Monoterpene production by the carotenogenic yeast Rhodosporidium toruloides.

BACKGROUND: Due to their high energy density and compatible physical properties, several monoterpenes have been investigated as potential renewable transportation fuels, either as blendstocks with petroleum or as drop-in replacements for use in vehicles (both heavy and light-weight) or in aviation. Sustainable microbial production of these biofuels requires the ability to utilize cheap and readily available feedstocks such as lignocellulosic biomass, which can be depolymerized into fermentable carbon sources such as glucose and xylose. However, common microbial production platforms such as the yeast Saccharomyces cerevisiae are not naturally capable of utilizing xylose, hence requiring extensive strain engineering and optimization to efficiently utilize lignocellulosic feedstocks. In contrast, the oleaginous red yeast Rhodosporidium toruloides is capable of efficiently metabolizing both xylose and glucose, suggesting that it may be a suitable host for the production of lignocellulosic bioproducts. In addition, R. toruloides naturally produces several carotenoids (C40 terpenoids), indicating that it may have a naturally high carbon flux through its mevalonate (MVA) pathway, providing pools of intermediates for the production of a wide range of heterologous terpene-based biofuels and bioproducts from lignocellulose. RESULTS: Sixteen terpene synthases (TS) originating from plants, bacteria and fungi were evaluated for their ability to produce a total of nine different monoterpenes in R. toruloides. Eight of these TS were functional and produced several different monoterpenes, either as individual compounds or as mixtures, with 1,8-cineole, sabinene, ocimene, pinene, limonene, and carene being produced at the highest levels. The 1,8-cineole synthase HYP3 from Hypoxylon sp. E74060B produced the highest titer of 14.94 ± 1.84 mg/L 1,8-cineole in YPD medium and was selected for further optimization and fuel properties study. Production of 1,8-cineole from lignocellulose was also demonstrated in a 2L batch fermentation, and cineole production titers reached 34.6 mg/L in DMR-EH (Deacetylated, Mechanically Refined, Enzymatically Hydorlized) hydrolysate. Finally, the fuel properties of 1,8-cineole were examined, and indicate that it may be a suitable petroleum blend stock or drop-in replacement fuel for spark ignition engines. CONCLUSION: Our results demonstrate that Rhodosporidium toruloides is a suitable microbial platform for the production of non-native monoterpenes with biofuel applications from lignocellulosic biomass.

Europe PMC in 2017.

Europe PMC (https://europepmc.org) is a comprehensive resource of biomedical research publications that offers advanced tools for search, retrieval, and interaction with the scientific literature. This article outlines new developments since 2014. In addition to delivering the core database and services, Europe PMC focuses on three areas of development: individual user services, data integration, and infrastructure to support text and data mining. Europe PMC now provides user accounts to save search queries and claim publications to ORCIDs, as well as open access profiles for authors based on public ORCID records. We continue to foster connections between scientific data and literature in a number of ways. All the data behind the paper - whether in structured archives, generic archives or as supplemental files - are now available via links to the BioStudies database. Text-mined biological concepts, including database accession numbers and data DOIs, are highlighted in the text and linked to the appropriate data resources. The SciLite community annotation platform accepts text-mining results from various contributors and overlays them on research articles as licence allows. In addition, text miners and developers can access all open content via APIs or via the FTP site.

High-efficiency homologous recombination in the oil-producing alga Nannochloropsis sp.

Algae have reemerged as potential next-generation feedstocks for biofuels, but strain improvement and progress in algal biology research have been limited by the lack of advanced molecular tools for most eukaryotic microalgae. Here we describe the development of an efficient transformation method for Nannochloropsis sp., a fast-growing, unicellular alga capable of accumulating large amounts of oil. Moreover, we provide additional evidence that Nannochloropsis is haploid, and we demonstrate that insertion of transformation constructs into the nuclear genome can occur by high-efficiency homologous recombination. As examples, we generated knockouts of the genes encoding nitrate reductase and nitrite reductase, resulting in strains that were unable to grow on nitrate and nitrate/nitrite, respectively. The application of homologous recombination in this industrially relevant alga has the potential to rapidly advance algal functional genomics and biotechnology.

