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Due to negative environmental influence and limited availability, petroleum-derived fuels need to be replaced by renewable biofuels. Biodiesel has attracted intensive attention as an important biofuel. Microalgae have numerous advantages for biodiesel production over many terrestrial plants. There are a series of consecutive processes for biodiesel production with microalgae as feedstock, including selection of adequate microalgal strains, mass culture, cell harvesting, oil extraction and transesterification. To reduce the overall production cost, technology development and process optimization are necessary. Genetic engineering also plays an important role in manipulating lipid biosynthesis in microalgae. Many approaches, such as sequestering carbon dioxide from industrial plants for the carbon source, using wastewater for the nutrient supply, and maximizing the values of by-products, have shown a potential for cost reduction. This review provides a brief overview of the process of biodiesel production with microalgae as feedstock. The methods associated with this process (e.g. lipid determination, mass culture, oil extraction) are also compared and discussed.

Background Thymol and carvacrol are natural phenolic compounds with various biological activities. They are widely used in the spice and medicine industries. Production of thymol and carvacrol using microbial cell factories is considered a viable alternative to extracting them from plants of the Thymus genus or the Lamiaceae family. Results In this study, the complete synthetic pathways of thymol and carvacrol were constructed using the oleaginous yeast Yarrowia lipolytica Po1f as a chassis. The total titer of thymol and carvacrol was increased 18.44-fold by enhancing the mevalonate pathway during the modification process and reducing the metabolic flux from geranyl diphosphate (GPP) to farnesyl diphosphate (FPP) through the modification of ERG20. Next, by increasing the copy number of the TvCYP71D507 gene combination ( TvCYP71D507 + TvTPS2 + TvSDR1 ) in the synthetic pathway, the total titer was increased by 1.75-fold. Finally, the engineered strain CT18, which was reintroduced the 3-isopropylmalate dehydrogenase ( LEU2 ) gene, achieved a titer of thymol and carvacrol of 7.14 mg/L in shake flasks and 61.31 mg/L in a 5-L bioreactor. Conclusion This study demonstrates the de novo synthesis of thymol and carvacrol in Y. lipolytica for the first time and provides a valuable reference for constructing microbial cell factories for phenolic monoterpenes biosynthesis.

Continuous research on obtaining an even more efficient production of very long-chain polyunsaturated fatty acids (VLC-PUFAs) in plants remains one of the main challenges of scientists working on plant lipids. Since crops are not able to produce these fatty acids due to the lack of necessary enzymes, genes encoding them must be introduced exogenously from native organisms producing VLC-PUFAs. In this study we reported, in tobacco leaves, the characterization of three distinct ∆ 6 -desaturases from diatom Phaeodactylum tricornutum , fungi Rhizopus stolonifer and microalge Osterococcus tauri and two different ∆ 5 -desaturases from P. tricornutum and single-celled saprotrophic eukaryotes Thraustochytrium sp . The in planta agroinfiltration of essential ∆ 6 -desaturases, ∆ 6 -elongases and ∆ 5 -desaturases allowed for successful introduction of eicosapentaenoic acid (20:5 ∆5,8,11,14,17 ) biosynthesis pathway. However, despite the desired, targeted production of ω3-fatty acids we detected the presence of ω6-fatty acids, indicating and confirming previous results that all tested desaturases are not specifically restricted to neither ω3- nor ω6-pathway. Nevertheless, the additional co-expression of acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT) from Phaeodactylum tricornutum boosted the proportion of ω3-fatty acids in newly synthesized fatty acid pools. For the most promising genes combinations the EPA content reached at maximum 1.4% of total lipid content and 4.5% of all fatty acids accumulated in the TAG pool. Our results for the first time describe the role of LPCAT enzyme and its effectiveness in alleviating a bottleneck called ‘substrate dichotomy’ for improving the transgenic production of VLC-PUFAs in plants.

