Malic Enzyme Gene Research Articles

Combined effects of nitrogen limitation and overexpression of malic enzyme gene on lipid accumulation in Dunaliella parva

Overexpression of Dunaliella parva (D. parva) malic enzyme (ME) gene (DpME) significantly increased DpME expression and ME enzyme activity in transgenic D. parva. Nitrogen limitation had an inhibitory effect on protein content, and DpME overexpression could improve protein content. Nitrogen limitation increased carbohydrate content, and Dunaliella parva overexpressing malic enzyme gene under nitrogen limitation (DpME-N−) group showed the lowest starch content among all groups. Dunaliella parva overexpressing malic enzyme gene under nitrogen sufficient condition (DpME) and DpME-N− groups showed considerably high mRNA levels of DpME. ME activity was significantly enhanced by DpME overexpression, and nitrogen limitation caused a smaller increase. DpME overexpression and nitrogen limitation obviously enhanced lipid accumulation, and DpME overexpression had more obvious effect. Compared with control (wild type), lipid content (68.97%) obviously increased in DpME-N− group. This study indicated that the combination of DpME overexpression and nitrogen limitation was favorable to the production of microalgae biodiesel.

TA-Cloning for Diabetes Treatment: Expressing Corynebacterium Malic Enzyme Gene in E. coli.

Diabetes mellitus represents a persistent metabolic condition marked by heightened levels of blood glucose, presenting a considerable worldwide health concern, and finding targeted treatment for it is a crucial priority for global health. Gram-positive aerobic bacteria, predominantly inhabiting water and soil, are known carriers of various enzyme-encoding genetic material, which includes the malic enzyme gene that plays a role in insulin secretion. Corynebacterium glutamicum bacteria (ATCC 21799) were acquired from the Pasteur Institute and confirmed using microbiological and molecular tests, including DNA extraction. After identification, gene purification and cloning of the maeB gene were performed using the TA Cloning method. Additionally, the enhancement of enzyme expression was assessed using the expression vector pET-28a, and validation of simulation results was monitored through a real-time PCR analysis. Based on previous studies, the malic enzyme plays a pivotal role in maintaining glucose homeostasis, and increased expression of this enzyme has been associated with enhanced insulin sensitivity. However, the production of malic enzyme has encountered numerous challenges and difficulties. This study successfully isolated the malic enzyme genes via Corynebacterium glutamicum and introduced them into Escherichia coli for high-yield production. According to the results, the optimum temperature for the activity of enzymes has been identified as 39°C.

Overexpression of malic enzyme is involved in breast cancer growth and is correlated with poor prognosis.

Malic enzyme (ME) genes are key functional metabolic enzymes playing a crucial role in carcinogenesis. However, the detailed effects of ME gene expression on breast cancer progression remain unclear. Here, our results revealed ME1 expression was significantly upregulated in breast cancer, especially in patients with oestrogen receptor/progesterone receptor-negative and human epidermal growth factor receptor 2-positive breast cancer. Furthermore, upregulation of ME1 was significantly associated with more advanced pathological stages (p < 0.001), pT stage (p < 0.001) and tumour grade (p < 0.001). Kaplan-Meier analysis revealed ME1 upregulation was associated with poor disease-specific survival (DSS: p = 0.002) and disease-free survival (DFS: p = 0.003). Multivariate Cox regression analysis revealed ME1 upregulation was significantly correlated with poor DSS (adjusted hazard ratio [AHR] = 1.65; 95% CI: 1.08-2.52; p = 0.021) and DFS (AHR, 1.57; 95% CI: 1.03-2.41; p = 0.038). Stratification analysis indicated ME1 upregulation was significantly associated with poor DSS (p = 0.039) and DFS (p = 0.038) in patients with non-triple-negative breast cancer (TNBC). However, ME1 expression did not affect the DSS of patients with TNBC. Biological function analysis revealed ME1 knockdown could significantly suppress the growth of breast cancer cells and influence its migration ability. Furthermore, the infiltration of immune cells was significantly reduced when they were co-cultured with breast cancer cells with ME1 knockdown. In summary, ME1 plays an oncogenic role in the growth of breast cancer; it may serve as a potential biomarker of progression and constitute a therapeutic target in patients with breast cancer.

