RESUMO
The diterpenoid sclareol is an industrially important precursor for alternative sustainable supply of ambergris. However, its current production from plant extraction is neither economical nor environmental-friendly, since it requires laborious and cost-intensive purification procedures and plants cultivation is susceptible to environmental factors. Engineering cell factories for bio-manufacturing can enable sustainable production of natural products. However, stringent metabolic regulation poses challenges to rewire cellular metabolism for overproduction of compounds of interest. Here we used a modular approach to globally rewire the cellular metabolism for improving sclareol production to 11.4 g/L in budding yeast Saccharomyces cerevisiae, the highest reported diterpenoid titer in microbes. Metabolic flux analysis showed that modular balanced metabolism drove the metabolic flux toward the biosynthesis of targeted molecules, and transcriptomic analysis revealed that the expression of central metabolism genes was shaped for a new balanced metabolism, which laid a foundation in extensive metabolic engineering of other microbial species for sustainable bio-production.
Assuntos
Diterpenos , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Diterpenos/metabolismo , Engenharia Metabólica/métodosRESUMO
Glucosylglycerol (GG) is an osmolyte found in a few bacteria (e.g., cyanobacteria) and plants grown in harsh environments. GG protects microbes and plants from salinity and desiccation stress. In the industry, GG is synthesized from a combination of ADP-glucose and glycerol-3-phosphate in a condensation reaction catalyzed by glucosylglycerol phosphate synthase. Proline, on the other hand, is an amino acid-based osmolyte that plays a key role in cellular reprograming. It functions as a protectant and a scavenger of reactive oxygen species. Studies on lifespan extension have focused on the use of Saccharomyces cerevisiae. Rhodosporidium toruloides, also known as Rhodotorula toruloides, is a basidiomycetous oleaginous yeast known to accumulate lipids to more than 70% of its dry cell weight. The oleaginous red yeast (R. toruloides) has not been intensely studied in the lifespan domain. We designed this work to investigate how GG and proline promote the longevity of this red yeast strain. The results obtained in our study confirmed that these molecules increased R. toruloides' viability, survival percentage, and lifespan upon supplementation. GG exerts the most promising effects at a relatively high concentration (100 mM), while proline functions best at a low level (2 mM). Elucidation of the processes underlying these favorable responses revealed that GG promotes the yeast chronological lifespan (CLS) through increased catalase activity, modulation of the culture medium pH, a rise in ATP, and an increase in reactive oxygen species (ROS) accumulation (mitohormesis). It is critical to understand the mechanisms of these geroprotector molecules, particularly GG, and the proclivity of its lifespan application; this will aid in offering clarity on its potential application in aging research.
Assuntos
Produtos Biológicos , Longevidade , Saccharomyces cerevisiae , Prolina , Espécies Reativas de Oxigênio , FosfatosRESUMO
OBJECTIVES: To better understand the unique inhibitory behavior of a non-natural cofactor preferred formaldehyde dehydrogenase (FalDH) mutant 9B2. RESULTS: We described our serendipitous observation that 9B2 was reversibly inhibited by residual imidazole introduced during protein preparation, while the wild-type enzyme was not sensitive to imidazole. Kinetic analysis showed that imidazole was a competitive inhibitor of formaldehyde with a Ki of 16 µM and an uncompetitive inhibitor of Nicotinamide Cytosine Dinucleotide for 9B2, indicating that formaldehyde and imidazole were combined in the same position. Molecular docking results of 9B2 showed that imidazole could favorably bind very close to the nicotinamide moiety of the cofactor, where formaldehyde was expected to reside for catalysis, which was in line with a competitive inhibition. CONCLUSION: The mutant 9B2 can be competitively inhibited by imidazole, suggesting that cautions should be taken to evaluate activities as protein mutants might attain unexpected sensitivity to a component in buffers for purification or activity assays.
Assuntos
Formaldeído , Imidazóis , Cinética , Simulação de Acoplamento Molecular , Imidazóis/farmacologia , NiacinamidaRESUMO
The enzyme formaldehyde dehydrogenase (FalDH) from Pseudomonas putida is of particular interest for biotechnological applications as it catalyzes the oxidation of formaldehyde independent of glutathione. However, the consumption of a stoichiometric amount of nicotinamide adenine dinucleotide (NAD) can be challenging at the metabolic level as this may affect many other NAD-linked processes. A potential solution is to engineer FalDH to utilize non-natural cofactors. Here we devised FalDH variants to favor nicotinamide cytosine dinucleotide (NCD) by structure-guided modification of the binding pocket for the adenine moiety of NAD. Several mutants were obtained and the best one FalDH 9B2 had over 150-fold higher preference for NCD than NAD. Molecular docking analysis indicated that the cofactor binding pocket shrunk to better fit NCD, a smaller-sized cofactor. FalDH 9B2 together with other NCD-linked enzymes offer opportunities to assemble orthogonal pathways for biological conversion of C1 molecules.
