Development of a growth-coupled selection platform for directed evolution of heme biosynthetic enzymes in Corynebacterium glutamicum.

Heme is an important tetrapyrrole compound, and has been widely applied in food and medicine industries. Although microbial production of heme has been developed with metabolic engineering strategies during the past 20 years, the production levels are relatively low due to the multistep enzymatic processes and complicated regulatory mechanisms of microbes. Previous studies mainly adopted the strategies of strengthening precursor supply and product transportation to engineer microbes for improving heme biosynthesis. Few studies focused on the engineering and screening of efficient enzymes involved in heme biosynthesis. Herein, a growth-coupled, high-throughput selection platform based on the detoxification of Zinc-protoporphyrin IX (an analogue of heme) was developed and applied to directed evolution of coproporphyrin ferrochelatase, catalyzing the insertion of metal ions into porphyrin ring to generate heme or other tetrapyrrole compounds. A mutant with 3.03-fold increase in k cat/K M was selected. Finally, growth-coupled directed evolution of another three key enzymes involved in heme biosynthesis was tested by using this selection platform. The growth-coupled selection platform developed here can be a simple and effective strategy for directed evolution of the enzymes involved in the biosynthesis of heme or other tetrapyrrole compounds.

Electroacupuncture Improves Blood-Brain Barrier and Hippocampal Neuroinflammation in SAMP8 Mice by Inhibiting HMGB1/TLR4 and RAGE/NADPH Signaling Pathways.

OBJECTIVE: To investigate the molecular mechanisms underlying the beneficial effect of electroacupuncture (EA) in experimental models of Alzheimer's disease (AD) in vivo. METHODS: Senescence-accelerated mouse prone 8 (SAMP8) mice were used as AD models and received EA at Yingxiang (LI 20, bilateral) and Yintang (GV 29) points for 20 days. For certain experiments, SAMP8 mice were injected intravenously with human fibrin (2 mg). The Morris water maze test was used to assess cognitive and memory abilities. The changes of tight junctions of blood-brain barrier (BBB) in mice were observed by transmission electron microscope. The expressions of fibrin, amyloid- ß (Aß), and ionized calcium-binding adapter molecule 1 (IBa-1) in mouse hippocampus (CA1/CA3) were detected by reverse transcription-quantitative polymerase chain reaction (qRT-PCR), Western blot or immunohistochemical staining. The expression of fibrin in mouse plasma was detected by enzyme-linked immunosorbent assay. The expressions of tight junction proteins zonula occludens-1 and claudin-5 in hippocampus were detected by qRT-PCR and immunofluorescence staining. Apoptosis of hippocampal neurons was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining. RESULTS: Fibrin was time-dependently deposited in the hippocampus of SAMP8 mice and this was inhibited by EA treatment (P<0.05 or P<0.01). Furthermore, EA treatment suppressed the accumulation of Aß in the hippocampus of SAMP8 mice (P<0.01), which was reversed by fibrin injection (P<0.05 or P<0.01). EA improved SAMP8 mice cognitive impairment and BBB permeability (P<0.05 or P<0.01). Moreover, EA decreased reactive oxygen species levels and neuroinflammation in the hippocampus of SAMP8 mice, which was reversed by fibrin injection (P<0.05 or P<0.01). Mechanistically, EA inhibited the promoting effect of fibrin on the high mobility group box protein 1 (HMGB1)/toll-like receptor 4 (TLR4) and receptor for advanced glycation end products (RAGE)/nicotinamide adenine dinucleotide phosphate (NADPH) signaling pathways (P<0.01). CONCLUSION: EA may potentially improve cognitive impairment in AD via inhibition of fibrin/A ß deposition and deactivation of the HMGB1/TLR4 and RAGE/NADPH signaling pathways.

Identification of a second follistatin gene in grass carp (Ctenopharyngodon idellus) and its regulatory function in myogenesis during embryogenesis.

Follistatin can antagonize the function of myostatin as a competitive binding protein and promote muscle growth in vivo. Here, we report the isolation and characterization of a second follistatin gene fst2 in grass carp (Ctenopharyngodon idellus). The grass carp fst2 cDNA was 1,376 bp in length, with an open reading frame (ORF) encoding 350 amino acid residues. A relatively low sequence identity of 78% was found between grass carp Fst2 and its paralog Fst1. Sequence and phylogenetic analyses suggest that the grass carp fst2 originated from fish-specific gene duplication. In adult fish, fst2 mRNA expression was observed in most tissues but was strongly expressed in the eyes, muscles, skin and ovary. Grass carp fst2 mRNA could be detected as early as 16 h post-fertilization (hpf), while fst1 mRNA was detected throughout embryogenesis. Using in situ hybridization, fst2 transcripts were detected in the anterior somites at 24 hpf and in the brain and posterior somites at 36 hpf. Meanwhile, fst1 mRNA was transcribed mainly in the optic vesicle and at the cephalic mesoderm at 12 hpf, in the eyes, cephalic mesoderm and at the lateral edge of most somites at 24 hpf, and mainly in the brain at 36 hpf. Furthermore, overexpression of fst2 mRNA markedly affected the formation of the embryonic midline and somite structures. Based on comparisons with fst1, our findings suggest that fst2 retained the ancestral functions of regulating muscle development and growth during embryogenesis in grass carp.