its interaction properties, improve its recombinant production, alter the characteristics of nanoparticles within or change its organic molecule carrier capacity. Finally, we will show how a similar approach has been integrated in an artificial intelligence classification scheme to identify somatic mutations in the human VHL gene that are related to renal clear-cell cancer and to predict the clinical outcome and prognosis of pVHL mutation and malfunction in humans, based on specific disruption of interactions with VHL binding partners. Clearly, our techniques show promising performance as a valuable and powerful bioinformatics tool to aid in the computer-aided design of engineered enzyme variants and in the understanding of function-structure, binding and affinity relationships in enzymes and other proteins

AAV gene therapy for alcoholism: Inhibition of mitochondrial aldehyde dehydrogenase enzyme expression in hepatoma cells

The process by which ethanol is metabolized in the liver occurs in two steps. The first step depends on the enzyme alcohol dehydrogenase (ADH) and the second step is catalyzed by the enzyme Aldehyde dehydrogenase (ALDH2). Some individuals of the Asian population who carry a mutation in the Aldehyde dehydrogenase gene (ALDH2*2) have a diminished capacity to metabolize acetaldehyde, producing strong effects including facial flushing, dizziness, hypotension, and palpitations. This results in an aversion to alcohol intake and protection against alcoholism. The large prevalence of this mutation in the human population strongly suggests that modulation of ALDH2*2 by genetic technologies could result in a similar phenotype. We utilized scAAV2 vectors encoding ALDH2 shRNA to validate this hypothesis by silencing ALDH2 gene expression in human cell lines. In the present study, we have shown that scAAV2 vectors encoding a single ALDH2 shRNA are effective in decreasing mitochondrial Aldehyde dehydrogenase expression in HEK-293/ALDH2, HepG2 and VL-17A HepG2 cell lines and increases acetaldehyde levels. Human cell lines 293 and HepG2 were transduced with scAAV2/shRNA showing a reduction in ALDH2 RNA and protein expression with the two viral concentrations assayed (1x104 and 1x105 vg/cell) at two different time points. In both cell lines ALDH2 RNA levels were reduced by 90% and protein expression was inhibited by 48% and 90%, respectively, five days post infection. ALDH2 silencing was evaluated in the VL-17A HepG2 cell line, which exhibits hepatocyte-like characteristics in response to ethanol. Cells were transduced with 1x105 vg/cell and ALDH2 expression was evaluated at the third day p.i. by Real Time RT-PCR and Western blot, showing an expression reduction of 40% as compared with the scramble control, in both analysis. As functional assay cells were incubated with ethanol (10, 25 and 100 mM) and acetaldehyde accumulation was measured by gas chromatography. Samples treated with the scAAV2/shRNA virus showed an increase of acetaldehyde levels of 50, 30 and 40% for each ethanol concentration assayed. Previously we have demonstrated a 50% decrease in ethanol consumption over 35 days with a similar gene therapy treatment of alcoholic mice that also inhibited the AlDH2 gene. These results suggest that gene therapy could be a useful tool for the treatment of alcoholism by knocking down ALDH2 expression using shRNA technology delivered by AAV vectors

The role of process variables in the design of multiproduct batch protein production plants

This work reports findings about the role of process variables in the design of multiproduct batch plants. Unlike continuous processes, batch processes are subject to size and time constraints which depend on the structure of the plant: the number of units at each stage and the provision of intermediate storage. We used simple process performance models (yet involving all the process variables with significant economic impact) to get explicit expressions for these size and time factors. The traditional approach uses fixed size and time factors. So the addition of those expressions to the original fixed factors model, permitted to simultaneously optimize the plant structure and process variables, and study the role of the latter in the design. We found that if the plant structure constraints are disregarded (with a Free Unlimited Storage operating policy), process variables behave just alike in continuous processes. They trade off cost components with the Total Annual Cost being quite insensitive to them in the neighborhood of the optimal solution. As setting the process variables sets the size and time factors, this means that near the optimal set of process variables, cycle times and size factors can be accommodated to the plant structure, with little effect on the cost of equipment.Fil: Montagna, Jorge Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo y Diseño. Universidad Tecnológica Nacional. Facultad Regional Santa Fe. Instituto de Desarrollo y Diseño; ArgentinaFil: Vecchietti, Aldo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo y Diseño. Universidad Tecnológica Nacional. Facultad Regional Santa Fe. Instituto de Desarrollo y Diseño; ArgentinaFil: Iribarren, Oscar Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo y Diseño. Universidad Tecnológica Nacional. Facultad Regional Santa Fe. Instituto de Desarrollo y Diseño; ArgentinaFil: Pinto, José M.. Universidade de Sao Paulo; BrasilFil: Asenjo, Juan A.. Universidad de Chile; Chil

Iron-meditated fungal starvation by lupine rhizosphere-associated and extremotolerant Streptomyces sp. S29 desferrioxamine production

Open Access via the RSC Open Access Agreement. Acknowledgements SAJ would like to thank the University of Aberdeen for funding doctoral studies through and Elphinstone Scholarship. DL would like to thank the Agencia Nacional de Investigación y Desarrollo (ANID) for funding doctoral studies through ‘Beca nacional de doctorado’ Scholarship. ED would like to thank the Ministry of Higher Education and Scientific Research – Sudan, together with the University of Khartoum, for joint funding of master's studies. We would like to thank to Valeria Razmilic and Jean Franco Castro for their valuable advice and work in the setup of Lupine Streptomyces culture collection. We would also like to thank the support team at GNPS and Justin J. J. van der Hooft at MS2LDA for help with data deposition and for help at any stage of running the relevant workflows.Peer reviewedPublisher PD

