Pentose Phosphate Pathway Genes Research Articles

The Roles of White Adipose Tissue and Liver NADPH in Dietary Restriction-Induced Longevity

Dietary restriction (DR) protocols frequently employ intermittent fasting. Following a period of fasting, meal consumption increases lipogenic gene expression, including that of NADPH-generating enzymes that fuel lipogenesis in white adipose tissue (WAT) through the induction of transcriptional regulators SREBP-1c and CHREBP. SREBP-1c knockout mice, unlike controls, did not show an extended lifespan on the DR diet. WAT cytoplasmic NADPH is generated by both malic enzyme 1 (ME1) and the pentose phosphate pathway (PPP), while liver cytoplasmic NADPH is primarily synthesized by folate cycle enzymes provided one-carbon units through serine catabolism. During the daily fasting period of the DR diet, fatty acids are released from WAT and are transported to peripheral tissues, where they are used for beta-oxidation and for phospholipid and lipid droplet synthesis, where monounsaturated fatty acids (MUFAs) may activate Nrf1 and inhibit ferroptosis to promote longevity. Decreased WAT NADPH from PPP gene knockout stimulated the browning of WAT and protected from a high-fat diet, while high levels of NADPH-generating enzymes in WAT and macrophages are linked to obesity. But oscillations in WAT [NADPH]/[NADP+] from feeding and fasting cycles may play an important role in maintaining metabolic plasticity to drive longevity. Studies measuring the WAT malate/pyruvate as a proxy for the cytoplasmic [NADPH]/[NADP+], as well as studies using fluorescent biosensors expressed in the WAT of animal models to monitor the changes in cytoplasmic [NADPH]/[NADP+], are needed during ad libitum and DR diets to determine the changes that are associated with longevity.

The Roles of White Adipose Tissue and Liver NADPH in Dietary Restriction-Induced Longevity

Dietary restriction (DR) protocols frequently employ intermittent fasting. Following a period of fasting, meal consumption increases lipogenic gene expression, including that of NADPH-generating enzymes that fuel lipogenesis in white adipose tissue (WAT) through the induction of transcriptional regulators SREBP-1c and CHREBP. SREBP-1c knockout mice, unlike controls, did not show an extended lifespan on the DR diet. WAT cytoplasmic NADPH is generated by both malic enzyme 1 (ME1) and the pentose phosphate pathway (PPP), while liver cytoplasmic NADPH is primarily synthesized by folate cycle enzymes provided one-carbon units through serine catabolism. During the daily fasting period of the DR diet, fatty acids are released from WAT and are transported to peripheral tissues, where they are used for beta-oxidation and for phospholipid and lipid droplet synthesis, where monounsaturated fatty acids (MUFAs) may activate Nrf1 and inhibit ferroptosis to promote longevity. Decreased WAT NADPH from PPP gene knockout stimulated the browning of WAT and protected from a high-fat diet, while high levels of NADPH-generating enzymes in WAT and macrophages are linked to obesity. But oscillations in WAT [NADPH]/[NADP+] from feeding and fasting cycles may play an important role in maintaining metabolic plasticity to drive longevity. Studies measuring the WAT malate/pyruvate as a proxy for the cytoplasmic [NADPH]/[NADP+], as well as studies using fluorescent biosensors expressed in the WAT of animal models to monitor the changes in cytoplasmic [NADPH]/[NADP+], are needed during ad libitum and DR diets to determine the changes that are associated with longevity.

New regulatory role of Znf1 in transcriptional control of pentose phosphate pathway and ATP synthesis for enhanced isobutanol and acid tolerance.

