Jingnan Lu

Roles of Multiple Acetoacetyl Coenzyme A Reductases in Polyhydroxybutyrate Biosynthesis in Ralstonia eutropha H16

The bacterium Ralstonia eutropha H16 synthesizes polyhydroxybutyrate (PHB) from acetyl coenzyme A (acetyl-CoA) through reactions catalyzed by a β-ketothiolase (PhaA), an acetoacetyl-CoA reductase (PhaB), and a polyhydroxyalkanoate synthase (PhaC). An operon of three genes encoding these enzymatic steps was discovered in R. eutropha and has been well studied. Sequencing and analysis of the R. eutropha genome revealed putative isologs for each of the PHB biosynthetic genes, many of which had never been characterized. In addition to the previously identified phaB1 gene, the genome contains the isologs phaB2 and phaB3 as well as 15 other potential acetoacetyl-CoA reductases. We have investigated the roles of the three phaB isologs by deleting them from the genome individually and in combination. It was discovered that the gene products of both phaB1 and phaB3 contribute to PHB biosynthesis in fructose minimal medium but that in plant oil minimal medium and rich medium, phaB3 seems to be unexpressed. This raises interesting questions concerning the regulation of phaB3 expression. Deletion of the gene phaB2 did not result in an observable phenotype under the conditions tested, although this gene does encode an active reductase. Addition of the individual reductase genes to the genome of the ΔphaB1 ΔphaB2 ΔphaB3 strain restored PHB production, and in the course of our complementation experiments, we serendipitously created a PHB-hyperproducing mutant. Measurement of the PhaB and PhaA activities of the mutant strains indicated that the thiolase reaction is the limiting step in PHB biosynthesis in R. eutropha H16 during nitrogen-limited growth on fructose.

Lipid and fatty acid metabolism in Ralstonia eutropha: relevance for the biotechnological production of value-added products

Lipid and fatty acid metabolism has been well studied in model microbial organisms like Escherichia coli and Bacillus subtilis. The major precursor of fatty acid biosynthesis is also the major product of fatty acid degradation (β-oxidation), acetyl-CoA, which is a key metabolite for all organisms. Controlling carbon flux to fatty acid biosynthesis and from β-oxidation allows for the biosynthesis of natural products of biotechnological importance. Ralstonia eutropha can utilize acetyl-CoA from fatty acid metabolism to produce intracellular polyhydroxyalkanoate (PHA). R. eutropha can also be engineered to utilize fatty acid metabolism intermediates to produce different PHA precursors. Metabolism of lipids and fatty acids can be rerouted to convert carbon into other value-added compounds like biofuels. This review discusses the lipid and fatty acid metabolic pathways in R. eutropha and how they can be used to construct reagents for the biosynthesis of products of industrial importance. Specifically, how the use of lipids or fatty acids as the sole carbon source in R. eutropha cultures adds value to these biotechnological products will be discussed here.

Ralstonia eutropha H16 as a Platform for the Production of Biofuels, Biodegradable Plastics, and Fine Chemicals from Diverse Carbon Resources

Abstract Ralstonia eutropha is capable of utilizing a plethora of simple and complex carbon-containing compounds. Growth has been demonstrated on carbon dioxide, glycerol, acetate, mixed organic acids, sugars, fatty acids, oils, and more. R. eutropha possesses oxygen-tolerant hydrogenases, which allow the bacterium to grow in aerobic lithoautotrophic conditions. When experiencing nutrient stress in the presence of excess carbon, R. eutropha can store carbon and energy in the form of polyhydroxyalkanoates (PHAs), a biodegradable and biocompatible plastic. Engineered R. eutropha strains are capable of producing PHAs with different chain lengths, resulting in different physical, biological, and chemical properties. These tailored PHAs, when depolymerized, can also serve as valuable chiral precursors for antibiotics, vitamins, perfumes, and pheromones. The native PHA carbon storage system can be genetically disabled and the carbon flux redirected to produce value-added chemicals such as biofuels and pharmaceuticals. Various strains of R. eutropha are also capable of bioremediation by breaking down phenol compounds and scavenging heavy metals in contaminated soils and water. Metabolic versatility and genetic tractability combined with its ability to convert a variety of carbon sources into storage molecules make R. eutropha an excellent platform organism for the production of value-added compounds.

Corrigendum to “Experimental evolution and gene knockout studies reveal AcrA-mediated isobutanol tolerance in Ralstonia eutropha” [J Biosci Bioeng 122 (2016) 64–69]

Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA, Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA, Engineering Systems Division, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA, and Department of Bioengineering, University of Massachusetts Dartmouth, 285 Old Westport Road, North Dartmouth, MA 02747, USA

Isobutanol tolerance in Ralstoniaeutropha

BackgroundRalstonia eutropha is bacterium known to naturally pro-duce polyhydroxybutyrate (PHB) as carbon storage duringnutrient starvation. Previously studies [1] showed that it ispossible through the incorporation of an engineered bio-synthetic pathway, to redirect carbon flux from PHB tothe production of Isobutanol (IBT), a biofuel largelystudied to replace the current fossil fuels in existing auto-mobile engines. However R. eutropha,isunabletogrowinthe presence of IBT at concentrations above 0.2% (v v

Periplasmic α-carbonic anhydrase plays an essential role in Ralstonia eutropha CO2 metabolism

Background Carbonic anhydrase (CA) enzymes catalyze the interconversion of CO2 and bicarbonate. These enzymes play important roles in cellular metabolism such as CO2 transport, ion transport, and internal pH regulation. Understanding the roles of CAs in the chemolithotropic betaproteobacteria Ralstonia eutropha is important for the development of fermentation processes based on the bacterium’s capacity for carbon fixation using the CalvinBenson-Bassham cycle. Of the five classes of CA, the alpha-CA is the best-characterized thus far. The gene encoding a periplasmic alpha-CA (caa, H16 B2403) has been identified in the R. eutropha H16 genome, along with three others CA from different classes. In this study, we evaluated the importance of Caa in the metabolism of R. eutropha by examination of CA activity and growth in caa gene deletion, complementation, and overexpression strains. Localization of Caa in the cell was accessed by fluorescent microscopy. Methods