High-level production of Rhodiola rosea characteristic component rosavin from D-glucose and L-arabinose in engineered Escherichia coli.

Rosavin is the characteristic component of Rhodiola rosea L., an important medicinal plant used widely in the world that has been reported to possess multiple biological activities. However, the endangered status of wild Rhodiola has limited the supply of rosavin. In this work, we successfully engineered an Escherichia coli strain to efficiently produce rosavin as an alternative production method. Firstly, cinnamate: CoA ligase from Hypericum calycinum, cinnamoyl-CoA reductase from Lolium perenne, and uridine diphosphate (UDP)-glycosyltransferase (UGT) from Bacillus subtilis (Bs-YjiC) were selected to improve the titer of rosin in E. coli. Subsequently, four UGTs from the UGT91R subfamily were identified to catalyze the formation of rosavin from rosin, with SlUGT91R1 from Solanum lycopersicum showing the highest activity level. Secondly, production of rosavin was achieved for the first time in E. coli by incorporating the SlUGT91R1 and UDP-arabinose pathway, including UDP-glucose dehydrogenase, UDP-xylose synthase, and UDP-xylose 4-epimerase, into the rosin-producing stain, and the titer reached 430.5 ± 91.4 mg/L. Thirdly, a two-step pathway derived from L-arabinose, composed of L-arabinokinase and UDP-sugar pyrophosphorylase, was developed in E. coli to further optimize the supply of the precursor UDP-arabinose. Furthermore, 1203.7 ± 32.1 mg/L of rosavin was produced from D-glucose and L-arabinose using shake-flask fermentation. Finally, the production of rosavin reached 7539.1 ± 228.7 mg/L by fed-batch fermentation in a 5-L bioreactor. Thus, the microbe-based production of rosavin shows great potential for commercialization. This work provides an effective strategy for the biosynthesis of other valuable natural products with arabinose-containing units from D-glucose and L-arabinose.

Evaluation of enzyme-constrained genome-scale model through metabolic engineering of anaerobic co-production of 2,3-butanediol and glycerol by Saccharomyces cerevisiae.

Enzyme-constrained genome-scale models (ecGEMs) have potential to predict phenotypes in a variety of conditions, such as growth rates or carbon sources. This study investigated if ecGEMs can guide metabolic engineering efforts to swap anaerobic redox-neutral ATP-providing pathways in yeast from alcoholic fermentation to equimolar co-production of 2,3-butanediol and glycerol. With proven pathways and low product toxicity, the ecGEM solution space aligned well with observed phenotypes. Since this catabolic pathway provides only one-third of the ATP of alcoholic fermentation (2/3 versus 2 ATP per glucose), the ecGEM predicted a growth decrease from 0.36 h-1 in the reference to 0.175 h-1 in the engineered strain. However, this <3-fold decrease would require the specific glucose consumption rate to increase. Surprisingly, after the pathway swap the engineered strain immediately grew at 0.15 h-1 with a glucose consumption rate of 29 mmol (g CDW)-1 h-1, which was indeed higher than reference (23 mmol (g CDW)-1 h-1) and one of the highest reported for S. cerevisiae. The accompanying 2,3-butanediol- (15.8 mmol (g CDW)-1 h-1) and glycerol (19.6 mmol (g CDW)-1 h-1) production rates were close to predicted values. Proteomics confirmed that this increased consumption rate was facilitated by enzyme reallocation from especially ribosomes (from 25.5 to 18.5 %) towards glycolysis (from 28.7 to 43.5 %). Subsequently, 200 generations of sequential transfer did not improve growth of the engineered strain, showing the use of ecGEMs in predicting opportunity space for laboratory evolution. The observations in this study illustrate both the current potential, as well as future improvements, of ecGEMs as a tool for both metabolic engineering and laboratory evolution.

Corynebacterium glutamicum cell factory design for the efficient production of cis, cis-muconic acid.