To develop a cost-effective microbial cell factory for the production of biofuels and biochemicals, an understanding of tolerant mechanisms is vital for the construction of robust host strains. Here, we characterized a new function of a key metabolic transcription factor named Znf1 and its involvement in stress response in Saccharomyces cerevisiae to enhance tolerance to advanced biofuel, isobutanol. RNA-sequencing analysis of the wild-type versus the znf1Δ deletion strains in glucose revealed a new role for transcription factor Znf1 in the pentose phosphate pathway (PPP) and energy generation. The gene expression analysis confirmed that isobutanol induces an adaptive cell response, resulting in activation of ATP1-3 and COX6 expression. These genes were Znf1 targets that belong to the electron transport chain, important to produce ATPs. Znf1 also activated PPP genes, required for the generation of key amino acids, cellular metabolites, and maintenance of NADP/NADPH redox balance. In glucose, Znf1 also mediated the upregulation of valine biosynthetic genes of the Ehrlich pathway, namely ILV3, ILV5, and ARO10, associated with the generation of key intermediates for isobutanol production.Using S. cerevisiae knockout collection strains, cells with deleted transcriptional regulatory gene ZNF1 or its targets displayed hypersensitivity to isobutanol and acid inhibitors; in contrast, overexpression of ZNF1 enhanced cell survival. Thus, the transcription factor Znf1 functions in the maintenance of energy homeostasis and redox balance at various checkpoints of yeast metabolic pathways. It ensures the rapid unwiring of gene transcription in response to toxic products/by-products generated during biofuel production. Importantly, we provide a new approach to enhance strain tolerance during the conversion of glucose to biofuels.

Efficient production of mycosporine-like amino acids, natural sunscreens, in Yarrowia lipolytica

BackgroundMycosporine-like amino acids (MAAs), including shinorine and porphyra-334, are gaining attention as safe natural sunscreens. The production of MAAs has been achieved in diverse microbial hosts, including Saccharomyces cerevisiae. While S. cerevisiae is the most extensively studied model yeast, the oleaginous yeast Yarrowia lipolytica has emerged as a promising candidate for the synthesis of valuable products. In this study, we explored the potential of Y. lipolytica as a host for producing MAAs, utilizing its advantages such as a robust pentose phosphate pathway flux and versatile carbon source utilization.ResultsWe produced MAAs in Y. lipolytica by introducing the MAA biosynthetic genes from cyanobacteria Nostoc punctiforme and Anabaena variabilis. These genes include mysA, mysB, and mysC responsible for producing mycosporine-glycine (MG) from sedoheptulose 7-phosphate (S7P). The two strains utilize different enzymes, D-Ala-D-Ala ligase homologue (MysD) in N. punctiforme and NRPS-like enzyme (MysE) in A. variabilis, for amino acid conjugation to MG. MysE specifically generated shinorine, a serine conjugate of MG, while MysD exhibited substrate promiscuity, yielding both shinorine and a small amount of porphyra-334, a threonine conjugate of MG. We enhanced MAAs production by selecting mysA, mysB, and mysC from A. variabilis and mysD from N. punctiforme based on their activities. We further improved production by strengthening promoters, increasing gene copies, and introducing the xylose utilization pathway. Co-utilization of xylose with glucose or glycerol increased MAAs production by boosting the S7P pool through the pentose phosphate pathway. Overexpressing GND1 and ZWF1, key genes in the pentose phosphate pathway, further enhanced MAAs production. The highest achieved MAAs level was 249.0 mg/L (207.4 mg/L shinorine and 41.6 mg/L of porphyra-334) in YP medium containing 10 g/L glucose and 10 g/L xylose.ConclusionsY. lipolytica was successfully engineered to produce MAAs, primarily shinorine. This achievement involved the introduction of MAA biosynthetic genes from cyanobacteria, establishing xylose utilizing pathway, and overexpressing the pentose phosphate pathway genes. These results highlight the potential of Y. lipolytica as a promising yeast chassis strain for MAAs production, notably attributed to its proficient expression of MysE enzyme, which remains non-functional in S. cerevisiae, and versatile utilization of carbon sources like glycerol.

Temporal changes in glucose metabolism reflect polarization in resident and monocyte-derived macrophages after myocardial infarction.