This study presents characterisation of diatom’s Pt LPCAT1 (acyl-CoA: lysophosphatidylcholine acyltransferase) activity in phospholipid remodelling. In this research microsomal fractions of yeast Δ ale1 mutant overexpressing Pt LPCAT1 were used as a source of the tested enzyme. In the assays evaluating remodelling of different phospholipids by Pt LPCAT1 not modified microsomal fractions of the tested yeast were used. The enzyme most intensively remodelled fatty acid composition of microsomal phosphatidylcholine (PC), however, it was also able to remodel phosphatidylethanolamine (PE) and phosphatidic acid (PA). To study the ability of the tested enzyme to remodel PC molecules containing fatty acids from the VLC-PUFA biosynthetic pathway the tested microsomes were enriched biochemically with: sn -1-18:1- sn -2-18:3(n-3)-PC, sn -1-18:1- sn -2-18:3(n-6)-PC, sn -1-18:1- sn -2-18:4(n-3)-PC, sn -1-18:1- sn -2-20:4(n-3)-PC and sn -1-18:1- sn -2-20:5(n-3)-PC. Further on it was shown that Pt LPCAT1 was able to remodel PC of such modified microsomes with higher intensity than PC of unmodified microsomes. The remodelling efficiency of Pt LPCAT1 was affected also by fatty acid donors; the process was most efficient when acyl-CoAs with unsaturated fatty acids were in the assays. In comparative studies the properties of Arabidopsis At LPCAT1 and yeast ALE1 were tested. Effect of the temperature and pH values on the remodelling activity of Pt LPCAT1 was also examined.

Microsomal fractions from yeast Δale1 cells harbouring the empty plasmid pYES2/CT and from yeast cells overexpressing PtATS2a (Phatr3_J11916) or PtATS2b (Phatr3_J43099) were used in the studies. When sn-1-18:1-LPA and [14C]16:0-CoA were used as exogenous substrates, both PtATS2a and PtATS2b showed the highest activity at 23 °C in the range of temperatures tested from 10 to 60 °C. Both enzymes showed the highest activity in alkaline pH. For PtATS2a, it was pH 10 while for PtATS2b, it was pH 11. At pH 6 and pH 12, the activities of both enzymes were very low. The calcium ions at concentrations of 0.05–1 mM drastically decreased the activity of both enzymes. The magnesium ions at a concentration of 0.05 mM had a little effect on the activity of both enzymes, while higher concentrations (0.5 mM and 1 mM) significantly inhibited their activity. To study the substrate specificity, seventeen different acyl-CoAs in combinations with sn-1-[14C]18:1-LPA were used. PtATS2a showed the highest preference for 18:4-CoA n-3 while PtATS2b for 18:1-CoA. The pattern of utilisation of other acyl-CoAs tested also differed between the two enzymes. The presented studies, for the first time, characterised LPAAT type enzymes from diatoms, organisms that naturally produced very-long-chain polyunsaturated fatty acids (VLC-PUFA).

Since methylmalonyl-CoA epimerase appears to be absent in the majority of photosynthetic organisms, including diatoms, (S)-methylmalonyl-CoA, the intermediate of isoleucine (Ile) catabolism, cannot be metabolized to (R)-methylmalonyl-CoA then to succinyl-CoA. In this study, propionyl-CoA carboxylase (PCC) RNAi silenced strains and 3-hydroxyisobutyryl-CoA hydrolase (HIBCH) overexpression strains were constructed to elucidate the Ile degradation pathway and its influence on lipid accumulation in Phaeodactylum tricornutum based on growth, neutral lipid content and metabolite profile analysis. Knockdown of PCC disturbed the metabolism of Ile through propionyl-CoA to methylmalonyl-CoA, as illustrated by much higher Ile content at day 6. However, Ile decreased to comparable levels to the wild-type at day 10. PCC silencing redirected propionyl-CoA to acetyl-CoA via a modified β-oxidation pathway, and transcript levels for some branched-chain amino acid (BCAA) degradation-related genes, especially HIBCH, significantly upregulated in the PCC mutant, which enhanced the BCAA degradations and thus resulted in higher triacylglycerol (TAG) content. Overexpression of HIBCH accelerates Ile degradation and results in a lowered Ile content in the overexpression strains, thus enhancing carbon skeletons to the tricarboxylic acid cycle and giving rise to increasing TAG accumulation. Our study provides a good strategy to obtain high-lipid-yield transgenic diatoms by modifying the propionyl-CoA metabolism. This article is part of the themed issue ‘The peculiar carbon metabolism in diatoms’.

Over the last few years microalgae have gained increasing interest as a natural source of valuable compounds and as bioreactors for recombinant protein production. Natural high-value compounds including pigments, long-chain polyunsaturated fatty acids, and polysaccharides, which have a wide range of applications in the food, feed, cosmetics, and pharmaceutical industries, are currently produced with nontransgenic microalgae. However, transgenic microalgae can be used as bioreactors for the production of therapeutic and industrially relevant recombinant proteins. This technology shows great promise to simplify the production process and significantly decrease the production costs. To date, a variety of recombinant proteins have been produced experimentally from the nuclear or chloroplast genome of transgenic Chlamydomonas reinhardtii. These include monoclonal antibodies, vaccines, hormones, pharmaceutical proteins, and others. In this review, we outline recent progress in the production of recombinant proteins with transgenic microalgae as bioreactors, methods for genetic transformation of microalgae, and strategies for highly efficient expression of heterologous genes. In particular, we highlight the importance of maximizing the value of transgenic microalgae through producing recombinant proteins together with recovery of natural high-value compounds. Finally, we outline some important issues that need to be addressed before commercial-scale production of high-value recombinant proteins and compounds from transgenic microalgae can be realized.

