Zebrafish models for human ALA-dehydratase-deficient porphyria (ADP) and hereditary coproporphyria (HCP) generated with TALEN and CRISPR-Cas9

ABSTRACTDefects in the enzymes involved in heme biosynthesis result in a group of human metabolic genetic disorders known as porphyrias. Using a zebrafish model for human hepatoerythropoietic porphyria (HEP), caused by defective uroporphyrinogen decarboxylase (Urod), the fifth enzyme in the heme biosynthesis pathway, we recently have found a novel aspect of porphyria pathogenesis. However, no hereditable zebrafish models with genetic mutations ofaladandcpox, encoding the second enzyme delta-aminolevulinate dehydratase (Alad) and the sixth enzyme coproporphyrinogen oxidase (Cpox), have been established to date. Here we employed site-specific genome-editing tools transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) to generate zebrafish mutants foraladandcpox. These zebrafish mutants display phenotypes of heme deficiency, hypochromia, abnormal erythrocytic maturation and accumulation of heme precursor intermediates, reminiscent of human ALA-dehydratase-deficient porphyria (ADP) and hereditary coproporphyrian (HCP), respectively. Further, we observed altered expression of genes involved in heme biosynthesis and degradation and particularly down-regulation of exocrine pancreatic zymogens in ADP (alad-/-) and HCP (cpox-/-) fishes. These two zebrafish porphyria models can survive at least 7 days and thus provide invaluable resources for elucidating novel pathological aspects of porphyrias, evaluating mutated forms of humanALADandCPOX, discovering new therapeutic targets and developing effective drugs for these complex genetic diseases. Our studies also highlight generation of zebrafish models for human diseases with two versatile genome-editing tools.

Effects of metal ions on biomass and 5-aminolevulinic acid production in Rhodopseudomonas palustris wastewater treatment

This work investigated the effects of eight metal ions on Rhodopseudomonas palustris growth and 5-aminolevulinic acid (ALA) yield in wastewater treatment. Results show that metal ions (Mg2+ of 15 mmol/L, Fe2+ of 400 μmol/L, Co2+ of 4 μmol/L, Ni2+ of 8 μmol/L and Zn2+ of 4 μmol/L) could effectively improve the chemical oxygen demand (COD) removal, Rp. palustris biomass and ALA yield. The highest ALA yield of 13.1 mg/g-biomass was achieved with Fe2+ of 400 μmol/L. ALA yields were differentially increased under different metal ions in the following order: Fe2+ group > Mg2+ group > Co2+ group = Ni2+ group > Zn2+ group = Mo2+ group > control. Cu2+ and Mn2+ inhibited Rp. palustris growth and ALA production. Mechanism analysis revealed that metal ions changed ALA yields by influencing the activities of ALA synthetase and ALA dehydratase.

Study of the Fermentation Conditions for 5-Aminolevulinic Acid Production by Rhodopseudomonas palustris

Culture conditions for Rhodopseudomonas palustris RB to produce 5-Aminolevulinic Acid (ALA) was investigated. As results, the best culture conditions for accumulation ALA were 10 % inoculating concentration, light intensity 3000 Lux and its pH value was 7.0. The production of ALA can be improved by useful of resisting the activity of ALA dehydratase in the biosynthetic pathway. And the experimental results showed that Gly was the key precursor substance for ALA. Under the optimal conditions, the production of ALA by this strain was as high as 9.0 mg/L when 45 mM LA and 60 mM were implanted, which was 2 times higher than the control.

Activity of 5-Aminolevulinic Acid Dehydratase Declines during Tomato Fruit Development and Ripening

Activity of 5-aminolevulinic acid (ALA) dehydratase [ALAD, (EC 4.2.1.24)] and soluble protein content were determined in `Rutgers' tomato (Lycopersicon esculentum Mill.) fruit pericarp extracts during development and ripening. ALAD activity in several organs of tomato plant also was determined. Fruit tissue was analyzed at 5-day intervals between days 10 and 60 postanthesis. ALAD activity in fruit tissue declined over time, with the most pronounced decrease occurring between days 10 and 25. At the mature green stage (day 40), and before the breaker stage (day 45), activity of ALAD had declined to a steady-state minimum, and it remained detectable at residual levels throughout ripening (days 40 to 60). Soluble protein content declined less rapidly than did ALAD activity. Immunoblot analysis showed that ALAD protein existed as a doublet of isozymes. One isozyme decreased in abundance, whereas the other isozyme remained constant during development and ripening. ALAD activity was greatest in extracts of chlorophyllous organs (stems, leaves, immature fruit) but only marginally detectable in extracts of nonchlorophyllous organs (roots, overripe fruit). The pH optimum and Km for tomato fruit ALAD were similar to those of ALAD isolated from other sources. Abbreviations: ALA, 5-aminolevulinic acid; ALAD, ALA dehydratase; PBG, porphobilinogen; SDS-PAGE, sodium dodecyl sulfate-polyacry - lamide gel electrophoresis.