Improved glycerol utilization by a triacylglycerol-producing Rhodococcus opacus strain for renewable fuels

Background: Glycerol generated during renewable fuel production processes is potentially an attractive substrate for the production of value-added materials by fermentation. An engineered strain MITXM-61 of the oleaginous bacterium Rhodococcus opacus produces large amounts of intracellular triacylglycerols (TAGs) for lipid-based biofuels on high concentrations of glucose and xylose. However, on glycerol medium, MITXM-61 does not produce TAGs and grows poorly. The aim of the present work was to construct a TAG-producing R. opacus strain capable of high-cell-density cultivation at high glycerol concentrations. Results: An adaptive evolution strategy was applied to improve the conversion of glycerol to TAGs in R. opacus MITXM-61. An evolved strain, MITGM-173, grown on a defined medium with 16 g L[superscript −1] glycerol, produced 2.3 g L[superscript −1] of TAGs, corresponding to 40.4% of the cell dry weight (CDW) and 0.144 g g[superscript −1] of TAG yield per glycerol consumed. MITGM-173 was able to grow on high concentrations (greater than 150 g L[superscript −1]) of glycerol. Cultivated in a medium containing an initial concentration of 20 g L[superscript −1] glycerol, 40 g L[superscript −1] glucose, and 40 g L[superscript −1] xylose, MITGM-173 was capable of simultaneously consuming the mixed substrates and yielding 13.6 g L[superscript −1] of TAGs, representing 51.2% of the CDM. In addition, when 20 g L[superscript −1] glycerol was pulse-loaded into the culture with 40 g L[superscript −1] glucose and 40 g L[superscript −1] xylose at the stationary growth phase, MITGM-173 produced 14.3 g L[superscript −1] of TAGs corresponding to 51.1% of the CDW although residual glycerol in the culture was observed. The addition of 20 g L[superscript −1] glycerol in the glucose/xylose mix resulted in a TAG yield per glycerol consumed of 0.170 g g[superscript −1] on the initial addition and 0.279 g g[superscript −1] on the pulse addition of glycerol. Conclusion: We have generated a TAG-producing R. opacus MITGM-173 strain that shows significantly improved glycerol utilization in comparison to the parental strain. The present study demonstrates that the evolved R. opacus strain shows significant promise for developing a cost-effective bioprocess to generate advanced renewable fuels from mixed sugar feedstocks supplemented with glycerol.Sweetwater Energy, Inc.MIT Energy Initiativ

Harnessing the Biosphere: Natural Products and Biotechnology

What do the organisms of the biosphere, specifically microorganisms, have to offer to biotechnological endeavors? In this course we will focus on the production of biomolecules using microbial systems. We will discuss potential growth substrates (such as agricultural waste and carbon dioxide) that can be used and learn about both established and cutting-edge manipulation techniques in the field of synthetic biology. We will also cover the production of biofuels, bioplastics, amino acids (e.g. lysine), food additives (e.g. monosodium glutamate, MSG), specialty chemicals (e.g. succinate), and biopharmaceuticals (e.g. plasmids for gene therapy). This course is one of many Advanced Undergraduate Seminars offered by the Biology Department at MIT. These seminars are tailored for students with an interest in using primary research literature to discuss and learn about current biological research in a highly interactive setting. Many instructors of the Advanced Undergraduate Seminars are postdoctoral scientists with a strong interest in teaching

Characterization and modification of enzymes in the 2-ketoisovalerate biosynthesis pathway of Ralstonia eutropha H16

2-Ketoisovalerate is an important cellular intermediate for the synthesis of branched-chain amino acids as well as other important molecules, such as pantothenate, coenzyme A, and glucosinolate. This ketoacid can also serve as a precursor molecule for the production of biofuels, pharmaceutical agents, and flavor agents in engineered organisms, such as the betaproteobacterium Ralstonia eutropha. The biosynthesis of 2-ketoisovalerate from pyruvate is carried out by three enzymes: acetohydroxyacid synthase (AHAS, encoded by ilvBH), acetohydroxyacid isomeroreductase (AHAIR, encoded by ilvC), and dihydroxyacid dehydratase (DHAD, encoded by ilvD). In this study, enzymatic activities and kinetic parameters were determined for each of the three R. eutropha enzymes as heterologously purified proteins. AHAS, which serves as a gatekeeper for the biosynthesis of all three branched-chain amino acids, demonstrated the tightest regulation through feedback inhibition by l-valine (IC[subscript 50] = 1.2 mM), l-isoleucine (IC[subscript 50] = 2.3 mM), and l-leucine (IC[subscript 50] = 5.4 mM). Intermediates in the valine biosynthesis pathway also exhibit feedback inhibitory control of the AHAS enzyme. In addition, AHAS has a very weak affinity for pyruvate (K[subscript M] = 10.5 μM) and is highly selective towards 2-ketobutyrate (R = 140) as a second substrate. AHAIR and DHAD are also inhibited by the branched-chain amino acids, although to a lesser extent when compared to AHAS. Experimental evolution and rational site-directed mutagenesis revealed mutants of the regulatory subunit of AHAS (IlvH) (N11S, T34I, A36V, T104S, N11F, G14E, and N29H), which, when reconstituted with wild-type IlvB, lead to AHAS having reduced valine, leucine, and isoleucine sensitivity. The study of the kinetics and inhibition mechanisms of R. eutropha AHAS, AHAIR, and DHAD has shed light on interactions between these enzymes and the products they produce; it, therefore, can be used to engineer R. eutropha strains with optimal production of 2-ketoisovalerate for value-added materials

