Enhanced BCAT1 activity and BCAA metabolism promotes RhoC activity in cancer progression.

Increased expression of branched-chain amino acid transaminase 1 or 2 (BCAT1 and BCAT2) has been associated with aggressive phenotypes of different cancers. Here we identify a gain of function of BCAT1 glutamic acid to alanine mutation at codon 61 (BCAT1E61A) enriched around 2.8% in clinical gastric cancer samples. We found that BCAT1E61A confers higher enzymatic activity to boost branched-chain amino acid (BCAA) catabolism, accelerate cell growth and motility and contribute to tumor development. BCAT1 directly interacts with RhoC, leading to elevation of RhoC activity. Notably, the BCAA-derived metabolite, branched-chain α-keto acid directly binds to the small GTPase protein RhoC and promotes its activity. BCAT1 knockout-suppressed cell motility could be rescued by expressing BCAT1E61A or adding branched-chain α-keto acid. We also identified that candesartan acts as an inhibitor of BCAT1E61A, thus repressing RhoC activity and cancer cell motility in vitro and preventing peritoneal metastasis in vivo. Our study reveals a link between BCAA metabolism and cell motility and proliferation through regulating RhoC activation, with potential therapeutic implications for cancers.

Co-production of 7-chloro-tryptophan and indole pyruvic acid based on an efficient FAD/FADH2 regeneration system.

Efficient FAD/FADH2 regeneration is vital for enzymatic biocatalysis and metabolic pathway optimization. Here, we constructed an efficient and simple FAD/FADH2 regeneration system through a combination of L-amino acid deaminase (L-AAD) and halogenase (CombiAADHa), which was applied for catalyzing the conversion of an L-amino acid to halide and an α-keto acid. For cell-free biotransformation, the optimal activity ratio of L-AAD and halogenase was set between 1:50 and 1:60. Within 6 h, 170 mg/L of 7-chloro-tryptophan (7-Cl-Trp) and 193 mg/L of indole pyruvic acid (IPA) were synthesized in the selected mono-amino acid system. For whole-cell biotransformation, 7-Cl-Trp and IPA synthesis was enhanced by 15% (from 96 to 110 mg/L) and 12% (from 115 to 129 mg/L), respectively, through expression fine-tuning and the strengthening of FAD/FADH2 supply. Finally, ultrasound treatment was applied to improve membrane permeability and adjust the activity ratio, resulting in 1.6-and 1.4-fold higher 7-Cl-Trp and IPA yields. The products were then purified. This system could also be applied to the synthesis of other halides and α-keto acids. KEY POINTS: • In this study, a whole cell FAD/FADH2 regeneration system co-expressing l-AAD and halogenase was constructed • This study found that the activity and ratio of enzyme and the concentration of cofactors had a significant effect on the catalytic process for the efficient co-production of 7-chlorotryptophan and indole pyruvate.

Branched-chain keto acids inhibit mitochondrial pyruvate carrier and suppress gluconeogenesis in hepatocytes.

Branched-chain amino acid (BCAA) metabolism is linked to glucose homeostasis, but the underlying signaling mechanisms are unclear. We find that gluconeogenesis is reduced in mice deficient of Ppm1k, a positive regulator of BCAA catabolism, which protects against obesity-induced glucose intolerance. Accumulation of branched-chain keto acids (BCKAs) inhibits glucose production in hepatocytes. BCKAs suppress liver mitochondrial pyruvate carrier (MPC) activity and pyruvate-supported respiration. Pyruvate-supported gluconeogenesis is selectively suppressed in Ppm1k-deficient mice and can be restored with pharmacological activation of BCKA catabolism by BT2. Finally, hepatocytes lack branched-chain aminotransferase that alleviates BCKA accumulation via reversible conversion between BCAAs and BCKAs. This renders liver MPC most susceptible to circulating BCKA levels hence a sensor of BCAA catabolism.

[Regulatory effects and mechanisms of branched chain amino acids and metabolic intermediates on insulin resistance].

