产油微生物油脂生物合成与代谢调控研究进展

自然界中少量微生物在适宜条件下产生并贮存质量超过其细胞干重 2 0 %的油脂 ,具有这种表型的菌种称为产油微生物。产油微生物利用可再生资源 ,得到的微生物油脂与植物油脂具有相似的脂肪酸组成 ,有的还含有丰富的多不饱和脂肪酸 ,具有广阔开发应用前景。简要介绍了产油微生物的种类和代谢特点 ,较详细地阐述了微生物产油机制和代谢调控途径的最新研究进展 ,并对微生物油脂研究的未来发展方向提出了初步见解     

Advancing oleaginous microorganisms to produce lipid via metabolic engineering technology

With the depletion of global petroleum and its increasing price, biodiesel has been becoming one of the most promising biofuels for global fuels market. Researchers exploit oleaginous microorganisms for biodiesel production due to their short life cycle, less labor required, less affection by venue, and easier to scale up. Many oleaginous microorganisms can accumulate lipids, especially triacylglycerols (TAGs), which are the main materials for biodiesel production. This review is covering the related researches on different oleaginous microorganisms, such as yeast, mold, bacteria and microalgae, which might become the potential oil feedstocks for biodiesel production in the future, showing that biodiesel from oleaginous microorganisms has a great prospect in the development of biomass energy. Microbial oils biosynthesis process includes fatty acid synthesis approach and TAG synthesis approach. In addition, the strategies to increase lipids accumulation via metabolic engineering technology, involving the enhancement of fatty acid synthesis approach, the enhancement of TAG synthesis approach, the regulation of related TAG biosynthesis bypass approaches, the blocking of competing pathways and the multi-gene approach, are discussed in detail. It is suggested that DGAT and ME are the most promising targets for gene transformation, and reducing PEPC activity is observed to be beneficial for lipid production.     

Metabolic engineering for the production of clinically important molecules: Omega-3 fatty acids, artemisinin, and taxol

Driven by requirements for sustainability as well as affordability and efficiency, metabolic engineering of plants and microorganisms is increasingly being pursued to produce compounds for clinical applications. This review discusses three such examples of the clinical relevance of metabolic engineering: the production of omega-3 fatty acids for the prevention of cardiovascular disease; the biosynthesis of artemisinic acid, an anti-malarial drug precursor, for the treatment of malaria; and the production of the complex natural molecule taxol, an anti-cancer agent. In terms of omega-3 fatty acids, bioengineering of fatty acid metabolism by expressing desaturases and elongases, both in soybeans and oleaginous yeast, has resulted in commercial-scale production of these beneficial molecules. Equal success has been achieved with the biosynthesis of artemisinic acid at low cost for developing countries. This is accomplished through channeling the flux of the isoprenoid pathway to the specific genes involved in artemisinin biosynthesis. Efficient coupling of the isoprenoid pathway also leads to the construction of an Escherichia coli strain that produces a high titer of taxadiene-the first committed intermediate for taxol biosynthesis. These examples of synthetic biology demonstrate the versatility of metabolic engineering to bring new solutions to our health needs.     

Metabolic engineering of fatty acid biosynthesis in plants.

Fatty acids are the most abundant form of reduced carbon chains available from nature and have diverse uses ranging from food to industrial feedstocks. Plants represent a significant renewable source of fatty acids because many species accumulate them in the form of triacylglycerol as major storage components in seeds. With the advent of plant transformation technology, metabolic engineering of oilseed fatty acids has become possible and transgenic plant oils represent some of the first successes in design of modified plant products. Directed gene down-regulation strategies have enabled the specific tailoring of common fatty acids in several oilseed crops. In addition, transfer of novel fatty acid biosynthetic genes from noncommercial plants has allowed the production of novel oil compositions in oilseed crops. These and future endeavors aim to produce seeds higher in oil content as well as new oils that are more stable, are healthier for humans, and can serve as a renewable source of industrial commodities. Large-scale new industrial uses of engineered plant oils are on the horizon but will require a better understanding of factors that limit the accumulation of unusual fatty acid structures in seeds.     

