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.
Highlights
In plant-derived animal feedstuffs, nearly 80 % of the total phosphorus content is stored as phytate
C. reinhardtii has been extensively used as a model protein factory, and here we show how a cell-wall mutant could be used in the administration of phytase into animal feedstuffs
Codon optimization of the PHYA E228K gene We chose the PhyA E228K mutant, developed by Kim, Mullaney [10], as the phytase gene to express in C. reinhardtii
Summary
In plant-derived animal feedstuffs, nearly 80 % of the total phosphorus content is stored as phytate. Phytate is poorly digested by monogastric animals such as poultry, swine and fish, as they lack the hydrolytic enzyme phytase; it is regarded as a nutritionally inactive compound from a phosphate bioavailability point of view. It chelates important dietary minerals and essential amino acids. Phytic acid (myo-inositol hexakisphosphate or IP6) is the major phosphate source in plant seeds, accounting for nearly 80 % of the total phosphate content. Mullaney [10] developed single and multiple mutants of the Aspergillus niger PHYA gene, resulting in a single mutant (E228K) with a shifted optimum pH (from 5.5 to 3.8) and 266 % greater hydrolysis of phytate at pH 3.5 compared to the wild-type version, the same pH as found in the gastrointestinal system of monogastric animals
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