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| Gene Synthesis | The?nucleotide sequences?for CDO (NCBI ID:?BAE73112) and CSAD (NCBI ID:?BAE73113) proteins were obtained for common carp (C. carpio) from the NCBI database. The corresponding nucleotide sequences were?codon?optimized for expression in?C. reinhardtii?chloroplast [20,21] as separate genes (cdo::csad), or as fused genes (cdo-csad). The two sequences were then joined with nucleotide sequence coding a GS linker (a 14 amino acid linker; GGGGSSGGGGGGSS) making the fused?cdo-csad?gene. Codon optimization was performed based on codon usage table for?C. reinhardtii?chloroplast genome. The codon-optimized?cdo-csad?(fused with GS linker) nucleotide sequence was synthesized by GenScript USA Inc. The chloroplast transformation vector for?C. reinhardtii?p322 with?aadA?cassette (spectinomycin resistance) under 5′ atpA and 3′ rbcL?untranslated regions?(UTRs) was used for transformation [11]. Sequences homologous to a 5.5?kb region of inverted repeats in the chloroplast genome were targeted for?homologous recombination?[20,21] (Fig. 2). For the?cdo-csad?construct, these regions flanked the?aadA?cassette and the?cdo-csad?sequence under 5′ and 3′?psbA?UTRs, resulting in p322::cdo-csad::aadA?construct (Fig. 2b). For the cdo::csad construct,?cdo?and?csadgenes were constructed with spacers to separate the synthetic?cdo-csad?product by using overlap extension PCR. The regions of?homology?flanked the?aadA?cassette, the?cdo?gene under the influence of 5′?psbA?and 3′?atpA?UTRs, and?csad?under 5′?psbDand 3′?psbA?UTRs (Fig. 2c). The resultant?plasmid?was called p322::cdo::csad::aadA. After cloning, the sequences were confirmed by?DNA sequencing. | Get A Quote |
Synthetic?taurine?is an important animal feed supplement critical for fish and cat nutrition. However, there is a demand for natural and renewable sources of this?nutraceutical?additive. The objective of this study was to increase the production of taurine using genetically engineered?Chlamydomonas reinhardtii?modified to have the?cysteine?dioxygenase/cysteine sulfinic acid decarboxylase (CDO/CSAD) taurine synthesis pathway from animals. Because unicellular green?microalgae?produce taurine at low levels, methods to increase taurine content may qualify the use of these?microbes?as a feed supplement. Here, increased microalgal taurine content was accomplished using a synthetic approach based on additi... More
