The bacterium Pseudomonas putida KT2440 is gaining considerable interest as a microbial platform for biotechnological valorization of polymeric organic materials, such as lignocellulosic residues or plastics. However, P. putida on its own cannot make much use of such complex substrates, mainly because it lacks an efficient extracellular depolymerizing apparatus. We seek to address this limitation by adopting a recombinant cellulosome strategy for this host. In this work, we report an essential step in this endeavor-a display of designer enzyme-anchoring protein "scaffoldins", encompassing cohesin binding domains from divergent cellulolytic bacterial species on the P. putida surface. Two P. putida chassis strains, EM42 and EM371, with streamlined genomes and differences in the composition of the outer membrane were employed in this study. Scaffoldin variants were optimally delivered to their surface with one of four tested autotransporter systems (Ag43 from Escherichia coli), and the efficient display was confirmed by extracellular attachment of chimeric β-glucosidase and fluorescent proteins. Our results not only highlight the value of cell surface engineering for presentation of recombinant proteins on the envelope of Gram-negative bacteria but also pave the way toward designer cellulosome strategies tailored for P. putida.
- MeSH
- beta-glukosidasa metabolismus MeSH
- celulosa metabolismus MeSH
- celulozómy metabolismus MeSH
- chromozomální proteiny, nehistonové chemie MeSH
- Escherichia coli metabolismus MeSH
- genom bakteriální * MeSH
- membránové proteiny metabolismus MeSH
- metabolické inženýrství metody MeSH
- proteinové domény MeSH
- proteiny buněčného cyklu chemie MeSH
- proteiny z Escherichia coli metabolismus MeSH
- Pseudomonas putida genetika metabolismus MeSH
- rekombinantní proteiny metabolismus MeSH
- vnější bakteriální membrána metabolismus MeSH
- zelené fluorescenční proteiny metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Anaerobic microorganisms (anaerobes) possess a fascinating metabolic versatility. This characteristic makes anaerobes interesting candidates for physiological studies and utilizable as microbial cell factories. To investigate the physiological characteristics of an anaerobic microbial population, yield, productivity, specific growth rate, biomass production, substrate uptake, and product formation are regarded as essential variables. The determination of those variables in distinct cultivation systems may be achieved by using different techniques for sampling, measuring of growth, substrate uptake, and product formation kinetics. In this review, a comprehensive overview of methods is presented, and the applicability is discussed in the frame of anaerobic microbiology and biotechnology.
Plant secondary metabolism evolved in the context of highly organized and differentiated cells and tissues, featuring massive chemical complexity operating under tight environmental, developmental and genetic control. Biotechnological demand for natural products has been continuously increasing because of their significant value and new applications, mainly as pharmaceuticals. Aseptic production systems of plant secondary metabolites have improved considerably, constituting an attractive tool for increased, stable and large-scale supply of valuable molecules. Surprisingly, to date, only a few examples including taxol, shikonin, berberine and artemisinin have emerged as success cases of commercial production using this strategy. The present review focuses on the main characteristics of plant specialized metabolism and their implications for current strategies used to produce secondary compounds in axenic cultivation systems. The search for consonance between plant secondary metabolism unique features and various in vitro culture systems, including cell, tissue, organ, and engineered cultures, as well as heterologous expression in microbial platforms, is discussed. Data to date strongly suggest that attaining full potential of these biotechnology production strategies requires being able to take advantage of plant specialized metabolism singularities for improved target molecule yields and for bypassing inherent difficulties in its rational manipulation.