Regulation of nif gene expression and the energetics of N2 fixation over the diel cycle in a hot spring microbial mat.

Nitrogen fixation, a prokaryotic, O2-inhibited process that reduces N2 gas to biomass, is of paramount importance in biogeochemical cycling of nitrogen. We analyzed the levels of nif transcripts of Synechococcus ecotypes, NifH subunit and nitrogenase activity over the diel cycle in the microbial mat of an alkaline hot spring in Yellowstone National Park. The results showed a rise in nif transcripts in the evening, with a subsequent decline over the course of the night. In contrast, immunological data demonstrated that the level of the NifH polypeptide remained stable during the night, and only declined when the mat became oxic in the morning. Nitrogenase activity was low throughout the night; however, it exhibited two peaks, a small one in the evening and a large one in the early morning, when light began to stimulate cyanobacterial photosynthetic activity, but O2 consumption by respiration still exceeded the rate of O2 evolution. Once the irradiance increased to the point at which the mat became oxic, the nitrogenase activity was strongly inhibited. Transcripts for proteins associated with energy-producing metabolisms in the cell also followed diel patterns, with fermentation-related transcripts accumulating at night, photosynthesis- and respiration-related transcripts accumulating during the day and late afternoon, respectively. These results are discussed with respect to the energetics and regulation of N2 fixation in hot spring mats and factors that can markedly influence the extent of N2 fixation over the diel cycle.

A novel two domain-fusion protein in cyanobacteria with similarity to the CAB/ELIP/HLIP superfamily: evolutionary implications and regulation.

Vascular plants contain abundant, light-harvesting complexes in the thylakoid membrane that are non-covalently associated with chlorophylls and carotenoids. These light-harvesting chlorophyll a/b binding (LHC) proteins are members of an extended CAB/ELIP/HLIP superfamily of distantly related polypeptides, which have between one and four transmembrane helices (TMH). This superfamily includes the single TMH, high-light-inducible proteins (Hlips), found in cyanobacteria that are induced by various stress conditions, including high light, and are considered ancestral to the LHC proteins. The roles of, and evolutionary relationships between, these superfamily members are of particular interest, since they function in both light harvesting and photoprotection and may have evolved through tandem gene duplication and fusion events. We have investigated the Hlips (hli gene family) in the thermophilic unicellular cyanobacterium Synechococcus OS-B'. The five hli genes present on the genome of Synechococcus OS-B' are relatively similar, but transcript analyses indicate that there are different patterns of transcript accumulation when the cells are exposed to various growth conditions, suggesting that different Hlips may have specific functions. Hlip5 has an additional TMH at the N-terminus as a result of a novel fusion event. This additional TMH is very similar to a conserved hypothetical, single membrane-spanning polypeptide present in most cyanobacteria. The evolutionary significance of these results is discussed.

Responses of a thermophilic Synechococcus isolate from the microbial mat of Octopus Spring to light.

Thermophilic cyanobacteria of the genus Synechococcus are major contributors to photosynthetic carbon fixation in the photic zone of microbial mats in Octopus Spring, Yellowstone National Park. Synechococcus OS-B' was characterized with regard to the ability to acclimate to a range of different light irradiances; it grows well at 25 to 200 micromol photons m(-2) s(-1) but dies when the irradiance is increased to 400 micromol photons m(-2) s(-1). At 200 micromol photons m(-2) s(-1) (high light [HL]), we noted several responses that had previously been associated with HL acclimation of cyanobacteria, including cell bleaching, reduced levels of phycobilisomes and chlorophyll, and elevated levels of a specific carotenoid. Synechococcus OS-B' synthesizes the carotenoids zeaxanthin and beta,beta-carotene and a novel myxol-anhydrohexoside. Interestingly, 77-K fluorescence emission spectra suggest that Synechococcus OS-B' accumulates very small amounts of photosystem II relative to that of photosystem I. This ratio further decreased at higher growth irradiances, which may reflect potential photodamage following exposure to HL. We also noted that HL caused reduced levels of transcripts encoding phycobilisome components, particularly that for CpcH, a 20.5-kDa rod linker polypeptide. There was enhanced transcript abundance of genes encoding terminal oxidases, superoxide dismutase, tocopherol cyclase, and phytoene desaturase. Genes encoding the photosystem II D1:1 and D1:2 isoforms (psbAI and psbAII/psbAIII, respectively) were also regulated according to the light regimen. The results are discussed in the context of how Synechococcus OS-B' may cope with high light irradiances in the high-temperature environment of the microbial mat.