Metabolic reprogramming from glycolysis to the mitochondrial tricarboxylic acid (TCA) cycle and oxidative phosphorylation may mediate macrophage polarization from the pro-inflammatory M1 to the anti-inflammatory M2 phenotype. We hypothesized that changes in cardiac macrophage glucose metabolism would reflect polarization status after myocardial infarction (MI), ranging from the early inflammatory phase to the later wound healing phase. MI was induced by permanent ligation of the left coronary artery in adult male C57BL/6J mice for 1 (D1), 3 (D3), or 7 (D7) days. Infarct macrophages were subjected to metabolic flux analysis or gene expression analysis. Monocyte versus resident cardiac macrophage metabolism was assessed using mice lacking the Ccr2 gene (CCR2 KO). By flow cytometry and RT-PCR, D1 macrophages exhibited an M1 phenotype while D7 macrophages exhibited an M2 phenotype. Macrophage glycolysis (extracellular acidification rate) was increased at D1 and D3, returning to basal levels at D7. Glucose oxidation (oxygen consumption rate) was decreased at D3, returning to basal levels at D7. At D1, glycolytic genes were elevated (Gapdh, Ldha, Pkm2), while TCA cycle genes were elevated at D3 (Idh1 and Idh2) and D7 (Pdha1, Idh1/2, Sdha/b). Surprisingly, Slc2a1 and Hk1/2 were increased at D7, as well as pentose phosphate pathway (PPP) genes (G6pdx, G6pd2, Pgd, Rpia, Taldo1), indicating increased PPP activity. Macrophages from CCR2 KO mice showed decreased glycolysis and increased glucose oxidation at D3, and decreases in Ldha and Pkm2 expression. Administration of dichloroacetate, a pyruvate dehydrogenase kinase inhibitor, robustly decreased pyruvate dehydrogenase phosphorylation in the non-infarcted remote zone, but did not affect macrophage phenotype or metabolism in the infarct zone. Our results indicate that changes in glucose metabolism and the PPP underlie macrophage polarization following MI, and that metabolic reprogramming is a key feature of monocyte-derived but not resident macrophages.

ATP13A2 activates the pentose phosphate pathway to promote colorectal cancer growth though TFEB-PGD axis.

The pentose phosphate pathway (PPP) is an important mechanism by which tumour cells resist stressful environments and maintain malignant proliferation. However, the mechanism by which the PPP regulates these processes in colorectal cancer (CRC) remains elusive. Closely related PPP genes were obtained from the TCGA and GEO databases. The effect of ATP13A2 on CRC cell proliferation was evaluated by performing in vitro assays. The connection between the PPP and ATP13A2 was explored by assessing proliferation and antioxidative stress. The molecular mechanism by which ATP13A2 regulates the PPP was investigated using chromatin immunoprecipitation and dual luciferase experiments. The clinical therapeutic potential of ATP13A2 was explored using patient-derived xenograft (PDX), patient-derived organoid (PDO) and AOM/DSS models. We identified ATP13A2 as a novel PPP-related gene. ATP13A2 deficiency inhibited CRC growth and PPP activity, as manifested by a decrease in the levels of PPP products and an increase in reactive oxygen species levels, whereas ATP13A2 overexpression induced the opposite effect. Mechanistically, ATP13A2 regulated the PPP mainly by affecting phosphogluconate dehydrogenase (PGD) mRNA expression. Subsequent studies showed that ATP13A2 overexpression promoted TFEB nuclear localization by inhibiting the phosphorylation of TFEB, thereby enhancing the transcription of PGD and ultimately affecting the activity of the PPP. Finally, ATP13A2 knockdown inhibited CRC growth in PDO and PDX models. ATP13A2- /- mice had a lower CRC growth capacity than ATP13A2+/+ in the AOM/DSS model.Our findings revealed that ATP13A2 overexpression-driven dephosphorylation of TFEB promotes PPP activation by increasing PGD transcription, suggesting that ATP13A2 may serve as a potential target for CRC therapy.

Engineering and adaptive laboratory evolution of Escherichia coli for improving methanol utilization based on a hybrid methanol assimilation pathway.

Engineering Escherichia coli for efficient methanol assimilation is important for developing methanol as an emerging next-generation feedstock for industrial biotechnology. While recent attempts to engineer E. coli as a synthetic methylotroph have achieved great success, most of these works are based on the engineering of the prokaryotic ribulose monophosphate (RuMP) pathway. In this study, we introduced a hybrid methanol assimilation pathway which consists of prokaryotic methanol dehydrogenase (Mdh) and eukaryotic xylulose monophosphate (XuMP) pathway enzyme dihydroxyacetone synthase (Das) into E. coli and reprogrammed E. coli metabolism to improve methanol assimilation by combining rational design and adaptive laboratory evolution. By deletion and down-regulation of key genes in the TCA cycle and glycolysis to increase the flux toward the cyclic XuMP pathway, methanol consumption and the assimilation of methanol to biomass were significantly improved. Further improvements in methanol utilization and cell growth were achieved via adaptive laboratory evolution and a final evolved strain can grow on methanol with only 0.1g/L yeast extract as co-substrate. 13C-methanol labeling assay demonstrated significantly higher labeling in intracellular metabolites in glycolysis, TCA cycle, pentose phosphate pathway, and amino acids. Transcriptomics analysis showed that the expression of fba, dhak, and part of pentose phosphate pathway genes were highly up-regulated, suggesting that the rational engineering strategies and adaptive evolution are effective for activating the cyclic XuMP pathway. This study demonstrated the feasibility and provided new strategies to construct synthetic methylotrophy of E. coli based on the hybrid methanol assimilation pathway with Mdh and Das.

