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Transkingdom genetic transfer from Escherichia coli to Saccharomyces cerevisiae as a simple gene introduction tool.

ABSTRACT: Transkingdom conjugation (TKC) permits transfer of DNA from bacteria to eukaryotic cells using a bacterial conjugal transfer system. However, it is not clear whether the process of DNA acceptance in a recipient eukaryote is homologous to the process of conjugation between bacteria. TKC transfer requires mobilizable shuttle vectors that are capable of conjugal transfer and replication in the donor and recipient strains. Here, we developed TKC vectors derived from plasmids belonging to the IncP and IncQ groups. We also investigated forms of transfer of these vectors from Escherichia coli into Saccharomyces cerevisiae to develop TKC as a simple gene introduction method. Both types of vectors were transferred precisely, conserving the origin of transfer (oriT) sequences, but IncP-based vectors appeared to be more efficient than an IncQ-based vector. Interestingly, unlike in agrobacterial T-DNA (transfer DNA) transfer, the efficiency of TKC transfer was similar between a wild-type yeast strain and DNA repair mutants defective in homologous recombination (rad51? and rad52?) or nonhomologous end joining (rad50?, yku70?, and lig4?). Lastly, a shuttle vector with two repeats of IncP-type oriT (oriT(P)) sequences flanking a marker gene was constructed. TKC transfer of this vector resulted in precise excision of both the oriT(P) loci as well as the marker gene, albeit at a low frequency of 17% of all transconjugants. This feature would be attractive in biotechnological applications of TKC. Taken together, these results strongly suggest that in contrast to agrobacterial T-DNA transfer, the circularization of vector single-stranded DNA occurs either before or after transfer but requires a factor(s) from the donor. TKC is a simple method of gene transfer with possible applications in yeast genetics and biotechnology.

SUBMITTER: Moriguchi K

PROVIDER: S-EPMC3697487 | biostudies-literature | 2013 Jul

REPOSITORIES: biostudies-literature

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Transkingdom genetic transfer from Escherichia coli to Saccharomyces cerevisiae as a simple gene introduction tool.

Moriguchi Kazuki K Edahiro Noritaka N Yamamoto Shinji S Tanaka Katsuyuki K Kurata Nori N Suzuki Katsunori K

Applied and environmental microbiology 20130510 14

Transkingdom conjugation (TKC) permits transfer of DNA from bacteria to eukaryotic cells using a bacterial conjugal transfer system. However, it is not clear whether the process of DNA acceptance in a recipient eukaryote is homologous to the process of conjugation between bacteria. TKC transfer requires mobilizable shuttle vectors that are capable of conjugal transfer and replication in the donor and recipient strains. Here, we developed TKC vectors derived from plasmids belonging to the IncP an ...[more]

PMID: 23666333

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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.

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Transkingdom genetic transfer from Escherichia coli to Saccharomyces cerevisiae as a simple gene introduction tool.

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Transkingdom genetic transfer from Escherichia coli to Saccharomyces cerevisiae as a simple gene introduction tool.

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