Protein methylation catalyzed by SAM-dependent methyltransferase represents a major PTM involved in many important biological processes. Because methylation can occur on nitrogen, oxygen and sulfur centers and multiple methylation states exist on the nitrogen centers, methylproteome remains poorly documented. Here we present the methylation by isotope labeled SAM (MILS) strategy for a highly-confident analysis of the methylproteome of the yeast Saccharomyces cerevisiae based on the online multidimensional ?HPLC/MS/MS technology. We identified 43 methylated proteins, containing 68 methylation events associated with 64 methylation sites. More than 90% of these methylation events were previously unannotated in Uniprot database. Our results indicated, 1) over 2.6% of identified S. cerevisiae proteins are methylated, 2) the amino acid residue preference of protein methylation follows the order Lys?Arg>Asp>Asn?Gln?His>Glu>Cys, and 3) the methylation state on nitrogen center is largely exclusive. As our dataset covers various types of methylation centers, it provides rich information about yeast methylproteome and should significantly contribute to the field of protein methylation.
Nicotinamide adenine dinucleotide (NAD) and its reduced form NADH are essential cofactors for many redox biocatalysts. Because these cofactors are consumed in stoichiometric amounts, whole-cell biocatalysts have been routinely employed in order to reduce the costs. To further improve the efficacy of redox biocatalysts, it is essential to maintain the stability of nicotinamide cofactors, for which it is attractive to block degradation pathways for NAD(H). While the biosynthesis of NAD(H) has been well studied, it is less understood how NAD(H) are degraded. Here we demonstrated that UshA was a major periplasmic enzyme for NAD degradation in Escherichia coli. Purified recombinant UshA showed high pyrophosphatase activity with the catalytic efficiencies for hydrolysis of NAD and NADH at 3.7?M(-1)s(-1) and 1.4?M(-1)s(-1), respectively. Deletion of the ushA gene from the chromosome led to faster cell growth and improved extracellular NAD stability by 3-fold under conditions similar to whole-cell biocatalysis. These results significantly enriched our understanding on NAD metabolism, and should facilitate many applications including designing more robust redox biocatalysts.
An activity screening between 1,2,3-triazole moiety-containing nicotinamide adenine dinucleotide (NAD) analogs and malic enzyme (ME) mutants identified some mutants capable of taking NAD analogs as the cofactor. One particular pair, ME-L310K/L404S and the analog B-8 had good catalytic efficiency and cofactor specificity. The new system gained about 1200-fold cofactor specificity shift from NAD toward B-8 in terms of oxidative decarboxylation of l-malate. Our results provided insightful information for the development of orthogonal redox system that is of particular important to precisely control engineered metabolic pathways.
Oligocelluloses and oligoxyloses are partially hydrolyzed products from lignocellulosic biomass hydrolysis. Biomass hydrolysates usually contain monosaccharides as well as various amounts of oligosaccharides. To utilize biomass hydrolysates more efficiently, it is important to identify microorganisms capable of converting biomass-derived oligosaccharides into biofuels or biochemicals.
The basidiomycetous yeast Rhodosporidium toruloides represents an excellent producer for microbial lipids and carotenoids. However, further rational engineering of this unconventional yeast remains challenging partially because of the absence of efficient and reliable transformation method. In this study, we developed an Agrobacterium-mediated transformation (ATMT) protocol for effective gene integration into the R. toruloides genome. Both haploid and diploid strains were successfully modified, and the integration was confirmed by colony PCR, Western blot analysis and genome walking. We further demonstrated that multiple genes could be integrated by consecutive ATMT, leading to engineered strains simultaneously resistant to multiple antibiotics. Our results provided a practical method for functional integration and expression of exogenous genes in R. toruloides, which should facilitate the development of genetic tools and the construction of superior strains to produce biofuel molecules and biochemicals.
With ever-increasing culture of yeasts for the production of biofuels and other metabolites, spent yeast cell mass exceeds its traditional market demands. Yeast cell mass contains glucose, mannose and other sugars that may be utilized for microbial culture. Here we demonstrated that the oleaginous yeast Lipomyces starkeyi could utilize glucose and mannose simultaneously for lipid production. Overall substrate consumption rates and lipid coefficients were 0.58 g/L/h and 0.18 g lipid/g sugar, respectively, in flask cultures regardless of glucose, mannose or a mixture of both as the carbon source. L. starkeyi grew well on the hydrolysates of spent cell mass of Rhodosporidium toruloides, consumed both glucose and mannose therein, and produced lipid at a yield of 0.12 g lipid/g total reducing sugars. This co-utilization strategy expands carbon sources for lipid production. It should provide an opportunity for recycling spent cell mass and be of significant interests to biorefinery and biofuel production.
Microbial lipids produced from lignocellulosic biomass hold great promise for the biodiesel industry. These lipids usually consist of three major processes: pretreatment, enzymatic hydrolysis and lipid production. However, the conventional strategy of using biomass hydrolysates as the feedstock for lipid production suffers from low lipid coefficient and prohibitively high costs. More cost-effective and integrated processes are required to advance lignocellulosic biomass-based microbial lipid technology.
Sclareol is a member of labdane type diterpenes mostly used as fragrance ingredient. To enable microbial production of sclareol, synthetic pathways were constructed by incorporating labdenediol diphosphate synthase (LPPS) and terpene synthase (TPS) of the plant Salvia sclarea into Saccharomyces cerevisiae. It was found that sclareol production could be benefited by overexpression of key enzyme for precursor biosynthesis, construction of fusion protein for substrate channeling, and removal of signal peptides from LPPS and TPS. Under optimal shake flask culture conditions, strain S6 produced 8.96 mg/L sclareol. These results provided useful information for development of heterologous hosts for production of terpenoids.
