Exploring linker's sequence diversity to fuse carotene cyclase and hydroxylase for zeaxanthin biosynthesis.

Fusion of catalytic domains can accelerate cascade reactions by bringing enzymes in close proximity. However, the design of a protein fusion and the choice of a linker are often challenging and lack of guidance. To determine the impact of linker parameters on fusion proteins, a library of linkers featuring various lengths, secondary structures, extensions and hydrophobicities was designed. Linkers were used to fuse the lycopene cyclase (crtY) and ß-carotene hydroxylase (crtZ) from Pantoea ananatis to create fusion proteins to produce zeaxanthin. The fusion efficiency was assessed by comparing the carotenoids content in a carotenoid-producer Escherichia coli strain. It was shown that in addition to the orientation of the enzymes and the size of the linker, the first amino acid of the linker is also a key factor in determining the efficiency of a protein fusion. The wide range of sequence diversity in our linker library enables the fine tuning of protein fusion and this approach can be easily transferred to other enzyme couples.

High-level de novo biosynthesis of glycosylated zeaxanthin and astaxanthin in Escherichia coli.

Because of wide applications in food, feed, pharmaceutical and cosmetic industries, the carotenoid market is growing rapidly. Most carotenoids are hydrophobic, which limits their bioavailability. Glycosylation is a natural route that substantially increases the water solubility, as well as the bioavailability, photostability and biological activities of carotenoids. Here, we report metabolic engineering efforts (e.g., promoter and RBS engineering, optimization of carbon sources and supplementation of bottleneck genes) to produce glycosylated carotenoids in Escherichia coli. By fine-tuning the carotenoid-biosynthetic genes (crtX, crtZ and crtY), our strain produced up to 47.2 mg/L (~ 11,670 ppm) of zeaxanthin glucosides, ~ 78% of the total carotenoids produced. In another construct with mevalonate, astaxanthin pathway and crtX genes, the strain produced a mixture of carotenoid glucosides including astaxanthin and adonixanthin glucosides with a total yield of 8.1 mg/L (1774 ppm). Our work demonstrated a proof-of-concept study for the microbial biosynthesis of glycosylated carotenoids.

Conservation of peripheral nervous system formation mechanisms in divergent ascidian embryos.

Ascidians with very similar embryos but highly divergent genomes are thought to have undergone extensive developmental system drift. We compared, in four species (Ciona and Phallusia for Phlebobranchia, Molgula and Halocynthia for Stolidobranchia), gene expression and gene regulation for a network of six transcription factors regulating peripheral nervous system (PNS) formation in Ciona. All genes, but one in Molgula, were expressed in the PNS with some differences correlating with phylogenetic distance. Cross-species transgenesis indicated strong levels of conservation, except in Molgula, in gene regulation despite lack of sequence conservation of the enhancers. Developmental system drift in ascidians is thus higher for gene regulation than for gene expression and is impacted not only by phylogenetic distance, but also in a clade-specific manner and unevenly within a network. Finally, considering that Molgula is divergent in our analyses, this suggests deep conservation of developmental mechanisms in ascidians after 390 My of separate evolution.