Branched chain amino acids, as essential amino acids, can be used to synthesize nitrogen-containing compounds and also act as signal molecules to regulate substance metabolism. Studies have shown that the elevated level of branched chain amino acids is closely related to insulin resistance and type 2 diabetes. It can affect insulin signal transduction by activating mammalian target of rapamycin (mTOR) signal pathway, and regulate insulin resistance by damaging lipid metabolism and affecting mitochondrial function. In addition, abnormal catabolism of branched amino acids can lead to the accumulation of metabolic intermediates, such as branched chain α-keto acids, 3-hydroxyisobutyrate and ß-aminoisobutyric acid. Branched chain α-keto acids and 3-hydroxyisobutyrate can induce insulin resistance by affecting insulin signaling pathway and damaging lipid metabolism. ß-aminoisobutyric acid can improve insulin resistance by reducing lipid accumulation and inflammatory reaction and enhancing fatty acid oxidation. This paper systematically reviewed the regulatory effects and mechanisms of branched chain amino acids and their metabolic intermediates on insulin resistance, which will provide a new direction for the prevention and treatment of insulin resistance and type 2 diabetes.

Branched-Chain Amino Acid Metabolism in the Failing Heart.

Branched-chain amino acids (BCAAs) are essential amino acids which have critical roles in protein synthesis and energy metabolism in the body. In the heart, there is a strong correlation between impaired BCAA oxidation and contractile dysfunction in heart failure. Plasma and myocardial levels of BCAA and their metabolites, namely branched-chain keto acids (BCKAs), are also linked to cardiac insulin resistance and worsening adverse remodelling in the failing heart. This review discusses the regulation of BCAA metabolism in the heart and the impact of depressed cardiac BCAA oxidation on cardiac energy metabolism, function, and structure in heart failure. While impaired BCAA oxidation in the failing heart causes the accumulation of BCAA and BCKA in the myocardium, recent evidence suggested that the BCAAs and BCKAs have divergent effects on the insulin signalling pathway and the mammalian target of the rapamycin (mTOR) signalling pathway. Dietary and pharmacological interventions that enhance cardiac BCAA oxidation and limit the accumulation of cardiac BCAAs and BCKAs have been shown to have cardioprotective effects in the setting of ischemic heart disease and heart failure. Thus, targeting cardiac BCAA oxidation may be a promising therapeutic approach for heart failure.

Regulatory effects and mechanisms of branched chain amino acids and metabolic intermediates on insulin resistance / 生理学报

Branched chain amino acids, as essential amino acids, can be used to synthesize nitrogen-containing compounds and also act as signal molecules to regulate substance metabolism. Studies have shown that the elevated level of branched chain amino acids is closely related to insulin resistance and type 2 diabetes. It can affect insulin signal transduction by activating mammalian target of rapamycin (mTOR) signal pathway, and regulate insulin resistance by damaging lipid metabolism and affecting mitochondrial function. In addition, abnormal catabolism of branched amino acids can lead to the accumulation of metabolic intermediates, such as branched chain α-keto acids, 3-hydroxyisobutyrate and β-aminoisobutyric acid. Branched chain α-keto acids and 3-hydroxyisobutyrate can induce insulin resistance by affecting insulin signaling pathway and damaging lipid metabolism. β-aminoisobutyric acid can improve insulin resistance by reducing lipid accumulation and inflammatory reaction and enhancing fatty acid oxidation. This paper systematically reviewed the regulatory effects and mechanisms of branched chain amino acids and their metabolic intermediates on insulin resistance, which will provide a new direction for the prevention and treatment of insulin resistance and type 2 diabetes.

BAY-069, a Novel (Trifluoromethyl)pyrimidinedione-Based BCAT1/2 Inhibitor and Chemical Probe.

The branched-chain amino acid transaminases (BCATs) are enzymes that catalyze the first reaction of catabolism of the essential branched-chain amino acids to branched-chain keto acids to form glutamate. They are known to play a key role in different cancer types. Here, we report a new structural class of BCAT1/2 inhibitors, (trifluoromethyl)pyrimidinediones, identified by a high-throughput screening campaign and subsequent optimization guided by a series of X-ray crystal structures. Our potent dual BCAT1/2 inhibitor BAY-069 displays high cellular activity and very good selectivity. Along with a negative control (BAY-771), BAY-069 was donated as a chemical probe to the Structural Genomics Consortium.

The sodium-glucose co-transporter-2 inhibitor ertugliflozin modifies the signature of cardiac substrate metabolism and reduces cardiac mTOR signalling, endoplasmic reticulum stress and apoptosis.