Comparative Genome Analysis of a Novel Alkaliphilic Actinobacterial Species Nesterenkonia haasae.

In the present study, a comparative genome analysis of the novel alkaliphilic actinobacterial Nesterenkonia haasae with other members of the genus Nesterenkonia was performed. The genome size of Nesterenkonia members ranged from 2,188,008 to 3,676,111 bp. N. haasae and Nesterenkonia members of the present study encode the essential glycolysis and pentose phosphate pathway genes. In addition, some Nesterenkonia members encode the crucial genes for Entner-Doudoroff pathways. Some Nesterenkonia members possess the genes responsible for sulfate/thiosulfate transport system permease protein/ ATP-binding protein and conversion of sulfate to sulfite. Nesterenkonia members also encode the genes for assimilatory nitrate reduction, nitrite reductase, and the urea cycle. All Nesterenkonia members have the genes to overcome environmental stress and produce secondary metabolites. The present study helps to understand N. haasae and Nesterenkonia members' environmental adaptation and niches specificity based on their specific metabolic properties. Further, based on genome analysis, we propose reclassifying Nesterenkonia jeotgali as a later heterotypic synonym of Nesterenkonia sandarakina.

Nutrition promotion of brewer's spent grain by symbiotic fermentation adding Bacillus velezensis and Levilactobacillus brevis

Brewer's spent grain (BSG) is the main byproduct of the beer industry and is commonly used as a low-value animal feed or food ingredient. This study investigated the role of symbiotic fermentation with Bacillus velezensis and Levilactobacillus brevis in improving the nutritional value of BSG. The addition of B. velezensis increased the cell growth of L. brevis by over thirty-fold higher than that in the single-strain system. Simultaneously, cellulase and protease activities were substantially increased, and more soluble sugars were produced. Total amino acid, glutamic acid, and γ-aminobutyric acid (GABA) levels increased by 52.2%, 155.1%, and 144.1%, respectively. Conversely, lactic acid production decreased. Genetic analysis showed that the glycolysis-related gene pykA, pentose phosphate pathway gene gnd, and GABA-related gene gad were upregulated, while the heterolactin-related gene xpkA was downregulated, explaining that B. velezensis might inhibit heterolactin fermentation while enhancing GABA biosynthesis. This study provides a potential strategy for the high-value utilization of BSG in the food or feed industry.

Metabolic Reprogramming Mediates Macrophage Polarization After Myocardial Infarction

