Enhancement of violaxanthin accumulation in Nannochloropsis oceanica by overexpressing a carotenoid isomerase gene from Phaeodactylum tricornutum

Yan Sun; Yi Xin; Luyao Zhang; Ying Wang; Ruolan Liu; Xiaohui Li; Chengxu Zhou; Lin Zhang; Jichang Han

doi:10.3389/fmicb.2022.942883

Enhancement of violaxanthin accumulation in Nannochloropsis oceanica by overexpressing a carotenoid isomerase gene from Phaeodactylum tricornutum

Front Microbiol. 2022 Aug 31:13:942883. doi: 10.3389/fmicb.2022.942883. eCollection 2022.

Authors

Yan Sun¹, Yi Xin², Luyao Zhang¹, Ying Wang¹, Ruolan Liu¹, Xiaohui Li¹, Chengxu Zhou¹, Lin Zhang³, Jichang Han^{1

4}

Affiliations

¹ College of Food and Pharmaceutical Sciences, Ningbo University, Ningbo, China.
² State Key Laboratory of Marine Resource Utilization in the South China Sea, College of Oceanology, Hainan University, Haikou, China.
³ Key Laboratory of Applied Marine Biotechnology of Ministry of Education of China, School of Marine Science, Ningbo University, Ningbo, China.
⁴ College of Marine Life Science, Ocean University of China, Qingdao, China.

Abstract

Nannochloropsis has been considered as a promising feedstock for the industrial production of violaxanthin. However, a rational breeding strategy for the enhancement of violaxanthin content in this microalga is still vacant, thereby limiting its industrial application. All-trans-lycopene locates in the first branch point of carotenogenesis. The carotenoid isomerase (CRTISO), catalyzing the lycopene formation, is thus regarded as a key enzyme for carotenogenesis. Phaeodactylum tricornutum can accumulate high-level carotenoids under optimal conditions. Therefore, it is feasible to improve violaxanthin level in Nannochloropsis by overexpression of PtCRTISO. Protein targeting analysis of seven PtCRTISO candidates (PtCRTISO1-6 and PtCRTISO-like) demonstrated that PtCRTISO4 was most likely the carotenoid isomerase of P. tricornutum. Moreover, the transcriptional pattern of PtCRTISO4 at different cultivation periods was quite similar to other known carotenogenesis genes. Thus, PtCRTISO4 was transformed into N. oceanica. Compared to the wild type (WT), all three transgenic lines (T1-T3) of N. oceanica exhibited higher levels of total carotenoid and violaxanthin. Notably, T3 exhibited the peak violaxanthin content of 4.48 mg g^-1 dry cell weight (DCW), which was 1.68-folds higher than WT. Interestingly, qRT-polymerase chain reaction (PCR) results demonstrated that phytoene synthase (NoPSY) rather than ζ-carotene desaturase (NoZDS) and lycopene β-cyclase (NoLCYB) exhibited the highest upregulation, suggesting that PtCRTISO4 played an additional regulatory role in terms of carotenoid accumulation. Moreover, PtCRTISO4 overexpression increased C18:1n-9 but decreased C16:1n-7, implying that C18:1 may serve as a main feedstock for xanthophyll esterification in Nannochloropsis. Our results will provide valuable information for the violaxanthin production from Nannochloropsis.

Keywords: Nannochloropsis oceanica; Phaeodactylum tricornutum; carotenoid isomerase; transgenic manipulation; violaxanthin.