Cryptococcus curvatus O3酵母菌培养及产油脂特性

生物柴油的发展, 导致全球油脂供求紧张。微生物油脂的甘三酯组成与植物油类似, 发展微生物油脂可部分缓解植物油脂供应压力。本文研究了Cryptococcus curvatus O3酵母利用葡萄糖为碳源生长和积累油脂的特性。Cryptococcus curvatus O3酵母在培养过程中能适应间歇式碳源流加方式达到高密度培养的目的, 但在相同培养条件下, 不同氮源能影响其代谢过程中糖到油脂转化的脂肪系数。Cryptococcus curvatus O3酵母利用葡萄糖作为碳源在30°C下摇瓶发酵, 菌体生物量为51.8 g/L, 油脂含量达65.1%。脂肪酸组成分析结果表明, 菌油富含饱和及低度不饱和长链脂肪酸, 其中饱和脂肪酸之和占总脂肪酸组成的64%左右, 其脂肪酸组成类似于可可脂, 这些结果对于利用产油微生物转化生物质获取如类可可脂等具有高附加值油脂的研究具有重要意义。     

新型可再生工业用油脂的代谢工程

植物种子油是一种可再生资源,亦用作生物燃油和化学工业原料. 一些野生植物能高水平合成积累羟化、环氧化和共轭脂肪酸等具有重要工业应用价值的特异脂肪酸.催化这些特异脂肪酸合成的酶主要是类脂肪酸去胞和酶2(类FAD2). 由特异脂肪酸合成到三酰基甘油脂 (TAG) 形成还需要酰基转移酶 (如DGAT) 的参与. 在油料作物种子中表达类FAD2酶及其相关基因(如DGAT),已培育出了能合成积累一定含量特异脂肪酸的工程油料品系,为基于农作物生产高附加值工业用油脂开辟了新途径. 本文论述了参与特异脂肪酸生物合成途径的关键酶基因、油料作物代谢工程策略,以及应用工程油料作物大规模生产重要工业用脂肪酸的研究进展、存在问题和应用前景等.     

Engineering oilseeds for sustainable production of industrial and nutritional feedstocks: solving bottlenecks in fatty acid flux

Oilseeds provide a unique platform for the production of high-value fatty acids that can replace non-sustainable petroleum and oceanic sources of specialty chemicals and aquaculture feed. However, recent efforts to engineer the seeds of crop and model plant species to produce new types of fatty acids, including hydroxy and conjugated fatty acids for industrial uses and long-chain omega-3 polyunsaturated fatty acids for farmed fish feed, have met with only modest success. The collective results from these studies point to metabolic 'bottlenecks' in the engineered plant seeds that substantially limit the efficient or selective flux of unusual fatty acids between different substrate pools and ultimately into storage triacylglycerol. Evidence is emerging that diacylglycerol acyltransferase 2, which catalyzes the final step in triacylglycerol assembly, is an important contributor to the synthesis of unusual fatty acid-containing oils, and is likely to be a key target for future oilseed metabolic engineering efforts.     

广谱碳源产油酵母菌的筛选

对10株酵母菌利用不同单糖为碳源条件下菌体内积累油脂的能力进行了初步考察,并对菌油进行了分离和脂肪酸组成分析。实验发现,以葡萄糖为唯一碳源时有9株菌油脂含量超过自身细胞干重的20%,可以界定为产油微生物。其中6#菌(T.cutaneumAS2.571)利用葡萄糖发酵菌体油脂含量达到65%(W/W)。所有实验菌株都能同化多种单糖,其中1#菌(L.starkeyiAS2.1390)、4#菌(R.toruloidesAS2.1389)和11#菌(L.starkeyiAS2.1608)表现出对碳源利用的广谱性,能转化五碳糖木糖和阿拉伯糖并在菌体内积累油脂,油脂含量最高达到26%。脂肪酸组成分析结果表明,菌油富含饱和及低度不饱和长链脂肪酸,其中棕榈酸、油酸和亚油酸三者之和占总脂肪酸组成的90%以上,脂肪酸组成分布类似于常见的植物油。这些结果对利用产油微生物转化木质纤维素水解混合糖获取油脂资源的研究具有重要意义。     