BackgroundMacrophages play a critical role in left ventricular (LV) remodeling after myocardial infarction (MI), and limiting macrophage‐mediated inflammatory responses while enhancing reparative roles is a promising therapeutic strategy. Monocyte‐derived M1 macrophages mediate the early inflammatory response, while M2 macrophages mediate the later wound healing and scar formation phase. Metabolic reprogramming from glycolysis to mitochondrial oxidative phosphorylation mediates M1 to M2 polarization. We hypothesized that metabolic reprogramming occurs in macrophages over the course of post‐MI remodeling, and that blocking macrophage glycolysis attenuates post‐MI inflammation.MethodsMI was induced in adult (16‐20 week old) male C57BL/6J mice by permanent ligation of the left coronary artery for 1, 3, or 7 days, and cardiac function was assessed by echocardiography. Macrophages were extracted from the LV infarct area by immunomagnetic sorting. Macrophage metabolic flux was assessed by Extracellular Flux Analysis (Seahorse); upon glucose administration, glycolysis was measured by extracellular acidification rate (ECAR) while glucose oxidation was measured by oxygen consumption (OCR). Leukocyte subtypes (neutrophils, monocytes, resident macrophages) were assessed by flow cytometry. Real‐time PCR was used to assess gene expression by relative mRNA levels. To specifically block macrophage glycolysis, LysMCre mice were crossed with mice floxed for Slc2a1, the major macrophage glucose transporter, to generate macrophage‐Slc2a1 null mice.ResultsMI led to LV contractile dysfunction (decreased ejection fraction; days 1, 3, and 7 versus day 0), thinning of the infarct wall (days 1, 3, and 7) and non‐infarct wall (day 7), and dilation (increased internal diameter at diastole and end‐diastolic volume; day 7). At day 1, increased neutrophils (Ly6G+) and monocytes (Ly6Chi) were observed in the infarcted LV; at day 7, neutrophils and monocytes returned to baseline levels and the majority of macrophages were resident M2 subtype (Ly6Clow versus day 1). Day 1 macrophages showed elevated glycolysis (ECAR versus day 0) and increased mRNA levels of the M1 marker Il1b (versus day 3 and 7); day 3 macrophages showed increased glycolysis (ECAR versus day 0) and decreased glucose oxidation (versus day 0); day 7 macrophages showed decreased glycolysis (ECAR versus day 1 and 3) and increased glucose oxidation (OCR versus day 1 and 3), and increased Slc2a1 mRNA, Krebs cycle genes (Pdha1, Idh1/2, Sdha/bmRNA), pentose phosphate pathway genes (G6pd2/x, Pgd, Rpia, Taldo1 mRNA), and Il10 (M2 marker) mRNA. In macrophage‐Slc2a1 null mice, macrophage glycolysis was decreased and glucose oxidation increased at day 3, and infarct levels of Il1b were decreased compared to floxed controls.ConclusionsOur results indicate that time‐dependent macrophage metabolic reprogramming occurs over the MI remodeling process, and that blocking macrophage glycolysis may be a promising strategy for limiting post‐MI inflammation and adverse remodeling.

Increasing NADPH Availability for Xylitol Production via Pentose-Phosphate-Pathway Gene Overexpression and Embden-Meyerhof-Parnas-Pathway Gene Deletion in Escherichia coli.

Cofactor availability is often a rate-limiting factor in the bioconversion of xylose to xylitol. The overexpression of pentose phosphate pathway genes and the deletion of Embden-Meyerhof-Parnas pathway genes can modulate the glucose metabolic flux and increase the intracellular NADPH supply, enabling Escherichia coli cells to produce xylitol from corncob hydrolysates. The effects of zwf and/or gnd overexpression and pfkA, pfkB, and/or pgi deletion on the intracellular redox environment and xylitol production were examined. The NADPH-enhanced strain 2bpgi produced 162 g/L xylitol from corncob hydrolysates after a 76 h fed-batch fermentation in a 15 L bioreactor, which was 13.3% greater than the 143 g/L xylitol produced by the IS5-d control strain. Additionally, the xylitol productivity and xylitol yield per glucose for 2bpgi were 2.13 g/L/h and 2.50 g/g, respectively. Thus, the genetic modifications in 2bpgi significantly enhanced NADPH regeneration, making 2bpgi a potentially useful strain for the industrial-scale production of xylitol from detoxified corncob hydrolysates.

Improving the Production of Riboflavin by Introducing a Mutant Ribulose 5-Phosphate 3-Epimerase Gene in Bacillus subtilis.

Ribulose 5-phosphate (Ru5P) and guanosine 5′-triphosphate (GTP) are two key precursors of riboflavin, whereby Ru5P is also a precursor of GTP. Ribulose 5-phosphate 3-epimerase (Rpe) catalyzes the conversion of ribulose 5-phosphate into xylulose 5-phosphate. Inactivation of Rpe can reduce the consumption of Ru5P, enhancing the carbon flux toward riboflavin biosynthesis. Here we investigated the effect of mutation of rpe and other related genes on riboflavin production, physiological and metabolic phenotypes in Bacillus subtilis LY (BSLY). Introducing single nucleotide deletion (generated BSR) or nonsense mutation (generated BSRN) on the genomic copy of rpe, resulting in more than fivefold increase of riboflavin production over the parental strain. BSR process 62% Rpe activity, while BSRN lost the entire Rpe activity and had a growth defect compared with the parent strain. BSR and BSRN exhibited increases of the inosine and guanine titers, in addition, BSRN exhibited an increase of inosine 5′-monophosphate titer in fermentation. The transcription levels of most oxidative pentose phosphate pathway and purine synthesis genes were unchanged in BSR, except for the levels of zwf and ndk, which were higher than in BSLY. The production of riboflavin was increased to 479.90 ± 33.21 mg/L when ribA was overexpressed in BSR. The overexpression of zwf, gntZ, prs, and purF also enhanced the riboflavin production. Finally, overexpression of the rib operon by the pMX45 plasmid and mutant gnd by pHP03 plasmid in BSR led to a 3.05-fold increase of the riboflavin production (977.29 ± 63.44 mg/L), showing the potential for further engineering of this strain.

