Effect of an Introduced Phytoene Synthase Gene Expression on Carotenoid Biosynthesis in the Marine Diatom Phaeodactylum tricornutum

Abstract

Carotenoids exert beneficial effects on human health through their excellent antioxidant activity. To increase carotenoid productivity in the marine Pennales Phaeodactylum tricornutum, we genetically engineered the phytoene synthase gene (psy) to improve expression because RNA-sequencing analysis has suggested that the expression level of psy is lower than other enzyme-encoding genes that are involved in the carotenoid biosynthetic pathway. We isolated psy from P. tricornutum, and this gene was fused with the enhanced green fluorescent protein gene to detect psy expression. After transformation using the microparticle bombardment technique, we obtained several P. tricornutum transformants and confirmed psy expression in their plastids. We investigated the amounts of PSY mRNA and carotenoids, such as fucoxanthin and β-carotene, at different growth phases. The introduction of psy increased the fucoxanthin content of a transformants by approximately 1.45-fold relative to the levels in the wild-type diatom. However, some transformants failed to show a significant increase in the carotenoid content relative to that of the wild-type diatom. We also found that the amount of PSY mRNA at log phase might contribute to the increase in carotenoids in the transformants at stationary phase.

Overexpression ofThalassiosira pseudonanaviolaxanthin de-epoxidase-like 2 (VDL2) increases fucoxanthin while stoichiometrically reducing diadinoxanthin cycle pigment abundance

“…Lavaud et al [2012] silenced the P. tricornutum gene that encodes Vx de-epoxidase (VDE), which performs de-epoxidation in both xanthophyll cycles, resulting in a Dtx deepoxidation deficiency (Vx de-epoxidation was not examined). Phytoene synthase (PSY), which catalyzes the first committed step of carotenoid biosynthesis, has been silenced [Kaur and Spillane 2014] and overexpressed [Kadono et al 2015] in P. tricornutum, resulting in reduced and increased cellular carotenoid content, respectively. Nevertheless, much remains to be understood about the post-β-car part of the carotenoid biosynthesis pathway in diatoms.…”

Expression Profile of Genes Involved in Isoprenoid Biosynthesis in the Marine Diatom <i>Phaeodactylum tricornutum</i>

“…In addition, powerful molecular biotechnological techniques such as RNA silencing (De Riso et al, 2009;Sakaguchi et al, 2011;Lavaud et al, 2012) and genome editing using transcription activator-like effector nucleases (TALENs) (Daboussi et al, 2014;Weyman et al, 2014) are available for manipulating target genes. These molecular tools have been used in some studies to create genetically modified P. tricornutum as a potential source of biofuels (Radakovits et al, 2011;Niu et al, 2013;Hamilton et al, 2014), carotenoids (Eilers et al, 2015;Kadono et al, 2015), and specialty chemicals (Hempel et al, 2011;Hempel and Maier, 2012).…”

“…In recent years, several endogenous gene promoters have become available for expressing transgenes in diatoms (Apt et al, 1996;Falciatore et al, 1999;Fischer et al, 1999;Harada et al, 2005;Poulsen and Kröger, 2005;Poulsen et al, 2006;Miyagawa-Yamaguchi et al, 2011;Muto et al, 2013;Ifuku et al, 2015;Seo et al, 2015). Of those endogenous gene promoters, the promoter for the fucoxanthin chlorophyll a/c-binding protein gene (fcp) has been commonly used in biotechnological applications in diatoms (Radakovits et al, 2011;Niu et al, 2013;Trentacoste et al, 2013;Hamilton et al, 2014;Kadono et al, 2015). However, some reports suggest that the activity of the fcp promoter may not be strong enough to overexpress introduced genes and promote significant increases in target products (Radakovits et al, 2011;Kadono et al, 2015).…”

“…Of those endogenous gene promoters, the promoter for the fucoxanthin chlorophyll a/c-binding protein gene (fcp) has been commonly used in biotechnological applications in diatoms (Radakovits et al, 2011;Niu et al, 2013;Trentacoste et al, 2013;Hamilton et al, 2014;Kadono et al, 2015). However, some reports suggest that the activity of the fcp promoter may not be strong enough to overexpress introduced genes and promote significant increases in target products (Radakovits et al, 2011;Kadono et al, 2015). Therefore, promoters useful for the high-level expression of transgenes in genetically modified P. tricornutum are desired.…”

Comparative analysis of structural and functional aspects of phytoene synthase from Meiothermus taiwanensis strain RP

“…The differences in localization, internal environmental conditions, the presence/absence of introns(Dibari et al 2012;Ahrazem et al 2019), and the presence/absence of signal peptides(Kadono et al 2015) might be the reason for the variation in sequence length among the different PSY proteins.The instability index of Meiothermus taiwanensis strain RP sequence is 31.09, which is well below the value of 40, above which proteins are deemed to be unstable. Thus, the RP PSY protein is stable in nature.…”