Effects of exogenous melatonin on sugar and organic acid metabolism in early-ripening peach fruits.

To evaluated the effects melatonin (MT) on the sugar and acid metabolism of early-ripening peach fruits, the concentration of 150 μmol/L MT was sprayed on the leaves of peach trees. MT increased the contents of total soluble sugar and sucrose in peach fruits during the whole ripening period, and increased the contents of glucose and sorbitol at the mature stage. During the whole ripening period, MT also increased the activities of sucrose synthase, sucrose phosphate synthase, neutral invertase, and acidic invertase and the relative expression levels of sucrose synthase, sucrose phosphate synthase, neutral invertase, and acidic invertase genes, while decreased the activity of sorbitol oxidase and the relative expression level of sorbitol dehydrogenase to some extent. Moreover, MT decreased the contents of total organic acid, malic acid, and citric acid at mature stage. At mature stage, MT decreased the activities of citrate synthetase and phosphoenolpyruvate carboxylase and the relative expression levels of citrate synthetase and phosphoenolpyruvate carboxylase genes, while increased the relative expression levels of Nicotinamide adenine dinucleotide phosphate (NADP+)-malic enzyme, malate dehydrogenase, and aconitase genes. Therefore, MT promotes the sugar accumulation and organic acid degradation in early-ripening peach fruits.

NADP-Dependent Malic Enzyme Genes in Sweet Pepper Fruits: Involvement in Ripening and Modulation by Nitric Oxide (NO).

NADPH is an indispensable cofactor in a wide range of physiological processes that is generated by a family of NADPH dehydrogenases, of which the NADP-dependent malic enzyme (NADP-ME) is a member. Pepper (Capsicum annuum L.) fruit is a horticultural product consumed worldwide that has great nutritional and economic relevance. Besides the phenotypical changes that pepper fruit undergoes during ripening, there are many associated modifications at transcriptomic, proteome, biochemical and metabolic levels. Nitric oxide (NO) is a recognized signal molecule with regulatory functions in diverse plant processes. To our knowledge, there is very scarce information about the number of genes encoding for NADP-ME in pepper plants and their expression during the ripening of sweet pepper fruit. Using a data mining approach to evaluate the pepper plant genome and fruit transcriptome (RNA-seq), five NADP-ME genes were identified, and four of them, namely CaNADP-ME2 to CaNADP-ME5, were expressed in fruit. The time course expression analysis of these genes during different fruit ripening stages, including green immature (G), breaking point (BP) and red ripe (R), showed that they were differentially modulated. Thus, while CaNADP-ME3 and CaNADP-ME5 were upregulated, CaNADP-ME2 and CaNADP-ME4 were downregulated. Exogenous NO treatment of fruit triggered the downregulation of CaNADP-ME4. We obtained a 50-75% ammonium-sulfate-enriched protein fraction containing CaNADP-ME enzyme activity, and this was assayed via non-denaturing polyacrylamide gel electrophoresis (PAGE). The results allow us to identify four isozymes designated from CaNADP-ME I to CaNADP-ME IV. Taken together, the data provide new pieces of information on the CaNADP-ME system with the identification of five CaNADP-ME genes and how the four genes expressed in pepper fruits are modulated during ripening and exogenous NO gas treatment.

A complex network regulating malate contents during fruit ripening in climacteric fruits.

This article is a Commentary on Wang et al. (2023), 239: 1014–1034.

