Background: Microalgae are ideal raw materials for biodiesel and bioactive compounds. Glycerol-3-phosphate is formed from dihydroxyacetone phosphate (DHAP) through the glycolytic pathway catalyzed by glycerol-3-phosphate dehydrogenase (GPDH). Results: GPDH was characterized in the marine diatom Phaeodactylum tricornutum. In the GPDH-overexpressing P. tricornutum cells, the glycerol concentration per cell in the transformed diatom increased by 6.8-fold compared with the wild type, indicating that the overexpression of GPDH promoted the conversion of DHAP to glycerol-3-phosphate. There was a 60% increase in neutral lipid content, reaching 39.7% of dry cell weight in transgenic cells in the stationary phase, despite a 20% decrease in cell concentration. Fatty acid profiling showed that the levels of 16-and 18-carbon monounsaturated fatty acids significantly increased. Conclusion: GPDH had a significant impact on numerous metabolic processes in diatom cells, including the biosynthesis of glycerol and neutral lipids. These findings are instructive for the metabolic engineering of microalgae for biofuel production.

Plastids are ideal subcellular hosts for the expression of transgenes and have been successfully used for the production of different biopolymers, therapeutic proteins and industrial enzymes. Phaeodactylum tricornutum is a widely used aquatic feed species. In this study, we focused on developing a high-efficiency plastid expression system for P. tricornutum. In the plastid transformation vector, the site selected for integration was the transcriptionally active intergenic region present between the trnI and trnA genes, located in the IR (inverted repeat) regions of the plastid genome. Initially, a CAT reporter gene (encoding chloramphenicol acetyltransferase) was integrated at this site in the plastid genome. The expression of CAT in the transformed microalgae conferred resistance to the antibiotic chloramphenicol, which enabled growth in the selection media. Overall, the plastid transformation efficiency was found to be approximately one transplastomic colony per 1,000 microalgae cells. Subsequently, a heterologous gene expression cassette for high-level expression of the target gene was created and cloned between the homologous recombination elements. A TA cloning strategy based on the designed XcmI-XcmI sites could conveniently clone the heterologous gene. An eGFP (green fluorescent protein) reporter gene was used to test the expression level in the plastid system. The relatively high-level expression of eGFP without codon optimisation in stably transformed microalgae was determined to account for 0.12 % of the total soluble protein. Thus, this study presents the first and convenient plastid gene expression system for diatoms and represents an interesting tool to study diatom plastids.Electronic supplementary materialThe online version of this article (doi:10.1007/s10126-014-9570-3) contains supplementary material, which is available to authorized users.

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Background and purpose The spread of the plasmid-mediated, colistin-resistance gene mcr-1 into New Delhi metallo-β-lactamase (NDM)-producing bacterial isolates can cause untreatable infections. In this study, we conducted a molecular characterization of a novel, conjugative, bla NDM-5 -positive IncFII plasmid (pNDM-EC16-50) together with an mcr-1 -bearing IncI2 plasmid in a single Escherichia coli ST167 clinical isolate EC16-50. Methods and results EC16-50, which belongs to the E. coli strain ST167 and phylogroup A, was identified to co-produce NDM-5 and MCR-1. S1-PFGE and Southern blotting showed that bla NDM-5 and mcr-1 genes were located oñ95 kb and ~65 kb plasmids, respectively. A conjugation assay revealed that both bla NDM-5 - and mcr-1 -bearing plasmids were self-transmissible. Comparative plasmid analysis suggested that bla NDM-5 -harboring F2:A-:B-plasmid might have evolved from the well-reported NDM-carrying pMC-NDM-like plasmid via recombination with a locally emerged pSJ_94 plasmid, whereas the mcr-1 -carrying IncI2 plasmid was similar to previously reported mcr-1 -bearing plasmids in China. Conclusion and impact This study represents the first report of the NDM-5 carrying Inc-FII- but not IncX3-type plasmid in an MCR-1-producing E. coli isolate. More striking was the dissemination of mcr-1 in a successful epidemic NDM-5-producing E. coli clone ST167, which could facilitate the spread of colistin resistance in carbapenemase-producing E. coli isolates.

Photosynthetic microalgae are of burgeoning interest in the generation of commercial bioproducts. Microalgae accumulate high lipid content under adverse conditions, which in turn compromise their growth and hinder their commercial potential. Hence, it is necessary to engineer microalgae to mitigate elevated lipid accumulation and biomass. In this study, we identified acetyl-CoA carboxylase (ACCase) in oleaginous microalga Phaeodactylum tricornutum (PtACC2) and expressed constitutively in the chloroplast to demonstrate the potential of chloroplast engineering. Molecular characterization of transplastomic microalgae revealed that PtACC2 was integrated, transcribed and expressed successfully, and localized in the chloroplast. Enzymatic activity of ACCase was elevated by 3.3-fold, and the relative neutral lipid content increased substantially by 1.77-fold, and lipid content reached up to 40.8% of dry weight. Accordingly, the number and size of oil bodies markedly increased. Fatty acid profiling showed that the content of monounsaturated fatty acids increased, while polyunsaturated fatty acids decreased. This method provides a valuable genetic engineering toolbox for microalgal bioreactors with industrial significance.