New Recombinant Escherichia coli Strain Tailored for the Production of Poly(3-Hydroxybutyrate) from Agroindustrial By-Products

A recombinant E. coli strain (K24K) was constructed and evaluated for poly(3-hydroxybutyrate) (PHB) production from whey and corn steep liquor as main carbon and nitrogen sources. This strain bears the pha biosynthetic genes from Azotobacter sp. strain FA8 expressed from a T5 promoter under the control of the lactose operator. K24K does not produce the lactose repressor, ensuring constitutive expression of genes involved in lactose transport and utilization. PHB was efficiently produced by the recombinant strain grown aerobically in fed-batch cultures in a laboratory scale bioreactor on a semisynthetic medium supplemented with the agroindustrial by-products. After 24 h, cells accumulated PHB to 72.9% of their cell dry weight, reaching a volumetric productivity of 2.13 g PHB per liter per hour. Physical analysis of PHB recovered from the recombinants showed that its molecular weight was similar to that of PHB produced by Azotobacter sp. strain FA8 and higher than that of the polymer from Cupriavidus necator and that its glass transition temperature was approximately 20°C higher than those of PHBs from the natural producer strains.     

Perspectives and limits of engineering the isoprenoid metabolism in heterologous hosts

Terpenoids belong to the largest class of natural compounds and are produced in all living organisms. The isoprenoid skeleton is based on assembling of C5 building blocks, but the biosynthesis of a great variety of terpenoids ranging from monoterpenoids to polyterpenoids is not fully understood today. Terpenoids play a fundamental role in human nutrition, cosmetics, and medicine. In the past 10 years, many metabolic engineering efforts have been undertaken in plants but also in microorganisms to improve the production of various terpenoids like artemisinin and paclitaxel. Recently, inverse metabolic engineering and combinatorial biosynthesis as main strategies in synthetic biology have been applied to produce high-cost natural products like artemisinin and paclitaxel in heterologous microorganisms. This review describes the recent progresses made in metabolic engineering of the terpenoid pathway with particular focus on fundamental aspects of host selection, vector design, and system biotechnology.     

Decreased mass specific respiration under experimental warming is robust to the microbial biomass method employed

Hartley et al. question whether reduction in R _mass, under experimental warming, arises because of the biomass method. We show the method they treat as independent yields the same result. We describe why the substrate-depletion hypothesis may not solely explain observed responses, and urge caution in interpretation of the seasonal data.     

Thermal adaptation of soil microbial respiration to elevated temperature

In the short‐term heterotrophic soil respiration is strongly and positively related to temperature. In the long‐term, its response to temperature is uncertain. One reason for this is because in field experiments increases in respiration due to warming are relatively short‐lived. The explanations proposed for this ephemeral response include depletion of fast‐cycling, soil carbon pools and thermal adaptation of microbial respiration. Using a > 15 year soil warming experiment in a mid‐latitude forest, we show that the apparent ‘acclimation’ of soil respiration at the ecosystem scale results from combined effects of reductions in soil carbon pools and microbial biomass, and thermal adaptation of microbial respiration. Mass‐specific respiration rates were lower when seasonal temperatures were higher, suggesting that rate reductions under experimental warming likely occurred through temperature‐induced changes in the microbial community. Our results imply that stimulatory effects of global temperature rise on soil respiration rates may be lower than currently predicted.     

Engineering of macrophages to produce IFN-gamma in response to hypoxia

Activation of murine macrophages (Mphi) requires the collaboration of signals derived from the immune system and the environment. In this study, we engineered a murine Mphi cell line to become activated in response to an environmental signal, hypoxia, as the sole stimulus. Hypoxia is a condition of low oxygen tension, occurring in several pathological tissues, which acts in synergy with IFN-gamma to induce full Mphi activation. We transfected the ANA-1 murine Mphi cell line with a construct containing the IFN-gamma gene controlled by a synthetic promoter inducible by hypoxia (HRE3x-Tk), and we characterized the cellular and molecular biology of the engineered Mphi under normoxia or hypoxia. Engineered Mphi in normoxia expressed basal levels of IFN-gamma mRNA and protein that were strongly augmented by shifting the cells to hypoxia. Furthermore, they responded to the synthesized IFN-gamma with induction of IFN-responsive factor-1 and 2'-5'-oligoadenylate synthase expression. Under normoxic conditions, the engineered Mphi had a significant constitutive level of Ia Ags and Fc receptors. Hypoxia induced further augmentation of Ia and Fc expression. Finally, hypoxia induced inducible NO synthase expression, and subsequent reoxygenation led to the production of NO. In conclusion, the engineered Mphi, which produce IFN-gamma in an inducible manner, express new biochemical and functional properties in response to low oxygen environment as the sole stimulus, thereby circumventing the need for costimulation by other immune system-derived signals.     

