Heterologous expression of phytase in Schizochytrium sp. as a fortified feed additive for the Livestock industry

Abstract

Phytates present in plant-derived feed can chelate nutrients and reduce their bioavailability for monogastric animals such as poultry and swine. The addition of hydrolase, phytase can alleviate this problem but is hindered by its cost. The goal of the current study is to clone, express and purify the phytase gene from Bacillus sp. (DS11) into Schizochytrium sp. ATCC 20888 is also a good producer of Docosahexaenoic acid (DHA). This is expected to enhance animal nutrition and reduce phosphate pollution. The DNA sequence analysis using multiple sequence alignments showed significant similarity to the phytase gene from Bacillus sp. (DS11). Subsequently, specific primers were designed based on the consensus sequence of the Bacillus phytase gene obtained from sequencing. The coding DNA sequence was determined to have a length of 1152 base pairs. Phytase gene was successfully cloned into the pRI201-AN DNA vector and transformed into Schyzochytrium sp. Screening on G418 plates showed 53 resistant colonies and from this 11 prominent colonies were chosen for further testing. Out of this, 8 colonies tested positive, with colony PCR having 1.5 kb with a phytase activity of 1.77 U/ml of crude lysate. Further purification with Ni-NTA affinity chromatography provided a specific activity of 15.59 U/mg. This appears to be the first ever reported recombinant phytase produced in Schizochytrium sp. The phytase recommendations are 250U/Kg of feed preparation for broiler & swine diets . It was also determined that 72.64 U/5.2 gm of wet biomass and 1.80% of w/w microalgae would fulfil these requirements per kg of feed preparation.