Metabolic engineering of Pichia pastoris to produce ricinoleic acid,a hydroxy fatty acid of industrial importance

Ricinoleic acid (12-hydroxyoctadec-cis-9-enoic acid) has many specialized uses in bioproduct industries, while castor bean is currently the only commercial source for the fatty acid. This report describes metabolic engineering of a microbial system (Pichia pastoris) to produce ricinoleic acid using a “push” (synthesis) and “pull” (assembly) strategy. CpFAH, a fatty acid hydroxylase from Claviceps purpurea, was used for synthesis of ricinoleic acid, and CpDGAT1, a diacylglycerol acyl transferase for the triacylglycerol synthesis from the same species, was used for assembly of the fatty acid. Coexpression of CpFAH and CpDGAT1 produced higher lipid contents and ricinoleic acid levels than expression of CpFAH alone. Coexpression in a mutant haploid strain defective in the Δ12 desaturase activity resulted in a higher level of ricinoleic acid than that in the diploid strain. Intriguingly, the ricinoleic acid produced was mainly distributed in the neutral lipid fractions, particularly the free fatty acid form, but with little in the polar lipids. This work demonstrates the effectiveness of the metabolic engineering strategy and excellent capacity of the microbial system for production of ricinoleic acid as an alternative to plant sources for industrial uses.     

高山被孢霉中二酰甘油酰基转移酶2同源基因的克隆、表达和活性分析

[背景] 高山被孢霉（Mortierella alpina）是一种可积累大量花生四烯酸（Arachidonic Acid,AA）的产油丝状真菌,其所产脂肪酸主要被组装到甘油骨架上以三酰甘油（Triacylglycerol,TAG）形式存在。二酰甘油酰基转移酶（Diacylglycerol Acyltransferase,DGAT）是TAG生物合成途径的关键酶,对于高山被孢霉TAG的生产具有重要意义。[目的] 通过探究高山被孢霉DGAT2在TAG生物合成方面的功能特点,以期为提高产油真菌的TAG产量及改善TAG的脂肪酸组成提供参考。[方法] 利用序列比对在高山被孢霉ATCC32222基因组中筛选出2个编码DGAT2的候选基因MaDGAT2A/2B,在酿酒酵母（Saccharomyces cerevisiae）中异源表达后进行功能分析,并在外源添加AA条件下通过检测TAG产量进一步分析MaDGAT2A/2B的活性,最后在高山被孢霉中同源过表达MaDGAT2A/2B,通过检测重组菌总脂肪酸产量及组分以分析MaDGAT2A/2B的体内活性。[结果] MaDGAT2A在S. cerevisiae中异源表达时,重组酵母菌TAG的产量达到细胞干重的3.06%,为对照组的4.91倍;而MaDGAT2B未明显提高重组酵母菌TAG的产量。在外源添加AA时,MaDGAT2A/2B均可显著促进重组酵母菌中TAG合成,表达MaDGAT2A的重组酵母菌TAG含量为对照组的3.67倍,表达MaDGAT2B的重组酵母菌TAG含量为对照组的2.61倍。MaDGAT2A/2B在高山被孢霉中过表达对其总脂肪酸产量无显著影响,但可显著提高总脂肪酸中AA的含量,AA占总脂肪酸比例最高达到39.15%,相比对照组提高16.14%。[结论] MaDGAT2A/2B可以参与TAG的生物合成,表明2个候选基因编码的蛋白具有DGAT活性,并且可提高高山被孢霉脂肪酸中AA的含量,对于改善产油真菌的脂肪酸组成从而提高其应用价值具有重要意义。     

Functional characterization of two diacylglycerol acyltransferase 1 genes in Mortierella alpina

Diacylglycerol acyltransferase (DGAT) is a crucial enzyme in the triacylglycerol (TAG) biosynthesis pathway. The oleaginous fungus Mortierella alpina can accumulate large amounts of arachidonic acid (ARA, C20:4) in the form of TAG. Therefore, it is important to study the functional characteristics of its DGAT. Two putative genes MaDGAT1A/1B encoding DGAT1 were identified in M. alpina ATCC 32222 genome by sequence alignment. Sequence alignment with identified DGAT1 homologs showed that MaDGAT1A/1B contain seven conserved motifs that are characteristic of the DGAT1 subfamily. Conserved domain analysis showed that both MaDGAT1A and MaDGAT1B belong to the Membrane-bound O-acyltransferases superfamily. The transforming with MaDGAT1A/1B genes could increase the accumulation of TAG in Saccharomyces cerevisiae to 4·47 and 7·48% of dry cell weight, which was 7·3-fold and 12·3-fold of the control group, respectively, but has no effect on the proportion of fatty acids in TAG. This study showed that MaDGAT1A/1B could effectively promote the accumulation of TAG and therefore may be used in metabolic engineering aimed to increase TAG production of oleaginous fungi.     