Oleaginous yeasts: Biodiversity and cultivation

Microbial lipids produced by oleaginous microorganisms, also called microbial oils and single cell oils (SCOs), are very promising sources for several oil industries. The exploration of efficient oleaginous yeast strains, meant to produce both high-quantity and high-quality lipids for the production of biodiesel, oleochemicals, and the other high value lipid products, have gained much attention. At present, the number of oleaginous yeast species that have been discovered is 8.2% of the total number of known yeast species, most of which have been isolated from their natural habitats. To explore high lipid producing yeasts, different methods, including high-throughput screening methods using colorimetric or fluorometric measures, have been developed. Understanding of the fatty acid composition profiles of lipids produced by oleaginous yeasts would help to define target lipid-related products. For lipid production, the employment of low-cost substrates suitable for yeast growth and lipid accumulation, and efficient cultivation processes are key factors for successfully increasing the amount of the accumulated lipid yield while decreasing the cost of production.     

Lipids from heterotrophic microbes: advances in metabolism research

Heterotrophic oleaginous microorganisms are capable of producing over 20% of their weight in single cell oils (SCOs) composed of triacylglycerols (TAGs). These TAGs contain fatty acids, such as palmitic, stearic and oleic acids, that are well-suited for biodiesel applications. Although some of these microbes are able to accumulate SCOs while growing on inexpensive agro-industrial biomass, the competition with plant oil resources means that a significant increase in productivity is desired. The present review aims to summarize recent details in lipid metabolism research and engineering (e.g. direct fatty acid ethyl ester production), as well as culture condition optimization and innovations, such as solid-state or semi-solid-state fermentation, that can all contribute to higher productivity and further advancement of the field.     

Molecular mechanisms for biosynthesis and assembly of nutritionally important very long chain polyunsaturated fatty acids in microorganisms

Very long chain polyunsaturated fatty acids (VLCPUFAs) such as docosahexaenoic acid (DHA, 22:6n-3), arachidonic acid (ARA, 20:4n-6) and eicosapentaenoic acid (EPA, 20:5-n3) are nutritionally important for humans and animals. De novo biosynthesis of these fatty acids mainly occurs in microorganisms and goes through either an aerobic pathway catalyzed by type I/II fatty acid synthase, desaturases and elongases or an anaerobic pathway catalyzed by a polyunsaturated fatty acid synthase. After synthesis, VLCPUFAs must be incorporated into glycerolipids for storage through acyl assembly processes. Understanding the mechanisms for the biosynthesis of VLCPUFAs and their incorporation into glycerolipids is important not only for developing a renewable, sustainable and environment-friendly source of these fatty acids in microorganisms, but also, for designing effective strategies for metabolic engineering of these fatty acids in heterologous systems. This review highlights recent findings which have increased our understanding of biosynthesis of VLCPUFAs and their incorporation into glycerolipids in microorganisms. Future directions in improving the production of VLCPUFAs in native microbial producers are also discussed along with transgenic production of these fatty acids in oleaginous microorganisms and oilseed crops for food and feed uses.     

Fatty acids from oleaginous yeasts and yeast-like fungi and their potential applications

Purpose: Oleaginous yeasts, fatty acids biosynthesis and regulation in the oleaginous yeasts and the fatty acids from the oleaginous yeasts and their applications are reviewed in this article.

Results: Oleaginous yeasts such as Rhodosporidium toruloides, Yarrowia lipolytica, Rhodotorula mucilaginosa, and Aureobasidium melanogenum, which can accumulate over 50% lipid of their cell dry weight, have many advantages over other oleaginous microorganisms. The fatty acids from the oleaginous yeasts have many potential applications. Many oleaginous yeasts have now been genetically modified to over-produce fatty acids and their derivatives. The most important features of the oleaginous yeasts are that they have special enzymatic systems for enhanced biosynthesis and regulation of fatty acids in their lipid particles. Recently, some oleaginous yeasts such as R. toruloides have been found to have a unique fatty acids synthetase and other oleaginous yeasts such as A. melanogenum have a unique highly reducing polyketide synthase (HR-PKS) involved in the biosynthesis of hydroxyl fatty acids.