Nitrilimine generated by photolysis of diaryltetrazole in aqueous phase under mild conditions was trapped by nucleophiles including amines and thioalcohols. The representative products were characterized, while products with all 20 natural amino acids and a peptide were observed by MALDI-TOF mass spectroscopy. Competitive studies showed that this reaction also occurred in the presence of acrylamide. These results provided new information for understanding the potential side reactions when tetrazole-alkene pairs were used as a bioorthogonal reaction in labeling proteins and related studies in buffered systems.
Fatty acid ethyl esters (FAEEs) are attractive biofuel molecules. Conventional FAEEs production process uses triglycerides and ethanol as feedstocks and is sensitive to water contents. In this work, we show that the oleaginous yeast Rhodosporidium toruloides cells are capable of converting lipids into FAEEs intracellularly in aqueous phase. Up to 73% of cellular neutral glycerides could be converted into FAEEs when lipid-rich cells were incubated for 84h at 35°C, pH 6.0 in a broth containing 10 vol% ethanol. It was found that neutral glycerides were first hydrolyzed to free fatty acids followed by esterification and that lipid droplets played important roles in the process. This new process provides a novel opportunity for integration of microbial lipid production technology with bioethanol fermentation for more efficient production of drop-in biofuels from renewable resources.
Whole-cell redox biocatalysis has been intensively explored for the production of valuable compounds because excellent selectivity is routinely achieved. Although the cellular cofactor level, redox state and the corresponding enzymatic activity are expected to have major effects on the performance of the biocatalysts, our ability remains limited to predict the outcome upon variation of those factors as well as the relationship among them.
Cytochrome P450 enzymes (CYPs) play major roles in generating highly functionalized terpenoids, but identifying the exact biotransformation step(s) catalyzed by plant CYP in terpenoid biosynthesis is extremely challenging. Tanshinones are abietane-type norditerpenoid naphthoquinones that are the main lipophilic bioactive components of the Chinese medicinal herb danshen (Salvia miltiorrhiza). Whereas the diterpene synthases responsible for the conversion of (E,E,E)-geranylgeranyl diphosphate into the abietane miltiradiene, a potential precursor to tanshinones, have been recently described, molecular characterization of further transformation of miltiradiene remains unavailable. Here we report stable-isotope labeling results that demonstrate the intermediacy of miltiradiene in tanshinone biosynthesis. We further use a next-generation sequencing approach to identify six candidate CYP genes being coregulated with the diterpene synthase genes in both the rhizome and danshen hairy roots, and demonstrate that one of these, CYP76AH1, catalyzes a unique four-electron oxidation cascade on miltiradiene to produce ferruginol both in vitro and in vivo. We then build upon the previous establishment of miltiradiene production in Saccharomyces cerevisiae, with incorporation of CYP76AH1 and phyto-CYP reductase genes leading to heterologous production of ferruginol at 10.5 mg/L. As ferruginol has been found in many plants including danshen, the results and the approaches that were described here provide a solid foundation to further elucidate the biosynthesis of tanshinones and related diterpenoids. Moreover, these results should facilitate the construction of microbial cell factories for the production of phytoterpenoids.
Microbial lipids are potential alternative feedstock for biofuel and oleochemical industries. The oleaginous yeast Rhodosporidium toruloides AS 2.1389 is an excellent lipid producer. To attain parameters for the understanding of the lipid production process, we performed continuous cultivation experiments under either carbon or nitrogen limitation. The maintenance coefficient and maximum cell mass yield for this yeast were determined as 5.7 mg glucose/g cell/h and 0.42 g cell/g glucose, respectively, under carbon limitation. Under nitrogen limitation, the highest lipid yield of 0.19 g/g was observed at the dilution rate of 0.02 h(-1) while the highest specific lipid formation rate of 0.058 g/g cell/h at the dilution rate of 0.08 h(-1). A kinetic model of lipid formation under steady state conditions was developed, parameters estimated, and optimal continuous cultivation conditions forecasted. These data should be very helpful to develop and design more efficient bioprocesses for microbial lipid production.
Protein allylation and fluorophore targeting: Arginine residues of the yeast nuclear ribonucleoprotein Npl3 were extensively modified by Hmt1-catalyzed allylation reaction with allyl-SAM as the allyl group donor. The allylated protein was further treated with tetrazole compounds under UV irradiation, leading to formation of protein-attached fluorescent products.
Microbial lipid production by using lignocellulosic biomass as the feedstock holds a great promise for biodiesel production and biorefinery. This usually involves hydrolysis of biomass into sugar-rich hydrolysates, which are then used by oleaginous microorganisms as the carbon and energy sources to produce lipids. However, the costs of microbial lipids remain prohibitively high for commercialization. More efficient and integrated processes are pivotal for better techno-economics of microbial lipid technology.
(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.
The intracellular aqueous volumes (V(in)) of microorganisms are fundamental data that can be used for estimating absolute cellular enzyme and metabolite concentrations. Because traditional methods are time-consuming and costly, the V(in) data have been largely estimated ambiguously. Here we developed an NAD(+) concentration-dependent method and demonstrated its usefulness for accurate estimation of the V(in) value of Escherichia coli cells. The V(in) value of E. coli BL21(DE3) cells was determined to be 1.9 ?L·mg(-1), which is 17% lower than that of the commonly assumed data. Similarly, the V(in) value of Agrobacterium tumefaciens AGL1 cells was determined to be 1.8 ?L·mg(-1). Because NAD(+) is routinely quantified during metabolite analysis, it may be integrated into metabolomic data collection with little additional time and labor expenditure. This method should also be applicable to estimate the V(in) data of other prokaryotic microorganisms.
