Keasling 5657 Metabolic engineering has the potential to produce from simple, readily available, inexpensive starting materials a large number of chemicals that are currently derived from nonrenewable resources or limited natural resources.
List of plasmids and strains used in this study Chemicals. Unless indicated otherwise, commercial reagents, enzymes, and coenzymes were supplied by Sigma Chemical Company St. The medium contained the following components: For agar plates, 0.
To make competent cells, to prepare cell lysates for enzyme assays and daily maintenance, and to determine isobutanol production on cellobiose, the strains were grown in modified VM medium.
Stock cultures of C. Cell transformation was conducted as described previously 11 with some modifications. Cells were grown for 17 to 24 h in ml cultures of modified VM medium to late exponential phase optical density at nm [OD], 0.
The cells were washed twice with cold electroporation buffer mM sucrose, 1 mM MgCl2, 5 mM sodium phosphate buffer, pH 7. The electroporated cells were transferred to 10 ml of fresh modified VM medium.
Bacterial growth was measured spectrophotometrically at nm. For cultures containing cellulose, the cellulose was allowed to settle for at least 2 h before samples were taken for measurement. The produced alcohol compounds were quantified by a gas chromatograph GC with a flame ionization detector.
Helium was used as the carrier gas, with 9. Supernatant of culture broth 0. Pentanol was used as the internal standard. The cells were grown for 17 to 24 h in ml cultures of modified VM medium to late exponential phase OD, 0. The cells were harvested, washed in 50 mM potassium phosphate buffer, pH 7.
Crude extract was prepared under aerobic conditions with 0. The concentration of acetoin produced was determined by a standard curve created using pure acetoin. To measure the reduction of 2-acetolactate to 2,3-dihydroxy-isovalerate, the oxidation of NADPH was monitored by a decrease in absorbance at nm.
The substrate, 2-acetolactate, was first produced in a separate reaction as described for the Als assay using purified, heterogeneously expressed Bacillus subtilis AlsS in E. The IlvD assay was performed as described previously The substrate, 2,3-dihydroxy-isovalerate, was synthesized as described previously 6.
The samples were then spun down for 1 min to remove coagulated protein. Sample absorbances were measured at nm. Standard curves based on known amounts of 2-ketoisovalerate were generated. The decarboxylation activity of Kivd was assayed as described previously 34with some modifications.
ADH6 was isolated as previously described The assay mixture contained 0. The reactions were started by the addition of the 2-ketoisovalerate. In order to achieve direct isobutanol production from pyruvate, the genes encoding B. These specific genes were chosen because they were the same genes utilized for isobutanol production in E.
The different combinations of the genes Fig. The pathway for isobutanol production in C. The asterisk indicates the adenine insertion in the alsS gene sequence.
The activities of the first three enzymes in the isobutanol pathway were examined by transforming plasmids expressing alsS or alsS ilvCD into C.
The same results were observed after repeated transformation efforts. Due to the fact that alsS and alsS ilvCD transformants could not be obtained, the complete isobutanol pathway was then examined.
While transformants were obtained, sequence confirmation of the plasmid revealed that a single adenine insertion, which is not found in the wild-type alsS sequence, was present 54 bp downstream of the start ATG.
This single insertion, by shifting the reading frame, results in a downstream premature stop codon TGA and, subsequently, a truncated amino-acid protein Fig. The first bp of the alsS sequence with the adenine insertion mutation.
The frameshift mutation in the alsS sequence was a cause for great concern because of the effect it could have on AlsS activity.
Thus, to determine the activities of AlsS and the other enzymes expressed from the synthetic operon, enzymatic assays were performed on lysates of the C. Thus, despite the insertion mutation, the mutant retained a significant level of activity.Feb 19, · The Metabolic Engineering Conference is a leading conference for sharing the state-of-the-art developments and achievements made in the field of metabolic engineering over the last two years.
The theme of this year's conference is biological design and synthesis. The Metabolic Engineering Conference is the world-leading conference to share premium developments and achievements in the field.
The theme of this year's conference was .
Welcome to the Gregory Stephanopoulos research group at MIT! WHO WE ARE WHAT WE DO. Metabolic Engineering (MBE) is devoted to the publication of original research papers on the directed modulation of metabolic pathways for metabolite over production or the improvement of cellular properties. Papers describing native pathway engineering and synthesis of heterologous pathways for converting microorganisms into microbial cell. 2 Metabolic engineering defined and successes. Metabolic engineering embodies the manipulation of enzymatic, transport, and regulatory functions of a cell through recombinant DNA technologies with the goal of improving a cellular phenotype, often yield of a desired product.
The Metabolic Engineering Conference is the leading conference for sharing the state-of-the-art developments and achievements made in the field of metabolic engineering. The theme of this year's conference is Design, Synthesis and System Integration for Metabolic Engineering.
Metabolic Engineering of Clostridium cellulolyticum for Production of 1 Department of Chemical and Biomolecular Engineering, University of California, Los Angeles, Los Angeles, California 2 BioEnergy Science Here we present a metabolic engineering example for the development of a Clostridium cellulolyticum.
The Plant Metabolic Engineering GRC will be held for the first time in Europe, at the Renaissance Tuscany, Il Ciocco, Italy between June 16th and June 21st, The objective is to build stronger networks between those working on plant metabolic engineering in the USA, Europe and in Asia, and the European venue will attract new participants.
The Microbiome Metabolic Engineering theme will integrate knowledge and experimental approaches from microbial physiology, microbial biochemistry, microbial ecology, enzymology, nutrition, animal model development, toxicology and environmental health, and systems biology to gain a better understanding of the microbiome’s role in health, develop new methods to assess host-microbe interactions.