Molecular elucidation for the variance of organic acid profile between citrus top and bottom canopy fruits

Organic acids are key metabolites for diverse fruit flavoring and vital for fruit quality measures. Their contents are significantly varied regarding the canopy position. However, the underlying mechanism is still unclear. In this study by using Citrus raticulata cv. Bendizao as research material, a significant 2-fold increase in citrate content and a 2-fold decrease in malate content were noticed in bottom-canopy fruits subjected to less light and low temperature compared to top-canopy fruits. Moreover, transcript levels of genes like citrate synthase (CS1), aconitate hydratase genes (ACO1 and ACO2), NAD-dependent malic enzyme gene (NAD-ME) and malate dehydrogenase genes (MDH1 and MDH2) were significantly higher, while transcript levels of other genes such as citrate utilization genes (ACL2, ACL3 and ACO3), malate synthase gene (MS) and cytosolic NAD-dependent malate dehydrogenase gene (cytNAD-MDH) were significantly lower in bottom-canopy fruits compared to top-canopy fruits. Additionally, transcript levels of proton pump genes (CsPHs and VHA), two transporter genes (MATE and ALMT9), and four transcription factor genes (MYB73, Noemi, MYC-N, and bHLH) were significantly higher, while mRNA levels of two transcription factor genes (MYB10 and WRKY14) and transporter genes (CsCit1 and ALMT9-like) were significantly lower in bottom-canopy fruits than those in top-canopy fruits. Taken together, the variance of organic acid profiles in the canopy could be attributed to the microclimate change, which comprehensively influences the organic acid accumulation-related genes’ transcript levels; reduced malate accumulation in bottom-canopy fruits is mainly due to the minimal expression of MS and cytNAD-MDH, and high expression of malate utilization genes (MDH and NAD-ME), while the higher citrate content in the bottom-canopy fruits is probably due to the enhanced vacuolar storage through increasing vacuolar proton pump genes and MATE transcript levels, and the minimal citrate degradation and efflux through decreasing ACL2, ACL3, ACO3 and CsCit1 transcript levels.

Overexpression of the Mitochondrial Malic Enzyme Genes (malC and malD) Improved the Lipid Accumulation in Mucor circinelloides WJ11

Mucor circinelloides serves as a model organism to investigate the lipid metabolism in oleaginous microorganisms. It is considered as an important producer of γ-linolenic acid (GLA) that has vital medicinal benefits. In this study, we used WJ11, a high lipid-producing strain of M. circinelloides (36% w/w lipid, cell dry weight, CDW), to examine the role in lipid accumulation of two mitochondrial malic enzyme (ME) genes malC and malD. The homologous overexpression of both malC and malD genes enhanced the total lipid content of WJ11 by 41.16 and 32.34%, respectively. In parallel, the total content of GLA was enhanced by 16.73 and 46.76% in malC and malD overexpressing strains, respectively, because of the elevation of total lipid content. The fact that GLA content was enhanced more in the strain with lower lipid content increase and vice versa, indicated that engineering of mitochondrial MEs altered the fatty acid profile. Our results reveal that mitochondrial ME plays an important role in lipid metabolism and suggest that future approaches may involve simultaneous overexpression of distinct ME genes to boost lipid accumulation even further.

Expression of Escherichia coli malic enzyme gene in Zymomonas mobilis for production of malic acid

Malic acid is one of the organic acids which is used in various industries including food and pharmaceuticals. Biotechnological production of malic acid by an efficient microorganism is highly desirable as the process will be eco-friendly and cost-effective. In this study, malic acid synthesis by Zymomonas mobilis was studied by expressing Escherichia coli malic enzyme gene under Pchap, Ptac and Ppdc promoters. The mae+ recombinants were obtained by recombineering-based genomic integration of Pchap-mae, Ptac-mae and Ppdc-mae sequences. The Ppdc promoter showed the highest expression of malic enzyme and the Pchap the lowest. However, cell growth was limited in mae+ recombinant containing Ppdc promoter. The metabolic analysis showed the highest level of malic acid in Ppdc-mae recombinant (2.84 g/L), which was about eight times higher than that in the wild type strain. The study showed that these three promoters can be used to produce organic acids in Z. mobilis.

Role of Cytosolic Malic Enzyme in Oleaginicity of High-Lipid-Producing Fungal Strain Mucor circinelloides WJ11.