Assuntos
Pseudomonas putida , Aldeído Oxirredutases , Citosina , Formaldeído , Simulação de Acoplamento Molecular , NAD/química , Niacinamida/químicaRESUMO
Molecular causes of aging and longevity interventions have witnessed an upsurge in the last decade. The resurgent interests in the application of small molecules as potential geroprotectors and/or pharmacogenomics point to nicotinamide adenine dinucleotide (NAD) and its precursors, nicotinamide riboside, nicotinamide mononucleotide, nicotinamide, and nicotinic acid as potentially intriguing molecules. Upon supplementation, these compounds have shown to ameliorate aging related conditions and possibly prevent death in model organisms. Besides being a molecule essential in all living cells, our understanding of the mechanism of NAD metabolism and its regulation remain incomplete owing to its omnipresent nature. Here we discuss recent advances and techniques in the study of chronological lifespan (CLS) and replicative lifespan (RLS) in the model unicellular organism Saccharomyces cerevisiae. We then follow with the mechanism and biology of NAD precursors and their roles in aging and longevity. Finally, we review potential biotechnological applications through engineering of microbial lifespan, and laid perspective on the promising candidature of alternative redox compounds for extending lifespan.
Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomycetales , Longevidade , NAD/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomycetales/metabolismoRESUMO
BACKGROUND: Resveratrol is a plant-derived phenylpropanoid with diverse biological activities and pharmacological applications. Plant-based extraction could not satisfy ever-increasing market demand, while chemical synthesis is impeded by the existence of toxic impurities. Microbial production of resveratrol offers a promising alternative to plant- and chemical-based processes. The non-conventional oleaginous yeast Rhodotorula toruloides is a potential workhorse for the production of resveratrol that endowed with an efficient and intrinsic bifunctional phenylalanine/tyrosine ammonia-lyase (RtPAL) and malonyl-CoA pool, which may facilitate the resveratrol synthesis when properly rewired. RESULTS: Resveratrol showed substantial stability and would not affect the R. toruloides growth during the yeast cultivation in flasks. The heterologus resveratrol biosynthesis pathway was established by introducing the 4-coumaroyl-CoA ligase (At4CL), and the stilbene synthase (VlSTS) from Arabidopsis thaliana and Vitis labrusca, respectively. Next, The resveratrol production was increased by 634% through employing the cinnamate-4-hydroxylase from A. thaliana (AtC4H), the fused protein At4CL::VlSTS, the cytochrome P450 reductase 2 from A. thaliana (AtATR2) and the endogenous cytochrome B5 of R. toruloides (RtCYB5). Then, the related endogenous pathways were optimized to affect a further 60% increase. Finally, the engineered strain produced a maximum titer of 125.2 mg/L resveratrol in YPD medium. CONCLUSION: The non-conventional oleaginous yeast R. toruloides was engineered for the first time to produce resveratrol. Protein fusion, co-factor channeling, and ARO4 and ARO7 overexpression were efficient for improving resveratrol production. The results demonstrated the potential of R. toruloides for resveratrol and other phenylpropanoids production.
Assuntos
Arabidopsis , Rhodotorula , Engenharia Metabólica/métodos , Resveratrol/metabolismo , Arabidopsis/genética , Rhodotorula/genética , Rhodotorula/metabolismo , Leveduras , PlantasRESUMO
Catalytic conversion of lignin into heteroatom functionalized chemicals is of great importance to bring the biorefinery concept into reality. Herein, a new strategy was designed for direct transformation of lignin ß-O-4 model compounds into benzylamines and phenols in moderate to excellent yields in the presence of organic amines. The transformation involves dehydrogenation of Cα -OH, hydrogenolysis of the Cß -O bond and reductive amination in the presence of Pd/C catalyst. Experimental data suggest that the dehydrogenation reaction proceeds over the other two reactions and secondary amines serve as both reducing agents and amine sources in the transformation. Moreover, the concept of "lignin to benzylamines" was demonstrated by a two-step process. This work represents a first example of synthesis of benzylamines from lignin, thus providing a new opportunity for the sustainable synthesis of benzylamines from renewable biomass, and expanding the products pool of biomass conversion to meet future biorefinery demands.