The peculiar distribution of class I and class II aldolases in diatoms and in red algae.

Diatom plastids probably evolved by secondary endocytobiosis from a red alga that was up by a eukaryotic host cell. Apparently, this process increased the complexity of the intracellular distribution of metabolic enzymes. We identified genes encoding fructose-bisphosphate aldolases (FBA) in two centric (Odontella sinensis, Thalassiosira pseudonana) and one pennate (Phaeodactylum tricornutum) diatoms and found that four different aldolases are present in both groups: two plastid targeted class II enzymes (FBAC1 and FBAC2), one cytosolic class II (FBA3) and one cytosolic class I (FBA4) enzyme. The pennate Phaeodactylum possesses an additional plastidic class I enzyme (FBAC5). We verified the classification of the different aldolases in the diatoms by enzymatic characterization of isolated plastids and whole cell extracts. Interestingly, our results imply that in plastids of centric and pennate diatoms mainly either class I or class II aldolases are active. We also identified genes for both class I and class II aldolases in red algal EST databases, thus presenting a fascinating example of the reutilization and recompartmentalization of different aldolase isoenzymes during secondary endocytobiosis but as well demonstrating the limited use of metabolic enzymes as markers for the interpretation of phylogenetic histories in algae.

Identification and characterization of a new conserved motif within the presequence of proteins targeted into complex diatom plastids.

Several groups of algae evolved by secondary endocytobiosis, which is defined as the uptake of a eukaryotic alga into a eukaryotic host cell and the subsequent transformation of the endosymbiont into an organelle. Due to this explicit evolutionary history such algae possess plastids that are surrounded by either three or four membranes. Protein targeting into plastids of these organisms depends on N-terminal bipartite presequences consisting of a signal and a transit peptide domain. This suggests that different protein targeting systems may have been combined during establishment of secondary endocytobiosis to enable the transport of proteins into the plastids. Here we demonstrate the presence of an apparently new type of transport into diatom plastids. We analyzed protein targeting into the plastids of diatoms and identified a conserved amino acid sequence motif within plastid preprotein targeting sequences. We expressed several diatom plastid presequence:GFP fusion proteins with or without modifications within that motif in the diatom Phaeodactylum tricornutum and found that a single conserved phenylalanine is crucial for protein transport into the diatom plastids in vivo, thus indicating the presence of a so far unknown new type of targeting signal. We also provide experimental data about the minimal requirements of a diatom plastid targeting presequence and demonstrate that the signal peptides of plastid preproteins and of endoplasmic reticulum-targeted preproteins in diatoms are functionally equivalent. Furthermore we show that treatment of the cells with Brefeldin A arrests protein transport into the diatom plastids suggesting that a vesicular transport step within the plastid membranes may occur.

Diatom genomics: genetic acquisitions and mergers.

Diatom algae arose by two-step endosymbiosis. The complete genome of the diatom Thalassiosira pseudonana has now been sequenced, allowing us to reconstruct the remarkable intracellular gene transfers that occurred during this convoluted cellular evolution.

Presequence acquisition during secondary endocytobiosis and the possible role of introns.

