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

Studies on the production of branched-chain alcohols in engineered Ralstonia eutropha

Wild-type Ralstonia eutropha H16 produces polyhydroxybutyrate (PHB) as an intracellular carbon storage material during nutrient stress in the presence of excess carbon. In this study, the excess carbon was redirected in engineered strains from PHB storage to the production of isobutanol and 3-methyl-1-butanol (branched-chain higher alcohols). These branched-chain higher alcohols can directly substitute for fossil-based fuels and be employed within the current infrastructure. Various mutant strains of R. eutropha with isobutyraldehyde dehydrogenase activity, in combination with the overexpression of plasmid-borne, native branched-chain amino acid biosynthesis pathway genes and the overexpression of heterologous ketoisovalerate decarboxylase gene, were employed for the biosynthesis of isobutanol and 3-methyl-1-butanol. Production of these branched-chain alcohols was initiated during nitrogen or phosphorus limitation in the engineered R. eutropha. One mutant strain not only produced over 180 mg/L branched-chain alcohols in flask culture, but also was significantly more tolerant of isobutanol toxicity than wild-type R. eutropha. After the elimination of genes encoding three potential carbon sinks (ilvE, bkdAB, and aceE), the production titer improved to 270 mg/L isobutanol and 40 mg/L 3-methyl-1-butanol. Semicontinuous flask cultivation was utilized to minimize the toxicity caused by isobutanol while supplying cells with sufficient nutrients. Under this semicontinuous flask cultivation, the R. eutropha mutant grew and produced more than 14 g/L branched-chain alcohols over the duration of 50 days. These results demonstrate that R. eutropha carbon flux can be redirected from PHB to branched-chain alcohols and that engineered R. eutropha can be cultivated over prolonged periods of time for product biosynthesis.United States. Dept. of EnergyUnited States. Advanced Research Projects Agency-Energ

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

Metabolic Engineering Corynebacterium glutamicum to Produce Triacylglycerols.

Plassmeier J, Li Y, Rückert C, Sinskey AJ. Metabolic Engineering Corynebacterium glutamicum to Produce Triacylglycerols. Metab Eng. 2016;33:86-97.In this study, we metabolically engineered Corynebacterium glutamicum to produce triacylglycerols (TAGs) by completing and constraining a de novo TAG biosynthesis pathway. First, the plasmid pZ8_TAG4 was constructed which allows the heterologous expression of four genes: three (atf1 and atf2, encoding the diacylglycerol acyltransferase; pgpB, encoding the phosphatidic acid phosphatase) to complete the TAG biosynthesis pathway, and one gene (tadA) for lipid body assembly. Second, we applied four metabolic strategies to increase TAGs accumulation: (i) boosting precursor supply by heterologous expression of tesA (encoding thioesterase to form free fatty acid to reduce the feedback inhibition by acyl-ACP) and fadD (encoding acyl-CoA synthetase to enhance acyl-CoA supply), (ii) reduction of TAG degradation and precursor consumption by deleting four cellular lipases (cg0109, cg0110, cg1676 and cg1320) and the diacylglycerol kinase (cg2849), (iii) enhancement of fatty acid biosynthesis by deletion of fasR (cg2737, TetR-type transcriptional regulator of genes for the fatty acid biosynthesis), and (iv) elimination of the observed by-product formation of organic acids by blocking the acetic acid (pqo) and lactic acid production (ldh) pathways. The final strain (CgTesRtcEfasEbp/pZ8_TAG4) achieved a 7.5% yield of total fatty acids (2.38±0.05g/L intracellular fatty acids and 0.64±0.09g/L extracellular fatty acids) from 4% glucose in shake flasks after process optimization. This corresponds to maximum intracellular fatty acids content of 17.8±0.5% of the dry cell

Metabolic Engineering Corynebacterium glutamicum to Produce Triacylglycerols.