Conclusions: It is necessary to further enhance lipid biosynthesis using metabolic engineering and explore new applications of fatty acids in biotechnology.     

Isolation and localization of a cytosolic 10 S triacylglycerol biosynthetic multienzyme complex from oleaginous yeast

Triacylglycerol is one of the major storage forms of metabolic energy in eukaryotic cells. Biosynthesis of triacylglycerol is known to occur in membranes. We report here the isolation, purification, and characterization of a catalytically active cytosolic 10 S multienzyme complex for triacylglycerol biosynthesis from Rhodotorula glutinis during exponential growth. The complex was characterized and was found to contain lysophosphatidic acid acyltransferase, phosphatidic acid phosphatase, diacylglycerol acyltransferase, acyl-acyl carrier protein synthetase, and acyl carrier protein. The 10 S triacylglycerol biosynthetic complex rapidly incorporates free fatty acids as well as fatty acyl-coenzyme A into triacylglycerol and its biosynthetic intermediates. Lysophosphatidic acid acyltransferase, phosphatidic acid phosphatase, and diacylglycerol acyltransferase from the complex were microsequenced. Antibodies were raised against the synthetic peptides corresponding to lysophosphatidic acid acyltransferase and phosphatidic acid phosphatase sequences. Immunoprecipitation and immunolocalization studies show the presence of a cytosolic multienzyme complex for triacylglycerol biosynthesis. Chemical cross-linking studies revealed that the 10 S multienzyme complex was held together by protein-protein interactions. These results demonstrate that the cytosol is one of the sites for triacylglycerol biosynthesis in oleaginous yeast.     

产油微生物利用餐厨垃圾生产油脂的研究进展

餐厨垃圾中含有丰富的营养物质,经生物转化过程可以合成对人类有用的化学品.某些产油微生物可以处理餐厨垃圾生产油脂,同时合成高附加值代谢产物如多不饱和脂肪酸、角鲨烯和类胡萝卜素等.这不仅能够降低生产成本,而且提高了产物的经济价值,具有极大的工业化应用潜力.文中主要概括了目前餐厨垃圾的处理研究现状,综述了产油微生物发酵餐厨垃...     

A single-host fermentation process for the production of flavor lactones from non-hydroxylated fatty acids

Lactone flavors with fruity, milky, coconut, and other aromas are widely used in the food and fragrance industries. Lactones are produced by chemical synthesis or by biotransformation of plant-sourced hydroxy fatty acids. We established a novel method to produce flavor lactones from abundant non-hydroxylated fatty acids using yeast cell factories. Oleaginous yeast Yarrowia lipolytica was engineered to perform hydroxylation of fatty acids and chain-shortening via β-oxidation to preferentially twelve or ten carbons. The strains could produce γ-dodecalactone from oleic acid and δ-decalactone from linoleic acid. Through metabolic engineering, the titer was improved 4-fold, and the final strain produced 282 mg/L γ-dodecalactone in a fed-batch bioreactor. The study paves the way for the production of lactones by fermentation of abundant fatty feedstocks.     

Oily yeasts as oleaginous cell factories

Oily yeasts have been described to be able to accumulate lipids up to 20% of their cellular dry weight. These yeasts represent a minor proportion of the total yeast population, and only 5% of them have been reported as able to accumulate more than 25% of lipids. The oily yeast genera include Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon, and Lipomyces. More specifically, examples of oleaginous yeasts include the species: Lipomyces starkeyi, Rhodosporidium toruloides, Rhodotorula glutinis, and Yarrowia lipolytica. Yeast do exhibit advantages for lipid production over other microbial sources, namely, their duplication times are usually lower than 1 h, are much less affected than plants by season or climate conditions, and their cultures are more easily scaled up than those of microalgae. Additionally, some oily yeasts have been reported to accumulate oil up to 80% of their dry weight and can indeed generate different lipids from different carbon sources or from lipids present in the culture media. Thus, they can vary their lipid composition by replacing the fatty acids present in their triglycerides. Due to the diversity of microorganisms and growth conditions, oily yeasts can be useful for the production of triglycerides, surfactants, or polyunsaturated fatty acids.     