RESUMO
Synthetic nicotinamide adenine dinucleotide (NAD) analogues are of great scientific and biotechnological interest. One such analogue, nicotinamide cytosine dinucleotide (NCD), has been successfully applied to creating bioorthogonal redox systems. Yet, only a few redox enzymes have been devised to favor NCD. We have engineered Lactobacillus helveticus-derived NAD-dependent d-lactate dehydrogenase (LhDLDH) to favor NCD by semirational design. Sequence alignment and structural analysis revealed that amino acid residues I177 and N213 form a "gate" guarding the NAD adenine moiety binding cavity. Saturated mutagenesis libraries were constructed by using the mutant LhDLDH-V152R as the parental sequence. Mutants were obtained with good catalytic efficiency, and NCD preference increased by up to 940-fold. Experiments showed that Escherichia coli cells expressing mutants with higher NCD preference afforded much less d-lactate, thus suggesting the potential to construct NCD-mediated orthogonal metabolism.
Assuntos
Lactato Desidrogenases/metabolismo , NAD/biossíntese , Engenharia de Proteínas , Sequência de Aminoácidos , Lactato Desidrogenases/química , Lactato Desidrogenases/genética , Lactobacillus helveticus/enzimologia , Modelos Moleculares , Conformação Molecular , Mutação , NAD/química , Alinhamento de SequênciaRESUMO
Formate dehydrogenase (FDH) has been widely used for the regeneration of the reduced nicotinamide adenine dinucleotide (NADH). To utilize nicotinamide cytosine dinucleotide (NCD) as a non-natural redox cofactor, it remains challenging as NCDH, the reduced form of NCD, has to be efficiently regenerated. Here we demonstrate successful engineering of FDH for NCDH regeneration. Guided by the structural information of FDH from Pseudomonas sp. 101 (pseFDH) and the NAD-pseFDH complex, semi-rational strategies were applied to design mutant libraries and screen for NCD-linked activity. The most active mutant reached a cofactor preference switch from NAD to NCD by 3700-fold. Homology modeling analysis showed that these mutants had reduced cofactor binding pockets and dedicated hydrophobic interactions for NCD. Efficient regeneration of NCDH was implemented by powering an NCD-dependent D-lactate dehydrogenase for stoichiometric and stereospecific reduction of pyruvate to D-lactate at the expense of formate.
Assuntos
Formiato Desidrogenases/química , Formiato Desidrogenases/metabolismo , NAD/metabolismo , Formiato Desidrogenases/genética , L-Lactato Desidrogenase/metabolismo , OxirreduçãoRESUMO
The red yeast Rhodosporidium toruloides naturally produces microbial lipids and carotenoids. In the past decade or so, many studies demonstrated R. toruloides as a promising platform for lipid production owing to its diverse substrate appetites, robust stress resistance and other favorable features. Also, significant progresses have been made in genome sequencing, multi-omic analysis and genome-scale modeling, thus illuminating the molecular basis behind its physiology, metabolism and response to environmental stresses. At the same time, genetic parts and tools are continuously being developed to manipulate this distinctive organism. Engineered R. toruloides strains are emerging for enhanced production of conventional lipids, functional lipids as well as other interesting metabolites. This review updates those progresses and highlights future directions for advanced biotechnological applications.
Assuntos
Microbiologia Industrial , Lipídeos/biossíntese , Engenharia Metabólica , Rhodotorula/metabolismo , Rhodotorula/genéticaRESUMO
A non-natural cofactor and formate driven system for reductive carboxylation of pyruvate is presented. A formate dehydrogenase (FDH) mutant, FDH*, that favors a non-natural redox cofactor, nicotinamide cytosine dinucleotide (NCD), for generation of a dedicated reducing equivalent at the expense of formate were acquired. By coupling FDH* and NCD-dependent malic enzyme (ME*), the successful utilization of formate is demonstrated as both CO2 source and electron donor for reductive carboxylation of pyruvate with a perfect stoichiometry between formate and malate. When 13 C-isotope-labeled formate was used in inâ vitro trials, up to 53 % of malate had labeled carbon atom. Upon expression of FDH* and ME* in the model host E.â coli, the engineered strain produced more malate in the presence of formate and NCD. This work provides an alternative and atom-economic strategy for CO2 fixation where formate is used in lieu of CO2 and offers dedicated reducing power.