Cis, cis-muconic acid (MA) is widely used as a key starting material in the synthesis of diverse polymers. The growing demand in these industries has led to an increased need for MA. Here, we constructed recombinant Corynebacterium glutamicum by systems metabolic engineering, which exhibit high efficiency in the production of MA. Firstly, the three major degradation pathways were disrupted in the MA production process. Subsequently, metabolic optimization strategies were predicted by computational design and the shikimate pathway was reconstructed, significantly enhancing its metabolic flux. Finally, through optimization and integration of key genes involved in MA production, the recombinant strain produced 88.2 g/L of MA with the yield of 0.30 mol/mol glucose in the 5 L bioreactor. This titer represents the highest reported titer achieved using glucose as the carbon source in current studies, and the yield is the highest reported for MA production from glucose in Corynebacterium glutamicum. Furthermore, to enable the utilization of more cost-effective glucose derived from corn straw hydrolysate, we subjected the strain to adaptive laboratory evolution in corn straw hydrolysate. Ultimately, we successfully achieved MA production in a high solid loading of corn straw hydrolysate (with the glucose concentration of 83.56 g/L), resulting in a titer of 19.9 g/L for MA, which is 4.1 times higher than that of the original strain. Additionally, the glucose yield was improved to 0.33 mol/mol. These provide possibilities for a greener and more sustainable production of MA.

Efficient production of an antitumor precursor actinocin and other medicinal molecules from kynurenine pathway in Escherichia coli.

Kynurenine pathway has a potential to convert L-tryptophan into multiple medicinal molecules. This study aims to explore the biosynthetic potential of kynurenine pathway for the efficient production of actinocin, an antitumor precursor selected as a proof-of-concept target molecule. Kynurenine pathway is first constructed in Escherichia coli by testing various combinations of biosynthetic genes from four different organisms. Metabolic engineering strategies are next performed to improve the production by inhibiting a competing pathway, and enhancing intracellular supply of a cofactor S-adenosyl-L-methionine, and ultimately to produce actinocin from glucose. Metabolome analysis further suggests additional gene overexpression targets, which finally leads to the actinocin titer of 719 mg/L. E. coli strain engineered to produce actinocin is further successfully utilized to produce 350 mg/L of kynurenic acid, a neuroprotectant, and 1401 mg/L of 3-hydroxyanthranilic acid, an antioxidant, also from glucose. These competitive production titers demonstrate the biosynthetic potential of kynurenine pathway as a source of multiple medicinal molecules. The approach undertaken in this study can be useful for the sustainable production of molecules derived from kynurenine pathway, which are otherwise chemically synthesized.

Controlling circuitry underlies the growth optimization of Saccharomyces cerevisiae.

Microbial growth emerges from coordinated synthesis of various cellular components from limited resources. In Saccharomyces cerevisiae, cyclic AMP (cAMP)-mediated signaling is shown to orchestrate cellular metabolism; however, it remains unclear quantitatively how the controlling circuit drives resource partition and subsequently shapes biomass growth. Here we combined experiment with mathematical modeling to dissect the signaling-mediated growth optimization of S. cerevisiae. We showed that, through cAMP-mediated control, the organism achieves maximal or nearly maximal steady-state growth during the utilization of multiple tested substrates as well as under perturbations impairing glucose uptake. However, the optimal cAMP concentration varies across cases, suggesting that different modes of resource allocation are adopted for varied conditions. Under settings with nutrient alterations, S. cerevisiae tunes its cAMP level to dynamically reprogram itself to realize rapid adaptation. Moreover, to achieve growth maximization, cells employ additional regulatory systems such as the GCN2-mediated amino acid control. This study establishes a systematic understanding of global resource allocation in S. cerevisiae, providing insights into quantitative yeast physiology as well as metabolic strain engineering for biotechnological applications.

Metabolic engineering of Halomonas bluephagenesis for high-level mevalonate production from glucose and acetate mixture.