The salt stress-induced LPA response in Chlamydomonas is produced via PLA₂ hydrolysis of DGK-generated phosphatidic acid

The unicellular green alga Chlamydomonas has frequently been used as a eukaryotic model system to study intracellular phospholipid signaling pathways in response to environmental stresses. Earlier, we found that hypersalinity induced a rapid increase in the putative lipid second messenger, phosphatidic acid (PA), which was suggested to be generated via activation of a phospholipase D (PLD) pathway and the combined action of a phospholipase C/diacylglycerol kinase (PLC/DGK) pathway. Lysophosphatidic acid (LPA) was also increased and was suggested to reflect a phospholipase A₂ (PLA₂) activity based on pharmacological evidence. The question of PA''s and LPA''s origin is, however, more complicated, especially as both function as precursors in the biosynthesis of phospho- and galactolipids. To address this complexity, a combination of fatty acid-molecular species analysis and in vivo ³²P-radiolabeling was performed. Evidence is provided that LPA is formed from a distinct pool of PA characterized by a high α-linolenic acid (18:3n-3) content. This molecular species was highly enriched in the polyphosphoinositide fraction, which is the substrate for PLC to form diacylglycerol. Together with differential ³²P-radiolabeling studies and earlier PLD-transphosphatidylation and PLA₂-inhibitor assays, the data were consistent with the hypothesis that the salt-induced LPA response is primarily generated through PLA₂-mediated hydrolysis of DGK-generated PA and that PLD or de novo synthesis [via endoplasmic reticulum - or plastid-localized routes] is not a major contributor.     

NADPH–Cytochrome P450 Reductase Mediates the Fatty Acid Desaturation of ω3 and ω6 Desaturases from Mortierella alpina

Fatty acid desaturases play an important role in maintaining the appropriate structure and function of biological membranes. The biochemical characterization of integral membrane desaturases, particularly ω3 and ω6 desaturases, has been limited by technical difficulties relating to the acquisition of large quantities of purified proteins, and by the fact that functional activities of these proteins were only tested in an NADH-initiated reaction system. The main aim of this study was to reconstitute an NADPH-dependent reaction system in vitro and investigate the kinetic properties of Mortierella alpina ω3 and ω6 desaturases in this system. After expression and purification of the soluble catalytic domain of NADPH–cytochrome P450 reductase, the NADPH-dependent fatty acid desaturation was reconstituted for the first time in a system containing NADPH, NADPH–cytochrome P450 reductase, cytochrome b5, M. alpina ω3 and ω6 desaturase and detergent. In this system, the maximum activity of ω3 and ω6 desaturase was 213.4 ± 9.0 nmol min⁻¹ mg⁻¹ and 10.0 ± 0.5 nmol min⁻¹ mg⁻¹, respectively. The highest k_cat/K_m value of ω3 and ω6 desaturase was 0.41 µM⁻¹ min⁻¹ and 0.09 µM⁻¹ min⁻¹ when using linoleoyl CoA (18:2 ω6) and oleoyl CoA (18:1 ω9) as substrates, respectively. M. alpina ω3 and ω6 desaturases were capable of using NADPH as reductant when mediated by NADPH–cytochrome P450 reductase; although, their efficiency is distinguishable from NADH-dependent desaturation. These results provide insights into the mechanisms underlying ω3 and ω6 fatty acid desaturation and may facilitate the production of important fatty acids in M. alpina.     