AIM: To investigate cardiac signalling pathways connecting substrate utilization with left ventricular remodelling in a murine pressure overload model. METHODS: Cardiac hypertrophy was induced by transverse aortic constriction surgery in 20-week-old C57BL/6J mice treated with or without the sodium-glucose co-transporter 2 (SGLT2) inhibitor ertugliflozin (225 mg kg-1 chow diet) for 10 weeks. RESULTS: Ertugliflozin improved left ventricular function and reduced myocardial fibrosis. This occurred simultaneously with a fasting-like response characterized by improved glucose tolerance and increased ketone body concentrations. While cardiac insulin signalling was reduced in response to SGLT2 inhibition, AMP-activated protein kinase (AMPK) signalling was increased with induction of the fatty acid transporter cluster of differentiation 36 and phosphorylation of acetyl-CoA carboxylase (ACC). Further, enzymes responsible for ketone body catabolism (ß-hydroxybutyrate dehydrogenase, succinyl-CoA:3-oxoacid-CoA transferase and acetyl-CoA acetyltransferase 1) were induced by SGLT2 inhibition. Ertugliflozin led to more cardiac abundance of fatty acids, tricarboxylic acid cycle metabolites and ATP. Downstream mechanistic target of rapamycin (mTOR) pathway, relevant for protein synthesis, cardiac hypertrophy and adverse cardiac remodelling, was reduced by SGLT2 inhibition, with alleviation of endoplasmic reticulum (ER) stress and unfolded protein response (UPR) providing a potential mechanism for abundant reduced left ventricular apoptosis and fibrosis. CONCLUSION: SGLT2 inhibition reduced left ventricular fibrosis in a murine model of cardiac hypertrophy. Mechanistically, this was associated with reduced cardiac insulin and increased AMPK signalling as a potential mechanism for less cardiac mTOR activation with alleviation of downstream ER stress, UPR and apoptosis.

Proteome reallocation enables the selective de novo biosynthesis of non-linear, branched-chain acetate esters.

The one-carbon recursive ketoacid elongation pathway is responsible for making various branched-chain amino acids, aldehydes, alcohols, ketoacids, and acetate esters in living cells. Controlling selective microbial biosynthesis of these target molecules at high efficiency is challenging due to enzyme promiscuity, regulation, and metabolic burden. In this study, we present a systematic modular design approach to control proteome reallocation for selective microbial biosynthesis of branched-chain acetate esters. Through pathway modularization, we partitioned the branched-chain ester pathways into four submodules including ketoisovalerate submodule for converting pyruvate to ketoisovalerate, ketoacid elongation submodule for producing longer carbon-chain ketoacids, ketoacid decarboxylase submodule for converting ketoacids to alcohols, and alcohol acyltransferase submodule for producing branched-chain acetate esters by condensing alcohols and acetyl-CoA. By systematic manipulation of pathway gene replication and transcription, enzyme specificity of the first committed steps of these submodules, and downstream competing pathways, we demonstrated selective microbial production of isoamyl acetate over isobutyl acetate. We found that the optimized isoamyl acetate pathway globally redistributed the amino acid fractions in the proteomes and required up to 23-31% proteome reallocation at the expense of other cellular resources, such as those required to generate precursor metabolites and energy for growth and amino acid biosynthesis. From glucose fed-batch fermentation, the engineered strains produced isoamyl acetate up to a titer of 8.8 g/L (>0.25 g/L toxicity limit), a yield of 0.22 g/g (61% of maximal theoretical value), and 86% selectivity, achieving the highest titers, yields and selectivity of isoamyl acetate reported to date.

Expressing 2-keto acid pathway enzymes significantly increases photosynthetic isobutanol production.

BACKGROUND: Cyanobacteria, photosynthetic microorganisms, are promising green cell factories for chemical production, including biofuels. Isobutanol, a four-carbon alcohol, is considered as a superior candidate as a biofuel for its high energy density with suitable chemical and physical characteristics. The unicellular cyanobacterium Synechocystis PCC 6803 has been successfully engineered for photosynthetic isobutanol production from CO2 and solar energy in a direct process. RESULTS: Heterologous expression of α-ketoisovalerate decarboxylase (KivdS286T) is sufficient for isobutanol synthesis via the 2-keto acid pathway in Synechocystis. With additional expression of acetolactate synthase (AlsS), acetohydroxy-acid isomeroreductase (IlvC), dihydroxy-acid dehydratase (IlvD), and alcohol dehydrogenase (Slr1192OP), the Synechocystis strain HX42, with a functional 2-keto acid pathway, showed enhanced isobutanol production reaching 98 mg L-1 in short-term screening experiments. Through modulating kivdS286T copy numbers as well as the composition of the 5'-region, a final Synechocystis strain HX47 with three copies of kivdS286T showed a significantly improved isobutanol production of 144 mg L-1, an 177% increase compared to the previously reported best producing strain under identical conditions. CONCLUSIONS: This work demonstrates the feasibility to express heterologous genes with a combination of self-replicating plasmid-based system and genome-based system in Synechocystis cells. Obtained isobutanol-producing Synechocystis strains form the base for further investigation of continuous, long-term-photosynthetic isobutanol production from solar energy and carbon dioxide.

