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2.
The non-conventional oleaginous yeast Yarrowia lipolytica shows great industrial promise. It naturally produces certain compounds of interest but can also artificially generate non-native metabolites, thanks to an engineering process made possible by the significant expansion of a dedicated genetic toolbox. In this review, we present recently developed synthetic biology tools that facilitate the manipulation of Y. lipolytica, including 1) DNA assembly techniques, 2) DNA parts for constructing expression cassettes, 3) genome-editing techniques, and 4) computational tools. 相似文献
3.
萜类化合物是一类广泛存在于植物中的天然产物,其在食品、药品和化工等多个领域中均有广泛的用途,市场潜力巨大.因此,开发生产萜类化合物等植物天然产物可再生的微生物资源来补充甚至代替原有稀少和珍贵的植物资源,具有重要的理论意义和潜在的应用价值.解脂耶氏酵母是目前使用最广泛的非常规酵母底盘细胞之一.近年来,利用代谢工程及合成生... 相似文献
4.
Yarrowia lipolytica is a biotechnological chassis for the production of a range of products, such as microbial oils and organic acids. However, it is unable to consume xylose, the major pentose in lignocellulosic hydrolysates, which are considered a preferred carbon source for bioprocesses due to their low cost, wide abundance and high sugar content.Here, we engineered Y. lipolytica to metabolize xylose to produce lipids or citric acid. The overexpression of xylose reductase and xylitol dehydrogenase from Scheffersomyces stipitis were necessary but not sufficient to permit growth. The additional overexpression of the endogenous xylulokinase enabled identical growth as the wild type strain in glucose. This mutant was able to produce up to 80 g/L of citric acid from xylose. Transferring these modifications to a lipid-overproducing strain boosted the production of lipids from xylose. This is the first step towards a consolidated bioprocess to produce chemicals and fuels from lignocellulosic materials. 相似文献
6.
Acid whey, a byproduct in cheese and yogurt production, demands high costs in disposal at large quantities. Nonetheless, it contains abundant sugars and nutrients that can potentially be utilized by microorganisms. Here we report a novel platform technology that converts acid whey into value-added products using Yarrowia lipolytica. Since wild type strains do not assimilate lactose, a major carbon source in whey, a secreted β-galactosidase was introduced. Additionally, to accelerate galactose metabolism, we overexpressed the relevant native four genes of the Leloir pathway. The engineered strain could achieve rapid total conversion of all carbon sources in acid whey, producing 6.61 g/L of fatty acids (FAs) with a yield of 0.146 g-FAs/g-substrates. Further engineering to introduce an omega-3 desaturase enabled the synthesis of α-linolenic acid from acid whey, producing 10.5 mg/gDCW within a short fermentation time. Finally, PEX10 knockout in our platform strain was shown to minimize hyphal formation in concentrated acid whey cultures, greatly improving fatty acid content. These results demonstrate the feasibility of using acid whey as a previously untapped resource for biotechnology. 相似文献
7.
In response to osmotic stress, the yeast Yarrowia lipolytica produces erythritol, a four‐carbon sugar alcohol, from erythrose‐P, an intermediate of the pentose phosphate pathway. Under non‐stressing conditions (isotonic environment), the produced erythritol is subsequently recycled into erythrose‐P that can feed the pentose phosphate pathway. Herein, gene YALI0F01584g was characterized as involved in the erythritol catabolic pathway. Several experimental evidences suggested that it encodes an erythrulose‐1P isomerase that converts erythrulose‐1P into erythrulose‐4P. On the basis of our previous reports and results gathered in this study with genetically modified strains, including ΔYALI0F01584g and ΔYALI0F01628g disrupted mutants, the entire erythritol catabolic pathway has been characterized. 相似文献
8.
