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

Funktion und Regulation des Propionatstoffwechsels in Corynebacterium glutamicum

Plassmeier J. Funktion und Regulation des Propionatstoffwechsels in Corynebacterium glutamicum. Bielefeld (Germany): Bielefeld University; 2010

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

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

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

Investigation of central carbon metabolism and the 2-methylcitrate cycle in Corynebacterium glutamicum by metabolic profiling using gas chromatography-mass spectrometry

Plassmeier J, Barsch A, Persicke M, Niehaus K, Kalinowski J. Investigation of central carbon metabolism and the 2-methylcitrate cycle in Corynebacterium glutamicum by metabolic profiling using gas chromatography-mass spectrometry. Journal of Biotechnology. 2007;130(4):354-363.The 2-methylcitrate cycle as the primary way to metabolize propionate was investigated using metabolic profiling. For this purpose, a fast harvesting procedure was applied in which cells growing in liquid minimal medium were harvested by a short centrifugation and freeze-dried. Subsequently, gas chromatography-mass spectrometry of polar extracts derivatized by MSTFA was employed for metabolite characterization. Routinely more than 300 different peaks were obtained in the chromatograms, and 74 substances were identified unequivocally by using pure standards. The procedure provided reliable data which closely relate to prior knowledge on flux distributions during growth on glucose and acetate as carbon sources. Propionate degradation via the 2-methylcitrate cycle was demonstrated on the metabolite level by the detection of the intermediates 2-methylcitrate and 2-methylisocitrate. Further characterization of the 2-methylcitrate cycle was carried out by comparing different mutant strains of this pathway. The growth deficit of a prpD2-mutant strain observed when propionate is added to a culture growing on acetate indicates that the toxic effect of propionate is based on the accumulation of 2-methylcitrate. It could also be shown that the 2-methylcitrate cycle is active in the absence of propionate and might fulfill house-keeping functions in the degradation of fatty acids or branched-chain amino acids. (c) 2007 Elsevier B.V. All rights reserved

Size exclusion chromatography-An improved method to harvest Corynebacterium glutamicum cells for the analysis of cytosolic metabolites

Persicke M, Plassmeier J, Neuweger H, Rückert C, Pühler A, Kalinowski J. Size exclusion chromatography-An improved method to harvest Corynebacterium glutamicum cells for the analysis of cytosolic metabolites. Journal of Biotechnology. 2011;154(2-3):171-178.The efficient separation of Corynebacterium glutamicum cells from culture medium by size exclusion chromatography (SEC) is presented. Residue analysis demonstrated that this method effectively depletes extracellular compounds. For evaluation, SEC was compared with the common methods cold methanol treatment, fast centrifugation and fast filtration. For this purpose, samples of C. glutamicum cells from fermenter cultures were harvested and subjected to a metabolome analysis. In particular, the wild type strain C. glutamicum ATCC13032 and the lysine production strain C. glutamicum DM1730 were grown in a minimal or in a complex medium. Comparison of metabolite pool sizes after harvesting C. glutamicum cells by the methods mentioned above by gas chromatography coupled to mass spectrometry (GC-MS) revealed that SEC is the most suitable method when intracellular metabolite pools are to be measured during growth in complex media or in the presence of significant amounts of secreted metabolites. In contrast to the other methods tested, the SEC method turned out to be fast and able to remove extracellular compounds almost completely. (C) 2010 Elsevier B.V. All rights reserved

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

Molecular characterization of PrpR, the transcriptional activator of propionate catabolism in Corynebacterium glutamicum

Plassmeier J, Persicke M, Pühler A, Sterthoff C, Rückert C, Kalinowski J. Molecular characterization of PrpR, the transcriptional activator of propionate catabolism in Corynebacterium glutamicum. Journal of Biotechnology. 2011;159(1-2):1-11.The 2-methylcitrate cycle is used to metabolize propionate in Corynebacterium glutamicum. The regulator, PrpR (Cg0800), of the prpDBC2 operon was identified and characterized. The regulator has no similarities to the up to now known PrpR regulators from other organisms. Growth of a ΔprpR mutant revealed severe growth deficits and a prolonged lag phase if propionate was present in the medium. Transcriptome analyses demonstrated the inability of the ΔprpR strain to induce the prpDBC2 genes in the presence of propionate indicating that PrpR represents a transcriptional activator. They also provided evidence that PrpR controls only the prpDBC2 operon while transcription of the prpR gene was found to be independent of the used carbon source. GC-MS based metabolic profiling of the wild type and the ΔprpR strain grown with propionate revealed smaller pool sizes of the metabolites of the 2-methylcitrate cycle in the mutant strain. The transcriptional start sites and their putative promoters of the prpDBC2 operon and the prpR gene were identified by RACE-PCR. Analyses of promoter test vector constructs led to the identification of a 121bp operator region upstream of prpDBC2, which is essential for a propionate-induced transcription by PrpR. Finally, EMSA studies revealed that 2-methylcitrate most probably acts as co-activator of PrpR