Unlocking microalgal host-exploring dark-growing microalgae transformation for sustainable high-value phytochemical production

Surumpa Jareonsin; Kanjana Mahanil; Kittiya Phinyo; Sirasit Srinuanpan; Jeeraporn Pekkoh; Masafumi Kameya; Hiroyuki Arai; Masaharu Ishii; Ruttaporn Chundet; Pachara Sattayawat; Chayakorn Pumas

doi:10.3389/fbioe.2023.1296216

Unlocking microalgal host-exploring dark-growing microalgae transformation for sustainable high-value phytochemical production

Front Bioeng Biotechnol. 2023 Nov 9:11:1296216. doi: 10.3389/fbioe.2023.1296216. eCollection 2023.

Authors

Surumpa Jareonsin^{1

2}, Kanjana Mahanil¹, Kittiya Phinyo^{1

3}, Sirasit Srinuanpan^{1

3

4}, Jeeraporn Pekkoh¹, Masafumi Kameya⁵, Hiroyuki Arai⁵, Masaharu Ishii⁵, Ruttaporn Chundet⁶, Pachara Sattayawat¹, Chayakorn Pumas^{1

7

8}

Affiliations

¹ Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai, Thailand.
² Applied Microbiology (International Program) in Faculty of Science, Chiang Mai University, Chiang Mai, Thailand.
³ Office of Research Administration, Chiang Mai University, Chiang Mai, Thailand.
⁴ Center of Excellence in Microbial Diversity and Sustainable Utilization, Chiang Mai University, Chiang Mai, Thailand.
⁵ Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan.
⁶ Division of Biotechnology, Faculty of Science, Maejo University, Chiangmai, Chiang Mai, Thailand.
⁷ Environmental Science Research Centre, Faculty of Science, Chiang Mai University, Chiang Mai, Thailand.
⁸ Multidisciplinary Research Institute, Chiang Mai University, Chiang Mai, Thailand.

Abstract

Microalgae have emerged as a promising, next-generation sustainable resource with versatile applications, particularly as expression platforms and green cell factories. They possess the ability to overcome the limitations of terrestrial plants, such non-arable land, water scarcity, time-intensive growth, and seasonal changes. However, the heterologous expression of interested genes in microalgae under heterotrophic cultivation (dark mode) remains a niche area within the field of engineering technologies. In this study, the green microalga, Chlorella sorokiniana AARL G015 was chosen as a potential candidate due to its remarkable capacity for rapid growth in complete darkness, its ability to utilize diverse carbon sources, and its potential for wastewater treatment in a circular bioeconomy model. The aims of this study were to advance microalgal genetic engineering via dark cultivation, thereby positioning the strain as promising dark-host for expressing heterologous genes to produce high-value phytochemicals and ingredients for food and feed. To facilitate comprehensive screening based on resistance, eleven common antibiotics were tested under heterotrophic condition. As the most effective selectable markers for this strain, G418, hygromycin, and streptomycin exhibited growth inhibition rates of 98%, 93%, and 92%, respectively, ensuring robust long-term transgenic growth. Successful transformation was achieved through microalgal cell cocultivation with Agrobacterium under complete darkness verified through the expression of green fluorescence protein and β-glucuronidase. In summary, this study pioneers an alternative dark-host microalgal platform, using, Chlorella, under dark mode, presenting an easy protocol for heterologous gene transformation for microalgal host, devoid of the need for expensive equipment and light for industrial production. Furthermore, the developed genetic transformation methodology presents a sustainable way for production of high-value nutrients, dietary supplements, nutraceuticals, proteins and pharmaceuticals using heterotrophic microalgae as an innovative host system.

Keywords: agrobacterium; chlorella; genetic engineering; heterotroph; microalgae; phytochemicals; transformation.

Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. The first author was partially supported by the Science Achievement Scholarship of Thailand (SAST), and the Graduate School of Chiang Mai University. This research project was partially supported by the Chiang Mai University.