Mucor circinelloides, an oleaginous filamentous fungus, is gaining popularity due to its ability to synthesize significant amounts of lipids containing γ-linolenic acid (GLA) that have important health benefits. Malic enzyme (ME), which serves as the main source of NADPH in some fungi, has been found to regulate lipid accumulation in oleaginous fungi. In the present study, the role of two cytosolic ME genes, cmalA and cmalB, in the lipid accumulation of the M. circinelloides high-lipid-producing strain WJ11, was evaluated. Strains overexpressing cmalA and cmalB showed a 9.8- and 6.4-fold rise in specific ME activity, respectively, and an elevation of the lipid content by 23.2% and 5.8%, respectively, suggesting that these genes are involved in lipid biosynthesis. Due to increased lipid accumulation, overall GLA content in biomass was observed to be elevated by 11.42% and 16.85% in cmalA and cmalB overexpressing strains, respectively. Our study gives an important insight into different studies exploring the role of the cmalA gene, while we have for the first time investigated the role of the cmalB gene in the M. circinelloides WJ11 strain.

The combined effect of nitrogen deprivation and overexpression of malic enzyme gene on growth and lipid accumulation in Phaeodactylum tricornutum

The combined effect of nitrogen deprivation and overexpression of malic enzyme gene on growth and lipid accumulation in Phaeodactylum tricornutum

Metabolic Engineering of Aspartic Acid Supply Modules for Enhanced Production of Bacitracin in Bacillus licheniformis.

Bacitracin, a type of cyclic dodecapeptide antibiotic mainly produced by Bacillus, is widely used in fields of veterinary drug and feed additive. Modularization of metabolic pathways based on the concept of synthetic biology has been widely used in the efficient synthesis of target products. Here, we want to improve bacitracin production through strengthening aspartic acid (Asp) supply in B. licheniformis DW2. First, exogenous Asp addition assays implied that strengthening Asp supply benefited bacitracin production. Second, Asp synthetic pathways were strengthened via overexpressing aspartate dehydrogenase AspD and asparaginase AnsB, attaining recombinant strain DW2-ASP2, and bacitracin yield produced by DW2-ASP2 was 862.81 U/mL, increased by 14.05% compared with that of DW2 (756.49 U/mL). Then, to improve precursor oxaloacetate (OAA) accumulation for Asp synthesis, pyruvate carboxylase PycA and carbonic anhydrase EcaA were co-overexpressed in DW2-ASP2, and malic enzyme gene malS was deleted to weak overflow metabolism of tricarboxylic acid, and the attained strain DW2-ASP7 showed further increased bacitracin production from 862.81 to 989.23 U/mL. Subsequently, transporter YveA was identified as an Asp exporter, and bacitracin yield was increased to 1025.26 U/mL via deleting yveA, attaining strain DW2-ASP9. Finally, Asp ammonia-lyase gene aspA was disrupted to weaken Asp degradation, and bacitracin yield of attained strain DW2-ASP10 reached 1059.86 U/mL, increased by 40.10% compared to DW2. Taken together, this research demonstrated that metabolic engineering of Asp metabolic modules is an efficient strategy for enhancing bacitracin production, and these strategies could also be applied in the production of other peptide-related metabolites.

Effects of Cabbage-Apple Juice Fermented by Lactobacillus plantarum EM on Lipid Profile Improvement and Obesity Amelioration in Rats.

This study aimed to investigate the potential of cabbage-apple juice, fermented by Lactobacillus plantarum EM isolated from kimchi, to protect against obesity and dyslipidemia that are induced by a high-fat diet in a rat model. Male rats were fed a modified AIN-93M high-fat diet (HFD), the same diet supplemented with non-fermented cabbage-apple juice, or the same diet supplemented with fermented cabbage-apple juice for eight weeks. In the HFD-fermented cabbage- apple juice administered groups the following parameters decreased: body weight, liver and white fat pad weights, serum triglyceride (TG), total cholesterol (TC), LDL-cholesterol, insulin, glucose and leptin levels, TG levels, while HDL-C and adiponectin levels in serum increased as compared with the HFD group. The HFD-fed rats that were supplemented with fermented cabbage-apple juice exhibited significantly lower fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC), and malic enzyme gene expression levels when compared to the exclusively HFD-fed rats. The anti-obesity and hypolipidemic effects were marginally greater in the fermented juice administered group than in the non-fermented juice administered group. These results suggest that cabbage-apple juice—especially fermented cabbage-apple juice—might have beneficial effects on lipid metabolism dysfunction and obesity-related abnormalities. However, further studies are necessary for analyzing the biochemical regulatory mechanisms of fermented juice for obesity amelioration and lipid metabolic homeostasis.

