The mTORC1 pathway stimulates glutamine metabolism and cell proliferation by repressing SIRT4.

Proliferating mammalian cells use glutamine as a source of nitrogen and as a key anaplerotic source to provide metabolites to the tricarboxylic acid cycle (TCA) for biosynthesis. Recently, mammalian target of rapamycin complex 1 (mTORC1) activation has been correlated with increased nutrient uptake and metabolism, but no molecular connection to glutaminolysis has been reported. Here, we show that mTORC1 promotes glutamine anaplerosis by activating glutamate dehydrogenase (GDH). This regulation requires transcriptional repression of SIRT4, the mitochondrial-localized sirtuin that inhibits GDH. Mechanistically, mTORC1 represses SIRT4 by promoting the proteasome-mediated destabilization of cAMP-responsive element binding 2 (CREB2). Thus, a relationship between mTORC1, SIRT4, and cancer is suggested by our findings. Indeed, SIRT4 expression is reduced in human cancer, and its overexpression reduces cell proliferation, transformation, and tumor development. Finally, our data indicate that targeting nutrient metabolism in energy-addicted cancers with high mTORC1 signaling may be an effective therapeutic approach.

Engineering peroxisomal biosynthetic pathways for maximization of triterpene production in Yarrowia lipolytica.

Constructing efficient cell factories for product synthesis is frequently hampered by competing pathways and/or insufficient precursor supply. This is particularly evident in the case of triterpenoid biosynthesis in Yarrowia lipolytica, where squalene biosynthesis is tightly coupled to cytosolic biosynthesis of sterols essential for cell viability. Here, we addressed this problem by reconstructing the complete squalene biosynthetic pathway, starting from acetyl-CoA, in the peroxisome, thus harnessing peroxisomal acetyl-CoA pool and sequestering squalene synthesis in this organelle from competing cytosolic reactions. This strategy led to increasing the squalene levels by 1,300-fold relatively to native cytosolic synthesis. Subsequent enhancement of the peroxisomal acetyl-CoA supply by two independent approaches, 1) converting cellular lipid pool to peroxisomal acetyl-CoA and 2) establishing an orthogonal acetyl-CoA shortcut from CO2-derived acetate in the peroxisome, further significantly improved local squalene accumulation. Using these approaches, we constructed squalene-producing strains capable of yielding 32.8 g/L from glucose, and 31.6 g/L from acetate by employing a cofeeding strategy, in bioreactor fermentations. Our findings provide a feasible strategy for protecting intermediate metabolites that can be claimed by multiple reactions by engineering peroxisomes in Y. lipolytica as microfactories for the production of such intermediates and in particular acetyl-CoA-derived metabolites.

Engineering a universal and efficient platform for terpenoid synthesis in yeast.

Engineering microbes for the production of valuable natural products is often hindered by the regulation of native competing metabolic networks in host. This is particularly evident in the case of terpenoid synthesis in yeast, where the canonical terpenoid precursors are tightly coupled to the biosynthesis of sterols essential for yeast viability. One way to circumvent this limitation is by engineering product pathways less connected to the host native metabolism. Here, we introduce a two-step isopentenol utilization pathway (IUP) in Saccharomyces cerevisiae to augment the native mevalonate pathway by providing a shortcut to the synthesis of the common terpenoid precursors, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). As such, the IUP was capable of elevating the IPP/DMAPP pool by 147-fold compared with the native pathway. We further demonstrate that cofeeding isoprenol and prenol enhances geranyl diphosphate (GPP) content for monoterpene biosynthesis. More importantly, we established a synthetic three-step route for efficient synthesis of di-and tetraterpene precursor geranylgeranyl diphosphate (GGPP), circumventing the competition with farnesyl diphosphate (FPP) for sterol biosynthesis and elevating the GGPP level by 374-fold. We combine these IUP-supported precursor-forming platforms with downstream terpene synthases to harness their potential and improve the production of industrially relevant terpenoids by several fold. Our exploration provides a universal and effective platform for supporting terpenoid synthesis in yeast.

Engineering carbon source division of labor for efficient α-carotene production in Corynebacterium glutamicum.