Differences in the levels of acetylcholinesterase (AChE) in ventral and dorsal spinal roots can be used to differentiate the spinal nerves. Although many methods are available to assay AChE, a rapid and sensitive method has not been previously developed. Here, we describe an antibody-based quartz crystal microbalance (QCM) assay and its application for the quantification of AChE in the solutions of ventral and dorsal spinal roots. The frequency variation of the QCM device corresponds to the level of AChE over a wide dynamic range (0.5-10 µg/ml), which is comparable to the response range of the ELISA method. The frequency shift caused by the ventral roots is 3-fold greater than that caused by the dorsal roots. The antibody-based QCM sensor was stable across many successive replicate samples, and the method required less than 10 min, including the AChE extraction and analysis steps. This method is a rapid and convenient means for the quantification of AChE in biological samples and may be applicable for distinguishing the ventral and dorsal roots during surgical operations.
Many enzymes catalyzing biological redox chemistry depend on the omnipresent cofactor, nicotinamide adenine dinucleotide (NAD). NAD is also involved in various nonredox processes. It remains challenging to disconnect one particular NAD-dependent reaction from all others. Here we present a bioorthogonal system that catalyzes the oxidative decarboxylation of l-malate with a dedicated abiotic cofactor, nicotinamide flucytosine dinucleotide (NFCD). By screening the multisite saturated mutagenesis libraries of the NAD-dependent malic enzyme (ME), we identified the mutant ME-L310R/Q401C, which showed excellent activity with NFCD, yet marginal activity with NAD. We found that another synthetic cofactor, nicotinamide cytosine dinucleotide (NCD), also displayed similar activity with the ME mutants. Inspired by these observations, we mutated d-lactate dehydrogenase (DLDH) and malate dehydrogenase (MDH) to DLDH-V152R and MDH-L6R, respectively, and both mutants showed fully active with NFCD. When coupled with DLDH-V152R, ME-L310R/Q401C required only a catalytic amount of NFCD to convert l-malate. Our results opened the window to engineer bioorthogonal redox systems for a wide variety of applications in systems biology and synthetic biology.
Biofuel is in high demand as an alternative energy source for petroleum and diesel. Fatty acid-based biofuel has higher energy density and better compatibility with existing infrastructures. Microbial fatty acid biosynthetic pathway is important to develop biofuel. In this article, recent progresses on the modification and reconstruction of fatty acid metabolism for the production of biofuel were reviewed, with a focus on micro-diesel, long chain fatty alcohol and alkane. Problems, solutions and directions for further development of fatty acid-based biofuel were also discussed in the respect of synthetic biology.
A new compound was detected during the production of 5-hydroxymethylfurfural (HMF) from glucose and cellulose in the ionic liquid 1-butyl-3-methylimidazolium chloride ([Bmim]Cl) at high temperatures. Further experiments found that it was derived from the reaction of HMF with [Bmim]Cl. The structure of new compound was established as 1-butyl-2-(5-methyl-2-furoyl)imidazole (BMI) based on nuclear magnetic resonance and mass spectrometry analysis, and a possible mechanism for its formation was proposed. Reactions of HMF with other imidazolium-based ionic liquids were performed to check the formation of BMI. Our results provided new insights in terms of side reactions between HMF and imidazolium-based ionic liquids, which should be valuable for designing better processes for the production of furans using biomass and related materials.
The oleaginous yeast Lipomyces starkeyi (L. starkeyi) is an excellent intracellular lipid producer. Thus, extraction of protein from lipid-rich L. starkeyi samples following conventional methods can be difficult, leading to poor data in terms of proteomic analysis. The presence of lipophilic components in those samples may also interfere with the extraction process and the downstream analysis. In this work, we developed a sequential extraction method for preparation and analysis of L. starkeyi proteome combining to an online multidimensional nano reversed-phase liquid chromatography-tandem mass spectrometry (microRPLC-MS/MS) strategy. Protein hits of high confidence reached 227 with false positive rate less than 0.1, twice of those identified from the one-buffer extraction preparation. Moreover, the protein hits related to primary metabolism was increased, which may be important to establish the molecular mechanism of lipid accumulation. The method should be valuable for protein extraction from oleaginous species.
NAD (NAD(+)) and its reduced form (NADH) are omnipresent cofactors in biological systems. However, it is difficult to determine the extremes of the cellular NAD(H) level in live cells because the NAD(+) level is tightly controlled by a biosynthesis regulation mechanism. Here, we developed a strategy to determine the extreme NAD(H) levels in Escherichia coli cells that were genetically engineered to be NAD(+) auxotrophic. First, we expressed the ntt4 gene encoding the NAD(H) transporter in the E. coli mutant YJE001, which had a deletion of the nadC gene responsible for NAD(+) de novo biosynthesis, and we showed NTT4 conferred on the mutant strain better growth in the presence of exogenous NAD(+). We then constructed the NAD(+)-auxotrophic mutant YJE003 by disrupting the essential gene nadE, which is responsible for the last step of NAD(+) biosynthesis in cells harboring the ntt4 gene. The minimal NAD(+) level was determined in M9 medium in proliferating YJE003 cells that were preloaded with NAD(+), while the maximal NAD(H) level was determined by exposing the cells to high concentrations of exogenous NAD(H). Compared with supplementation of NADH, cells grew faster and had a higher intracellular NAD(H) level when NAD(+) was fed. The intracellular NAD(H) level increased with the increase of exogenous NAD(+) concentration, until it reached a plateau. Thus, a minimal NAD(H) level of 0.039 mM and a maximum of 8.49 mM were determined, which were 0.044× and 9.6× those of wild-type cells, respectively. Finally, the potential application of this strategy in biotechnology is briefly discussed.