The phosphoenolpyruvate-pyruvate-oxaloacetate node genes and enzymes in Streptomyces coelicolor M-145.

The phosphoenolpyruvate-pyruvate-oxaloacetate node is a major branch within the central carbon metabolism and acts as a connection point between glycolysis, gluconeogenesis, and the TCA cycle. Phosphoenolpyruvate carboxylase, pyruvate carboxylase, phosphoenolpyruvate carboxykinase, malic enzymes, and pyruvate kinase, among others, are enzymes included in this node. We determined the mRNA levels and specific activity profiles of some of these genes and enzymes in Streptomyces coelicolor M-145. The results obtained in the presence of glucose demonstrated that all genes studied of the phosphoenolpyruvate-pyruvate-oxaloacetate node were expressed, although at different levels, with 10- to 100-fold differences. SCO3127 (phosphoenolpyruvate carboxylase gene) and SCO5261 (NADP+-dependent malic enzyme gene) showed the highest expression in the rapid growth phase, and the mRNA levels corresponding to SCO5896 (phosphoenolpyruvate-utilizing enzyme gene), and SCO0546 (pyruvate carboxylase gene) increased 5- to 10-fold towards the stationary phase. In casamino acids, in general mRNA levels of S. coelicolor were lower than in glucose, however, results showed greater mRNA expression of SCO4979 (PEP carboxykinase), SCO0208 (pyruvate phosphate dikinase gene), and SCO5261 (NADP+-dependent malic enzyme). These results suggest that PEP carboxylase (SCO3127) is an important enzyme during glucose catabolism and oxaloacetate replenishment. On the other hand, phosphoenolpyruvate carboxykinase, pyruvate phosphate dikinase, and NADP+-malic enzyme could have an important role in gluconeogenesis in S. coelicolor.

Engineering a malic enzyme to enhance lipid accumulation in Chlorella protothecoides and direct production of biodiesel from the microalgal biomass

Genetic engineering of the eukaryotic microalga Chlorella protothecoides remains in the early stages of development. There is also an urgent need to develop cost-effective biodiesel production processes from wet and lipid-rich C. protothecoides. Malic enzyme is a potential key enzyme responsible for a rate-limiting step associated with lipid accumulation in some microorganisms. In this study, we genetically engineered C. protothecoides by overexpressing a key malic enzyme to improve lipid accumulation for direct biodiesel production from microalgal biomass. An open reading frame encoding 509 amino acid residues of a putative malic enzyme was obtained and identified. Cloning and overexpression of the malic enzyme gene allowed genetic engineering of C. protothecoides for lipid accumulation and further direct production of biodiesel without the need for lipid extraction steps. The total lipid accumulation in genetically engineered C. protothecoides increased 2.8-fold compared to the wild type. A dehydration step using methanol pretreatment coupled with H2SO4 or lipase catalyzed biodiesel production enabled 93–98% biodiesel yields directly from microalgal biomass without extraction of lipid under optimal conditions. The efficient direct dehydration and chemical and enzymatic transformation processes could significantly reduce the overall cost of biodiesel production from wet and lipid-rich microalgal feedstocks.