Effective utilization of glucose, xylose, and acetate, common carbon sources in lignocellulose hydrolysate, can boost biomanufacturing economics. However, carbon leaks into biomass biosynthesis pathways instead of the intended target product remain to be optimized. This study aimed to enhance α-carotene production by optimizing glucose, xylose, and acetate utilization in a high-efficiency Corynebacterium glutamicum cell factory. Heterologous xylose pathway expression in C. glutamicum resulted in strain m4, exhibiting a two-fold increase in α-carotene production from xylose compared to glucose. Xylose utilization was found to boost the biosynthesis of pyruvate and acetyl-CoA, essential precursors for carotenoid biosynthesis. Additionally, metabolic engineering including pck, pyc, ppc, and aceE deletion, completely disrupted the metabolic connection between glycolysis and the TCA cycle, further enhancing α-carotene production. This strategic intervention directed glucose and xylose primarily towards target chemical production, while acetate supplied essential metabolites for cell growth recovery. The engineered strain C. glutamicum m8 achieved 30 mg/g α-carotene, 67% higher than strain m4. In fed-batch fermentation, strain m8 produced 1802 mg/L of α-carotene, marking the highest titer reported to date in microbial fermentation. Moreover, it exhibited excellent performance in authentic lignocellulosic hydrolysate, producing 216 mg/L α-carotene, 1.45 times higher than the initial strain (m4). These labor-division strategies significantly contribute to the development of clean processes for producing various valuable chemicals from lignocellulosic resources.

Proton export alkalinizes intracellular pH and reprograms carbon metabolism to drive normal and malignant cell growth.

Proton export is often considered a detoxifying process in animal cells, with monocarboxylate symporters coexporting excessive lactate and protons during glycolysis or the Warburg effect. We report a novel mechanism by which lactate/H+ export is sufficient to induce cell growth. Increased intracellular pH selectively activates catalysis by key metabolic gatekeeper enzymes HK1/PKM2/G6PDH, thereby enhancing glycolytic and pentose phosphate pathway carbon flux. The result is increased nucleotide levels, NADPH/NADP+ ratio, and cell proliferation. Simply increasing the lactate/proton symporter monocarboxylate transporter 4 (MCT4) or the sodium-proton antiporter NHE1 was sufficient to increase intracellular pH and give normal hematopoietic cells a significant competitive growth advantage in vivo. This process does not require additional cytokine triggers and is exploited in malignancy, where leukemogenic mutations epigenetically increase MCT4. Inhibiting MCT4 decreased intracellular pH and carbon flux and eliminated acute myeloid leukemia-initiating cells in mice without cytotoxic chemotherapy. Intracellular alkalization is a primitive mechanism by which proton partitioning can directly reprogram carbon metabolism for cell growth.

Biosynthesis of geranate via isopentenol utilization pathway in Escherichia coli.

Isoprenoids are a large family of natural products with diverse structures, which allow them to play diverse and important roles in the physiology of plants and animals. They also have important commercial uses as pharmaceuticals, flavoring agents, fragrances, and nutritional supplements. Recently, metabolic engineering has been intensively investigated and emerged as the technology of choice for the production of isoprenoids through microbial fermentation. Isoprenoid biosynthesis typically originates in plants from acetyl-coA in central carbon metabolism, however, a recent study reported an alternative pathway, the isopentenol utilization pathway (IUP), that can provide the building blocks of isoprenoid biosynthesis from affordable C5 substrates. In this study, we expressed the IUP in Escherichia coli to efficiently convert isopentenols into geranate, a valuable isoprenoid compound. We first established a geraniol-producing strain in E. coli that uses the IUP. Then, we extended the geraniol synthesis pathway to produce geranate through two oxidation reactions catalyzed by two alcohol/aldehyde dehydrogenases from Castellaniella defragrans. The geranate titer was further increased by optimizing the expression of the two dehydrogenases and also parameters of the fermentation process. The best strain produced 764 mg/L geranate in 24 h from 2 g/L isopentenols (a mixture of isoprenol and prenol). We also investigated if the dehydrogenases could accept other isoprenoid alcohols as substrates.