Mevalonate (MVA) plays a crucial role as a building block for the biosynthesis of isoprenoids. In this study, we engineered Halomonas bluephagenesis to efficiently produce MVA. Firstly, by screening MVA synthetases from eight different species, the two efficient candidate modules, specifically NADPH-dependent mvaESEfa from Enterococcus faecalis and NADH-dependent mvaESLca from Lactobacillus casei, were integrated into the chromosome, leading to the construction of the H. bluephagenesis MVA11. Through the synergetic utilization of glucose and acetate as mixed carbon sources, MVA11 produced 11.2 g/L MVA with a yield of 0.45 g/g (glucose + acetic acid) in the shake flask. Subsequently, 10 beneficial genes out of 50 targets that could promote MVA production were identified using CRISPR interference. The simultaneous repression of rpoN (encoding RNA polymerase sigma-54 factor) and IldD (encoding L-lactate dehydrogenase) increased MVA titer (13.3 g/L) by 19.23% and yield (0.53 g/g (glucose + acetic acid)) by 17.78%, respectively. Furthermore, introducing the non-oxidative glycolysis (NOG) pathway into MVA11 enhanced MVA yield by 12.20%. Ultimately, by combining these strategies, the resultant H. bluephagenesis MVA13/pli-63 produced 13.9 g/L MVA in the shake flask, and the yield increased to 0.56 g/g (glucose + acetic acid), which was the highest reported so far. Under open fed-batch fermentation conditions, H. bluephagenesis MVA13/pli-63 produced 121 g/L of MVA with a yield of 0.42 g/g (glucose + acetic acid), representing the highest reported titer and yield in the bioreactor to date. This study demonstrates that H. bluephagenesis is one of the most favorable chassis for MVA production.

Designing glucose utilization "highway" for recombinant biosynthesis.

cAMP receptor protein (CRP) is known as a global regulatory factor mainly mediating carbon source catabolism. Herein, we successfully engineered CRP to develop microbial chassis cells with improved recombinant biosynthetic capability in minimal medium with glucose as single carbon source. The obtained best-performing cAMP-independent CRPmu9 mutant conferred both faster cell growth and a 133-fold improvement in expression level of lac promoter in presence of 2% glucose, compared with strain under regulation of CRPwild-type. Promoters free from "glucose repression" are advantageous for recombinant expression, as glucose is a frequently used inexpensive carbon source in high-cell-density fermentations. Transcriptome analysis demonstrated that the CRP mutant globally rewired cell metabolism, displaying elevated tricarboxylic acid cycle activity; reduced acetate formation; increased nucleotide biosynthesis; and improved ATP synthesis, tolerance, and stress-resistance activity. Metabolites analysis confirmed the enhancement of glucose utilization with the upregulation of glycolysis and glyoxylate-tricarboxylic acid cycle. As expected, an elevated biosynthetic capability was demonstrated with vanillin, naringenin and caffeic acid biosynthesis in strains regulated by CRPmu9. This study has expanded the significance of CRP optimization into glucose utilization and recombinant biosynthesis, beyond the conventionally designated carbon source utilization other than glucose. The Escherichiacoli cell regulated by CRPmu9 can be potentially used as a beneficial chassis for recombinant biosynthesis.

High-level and -yield production of L-leucine in engineered Escherichia coli by multistep metabolic engineering.

L-leucine is an essential amino acid widely used in food and pharmaceutical industries. However, the relatively low production efficiency limits its large-scale application. In this study, we rationally developed an efficient L-leucine-producing Escherichia coli strain. Initially, the L-leucine synthesis pathway was enhanced by overexpressing feedback-resistant 2-isopropylmalate synthase and acetohydroxy acid synthase both derived from Corynebacterium glutamicum, along with two other native enzymes. Next, the pyruvate and acetyl-CoA pools were enriched by deleting competitive pathways, employing the nonoxidative glycolysis pathway, and dynamically modulating the citrate synthase activity, which significantly promoted the L-leucine production and yield to 40.69 g/L and 0.30 g/g glucose, respectively. Then, the redox flux was improved by substituting the native NADPH-dependent acetohydroxy acid isomeroreductase, branched chain amino acid transaminase, and glutamate dehydrogenase with their NADH-dependent equivalents. Finally, L-leucine efflux was accelerated by precise overexpression of the exporter and deletion of the transporter. Under fed-batch conditions, the final strain LXH-21 produced 63.29 g/L of L-leucine, with a yield and productivity of 0.37 g/g glucose and 2.64 g/(L h), respectively. To our knowledge, this study achieved the highest production efficiency of L-leucine to date. The strategies presented here will be useful for engineering E. coli strains for producing L-leucine and related products on an industrial scale.

