Dataset Information

Trans-Kingdom Conjugation within Solid Media from Escherichia coli to Saccharomyces cerevisiae.

ABSTRACT: Conjugation is a bacterial mechanism for DNA transfer from a donor cell to a wide range of recipients, including both prokaryotic and eukaryotic cells. In contrast to conventional DNA delivery techniques, such as electroporation and chemical transformation, conjugation eliminates the need for DNA extraction, thereby preventing DNA damage during isolation. While most established conjugation protocols allow for DNA transfer in liquid media or on a solid surface, we developed a procedure for conjugation within solid media. Such a protocol may expand conjugation as a tool for DNA transfer to species that require semi-solid or solid media for growth. Conjugation within solid media could also provide a more stable microenvironment in which the conjugative pilus can establish and maintain contact with recipient cells for the successful delivery of plasmid DNA. Furthermore, transfer in solid media may enhance the ability to transfer plasmids and chromosomes greater than 100 kbp. Using our optimized method, plasmids of varying sizes were tested for transfer from Escherichia coli to Saccharomyces cerevisiae. We demonstrated that there was no significant change in conjugation frequency when plasmid size increased from 56.5 to 138.6 kbp in length. Finally, we established an efficient PCR-based synthesis protocol to generate custom conjugative plasmids.

SUBMITTER: Soltysiak MPM

PROVIDER: S-EPMC6829330 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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Trans-Kingdom Conjugation within Solid Media from <i>Escherichia coli</i> to <i>Saccharomyces cerevisiae</i>.

Soltysiak Maximillian P M MPM Meaney Rebecca S RS Hamadache Samir S Janakirama Preetam P Edgell David R DR Karas Bogumil J BJ

International journal of molecular sciences 20191021 20

Conjugation is a bacterial mechanism for DNA transfer from a donor cell to a wide range of recipients, including both prokaryotic and eukaryotic cells. In contrast to conventional DNA delivery techniques, such as electroporation and chemical transformation, conjugation eliminates the need for DNA extraction, thereby preventing DNA damage during isolation. While most established conjugation protocols allow for DNA transfer in liquid media or on a solid surface, we developed a procedure for conjug ...[more]

PMID: 31640164

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Project description:Heritable variation in gene expression is common within species. Much of this variation is due to genetic differences outside of the gene with altered expression and is trans-acting. This trans-regulatory variation is often polygenic, with individual variants typically having small effects, making the genetic architecture and evolution of trans-regulatory variation challenging to study. Consequently, key questions about trans-regulatory variation remain, including the variability of trans-regulatory variation within a species, how selection affects trans-regulatory variation, and how trans-regulatory variants are distributed throughout the genome and within a species. To address these questions, we isolated and measured trans-regulatory differences affecting TDH3 promoter activity among 56 strains of Saccharomyces cerevisiae, finding that trans-regulatory backgrounds varied approximately twofold in their effects on TDH3 promoter activity. Comparing this variation to neutral models of trans-regulatory evolution based on empirical measures of mutational effects revealed that despite this variability in the effects of trans-regulatory backgrounds, stabilizing selection has constrained trans-regulatory differences within this species. Using a powerful quantitative trait locus mapping method, we identified ∼100 trans-acting expression quantitative trait locus in each of three crosses to a common reference strain, indicating that regulatory variation is more polygenic than previous studies have suggested. Loci altering expression were located throughout the genome, and many loci were strain specific. This distribution and prevalence of alleles is consistent with recent theories about the genetic architecture of complex traits. In all mapping experiments, the nonreference strain alleles increased and decreased TDH3 promoter activity with similar frequencies, suggesting that stabilizing selection maintained many trans-acting variants with opposing effects. This variation may provide the raw material for compensatory evolution and larger scale regulatory rewiring observed in developmental systems drift among species.

