Development of phytase-expressing chlamydomonas reinhardtii for monogastric animal nutrition

Abstract

BackgroundIn plant-derived animal feedstuffs, nearly 80 % of the total phosphorus content is stored as phytate. However, phytate is poorly digested by monogastric animals such as poultry, swine and fish, as they lack the hydrolytic enzyme phytase; hence it is regarded as a nutritionally inactive compound from a phosphate bioavailability point of view. In addition, it also chelates important dietary minerals and essential amino acids. Therefore, dietary supplementation with bioavailable phosphate and exogenous phytases are required to achieve optimal animal growth. In order to simplify the obtaining and application processes, we developed a phytase expressing cell-wall deficient Chlamydomonas reinhardtii strain.ResultsIn this work, we developed a transgenic microalgae expressing a fungal phytase to be used as a food supplement for monogastric animals. A codon optimized Aspergillus niger PhyA E228K phytase (mE228K) with improved performance at pH 3.5 was transformed into the plastid genome of Chlamydomonas reinhardtii in order to achieve optimal expression. We engineered a plastid-specific construction harboring the mE228K gene, which allowed us to obtain high expression level lines with measurable in vitro phytase activity. Both wild-type and cell-wall deficient strains were selected, as the latter is a suitable model for animal digestion. The enzymatic activity of the mE228K expressing lines were approximately 5 phytase units per gram of dry biomass at pH 3.5 and 37 °C, similar to physiological conditions and economically competitive for use in commercial activities.ConclusionsA reference basis for the future biotechnological application of microalgae is provided in this work. A cell-wall deficient transgenic microalgae with phytase activity at gastrointestinal pH and temperature and suitable for pellet formation was developed. Moreover, the associated microalgae biomass costs of this strain would be between US$5 and US$60 per ton of feedstuff, similar to the US$2 per ton of feedstuffs of commercially available phytases. Our data provide evidence of phytate-hydrolyzing microalgae biomass for use as a food additive without the need for protein purification.

The algal chloroplast as a synthetic biology platform for production of therapeutic proteins

“…Therefore, dietary supplementation with bioavailable phosphate and exogenous phytases are required to achieve optimal animal growth [53]. Two separate studies have produced recombinant phytases in C. reinhardtii [54,55], with the earlier study demonstrating that dried algal biomass fed to broiler chicks significantly reduced phytate excretion, and the latter study calculating that the costs of production in microalgae are comparable to those for commercial supplies of phytase. It is possible to envisage further cost savings in, for example, pig feed by 'pyramiding' different gut-active proteins such that a single alga produces multiple recombinant products such as phytase, M-SAA, vaccines and anti-bacterials.…”

Synthetic gene design—The rationale for codon optimization and implications for molecular pharming in plants

“…marginale (Pierlé et al 2013), R. pomeroyi (Green et al 2013), L. acidophilus (Askelson et al 2014), C. reinhardtir (Erpel et al 2016), Y. lipolytica (Matthaus et al 2014), S. elongates (van der Woude et al 2016) & baculovirus (Maghodia et al 2016). While it is encouraging that codon optimization software programs are used for a variety of species and purposes, most of the papers…”

“…Many of the codon optimization software lack citations. Of these software packages, citations have been found for JCat and OPTIMIZER for expression systems such as E. coli (Fahimi et al, 2016;Guo et al, 2016;Karkhah and Amani, 2016;Zhao et al, 2016), S. cerevisiae (Ask et al, 2013;Guadalupe-Medina et al, 2013;Li et al, 2015;Milne et al, 2015;Solis-Escalante et al, 2013), N. benthamiana (Binder et al, 2014), HEK293 cells (Shah et al, 2015), B. subtilis (Reilman et al, 2014), Caulobacter crescentus (Ko et al, 2013), Pseudomonas putida (Dammeyer et al, 2011(Dammeyer et al, , 2013, Salmonella typhimurium (Manuel et al, 2011), S. lividans (Dubeau et al, 2009), wheat (Mih alik et al, 2015, transplastomic tobacco plants (chloroplast translation system) (Occhialini et al, 2015), Plasmodium berghei (Singer et al, 2015), Spodoptera frugiperda (Geisler et al, 2015), S. pneumoniae (Overkamp et al, 2013), A. marginale (Pierl e et al, 2013), R. pomeroyi (Green et al, 2013), L. acidophilus (Askelson et al, 2014), C. reinhardtir (Erpel et al, 2016), Y. lipolytica (Matthaus et al, 2014), S. elongates (van der Woude et al, 2016), and baculovirus (Maghodia et al, 2016). While it is encouraging that codon optimization software programs are used for a variety of species and purposes, most of the papers do not compare yields of native and codon optimized sequences, so yield comparisons cannot be made.…”

Engineering the unicellular alga Phaeodactylum tricornutum for high‐value plant triterpenoid production

“…Often these genes are prone to gene silencing mechanisms (Cerutti et al, 1997;Rasala et al, 2014). Therefore, extensive screening needs to be undertaken to weed out lines that only express the resistance marker and identify those which express the transgene to sufficient levels (Erpel et al, 2016;Lauersen et al, 2016;Shin et al, 2016). While C. reinhardtii has proven to be a good model organism for the elucidation of physiological processes, other algal systems are better suited for both the expression of heterologous pathways and for deployment in industrial processes.…”