Exploiting genetic polymorphisms in metabolic enzymes for rapid screening of Leishmania infantum genotypes

BackgroundLeishmania infantum is the aetiological agent of visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). Numerous strains and/or zymodemes have been identified and characterized by multilocus enzyme electrophoresis (MLEE). MLEE is considered the reference method for L. infantum parasite typing and it is based upon enzyme electrophoretic mobility analysis from promastigote cultures. However, the MLEE technique is cumbersome, time-consuming and does not detect silent genetic mutations or nucleotide changes that give rise to amino acid changes that do not alter electrophoretic mobility. As a result of these difficulties, many DNA-based typing methods have been developed over the past few years. However, relative to the enzymes utilized in MLEE analysis, we observed a shortage of DNA sequences available in the GenBank database or an absolute lack of sequences belonging to specific zymodemes. The aims of the present study were to (i) implement the number of sequences coding for metabolic enzymes used in MLEE; (ii) identify polymorphisms that characterize L. infantum zymodemes most prevalent in the Mediterranean basin; and (iii) exploit these polymorphisms to develop a rapid screening test that would give results comparable with existing MLEE typing.ResultsPartial sequences of seven metabolic enzyme genes (malic enzyme, 6-phosphogluconate dehydrogenase, mitochondrial isocitrate dehydrogenase, glucose-6-phosphate isomerase, glucose-6-phosphate dehydrogenase, phosphoglucomutase and mannose phosphate isomerase) were obtained from 11 L. infantum strains. The comparison of these sequences with those obtained from GenBank allowed for the identification of a few polymorphisms that could distinguish several zymodemes. In particular, the polymorphism 390T>G in the malic enzyme gene has been exploited to develop a high-resolution melt (HRM)-based assay to rapidly differentiate the genotype 390T, associated with zymodemes MON-1, MON-72 and MON-201, evidencing a partial agreement between genotyping results and MLEE. The assay has been successfully applied to L. infantum clinical isolates and clinical samples.ConclusionsA HRM-based assay for rapid identification of genotypes associated with the most common L. infantum zymodemes in the Mediterranean basin has been developed and its potential application in epidemiological research for L. infantum population screening, without parasite isolation and culturing, has been demonstrated.

Characterization of the Genes Involved in Malic Acid Metabolism from Pear Fruit and Their Expression Profile after Postharvest 1-MCP/Ethrel Treatment.

In this study, five genes involved in malic acid (MA) metabolism, including a cytosolic NAD-dependent malate dehydrogenase gene ( cyNAD-MDH), a cytosolic NADP-dependent malic enzyme gene ( cyNADP-ME), two vacuolar H+-ATPase genes ( vVAtp1 and vVAtp2), and one vacuolar inorganic pyrophosphatase gene ( vVPp), were characterized from pear fruit based on bioinformatic and experimental analysis. Their expression profile in "Housui" pear was tissue-specific, and their expression patterns during fruit development were diverse. During "Housui" pear storage, MA content decreased, which was associated with the downregulated transcripts of MA metabolism-related genes and cyNAD-MDH activity and higher cyNADP-ME activity. The response of MA metabolism to postharvest 1.5 μL L-1 1-MCP fumigation and 0.5 mL L-1 ethrel dipping was distinct: 1-MCP fumigation upregulated gene expression and cyNAD-MDH activity and suppressed cyNADP-ME activity, and thus maintained higher MA abundance when compared with those in the control; on the other hand, an opposite behavior was observed in ethrel-treated fruit.

Synergistic effect of eicosapentaenoic acid-enriched phospholipids and sea cucumber saponin on orotic acid-induced non-alcoholic fatty liver disease in rats.

Non-alcoholic fatty liver disease (NAFLD) is becoming an increasingly prevalent chronic liver disease all over the world. The present study was undertaken to explore the synergistic effects of sea cucumber saponins (SCS) and eicosapentaenoic acid-enriched phospholipids (EPA-PL) at ratios of 0.5 : 0.5 and 1 : 1 on NAFLD and demonstrate possible protective mechanisms. It was found that the combination of EPA-PL and SCS at half dose exhibited better effects than EPA-PL or SCS alone and the combination of EPA-PL and SCS at full dose in alleviating orotic acid (OA)-induced symptoms including growth parameters, serum parameters and liver function. Further evaluation of the mechanism illustrated that EPA-PL and SCS combination at the ratio of 0.5 : 0.5 could markedly reduce the mRNA expressions of fatty acid synthase, acetyl-CoA carboxylase, glucose-6-phosphate dehydrogenase and malic enzyme genes and significantly increase expression of genes relevant to fatty acid β-oxidation including peroxisome proliferator-activated receptor and its target genes (CPT1, CPT2 and ACOX1), suggesting that the protection of the EPA-PL and SCS combination at the ratio of 0.5 : 0.5 against OA-induced NAFLD might be mainly via lipogenesis inhibition and β-oxidation enhancement in the liver. The synergistic effects of EPA-PL and SCS make it possible to reduce the doses of EPA-PL or SCS to avoid side effects, which is of value for the development of dietary supplements or functional foods for preventing or treating NAFLD.

