Design, construction and performance of the most efficient biomass producing E. coli bacterium

Inverse metabolic engineering based on elementary mode analysis was applied to maximize the biomass yield of Escherchia coli MG1655. Elementary mode analysis was previously employed to identify among 1691 possible pathways for cell growth the most efficient pathway with maximum biomass yield. The metabolic network analysis predicted that deletion of only 6 genes reduces the number of possible elementary modes to the most efficient pathway. We have constructed a strain containing these gene deletions and we evaluated its properties in batch and in chemostat growth experiments. The results show that the theoretical predictions are closely matched by the properties of the designed strain.     

Replication Fork Stalling and Checkpoint Activation by a PKD1 Locus Mirror Repeat Polypurine-Polypyrimidine (Pu-Py) Tract

DNA sequences prone to forming noncanonical structures (hairpins, triplexes, G-quadruplexes) cause DNA replication fork stalling, activate DNA damage responses, and represent hotspots of genomic instability associated with human disease. The 88-bp asymmetric polypurine-polypyrimidine (Pu-Py) mirror repeat tract from the human polycystic kidney disease (PKD1) intron 21 forms non-B DNA secondary structures in vitro. We show that the PKD1 mirror repeat also causes orientation-dependent fork stalling during replication in vitro and in vivo. When integrated alongside the c-myc replicator at an ectopic chromosomal site in the HeLa genome, the Pu-Py mirror repeat tract elicits a polar replication fork barrier. Increased replication protein A (RPA), Rad9, and ataxia telangiectasia- and Rad3-related (ATR) checkpoint protein binding near the mirror repeat sequence suggests that the DNA damage response is activated upon replication fork stalling. Moreover, the proximal c-myc origin of replication was not required to cause orientation-dependent checkpoint activation. Cells expressing the replication fork barrier display constitutive Chk1 phosphorylation and continued growth, i.e. checkpoint adaptation. Excision of the Pu-Py mirror repeat tract abrogates the DNA damage response. Adaptation to Chk1 phosphorylation in cells expressing the replication fork barrier may allow the accumulation of mutations that would otherwise be remediated by the DNA damage response.     

Contributions of Microorganisms to Industrial Biology

Life on earth is not possible without microorganisms. Microbes have contributed to industrial science for over 100 years. They have given us diversity in enzymatic content and metabolic pathways. The advent of recombinant DNA brought many changes to industrial microbiology. New expression systems have been developed, biosynthetic pathways have been modified by metabolic engineering to give new metabolites, and directed evolution has provided enzymes with modified selectability, improved catalytic activity and stability. More and more genomes of industrial microorganisms are being sequenced giving valuable information about the genetic and enzymatic makeup of these valuable forms of life. Major tools such as functional genomics, proteomics, and metabolomics are being exploited for the discovery of new valuable small molecules for medicine and enzymes for catalysis.     

Glucose catabolism by Thiobacillus A2 grown in chemostat culture under carbon or nitrogen limitation

Thiobacillus A2 was grown in glucose- or ammonium-limited chemostats and relative contributions of the Embden-Meyerhof (EM), Entner-Doudoroff (ED) and pentose phosphate (PP) pathways to glucose catabolism estimated by ¹⁴C-glucose radiorespirometry. In fast growing strain GFI, the EM pathway predominated (41–79%) under all growth conditions with the PP pathway contributing 18–30%. The ED pathway was apparently absent under some conditions of glucose limitation. In contrast, wild type Thiobacillus A2 exhibited predominance of the EM pathway (43–48%) under ammonium-limitation but apparent predominance of the PP pathway (43–55%) under glucose-limitation, although all three pathways were calculated to operate. Under some conditions of glucose limitation the EM pathway was possibly considerably depressed. No clear pattern of response of the three pathways to altered environmental conditions could be deduced, although marked change in pathway activities were obviously induced. Growth yield was apparently unaffected by variation in pathways. The problems of interpreting such complex radiorespirometric data are discussed.Abbreviations EM Embden-Meyerhof - ED Entner-Doudoroff - KDPG 2-keto-3-deoxy-6-phosphogluconate - 6-PG 6-phosphogluconate - PK phosphoketolase - PP pentose phosphate     

Net photosynthetic recovery in subarctic lichens with contrasting water relations

Summary The rates of net photosynthetic recovery after wetting for six subarctic lichens were related to their drying rates under laboratory conditions. Net photosynthetic recovery was described by the three parameters of the Von-Bertalanffy equation: T _o and K, related to the rates of resaturation respiration and gross photosynthetic recovery, and P _max, the photosynthetic maximum attained at full recovery. The time to full photosynthetic recovery ranged from 143 to 510 min and was positively correlated with the drying rate of the thallus. In order from most to least rapid recovery, the species are Coelocaulon divergens, Cetraria cucullata, Alectoria ochroleuca, Cladina stellaris, Nephroma arcticum, and Cladonia sulphurina.In nature the high evaporative resistance or low waterholding capacity characterizing fast-drying species will result in short, frequent cycles of wetting and drying which induce carbon losses. In such situations a rapid photosynthetic recovery should be adaptive since it increases photosynthetic carbon gain during a period of metabolic activity. We hypothesize that fast-drying species achieve their rapid photosynthetic recovery by an increased desiccation-tolerance which has a metabolic cost associated with it. In slowdrying species a rapid recovery is not favored by natural selection since these species can take advantage of longer photosynthetic activity periods and are exposed less frequently to deleterious wetting and drying cycles. Future studies of lichen distribution and productivity should take into account the recovery phenomena.     

