The role of the two key enzymes of fatty acid (FA) synthesis, ATP-citrate lyase (Acl) and malic enzyme (Mae), was analyzed in the oleaginous yeast Yarrowia lipolytica. In most oleaginous yeasts, Acl ...and Mae are proposed to provide, respectively, acetyl-CoA and NADPH for FA synthesis. Acl was mainly studied at the biochemical level but no strain depleted for this enzyme was analyzed in oleaginous microorganisms. On the other hand the role of Mae in FA synthesis in Y. lipolytica remains unclear since it was proposed to be a mitochondrial NAD(H)-dependent enzyme and not a cytosolic NADP(H)-dependent enzyme. In this study, we analyzed for the first time strains inactivated for corresponding genes. Inactivation of ACL1 decreases FA synthesis by 60 to 80%, confirming its essential role in FA synthesis in Y. lipolytica. Conversely, inactivation of MAE1 has no effects on FA synthesis, except in a FA overaccumulating strain where it improves FA synthesis by 35%. This result definitively excludes Mae as a major key enzyme for FA synthesis in Y. lipolytica. During the analysis of both mutants, we observed a negative correlation between FA and mannitol level. As mannitol and FA pathways may compete for carbon storage, we inactivated YlSDR, encoding a mannitol dehydrogenase converting fructose and NADPH into mannitol and NADP+. The FA content of the resulting mutant was improved by 60% during growth on fructose, demonstrating that mannitol metabolism may modulate FA synthesis in Y. lipolytica.
•We analyzed two key enzymes for FA synthesis Acl and Mae, in Y. lipolytica.•Δacl1 mutant presents lower FA content and a higher citrate and mannitol production.•MAE1 inactivation increases FA content in obese mutant decreasing mannitol content.•Inactivation of mannitol dehydrogenase, improves FA content on fructose.•Mannitol metabolism may modulate FA synthesis in Y. lipolytica.
Although the presence of mannitol in organisms as diverse as plants and fungi clearly suggests that this compound has important roles, our understanding of fungal mannitol metabolism and its ...interaction with mannitol metabolism in plants is far from complete. Despite recent inroads into understanding the importance of mannitol and its metabolic roles in salt, osmotic, and oxidative stress tolerance in plants and fungi, our current understanding of exactly how mannitol protects against reactive oxygen is also still incomplete. In this opinion, we propose a new model of the interface between mannitol metabolism in plants and fungi and how it impacts plant–pathogen interactions.
Mannitol is proposed to have a role(s) in protecting cells and cellular structures against damage by reactive oxygen that is unrelated to previous models of radical scavenging.
The application of newer techniques, such as targeted gene disruption, now allows for new interpretations of the nature and role of mannitol metabolism in fungi.
Increased intestinal permeability has been associated with Crohn’s disease (CD), but it is not clear whether it is a cause or result of the disease. We performed a prospective study to determine ...whether increased intestinal permeability is associated with future development of CD.
We assessed the intestinal permeability, measured by the urinary fractional excretion of lactulose-to-mannitol ratio (LMR) at recruitment in 1420 asymptomatic first-degree relatives (6–35 years old) of patients with CD (collected from 2008 through 2015). Participants were then followed up for a diagnosis of CD from 2008 to 2017, with a median follow-up time of 7.8 years. We analyzed data from 50 participants who developed CD after a median of 2.7 years during the study period, along with 1370 individuals who remained asymptomatic until October 2017. We used the Cox proportional hazards model to evaluate time-related risk of CD based on the baseline LMR.
An abnormal LMR (>0.03) was associated with a diagnosis of CD during the follow-up period (hazard ratio, 3.03; 95% CI, 1.64–5.63; P = 3.97 × 10–4). This association remained significant even when the test was performed more than 3 years before the diagnosis of CD (hazard ratio, 1.62; 95% CI, 1.051–2.50; P = .029).
Increased intestinal permeability is associated with later development of CD; these findings support a model in which altered intestinal barrier function contributes to pathogenesis. Abnormal gut barrier function might serve as a biomarker for risk of CD onset.
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The proposed pathways of D-mannitol production via hyperthermophilic archaeal.
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•Purified MtDH is an exceptional promoter for mannitol synthesis.•MtDH with unique properties is ...proposed as an industrial enzyme.•MtDH is a promised biocatalyst to fulfill the gab in food and medical applications.