Small but mighty: microexons in glucose homeostasis.

Many molecular mechanisms underlying blood glucose homeostasis remain elusive. Juan-Mateu et al. find that pancreatic islet cells utilize a regulatory program, originally identified in neurons, that involves alternative splicing of microexons in genes important for insulin secretion or diabetes risk.

Systems metabolic engineering upgrades Corynebacterium glutamicum for selective high-level production of the chiral drug precursor and cell-protective extremolyte L-pipecolic acid.

The nonproteinogenic cyclic metabolite l-pipecolic acid is a chiral precursor for the synthesis of various commercial drugs and functions as a cell-protective extremolyte and mediator of defense in plants, enabling high-value applications in the pharmaceutical, medical, cosmetic, and agrochemical markets. To date, the production of the compound is unfavorably fossil-based. Here, we upgraded the strain Corynebacterium glutamicum for l-pipecolic acid production using systems metabolic engineering. Heterologous expression of the l-lysine 6-dehydrogenase pathway, apparently the best route to be used in the microbe, yielded a family of strains that enabled successful de novo synthesis from glucose but approached a limit of performance at a yield of 180 mmol mol-1. Detailed analysis of the producers at the transcriptome, proteome, and metabolome levels revealed that the requirements of the introduced route were largely incompatible with the cellular environment, which could not be overcome after several further rounds of metabolic engineering. Based on the gained knowledge, we based the strain design on l-lysine 6-aminotransferase instead, which enabled a substantially higher in vivo flux toward l-pipecolic acid. The tailormade producer C. glutamicum PIA-7 formed l-pipecolic acid up to a yield of 562 mmol mol-1, representing 75% of the theoretical maximum. Ultimately, the advanced mutant PIA-10B achieved a titer of 93 g L-1 in a fed-batch process on glucose, outperforming all previous efforts to synthesize this valuable molecule de novo and even approaching the level of biotransformation from l-lysine. Notably, the use of C. glutamicum allows the safe production of GRAS-designated l-pipecolic acid, providing extra benefit toward addressing the high-value pharmaceutical, medical, and cosmetic markets. In summary, our development sets a milestone toward the commercialization of biobased l-pipecolic acid.

Establishing biosynthetic pathway for the production of p-hydroxyacetophenone and its glucoside in Escherichia coli.

p-Hydroxyacetophenone (p-HAP) and its glucoside picein are plant-derived natural products that have been extensively used in chemical, pharmaceutical and cosmetic industries owing to their antioxidant, antibacterial and antiseptic activities. However, the natural biosynthetic pathways for p-HAP and picein have yet been resolved so far, limiting their biosynthesis in microorganisms. In this study, we design and construct a biosynthetic pathway for de novo production of p-HAP and picein from glucose in E. coli. First, screening and characterizing pathway enzymes enable us to successfully establish functional biosynthetic pathway for p-HAP production. Then, the rate-limiting step in the pathway caused by a reversible alcohol dehydrogenase is completely eliminated by modulating intracellular redox cofactors. Subsequent host strain engineering via systematic increase of precursor supplies enables production enhancement of p-HAP with a titer of 1445.3 mg/L under fed-batch conditions. Finally, a novel p-HAP glucosyltransferase capable of generating picein from p-HAP is identified and characterized from a series of glycosyltransferases. On this basis, de novo biosynthesis of picein from glucose is achieved with a titer of 210.7 mg/L under fed-batch conditions. This work not only demonstrates a microbial platform for p-HAP and picein synthesis, but also represents a generalizable pathway design strategy to produce value-added compounds.

Metabolic engineering of Escherichia coli for the production of an antifouling agent zosteric acid.

