Recombinant protein production provoked accumulation of ATP, fructose-1,6-bisphosphate and pyruvate in E. coli K12 strain TG1

Background Recently it was shown that production of recombinant proteins in E. coli BL21(DE3) using pET based expression vectors leads to metabolic stress comparable to a carbon overfeeding response. Opposite to original expectations generation of energy as well as catabolic provision of precursor metabolites were excluded as limiting factors for growth and protein production. On the contrary, accumulation of ATP and precursor metabolites revealed their ample formation but insufficient withdrawal as a result of protein production mediated constraints in anabolic pathways. Thus, not limitation but excess of energy and precursor metabolites were identified as being connected to the protein production associated metabolic burden. Results Here we show that the protein production associated accumulation of energy and catabolic precursor metabolites is not unique to E. coli BL21(DE3) but also occurs in E. coli K12. Most notably, it was demonstrated that the IPTG-induced production of hFGF-2 using a tac-promoter based expression vector in the E. coli K12 strain TG1 was leading to persistent accumulation of key regulatory molecules such as ATP, fructose-1,6-bisphosphate and pyruvate. Conclusions Excessive energy generation, respectively, accumulation of ATP during recombinant protein production is not unique to the BL21(DE3)/T7 promoter based expression system but also observed in the E. coli K12 strain TG1 using another promoter/vector combination. These findings confirm that energy is not a limiting factor for recombinant protein production. Moreover, the data also show that an accelerated glycolytic pathway flux aggravates the protein production associated “metabolic burden”. Under conditions of compromised anabolic capacities cells are not able to reorganize their metabolic enzyme repertoire as required for reduced carbon processing.

Designing microbial communities to maximize the thermodynamic driving force for the production of chemicals

Microbial communities have become a major research focus due to their importance for biogeochemical cycles, biomedicine and biotechnological applications. While some biotechnological applications, such as anaerobic digestion, make use of naturally arising microbial communities, the rational design of microbial consortia for bio-based production processes has recently gained much interest. One class of synthetic microbial consortia is based on specifically designed strains of one species. A common design principle for these consortia is based on division of labor, where the entire production pathway is divided between the different strains to reduce the metabolic burden caused by product synthesis. We first show that classical division of labor does not automatically reduce the metabolic burden when metabolic flux per biomass is analyzed. We then present ASTHERISC (Algorithmic Search of THERmodynamic advantages in Single-species Communities), a new computational approach for designing multi-strain communities of a single-species with the aim to divide a production pathway between different strains such that the thermodynamic driving force for product synthesis is maximized. ASTHERISC exploits the fact that compartmentalization of segments of a product pathway in different strains can circumvent thermodynamic bottlenecks arising when operation of one reaction requires a metabolite with high and operation of another reaction the same metabolite with low concentration. We implemented the ASTHERISC algorithm in a dedicated program package and applied it on E. coli core and genome-scale models with different settings, for example, regarding number of strains or demanded product yield. These calculations showed that, for each scenario, many target metabolites (products) exist where a multi-strain community can provide a thermodynamic advantage compared to a single strain solution. In some cases, a production with sufficiently high yield is thermodynamically only feasible with a community. In summary, the developed ASTHERISC approach provides a promising new principle for designing microbial communities for the bio-based production of chemicals.

Impact of changes in waist-to-hip ratio after kidney transplantation on cardiovascular outcomes

Recently, waist to hip ratio (WHR) has been reported to be a better indicator of predicting cardiovascular outcomes than body mass index (BMI). We evaluated the effects of pre or post-transplant changes of WHR or BMI on the new onset cardiovascular diseases (CVD) in recipients of kidney transplantation (KT). A total of 572 patients were enrolled from a multicenter observational cohort (KNOW-KT). Measurement of WHR and BMI was done at pre-KT, first and last visit year after KT, and the changes of these parameters and their effect on the incident CVD were analyzed. During the median follow up period of 32.73 ± 15.26 months, the new onset CVD developed in 31 out of 572 patients. The older age, diabetes mellitus and increase of WHR from pre KT or previous follow up year were found to be independent factors predicting the new onset CVD in these patients. However, baseline BMI, WHR prior to KT did not predict the incident CVD. The new metabolic burden, presented as increase of WHR in KT patients has a critical impact on the development of new onset CVD. Strategies to prevent the metabolic burden after KT might improve cardiovascular outcomes and patient’s survival.

A shift away from mutualism under food-deprived conditions in an anemone-dinoflagellate association

The mutualistic symbiosis between anthozoans and intra-gastrodermal dinoflagellates of the family Symbiodiniaceae is the functional basis of all coral reef ecosystems, with the latter providing up to 95% of their fixed photosynthate to their hosts in exchange for nutrients. However, recent studies of sponges, jellyfish, and anemones have revealed the potential for this mutualistic relationship to shift to parasitism under stressful conditions. Over a period of eight weeks, we compared the physiological conditions of both inoculated and aposymbiotic anemones (Exaiptasia pallida) that were either fed or starved. By the sixth week, both fed groups of anemones were significantly larger than their starved counterparts. Moreover, inoculated and starved anemones tended to disintegrate into “tissue balls” within eight weeks, and 25% of the samples died; in contrast, starved aposymbiotic anemones required six months to form tissue balls, and no anemones from this group died. Our results show that the dinoflagellates within inoculated anemones may have posed a fatal metabolic burden on their hosts during starvation; this may be because of the need to prioritize their own metabolism and nourishment at the expense of their hosts. Collectively, our study reveals the potential of this dynamic symbiotic association to shift away from mutualism during food-deprived conditions.

Census of Candidatus Liberibacter asiaticus population inside the phloem of citrus trees

Candidatus Liberibacter asiaticus (CLas) is the predominant causal agent of citrus huanglongbing (HLB). The pathogen population size in local tissues and the whole plant are critical for the development of disease symptoms via pathogenicity factors and causing metabolic burden to the host. However, the total population size of CLas in a whole plant and the ratio of CLas vs. citrus cells in local tissues have not been addressed previously. The total CLas population size for 2.5-year-old Valencia sweet orange trees was quantified using quantitative PCR to be approximately 1.74 x 109, whereas that of 7 and 20-year-old sweet orange trees were estimated to be 4.3 x 1010, and 6.0 x 1010, respectively. The majority of CLas cells were distributed in the leaf tissues (55.58%), followed by that in the branch tissues (36.78%), feeder roots (4.75%), trunk (2.39%), and structural root (0.51%) tissues. The ratios of citrus cells vs. CLas cells for branch, leaf, trunk, feeder root, and structural root samples were approximately 39, 44, 153, 191, and 561, respectively, representing the metabolic burden of CLas in different organs. Approximately 0.01% of the total citrus phloem volume was estimated to be occupied by CLas. The CLas titer inside the leaf was estimated to be approximately 1.64 x 106 cells/leaf or 9.2 x 104 cells cm-2 in leaves, approximately 104 times less than that of typical apoplastic bacterial pathogens. This study provides quantitative estimates of phloem colonization by bacterial pathogens and further understands the biology and virulence mechanism of CLas.