Recently, the term healthy lifestyle connected to low-calorie diets, although it is not possible to get rid of added sugars as a source of energy, despite the close relation of added sugars to some diseases such as obesity, diabetes, etc. As a result, the sweetener market has flourished, which has led to increased demand for natural sweeteners such as polyols, including d-mannitol. Various methods have been developed to produce d-mannitol to achieve high productivity and low cost. In particular, metabolic engineering for d-mannitol considers one of the most promising approaches for d-mannitol production on the industrial scale. To date, the chemical process is not ideal for large-scale production because of its multistep mechanism involving hydrogenation and high cost. In this review, we highlight and present a comparative evaluation of the biochemical parameters that affecting d-mannitol synthesis from Thermotoga neapolitana and Thermotoga maritima mannitol dehydrogenase (MtDH) as a potential contribution for d-mannitol bio-synthesis. These species were selected because purified mannitol dehydrogenases from both strains have been reported to produce d-mannitol with no sorbitol formation under temperatures (90–120 °C).
Mannitol, a naturally occurring polyol (sugar alcohol), is widely used in the food, pharmaceutical, medical, and chemical industries. The production of mannitol by fermentation has become attractive ...because of the problems associated with its production chemically. A number of homo- and heterofermentative lactic acid bacteria (LAB), yeasts, and filamentous fungi are known to produce mannitol. In particular, several heterofermentative LAB are excellent producers of mannitol from fructose. These bacteria convert fructose to mannitol with 100% yields from a mixture of glucose and fructose (1:2). Glucose is converted to lactic acid and acetic acid, and fructose is converted to mannitol. The enzyme responsible for conversion of fructose to mannitol is NADPH- or NADH-dependent mannitol dehydrogenase (MDH). Fructose can also be converted to mannitol by using MDH in the presence of the cofactor NADPH or NADH. A two enzyme system can be used for cofactor regeneration with simultaneous conversion of two substrates into two products. Mannitol at 180 g l⁻¹ can be crystallized out from the fermentation broth by cooling crystallization. This paper reviews progress to date in the production of mannitol by fermentation and using enzyme technology, downstream processing, and applications of mannitol.
Mannitol is a natural low-calorie sugar alcohol produced by certain (micro)organisms applicable in foods for diabetics due to its zero glycemic index. In this work, we evaluated mannitol production ...and yield by the fruit origin strain
Fructobacillus tropaeoli
CRL 2034 using response surface methodology with central composite design (CCD) as optimization strategy. The effect of the total saccharide (glucose + fructose, 1:2) content (TSC) in the medium (75, 100, 150, 200, and 225 g/l) and stirring (S; 50, 100, 200, 300 and 350 rpm) on mannitol production and yield by this strain was evaluated by using a 2
2
full-factorial CCD with 4 axial points (
α
= 1.5) and four replications of the center point, leading to 12 random experimental runs. Fermentations were carried out at 30 °C and pH 5.0 for 24 h. Minitab-15 software was used for experimental design and data analyses. The multiple response prediction analysis established 165 g/l of TSC and 200 rpm of S as optimal culture conditions to reach 85.03 g/l 95% CI (78.68, 91.39) of mannitol and a yield of 82.02% 95% CI (71.98, 92.06). Finally, a validation experiment was conducted at the predicted optimum levels. The results obtained were 81.91 g/l of mannitol with a yield of 77.47% in outstanding agreement with the expected values. The mannitol 2-dehydrogenase enzyme activity was determined with 4.6–4.9 U/mg as the highest value found. To conclude,
F. tropaeoli
CRL 2034 produced high amounts of high-quality mannitol from fructose, being an excellent candidate for this polyol production.
The intracellular accumulation of organic compatible solutes functioning as osmoprotectants, such as polyols, is an important response mechanism of several plants to drought and salinity. In Olea ...europaea a mannitol transport system (OeMaT1) was previously characterized as a key player in plant response to salinity. In the present study, heterotrophic sink models, such as olive cell suspensions and fruit tissues, and source leaves were used for analytical, biochemical and molecular studies. The kinetic parameters of mannitol dehydrogenase (MTD) determined in cells growing in mannitol, at 25°C and pH 9.0, were as follows: K
m, 54.5 mM mannitol; and V
max, 0.47 μmol h−1 mg−1 protein. The corresponding cDNA was cloned and named OeMTD1. OeMTD1 expression was correlated with MTD activity, OeMaT1 expression and carrier-mediated mannitol transport in mannitol- and sucrose-grown cells. Furthermore, sucrose-grown cells displayed only residual OeMTD activity, even though high levels of OeMTD1 transcription were observed. There is evidence that OeMTD is regulated at both transcriptional and post-transcriptional levels. MTD activity and OeMTD1 expression were repressed after Na+, K+ and polyethylene glycol (PEG) treatments, in both mannitol- and sucrose-grown cells. In contrast, salt and drought significantly increased mannitol transport activity and OeMaT1 expression. Taken together, these studies support that olive trees cope with salinity and drought by coordinating mannitol transport with intracellular metabolism.
