Improving the production of carbamoyltobramycin by an industrial Streptoalloteichus tenebrarius through metabolic engineering.

Tobramycin is an essential and extensively used broad-spectrum aminoglycoside antibiotic obtained through alkaline hydrolysis of carbamoyltobramycin, one of the fermentation products of Streptoalloteichus tenebrarius. To simplify the composition of fermentation products from industrial strain, the main byproduct apramycin was blocked by gene disruption and constructed a mutant mainly producing carbamoyltobramycin. The generation of antibiotics is significantly affected by the secondary metabolism of actinomycetes which could be controlled by modifying the pathway-specific regulatory proteins within the cluster. Within the tobramycin biosynthesis cluster, a transcriptional regulatory factor TobR belonging to the Lrp/AsnC family was identified. Based on the sequence and structural characteristics, tobR might encode a pathway-specific transcriptional regulatory factor during biosynthesis. Knockout and overexpression strains of tobR were constructed to investigate its role in carbamoyltobramycin production. Results showed that knockout of TobR increased carbamoyltobramycin biosynthesis by 22.35%, whereas its overexpression decreased carbamoyltobramycin production by 10.23%. In vitro electrophoretic mobility shift assay (EMSA) experiments confirmed that TobR interacts with DNA at the adjacent tobO promoter position. Strains overexpressing tobO with ermEp* promoter exhibited 36.36% increase, and tobO with kasOp* promoter exhibited 22.84% increase in carbamoyltobramycin titer. When the overexpressing of tobO and the knockout of tobR were combined, the production of carbamoyltobramycin was further enhanced. In the shake-flask fermentation, the titer reached 3.76 g/L, which was 42.42% higher than that of starting strain. Understanding the role of Lrp/AsnC family transcription regulators would be useful for other antibiotic biosynthesis in other actinomycetes. KEY POINTS: • The transcriptional regulator TobR belonging to the Lrp/AsnC family was identified.  • An oxygenase TobO was identified within the tobramycin biosynthesis cluster. • TobO and TobR have significant effects on the synthesis of carbamoyltobramycin.

Newtic1 Is a Component of Globular Structures That Accumulate along the Marginal Band of Erythrocytes in the Limb Blastema of Adult Newt, Cynops pyrrhogaster.

In adult newts, when a limb is amputated, a mesenchymal cell mass called the blastema is formed on the stump, where blood vessels filled with premature erythrocytes, named polychromatic normoblasts (PcNobs), elongate. We previously demonstrated that PcNobs in the blastema express an orphan gene, Newtic1, and that they secrete growth factors such as BMP2 and TGFß1 into the surrounding tissues. However, the relationship between Newtic1 expression and growth factor secretion was not clear since Newtic1 was thought to encode a membrane protein. In this study, we addressed this issue using morphological techniques and found that the Newtic1 protein is a component of globular structures that accumulate at the marginal band in the cytoplasm along the equator of PcNobs. Newtic1-positive (Newtic1(+)) globular structures along the equator were found only in PcNobs with a well-developed marginal band in the blastema. Newtic1(+) globular structures were associated with microtubules and potentially incorporated TGFß1. Based on these observations, we propose a hypothesis that the Newtic1 protein localizes to the membrane of secretory vesicles that primarily carry TGFß1 and binds to microtubules, thereby tethering secretory vesicles to microtubules and transporting them to the cell periphery as the marginal band develops.

Effects of acute cold exposure on oxidative balance and total antioxidant capacity in juvenile Chinese soft-shelled turtle, Pelodiscus sinensis.

Acute cold exposure may disturb the physiological homeostasis of the body in ectotherms. To date, there has been no information on the effects of cold exposure on homeostasis of reactive oxygen species (ROS) or antioxidant defense response in the Chinese soft-shelled turtle, Pelodiscus sinensis. In this study, P. sinensis juveniles were acclimated at 28 °C, transferred to 8 °C as cold exposure for 12 h, then moved back to 28 °C rewarming for 24 h. We measured the ROS level and total antioxidant capacity (TAC) in the brain, liver, kidney and spleen at 2 and 12 h cold exposure, and at the end of the rewarming period. Malonaldehyde (MDA) and carbonyl protein were used as markers of oxidative damage. Turtles being maintained simultaneously at 28 °C were used as the control group. Cold exposure did not disturb the ROS balance in all 4 tissues, while rewarming raised the ROS level in the brain and kidney of P. sinensis. Cold exposure and rewarming decreased the TAC in the brain, liver and spleen but did not change the TAC in the kidney. MDA and carbonyl protein levels did not increase during the treatment, indicating no oxidative damage in all 4 tissues of P. sinensis. Our results indicated that extreme cold exposure did not impact the inner oxidative balance of P. sinensis, but more ROS was produced during rewarming. P. sinensis showed good tolerance to the harsh temperature change through effective protection of its antioxidant defense system to oxidative damage. This study provides basic data on the stress biology of P. sinensis.