Microbial lipid is a potential raw material for biofuel industry. In this review, we summarized recent progress in microbial lipid production by oleaginous fungi in terms of identifying cheap feedstock, developing robust lipid producer, establishing novel strategies and better culture modes for cellular lipid accumulation, as well as revealing the molecular mechanism of oleaginity. We discussed issues, solutions and directions for further development of microbial lipid technology.
The glycoside hydrolase ?-1,3-glucomannanase is an enzyme that specifically breaks the ?-1,3 glycosidic bond of the glucomannan, the main cell wall constituent of some yeasts. In this work, a codon optimized DNA sequence of the MAN5C gene from Penicillium lilacinum ATCC 36010 was expressed in the yeast Pichia pastoris under the control of AOX1 promoter. The recombinant protein plMAN5C was purified from the shake flask culture and the stirred-tank bioreactor culture in yields of 30.0mg/l and 224.0mg/l, respectively. The purified protein had a specific activity of 14.6 U/mg at 37 °C, pH 4.5. Biochemical analysis showed that the optimal temperature and pH for plMAN5C were 50 °C and 4.5, respectively. The recombinant plMAN5C was efficient in lysis of the cell wall of the red yeast Rhodosporidium toruloides to form protoplast. Our work provided an effective system for heterogeneous production of ?-1,3-glucomannanase, which should facilitate a more convenient application of this enzyme in biotechnology and other related areas.
Biochemical conversion of lignocellulose hydrolysates remains challenging, largely because most microbial processes have markedly reduced efficiency in the presence of both hexoses and pentoses. Thus, identification of microorganisms capable of efficient and simultaneous utilization of both glucose and xylose is pivotal to improving this process.
Two-stage fermentation process was used for lipid production by Lipomyces starkeyi AS 2.1560 in glucose solution without auxiliary nutrients. In the first stage, cells were cultivated in a nutrient-rich medium for propagation. In the second stage, cells were resuspended in glucose solution to achieve high cellular lipid contents. The effects of the inocula age, cell density and initial glucose concentration on lipid production were briefly studied. When high cell density fermentation was performed in a 7-L stirred-tank bioreactor for 40h using non-sterile glucose solution as carbon source, the biomass, lipid and lipid content reached 104.6g/L, 67.9g/L and 64.9%, respectively. More significantly, lipid productivity reached 2.0g/Lh during the initial 16h-period and 1.6g/Lh for the entire culture. Our results demonstrated that cell propagation and lipid accumulation processes can be spatially separated, allowing further optimization to improve both processes. The two-stage fermentation method should have a great potential to develop more efficient processes to convert renewable materials into biofuel and related products.
Eleven triazole moiety-containing nucleotide analogs were synthesized starting form tetra-O-acetylribose in 55-63% total yields. The synthesis involved two key steps, the lipase-mediated selective deacylation of 1-azido-2,3,5-tri-O-acetyl-?-D-ribofuranoside and the Huisgen 1,3-dipolar cycloaddition between terminal alkynes and the 1-azido ribofuranoside derivative. These analogs showed inhibitory effects against a recombinant Escherichia coli NAD-dependent malic enzyme.
A clean, facile, and environment-friendly catalytic method has been developed for the conversion of furfuryl alcohol into alkyl levulinates making use of the novel solid catalyst methylimidazolebutylsulfate phosphotungstate ([MIMBS]?PW??O??). The solid catalyst is an organic-inorganic hybrid material, which consists of an organic cation and an inorganic anion. A study for optimizing the reaction conditions such as the reaction time, the temperature and the catalyst loading has been performed. Under optimal conditions, a high n-butyl levulinate yield of up to 93?% is obtained. Furthermore, the kinetics of the reaction pathways and the mechanism for the alcoholysis of furfuryl alcohol are discussed. This method is environmentally benign and economical for the conversion of biomass-based derivatives into fine chemicals.
Sepiolite--an inexpensive, resourceful, fibrous yet inoffensive mineral--made DNA transformation rapid, simple and efficient but the mechanism for DNA transformation was still unclear. Through RNA competition test, we proposed the different transforming mechanisms from the previous report. Meanwhile, we optimized the transforming method and could transfer a colony stored at 4 degrees C for a month with plasmid through sepiolite fibers. The cells could be transformed well without competent cells preparation or incubation process. In sum, this was a novel potential transforming method, which could be explored further if the chemical method and electroporation could not be used.
To shorten the cultivation time and reduce the consumption of raw materials for microbial lipid production, oleaginous yeast Rhodosporidium toruloides AS 2.1389 was cultivated using a two-stage culture mode, in which the cell propagation and lipid accumulation were separated. The yeast cells recovered from the propagation culture were re-suspended in glucose solution for lipid accumulation, through which lipid content over 55% of the dry cell weight was achieved, the longer the propagation stage was, the higher the lipid content. Analysis of the lipid indicated that the long-chain fatty acids with 16 and 18 carbon atoms were major components, suggesting that the lipid can be an alternative feedstock for biodiesel production.
