Characterization of endogenous promoters of GapC1 and GS for recombinant protein expression in Phaeodactylum tricornutum.

Although diatoms have been utilized as a cellular factory to produce biopharmaceuticals, recombinant proteins, and biofuels, only a few numbers of gene promoters are available. Therefore, the development of novel endogenous promoters is essential for the production of a range of bioactive substances. Here, we characterized the activities of endogenous promoters glyceraldehyde-3-phosphate dehydrogenase (GapC1) and glutamine synthetase (GS) of Phaeodactylum tricornutum using green fluorescent protein (GFP) under different culture conditions. Compared with the widely used fucoxanthin chlorophyll-binding protein A (fcpA) promoter, the GS promoter constitutively drove the expression of GFP throughout all growth phases of P. tricornutum, regardless of culture conditions. Additionally, the GFP level driven by the GapC1 promoter was the highest at the log phase, similar to the fcpA promoter, and increased light and nitrogen-starvation conditions reduced GFP levels by inhibiting promoter activity. These results suggested that the GS promoter could be utilized as a strong endogenous promoter for the genetic engineering of P. tricornutum.

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

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.

Exceptionally high percentage of IPP synthesis by Ginkgo biloba IspH is mainly due to Phe residue in the active site.

(E)-4-Hydroxy-3-methylbut-2-enyl diphosphate (HMBPP) reductase (IspH, HDR or LytB) is an Fe/S enzyme catalyzing the reductive dehydroxylation of HMBPP to isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) in the last step of methylerythritol phosphate (MEP) pathway. The MEP pathway is known to produce 4-6:1 ratio of IPP and DMAPP mixture by the last enzyme, IspH. Plant IspH in plastids follows same catalytic mechanism as others, but GbIspH (Ginkgo biloba IspH) was reported to produce a mixture of IPP and DMAPP in a ratio of 16:1. Present catalytic mechanisms of IspH involve a common allyl anion intermediate, and the intramolecular proton transfer to the allyl moiety is considered as the key reaction step determining the product between IPP and DMAPP. The F212 residue in plant IspH was found as a potential amino acid residue that could mediate the proton transfer to the allyl anion intermediate before the product release. In this report, catalytic function of GbIspH F212 residue (H74 in E. coli), especially during the product formation in the active site, was studied by means of site-directed mutation. The product ratio of IPP/DMAPP was measured as 6.5 ± 0.1 for F212H GbIspH, and the value was close to the reported bacterial IspH having His residue on that specific position. Along with the other F212Y mutant, of which ratio was determined as 10.9 ± 0.1, the results strongly support that the Phe residue in plant IspH is the key amino acid residue that allows exclusive production of IPP in plant chloroplast.

N-Terminal Region of GbIspH1, Ginkgo biloba IspH Type 1, May Be Involved in the pH-Dependent Regulation of Enzyme Activity.

GbIspH1, IspH type 1 in Ginkgo biloba chloroplast, is the Fe/S enzyme catalyzing the reductive dehydroxylation of HMBPP to isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) at the final step of methylerythritol phosphate pathway in chloroplast. Compared to the bacterial IspH, plant IspH, including GbIspH1, has an additional polypeptide chain at the N-terminus. Here, biochemical function of the N-terminal region of GbIspH1 was investigated with the N-terminal truncated GbIspH1 (GbIspH1-truncated). Both wild type GbIspH1 (GbIspH1-full) and GbIspH1-truncated were catalytically active and produced IPP and DMAPP in a ratio of 15 : 1. Kinetic parameters of K M (17.3 ± 1.9 and 14.9 ± 2.3 µM) and k cat (369 ± 10 and 347 ± 12 min(-1)) at pH 8.0 were obtained for GbIspH1-full and GbIspH1-truncated, respectively. Interestingly, GbIspH1-full and GbIspH1-truncated showed significantly different pH-dependent activities, and the maximum enzyme activities were obtained at pH 8.0 and 7.5, respectively. However, catalytic activation energies (E a ) of GbIspH1-full and GbIspH1-truncated were almost the same with 36.5 ± 1.6 and 35.0 ± 1.9 kJ/mol, respectively. It was suggested that the N-terminal region of GbIspH1 is involved in the pH-dependent regulation of enzyme activity during photosynthesis.

Absolute configurations of isoflavan-4-ol stereoisomers.

Isoflavan-4-ol has been synthesized quantitatively from the reduction of isoflavone in the presence of Pd/C and ammonium formate under N(2) atmosphere. Isolation of cis- and trans-isomers was achieved by flash column chromatography and each enantiomer was separated by Sumi-Chiral column chromatography. Absolute configurations of four stereoisomers were determined by circular dichroism spectroscopy.