[Removal of Antibiotics and Antibiotic Resistance Genes from Urban Rivers Using Artificial Ecosystems].

Four antibiotics[azithromycin (AZM), sulfamethoxazole (SMZ), ciprofloxacin (CIP), and tetracycline (TCY)], and the antibiotic resistance genes (ARGs)[sulfonamides (sul1 and sul2), tetracyclines (tetX and tetM), quinolones (qnrS and qnrD), macrolides (ermB), and 16S rDNA] were selected as target compounds. Artificial ecosystems were constructed with combinations of two emergent plants and Microcystis aeruginosa (Acorus calamus+Cordyceps, algae+Cordyceps, algae+Acorus calamus, and algae+Acorus calamus+Cordyceps) in an indoor-simulated river system. Throughout the artificial ecosystems, changes in antibiotic concentrations and other pollution indicators (i.e., COD, NH4+-N, TP, and TN) were monitored in different media (the aqueous phase, sediment phase, and in plants), and the distribution and removal of ARGs in aqueous and sediment phases were explored. Removal of the target compounds was calculated based on mass balance, and the correlation between ARG abundance and environmental factors in the aqueous and sediment phases was analyzed. The results showed that the constructed artificial ecosystem achieved removal rates of COD, NH4+-N, TP, and TN ranging from 60.2% to 74.8%, 63.4% to 77.4%, 64.0% to 73.2%, and 46.8% to 54.8%, respectively. The antibiotics in the aqueous phase were notably removed and the artificial ecosystem 'algae+Acorus calamus+Cordyceps' achieved the best removal efficiency for the four antibiotics. Removal rates of the antibiotics in the sediment phase were ranked in the order TCY>CIP>AZM>SMZ; the removal efficiency of TCY in the 'algae+Acorus calamus+Cordyceps' system reached up to 53.5%. The total removal rates of antibiotics obtained by the ecosystems were ranked in the following order:algae+Acorus calamus+Cordyceps > algae+Cordyceps > algae+Acorus calamus > Acorus calamus+Cordyceps. Removal of the four ARGs was very efficient and was higher in the aqueous phase than in the sediment phase. Correlations between the ARGs, the other pollution indicators, and the antibiotics were variable; tetX and environmental factors were correlated in the aqueous phase, while AZM and its corresponding ARGs were not significantly correlated in the sediment phase. The results showed that ARGs can be targeted under corresponding antibiotic pressure and other types of environmental pressure. In the study system, the concentrations of antibiotics did not directly affect the transmission of ARGs. Overall, this study shows that artificial ecosystems constructed with emergent plants and Microcystis aeruginosa can be effective at purifying water and reducing the environmental risks of antibiotics in urban rivers.

Biosensor-assisted transcriptional regulator engineering for Methylobacterium extorquens AM1 to improve mevalonate synthesis by increasing the acetyl-CoA supply.

Acetyl-CoA is not only an important intermediate metabolite for cells but also a significant precursor for production of industrially interesting metabolites. Methylobacterium extorquens AM1, a model strain of methylotrophic cell factories using methanol as carbon source, is of interest because it produces abundant coenzyme A compounds capable of directing to synthesis of different useful compounds from methanol. However, acetyl-CoA is not always efficiently accumulated in M. extorquens AM1, as it is located in the center of three cyclic central metabolic pathways. Here we successfully demonstrated a strategy for sensor-assisted transcriptional regulator engineering (SATRE) to control metabolic flux re-distribution to increase acetyl-CoA flux from methanol for mevalonate production in M. extorquens AM1 with introduction of mevalonate synthesis pathway. A mevalonate biosensor was constructed and we succeeded in isolating a mutated strain (Q49) with a 60% increase in mevalonate concentration (an acetyl-CoA-derived product) following sensor-based high-throughput screening of a QscR transcriptional regulator library. The mutated QscR-49 regulator (Q8*,T61S,N72Y,E160V) lost an N-terminal α-helix and underwent a change in the secondary structure of the RD-I domain at the C terminus, two regions that are related to its interaction with DNA. 13C labeling analysis revealed that acetyl-CoA flux was improved by 7% and transcriptional analysis revealed that QscR had global effects and that two key points, NADPH generation and fumC overexpression, might contribute to the carbon flux re-distribution. A fed-batch fermentation in a 5-L bioreactor for QscR-49 mutant yielded a mevalonate concentration of 2.67g/L, which was equivalent to an overall yield of 0.055mol acetyl-CoA/mol methanol, the highest yield among engineered strains of M. extorquens AM1. This work was the first attempt to regulate M. extorquens AM1 on transcriptional level and provided molecular insights into the mechanism of carbon flux regulation.