Oleaginous yeast Lipomyces starkeyi, a species in the Saccharomycetales order, has the capability to accumulate over 70% of its cell biomass as lipid under defined culture conditions. In this study, analysis of L. starkeyi AS 2.1560 proteome samples from different culture stages during a typical lipid production process was performed using an online multidimensional ?RPLC/MS/MS method. Data searching against the proteome database of the yeast Saccharomyces cerevisiae led to the identification of 289 protein hits. Further comparative and semi-quantitative analysis under more stringent criteria revealed 81 proteins with significant expression-level changes. Among them, 52 proteins were upregulated and 29 proteins were downregulated. Gene ontology annotation indicated that global responses occurred when cells were exposed to the nitrogen deficiency environment for lipid production. Protein hits were annotated and largely concerned metabolic processes for alternative nitrogen sources usage or lipid accumulation. Many of the downregulated proteins were related to glycolysis, whereas the majority of the upregulated proteins were involved in proteolysis and peptidolysis, carbohydrate metabolism and lipid metabolism. Insights were provided in terms of cellular responses to nutrient availability as well as the basic biochemistry of lipid accumulation. This work presented potentially valuable information for understanding the biochemical events related to microbial oleaginity and rational engineering of oleaginous yeasts.
Production of 5-hydroxymethylfurfural (HMF) from glucose was studied in ionic liquids in the presence of hydroxyapatite supported chromium chloride (Cr-HAP) using oil-bath heating and microwave irradiation (MI). Compared with oil-bath heating, the MI way obviously increased HMF yield and reduced the reaction time from days to several minutes. A maximum HMF yield of 40% was obtained from the dehydration of glucose under MI in 2.5 min. This method is potential as an energy-efficient and cost-effective approach for the conversion of biomass into platform chemicals.
Direct conversion of carbohydrates into 5-hydroxymethylfurfural (HMF) catalyzed by germanium(IV) chloride in ionic liquids has been investigated in search of an efficient and environmentally friendly process. Monosaccharides D-fructose and D-glucose, disaccharides sucrose and maltose, and even the polysaccharide cellulose were successfully converted into HMF with good yields under mild conditions (yield up to 92?% in 5?min in the case of fructose). The structure of ionic liquids, catalyst loading, reaction temperature and water content had noticeable effects on this catalytic system. Addition of 5?Å molecular sieves during the dehydration of glucose resulted in an increase in HMF yield from 38.4?% to 48.4?%. A mechanism for glucose conversion to HMF catalyzed by germanium(IV) chloride was proposed according to ¹³C?NMR spectra obtained in?situ under different conditions using D-glucose-2-¹³C as the substrate.
Microbial lipid is a potential alternative feedstock for the biodiesel industry. New culture strategies remain to be developed to improve the economics of microbial lipid technology. This work describes lipid production by the oleaginous yeast Rhodosporidium toruloides Y4 using a 15-l bioreactor with different substrate feeding strategies. Among these strategies, the intermittent feeding mode gave a lipid productivity of 0.36 g l(-1) h(-1), whereas the constant glucose concentration II (CC-II) mode gave the highest lipid productivity of 0.57 g l(-1) h(-1). The repeated fed-batch mode according to the CC-II mode was performed with a duration time of 358 h, and the overall lipid productivity was 0.55 g l(-1) h(-1). Our results suggested that substrate feeding modes had a great impact on lipid productivity and that the repeated fed-batch process was the most appealing method by which to enhance microbial lipid production.
Acid-promoted, selective production of 5-hydroxymethylfurfural (HMF) under high fructose concentration conditions was achieved in ionic liquids (ILs) at 80 degrees C. A HMF yield up to 97% was obtained in 8min using 1-butyl-3-methylimidazolium chloride ([C(4)mim]Cl) catalyzed with 9mol% hydrochloric acid. More significantly, an HMF yield of 51% was observed when fructose was loaded at a high concentration of 67wt% in [C(4)mim]Cl. Water content below 15.4% in the system had little effect on HMF yield, whereas a higher water content was detrimental to both reaction rate and HMF yield. In situ NMR analysis suggested that the transformation of fructose to HMF was a highly selective reaction that proceeded through the cyclic fructofuranosyl intermediate pathway. This work increased our capacity to produce HMF, and should be valuable to facilitate cost-efficient conversion of biomass into biofuels and bio-based products.
Novel biochemical approaches remain to be developed to improve microbial lipid technology. This study demonstrated that sulfate limitation was effective to promote accumulating substantial amounts of intracellular lipid by the oleaginous yeast Rhodosporidium toruloides Y4. When it was cultivated using a medium with an initial carbon-to-sulfur (C/S) molar ratio of 46,750, cellular lipid content reached up to 58.3%. The time courses of cell growth, lipid accumulation and nutrient depletion were analyzed and discussed in terms of lipid biosynthesis. Moreover, lipid accumulation under sulfate-limited conditions was effective regardless of the presence of a high concentration of nitrogen sources. Thus, lipid contents almost held constant at near 57% in the media with an initial C/S molar ratio of 11,380 although the carbon-to-nitrogen molar ratio ranged from 28.3 to 5.7. Taken together, our results established the sulfate-limitation approach to control lipid biosynthesis, which should be valuable to explore nitrogen-rich raw materials as the feedstock for lipid production.
The malic enzyme-encoding cDNA (GQ372891) from the oleaginous yeast Lipomyces starkeyi AS 2.1560 was isolated, which has an 1719-bp open reading frame flanked by a 290-bp 5 untranslated sequence and a 92-bp 3 untranslated sequence. The proposed gene, LsME1, encoded a protein with 572 amino acid residues. The protein presented 58% sequence identity with the malic enzymes from Yarrowia lipolytica CLIB122 and Aspergillus fumigatus Af293. The LsME1 gene was cloned into the vector pMAL-p4x to express a fusion protein (MBP-LsME1) in Escherichia coli TB1. The fusion protein was purified and then cleaved by Factor Xa to give the recombinant LsME1. This purified enzyme took either NAD(+) or NADP(+) as the coenzyme but preferred NAD(+). The K (m) values for malic acid, NAD(+) and NADP(+) were 0.85 +/- 0.05 mM, 0.34 +/- 0.08 mM, and 7.4 +/- 0.32 mM, respectively, at pH 7.3.