Zosteric acid (ZA) is a Zostera species-derived, sulfated phenolic acid compound with antifouling activity and has gained much attention due to its nontoxic and biodegradable characteristics. However, the yield of Zostera species available for ZA extraction is limited by natural factors, such as season, latitude, light, and temperature. Here we report the development of metabolically engineered Escherichia coli strains capable of producing ZA from glucose and glycerol. First, intracellular availability of the sulfur donor 3'-phosphoadenosine-5'-phosphosulfate (PAPS) was enhanced by knocking out the cysH gene responsible for PAPS consumption and overexpressing the genes required for PAPS biosynthesis. Co-overexpression of the genes encoding tyrosine ammonia-lyase, sulfotransferase 1A1, ATP sulfurylase, and adenosine 5'-phosphosulfate kinase constructed ZA producing strain with enhanced PAPS supply. Second, the feedback-resistant forms of aroG and tyrA genes (encoding 3-deoxy-d-arabinoheptulosonate 7-phosphate synthase and chorismate mutase, respectively) were overexpressed to relieve the feedback regulation of L-tyrosine biosynthesis. Third, the pykA gene involved in phosphoenolpyruvate-consuming reaction, the regulator gene tyrR, the competing pathway gene pheA, and the ptsHIcrr genes essential for the PEP:carbohydrate phosphotransferase system were deleted. Moreover, all genes involved in the shikimate pathway and the talA, tktA, and tktB genes in the pentose phosphate pathway were examined for ZA production. The PTS-independent glucose uptake system, the expression vector system, and the carbon source were also optimized. As a result, the best-performing strain successfully produced 1.52 g L-1 ZA and 1.30 g L-1p-hydroxycinnamic acid from glucose and glycerol in a 700 mL fed-batch bioreactor.

Tup1 is critical for transcriptional repression in Quiescence in S. cerevisiae.

Upon glucose starvation, S. cerevisiae shows a dramatic alteration in transcription, resulting in wide-scale repression of most genes and activation of some others. This coincides with an arrest of cellular proliferation. A subset of such cells enters quiescence, a reversible non-dividing state. Here, we demonstrate that the conserved transcriptional corepressor Tup1 is critical for transcriptional repression after glucose depletion. We show that Tup1-Ssn6 binds new targets upon glucose depletion, where it remains as the cells enter the G0 phase of the cell cycle. In addition, we show that Tup1 represses a variety of glucose metabolism and transport genes. We explored how Tup1 mediated repression is accomplished and demonstrated that Tup1 coordinates with the Rpd3L complex to deacetylate H3K23. We found that Tup1 coordinates with Isw2 to affect nucleosome positions at glucose transporter HXT family genes during G0. Finally, microscopy revealed that a quarter of cells with a Tup1 deletion contain multiple DAPI puncta. Taken together, these findings demonstrate the role of Tup1 in transcriptional reprogramming in response to environmental cues leading to the quiescent state.

Metabolic engineering of Escherichia coli for high production of 1,5-pentanediol via a cadaverine-derived pathway.

1,5-Pentanediol (1,5-PDO) is a high value-added chemical which is widely used as a monomer in the polymer industry. There are no natural organisms that could directly produce 1,5-PDO from renewable carbon sources. In this study, we report metabolic engineering of Escherichia coli for high-level production of 1,5-PDO from glucose via a cadaverine-derived pathway. In the newly proposed pathway, cadaverine can be converted to 1,5-PDO via 5-hydroxyvalerate (5-HV) by introducing only one heterologous enzyme in E. coli. Different endogenous genes of E. coli were screened and heterologous carboxylic acid reductase genes were tested to build a functional pathway. Compared to the previously reported pathways, the engineered cadaverine-based pathway has a higher theoretical yield (0.70 mol/mol glucose) and higher catalytic efficiency. By further combining strategies of pathway engineering and process engineering, we constructed an engineered E. coli strain that could produce 2.62 g/L 1,5-PDO in shake-flask and 9.25 g/L 1,5-PDO with a yield of 0.28 mol/mol glucose in fed-batch fermentation. The proposed new pathway and engineering strategies reported here should be useful for developing biological routes to produce 1,5-PDO for real application.

A synthetic C2 auxotroph of Pseudomonas putida for evolutionary engineering of alternative sugar catabolic routes.