Production of arachidonic acid and dihomo-γ-linolenic acid from glycerol by oil-producing filamentous fungi, Mortierella in the ARS culture collection

The filamentous fungi of the genus Mortierella are known to produce arachidonic acid from glucose, and the species alpina is currently used in industrial production of arachidonic acid in Japan. In anticipation of a large excess of the co-product glycerol from the national biodiesel program, we are trying to find new uses for bioglycerin. We screened 12 Mortierella species: M. alpina NRRL 6302, M. claussenii NRRL 2760, M. elongata NRRL 5246, M. epigama NRRL 5512, M. humilis NRRL 6369, M. hygrophila NRRL 2591, M. minutissima NRRL 6462, M. multidivaricata NRRL 6456, M. nantahalensis NRRL 5216, M. parvispora NRRL 2941, M. sepedonioides NRRL 6425, and M. zychae NRRL 2592 for their production of arachidonic acid (AA) and dihomo-γ-linolenic acid (DGLA) from glycerol. With glucose as substrate all of the strains tested produced AA and DGLA. The total fatty acid content of 125 mg/g cell dry weight (CDW) and fatty acid composition for AA (19.63%) and DGLA (5.95%) in the mycelia of M. alpina grown on glucose were comparable with those reported by Takeno et al. (Appl Environ Microbiol 71:5124–5128, 2005). With glycerol as substrate all species tested grew on glycerol and produced AA and DGLA except M. nantahalensis NRRL 5216, which could not grow on glycerol. The amount of AA and DGLA produced were comparable with those obtained with glucose-grown mycelia. The top five AA producers (mg AA/CDW) from glycerol were in the following order: M. parvispora > M. claussenii > M. alpina > M. zychae > M. minutissima. The top five dry mycelia weights were: M. zychae > M. epigama > M. hygrophila > M. humilis > M. minutissima. The top five species for total fatty acids production (mg /g CDW) were: M. claussenii > M. parvispora > M. minutissima > M. hygrophila > M. maltidivaricata. We selected two species, M. alpina and M. zychae for further studies with glycerol substrate. Their optimum production conditions were determined. Time course studies showed that the maximum cell growth and AA production for both species were at 6 days of incubation. Therefore, glycerol can be considered for industrial use in the production of AA and DGLA.     

Improved production of various polyunsaturated fatty acids through filamentous fungus Mortierella alpina breeding

Studies on the application of functional lipids such as polyunsaturated fatty acids (PUFAs) have proceeded in various fields regarding health and dietary requirements in a search for novel and rich sources. Filamentous fungus Mortierella alpina 1S-4 produces triacylglycerols rich in arachidonic acid, ones reaching 20 g/L and containing 30–70% arachidonic acid as to the total fatty acids. Mutants derived from M. alpina 1S-4, defective in Δ5 and Δ6 desaturases, accumulate triacylglycerols rich in unique PUFAs, i.e., dihomo-γ-linolenic acid and Mead acid, respectively. Furthermore, various mutants derived from M. alpina 1S-4 have led to the production of oils containing n−1, n−3, n−4, n−6, n−7, and n−9 PUFAs. A variety of genes encoding fatty acid desaturases and elongases involved in PUFA biosynthesis in M. alpina 1S-4 has been isolated and characterized. Molecular breeding of M. alpina strains by means of manipulation of these genes facilitates improvement of PUFA productivity and elucidation of the functions of enzymes involved in PUFA biosynthesis.     

Expression of <Emphasis Type="Italic">Mucor circinelloides</Emphasis> gene for Δ6 desaturase results in the accumulation of high levels of γ-linolenic acid in transgenic tobacco

γ-Linolenic acid (GLA; C18:3 Δ6,9,12) is a nutritionally important fatty acid (FA) playing a vital role in biological structures and cellular functions, which is not produced in oil seed crops. Many oil seed plants, however, produce significant quantities of linoleic acid, a FA that could be converted into GLA by the enzyme Δ6 desaturase, if it is present. As a first step to produce GLA in oil seed crops, we isolated a cDNA encoding the Δ6-FA desaturase from filamentous fungus Mucor circinelloides M29. Expression of this gene in transgenic tobacco resulted in the accumulation of GLA to the levels of 23.1% of the total FA. The results suggested that it is feasible to introduce the M. circinelloides Δ6 desaturase gene into conventional oil crop to produce a large amount of GLA for functional foods and pharmaceutical products. This text was submitted by the authors in English. Y.L. Hao, X.H. Mei, and Y.B. Luo contributed equally to this work.     