Nitrogen-limited conditions have been routinely prepared for efficient lipid production by oleaginous microorganisms. However, it is difficult to attain high cellular lipid contents with natural nitrogen-rich substrates. In the present study, we showed that lipid accumulation by Rhodosporidium toruloides Y4 was directly linked to the carbon to phosphorus (C/P) molar ratios of the culture media. Moreover, such lipid accumulation phenomena were effective regardless of the presence of high amounts of nitrogen sources. Thus, cellular lipid content and lipid yield were 62.2% and 0.205 g/g glucose, respectively, using a medium with a carbon to nitrogen (C/N) molar ratio of 6.1 and a C/P molar ratio of 9552. This work suggested that phosphorus limitation can be equally effective and efficient to mediate lipid accumulation, which in turn, provides opportunities to produce microbial lipid more economically using natural or waste materials with high nitrogen content.
Jerusalem artichoke (JA) is a perennial herbaceous plant widely available as non-grain raw material. Microbial lipid has been suggested as a potential feedstock for large scale biodiesel production. This paper describes lipid production using JA tuber processed by oleaginous yeast Rhodosporidium toruloides Y4. Batch and fed-batch modes were tested with feeding of concentrated JA extracts or JA hydrolysates. Cultivation of R. toruloides Y4 with JA extracts gave a moderate cellular lipid content of 40% (w/w), whereas lipid titer and cellular lipid content reached 39.6 g l(-1) and 56.5% (w/w), respectively, when JA hydrolysates were fed. Our results suggested that JA tubers may be further explored as raw material for large scale microbial lipid production.
The oleaginous yeast Lipomyces starkeyi can accumulate intracellular lipids to over 60% of its cell dry mass under a nitrogen-limited condition. We showed that extracellular and intracellular citrate concentrations of L. starkeyi AS 2.1560 increased and the nicotinamide adenine dinucleotide - isocitrate dehydrogenase (NAD+-IDH) activity decreased at the beginning of the lipid accumulation, suggesting that the attenuation of the NAD+-IDH activity might initiate lipid storage. We next cloned the IDH gene by the methods of degenerate PCR and rapid amplification of cDNA ends. Phylogenetic analyses of the evolutionary relationships among LsIDH1, LsIDH2, and other yeast NAD+-IDHs revealed that the L. starkeyi IDH had a closer relationship with the IDHs of Yarrowia lipolytica. Further real-time PCR analysis showed that the expression levels of both LsIDH1 and LsIDH2 decreased concurrently with the evolution of cellular lipids. Our data should be valuable for understanding the biology of oleaginous yeasts and for further strain engineering of L. starkeyi.
Intracellular lipid accumulation is a common biological process for some eukaryotic microorganisms under specific growth conditions, yet study on this phenomenon at an -omics level remains rare. In this study we induced lipid accumulation by the oleaginous yeast Rhodosporidium toruloides by transferring cells into a nitrogen-limited medium and performed a comparative and semi-quantitative proteomic analysis of cell samples obtained thereafter by a 2D-LC-MS/MS approach. A total of 184 proteins were identified, based on the database of yeast Saccharomyces cerevisiae. Semi-quantitative analysis suggested that 46 proteins were notably changed during the lipid production process. Among them, seven, three and four proteins were significantly upregulated only at the late stage, the early stage and both stages, respectively. There were 26 proteins drastically downregulated at both stages. The majority of the downregulated proteins are related to protein metabolism and carbohydrate metabolism, whereas the upregulated proteins are mainly involved in alternative nitrogen sources metabolism and lipid biosynthesis. Our data indicated that a nitrogen deficiency environment had a key impact on cellular metabolism that likely stimulated the lipid accumulation process by R. toruloides. This work provids valuable information for further exploration of the molecular mechanism of cellular lipid metabolism and should be of great interest in oleaginous microorganisms engineering.
Production of 5-hydroxymethylfurfural (HMF) and furfural from lignocellulosic biomass was studied in ionic liquid in the presence of CrCl(3) under microwave irradiation. Corn stalk, rice straw and pine wood treated under typical reaction conditions produced HMF and furfural in yields of 45-52% and 23-31%, respectively, within 3 min. This method should be valuable to facilitate energy-efficient and cost-effective conversion of biomass into biofuels and platform chemicals.
Solid acid-catalyzed hydrolysis of cellulose in ionic liquid was greatly promoted by microwave heating. H-form zeolites with a lower Si/Al molar ratio and a larger surface area showed a relatively higher catalytic activity. These solid catalysts exhibited better performance than the sulfated ion-exchanging resin NKC-9. Compared with conventional oil bath heating mode, microwave irradiation at an appropriate power significantly reduced the reaction time and increased the yields of reducing sugars. A typical hydrolysis reaction with Avicel cellulose produced glucose in around 37% yield within 8min.
Two novel chemical probes each carrying an intact isoprenoid chain, a biotin tag and a benzophenone moiety were synthesized. Photoaffinity labeling of the Saccharomyces cerevisiae cell lysate revealed that these probes could selectively trap some proteins, and proteins with molecular weight of approximately 70 KDa appeared as a major band upon Streptavidin blot analysis.