Guanosine 5′-diphosphate (GDP)-fucose is the indispensible donor substrate for fucosyltransferase-catalyzed synthesis of fucose-containing biomolecules, which have been found involving in various biological functions. In this work, the salvage pathway for GDP-fucose biosynthesis from Bacterioides fragilis was introduced into Escherichia coli. Besides, the biosynthesis of guanosine 5′-triphosphate (GTP), an essential substrate for GDP-fucose biosynthesis, was enhanced via overexpression of enzymes involved in the salvage pathway of GTP biosynthesis. The production capacities of metabolically engineered strains bearing different combinations of recombinant enzymes were compared. The shake flask fermentation of the strain expressing Fkp, Gpt, Gmk and Ndk obtained the maximum GDP-fucose content of 4.6 ± 0.22 μmol/g (dry cell mass), which is 4.2 fold that of the strain only expressing Fkp. Through fed-batch fermentation, the GDP-fucose content further rose to 6.6 ± 0.14 μmol/g (dry cell mass). In addition to a better productivity than previous fermentation processes based on the de novo pathway for GDP-fucose biosynthesis, the established schemes in this work also have the advantage to be a potential avenue to GDP-fucose analogs encompassing chemical modification on the fucose residue. 相似文献
9.
In this study, crude glycerol with high potassium concentration was purified using acid treatment and used as carbon source for lipid production using Yarrowia lipolytica SKY7. The crude glycerol was purified using phosphoric acid (pH 2) followed by centrifugation. When purified glycerol was used as carbon source for fermentation, higher biomass productivity (0.54 g/L/h) and lipid productivity (0.2 g/L/h) was observed at 96 h compared to crude glycerol. Results indicated that 6.32 g/L potassium in crude glycerol medium was inhibitory for cell growth and lipid production by Y. lipolytica. Yield coefficients, productivities and specific growth rates were calculated for each glycerol medium. The process performance with purified glycerol medium was comparable to that of pure glycerol medium. A higher lipid yield was obtained in purified glycerol medium (0.21 g/g glycerol) than crude glycerol medium (0.124 g/g glycerol). During purification of crude glycerol, KH 2PO 4 was also produced as by-product. This study provides a way for valorization of crude glycerol with high potassium concentration for microbial lipid production. 相似文献
10.
ObjectiveErythritol (1,2,3,4-butanetetrol) is a 4-carbon sugar alcohol that occurs in nature as a metabolite or storage compound. In this study, a multiple gene integration strategy was employed to enhance erythritol production in Y. lipolytica. ResultsThe effects on the production of erythritol in Y. lipolytica of seven key genes involved in the erythritol synthesis pathway were evaluated individually, among which transketolase (TKL1) and transaldolase (TAL1) showed important roles in enhancing erythritol production. The combined overexpression of four genes (GUT1, TPI1, TKL1, TAL1) and disruption of the EYD1 gene (encoding erythritol dehydrogenase), resulted in produce approximately 40 g/L erythritol production from glycerol. Further enhanced erythritol synthesis was obtained by overexpressing the RKI1 gene (encoding ribose 5-phosphate isomerase) and the AMPD gene (encoding AMP deaminase), indicating for the first time that these two genes are also related to the enhancement of erythritol production in Y. lipolytica. ConclusionsA combined gene overexpression strategy was developed to efficiently improve the production of erythritol in Y. lipolytica, suggesting a great capacity and promising potential of this non-conventional yeast in converting glycerol into erythritol. 相似文献
11.
中长链聚羟基脂肪酸酯(mcl-PHA)是一大类由微生物合成的天然生物聚酯,因具有可再生性和生物降解性越来越受到人们的关注。Mcl-PHA可由一些假单胞菌类利用自身的脂肪酸合成途径或β-氧化途径来合成。耶氏解脂酵母具有很好的脂/脂肪酸分解代谢能力,但是它体内缺乏PHA合成酶不能合成mcl-PHA。采用代谢工程策略构建重组解脂酵母,外源表达来自铜绿假单胞菌PAO1(Pseudomonas aeruginosa PAO1)的PHA合成酶。在PHA合成酶的C端添加PTS1过氧化物酶体定位信号序列,使其在过氧化物酶体内发挥功能,并对其编码基因PhaC1进行密码子优化得到oPhaC1。利用pINA1312载体构建表达框,借助载体上的zeta序列元件将oPhaC1基因表达框整合至酵母基因组,完成基因的稳定表达。重组菌PSOC在葡萄糖为唯一碳源的培养基中几乎不产PHA,添加0.5%的油酸时可合成占细胞干重0.67%的mcl-PHA。在含三油酸甘油酯的培养基中发酵72h产生1.51% mcl-PHA(wt%)。实验结果充分证明重组解脂酵母作为有潜力的微生物细胞工厂可以用于生产mcl-PHA,也为将来利用富含油脂和其他营养的餐厨垃圾水解液等廉价资源生产mcl-PHA打下基础。 相似文献
12.