- MeSH
- artemisininy izolace a purifikace metabolismus MeSH
- axenická kultura MeSH
- berberin izolace a purifikace metabolismus MeSH
- biologické přípravky izolace a purifikace metabolismus MeSH
- biotechnologie metody MeSH
- buněčné kultury MeSH
- fytonutrienty biosyntéza izolace a purifikace MeSH
- metabolické inženýrství metody MeSH
- naftochinony izolace a purifikace metabolismus MeSH
- paclitaxel biosyntéza izolace a purifikace MeSH
- rostlinné buňky chemie metabolismus MeSH
- rostliny chemie genetika metabolismus MeSH
- sekundární metabolismus MeSH
- techniky tkáňových kultur MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
Beer production is one of the oldest known traditional biotechnological processes, but is nowadays facing increasing demands not only for enhanced product quality, but also for improved production economics. Targeted genetic modification of a yeast strain is one way to increase beer quality and to improve the economics of beer production. In this review we will present current knowledge on traditional approaches for improving brewing strains and for rational metabolic engineering. These research efforts will, in the near future, lead to the development of a wider range of industrial strains that should increase the diversity of commercial beers.
- MeSH
- CRISPR-Cas systémy MeSH
- fermentace MeSH
- genetické inženýrství metody MeSH
- geneticky modifikované mikroorganismy genetika fyziologie MeSH
- genom fungální MeSH
- lidé MeSH
- metabolické inženýrství metody MeSH
- pivo * MeSH
- plazmidy genetika MeSH
- regulace genové exprese u hub MeSH
- Saccharomyces cerevisiae genetika fyziologie MeSH
- veřejné mínění MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
The synthesis of renewable bioproducts using photosynthetic microorganisms holds great promise. Sustainable industrial applications, however, are still scarce and the true limits of phototrophic production remain unknown. One of the limitations of further progress is our insufficient understanding of the quantitative changes in photoautotrophic metabolism that occur during growth in dynamic environments. We argue that a proper evaluation of the intra- and extracellular factors that limit phototrophic production requires the use of highly-controlled cultivation in photobioreactors, coupled to real-time analysis of production parameters and their evaluation by predictive computational models. In this addendum, we discuss the importance and challenges of systems biology approaches for the optimization of renewable biofuels production. As a case study, we present the utilization of a state-of-the-art experimental setup together with a stoichiometric computational model of cyanobacterial metabolism for quantitative evaluation of ethylene production by a recombinant cyanobacterium Synechocystis sp. PCC 6803.
Anthropogenic halogenated compounds were unknown to nature until the industrial revolution, and microorganisms have not had sufficient time to evolve enzymes for their degradation. The lack of efficient enzymes and natural pathways can be addressed through a combination of protein and metabolic engineering. We have assembled a synthetic route for conversion of the highly toxic and recalcitrant 1,2,3-trichloropropane to glycerol in Escherichia coli, and used it for a systematic study of pathway bottlenecks. Optimal ratios of enzymes for the maximal production of glycerol, and minimal toxicity of metabolites were predicted using a mathematical model. The strains containing the expected optimal ratios of enzymes were constructed and characterized for their viability and degradation efficiency. Excellent agreement between predicted and experimental data was observed. The validated model was used to quantitatively describe the kinetic limitations of currently available enzyme variants and predict improvements required for further pathway optimization. This highlights the potential of forward engineering of microorganisms for the degradation of toxic anthropogenic compounds.
- MeSH
- bakteriální proteiny MeSH
- biodegradace * MeSH
- Escherichia coli genetika metabolismus MeSH
- genetické inženýrství MeSH
- glycerol analýza metabolismus MeSH
- látky znečišťující životní prostředí analýza metabolismus MeSH
- metabolické inženýrství metody MeSH
- metabolické sítě a dráhy MeSH
- počítačová simulace MeSH
- propan analogy a deriváty analýza metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- MeSH
- Actinomycetales * metabolismus růst a vývoj MeSH
- herbicidy farmakologie izolace a purifikace MeSH
- kokcidiostatika farmakologie izolace a purifikace MeSH
- metabolické inženýrství * metody MeSH
- organofosforové sloučeniny farmakologie MeSH
- protinádorová antibiotika izolace a purifikace farmakologie izolace a purifikace MeSH
- protinádorové antimetabolity farmakologie izolace a purifikace MeSH
- vztahy mezi strukturou a aktivitou MeSH