A sensor array system consisting of five quartz crystal microbalance (QCM) sensors (four for measuring and one for reference) and an artificial neural network (ANN) method is presented for on-line detection of volatile organic compounds. Three ionic liquids, 1-butyl-3-methylimidazolium chloride (C(4)mimCl), 1-butyl-3-methylimidazolium hexafluorophosphate (C(4)mimPF(6)), 1-dedocyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide (C(4)mimNTf(2)), and silicone oil II, which is widely used as gas chromatographic stationary phase, have been selected as sensitive coatings on the quartz surface allowing the sensor array effective to identify chemical vapors, such as toluene, ethanol, acetone and dichloromethane. The success rate for the qualitative recognition reached 100%. Quantitative analysis has also been investigated, within the concentration range of 0.6-6.1 mg/L for toluene, 0.9-7.5 mg/L for ethanol, 2.8-117 mg/L for dichloromethane, and 0.7-38 mg/L for acetone, with a prediction error lower than 8%.
Lignocellulosic biomass hydrolysis inevitably coproduces byproducts that may have various affects on downstream biotransformation. It is imperative to document the inhibitor tolerance ability of microbial strain in order to utilize biomass hydrolysate more effectively. To achieve better lipid production by Rhodosporidium toruloides Y4, we performed fermentation experiments in the presence of some representative inhibitors. We found that acetate, 5-hydroxymethylfurfural and syringaldehyde had slightly inhibitory effects; p-hydroxybenzaldehyde and vanillin were toxic at a concentration over 10 mM; and furfural and its derivatives furfuryl alcohol and furoic acid inhibited cell growth by 45% at around 1 mM. We further demonstrated that inhibition is generally additive, although strong synergistic inhibitions were also observed. Finally, lipid production afforded good results in the presence of six inhibitors at their respective concentrations usually found in biomass hydrolysates. Fatty acid compositional profile of lipid samples indicated that those inhibitors had little effects on lipid biosynthesis. Our work will be useful for optimization of biomass hydrolysis processes and lipid production using lignocellulosic materials.
Lignocellulosic biomass pretreatment with ionic liquids (ILs) has been emerged as a new technology, but the effects of residual ILs on the downstream biotransformation remain largely unknown. Here, three typical ILs were tested for their effects on lipid production by the oleaginous yeast Rhodosporidium toruloides AS 2.1389. When cultures were maintained at pH 6.0 in the presence of 30mM ILs, [Emim]Cl, [Emim][DEP], or [Emim][OAc], minor inhibition effects were observed. When cultures were performed in the presence of 60mM ILs or without pH control, inhibition was largely dependent on ILs. Detailed analysis indicated that the anion of [Emim][OAc] was assimilated, leading to a rapid alkaline-pH shift and enhanced inhibition on cell growth and lipid production. Our results demonstrated that R. toruloides is a robust lipid producer tolerating ILs at low concentrations, and that care should be taken in bioprocess control and data analysis when ILs are involved.
Red yeasts hold great promise in the production of microbial lipids and carotenoids. Genetic study of red yeasts has attracted much attention; however, rapid amplification of genes from red yeast samples remains technically challenging. Here a highly efficient method for the preparation of genomic DNA (gDNA) template, which could be directly used for PCR, was developed. Cells from colonies or liquid cultures were collected and sequentially treated by microwave, plMAN5C, proteinase K and boiling (MMPB) in a single tube to give cell lysates that were qualified as PCR templates. Single-copied gDNA fragments o up to 2.8?kb were successfully amplified. We also demonstrated successful application of this method for species in the Ascomycetes and Basidiomycetes and identification of two leucine auxotroph mutants of Rhodotorula glutinis. This method could be widely employed for the screening and genetic engineering of various yeasts.
Triacylglycerols are among the most attractive alternative raw materials for biofuel development. Current oil plant-based technologies are limited in terms of triacylglycerol production capacity and rate. These limitations may be circumvented by biotransformation of carbohydrates into lipids; however, our understanding of microbial oleaginicity remains limited. Here we present the results of a multi-omic analysis of Rhodosporidium toruloides, a robust triacylglycerol-producing fungus. The assembly of genome and transcriptome sequencing data reveals a genome of 20.2 Mb containing 8,171 protein-coding genes, the majority of which have multiple introns. Genes including a novel fatty acid synthase are predicted to participate in metabolic pathways absent in non-oleaginous yeasts. Transcriptomic and proteomic data suggest that lipid accumulation under nitrogen-limited conditions correlates with the induction of lipogenesis, nitrogenous compound recycling, macromolecule metabolism and autophagy. The multi-omic map of R. toruloides therefore provides a valuable resource for efforts to rationally engineer lipid-production pathways.
The objective of this work is to investigate how dilution rate and carbon-to-nitrogen (C/N) ratio affects lipid accumulation by Rhodosporidium toruloides AS 2.138 9 in continuous culture. Under steady-state conditions, the increase in dilution rate led to the decrease in lipid content and lipid yield. The highest lipid yield and lipid content at D = 0.02 h(-1) were 0.18 g lipid/g sugar and 57.1%, respectively, while the highest lipid productivity and biomass productivity were obtained at D = 0.14 h(-1). The increase in C/N ratio led to the increase in lipid content. The highest lipid content of 38% was obtained at C/N = 237. The highest lipid yield of 0.12 g lipid/g sugar was obtained at C/N = 92. However, the highest lipid productivity of 0.12 g/(L x h) was obtained at C/N = 32. No significant changes were observed in terms of fatty acid composition of the lipid produced under different C/N ratios, and these three fatty acids, palmitic acid, stearic acid and oleic acid, took over 85% in all samples.
Hydrolysates of lignocellulosic biomass contain glucose, xylose, arabinose, cellobiose, among other sugars. Effective utilization of these sugars remains challenging for microbial conversion, because most microorganisms consume such sugars sequentially with a strong preference for glucose. In the present study, the oleaginous yeast, Lipomyces starkeyi, was shown to consume cellobiose and xylose simultaneously and to produce intracellular lipids from cellobiose, xylose and glucose. In flask cultures with glucose, cellobiose or a mixture of cellobiose/xylose as carbon sources, overall substrate consumption rates were close to 0.6 g/L/h, and lipid coefficients were 0.19 g lipid/g sugar, respectively. This cellobiose/xylose co-fermentation strategy provides an opportunity to efficiently utilize lignocellulosic biomass for microbial lipid production, which is important for biorefinery and biofuel production.