RESUMO
Fungal type I fatty acid synthases (FASs) are mega-enzymes with two separated, identical compartments, in which the acyl carrier protein (ACP) domains shuttle substrates to catalytically active sites embedded in the chamber wall. We devised synthetic FASs by integrating heterologous enzymes into the reaction chambers and demonstrated their capability to convert acyl-ACP or acyl-CoA from canonical fatty acid biosynthesis to short/medium-chain fatty acids and methyl ketones.
Assuntos
Basidiomycota/enzimologia , Ácido Graxo Sintase Tipo I/metabolismo , Ácidos Graxos/metabolismo , Cetonas/metabolismo , Saccharomyces cerevisiae/enzimologia , Biocatálise , Ácido Graxo Sintase Tipo I/química , Ácidos Graxos/química , Cetonas/química , Modelos Moleculares , Estrutura MolecularRESUMO
Many alcohol dehydrogenases (ADHs) catalyze oxidation of a broad scope of alcohols. When an NAD-dependent ADH oxidizes methanol, albeit at a poor rate, it may be treated as methanol dehydrogenase (MDH). One ADH from Geobacillus stearothermophilus DSM 2334 (GsADH) has been widely used as MDH, but its actual substrate scope remains less characterized. Here we purified recombinant GsADH from Escherichia coli and determined its crystal structure. We collected kinetics data of this enzyme towards a number of short chain alcohols, and found that isopropanol is by far the most favorable substrate. Moreover, molecular docking analysis suggested that substrate preference is mainly attributed to the conformer energy of the protein-substrate complex. Our data clarified the substrate scope of GsADH and provided structural insights, which may facilitate more efficient cofactor regeneration and rational metabolic engineering.
Assuntos
Álcool Desidrogenase/metabolismo , Oxirredutases do Álcool/metabolismo , Sequência de Aminoácidos , Humanos , Simulação de Acoplamento MolecularRESUMO
(S)-2-hydroxypropylphosphonate ((S)-2-HPP) epoxidase (HppE) is a mononuclear non-haem-iron-dependent enzyme responsible for the final step in the biosynthesis of the clinically useful antibiotic fosfomycin. Enzymes of this class typically catalyse oxygenation reactions that proceed via the formation of substrate radical intermediates. By contrast, HppE catalyses an unusual dehydrogenation reaction while converting the secondary alcohol of (S)-2-HPP to the epoxide ring of fosfomycin. Here we show that HppE also catalyses a biologically unprecedented 1,2-phosphono migration with the alternative substrate (R)-1-HPP. This transformation probably involves an intermediary carbocation, based on observations with additional substrate analogues, such as (1R)-1-hydroxyl-2-aminopropylphosphonate, and model reactions for both radical- and carbocation-mediated migration. The ability of HppE to catalyse distinct reactions depending on the regio- and stereochemical properties of the substrate is given a structural basis using X-ray crystallography. These results provide compelling evidence for the formation of a substrate-derived cation intermediate in the catalytic cycle of a mononuclear non-haem-iron-dependent enzyme. The underlying chemistry of this unusual phosphono migration may represent a new paradigm for the in vivo construction of phosphonate-containing natural products that can be exploited for the preparation of new phosphonate derivatives.
Assuntos
Biocatálise , Fosfomicina/biossíntese , Organofosfonatos/metabolismo , Oxirredutases/metabolismo , Produtos Biológicos/química , Produtos Biológicos/metabolismo , Cristalografia por Raios X , Fosfomicina/química , Fosfomicina/metabolismo , Hidrogenação , Ferro , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Ferroproteínas não Heme/química , Ferroproteínas não Heme/metabolismo , Organofosfonatos/química , Oxirredutases/química , Especificidade por Substrato , Fatores de TempoRESUMO
OBJECTIVES: To establish a recombinase flippase (FLP) and flippase recognition target (FRT) system-mediated protocol for post-integration excision of exogenous DNA fragments in the oleaginous yeast Rhodosporidium toruloides. RESULTS: Binary vectors were constructed to harbor FLP expressing cassette together with the hygromycin-resistance marker. Results showed that R. toruloides transformants produced FLP, but failed to mediate removal of the bleomycin-resistance marker within two FRT sites. When FLP was fused with a native nuclear localization signal (NLS) peptide, the system was found functional. Moreover, R. toruloides recombinant strains expressing the NLS-fused FLP under the control of PADH2, an promoter of alcohol dehydrogenase 2 gene (RHTO_03062), were obtained to realize homologous recombination upon growing in glucose-deficient medium. CONCLUSIONS: We have devised a homologous recombination method for R. toruloides based on the FLP/FRT system, which may facilitate further metabolic engineering and designing advanced cell factories for value-added chemicals.