Pyruvate kinase isoform expression alters nucleotide synthesis to impact cell proliferation.

Metabolic regulation influences cell proliferation. The influence of pyruvate kinase isoforms on tumor cells has been extensively studied, but whether PKM2 is required for normal cell proliferation is unknown. We examine how PKM2 deletion affects proliferation and metabolism in nontransformed, nonimmortalized PKM2-expressing primary cells. We find that deletion of PKM2 in primary cells results in PKM1 expression and proliferation arrest. PKM1 expression, rather than PKM2 loss, is responsible for this effect, and proliferation arrest cannot be explained by cell differentiation, senescence, death, changes in gene expression, or prevention of cell growth. Instead, PKM1 expression impairs nucleotide production and the ability to synthesize DNA and progress through the cell cycle. Nucleotide biosynthesis is limiting, as proliferation arrest is characterized by severe thymidine depletion, and supplying exogenous thymine rescues both nucleotide levels and cell proliferation. Thus, PKM1 expression promotes a metabolic state that is unable to support DNA synthesis.

Insulin resistance rewires the metabolic gene program and glucose utilization in human white adipocytes.

BACKGROUND: In obesity, adipose tissue dysfunction resulting from excessive fat accumulation leads to systemic insulin resistance (IR), the underlying alteration of Type 2 Diabetes. The specific pathways dysregulated in dysfunctional adipocytes and the extent to which it affects adipose metabolic functions remain incompletely characterized. METHODS: We interrogated the transcriptional adaptation to increased adiposity in association with insulin resistance in visceral white adipose tissue from lean men, or men presenting overweight/obesity (BMI from 19 to 33) and discordant for insulin sensitivity. In human adipocytes in vitro, we investigated the direct contribution of IR in altering metabolic gene programming and glucose utilization using 13C-isotopic glucose tracing. RESULTS: We found that gene expression associated with impaired glucose and lipid metabolism and inflammation represented the strongest association with systemic insulin resistance, independently of BMI. In addition, we showed that inducing IR in mature human white adipocytes was sufficient to reprogram the transcriptional profile of genes involved in important metabolic functions such as glycolysis, the pentose phosphate pathway and de novo lipogenesis. Finally, we found that IR induced a rewiring of glucose metabolism, with higher incorporation of glucose into citrate, but not into downstream metabolites within the TCA cycle. CONCLUSIONS: Collectively, our data highlight the importance of obesity-derived insulin resistance in impacting the expression of key metabolic genes and impairing the metabolic processes of glucose utilization, and reveal a role for metabolic adaptation in adipose dysfunction in humans.

Aldehyde dehydrogenase 3a2 protects AML cells from oxidative death and the synthetic lethality of ferroptosis inducers.

Metabolic alterations in cancer represent convergent effects of oncogenic mutations. We hypothesized that a metabolism-restricted genetic screen, comparing normal primary mouse hematopoietic cells and their malignant counterparts in an ex vivo system mimicking the bone marrow microenvironment, would define distinctive vulnerabilities in acute myeloid leukemia (AML). Leukemic cells, but not their normal myeloid counterparts, depended on the aldehyde dehydrogenase 3a2 (Aldh3a2) enzyme that oxidizes long-chain aliphatic aldehydes to prevent cellular oxidative damage. Aldehydes are by-products of increased oxidative phosphorylation and nucleotide synthesis in cancer and are generated from lipid peroxides underlying the non-caspase-dependent form of cell death, ferroptosis. Leukemic cell dependence on Aldh3a2 was seen across multiple mouse and human myeloid leukemias. Aldh3a2 inhibition was synthetically lethal with glutathione peroxidase-4 (GPX4) inhibition; GPX4 inhibition is a known trigger of ferroptosis that by itself minimally affects AML cells. Inhibiting Aldh3a2 provides a therapeutic opportunity and a unique synthetic lethality to exploit the distinctive metabolic state of malignant cells.

Two-step pathway for isoprenoid synthesis.

