Engineering Haloferax mediterranei as an Efficient Platform for High Level Production of Lycopene.

Lycopene attracts increasing interests in the pharmaceutical, food, and cosmetic industries due to its anti-oxidative and anti-cancer properties. Compared with other lycopene production methods, such as chemical synthesis or direct extraction from plants, the biosynthesis approach using microbes is more economical and sustainable. In this work, we engineered Haloferax mediterranei, a halophilic archaeon, as a new lycopene producer. H. mediterranei has the de novo synthetic pathway for lycopene but cannot accumulate this compound. To address this issue, we reinforced the lycopene synthesis pathway, blocked its flux to other carotenoids and disrupted its competitive pathways. The reaction from geranylgeranyl-PP to phytoene catalyzed by phytoene synthase (CrtB) was identified as the rate-limiting step in H. mediterranei. Insertion of a strong promoter PphaR immediately upstream of the crtB gene, or overexpression of the heterologous CrtB and phytoene desaturase (CrtI) led to a higher yield of lycopene. In addition, blocking bacterioruberin biosynthesis increased the purity and yield of lycopene. Knock-out of the key genes, responsible for poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) biosynthesis, diverted more carbon flux into lycopene synthesis, and thus further enhanced lycopene production. The metabolic engineered H. mediterranei strain produced lycopene at 119.25 ± 0.55 mg per gram of dry cell weight in shake flask fermentation. The obtained yield was superior compared to the lycopene production observed in most of the engineered Escherichia coli or yeast even when they were cultivated in pilot scale bioreactors. Collectively, this work offers insights into the mechanism involved in carotenoid biosynthesis in haloarchaea and demonstrates the potential of using haloarchaea for the production of lycopene or other carotenoids.

Biodegradation and biocompatibility of haloarchaea-produced poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymers.

We previously reported that the tailor-made random poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (R-PHBHV) and higher-order PHBHV (O-PHBHV) produced by haloarchaea possessed unique material properties to meet biomedical application-specific requirements. Here, we further investigated the biocompatibility and biodegradation of these novel materials. Cell biocompatibility of solution-cast films, assessed using rat fibroblast and osteoblast cells, revealed that R-PHBHV and O-PHBHV exhibited better support for cell attachment and proliferation compared with the bacteria-produced poly-3-hydroxybutyrate (PHB) and PHBHV or polylactic acid (PLA). In vitro and in vivo biodegradation of these materials were evaluated in lipase-containing phosphate buffered solution (LPBS) at pH 7.4 and by implantation in the rabbit dorsal subcutis, respectively. As expected, the R-PHBHV and O-PHBHV films degraded much faster in vivo than those observed in vitro, as demonstrated by obvious weight loss, heavy surface erosion, and fast molecular weight drop under implantation condition. These films showed diverse in vivo degradation rates. Among them, the O-PHBHV-1 film degraded fastest and even faster than PLA. Generally, the tissue response was mild for R-PHBHV and O-PHBHV compared with the controls during the implantation period. Taken together, these data revealed that R-PHBHV and O-PHBHV copolyesters had a wild range of biodegradation profiles and excellent biocompatibility. Thus, haloarchaea-produced PHBHV materials would have great potential for use in different biomedical applications.

Identification of 12 animal species meat by T-RFLP on the 12S rRNA gene.

The verification of authenticity of meat products is relevant for economical, religious or public health concerning reasons. A molecular approach using terminal restriction fragment length polymorphism (T-RFLP) was developed to distinguish 12 common economically important meat species. The partial 12S rRNA gene was amplified with double-fluorescently labeled primers. The amplified fragments were digested with two endonucleases and only the terminal restriction fragment containing labeled primer was detected on capillary electrophoresis system ABI3100. AluI and Tru9I generated differently-sized terminal fragments in different species. Pig and buffalo can be separated by 3'-terminal fragment of AluI digestion. Horse, turkey, goat, sheep, deer, and cattle can be further separated by 5'-terminal fragment of Tru9I digestion. Dog and chicken, sturgeon and salmon can finally be separated by 5'-terminal fragment of AluI digestion and 3'-terminal fragment of Tru9I digestion. Our results demonstrated the potential feasibility and applicability of T-RFLP method for rapid and accurate identification of animal species.