Alkaline phosphatase promoter as an efficient driving element for exogenic recombinant in the marine diatom Phaeodactylum tricornutum

Lin, Hung-Yun; Yen, Shao-Chieh; Kuo, Po-Chih; Chung, Chih-Yu; Yeh, Kuo-Liang; Huang, Ching-Huei; Chang, Jeng Kuei; Lin, Han‐Jia

doi:10.1016/j.algal.2017.01.007

Cited by 23 publications

(19 citation statements)

References 38 publications

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“…Both AP1p_CrGES-mVenus constructs contained the CrGES gene fused at the carboxyl-terminus to a mVenus yellow fluorescent protein (YFP) (Kremers et al, 2006) for rapidly screening the cell lines by flow cytometry. The CrGES-mVenus fusion gene was driven by the P. tricornutum native alkaline phosphatase (AP1, Phatr3_J49678) promoter (hereafter AP1p), which is induced in low phosphate conditions for controllable expression (Lin et al, 2017).…”

Section: Resultsmentioning

confidence: 99%

Metabolic Engineering Strategies in Diatoms Reveal Unique Phenotypes and Genetic Configurations With Implications for Algal Genetics and Synthetic Biology

George

Kahlke

Abbriano

et al. 2020

Front. Bioeng. Biotechnol.

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Section: Resultsmentioning

confidence: 99%

Metabolic Engineering Strategies in Diatoms Reveal Unique Phenotypes and Genetic Configurations With Implications for Algal Genetics and Synthetic Biology

George

Kahlke

Abbriano

et al. 2020

Front. Bioeng. Biotechnol.

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“…The fourth most abundant protein (PHATRDRAFT_54681) was annotated via homology as the enzyme endo-1,3-beta-glucanase which is involved in carbohydrate metabolism 28 . In addition, the fifth most abundant protein (PHATRDRAFT_49678) encodes an alkaline phosphatase, whose activity has been experimentally proven 29 . The protein levels of HASP1 were drastically higher than those of any of the other protein in the culture medium.…”

Section: Resultsmentioning

confidence: 99%

Identification and characterisation of the novel endogenous promoter HASP1 and its signal peptide from Phaeodactylum tricornutum

Erdene-Ochir

Shin²,

Kwon³

et al. 2019

Sci Rep

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Although diatoms have been extensively studied as bioreactors, only a limited number of efficient gene promoters are available. Therefore, the development of new endogenous promoters is important for the heterologous production of a variety of recombinant proteins. Herein, we identified the most abundant secreted protein in Phaeodactylum tricornutum , designated ‘highly abundant secreted protein 1’ (HASP1), and characterised the activities of its promoter and signal peptide using green fluorescent protein (GFP) as a reporter. The HASP1 promoter strongly drove GFP expression during all growth phases of P . tricornutum in culture, in contrast to the commonly used fcpA promoter, which is less active during the stationary phase. The HASP1 signal peptide was also sufficient for facilitating efficient secretion of GFP by P . tricornutum . Our findings suggest that both the promoter and the signal peptide of HASP1 can be utilized as novel tools for the overexpression and secretion of recombinant proteins in P . tricornutum .

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“…The subcellular localization of GPAT1 and LPAT1 was determined by fusing EGFP with the 3′-terminus of the target gene in pPhAP1-EGFP following the previous method [ 30 ]. The recombinant plasmids containing the target genes were electroporated into algal cells.…”

Section: Methodsmentioning

confidence: 99%

Dual expression of plastidial GPAT1 and LPAT1 regulates triacylglycerol production and the fatty acid profile in Phaeodactylum tricornutum

Wang

Dong

Wei

et al. 2018

Biotechnol Biofuels

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BackgroundMetabolic engineering has emerged as a potential strategy for improving microalgal lipid content through targeted changes to lipid metabolic networks. However, the intricate nature of lipogenesis has impeded metabolic engineering. Therefore, it is very important to identify the crucial metabolic nodes and develop strategies to exploit multiple genes for transgenesis. In an attempt to unravel the microalgal triacylglycerol (TAG) pathway, we overexpressed two key lipogenic genes, glycerol-3-phosphate acyltransferase (GPAT1) and lysophosphatidic acid acyltransferase (LPAT1), in oleaginous Phaeodactylum tricornutum and determined their roles in microalgal lipogenesis.ResultsEngineered P. tricornutum strains showed enhanced growth and photosynthetic efficiency compared with that of the wild-type during the growth phase of the cultivation period. However, both the cell types reached stationary phase on day 7. Overexpression of GPAT1 and LPAT1 increased the TAG content by 2.3-fold under nitrogen-replete conditions without compromising cell growth, and they also orchestrated the expression of other key genes involved in TAG synthesis. The transgenic expression of GPAT1 and LPAT1 influenced the expression of malic enzyme and glucose 6-phosphate dehydrogenase, which enhanced the levels of lipogenic NADPH in the transgenic lines. In addition, GPAT1 and LPAT1 preferred C16 over C18 at the sn-2 position of the glycerol backbone.ConclusionOverexpression of GPAT1 together with LPAT1 significantly enhanced lipid content without affecting growth and photosynthetic efficiency, and they orchestrated the expression of other key photosynthetic and lipogenic genes. The lipid profile for elevated fatty acid content (C16-CoA) demonstrated the involvement of the prokaryotic TAG pathway in marine diatoms. The results suggested that engineering dual metabolic nodes should be possible in microalgal lipid metabolism. This study also provides the first demonstration of the role of the prokaryotic TAG biosynthetic pathway in lipid overproduction and indicates that the fatty acid profile can be tailored to improve lipid production.Electronic supplementary materialThe online version of this article (10.1186/s13068-018-1317-3) contains supplementary material, which is available to authorized users.

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Alkaline phosphatase promoter as an efficient driving element for exogenic recombinant in the marine diatom Phaeodactylum tricornutum

Cited by 23 publications

References 38 publications

Metabolic Engineering Strategies in Diatoms Reveal Unique Phenotypes and Genetic Configurations With Implications for Algal Genetics and Synthetic Biology

Metabolic Engineering Strategies in Diatoms Reveal Unique Phenotypes and Genetic Configurations With Implications for Algal Genetics and Synthetic Biology

Identification and characterisation of the novel endogenous promoter HASP1 and its signal peptide from Phaeodactylum tricornutum

Dual expression of plastidial GPAT1 and LPAT1 regulates triacylglycerol production and the fatty acid profile in Phaeodactylum tricornutum

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