Nrf2 deficiency decreases NADPH from impaired IDH shuttle and pentose phosphate pathway in retinal pigmented epithelial cells to magnify oxidative stress-induced mitochondrial dysfunction.

The nuclear factor‐erythroid 2‐related factor‐2 (Nrf2), a major antioxidant transcription factor, is decreased in several age‐related diseases including age‐related macular degeneration (AMD), the most common cause of blindness among the elderly in western society. Since Nrf2’s mito‐protective response is understudied, we investigated its antioxidant response on mitochondria. Control and Nrf2‐deficient retinal pigmented epithelial (RPE) cells were compared after treating with cigarette smoke extract (CSE). Mitochondrial antioxidant abundance and reactive oxygen species (ROS) were quantified. Mitochondrial function was assessed by TMRM assay, NADPH, electron transport chain activity, and Seahorse. Results were corroborated in Nrf2−/− mice and relevance to AMD was provided by immunohistochemistry of human globes. CSE induced mitochondrial ROS to impair mitochondrial function. H2O2 increase in particular, was magnified by Nrf2 deficiency, and corresponded with exaggerated mitochondrial dysfunction. While Nrf2 did not affect mitochondrial antioxidant abundance, oxidized PRX3 was magnified by Nrf2 deficiency due to decreased NADPH from decreased expression of IDH2 and pentose phosphate pathway (PPP) genes. With severe CSE stress, intrinsic apoptosis was activated to increase cell death. PPP component TALDO1 immunolabeling was decreased in dysmorphic RPE of human AMD globes. Despite limited regulation of mitochondrial antioxidant expression, Nrf2 influences PPP and IDH shuttle activity that indirectly supplies NADPH for the TRX2 system. These results provide insight into how Nrf2 deficiency impacts the mitochondrial antioxidant response, and its role in AMD pathobiology.

Transcriptomic Changes Induced by Deletion of Transcriptional Regulator GCR2 on Pentose Sugar Metabolism in Saccharomyces cerevisiae.

Being a microbial host for lignocellulosic biofuel production, Saccharomyces cerevisiae needs to be engineered to express a heterologous xylose pathway; however, it has been challenging to optimize the engineered strain for efficient and rapid fermentation of xylose. Deletion of PHO13 (Δpho13) has been reported to be a crucial genetic perturbation in improving xylose fermentation. A confirmed mechanism of the Δpho13 effect on xylose fermentation is that the Δpho13 transcriptionally activates the genes in the non-oxidative pentose phosphate pathway (PPP). In the current study, we found a couple of engineered strains, of which phenotypes were not affected by Δpho13 (Δpho13-negative), among many others we examined. Genome resequencing of the Δpho13-negative strains revealed that a loss-of-function mutation in GCR2 was responsible for the phenotype. Gcr2 is a global transcriptional factor involved in glucose metabolism. The results of RNA-seq confirmed that the deletion of GCR2 (Δgcr2) led to the upregulation of PPP genes as well as downregulation of glycolytic genes, and changes were more significant under xylose conditions than those under glucose conditions. Although there was no synergistic effect between Δpho13 and Δgcr2 in improving xylose fermentation, these results suggested that GCR2 is a novel knockout target in improving lignocellulosic ethanol production.

Minimize the Xylitol Production in Saccharomyces cerevisiae by Balancing the Xylose Redox Metabolic Pathway.