Assuntos
Basidiomycota/genética , DNA Nucleotidiltransferases/genética , Engenharia Genética/métodos , Recombinação Homóloga/genética , Basidiomycota/metabolismo , Biotecnologia , Proteínas Fúngicas/genética , Regiões Promotoras Genéticas/genética , Transformação Genética/genéticaRESUMO
NAD and its reduced form NADH function as essential redox cofactors and have major roles in determining cellular metabolic features. NAD can be synthesized through the deamidated and amidated pathways, for which the key reaction involves adenylylation of nicotinic acid mononucleotide (NaMN) and nicotinamide mononucleotide (NMN), respectively. In Escherichia coli, NAD de novo biosynthesis depends on the protein NadD-catalyzed adenylylation of NaMN to nicotinic acid adenine dinucleotide (NaAD), followed by NAD synthase-catalyzed amidation. In this study, we engineered NadD to favor NMN for improved amidated pathway activity. We designed NadD mutant libraries, screened by a malic enzyme-coupled colorimetric assay, and identified two variants, 11B4 (Y84V/Y118D) and 16D8 (A86W/Y118N), with a high preference for NMN. Whereas in the presence of NMN both variants were capable of enabling the viability of cells of E. coli BW25113-derived NAD-auxotrophic strain YJE003, for which the last step of the deamidated pathway is blocked, the 16D8 expression strain could grow without exogenous NMN and accumulated a higher cellular NAD(H) level than BW25113 in the stationary phase. These mutants established fully active amidated NAD biosynthesis and offered a new opportunity to manipulate NAD metabolism for biocatalysis and metabolic engineering.IMPORTANCE Adenylylation of nicotinic acid mononucleotide (NaMN) and adenylylation of nicotinamide mononucleotide (NMN), respectively, are the key steps in the deamidated and amidated pathways for NAD biosynthesis. In most organisms, canonical NAD biosynthesis follows the deamidated pathway. Here we engineered Escherichia coli NaMN adenylyltransferase to favor NMN and expressed the mutant enzyme in an NAD-auxotrophic E. coli strain that has the last step of the deamidated pathway blocked. The engineered strain survived in M9 medium, which indicated the implementation of a functional amidated pathway for NAD biosynthesis. These results enrich our understanding of NAD biosynthesis and are valuable for manipulation of NAD homeostasis for metabolic engineering.
Assuntos
Escherichia coli/enzimologia , NAD/biossíntese , Nicotinamida-Nucleotídeo Adenililtransferase/genética , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Mutação , NAD/análogos & derivados , NAD/metabolismo , Mononucleotídeo de Nicotinamida/análogos & derivados , Mononucleotídeo de Nicotinamida/metabolismo , Nicotinamida-Nucleotídeo Adenililtransferase/química , Nicotinamida-Nucleotídeo Adenililtransferase/metabolismo , Engenharia de Proteínas , Especificidade por SubstratoRESUMO
Lipid production by the red yeast Rhodosporidium toruloides was explored under nutrient limitation. To determine the compositional profiles of R. toruloides cells, samples were prepared using a continuous cultivation process under nutrient limitation and analyzed via several methods, including Fourier transform infrared spectroscopy and elemental analysis. Under nitrogen limitation, as the dilution rate increased, the cellular lipid content decreased but the carbohydrate and protein contents increased. Under carbon limitation, the cellular lipid, protein, and carbohydrate contents remained relatively constant at the different dilution rates. Moreover, the cellular elemental composition was essentially identical under nitrogen and carbon limitation at a high dilution rate of 0.20 h-1. We also analyzed the consumed carbon to nitrogen (C/N) under different nutrition conditions. The results indicated that the consumed C/N had a major influence on cell metabolism and product formation, which contributed to our understanding of the physiological characteristics of R. toruloides.
