Method for assembling and expressing multiple genes in the nucleus of microalgae.

The green alga, Chlamydomonas reinhardtii, is a model organism used in the study of photosynthesis and biotechnological research. Despite its importance, a complete set of genetic tools has yet to be developed. Here, we report the development of a new method for constructing a multi-gene pathway in Saccharomyces cerevisiae and integrating the assembled pathway into the nuclear genome of C.

Production of xylitol by recombinant microalgae.

Microalgae have received significant attention recently as a potential low-cost host for the production of next-generation biofuels and natural products. Here we show that the chloroplast genome of the eukaryotic green microalga Chlamydomonas reinhardtii can be genetically engineered to produce xylitol through the introduction of a gene encoding a xylose reductase (XR) from the fungi Neurospora crassa. Increased levels of heterologous protein accumulation and xylitol production were achieved by synthesizing the XR gene in the chloroplast codon bias and by driving expression of the codon-optimized XR gene using a 16S/atpA promoter/5'-UTR fusion.

Method to assemble and integrate biochemical pathways into the chloroplast genome of Chlamydomonas reinhardtii.

Recombinant protein expression in the chloroplasts of green algae has recently become more routine; however, the heterologous expression of multiple proteins or complete biosynthetic pathways remains a significant challenge. Here, we show that a modified DNA Assembler approach can be used to rapidly assemble multiple-gene biosynthetic pathways in yeast and then integrate these assembled pathways at a site-specific location in the chloroplast genome of the microalgal species Chlamydomonas reinhardtii. As a proof of concept, this method was used to successfully integrate and functionally express up to three reporter proteins (AphA6, AadA, and GFP) in the chloroplast of C.

Directed evolution: selection of the host organism.

Directed evolution has become a well-established tool for improving proteins and biological systems. A critical aspect of directed evolution is the selection of a suitable host organism for achieving functional expression of the target gene. To date, most directed evolution studies have used either Escherichia coli or Saccharomyces cerevisiae as a host; however, other bacterial and yeast species, as well as mammalian and insect cell lines, have also been successfully used.