Bioconversion of methanol to value-added mevalonate by engineered Methylobacterium extorquens AM1 containing an optimized mevalonate pathway.

Methylotrophic biosynthesis using methanol as a feedstock is a promising and attractive method to solve the over-dependence of the bioindustry on sugar feedstocks derived from grains that are used for food. In this study, we introduced and engineered the mevalonate pathway into Methylobacterium extorquens AM1 to achieve high mevalonate production from methanol, which could be a platform for terpenoid synthesis. We first constructed a natural operon (MVE) harboring the mvaS and mvaE genes from Enterococcus faecalis as well as an artificial operon (MVH) harboring the hmgcs1 gene from Blattella germanica and the tchmgr gene from Trypanosoma cruzi that encoded enzymes with the highest reported activities. We achieved mevalonate titers of 56 and 66 mg/L, respectively, in flask cultivation. Introduction of the phaA gene from Ralstonia eutropha into the operon MVH increased the mevalonate titer to 180 mg/L, 3.2-fold higher than that of the natural operon MVE. Further modification of the expression level of the phaA gene by regulating the strength of the ribosomal binding site resulted in an additional 20 % increase in mevalonate production to 215 mg/L. A fed-batch fermentation of the best-engineered strain yielded a mevalonate titer of 2.22 g/L, which was equivalent to an overall yield and productivity of 28.4 mg mevalonate/g methanol and 7.16 mg/L/h, respectively. The production of mevalonate from methanol, which is the initial, but critical step linking methanol with valuable terpenoids via methylotrophic biosynthesis, represents a proof of concept for pathway engineering in M. extorquens AM1.

High crude violacein production from glucose by Escherichia coli engineered with interactive control of tryptophan pathway and violacein biosynthetic pathway.

BACKGROUND: As bacteria-originated crude violacein, a natural indolocarbazole product, consists of violacein and deoxyviolacein, and can potentially be a new type of natural antibiotics, the reconstruction of an effective metabolic pathway for crude violacein (violacein and deoxyviolacein mixture) synthesis directly from glucose in Escherichia coli was of importance for developing industrial production process. RESULTS: Strains with a multivariate module for varied tryptophan productivities were firstly generated by combinatorial knockout of trpR/tnaA/pheA genes and overexpression of two key genes trpEfbr /trpD from the upstream tryptophan metabolic pathway. Then, the gene cluster of violacein biosynthetic pathway was introduced downstream of the generated tryptophan pathway. After combination of these two pathways, maximum crude violacein production directly from glucose by E. coli B2/pED+pVio was realized with a titer of 0.6±0.01 g L(-1) in flask culture, which was four fold higher than that of the control without the tryptophan pathway up-regulation. In a 5-L bioreactor batch fermentation with glucose as the carbon source, the recombinant E. coli B2/pED+pVio exhibited a crude violacein titer of 1.75 g L(-1) and a productivity of 36 mg L(-1) h(-1), which was the highest titer and productivity reported so far under the similar culture conditions without tryptophan addition. CONCLUSION: Metabolic pathway analysis using 13C labeling illustrated that the up-regulated tryptophan supply enhanced tryptophan metabolism from glucose, whereas the introduction of violacein pathway drew more carbon flux from glucose to tryptophan, thereby contributing to the effective production of crude violacein in the engineered E. coli cell factory.