Heterologous expression of a cryptic gene cluster from Streptomyces leeuwenhoekii C34T yields a novel lasso peptide, leepeptin

ACKNOWLEDGEMENTS. We are grateful to Michael Goodfellow and Alan Bull for providing S. leeuwenhoekii C34T , and to Michael Fischbach and Jan Claesen for S. viridochromogenes and S. pristinaspiralis, Matthias Mach for S. davawensis, and Kristian Apel on October 31, 2019 at University of Aberdeen http://aem.asm.org/ Downloaded from 17 for S. roseochromogenes. We thank Govind Chandra for advice on blastP analyses of the lasso peptide data sets, Solène Rollet for technical support in the isolation of leepeptin and Andrew Truman for his comments on the manuscript. J.F.C. and V.R. received National PhD Scholarships (#21110356 and #21110384, respectively) and Visiting Student Scholarships (Becas Chile, 2013–2014) from the National Commission for Scientific and Technological Research (CONICYT). S.A.J. thanks the University of Aberdeen for an Elphinstone Scholarship. This work was supported financially by the Biotechnological and Biological Sciences Research Council (BBSRC, United Kingdom) Institute Strategic Programme Grant “Understanding and Exploiting Plant and Microbial Secondary Metabolism” (BB/J004561/1), the Basal Programme of CONICYT (Chile) for funding of the Centre for Biotechnology and Bioengineering, CeBiB (project FB0001) and the UK Newton Project for UK–Chile collaboration (grant JIC CA586).Peer reviewedPublisher PD

Microbial engineering of new streptomyces sp. from extreme environments for novel antibiotics and anticancer drugs

Today there is a tremendous need for new antibiotics and novel cytotoxic compounds against cancer cells to develop efficient alternative treatment to chemotherapy. We have searched for highly active Streptomyces strains in the driest desert in the world, the Atacama desert in northern Chile. We have identified several new strains and found many novel antibiotics and anticancer agents (“Chaxamycins”, “Chaxalactins” and “Atacamycins”) from Streptomyces C34 and C38. A genome scale model of the metabolism of Streptomyces leeuwenhoekii C34 has been developed from its genome sequence. The model, iVR1007, has 1726 reactions including 239 for transport, reactions for secondary metabolite biosynthesis, 1463 metabolites and 1007 genes. The model was validated with experimental data of growth in 89, 54 and 23 sole carbon, nitrogen and phosphorous sources, respectively, and showed a high level of accuracy (82.5 %). We have included reactions for desferrioxamines, ectoine, Chaxamycins, Chaxalactins and for the hybrid polyketides/non-ribosomal peptide synthesized by the halogenase cluster. A detailed Metabolic Flux Balance Analysis was carried out in order to study the metabolic pathways of Chaxalactins, Chaxamycins and the product of the halogenase cluster, by recognizing overexpression targets and useful knock-out sites to increase production of these secondary metabolites. Alternatively we have identified the gene cluster in S. leeuwenhoekii C34 responsible for the biosynthesis of the Chaxamycins and Chaxalactins and have cloned the whole gene cluster in a much more efficient strain of Streptomyces, namely S. coelicolor A3 whose heterologous expression of gene clusters from other Streptomyces strains has been successfully tested. Our recent results concerning these two alternative strategies for identification and overproduction of these important secondary metabolites will be presented and discussed in this presentation

Cutting the Gordian knot : early and complete amino acid sequence confirmation of class II lasso peptides by HCD fragmentation

SAJ would like to thank the University of Aberdeen for an Elphinstone Scholarship. CC-A thanks CONICYT PFCHA/DOCTORADO BECAS CHILE/2016 (#21160585) fellowship and CONICYT Basal Centre Grant for the Centre for Biotechnology and Bioengineering, CeBiB (FB0001). JFC also thanks CONICYT for a National PhD Scholarship (#21110356) and a Visiting Student Scholarship.Peer reviewedPostprin

Mutactimycin AP, a New Mutactimycin Isolated from an Actinobacteria from the Atacama Desert

Funding: This work was supported by national funds from FCT—Fundação para a Ciência e a Tecnologia, I.P., within the projects UIDB/04564/2020 and UIDP/04564/2020.Peer reviewedPublisher PD

New genus-specific primers for PCR identification of Rubrobacter strains

A set of oligonucleotide primers, Rubro223f and Rubro454r, were found to amplify a 267 nucleotide sequence of 16S rRNA genes of Rubrobacter type strains. The primers distinguished members of this genus from other deeply-rooted actinobacterial lineages corresponding to the genera Conexibacter, Gaiella, Parviterribacter, Patulibacter, Solirubrobacter and Thermoleophilum of the class Thermoleophilia. Amplification of DNA bands of about 267 nucleotides were generated from environmental DNA extracted from soil samples taken from two locations in the Atacama Desert. Sequencing of a DNA library prepared from the bands showed that all of the clones fell within the evolutionary radiation occupied by the genus Rubrobacter. Most of the clones were assigned to two lineages that were well separated from phyletic lines composed of Rubrobacter type strains. It can be concluded that primers Rubro223f and Rubro454r are specific for the genus Rubrobacter and can be used to detect the presence and abundance of members of this genus in the Atacama Desert and other biomes