Chronic Pancreatitis and Systemic Inflammatory Response Syndrome Prevent Impact of Chemotherapy with Gemcitabine in a Genetically Engineered Mouse Model of Pancreatic Cancer

BACKGROUND AND AIMS: BACKGROUND AND AIMSGemcitabine is the standard therapy for patients with pancreatic cancer with metastatic disease. Patients with metastatic pancreatic cancer presenting with increased values of C-reactive protein do not respond to gemcitabine. So far, no studies have evaluated the correlation between chronic pancreatitis, systemic inflammatory response syndrome, and the loss of chemotherapeutic benefit. METHODS: Pdx-1-Cre;LSL-KrasG12D/+;LSL-Trp53R172H/+ mice were assigned into four groups: 1) Sixteen animals received a daily intraperitoneal injection of caerulein from their ninth week of life on. 2) Sixteen mice were additionally given gemcitabine. 3) Twelve animals received gemcitabine only. 4) Saline-treated control group. Furthermore, human Paca44 pancreatic ductal adenocarcinoma cells were seeded and cultured in 0.5% FBS containing growth medium plus/minus 1 μM gemcitabine plus/minus recombinant human interleukin (IL)-6. RESULTS: Induced systemic inflammatory response syndrome and a mild chronic pancreatitis diminished the beneficial effects of gemcitabine upon median overall survival. In median, the monogemcitabine group survived 191 days, whereas the caerulein-mono group survived 114, the control group 121, and the caerulein gemcitabine group 127 days (P < .05). In vitro, the induction of STAT3 phosphorylation by recombinant human IL-6 promoted pancreatic ductal adenocarcinoma cell survival during gemcitabine treatment. CONCLUSION: We could demonstrate for the first time that an improvement in median overall survival with gemcitabine is significantly abolished by a persistent mild chronic pancreatitis and a systemic inflammatory response syndrome. In particular, the inflammation biomarkers C-reactive protein, IL-6, and IL-1α could indicate the prognostic benefit of gemcitabine chemotherapy and should now be tested in prospective patient-controlled trials

[clc] Kinetic and stoichiometric characterization of organoautotrophic growth of Ralstonia eutropha on formic acid in fed-batch and continuous cultures

Formic acid, acting as both carbon and energy source, is a safe alternative to a carbon dioxide, hydrogen and dioxygen mix for studying the conversion of carbon through the Calvin–Benson–Bassham (CBB) cycle into value-added chemical compounds by non-photosynthetic microorganisms. In this work, organoautotrophic growth of Ralstonia eutropha on formic acid was studied using an approach combining stoichiometric modeling and controlled cultures in bioreactors. A strain deleted of its polyhydroxyalkanoate production pathway was used in order to carry out a physiological characterization. The maximal growth yield was determined at 0.16 Cmole Cmole[superscript −1] in a formate-limited continuous culture. The measured yield corresponded to 76% to 85% of the theoretical yield (later confirmed in pH-controlled fed-batch cultures). The stoichiometric study highlighted the imbalance between carbon and energy provided by formic acid and explained the low growth yields measured. Fed-batch cultures were also used to determine the maximum specific growth rate (μ[subscript max] = 0.18 h[superscript −1]) and to study the impact of increasing formic acid concentrations on growth yields. High formic acid sensitivity was found in R eutropha since a linear decrease in the biomass yield with increasing residual formic acid concentrations was observed between 0 and 1.5 g l[superscript −1].United States. Advanced Research Projects Agency-EnergyMIT-France Seed Fund (Grant)Centre National de la Recherche Scientifique (France) (French Ministry of Higher Education and Research. Post-doctoral Grant