BCAA-BCKA axis regulates WAT browning through acetylation of PRDM16.

The link between branched-chain amino acids (BCAAs) and obesity has been known for decades but the functional role of BCAA metabolism in white adipose tissue (WAT) of obese individuals remains vague. Here, we show that mice with adipose tissue knockout of Bcat2, which converts BCAAs to branched-chain keto acids (BCKAs), are resistant to high-fat diet-induced obesity due to increased inguinal WAT browning and thermogenesis. Mechanistically, acetyl-CoA derived from BCKA suppresses WAT browning by acetylation of PR domain-containing protein 16 (PRDM16) at K915, disrupting the interaction between PRDM16 and peroxisome proliferator-activated receptor-γ (PPARγ) to maintain WAT characteristics. Depletion of BCKA-derived acetyl-CoA robustly prompts WAT browning and energy expenditure. In contrast, BCKA supplementation re-establishes high-fat diet-induced obesity in Bcat2 knockout mice. Moreover, telmisartan, an anti-hypertension drug, significantly represses Bcat2 activity via direct binding, resulting in enhanced WAT browning and reduced adiposity. Strikingly, BCKA supplementation reverses the lean phenotype conferred by telmisartan. Thus, we uncover the critical role of the BCAA-BCKA axis in WAT browning.

Expanding the Reactive Sulfur Metabolome: Intracellular and Efflux Measurements of Small Oxoacids of Sulfur (SOS) and H2S in Human Primary Vascular Cell Culture.

Hydrogen sulfide (H2S) is an endogenous signaling molecule which is important for cardiovascular health, but its mechanism of action remains poorly understood. Here, we report measurements of H2S as well as its oxidized metabolites, termed small oxoacids of sulfur (SOS = HSOH and HOSOH), in four human primary vascular cell lines: smooth muscle and endothelial cells derived from both human arterial and coronary tissues. We use a methodology that targets small molecular weight sulfur species; mass spectrometric analysis allows for species quantification to report cellular concentrations based on an H2S calibration curve. The production of H2S and SOS is orders of magnitude higher in smooth muscle (nanomolar) as compared to endothelial cell lines (picomolar). In all the primary lines measured, the distributions of these three species were HOSOH >H2S > HSOH, with much higher SOS than seen previously in non-vascular cell lines. H2S and SOS were effluxed from smooth muscle cells in higher concentrations than endothelial cells. Aortic smooth muscle cells were used to examine changes under hypoxic growth conditions. Hypoxia caused notable increases in HSOH and ROS, which we attribute to enhanced sulfide quinone oxidase activity that results in reverse electron transport.

Transamination-Like Reaction Catalyzed by Leucine Dehydrogenase for Efficient Co-Synthesis of α-Amino Acids and α-Keto Acids.

α-Amino acids and α-keto acids are versatile building blocks for the synthesis of several commercially valuable products in the food, agricultural, and pharmaceutical industries. In this study, a novel transamination-like reaction catalyzed by leucine dehydrogenase was successfully constructed for the efficient enzymatic co-synthesis of α-amino acids and α-keto acids. In this reaction mode, the α-keto acid substrate was reduced and the α-amino acid substrate was oxidized simultaneously by the enzyme, without the need for an additional coenzyme regeneration system. The thermodynamically unfavorable oxidation reaction was driven by the reduction reaction. The efficiency of the biocatalytic reaction was evaluated using 12 different substrate combinations, and a significant variation was observed in substrate conversion, which was subsequently explained by the differences in enzyme kinetics parameters. The reaction with the selected model substrates 2-oxobutanoic acid and L-leucine reached 90.3% conversion with a high total turnover number of 9.0 × 106 under the optimal reaction conditions. Furthermore, complete conversion was achieved by adjusting the ratio of addition of the two substrates. The constructed reaction mode can be applied to other amino acid dehydrogenases in future studies to synthesize a wider range of valuable products.

Cobalt Phosphate Nanocrystals: A Catalase-Like Nanozyme and In Situ Enzyme-Encapsulating Carrier for Efficient Chemoenzymatic Synthesis of α-Keto Acid.