In this work, we found that lignosulfonic acid (LS), which is a waste byproduct from the paper industry, in ionic liquids (ILs) can catalyze the dehydration of fructose and inulin into 5-hydroxymethylfurfural (HMF) efficiently, which is a promising potential substitute for petroleum-based building blocks. The effects of reaction time, temperature, catalyst loading, and reusability of the catalytic system were studied. It was found that a 94.3% yield of HMF could be achieved in only 10 min at 100?°C under mild conditions. The reusability study of the LS-IL catalytic system after removal of HMF by ethyl acetate extraction demonstrated that the catalytic activity decreased from 77.4 to 62.9% after five cycles and the catalytic activity could be recovered after simply removing the accumulated humins by filtration after adding ethanol to the LS-ILs. The integrated utilization of a biorenewable feedstock, catalyst, and ILs is an example of an ideal green chemical process.
In this paper, the kinetics of acid-catalyzed cellulose hydrolysis in ionic liquids (ILs) was investigated by using 1-butyl-3-methylimidazolium chloride ([Bmim]Cl) as the model IL. General kinetic equations for the formation of glucose as well as cellooligomers were constructed at a molecular level, assuming that cellulose is fully dissolved to form a homogenous solution and that the scission of the glycosidic bond occurs randomly within the cellulose chain. Experimental data were well fitted according to these equations. Variations of kinetic parameters in the presence of different water content indicated that water behaved also as a base to decrease the acidity of the reaction medium. More importantly, it offered a profile of the evolution of cellooligomers. These results provided insights into the detailed mechanisms of cellulose hydrolysis in a non-aqueous, homogenous environment and should be valuable for developing strategies to depolymerize lignocellulosic biomass.
Lipids produced by oleaginous microorganisms are a potential feedstock for biodiesel production and chemical synthesis. Yet, the costs of microbial lipids remain high, partially because the lipid recovery process is tedious and costly. In the present study, enzyme-assisted extraction of lipids from the culture of the yeast Rhodosporidium toruloides was carried out. With a heat pre-treatment with microwave, enzymatic treatment with the recombinant ?-1,3-glucomannanase, plMAN5C, and extraction with ethyl acetate, 96.6% of the total lipids were extracted from R. toruloides cells at room temperature and atmospheric pressure directly from the culture without dewatering. Therefore, this process could significantly reduce energy consumption and costs for lipids extraction from the yeast.
Microbial production can be advantageous over the extraction of phytoterpenoids from natural plant sources, but it remains challenging to rationally and rapidly access efficient pathway variants. Previous engineering attempts mainly focused on the mevalonic acid (MVA) or methyl-d-erythritol phosphate (MEP) pathways responsible for the generation of precursors for terpenoids biosynthesis, and potential interactions between diterpenoids synthases were unexplored. Miltiradiene, the product of the stepwise conversion of (E,E,E)-geranylgeranyl diphosphate (GGPP) catalyzed by diterpene synthases SmCPS and SmKSL, has recently been identified as the precursor to tanshionones, a group of abietane-type norditerpenoids rich in the Chinese medicinal herb Salvia miltiorrhiza . Here, we present the modular pathway engineering (MOPE) strategy and its application for rapid assembling synthetic miltiradiene pathways in the yeast Saccharomyces cerevisiae . We predicted and analyzed the molecular interactions between SmCPS and SmKSL, and engineered their active sites into close proximity for enhanced metabolic flux channeling to miltiradiene biosynthesis by constructing protein fusions. We show that the fusion of SmCPS and SmKSL, as well as the fusion of BTS1 (GGPP synthase) and ERG20 (farnesyl diphosphate synthase), led to significantly improved miltiradiene production and reduced byproduct accumulation. The MOPE strategy facilitated a comprehensive evaluation of pathway variants involving multiple genes, and, as a result, our best pathway with the diploid strain YJ2X reached miltiradiene titer of 365 mg/L in a 15-L bioreactor culture. These results suggest that terpenoids synthases and the precursor supplying enzymes should be engineered systematically to enable an efficient microbial production of phytoterpenoids.
Early biochemical studies have demonstrated that lipid accumulation by oleaginous yeasts is linked to the activity of the NAD(+)-dependent isocitrate dehydrogenase (Idh). However, molecular study of Idh of oleaginous microorganisms remains limited. Here, we present the cloning of a mitochondrial NAD(+)-specific Idh from Rhodosporidium toruloides (RtIdh), an excellent microbial lipid producer that uses carbohydrates as the carbon source. The evolutionary relationship analyses among RtIdhs and other yeast Idhs revealed that RtIdh had a closer relationship with the Idhs of Ustilago maydis and Schizophyllum commune. We expressed the RtIDH gene in the yeast Saccharomyces cerevisiae idh? mutant. Under the nitrogen-limited condition, the intracellular lipid content and extracellular citrate concentration of the culture of the S. cerevisiae idh? carrying the RtIDH gene increased as the carbon/nitrogen molar ratio of the media increased, while the wild-type S. cerevisiae strain showed no correlation. Our data provided valuable information for elucidating the molecular mechanism of microbial oleaginicity and for engineering microorganisms to produce metabolites of fatty acid pathway.
Related JoVE Video
Journal of Visualized Experiments
What is Visualize?
JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.
How does it work?
We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.
Video X seems to be unrelated to Abstract Y...
In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.