Isoprenoids comprise a large class of chemicals of significant interest due to their diverse properties. Biological production of isoprenoids is considered to be the most efficient way for their large-scale production. Isoprenoid biosynthesis has thus far been dependent on pathways inextricably linked to glucose metabolism. These pathways suffer from inherent limitations due to their length, complex regulation, and extensive cofactor requirements. Here, we present a synthetic isoprenoid pathway that aims to overcome these limitations. This isopentenol utilization pathway (IUP) can produce isopentenyl diphosphate or dimethylallyl diphosphate, the main precursors to isoprenoid synthesis, through sequential phosphorylation of isopentenol isomers isoprenol or prenol. After identifying suitable enzymes and constructing the pathway, we attempted to probe the limits of the IUP for producing various isoprenoid downstream products. The IUP flux exceeded the capacity of almost all downstream pathways tested and was competitive with the highest isoprenoid fluxes reported.

Targeting pathway expression to subcellular organelles improves astaxanthin synthesis in Yarrowia lipolytica.

Metabolic engineering approaches for the production of high-value chemicals in microorganisms mostly use the cytosol as general reaction vessel. However, sequestration of enzymes and substrates, and metabolic cross-talk frequently prevent efficient synthesis of target compounds in the cytosol. Organelle compartmentalization in eukaryotic cells suggests ways for overcoming these challenges. Here we have explored this strategy by expressing the astaxanthin biosynthesis pathway in sub-organelles of the oleaginous yeast Yarrowia lipolytica. We first showed that fusion of the two enzymes converting ß-carotene to astaxanthin, ß-carotene ketolase and hydroxylase, performs better than the expression of individual enzymes. We next evaluated the pathway when expressed in compartments of lipid body, endoplasmic reticulum or peroxisome, individually and in combination. Targeting the astaxanthin pathway to subcellular organelles not only accelerated the conversion of ß-carotene to astaxanthin, but also significantly decreased accumulation of the ketocarotenoid intermediates. Anchoring enzymes simultaneously to all three organelles yielded the largest increase of astaxanthin synthesis, and ultimately produced 858 mg/L of astaxanthin in fed-batch fermentation (a 141-fold improvement over the initial strain). Our study is expected to help unlock the full potential of subcellular compartments and advance LB-based compartmentalized isoprenoid biosynthesis in Y. lipolytica.

Constructing an ethanol utilization pathway in Escherichia coli to produce acetyl-CoA derived compounds.

Engineering microbes to utilize non-conventional substrates could create short and efficient pathways to convert substrate into product. In this study, we designed and constructed a two-step heterologous ethanol utilization pathway (EUP) in Escherichia coli by using acetaldehyde dehydrogenase (encoded by ada) from Dickeya zeae and alcohol dehydrogenase (encoded by adh2) from Saccharomyces cerevisiae. This EUP can convert ethanol into acetyl-CoA without ATP consumption, and generate two molecules of NADH per molecule of ethanol. We optimized the expression of these two genes and found that ethanol consumption could be improved by expressing them in a specific order (ada-adh2) with a constitutive promoter (PgyrA). The engineered E. coli strain with EUP consumed approximately 8 g/L of ethanol in 96 h when it was used as sole carbon source. Subsequently, we combined EUP with the biosynthesis of polyhydroxybutyrate (PHB), a biodegradable polymer derived from acetyl-CoA. The engineered E. coli strain carrying EUP and PHB biosynthetic pathway produced 1.1 g/L of PHB from 10 g/L of ethanol and 1 g/L of aspartate family amino acids in 96 h. We also engineered a E. coli strain to produce 24 mg/L of prenol in an ethanol-containing medium, supporting the feasibility of converting ethanol into different classes of acetyl-CoA derived compounds.

Partitioning metabolism between growth and product synthesis for coordinated production of wax esters in Acinetobacter baylyi ADP1.