Chemoenzymatic catalysis combining the traits of chemical and enzymatic catalysis provides tremendous possibilities for the design of biosynthetic pathways utilizing inorganic catalysts and enzymes. However, the efficiency of chemoenzymatic catalysis is usually governed by the synergy and compatibility of the two catalysts. Here, we report for the first time the catalase-like activity of cobalt phosphate nanocrystals (CoPs). By a one-pot biomimetic mineralization with CoPs and l-amino acid oxidase (LAAO) under a mild condition, we have fabricated a hybrid nanobiocatalyst, LAAO@CoPs, for the chemoenzymatic synthesis of α-keto acid. The as-fabricated nanobiocatalyst with directly contacted catalytic sites of the enzyme and nanozyme maximizes the substrate channeling effects for in situ chemical decomposition of the oxidative intermediate, H2O2, during the enzymatic oxidation of l-tryptophan (l-Trp), thus minimizing the H2O2 accumulation and byproduct generation. Benefiting from the superiority of LAAO@CoPs, complete conversion (100.0%) of l-Trp to indole pyruvic acid is achieved, over two times higher than the yield of the free LAAO system (47.6%). Meanwhile, LAAO@CoPs show high stabilities against heat and proteolytic treatments. This work offers a new design approach for constructing a high-performance nanobiocatalyst for cascade reactions, especially for those systems with toxic or reactive intermediates.

Characteristics of Biotin lipoyl attachment and 2-oxoacid dehydrogenase acyltransferase of the parasitic nematode Haemonchus contortus and its modulatory functions on goat PBMCs in vitro.

Biotin lipoyl attachment and 2-oxoacid dehydrogenase acyltransferase (BLAODA), as an essential excretion of Haemonchus contortus (HcESPs), was identified to have antigenic functions. T helper-9 (Th9) cells secrete interleukin (IL)-9, a signature cytokine associated with tumour immunology, allergy and autoimmunity. Nonetheless, the understanding of modulatory functions of BLAODA on Th9 and other immune cells is limited. In this study, the BLAODA gene was cloned, and the recombinant (r) protein of BLAODA (rHcBLAODA) was expressed and immunoblotting was performed. The results revealed that HcBLAODA gene was successfully cloned and rHcBLAODA protein was expressed. The localization of rHcBLAODA was confirmed on the surface of gut sections from adult H. contortus. The rHcBLAODA protein capability to react precisely with anti-H. contortus antibodies were confirmed by immunoblotting and immunofluorescence assay (IFA). Further functional analysis showed that interaction of rHcBLAODA with host cells significantly enhanced Th9 cells generation, IL-9 expression, nitric oxide production and cell apoptosis while suppressing the cells proliferation and cells migration depending on the concentration. Overall, these findings suggest that rHcBLAODA protein could modulate the host immune response by inducing Th9 cells to secrete IL-9 cytokine in vitro.

Convenient Genetic Encoding of Phenylalanine Derivatives through Their α-Keto Acid Precursors.

The activity and function of proteins can be improved by incorporation of non-canonical amino acids (ncAAs). To avoid the tedious synthesis of a large number of chiral phenylalanine derivatives, we synthesized the corresponding phenylpyruvic acid precursors. Escherichia coli strain DH10B and strain C321.ΔA.expΔPBAD were selected as hosts for phenylpyruvic acid bioconversion and genetic code expansion using the MmPylRS/pyltRNACUA system. The concentrations of keto acids, PLP and amino donors were optimized in the process. Eight keto acids that can be biotransformed and their coupled genetic code expansions were identified. Finally, the genetic encoded ncAAs were tested for incorporation into fluorescent proteins with keto acids.

Loss of YggS (COG0325) impacts aspartate metabolism in Salmonella enterica.

YggS is a pyridoxal 5'-phosphate (PLP)-binding protein of the conserved COG0325 family. Despite a connection with vitamin B6 homeostasis in many species, neither a precise biochemical activity nor the molecular mechanism of how YggS contributes to cellular function has been described. In a transposon mutagenesis screen, we found that insertions in aspC (encoding a PLP-dependent aspartate aminotransferase, EC 2.6.1.1) in a Salmonella enterica strain lacking yggS caused a synthetic growth defect, which could be rescued by the addition of exogenous aspartate. Characterization of spontaneous suppressors which improved the growth of the yggS aspC double mutant suggested that this synthetic aspartate limitation was dependent on TyrB, a PLP-dependent aromatic amino acid aminotransferase (EC 2.6.1.57). Genetic and biochemical data were consistent with the hypothesis that TyrB activity was inhibited by accumulated pyridoxine 5'-phosphate and α-keto acids caused by a yggS mutation. This study provides data consistent with a working model implicating YggS in modulating concentrations of B6 vitamers via transamination.