Molecular mechanism of substrate specificity for delta 6 desaturase from Mortierella alpina and Micromonas pusilla

The ω6 and ω3 pathways are two major pathways in the biosynthesis of PUFAs. In both of these, delta 6 desaturase (FADS6) is a key bifunctional enzyme desaturating linoleic acid or α-linolenic acid. Microbial species have different propensity for accumulating ω6- or ω3-series PUFAs, which may be determined by the substrate preference of FADS6 enzyme. In the present study, we analyzed the molecular mechanism of FADS6 substrate specificity. FADS6 cDNAs were cloned from Mortierella alpina (ATCC 32222) and Micromonas pusilla (CCMP1545) that synthesized high levels of arachidonic acid and EPA, respectively. M. alpina FADS6 (MaFADS6-I) showed substrate preference for LA; whereas, M. pusilla FADS6 (MpFADS6) preferred ALA. To understand the structural basis of substrate specificity, MaFADS6-I and MpFADS6 sequences were divided into five sections and a domain swapping approach was used to examine the role of each section in substrate preference. Our results showed that sequences between the histidine boxes I and II played a pivotal role in substrate preference. Based on our domain swapping results, nine amino acid (aa) residues were targeted for further analysis by site-directed mutagenesis. G194L, E222S, M227K, and V399I/I400E substitutions interfered with substrate recognition, which suggests that the corresponding aa residues play an important role in this process.     

Effects of aeration on metabolic profiles of <Emphasis Type="Italic">Mortierella alpina</Emphasis> during the production of arachidonic acid

To investigate the metabolic regulation against oxygen supply, comparative metabolomics was performed to explore the metabolic responses of Mortierella alpina in the process of arachidonic acid (ARA) production. More than 110 metabolites involved in Embden–Meyerhof–Parnas pathway, pentose phosphate pathway, tricarboxylic acid cycle, inositol phosphate metabolism, fatty acid biosynthesis, and amino acid metabolism were identified by gas chromatography–mass spectrometry. Samples at different aeration rates were clearly distinguished by principal components analysis and partial least squares analysis, indicating that oxygen supply had a profound effect on the metabolism of M. alpina. Eleven major metabolites were identified as potential biomarkers to be primarily responsible for the difference of metabolism. Further study of metabolic changes with the relevant pathways demonstrated that the levels of several intermediate metabolites in relation to central carbon metabolism changed remarkably via both processes and citrate and malate was supposed to play vital roles in polyunsaturated acid (PUFA) synthesis. Increase of myo-inositol and sorbitol were probably for osmo-regulation and redox balance, while enhanced phosphoric acid and pyroglutamic acid were supposed to have function in the activation of signal transduction pathway for stress resistance. The present study provides a novel insight into the metabolic responses of M. alpina to aeration rates and the metabolic characteristics during the ARA fermentation.     

Glucose‐6‐Phosphate Dehydrogenase from the Oleaginous Microalga Nannochloropsis Uncovers Its Potential Role in Promoting Lipogenesis

Microalgae have long been considered as potential biological feedstock for the production of wide array of bioproducts, such as biofuel feedstock because of their lipid accumulating capability. However, lipid productivity of microalgae is still far below commercial viability. Here, a glucose‐6‐phosphate dehydrogenase from the oleaginous microalga Nannochloropsis oceanica is identified and heterologously expressed in the green microalga Chlorella pyrenoidosa to characterize its function in the pentose phosphate pathway. It is found that the G6PD enzyme activity toward NADPH production is increased by 2.19‐fold in engineered microalgal strains. Lipidomic analysis reveals up to 3.09‐fold increase of neutral lipid content in the engineered strains, and lipid yield is gradually increased throughout the cultivation phase and saturated at the stationary phase. Moreover, cellular physiological characteristics including photosynthesis and growth rate are not impaired. Collectively, these results reveal the pivotal role of glucose‐6‐phosphate dehydrogenase from N. oceanica in NADPH supply, demonstrating that provision of reducing power is crucial for microalgal lipogenesis and can be a potential target for metabolic engineering.     

Role of Malic Enzyme during Fatty Acid Synthesis in the Oleaginous Fungus Mortierella alpina

The generation of NADPH by malic enzyme (ME) was postulated to be a rate-limiting step during fatty acid synthesis in oleaginous fungi, based primarily on the results from research focusing on ME in Mucor circinelloides. This hypothesis is challenged by a recent study showing that leucine metabolism, rather than ME, is critical for fatty acid synthesis in M. circinelloides. To clarify this, the gene encoding ME isoform E from Mortierella alpina was homologously expressed. ME overexpression increased the fatty acid content by 30% compared to that for a control. Our results suggest that ME may not be the sole rate-limiting enzyme, but does play a role, during fatty acid synthesis in oleaginous fungi.     