Assuntos
Meios de Cultura , Lipídeos/análise , Rhodotorula/química , Rhodotorula/crescimento & desenvolvimento , Carboidratos/análise , Carbono/análise , Elementos Químicos , Nitrogênio/análise , Proteínas/análise , Rhodotorula/metabolismo , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
OBJECTIVES: To target a carotenoid biosynthetic gene in the oleaginous yeast Rhodosporidium toruloides by using the Agrobacterium-mediated transformation (AMT) method. RESULTS: The RHTO_04602 locus of R. toruloides NP11, previously assigned to code the carotenoid biosynthetic gene CRTI, was amplified from genomic DNA and cloned into the binary plasmid pZPK-mcs, resulting in pZPK-CRT. A HYG-expression cassette was inserted into the CRTI sequence of pZPK-CRT by utilizing the restriction-free clone strategy. The resulted plasmid was used to transform R. toruloides cells according to the AMT method, leading to a few white transformants. Sequencing analysis of those transformants confirmed homologous recombination and insertional inactivation of CRTI. When the white variants were transformed with a CRTI-expression cassette, cells became red and produced carotenoids as did the wild-type strain NP11. CONCLUSIONS: Successful homologous targeting of the CrtI locus confirmed the function of RHTO_04602 in carotenoids biosynthesis in R. toruloides. It provided valuable information for metabolic engineering of this non-model yeast species.
Assuntos
Agrobacterium/genética , Vias Biossintéticas/genética , Carotenoides/biossíntese , Marcação de Genes , Rhodotorula/genética , Rhodotorula/metabolismo , Transformação Genética , Técnicas de Inativação de Genes , Recombinação Homóloga , Análise de Sequência de DNARESUMO
Cytochromes P450 (CYPs) play a key role in generating the structural diversity of terpenoids, the largest group of plant natural products. However, functional characterization of CYPs has been challenging because of the expansive families found in plant genomes, diverse reactivity and inaccessibility of their substrates and products. Here we present the characterization of two CYPs, CYP76AH3 and CYP76AK1, which act sequentially to form a bifurcating pathway for the biosynthesis of tanshinones, the oxygenated diterpenoids from the Chinese medicinal plant Danshen (Salvia miltiorrhiza). These CYPs had similar transcription profiles to that of the known gene responsible for tanshinone production in elicited Danshen hairy roots. Biochemical and RNA interference studies demonstrated that both CYPs are promiscuous. CYP76AH3 oxidizes ferruginol at two different carbon centers, and CYP76AK1 hydroxylates C-20 of two of the resulting intermediates. Together, these convert ferruginol into 11,20-dihydroxy ferruginol and 11,20-dihydroxy sugiol en route to tanshinones. Moreover, we demonstrated the utility of these CYPs by engineering yeast for heterologous production of six oxygenated diterpenoids, which in turn enabled structural characterization of three novel compounds produced by CYP-mediated oxidation. Our results highlight the incorporation of multiple CYPs into diterpenoid metabolic engineering, and a continuing trend of CYP promiscuity generating complex networks in terpenoid biosynthesis.
Assuntos
Abietanos/metabolismo , Vias Biossintéticas , Sistema Enzimático do Citocromo P-450/metabolismo , Proteínas de Plantas/metabolismo , Salvia miltiorrhiza/metabolismo , Abietanos/química , Cromatografia Líquida , Sistema Enzimático do Citocromo P-450/química , Regulação da Expressão Gênica de Plantas , Engenharia Genética , Espectrometria de Massas , Simulação de Acoplamento Molecular , Proteínas de Plantas/química , Saccharomyces cerevisiae/metabolismo , Salvia miltiorrhiza/enzimologia , Salvia miltiorrhiza/genética , Homologia Estrutural de ProteínaRESUMO
Lipid droplets (LDs) are ubiquitous organelles that serve as a neutral lipid reservoir and a hub for lipid metabolism. Manipulating LD formation, evolution, and mobilization in oleaginous species may lead to the production of fatty acid-derived biofuels and chemicals. However, key factors regulating LD dynamics remain poorly characterized. Here we purified the LDs and identified LD-associated proteins from cells of the lipid-producing yeast Rhodosporidium toruloides cultured under nutrient-rich, nitrogen-limited, and phosphorus-limited conditions. The LD proteome consisted of 226 proteins, many of which are involved in lipid metabolism and LD formation and evolution. Further analysis of our previous comparative transcriptome and proteome data sets indicated that the transcription level of 85 genes and protein abundance of 77 proteins changed under nutrient-limited conditions. Such changes were highly relevant to lipid accumulation and partially confirmed by reverse transcription-quantitative PCR. We demonstrated that the major LD structure protein Ldp1 is an LD marker protein being upregulated in lipid-rich cells. When overexpressed in Saccharomyces cerevisiae, Ldp1 localized on the LD surface and facilitated giant LD formation, suggesting that Ldp1 plays an important role in controlling LD dynamics. Our results significantly advance the understanding of the molecular basis of lipid overproduction and storage in oleaginous yeasts and will be valuable for the development of superior lipid producers.