Background Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), belonging to ω-3 long-chain polyunsaturated fatty acids (ω3-LC-PUFAs), are essential components of human diet. They are mainly supplemented by marine fish consumption, although their native producers are oleaginous microalgae. Currently, increasing demand for fish oils is insufficient to meet the entire global needs, which puts pressure on searching for the alternative solutions. One possibility may be metabolic engineering of plants with an introduced enzymatic pathway producing ω3-LC-PUFAs. Result In this study we focused on the acyl-CoA:diacylglycerol acyltransferase2b ( Pt DGAT2b) from the diatom Phaeodactylum tricornutum , an enzyme responsible for triacylglycerol (TAG) biosynthesis via acyl-CoA-dependent pathway. Gene encoding Pt DGAT2b, incorporated into TAG-deficient yeast strain H1246, was used to confirm its activity and conduct biochemical characterization. Pt DGAT2b exhibited a broad acyl-CoA preference with both di-16:0-DAG and di-18:1-DAG, whereas di-18:1-DAG was favored. The highest preference for acyl donors was observed for 16:1-, 10:0- and 12:0-CoA. Pt DGAT2b also very efficiently utilized CoA-conjugated ω-3 LC-PUFAs (stearidonic acid, eicosatetraenoic acid and EPA). Additionally, verification of the potential role of Pt DGAT2b in planta , through its transient expression in tobacco leaves, indicated increased TAG production with its relative amount increasing to 8%. Its co-expression with the gene combinations aimed at EPA biosynthesis led to, beside elevated TAG accumulation, efficient accumulation of EPA which constituted even 25.1% of synthesized non-native fatty acids (9.2% of all fatty acids in TAG pool). Conclusions This set of experiments provides a comprehensive biochemical characterization of DGAT enzyme from marine microalgae. Additionally, this study elucidates that Pt DGAT2b can be used successfully in metabolic engineering of plants designed to obtain a boosted TAG level, enriched not only in ω-3 LC-PUFAs but also in medium-chain and ω-7 fatty acids.

Phaeodactylum tricornutum (Pt) is a critical microbial cell factory to produce a wide spectrum of marketable products including recombinant biopharmaceutical N-glycoproteins. N-glycosylation modification of proteins is important for their activity, stability, and half-life, especially some special modifications, such as fucose-modification by fucosyltransferase (FucT). Three PtFucTs were annotated in the genome of P. tricornutum , PtFucT1 was located on the medial/trans-Golgi apparatus and PtFucT2-3 in the plastid stroma. Algal growth, biomass and photosynthesis efficiency were significantly inhibited in a knockout mutant of PtFucT1 (PtFucT1-KO). PtFucT1 played a role in non-core fucose modification of N-glycans. The knockout of PtFucT1 might affect the activity of PtGnTI in the complex and change the complex N-glycan to mannose type N-glycan. The study provided critical information for understanding the mechanism of protein N-glycosylation modification and using microalgae as an alternative ecofriendly cell factory to produce biopharmaceuticals.

Located in Pujiang, Chengdu, China, more than four hundred artifacts were unearthed during the excavation of the Warring States (the Warring States period lasts from 475 to 221 BC) ship-shaped graves in 2016. The compositional and metallurgical feature of the unearthed vessels are still unclear. Archaeologists and conservators are puzzled by such problems that need to be answered by systematic scientific studies. Fifty one samples, either from the matrix (26 samples), or from the corrosion part (25 samples) of 25 unearthed bronze vessels, were carefully collected from the fallen part of the bronze vessels for analysis. Technical methods, such as OM, SEM-EDS, XRD and Raman Spectroscopy were adopted for compositional and metallurgical characterization of the bronzes, as well as identification of the corrosion products. The bronzes are Cu–Sn–Pb trinary alloys with typical casting microstructure. The proportion of Sn are in accordance with excavated bronze vessels in surrounding areas. Recycling isn’t involved with the production of such vessels. Layered structure of the corrosion products was observed. Different corrosion products were identified as Cu2CO3(OH)2, Cu2O, CuSO4·5H2O SnO2, PbSO4 and PbCO3. The findings help us gain insights into the bronze manufacturing in Bashu district, as well as the corrosion behavior in certain condition.