Pivotal role of the RB family proteins in in vitro thyroid cell transformation

Rat thyroid differentiated cells (PC Cl 3) are an excellent model system with which to study the interaction between differentiation and cell transformation. We previously demonstrated that PC Cl 3 cells expressing the adenovirus E1A gene no longer depend on thyrotropin for growth and do not express thyroid differentiation markers. Here we show that an E1A mutant unable to bind the RB protein failed to transform the PC Cl 3 cells. Conversely, mutations in the E1A p300 interacting region did not affect its transforming ability. The pivotal role of RB family proteins in the thyroid cell transformation is supported by the thyrotropin independence induced by the E7 gene of human papilloma virus type 16, but not by a mutated form in the RB-binding region.     

Climatic and biotic controls on annual carbon storage in Amazonian ecosystems

• 1 The role of undisturbed tropical land ecosystems in the global carbon budget is not well understood. It has been suggested that interannual climate variability can affect the capacity of these ecosystems to store carbon in the short term. In this paper, we use a transient version of the Terrestrial Ecosystem Model (TEM) to estimate annual carbon storage in undisturbed Amazonian ecosystems during the period 1980–94, and to understand the underlying causes of the year‐to‐year variations in net carbon storage for this region.
  • 2 We estimate that the total carbon storage in the undisturbed ecosystems of the Amazon Basin in 1980 was 127.6 Pg C, with about 94.3 Pg C in vegetation and 33.3 Pg C in the reactive pool of soil organic carbon. About 83% of the total carbon storage occurred in tropical evergreen forests. Based on our model’s results, we estimate that, over the past 15 years, the total carbon storage has increased by 3.1 Pg C (+ 2%), with a 1.9‐Pg C (+2%) increase in vegetation carbon and a 1.2‐Pg C (+4%) increase in reactive soil organic carbon. The modelled results indicate that the largest relative changes in net carbon storage have occurred in tropical deciduous forests, but that the largest absolute changes in net carbon storage have occurred in the moist and wet forests of the Basin.
    • 3 Our results show that the strength of interannual variations in net carbon storage of undisturbed ecosystems in the Amazon Basin varies from a carbon source of 0.2 Pg C/year to a carbon sink of 0.7 Pg C/year. Precipitation, especially the amount received during the drier months, appears to be a major controller of annual net carbon storage in the Amazon Basin. Our analysis indicates further that changes in precipitation combine with changes in temperature to affect net carbon storage through influencing soil moisture and nutrient availability.
      • 4 On average, our results suggest that the undisturbed Amazonian ecosystems accumulated 0.2 Pg C/year as a result of climate variability and increasing atmospheric CO₂ over the study period. This amount is large enough to have compensated for most of the carbon losses associated with tropical deforestation in the Amazon during the same period.
        • 5 Comparisons with empirical data indicate that climate variability and CO₂ fertilization explain most of the variation in net carbon storage for the undisturbed ecosystems. Our analyses suggest that assessment of the regional carbon budget in the tropics should be made over at least one cycle of El Niño–Southern Oscillation because of interannual climate variability. Our analyses also suggest that proper scaling of the site‐specific and subannual measurements of carbon fluxes to produce Basin‐wide flux estimates must take into account seasonal and spatial variations in net carbon storage.

     

Carbon and nitrogen dynamics along the decay continuum: Plant litter to soil organic matter

Decay processes in an ecosystem can be thought of as a continuum beginning with the input of plant litter and leading to the formation of soil organic matter. As an example of this continuum, we review a 77-month study of the decay of red pine (Pinus resinosa Ait.) needle litter. We tracked the changes in C chemistry and the N pool in red pine (Pinus resinosa Ait.) needle litter during the 77-month period using standard chemical techniques and stable isotope, analyses of C and N.Mass loss is best described by a two-phase model: an initial phase of constant mass loss and a phase of very slow loss dominated by degradation of lignocellulose (acid soluble sugars plus acid insoluble C compounds). As the decaying litter enters the second phase, the ratio of lignin to lignin and cellulose (the lignocellulose index, LCI) approaches 0.7. Thereafter, the LCI increases only slightly throughout the decay continuum indicating that acid insoluble materials (lignin) dominate decay in the latter part of the continuum.Nitrogen dynamics are also best described by a two-phase model: a phase of N net immobilization followed by a phase of N net mineralization. Small changes in C and N isotopic composition were observed during litter decay. Larger changes were observed with depth in the soil profile.An understanding of factors that control lignin degradation is key to predicting the patterns of mass loss and N dynamics late in decay. The hypothesis that labile C is needed for lignin degradation must be evaluated and the sources of this C must be identified. Also, the hypothesis that the availability of inorganic N slows lignin decay must be evaluated in soil systems.