Xylose is the second most abundant sugar in lignocellulose, but it cannot be used as carbon source by budding yeast Saccharomyces cerevisiae. Rational promoter elements engineering approaches were taken for efficient xylose fermentation in budding yeast. Among promoters surveyed, HXT7 exhibited the best performance. The HXT7 promoter is suppressed in the presence of glucose and derepressed by xylose, making it a promising candidate to drive xylose metabolism. However, simple ectopic expression of both key xylose metabolic genes XYL1 and XYL2 by the HXT7 promoter resulted in massive accumulation of the xylose metabolic byproduct xylitol. Through the HXT7-driven expression of a reported redox variant, XYL1-K270R, along with optimized expression of XYL2 and the downstream pentose phosphate pathway genes, a balanced xylose metabolism toward ethanol formation was achieved. Fermented in a culture medium containing 50 g/L xylose as the sole carbon source, xylose is nearly consumed, with less than 3 g/L xylitol, and more than 16 g/L ethanol production. Hence, the combination of an inducible promoter and redox balance of the xylose utilization pathway is an attractive approach to optimizing fuel production from lignocellulose.

Engineered Pseudomonas putida simultaneously catabolizes five major components of corn stover lignocellulose: Glucose, xylose, arabinose, p-coumaric acid, and acetic acid

Valorization of all major lignocellulose components, including lignin, cellulose, and hemicellulose is critical for an economically viable bioeconomy. In most biochemical conversion approaches, the standard process separately upgrades sugar hydrolysates and lignin. Here, we present a new process concept based on an engineered microbe that could enable simultaneous upgrading of all lignocellulose streams, which has the ultimate potential to reduce capital cost and enable new metabolic engineering strategies. Pseudomonas putida is a robust microorganism capable of natively catabolizing aromatics, organic acids, and D-glucose. We engineered this strain to utilize D-xylose by tuning expression of a heterologous D-xylose transporter, catabolic genes xylAB, and pentose phosphate pathway (PPP) genes tal-tkt. We further engineered L-arabinose utilization via the PPP or an oxidative pathway. This resulted in a growth rate on xylose and arabinose of 0.32 h−1 and 0.38 h−1, respectively. Using the oxidative L-arabinose pathway with the PPP xylose pathway enabled D-glucose, D-xylose, and L-arabinose co-utilization in minimal medium using model compounds as well as real corn stover hydrolysate, with a maximum hydrolysate sugar consumption rate of 3.3 g/L/h. After modifying catabolite repression, our engineered P. putida simultaneously co-utilized five representative compounds from cellulose (D-glucose), hemicellulose (D-xylose, L-arabinose, and acetic acid), and lignin-related compounds (p-coumarate), demonstrating the feasibility of simultaneously upgrading total lignocellulosic biomass to value-added chemicals.

Drosophila estrogen-related receptor directs a transcriptional switch that supports adult glycolysis and lipogenesis.

Metabolism and development must be closely coupled to meet the changing physiological needs of each stage in the life cycle. The molecular mechanisms that link these pathways, however, remain poorly understood. Here we show that the Drosophila estrogen-related receptor (dERR) directs a transcriptional switch in mid-pupae that promotes glucose oxidation and lipogenesis in young adults. dERR mutant adults are viable but display reduced locomotor activity, susceptibility to starvation, elevated glucose, and an almost complete lack of stored triglycerides. Molecular profiling by RNA-seq, ChIP-seq, and metabolomics revealed that glycolytic and pentose phosphate pathway genes are induced by dERR, and their reduced expression in mutants is accompanied by elevated glycolytic intermediates, reduced TCA cycle intermediates, and reduced levels of long chain fatty acids. Unexpectedly, we found that the central pathways of energy metabolism, including glycolysis, the tricarboxylic acid cycle, and electron transport chain, are coordinately induced at the transcriptional level in mid-pupae and maintained into adulthood, and this response is partially dependent on dERR, leading to the metabolic defects observed in mutants. Our data support the model that dERR contributes to a transcriptional switch during pupal development that establishes the metabolic state of the adult fly.