Isolation and characterization of fatty acid auxotrophs from the oleaginous yeast Apiotrichum curvatum

Summary In order to improve the economic value of lipids produced by the oleaginous yeast strain Apiotrichum curvatum ATCC 20509, a search was made for mutants defective in the conversion of stearic acid to oleic acid. Mutants could be selected as unsaturated fatty acid auxotrophs, since unsaturated fatty acids are essential componenets in membrane lipids. After treatment of A. curvatum wild-type with N-methyl-N-nitro-N-nitrosoguanidine, 58 fatty-acid-requiring mutants were isolated. On the basis of (1) the growth response to saturated and unsaturated fatty acids and (2) the fatty acid composition of lipids produced by these mutants, it was concluded that only 18 of them were real unsaturated fatty acid (Ufa) mutants, while the other 40 were designated as fatty acid synthetase (Fas) mutants. It is further shown that Ufa mutants of A. curvatum are able to produce high amounts of lipids consisting of more than 90% triacylglycerols with a percentage of saturated fatty acids resembling that of cocoa butter, when grown in the presence of relatively small amounts of oleic acid in the growth medium. This may offer an economically favourable alternative in comparison with other methods that have been developed for the production of cocoa butter equivalents by microorganisms.Offprint requests to: H. Smit     

Lipid production of revertants of Ufa mutants from the oleaginous yeast Apiotrichum curvatum

Summary From six unsaturated fatty acid auxotrophs (Ufa mutants) of the oleaginous yeast Apiotrichum curvatum blocked in the conversion of stearic to oleic acid, were isolated revertants able to grow in the absence of unsaturated fatty acids, in a search for strains that can produce cocoa butter equivalents. A broad range in the percentage of saturated fatty acids (%SFA) was observed in the lipids of individual revertants (varying from 27%–86% SFA), compared with the wild-type (44% SFA). Further analysis of fatty acid composition indicated that: (i) not all six Ufa mutants had the same genetic background and (ii) one specific Ufa mutation could be reverted in more than one way. Revertants that produced lipids with a %SFA>56%, were examined further. These strains were cultivated for 50 generations and half of them produced lipids with high %SFA after that time and were defined as stable. The viability of revertant strains with extremely high %SFA (>80%) may be explained by our finding that polar lipids, which are part of yeast membranes, contained much more polyunsaturated fatty acids and a significantly lower %SFA than neutral (storage) lipids. One revertant (R25.75) was selected that was able to produce lipids in whey permeate at a rate comparable with wild-type A. curvatum and with a fatty acid composition and congelation curve comparable with cocoa butter. Offprint requests to: A. Ykema     

Eighteen new oleaginous yeast species

Of 1600 known species of yeasts, about 70 are known to be oleaginous, defined as being able to accumulate over 20 % intracellular lipids. These yeasts have value for fundamental and applied research. A survey of yeasts from the Phaff Yeast Culture Collection, University of California Davis was performed to identify additional oleaginous species within the Basidiomycota phylum. Fifty-nine strains belonging to 34 species were grown in lipid inducing media, and total cell mass, lipid yield and triacylglycerol profiles were determined. Thirty-two species accumulated at least 20 % lipid and 25 species accumulated over 40 % lipid by dry weight. Eighteen of these species were not previously reported to be oleaginous. Triacylglycerol profiles were suitable for biodiesel production. These results greatly expand the number of known oleaginous yeast species, and reveal the wealth of natural diversity of triacylglycerol profiles within wild-type oleaginous Basidiomycetes.