Taxus metabolomics: methyl jasmonate preferentially induces production of taxoids oxygenated at C-13 in Taxus x media cell cultures

Cells from suspension cultures of Taxus cuspidata were extracted with pentane as a source of relatively non-polar taxoids. Of the 13 taxoids identified in this fraction, eight were oxygenated at C-14 and two had not been previously described. These taxoids, along with existing taxoid standards, were employed to profile the metabolites of Taxus x media cv. Hicksii cell suspension cultures induced with methyl jasmonate to produce paclitaxel (Taxol). The majority of the taxoid metabolites produced in these induced cultures were oxygenated at C-13, and not C-14.     

System-wide studies of N-lysine acetylation in Rhodopseudomonas palustris reveal substrate specificity of protein acetyltransferases

N-lysine acetylation is a posttranslational modification that has been well studied in eukaryotes and is likely widespread in prokaryotes as well. The central metabolic enzyme acetyl-CoA synthetase is regulated in both bacteria and eukaryotes by acetylation of a conserved lysine residue in the active site. In the purple photosynthetic α-proteobacterium Rhodopseudomonas palustris, two protein acetyltransferases (RpPat and the newly identified RpKatA) and two deacetylases (RpLdaA and RpSrtN) regulate the activities of AMP-forming acyl-CoA synthetases. In this work, we used LC/MS/MS to identify other proteins regulated by the N-lysine acetylation/deacetylation system of this bacterium. Of the 24 putative acetylated proteins identified, 14 were identified more often in a strain lacking both deacetylases. Nine of these proteins were members of the AMP-forming acyl-CoA synthetase family. RpPat acetylated all nine of the acyl-CoA synthetases identified by this work, and RpLdaA deacetylated eight of them. In all cases, acetylation occurred at the conserved lysine residue in the active site, and acetylation decreased activity of the enzymes by >70%. Our results show that many different AMP-forming acyl-CoA synthetases are regulated by N-lysine acetylation. Five non-acyl-CoA synthetases were identified as possibly acetylated, including glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and Rpa1177, a putative 4-oxalocrotonate tautomerase. Neither RpPat nor RpKatA acetylated either of these proteins in vitro. It has been reported that Salmonella enterica Pat (SePat) can acetylate a number of metabolic enzymes, including GAPDH, but we were unable to confirm this claim, suggesting that the substrate range of SePat is not as broad as suggested previously.     

Metabolic engineering of Bacillus sp. for diacetyl production

Diacetyl, a highly valuable product that is extensively used as an ingredient of food, tobacco, and daily chemicals such as perfumes, can be produced from the nonenzymatic oxidative decarboxylation of α-acetolactate during bacterial fermentation and converted to acetoin and 2,3-butanediol by 2,3-butanediol dehydrogenase. In the present study, Bacillus sp. DL01, which gives high acetoin production, was metabolically engineered to improve diacetyl production. After the deletion of α-acetolactate decarboxylase (ALDC)-encoding gene (alsD) by homologous recombination, the engineered strain, named Bacillus sp. DL01-ΔalsD, lost ALDC activity and produced 1.53 g/L diacetyl without acetoin and 2,3-butanediol accumulation. The channeling of carbon flux into diacetyl biosynthetic pathway was amplified by an overexpressed α-acetolactate synthase (ALS)-encoding gene (alsS) in Bacillus sp. DL01-ΔalsD-alsS, which produced 4.02 g/L α-acetolactate and 1.94 g/L diacetyl, and the conversion from α-acetolactate to diacetyl was increased by 1-fold after 20 mM Fe³⁺ was added to the fermentation medium. A titer of 8.69 g/L diacetyl, the highest reported diacetyl production, was achieved by fed-batch fermentation in optimal conditions using the metabolically engineered strain of Bacillus sp. DL01-ΔalsD-alsS. These results are of great importance as a new method for the efficient production of diacetyl by food-safe bacteria.     

Role of the malate-aspartate shuttle on the metabolic response to myocardial ischemia

The malate-aspartate (M-A) shuttle provides an important mechanism to regulate glycolysis and lactate metabolism in the heart by transferring reducing equivalents from cytosol into mitochondria. However, experimental characterization of the M-A shuttle has been incomplete because of limitations in quantifying cytosolic and mitochondrial metabolites. In this study, we developed a multi-compartment model of cardiac metabolism with detailed presentation of the M-A shuttle to quantitatively predict non-observable fluxes and metabolite concentrations under normal and ischemic conditions in vivo. Model simulations predicted that the M-A shuttle is functionally localized to a subdomain that spans the mitochondrial and cytosolic spaces. With the onset of ischemia, the M-A shuttle flux rapidly decreased to a new steady state in proportion to the reduction in blood flow. Simulation results suggest that the reduced M-A shuttle flux during ischemia was not due to changes in shuttle-associated enzymes and transporters. However, there was a redistribution of shuttle-associated metabolites in both cytosol and mitochondria. Therefore, the dramatic acceleration in glycolysis and the switch to lactate production that occur immediately after the onset of ischemia is mediated by reduced M-A shuttle flux through metabolite redistribution of shuttle associated species across the mitochondrial membrane.