Glycerol is a by-product generated in large amounts during the production of biofuels. This study presents an alternative means of crude glycerol valorization through the production of erythritol and mannitol. In a shake-flasks experiment in a buffered medium, nine Yarrowia lipolytica strains were examined for polyols production. Three strains (A UV'1, A-15 and Wratislavia K1) were selected as promising producers of erythritol or/and mannitol and used in bioreactor batch cultures and fed-batch mode. Pure and biodiesel-derived crude glycerol media both supplemented (to 2.5 and 3.25?%) and not-supplemented with NaCl were applied. The best results for erythritol biosynthesis were achieved in medium with crude glycerol supplemented with 2.5?% NaCl. Wratislavia K1 strain produced up to 80.0?g?l(-1) erythritol with 0.49?g?g(-1) yield and productivity of 1.0?g?l(-1)?h(-1). Erythritol biosynthesis by A UV'1 and A-15 strains was accompanied by the simultaneous production of mannitol (up to 27.6?g?l(-1)). Extracellular as well as intracellular erythritol and mannitol ratios depended on the glycerol used and the presence of NaCl in the medium. The results from this study indicate that NaCl addition to the medium improves erythritol biosynthesis, and simultaneously inhibits mannitol formation. 相似文献
13.
Erythritol is an important natural sweetener, industrially produced only by fermentation on glucose media. Glycerol is an important renewable feedstock as it is the major by-product of the biodiesel production process; here we present an alternative way to convert this low-cost substrate into value-added products, such as erythritol. Repeated batch cultures (RBC) were performed to improve the productivity of erythritol from pure and crude glycerol. An acetate negative mutant of Yarrowia lipolytica Wratislavia K1 was found to be applicable for the production of high amounts of erythritol in RBC. When 20 % of fresh replaced medium was added, the strain Wratislavia K1 was able to produce 220 g l ?1 erythritol, which corresponded to a 0.43 g g ?1 yield and a productivity of 0.54 g l ?1 h ?1. Additionally, the activity of the culture remained stable for more than 1,000 h, i.e., 11 cycles of the repeated batch bioreactors. 相似文献
14.
Erythritol is a natural sweetener commonly used in the food and pharmaceutical industries. Produced by microorganisms as an osmoprotectant, it is an ideal sucrose substitute for diabetics or overweight persons due to its almost zero calorie content. Currently, erythritol is produced on an industrial scale through the fermentation of sugars by some yeasts, such as Moniliella sp. However, the popularity of erythritol as a sweetener is still small because of its high retail price. This creates an opportunity for further process improvement. Recent years have brought the rapid development of erythritol biosynthesis methods from the low-cost substrates, and a better understanding of the metabolic pathways leading to erythritol synthesis. The yeast Yarrowia lipolytica emerges as an organism effectively producing erythritol from pure or crude glycerol. Moreover, novel erythritol producing organisms and substrates may be taken into considerations due to metabolic engineering. This review focuses on the modification of erythritol production to use low-cost substrates and metabolic engineering of the microorganisms in order to improve yield and productivity. 相似文献
15.
Polyketides are a diverse class of molecules sought after for their valuable properties, including as potential pharmaceuticals. Previously, we demonstrated that the oleaginous yeast Yarrowia lipolytica is an optimal host for production of the simple polyketide, triacetic acid lactone (TAL). We here expand the capacities of this host by overcoming previous media challenges and enabling production of more complex polyketides. Specifically, we employ a β-oxidation related strategy to improve polyketide production directly from defined media. Beyond TAL production, we establish biosynthesis of the 4-coumaroyl-CoA derived polyketides: naringenin, resveratrol, and bisdemethoxycurcumin, as well as the diketide intermediate, ( E)-5-(4-hydroxyphenyl)-3-oxopent-4-enoic acid. In this background, we enable high-level de novo production of naringenin through import of both a heterologous pathway and a mutant Y. lipolytica allele. In doing so, we generated an averaged maximum titer of 898 mg/L naringenin, the highest titer reported to date in any host. These results demonstrate that Y. lipolytica is an ideal polyketide production host for more complex 4-coumaroyl-CoA derived products. 相似文献
16.