Plassmeier J, Li Y, Rückert C, Sinskey AJ. Metabolic Engineering Corynebacterium glutamicum to Produce Triacylglycerols. Metab Eng. 2016;33:86-97.In this study, we metabolically engineered Corynebacterium glutamicum to produce triacylglycerols (TAGs) by completing and constraining a de novo TAG biosynthesis pathway. First, the plasmid pZ8_TAG4 was constructed which allows the heterologous expression of four genes: three (atf1 and atf2, encoding the diacylglycerol acyltransferase; pgpB, encoding the phosphatidic acid phosphatase) to complete the TAG biosynthesis pathway, and one gene (tadA) for lipid body assembly. Second, we applied four metabolic strategies to increase TAGs accumulation: (i) boosting precursor supply by heterologous expression of tesA (encoding thioesterase to form free fatty acid to reduce the feedback inhibition by acyl-ACP) and fadD (encoding acyl-CoA synthetase to enhance acyl-CoA supply), (ii) reduction of TAG degradation and precursor consumption by deleting four cellular lipases (cg0109, cg0110, cg1676 and cg1320) and the diacylglycerol kinase (cg2849), (iii) enhancement of fatty acid biosynthesis by deletion of fasR (cg2737, TetR-type transcriptional regulator of genes for the fatty acid biosynthesis), and (iv) elimination of the observed by-product formation of organic acids by blocking the acetic acid (pqo) and lactic acid production (ldh) pathways. The final strain (CgTesRtcEfasEbp/pZ8_TAG4) achieved a 7.5% yield of total fatty acids (2.38±0.05g/L intracellular fatty acids and 0.64±0.09g/L extracellular fatty acids) from 4% glucose in shake flasks after process optimization. This corresponds to maximum intracellular fatty acids content of 17.8±0.5% of the dry cell

Engineering L-arabinose metabolism in triacylglycerol-producing Rhodococcus opacus for lignocellulosic fuel production

Kurosawa K, Plassmeier J, Kalinowski J, Rückert C, Sinskey AJ. Engineering L-arabinose metabolism in triacylglycerol-producing Rhodococcus opacus for lignocellulosic fuel production. Metabolic engineering. 2015;30:89-95.: Advanced biofuels from lignocellulosic biomass have been considered as a potential solution for the issues of energy sustainability and environmental protection. Triacylglycerols (TAGs) are potential precursors for the production of lipid-based liquid biofuels. Rhodococcus opacus PD630 can accumulate large amounts of TAGs when grown under physiological conditions of high carbon and low nitrogen. However, R. opacus PD630 does not utilize the sugar L-arabinose present in lignocellulosic hydrolysates. Here, we report the engineering of R. opacus to produce TAGs on L-arabinose. We constructed a plasmid (pASC8057) harboring araB, araD and araA genes derived from a Streptomyces bacterium, and introduced the genes into R. opacus PD630. One of the engineered strains, MITAE-348, was capable of growing on high concentrations (up to 100g/L) of L-arabinose. MITAE-348 was grown in a defined medium containing 16g/L L-arabinose or a mixture of 8g/L L-arabinose and 8g/L D-glucose. In a stationary phase occurring 3 days post-inoculation, the strain was able to completely utilize the sugar, and yielded 2.0g/L for L-arabinose and 2.2g/L for L-arabinose/D-glucose of TAGs, corresponding to 39.7% or 42.0%, respectively, of the cell dry weight

[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

FarR, a putative regulator of amino acid metabolism in Corynebacterium glutamicum

Haenssler E, Mueller T, Jessberger N, et al. FarR, a putative regulator of amino acid metabolism in Corynebacterium glutamicum. APPLIED MICROBIOLOGY AND BIOTECHNOLOGY. 2007;76(3):625-632.With the publication of the Corynebacterium glutamicum genome sequence, a global characterization of genes controlled by functionally uncharacterized transcriptional regulators became possible. We used DNA microarrays in combination with gel retardation experiments to study gene regulation by FarR, a HutC/FarR-type regulator of the GntR family. Based on our results, FarR seems to be involved in the regulation of amino acid biosynthesis in C. glutamicum. Especially, transcript levels of the arg cluster and the gdh gene are influenced by deletion of farR