Electrochemical Molecular Conversion of α-Keto Acid to Amino Acid at a Low Overpotential Using a Nanoporous Gold Catalyst.

A nanoporous gold (NPG) electrode prepared through a facile anodization technique was employed in the electrochemical reductive amination of biomass-derivable α-keto acids in the presence of a nitrogen source to produce the corresponding amino acids. NPG showed a clear reductive current in the presence of α-keto acid and NH2OH, and the electrolysis experiments confirmed the production of L-amino acid. A reductive voltammetric signal at the NPG electrode appeared at a more positive potential by 0.18-0.79 V, compared with those at the planar-gold electrode without anodization and other previously reported electrode systems, indicating the high activity of the prepared nanostructure for the electrochemical reaction. Maximum Faradaic efficiencies (FEs) of 74-93% in the reductive molecular conversion to amino acids of Ala, Asp, Glu, Gly, and Leu were obtained under the optimized conditions. The FE values were strongly dependent on the applied potential in the electrolysis, suggesting that the hydrogen evolution reaction at the electrode surface was more significant as the applied potential became more negative. The effect of potential at the NPG was lower than that at the planar-gold electrode. These results indicate that nanostructurization decreases the overpotential for the electrochemical reductive amination, resulting in high FE.

Insulin action, type 2 diabetes, and branched-chain amino acids: A two-way street.

BACKGROUND: A strong association of obesity and insulin resistance with increased circulating levels of branched-chain and aromatic amino acids and decreased glycine levels has been recognized in human subjects for decades. SCOPE OF REVIEW: More recently, human metabolomics and genetic studies have confirmed and expanded upon these observations, accompanied by a surge in preclinical studies that have identified mechanisms involved in the perturbation of amino acid homeostasis- how these events are connected to dysregulated glucose and lipid metabolism, and how elevations in branched-chain amino acids (BCAA) may participate in the development of insulin resistance, type 2 diabetes (T2D), and other cardiometabolic diseases and conditions. MAJOR CONCLUSIONS: In human cohorts, BCAA and related metabolites are now well established as among the strongest biomarkers of obesity, insulin resistance, T2D, and cardiovascular diseases. Lowering of BCAA and branched-chain ketoacid (BCKA) levels by feeding BCAA-restricted diet or by the activation of the rate-limiting enzyme in BCAA catabolism, branched-chain ketoacid dehydrogenase (BCKDH), in rodent models of obesity have clear salutary effects on glucose and lipid homeostasis, but BCAA restriction has more modest effects in short-term studies in human T2D subjects. Feeding of rats with diets enriched in sucrose or fructose result in the induction of the ChREBP transcription factor in the liver to increase expression of the BCKDH kinase (BDK) and suppress the expression of its phosphatase (PPM1K) resulting in the inactivation of BCKDH and activation of the key lipogenic enzyme ATP-citrate lyase (ACLY). These and other emergent links between BCAA, glucose, and lipid metabolism motivate ongoing studies of possible causal actions of BCAA and related metabolites in the development of cardiometabolic diseases.

Role of beta-isopropylmalate dehydrogenase in lipid biosynthesis of the oleaginous fungus Mortierella alpina.

Branched-chain amino acids (BCAAs) play an important role in lipid metabolism by serving as signal molecules as well as a potential acetyl-CoA source. Our previous study found that in the oleaginous fungus Mucor circinelloides, beta-isopropylmalate dehydrogenase (IPMDH), an important enzyme participating in the key BCAA leucine biosynthesis, was differentially expressed during lipid accumulation phase and has a positive role on lipogenesis. To further analyze its effects on lipogenesis in another oleaginous fungus Mortierella alpina, the IPMDH-encoding gene MaLeuB was homologously expressed. It was found that the total fatty acid content in the recombinant strain was increased by 20.2% compared with the control strain, which correlated with a 4-fold increase in the MaLeuB transcriptional level. Intracellular metabolites analysis revealed significant changes in amino acid biosynthesis and metabolism, tricarboxylic acid cycle and butanoate metabolism; specifically, leucine and isoleucine levels were upregulated by 6.4-fold and 2.2-fold, respectively. Our genetic engineering approach and metabolomics study demonstrated that MaLeuB is involved in fatty acid metabolism in M. alpina by affecting BCAAs metabolism, and this newly discovered role of IPMDH provides a potential bypass route to increase lipogenesis in oleaginous fungi.