Recombinant xylose-fermenting yeast construction for the co-production of ethanol and cis,cis-muconic acid from lignocellulosic biomass

New exogenous cis,cis-muconic acid biosynthetic pathway genes were expressed in Saccharomyces cerevisiae. The xylose isomerase gene from Bacteroides valgutus and pentose phosphate pathway genes from S. cerevisiae were overexpressed in the yeast strain. The strain was further modified by the overexpression of gene Aro1 (with a stop codon of AroE) and a feedback-resistant Aro4opt mutant gene from S. cerevisiae. Under oxygen-limited conditions, it produced 65 mg/L cis,cis-muconic acid from xylose. Co-fermentation of 88 g/L glucose and 50 g/L xylose generated 54 g/L ethanol and 248 mg/L cis,cis-muconic acid. Under aerobic conditions, muconic acid titer reached 424 mg/L. With the supplement of 1 g/L catechol, 1286 mg/L muconic acid was produced. Fermentation of an oil palm empty fruit bunch hydrolysate resulted in 31.3 g/L ethanol and 53.4 mg/L muconic acid. This is the first report on the production of muconic acid from lignocellulosic biomass hydrolysate using a recombinant xylose-fermenting yeast.

Transcriptomic profiling sheds light on the blue-light and red-light response of oyster mushroom (Pleurotus ostreatus)

Blue light is an important environmental factor that induces mushroom primordium differentiation and fruiting body development. Although blue-light treatment has been applied for the production of oyster mushroom (Pleurotus ostreatus), the blue-light response mechanisms of P. ostreatus still remain unclear. In the present study, we exposed the primordium of P. ostreatus to blue-light, red-light, and dark conditions for 7 days. Subsequently, comparative transcriptomics analysis of the stipe, pileus, and gill under the three light conditions was performed to reveal the gene expression response mechanism of P. ostreatus to blue light and red light. The results showed that blue light enhanced the growth and development of all the three organs of P. ostreatus, especially the pileus. In contrast, red light slightly (non-significantly) inhibited pileus growth. When compared with red-light and dark treatments, blue-light treatment significantly upregulated gene expression involved in glycolysis/gluconeogenesis, the pentose phosphate pathway and the peroxisome in the pileus, but not in the gill or stipe. Most of the glycolysis and pentose phosphate pathway genes were upregulated in the pileus by blue light. When compared with dark treatment, red-light treatment downregulated the expression of many respiration metabolism genes in the pileus. These results revealed that blue light enhanced the activation of glycolysis and the pentose phosphate pathway, whereas red light weakened glycolysis and pentose phosphate pathway activation. The conclusion can be drawn that blue light improved P. ostreatus fruiting body (particularly, the pileus) growth rate via enhancement of glycolysis and the pentose phosphate pathway.

Improving synthetic methylotrophy via dynamic formaldehyde regulation of pentose phosphate pathway genes and redox perturbation

Escherichia coli is an ideal choice for constructing synthetic methylotrophs capable of utilizing the non-native substrate methanol as a carbon and energy source. All current E. coli-based synthetic methylotrophs require co-substrates. They display variable levels of methanol-carbon incorporation due to a lack of native regulatory control of biosynthetic pathways, as E. coli does not recognize methanol as a proper substrate despite its ability to catabolize it. Here, using the E. coli formaldehyde-inducible promoter Pfrm, we implement dynamic expression control of select pentose-phosphate genes in response to the formaldehyde produced upon methanol oxidation. Genes under Pfrm control exhibited 8- to 30-fold transcriptional upregulation during growth on methanol. Formaldehyde-induced episomal expression of the B. methanolicus rpe and tkt genes involved in the regeneration of ribulose 5-phosphate required for formaldehyde fixation led to significantly improved methanol assimilation into intracellular metabolites, including a 2-fold increase of 13C-methanol into glutamate. Using a simple strategy for redox perturbation by deleting the E. coli NAD-dependent malate dehydrogenase gene maldh, we demonstrate 5-fold improved biomass formation of cells growing on methanol in the presence of a small concentration of yeast extract. Further improvements in methanol utilization are achieved via adaptive laboratory evolution and heterologous rpe and tkt expression. A short-term in vivo13C-methanol labeling assay was used to determine methanol assimilation activity for Δmaldh strains, and demonstrated dramatically higher labeling in intracellular metabolites, including a 6-fold and 1.8-fold increase in glycine labeling for the rpe/tkt and evolved strains, respectively. The combination of formaldehyde-controlled pentose phosphate pathway expression and redox perturbation with the maldh knock-out greatly improved both growth benefit with methanol and methanol carbon incorporation into intracellular metabolites.