Vitamin E is important for human and animal health. Many human diseases, such as certain cancers and neurodegenerative and cardiovascular disease, are associated with the insufficient intake of vitamin E. The daily requirements for vitamin E in men and women have been increased to 15–30 mg. Because the primary source of dietary vitamin E comes from plants, there is a need to increase vitamin E production through plant engineering in order to meet the demand in human consumption. Numerous studies have been carried out in this field, leading to many successful examples. In this review, we summarized the recent progress in vitamin E metabolic engineering in plants aimed at improving the vitamin E content and regulating composition of vitamin E. 相似文献
18.
BackgroundOver the last years, several methods for the phenotype simulation of microorganisms, under specified genetic and environmental conditions have been proposed, in the context of Metabolic Engineering (ME). These methods provided insight on the functioning of microbial metabolism and played a key role in the design of genetic modifications that can lead to strains of industrial interest. On the other hand, in the context of Systems Biology research, biological network visualization has reinforced its role as a core tool in understanding biological processes. However, it has been scarcely used to foster ME related methods, in spite of the acknowledged potential. ResultsIn this work, an open-source software that aims to fill the gap between ME and metabolic network visualization is proposed, in the form of a plugin to the OptFlux ME platform. The framework is based on an abstract layer, where the network is represented as a bipartite graph containing minimal information about the underlying entities and their desired relative placement. The framework provides input/output support for networks specified in standard formats, such as XGMML, SBGN or SBML, providing a connection to genome-scale metabolic models. An user-interface makes it possible to edit, manipulate and query nodes in the network, providing tools to visualize diverse effects, including visual filters and aspect changing (e.g. colors, shapes and sizes). These tools are particularly interesting for ME, since they allow overlaying phenotype simulation results or elementary flux modes over the networks. ConclusionsThe framework and its source code are freely available, together with documentation and other resources, being illustrated with well documented case studies. Electronic supplementary materialThe online version of this article (doi:10.1186/s12859-014-0420-0) contains supplementary material, which is available to authorized users. 相似文献
19.
In recent years, bio‐based production of free fatty acids from renewable resources has attracted attention for their potential as precursors for the production of biofuels and biochemicals. In this study, the oleaginous yeast Yarrowia lipolytica was engineered to produce free fatty acids by eliminating glycerol metabolism. Free fatty acid production was monitored under lipogenic conditions with glycerol as a limiting factor. Firstly, the strain W29 (Δgpd1), which is deficient in glycerol synthesis, was obtained. However, W29 (Δgpd1) showed decreased biomass accumulation and glucose consumption in lipogenic medium containing a limiting supply of glycerol. Analysis of substrate utilization from a mixture of glucose and glycerol by the parental strain W29 revealed that glycerol was metabolized first and glucose utilization was suppressed. Thus, the Δgpd1Δgut2 double mutant, which is deficient also in glycerol catabolism, was constructed. In this genetic background, growth was repressed by glycerol. Oleate toxicity was observed in the Δgpd1Δgut2Δpex10 triple mutant strain which is deficient additionally in peroxisome biogenesis. Consequently, two consecutive rounds of selection of spontaneous mutants were performed. A mutant released from growth repression by glycerol was able to produce 136.8 mg L?1 of free fatty acids in a test tube, whereas the wild type accumulated only 30.2 mg L?1. Next, an isolated oleate‐resistant strain produced 382.8 mg L?1 of free fatty acids. Finely, acyl‐CoA carboxylase gene (ACC1) over‐expression resulted to production of 1436.7 mg L?1 of free fatty acids. The addition of dodecane promoted free fatty acid secretion and enhanced the level of free fatty acids up to 2033.8 mg L?1 during test tube cultivation. 相似文献
20.
A cyclic fed-batch bioprocess is designed and a significant improvement of rice alpha-amylase productivity of recombinant Yarrowia lipolytica is illustrated. A bioprocess control strategy developed and reported here entails use of a genetically stable recombinant cloned for heterologous protein, use of optimized media for cell growth and enzyme production phases, and process control strategy enabling high cell-density culture and high alpha-amylase productivity. This process control can be achieved through maintaining a constant optimal specific cell growth rate at a predetermined value (i.e., 0.1 h-1), controlling medium feed rate commensurate with the cell growth rate, and maintaining a high cell-density culture (i.e., 60-70 g/L) for high productivity of cloned heterologous protein. The volumetric enzyme productivity (1, 960 units/L. h) achieved from the cyclic fed-batch process was about 3-fold higher than that of the fed-batch culture process (630 units/L. h). 相似文献
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