Journal of Marine Science and Technology

Journal of Marine Science and Technology

The potential of phytase enzyme production in microalgae using CRISPR method for application in aquaculture

Document Type : Original Manuscript

Authors
Hassan Zadabbas Shahabadi 1
Arash Akbarzadeh 1
Hamideh Ofoghi 2
Sabihe Soleimanian-Zad 3
Saeid Kadkhodaei 4
1 Department of Fisheries, Faculty of Marine Science and Technology, University of Hormozgan, Bandar Abbas, Iran.
2 Department of Biotechnology, Medical and Pharmaceutical Biotechnology Iranian Research Organization for Science and Technology (IROST), Tehran, Iran.
3 Department of Food Science and Technology, College of Agriculture, Isfahan University of Technology, Isfahan, Iran.
4 Genetic Engineering- Genomix, Agricultural Biotechnology Research Institute of Iran (ABRII), Isfahan branch, Isfahan, Iran.
10.22113/jmst.2023.417019.2549
Abstract
ABSTRACT
In modern aquaculture, energy consumption, raw materials used, and environmental effects should be considered, therefore, during the last decade, phytase has been used by aquatic feed industries as an enzyme supplement to increase the digestibility and absorption of plant compounds and improve growth performance along with reducing phosphorus pollution in the aquatic environment. In this study, by using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system, the bacterial phytase gene was inserted into the model microalgae Chlamydomonas reinhardtii in the designed position of the NR gene, and the correct editing was checked and confirmed by PCR. Confirmation of the efficiency and expression of the phytase gene placed in the NR site was investigated using SDS Page gel.  Also, the translation of the phytase gene was confirmed by SDS PAGE on five colonies containing the correct editing and one control sample colony as confirmation of the expression of the transgene inserted at the protein level, which indicated the successful transcription and translation of the phytase gene inserted in the exon 2 position of the microalgae NR gene. considering the benefits of producing and using phytase enzymes in aquatic diets, reducing production costs and eutrophication of aquatic ecosystems will be achieved and it can be a new perspective to accelerate the commercial production of other recombinant proteins in organisms with nutritional value "C. reinhardtii" and provide the development of the fish feed industry and modern aquaculture.
1. INTRODUCTION 
The rising cost and scarcity of fish meal have prompted the exploration of affordable alternative protein sources, such as plant derivatives (Morales et al., 2014). However, the utilization of plant by-products as protein sources is hindered by the presence of anti-nutritional factors, particularly phytate (von Danwitz et al., 2016). Phytate inhibits phosphorus absorption and forms complexes with essential minerals like magnesium, manganese, copper, iron, and calcium, thereby reducing their bioavailability (Dawood et al., 2015), ultimately impacting fish growth performance (Council and others, 1993). Numerous studies highlight the effectiveness of incorporating phytase into the diet to counteract the negative effects of phytate. Phytase hydrolyzes phytate, cleaving phosphate groups and making phosphorus and other vital nutrients available for absorption (Lei et al., 2013). As a significant feed additive, phytase plays a crucial role in providing access to essential nutrients involved in the phytate complex. 
2. MATERIALS AND METHODS
Cultures of Chlamydomonasreinhardtii lacking the cell wall "UVM11" in TAP medium, as outlined by Andersen in 2005, were employed in this study. Phytase proteins from various natural bacterial and fungal sources were investigated and analyzed, leading to the selection of a bacterial phytase gene from Buttiauxellasp., engineered by Danisco. 
The transfection process involved introducing the RNP complex and a donor plasmid containing the phytase gene (P1Chlamyck-I/O) into C. reinhardtii using the glass bead method described by Zadabbas Shahabadi et al. in 2023. Verification of the edited (knocked-in) microalgae was conducted using the PCR method, wherein genomic DNA was extracted from positive colonies utilizing the CTAB method as outlined by Zadabbas Shahabadi et al. in 2023. The accuracy of the editing was confirmed through molecular methods using a PCR device as detailed in the same study. To analyze the expression of phytase, the protein was extracted and subsequent analysis was carried out through SDS PAGE.
3. RESULTS 
In this study, the direct transfer of the RNP complex, in conjunction with the DNA donor plasmid "p1ChlamyCK-I/O," to C. reinhardtii cells was successfully executed, leading to the screening of positive colonies. Confirmation of the phytase gene's translation, indicating the expression of the foreign gene on the protein surface, was verified through SDS PAGE. The results unequivocally demonstrate the successful transcription and translation of the phytase gene, showcasing a molecular weight of 47.5 kDa. 
Moreover, the study findings indicate the successful localization of the phytase gene in the desired position (exon 2 of the nitrate reductase gene) with a consistent expression level. Conversely, the control sample (gene transfer without using the RNP complex) did not exhibit stable expression of the phytase gene.
4. DISCUSSION AND CONCLUSION 
This study achieved successful production of the phytase enzyme by C. reinhardtii using the CRISPR method. The bacterial phytase gene was inserted into the nitrate reductase gene via CRISPR/Cas9 RNP. The efficiency of the gene insertion at the exon 2 position of the nitrate reductase gene was confirmed through SDS PAGE data analysis. Importantly, the study demonstrated the continuity of expression and translation even in the 15th generation of genome-edited clones, confirming the stability of phytase expression at the nitrate reductase (NR) locus. The suitability of the NR gene for inserting foreign target genes was also underscored. Furthermore, cultivation and selection were conducted on all positively edited colonies over 15 generations, spanning 150 days. Results indicated correct and stable editing without the negative effects of gene silencing or insertion sites. Considering the edibility of C. reinhardtii and its FDA approval, these findings have potential implications for the poultry and aquatic industries, where phytase enzyme is a significant dietary component. 
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Keywords
Chlamydomonas reinhardtii microalgae
Phytase
CRISPR system
Nitrate Reductase gene

Subjects

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Journal of Marine Science and Technology
Volume 24, Issue 2
Autumn 2025
Pages 25-39

  • Receive Date 27 September 2023
  • Revise Date 23 October 2023
  • Accept Date 06 December 2023
  • Publish Date 23 August 2025