One-step utilization of inulin for docosahexaenoic acid (DHA) production by recombinant Aurantiochytrium sp. carrying Kl
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RESEARCH PAPER
One‑step utilization of inulin for docosahexaenoic acid (DHA) production by recombinant Aurantiochytrium sp. carrying Kluyveromyces marxianus inulinase Xin‑Jun Yu1 · Zhi‑Peng Wang2 · Miao‑Jie Liang1 · Zhao Wang1 · Xiao‑Yan Liu3 · Lei Hu3 · Jun Xia3 Received: 3 March 2020 / Accepted: 30 April 2020 © Springer-Verlag GmbH Germany, part of Springer Nature 2020
Abstract This study aimed to express an inulinase gene from the yeast Kluyveromyces marxianus (KmINU) in Aurantiochytrium sp. and realized one-step utilization of inulin resource for DHA production without any chemical pretreatment. An expression cassette with a length of 6052 bp for expressing the inulinase gene was constructed by a fast two-step PCR method and then was transferred into the Aurantiochytrium sp. cells. The Aurantiochytrium sp. recombinant T39 was selected with an inulinase activity up to 50.1 U/mL in 72 h. In a 5-l fed-batch fermentation, as high as 148.9 g/L of inulin was directly used within 120 h, and only 1.2 g/L of total sugar was left in the medium at the end of fermentation. The biomass of 51.4 g/L with a lipid content of 69.2% DCW and a DHA yield of 14.9 g/L was obtained. Keywords Aurantiochytrium sp. · Inulinase · Kluyveromyces marxianus · Docosahexaenoic acid (DHA) · Overlap PCR · Inulin Abbreviations DHA Docosahexaenoic acid IOS Inulooligosaccharide SP Signal peptide Pac Actin promoter Pubi Ubiquitin promoter LB Luria–Bertani medium Torfc OrfC terminator Xin-Jun Yu and Zhi-Peng Wang contributed equally to this paper. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00449-020-02371-z) contains supplementary material, which is available to authorized users. * Xin‑Jun Yu [email protected] 1
Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, No.18, Chaowang Road, Hangzhou 310014, People’s Republic of China
2
Marine Science and Engineering College, Qingdao Agricultural University, Qingdao 266109, People’s Republic of China
3
Jiangsu Key Laboratory for Biomass‑Based Energy and Enzyme Technology, Huaiyin Normal University, Huaian 223300, People’s Republic of China
KmINU The inulinase gene from Kluyveromyces marxianus DCW Dry cell weight
Introduction Docosahexaenoic acid (DHA) belongs to a typical ω-3 polyunsaturated fatty acid (PUFA) with various physiological and functional effects for humans, such as improvements of neural and retinal developing in infants and decline in atherosclerosis, hypertriglyceridemia, hypertension, schizophrenia, and cancers [1, 2]. Thus, DHA has been widely applied in the food, medical, and feed industries with significant market potentials. Aurantiochytrium sp. is a heterotrophic thraustochytrid and derived from the marine ecology. Utilizing numerous substrates, Aurantiochytrium sp. can grow at a high rate and accumulate a large amount of lipid rich in ω-3 PUFA (mainly DHA) in its cell. Thus, Aurantiochytrium sp. is well known as a typ
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