Acetyl-coenzyme A (AcCoA) is a metabolic hub in virtually all living cells, serving as both a key precursor of essential biomass components and a metabolic sink for catabolic pathways for a large variety of substrates. Owing to this dual role, tight growth-production coupling schemes can be implemented around the AcCoA node. Building on this concept, a synthetic C2 auxotrophy was implemented in the platform bacterium Pseudomonas putida through an in silico-informed engineering approach. A growth-coupling strategy, driven by AcCoA demand, allowed for direct selection of an alternative sugar assimilation route-the phosphoketolase (PKT) shunt from bifidobacteria. Adaptive laboratory evolution forced the synthetic P. putida auxotroph to rewire its metabolic network to restore C2 prototrophy via the PKT shunt. Large-scale structural chromosome rearrangements were identified as possible mechanisms for adjusting the network-wide proteome profile, resulting in improved PKT-dependent growth phenotypes. 13C-based metabolic flux analysis revealed an even split between the native Entner-Doudoroff pathway and the synthetic PKT bypass for glucose processing, leading to enhanced carbon conservation. These results demonstrate that the P. putida metabolism can be radically rewired to incorporate a synthetic C2 metabolism, creating novel network connectivities and highlighting the importance of unconventional engineering strategies to support efficient microbial production.

Metabolic engineering of E. coli for ß-alanine production using a multi-biosensor enabled approach.

ß-alanine is an important biomolecule used in nutraceuticals, pharmaceuticals, and chemical synthesis. The relatively eco-friendly bioproduction of ß-alanine has recently attracted more interest than petroleum-based chemical synthesis. In this work, we developed two types of in vivo high-throughput screening platforms, wherein one was utilized to identify a novel target ribonuclease E (encoded by rne) as well as a redox-cofactor balancing module that can enhance de novo ß-alanine biosynthesis from glucose, and the other was employed for screening fermentation conditions. When combining these approaches with rational upstream and downstream module engineering, an engineered E. coli producer was developed that exhibited 3.4- and 6.6-fold improvement in ß-alanine yield (0.85 mol ß-alanine/mole glucose) and specific ß-alanine production (0.74 g/L/OD600), respectively, compared to the parental strain in a minimal medium. Across all of the strains constructed, the best yielding strain exhibited 1.08 mol ß-alanine/mole glucose (equivalent to 81.2% of theoretic yield). The final engineered strain produced 6.98 g/L ß-alanine in a batch-mode bioreactor and 34.8 g/L through a whole-cell catalysis. This approach demonstrates the utility of biosensor-enabled high-throughput screening for the production of ß-alanine.

Nab3 nuclear granule accumulation is driven by respiratory capacity.

Numerous biological processes involve proteins capable of transiently assembling into subcellular compartments necessary for cellular functions. One process is the RNA polymerase II transcription cycle which involves initiation, elongation, co-transcriptional modification of nascent RNA, and termination. The essential yeast transcription termination factor Nab3 is required for termination of small non-coding RNAs and accumulates into a compact nuclear granule upon glucose removal. Nab3 nuclear granule accumulation varies in penetrance across yeast strains and a higher Nab3 granule accumulation phenotype is associated with petite strains, suggesting a possible ATP-dependent mechanism for granule disassembly. Here, we demonstrate the uncoupling of mitochondrial oxidative phosphorylation by drug treatment or deletions of nuclear-encoded ATP synthase subunit genes were sufficient to increase Nab3 granule accumulation and led to an inability to proliferate during prolonged glucose deprivation, which requires respiration. Additionally, by enriching for respiration competent cells from a petite-prone strain, we generated a low granule-accumulating strain from a relatively high one, providing another link between respiratory competency and Nab3 granules. Consistent with the resulting idea that ATP is involved in granule accumulation, the addition of extracellular ATP to semi-permeabilized cells was sufficient to reduce Nab3 granule accumulation. Deleting the SKY1 gene, which encodes a kinase that phosphorylates nuclear SR repeat-containing proteins and is involved in efficient stress granule disassembly, also resulted in increased granule accumulation. This observation implicates Sky1 in Nab3 granule biogenesis. Taken together, these findings suggest there is normally an equilibrium between termination factor granule assembly and disassembly mediated by ATP-requiring nuclear machinery.

Metabolic engineering of Escherichia coli W3110 for efficient production of homoserine from glucose.