Oligounsaturated fatty acid production by selected strains of micromycetes

Fifteen strains of filamentous fungi from theCulture Collection of Fungi (Charles University, Prague) were tested for their lipid production, fatty acid composition with emphasis on accumulation of oligounsaturated fatty acids. All cultures contained palmitic (16:0), palmitoleic (16:1), stearic (18:0), oleic (18:1), linoleic (18:2) and γ-linolenic (18:3) acid (GLA). The mycelium ofCunninghamella elegans, Rhizopus arrhizus, Mortierella parvispora, M. elongata andM. alpina contained arachidonic acid (ARA) in the range of 2.3–33.5% of the total fatty acids. The strains used in our experiment were capable to accumulate a relatively high amount of intracellular lipid (9.6–20.1% in dry biomass). The highest content of GLA (22.3 mg/g) was found inMucor circinelloides. The strain ofM. alpina containing 47.1 mg/g of ARA could be considered as the best producer of ARA.     

Production of shikimic acid

Shikimic acid is a key intermediate for the synthesis of the antiviral drug oseltamivir (Tamiflu®). Shikimic acid can be produced via chemical synthesis, microbial fermentation and extraction from certain plants. An alternative production route is via biotransformation of the more readily available quinic acid. Much of the current supply of shikimic acid is sourced from the seeds of Chinese star anise (Illicium verum). Supply from star anise seeds has experienced difficulties and is susceptible to vagaries of weather. Star anise tree takes around six-years from planting to bear fruit, but remains productive for long. Extraction and purification from seeds are expensive. Production via fermentation is increasing. Other production methods are too expensive, or insufficiently developed. In the future, production in recombinant microorganisms via fermentation may become established as the preferred route. Methods for producing shikimic acid are reviewed.     

The role of acyl-CoA thioesterase ACOT8I in mediating intracellular lipid metabolism in oleaginous fungus <Emphasis Type="Italic">Mortierella alpina</Emphasis>

Thioesterases (TEs) play an essential role in the metabolism of fatty acids (FAs). To explore the role of TEs in mediating intracellular lipid metabolism in the oleaginous fungus Mortierella alpina, the acyl-CoA thioesterase ACOT8I was overexpressed. The contents of total fatty acids (TFAs) were the same in the recombinant strains as in the wild-type M. alpina, whilst the production of free fatty acids (FFAs) was enhanced from about 0.9% (wild-type) to 2.8% (recombinant), a roughly threefold increase. Linoleic acid content in FFA form constituted about 9% of the TFAs in the FFA fraction in the recombinant strains but only about 1.3% in the wild-type M. alpina. The gamma-linolenic acid and arachidonic acid contents in FFA form accounted for about 4 and 25%, respectively, of the TFAs in the FFA fraction in the recombinant strains, whilst neither of them in FFA form were detected in the wild-type M. alpina. Overexpression of the TE ACOT8I in the oleaginous fungus M. alpina reinforced the flux from acyl-CoAs to FFAs, improved the production of FFAs and tailored the FA profiles of the lipid species.     

微藻三酰甘油合成途径及其最新研究进展

三酰甘油(triacylglycerols,TAGs)是动物、植物、微生物和微藻细胞主要的储藏性脂类,它可应用于食品、轻工业和生物燃料等方面,是一种新型可再生能源——生物柴油生产的重要原料。与高等油料作物相比,微藻具有光合作用效率高、生长速度快、油脂产量高、不占用农业耕地和适应多种生长环境等优势,是一种潜在的新型生物柴油生产原料。然而,目前人们对有机体,尤其是微藻细胞内TAG合成与积累的分子机制及细胞的代谢调控机制还知之甚少。对TAG合成的一系列重要过程,包括脂肪酸的合成,TAG生物合成的主要途径和旁路途径,以及与TAG合成相关的关键酶和重要基因等进行了综述,特别对微藻细胞中与TAG合成相关的关键基因的最新研究进展进行了总结,旨在更好地了解油脂代谢的调控途径,为最大限度地供应生物柴油的生产原料提供理论基础。