Project description:BACKGROUND: Heterologous microbial production of rare plant terpenoids of medicinal or industrial interest is attracting more and more attention but terpenoid yields are still low. Escherichia coli and Saccharomyces cerevisiae are the most widely used heterologous hosts; a direct comparison of both hosts based on experimental data is difficult though. Hence, the terpenoid pathways of E. coli (via 1-deoxy-D-xylulose 5-phosphate, DXP) and S. cerevisiae (via mevalonate, MVA), the impact of the respective hosts metabolism as well as the impact of different carbon sources were compared in silico by means of elementary mode analysis. The focus was set on the yield of isopentenyl diphosphate (IPP), the general terpenoid precursor, to identify new metabolic engineering strategies for an enhanced terpenoid yield. RESULTS: Starting from the respective precursor metabolites of the terpenoid pathways (pyruvate and glyceraldehyde-3-phosphate for the DXP pathway and acetyl-CoA for the MVA pathway) and considering only carbon stoichiometry, the two terpenoid pathways are identical with respect to carbon yield. However, with glucose as substrate, the MVA pathway has a lower potential to supply terpenoids in high yields than the DXP pathway if the formation of the required precursors is taken into account, due to the carbon loss in the formation of acetyl-CoA. This maximum yield is further reduced in both hosts when the required energy and reduction equivalents are considered. Moreover, the choice of carbon source (glucose, xylose, ethanol or glycerol) has an effect on terpenoid yield with non-fermentable carbon sources being more promising. Both hosts have deficiencies in energy and redox equivalents for high yield terpenoid production leading to new overexpression strategies (heterologous enzymes/pathways) for an enhanced terpenoid yield. Finally, several knockout strategies are identified using constrained minimal cut sets enforcing a coupling of growth to a terpenoid yield which is higher than any yield published in scientific literature so far. CONCLUSIONS: This study provides for the first time a comprehensive and detailed in silico comparison of the most prominent heterologous hosts E. coli and S. cerevisiae as terpenoid factories giving an overview on several promising metabolic engineering strategies paving the way for an enhanced terpenoid yield.

Project description:BackgroundEndoglucanases from thermophilic microorganisms are a valuable resource as they can be used in a wide variety of biotechnological applications including the valorisation of biomass and the production of biofuels. In the present work we analysed the metagenome from the hot spring Muiño da Veiga, located in the northwest of Spain (in the Galicia region), in search for novel thermostable endoglucanases.ResultsSequence analysis of the metagenome revealed a promising enzyme (Cel776). Predictions on protein structure and conserved amino acid sequences were conducted, as well as expression in heterologous systems with Escherichia coli and Saccharomyces cerevisiae as the host. Cel776Ec was correctly expressed and purified by taking advantage of the His-Tag system, with a yield of 0.346 U/mL in the eluted fraction. Cel776Sc was expressed extracellulary and was easily recovered from the supernatant without the need of further purification, requiring only a concentration step by ultrafiltration, with a significantly higher yield of 531.95 U/mL, revealing a much more suitable system for production of large amounts of the enzyme. Their biochemical characterization revealed biotechnologically interesting enzymes. Both Cel776Ec and Cel776Sc had an optimal temperature of 80 °C and optimal pH of 5. Cel776Ec exhibited high thermostability maintaining its activity for 24 h at 60 °C and maintained its activity longer than Cel776Sc at increasing incubation temperatures. Moreover, its substrate specificity allowed the degradation of both cellulose and xylan. Whereas Cel776Ec was more active in the presence of calcium and magnesium, manganese was found to increase Cel776Sc activity. A stronger inhibitory effect was found for Cel776Ec than Cel776Sc adding detergent SDS to the reaction mix, whereas EDTA only significantly affected Cel776Sc activity.ConclusionsOur study reports the discovery of a new promising biocatalyst for its application in processes, such as the production of biofuel and the saccharification of plant biomass, due to its bifunctional enzymatic activity as an endoglucanase and as a xylanase, as well as highlights the advantages of a yeast expression system over bacteria.

Dataset Information

Trans-Kingdom Conjugation within Solid Media from Escherichia coli to Saccharomyces cerevisiae.

Publications

Trans-Kingdom Conjugation within Solid Media from <i>Escherichia coli</i> to <i>Saccharomyces cerevisiae</i>.

Similar Datasets

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