Mannitol is a naturally occurring six-carbon sugar alcohol that has wide applications in the food and pharmaceutical industry because of its many properties, namely being a natural sweetener with a ...low metabolism and no glycemic index. The increasing demand for mannitol has spurred many studies of its production. Compared with its chemical synthesis and extraction from plants, both of which are difficult to satisfy for industrial requirements, biotechnological production of mannitol has received considerably more attention and interest from scientists because of its known advantages over those two methods. Accordingly, in this review, we summarize recent advances made in the production of mannitol through various biotechnological methods. The physicochemical properties, sources, and physiological functionalities and applications of mannitol are systematically covered and presented. Then, different determination methods for mannitol are also described and compared. Furthermore, different biotechnological strategies for the production of mannitol via fermentation engineering, protein engineering, and metabolic engineering receive a detailed overview in terms of mannitol-producing strains, enzymes, and their key reaction parameters and conditions.
Key points
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Physiological functionalities and applications of mannitol are presented in detail.
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Different determination methods for mannitol are also described and compared.
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Various biotechnological strategies for the production of mannitol are reviewed.
Rapidly proliferating leukemic progenitor cells consume substantial glucose, which may lead to glucose insufficiency in bone marrow. We show that acute myeloid leukemia (AML) cells are prone to ...fructose utilization with an upregulated fructose transporter GLUT5, which compensates for glucose deficiency. Notably, AML patients with upregulated transcription of the GLUT5-encoding gene SLC2A5 or increased fructose utilization have poor outcomes. Pharmacological blockage of fructose uptake ameliorates leukemic phenotypes and potentiates the cytotoxicity of the antileukemic agent, Ara-C. In conclusion, this study highlights enhanced fructose utilization as a metabolic feature of AML and a potential therapeutic target.
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•AML cells are prone to fructose utilization to offset glucose insufficiency•Increased SLC2A5 transcription and fructose utilization predict poor patient outcomes•Enhanced fructose utilization exacerbates leukemic cell phenotypes•Inhibition of fructose uptake shows therapeutic potential for AML
Chen et al. show that AML cells exhibit enhanced fructose utilization under low-glucose conditions via upregulating the fructose transporter GLUT5, exacerbating leukemic phenotypes. Pharmacologic blockade of fructose utilization selectively eliminates AML cells and enhances the efficacy of Ara-C.
Enzymatic synthesis of biochemicals in vitro is vital in synthetic biology for its efficiency, minimal by-products, and easy product separation. However, challenges like enzyme preparation, ...stability, and reusability persist. Here, we introduced a protein scaffold and biosilicification coupled system, providing a singular process for the purification and immobilization of multiple enzymes. Using d-mannitol as a model, we initially constructed a self-assembling EE/KK protein scaffold for the co-immobilization of glucose dehydrogenase and mannitol dehydrogenase. Under an enzyme-to-scaffold ratio of 1:8, a d-mannitol yield of 0.692 mol/mol was achieved within 4 h, 2.16-fold higher than the free enzymes. The immobilized enzymes retained 70.9 % of the initial joint activity while the free ones diminished nearly to inactivity after 8 h. Furthermore, we incorporated the biosilicification peptide CotB into the EE/KK scaffold, inducing silica deposition, which enabled the one-step purification and immobilization process assisted by Spy/Snoop protein-peptide pairs. The coupled system demonstrated a comparable d-mannitol yield to that of EE/KK scaffold and 1.34-fold higher remaining activities after 36 h. Following 6 cycles of reaction, the immobilized system retained the capability to synthesize 56.4 % of the initial d-mannitol titer. The self-assembly co-immobilization platform offers an effective approach for enzymatic synthesis of d-mannitol and other biochemicals.
•An integrated co-immobilization system was constructed by coupling protein scaffold and biosilicification.•The system could realize one-step purification, immobilization and reuse of multienzymes.•Enzymes immobilized by the system showed superior performances to the free enzymes.
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