Targeting of nucleus-encoded proteins into chloroplasts is mediated by N-terminal presequences. During evolution of plastids from formerly free-living cyanobacteria by endocytobiosis, genes for most plastid proteins have been transferred from the plastid genome to the nucleus and subsequently had to be equipped with such plastid targeting sequences. So far it is unclear how the gene domains coding for presequences and the respective mature proteins may have been assembled. While land plant plastids are supposed to originate from a primary endocytobiosis event (a prokaryotic cyanobacterium was taken up by a eukaryotic cell), organisms with secondary plastids like diatoms experienced a second endocytobiosis step involving a eukaryotic alga taken up by a eukaryotic host cell. In this group of algae, apparently most genes encoding chloroplast proteins have been transferred a second time (from the nucleus of the endosymbiont to the nucleus of the secondary host) and thus must have been equipped with additional targeting signals. We have analyzed cDNAs and the respective genomic DNA fragments of seven plastid preproteins from the diatom Phaeodactylum tricornutum. In all of these genes we found single spliceosomal introns, generally located within the region coding for the N-terminal plastid targeting sequences or shortly downstream of it. The positions of the introns can be related to the putative phylogenetic histories of the respective genes, indicating that the bipartite targeting sequences in these secondary algae might have evolved by recombination events via introns.

New insight into Phaeodactylum tricornutum fatty acid metabolism. Cloning and functional characterization of plastidial and microsomal delta12-fatty acid desaturases.

In contrast to 16:3 plants like rapeseed (Brassica napus), which contain alpha-linolenic acid (18:3(Delta9,12,15)) and hexadecatrienoic acid (16:3(Delta7,10,13)) as major polyunsaturated fatty acids in leaves, the silica-less diatom Phaeodactylum tricornutum contains eicosapentaenoic acid (EPA; 20:5(Delta5,8,11,14,17)) and a different isomer of hexadecatrienoic acid (16:3(Delta6,9,12)). In this report, we describe the characterization of two cDNAs having sequence homology to Delta12-fatty acid desaturases from higher plants. These cDNAs were shown to code for a microsomal and a plastidial Delta12-desaturase (PtFAD2 and PtFAD6, respectively) by heterologous expression in yeast (Saccharomyces cerevisiae) and Synechococcus, respectively. Using these systems in the presence of exogenously supplied fatty acids, the substrate specificities of the two desaturases were determined and compared with those of the corresponding rapeseed enzymes (BnFAD2 and BnFAD6). The microsomal desaturases were similarly specific for oleic acid (18:1(Delta9)), suggesting that PtFAD2 is involved in the biosynthesis of EPA. In contrast, the plastidial desaturase from the higher plant and the diatom clearly differed. Although the rapeseed plastidial desaturase showed high activity toward the omega9-fatty acids 18:1(Delta9) and 16:1(Delta7), in line with the fatty acid composition of rapeseed leaves, the enzyme of P. tricornutum was highly specific for 16:1(Delta9). Our results indicate that in contrast to EPA, which is synthesized in the microsomes, the hexadecatrienoic acid isomer found in P. tricornutum (16:3(Delta6,9,12)) is of plastidial origin.

In vivo characterization of diatom multipartite plastid targeting signals.

Plastids of diatoms and related algae are delineated by four membranes: the outermost membrane (CER) is continuous with the endoplasmic reticulum while the inner two membranes are homologous to plastid envelope membranes of vascular plants and green algae. Proteins are transported into these plastids by pre-sequences that have two recognizable domains. To characterize targeting of polypeptides within diatom cells, we generated constructs encoding green fluorecent protein (GFP) fused to leader sequences. A fusion of GFP to the pre-sequence of BiP [an endoplasmic reticulum (ER)-localized chaperone] resulted in accumulation of GFP within the ER; a construct encoding the pre-sequence of a plastid protein fused to GFP was directed into the plastids. Additional constructs demonstrated that the N-terminal region of the bipartite plastid targeting pre-sequence was necessary for transport of polypeptides to the lumen of the ER, while the C-terminal region was shown to enable the proteins to traverse the plastid double envelope membrane. Our data strongly support the hypothesis of a multi-step plastid targeting process in chromophytic algae and raises questions about the continuity of the ER and CER and the function of the latter in polypeptide trafficking.