Microbial storage compounds, such as wax esters (WE), are potential high-value lipids for the production of specialty chemicals and medicines. Their synthesis, however, is strictly regulated and competes with cell growth, which leads to trade-offs between biomass and product formation. Here, we use metabolic engineering and synergistic substrate cofeeding to partition the metabolism of Acinetobacter baylyi ADP1 into two distinct modules, each dedicated to cell growth and WE biosynthesis, respectively. We first blocked the glyoxylate shunt and upregulated the WE synthesis pathway to direct the acetate substrate exclusively for WE synthesis, then we controlled the supply of gluconate so it could be used exclusively for cell growth and maintenance. We show that the two modules are functionally independent from each other, allowing efficient lipid accumulation while maintaining active cell growth. Our strategy resulted in 7.2- and 4.2-fold improvements in WE content and productivity, respectively, and the product titer was enhanced by 8.3-fold over the wild type strain. Notably, during a 24-h cultivation, a yield of 18% C-WE/C-total-substrates was achieved, being the highest reported for WE biosynthesis. This study provides a simple, yet powerful, means of controlling cellular operations and overcoming some of the fundamental challenges in microbial storage lipid production.

Transcriptional control of autophagy-lysosome function drives pancreatic cancer metabolism.

Activation of cellular stress response pathways to maintain metabolic homeostasis is emerging as a critical growth and survival mechanism in many cancers. The pathogenesis of pancreatic ductal adenocarcinoma (PDA) requires high levels of autophagy, a conserved self-degradative process. However, the regulatory circuits that activate autophagy and reprogram PDA cell metabolism are unknown. Here we show that autophagy induction in PDA occurs as part of a broader transcriptional program that coordinates activation of lysosome biogenesis and function, and nutrient scavenging, mediated by the MiT/TFE family of transcription factors. In human PDA cells, the MiT/TFE proteins--MITF, TFE3 and TFEB--are decoupled from regulatory mechanisms that control their cytoplasmic retention. Increased nuclear import in turn drives the expression of a coherent network of genes that induce high levels of lysosomal catabolic function essential for PDA growth. Unbiased global metabolite profiling reveals that MiT/TFE-dependent autophagy-lysosome activation is specifically required to maintain intracellular amino acid pools. These results identify the MiT/TFE proteins as master regulators of metabolic reprogramming in pancreatic cancer and demonstrate that transcriptional activation of clearance pathways converging on the lysosome is a novel hallmark of aggressive malignancy.

Monoterpenoid biosynthesis by engineered microbes.

Monoterpenoids are C10 isoprenoids and constitute a large family of natural products. They have been used as ingredients in food, cosmetics, and therapeutic products. Many monoterpenoids such as linalool, geraniol, limonene, and pinene are volatile and can be found in plant essential oils. Conventionally, these bioactive compounds are obtained from plant extracts by using organic solvents or by distillation method, which are costly and laborious if high-purity product is desired. In recent years, microbial biosynthesis has emerged as alternative source of monoterpenoids with great promise for meeting the increasing global demand for these compounds. However, current methods of production are not yet at levels required for commercialization. Production efficiency of monoterpenoids in microbial hosts is often restricted by high volatility of the monoterpenoids, a lack of enzymatic activity and selectivity, and/or product cytotoxicity to the microbial hosts. In this review, we summarize advances in microbial production of monoterpenoids over the past 3 years with particular focus on the key metabolic engineering strategies for different monoterpenoid products. We also provide our perspective on the promise of future endeavors to improve monoterpenoid productivity.

Loss of RBF1 changes glutamine catabolism.

Inactivation of the retinoblastoma tumor suppressor (pRB) alters the expression of a myriad of genes. To understand the altered cellular environment that these changes create, we took advantage of the Drosophila model system and used targeted liquid chromatography tandem mass spectrometry (LC-MS/MS) to profile the metabolic changes that occur when RBF1, the fly ortholog of pRB, is removed. We show that RBF1-depleted tissues and larvae are sensitive to fasting. Depletion of RBF1 causes major changes in nucleotide synthesis and glutathione metabolism. Under fasting conditions, these changes interconnect, and the increased replication demand of RBF1-depleted larvae is associated with the depletion of glutathione pools. In vivo (13)C isotopic tracer analysis shows that RBF1-depleted larvae increase the flux of glutamine toward glutathione synthesis, presumably to minimize oxidative stress. Concordantly, H(2)O(2) preferentially promoted apoptosis in RBF1-depleted tissues, and the sensitivity of RBF1-depleted animals to fasting was specifically suppressed by either a glutamine supplement or the antioxidant N-acetyl-cysteine. Effects of pRB activation/inactivation on glutamine catabolism were also detected in human cell lines. These results show that the inactivation of RB proteins causes metabolic reprogramming and that these consequences of RBF/RB function are present in both flies and human cell lines.