A novel malic enzyme gene, Mime2, from Mortierella isabellina M6-22 contributes to lipid accumulation.

This study was aimed at cloning and characterizing a novel malic enzyme (ME) gene of Mortierella isabellina M6-22 and identifying its relation with lipid accumulation. Mime2 was cloned from strain M6-22. Plasmid pET32aMIME2 was constructed to express ME of MIME2 in Escherichia coli BL21. After purification, the optimal pH and temperature of MIME2, as well as Km and Vmax for NADP+ were determined. The effects of EDTA or metal ions (Mn2+, Mg2+, Co2+, Cu2+, Ca2+, or Zn2+) on the enzymatic activity of MIME2 were evaluated. Besides, plasmid pRHMIME2 was created to express MIME2 in Rhodosporidium kratochvilovae YM25235, and its cell lipid content was measured by the acid-heating method. The optimal pH and temperature of MIME2 are 5.8 and 30°C, respectively. The act ivity of MIME2 was significantly increased by Mg2+, Ca2+, or Mn2+ at 0.5mM but inhibited by Cu2+ or Zn2+ (p < 0.05). The optimal enzymatic activity of MIME2 is 177.46 U/mg, and the Km and Vmax for NADP+ are 0.703mM and 156.25μg/min, respectively. Besides, Mime2 transformation significantly increased the cell lipid content in strain YM25235 (3.15 ± 0.24 vs. 2.17 ± 0.31g/L, p < 0.01). The novel ME gene Mime2 isolated from strain M6-22 contributes to lipid accumulation in strain YM25235.

Genome-scale model-driven strain design for dicarboxylic acid production in Yarrowia lipolytica

BackgroundRecently, there have been several attempts to produce long-chain dicarboxylic acids (DCAs) in various microbial hosts. Of these, Yarrowia lipolytica has great potential due to its oleaginous characteristics and unique ability to utilize hydrophobic substrates. However, Y. lipolytica should be further engineered to make it more competitive: the current approaches are mostly intuitive and cumbersome, thus limiting its industrial application.ResultsIn this study, we proposed model-guided metabolic engineering strategies for enhanced production of DCAs in Y. lipolytica. At the outset, we reconstructed genome-scale metabolic model (GSMM) of Y. lipolytica (iYLI647) by substantially expanding the previous models. Subsequently, the model was validated using three sets of published culture experiment data. It was finally exploited to identify genetic engineering targets for overexpression, knockout, and cofactor modification by applying several in silico strain design methods, which potentially give rise to high yield production of the industrially relevant long-chain DCAs, e.g., dodecanedioic acid (DDDA). The resultant targets include (1) malate dehydrogenase and malic enzyme genes and (2) glutamate dehydrogenase gene, in silico overexpression of which generated additional NADPH required for fatty acid synthesis, leading to the increased DDDA fluxes by 48% and 22% higher, respectively, compared to wild-type. We further investigated the effect of supplying branched-chain amino acids on the acetyl-CoA turn-over rate which is key metabolite for fatty acid synthesis, suggesting their significance for production of DDDA in Y. lipolytica.ConclusionIn silico model-based strain design strategies allowed us to identify several metabolic engineering targets for overproducing DCAs in lipid accumulating yeast, Y. lipolytica. Thus, the current study can provide a methodological framework that is applicable to other oleaginous yeasts for value-added biochemical production.