Efficient microbial cell factory for the production of homoserine from glucose has been developed by iterative and rational engineering of Escherichia coli W3110. The whole pathway from glucose to homoserine was divided into three groups, namely, glucose transport and glycolysis ('up-stream'), TCA and glyoxylate cycles ('mid-stream'), and homoserine module (conversion of aspartate to homoserine and its secretion; 'down-stream'), and the carbon flux in each group as well as between the groups were accelerated and balanced. Altogether, ∼18 genes were modified for active and consistent production of homoserine during both the actively-growing and non-growing stages of cultivation. Finally, fed-batch, two-stage bioreactor experiments, separating the growth from the production stage, were conducted for 61 h, which gave the high titer of 110.8 g/L, yield of 0.64 g/g glucose and volumetric productivity of 1.82 g/L/h, with an insignificant amount of acetate (<0.5 g/L) as the only noticeable byproduct. The metabolic engineering strategy employed in this study should be applicable for the biosynthesis of other amino acids or chemicals derived from aspartic acid.

Systems genetics in the rat HXB/BXH family identifies Tti2 as a pleiotropic quantitative trait gene for adult hippocampal neurogenesis and serum glucose.

Neurogenesis in the adult hippocampus contributes to learning and memory in the healthy brain but is dysregulated in metabolic and neurodegenerative diseases. The molecular relationships between neural stem cell activity, adult neurogenesis, and global metabolism are largely unknown. Here we applied unbiased systems genetics methods to quantify genetic covariation among adult neurogenesis and metabolic phenotypes in peripheral tissues of a genetically diverse family of rat strains, derived from a cross between the spontaneously hypertensive (SHR/OlaIpcv) strain and Brown Norway (BN-Lx/Cub). The HXB/BXH family is a very well established model to dissect genetic variants that modulate metabolic and cardiovascular diseases and we have accumulated deep phenome and transcriptome data in a FAIR-compliant resource for systematic and integrative analyses. Here we measured rates of precursor cell proliferation, survival of new neurons, and gene expression in the hippocampus of the entire HXB/BXH family, including both parents. These data were combined with published metabolic phenotypes to detect a neurometabolic quantitative trait locus (QTL) for serum glucose and neuronal survival on Chromosome 16: 62.1-66.3 Mb. We subsequently fine-mapped the key phenotype to a locus that includes the Telo2-interacting protein 2 gene (Tti2)-a chaperone that modulates the activity and stability of PIKK kinases. To verify the hypothesis that differences in neurogenesis and glucose levels are caused by a polymorphism in Tti2, we generated a targeted frameshift mutation on the SHR/OlaIpcv background. Heterozygous SHR-Tti2+/- mutants had lower rates of hippocampal neurogenesis and hallmarks of dysglycemia compared to wild-type littermates. Our findings highlight Tti2 as a causal genetic link between glucose metabolism and structural brain plasticity. In humans, more than 800 genomic variants are linked to TTI2 expression, seven of which have associations to protein and blood stem cell factor concentrations, blood pressure and frontotemporal dementia.

Stanniocalcin2 inhibits the epithelial-mesenchymal transition and invasion of trophoblasts via activation of autophagy under high-glucose conditions.

Maternal pregnancy hyperglycemia is often accompanied by placental dysfunction. During placental development, epithelial-mesenchymal transition (EMT) contributes to the transformation of relatively noninvasive trophoblasts into highly invasive extravillous trophoblasts (EVTs). However, the specific role of EMT in placentas under hyperglycemia environments remains relatively unexplored. Stanniocalcin2 (STC2) regulates EMT in many cancers. In this study, we first demonstrated that STC2 expression was upregulated in GDM placenta. We found that STC2 activated autophagy and suppressed EMT in high-glucose-treated EVTs and was associated with a lack of invasiveness. Specifically, STC2 inhibited the interactions between p62/SQSTM1 (p62) and EMT transcription factors to promote the degradation of Twist1 and Snail via a proteasome-dependent pathway. Furthermore, the PI3K/AKT/AMPK signaling pathway was involved in the regulation of autophagy and EMT by STC2. Taken together, our results reveal that STC2 may serve as a potential prognostic biomarker in GDM and sheds light on the regulatory mechanisms of trophoblast invasion.