Engineering Yarrowia lipolytica for the utilization of acid whey.

Acid whey, a byproduct in cheese and yogurt production, demands high costs in disposal at large quantities. Nonetheless, it contains abundant sugars and nutrients that can potentially be utilized by microorganisms. Here we report a novel platform technology that converts acid whey into value-added products using Yarrowia lipolytica. Since wild type strains do not assimilate lactose, a major carbon source in whey, a secreted ß-galactosidase was introduced. Additionally, to accelerate galactose metabolism, we overexpressed the relevant native four genes of the Leloir pathway. The engineered strain could achieve rapid total conversion of all carbon sources in acid whey, producing 6.61 g/L of fatty acids (FAs) with a yield of 0.146 g-FAs/g-substrates. Further engineering to introduce an omega-3 desaturase enabled the synthesis of α-linolenic acid from acid whey, producing 10.5 mg/gDCW within a short fermentation time. Finally, PEX10 knockout in our platform strain was shown to minimize hyphal formation in concentrated acid whey cultures, greatly improving fatty acid content. These results demonstrate the feasibility of using acid whey as a previously untapped resource for biotechnology.

Enhancing isoprenoid synthesis in Yarrowia lipolytica by expressing the isopentenol utilization pathway and modulating intracellular hydrophobicity.

The abundant supply of biosynthetic precursors and product compatibility with the intracellular environment play important roles for microbial isoprenoid production. In this study, we tailor to both of these requirements by introducing the two-step isopentenol utilization pathway (IUP) to augment the native pathway in the oleaginous yeast Yarrowia lipolytica. With shortcut access to the common isoprenoid precursor, isopentenyl pyrophosphate (IPP) and its isomer dimethylallyl pyrophosphate (DMAPP), IUP is capable of elevating IPP + DMAPP levels by 15.7-fold compared to the mevalonate pathway alone. The increase in IPP + DMAPP levels can directly lead to better isoprenoid synthesis, which is illustrated using lycopene as a model compound. Moreover, we also demonstrate that higher lipid contents in the cells correlate with improved intracellular lycopene production, suggesting the importance of having a substantial hydrophobic environment to sequester isoprenoids. Combining these strategies with further genetic and fermentation optimizations, we achieved a final lycopene titer of 4.2 g/L. Overall, these strategies hold great potential for strengthening the synthesis of long-chain isoprenoids and fat-soluble natural products in microbes.

Using biopolymer bodies for encapsulation of hydrophobic products in bacterium.

Producing some small hydrophobic molecules in microbes is challenging. Often these molecules cannot cross membranes, and thus their production may be limited by lack of storage space in the producing organism. This study reports a new technology for in vivo storage of valuable hydrophobic products in/on biopolymer bodies in Escherichia coli. A biodegradable and biocompatible polyester - poly (3-hydroxybutyrate) (PHB) - was selected as the intracellular storage vessel to encapsulate lycopene, which is a chromogenic model compound. The hydrophobic interaction between lycopene and PHB was verified by using in vitro binding test and sucrose density gradient centrifugation. Further in vivo characterization was performed by using Confocal Laser Scanning Microscopy (CLSM). The images validated the in vivo co-localization between PHB granules and lycopene. The images also showed that lycopene aggregated in bacteria that did not produce PHB, which may challenge the commonly accepted hypothesis that most lycopene molecules are stored in cell membranes of recombinant host. We also confirmed that producing PHB did not negatively affect lycopene biosynthesis in the E. coli strains and collected data suggesting that PHB titer and lycopene titer were positively correlated when the cells were engineered to co-produce them. The biopolymers that encapsulated hydrophobic molecules could have many useful applications, especially in controlled release because the polymers are biodegradable, and the encapsulated products would be released during the polymer degradation.