CRISPR-assisted rational flux-tuning and arrayed CRISPRi screening of an L-proline exporter for L-proline hyperproduction.

Development of hyperproducing strains is important for biomanufacturing of biochemicals and biofuels but requires extensive efforts to engineer cellular metabolism and discover functional components. Herein, we optimize and use the CRISPR-assisted editing and CRISPRi screening methods to convert a wild-type Corynebacterium glutamicum to a hyperproducer of L-proline, an amino acid with medicine, feed, and food applications. To facilitate L-proline production, feedback-deregulated variants of key biosynthetic enzyme γ-glutamyl kinase are screened using CRISPR-assisted single-stranded DNA recombineering. To increase the carbon flux towards L-proline biosynthesis, flux-control genes predicted by in silico analysis are fine-tuned using tailored promoter libraries. Finally, an arrayed CRISPRi library targeting all 397 transporters is constructed to discover an L-proline exporter Cgl2622. The final plasmid-, antibiotic-, and inducer-free strain produces L-proline at the level of 142.4 g/L, 2.90 g/L/h, and 0.31 g/g. The CRISPR-assisted strain development strategy can be used for engineering industrial-strength strains for efficient biomanufacturing.

Production of proline and protease with different organic wastes in bacteria (Production proline and protease with organic wastes) / Produção de prolina e protease com diferentes resíduos orgânicos em bactérias (produção de prolina e protease com resíduos orgânicos)

In this study, we investigated the proline and protease production of different bacteria in several organic waste materials. Our aim was to produce proline and protease economically in waste that is abundantly available while reducing its environmental impact. 5 ml of different organic waste materials (OWW: Olive waste water; N.B: Nutrient Broth; EW: Eggshell; PBS: PBS buffer; PLW: Peach leaf wastes; TCW: Turkish coffee wastes; TWW: Tea waste water; WCW: Waste cheese whey; WFO: Waste frying oil) were placed in 10 ml grow tubes, inoculated and incubated for 24 h. Phosphate-buffered saline and 10% solutions of different organic wastes were added. These cultures were subsequently incubated at 37°C for 24 h. Cells were harvested at 24 h for L-proline assay. 1 ml of culture was transferred by pipette into an Eppendorf tube and centrifuged at 14,000 rpm for 20 min at room temperature. Cellular debris was removed by centrifuge and the supernatant was used for proline activity assays. Protease activity was determined using a modified method with casein as the substrate. We found that proline and protease can easily be produced economically using Turkish coffee wastes (TCW), Waste cheese whey (WCW) and Olive waste water (OWW) organic waste. We believe that this study will result in similar research leading to the economical use of these waste materials thus reducing their impact on the environment.

Neste estudo, investigamos a produção de prolina e protease de diferentes bactérias em diversos resíduos orgânicos. Nosso objetivo era produzir prolina e protease economicamente em resíduos que estão disponíveis em abundância, reduzindo seu impacto ambiental. Cinco ml de diferentes materiais de resíduos orgânicos (OWW: resíduos de azeitona; NB: caldo nutriente; EW: casca de ovo; PBS: tampão PBS; PLW: resíduos de folhas de pêssego; TCW: resíduos de café turco; TWW: resíduos de chá; WCW: resíduos de queijo soro de leite; WFO: óleo de fritura residual) foram colocados em tubos de cultivo de 10 ml, inoculados e incubados por 24 horas. Adicionaram-se solução salina tamponada com fosfato e soluções a 10% de diferentes resíduos orgânicos. Essas culturas foram subsequentemente incubadas a 37° C durante 24 h. As células foram colhidas às 24 h para o ensaio de L-prolina. Um ml de cultura foi transferido por pipeta para um tubo Eppendorf e centrifugado a 14.000 rpm, por 20 min, em temperatura ambiente. Os detritos celulares foram removidos por centrifugação e o sobrenadante foi usado para ensaios de atividade de prolina. A atividade da protease foi determinada usando um método modificado com caseína como substrato. Descobrimos que a prolina e a protease podem ser facilmente produzidas economicamente, usando resíduos de café turco (TCW), resíduos de soro de queijo (WCW) e resíduos orgânicos de água de oliva (OWW). Acreditamos que este estudo resultará em pesquisas semelhantes, levando ao uso econômico desses materiais residuais, reduzindo, assim, seu impacto no meio ambiente.

Phenyl-substituted aminomethylene-bisphosphonates inhibit human P5C reductase and show antiproliferative activity against proline-hyperproducing tumour cells.

In certain cancers, such as breast, prostate and some lung and skin cancers, the gene for the enzyme catalysing the second and last step in proline synthesis, δ1-pyrroline-5-carboxylate (P5C) reductase, has been found upregulated. This leads to a higher proline content that exacerbates the effects of the so-called proline-P5C cycle, with tumour cells effectively using this method to increase cell survival. If a method of reducing or inhibiting P5C reductase could be discovered, it would provide new means of treating cancer. To address this point, the effect of some phenyl-substituted derivatives of aminomethylene-bisphosphonic acid, previously found to interfere with the catalytic activity of plant and bacterial P5C reductases, was evaluated in vitro on the human isoform 1 (PYCR1), expressed in E. coli and affinity purified. The 3.5-dibromophenyl- and 3.5-dichlorophenyl-derivatives showed a remarkable effectiveness, with IC50 values lower than 1 µM and a mechanism of competitive type against both P5C and NADPH. The actual occurrence in vivo of enzyme inhibition was assessed on myelogenous erythroleukemic K562 and epithelial breast cancer MDA-MB-231 cell lines, whose growth was progressively impaired by concentrations of the dibromo derivative ranging from 10-6 to 10-4 M. Interestingly, growth inhibition was not relieved by the exogenous supply of proline, suggesting that the effect relies on the interference with the proline-P5C cycle, and not on proline starvation.

Effects of cadmium stress on growth and physiological characteristics of sassafras seedlings.

The effects of cadmium stress on the growth and physiological characteristics of Sassafras tzumu Hemsl. were studied in pot experiments. Five Cd levels were tested [CT(Control Treatment) : 0 mg/kg, Cd5: 5 mg/kg, Cd20: 20 mg/kg, Cd50: 50 mg/kg, and Cd100: 100 mg/kg]. The growth and physiological characteristics of the sassafras seedlings in each level were measured. The results showed that soil Cd had negative influences on sassafras growth and reduced the net growth of plant height and the biomass of leaf, branch and root. Significant reductions were recorded in root biomass by 18.18%(Cd5), 27.35%(Cd20), 27.57%(Cd50) and 28.95%(Cd100). The contents of hydrogen peroxide decreased first then increased while malondialdehyde showed the opposite trend with increasing cadmium concentration. Decreases were found in hydrogen peroxide contents by 10.96%(Cd5), 11.82%(Cd20) and 7.02%(Cd50); increases were found in malondialdehyde contents by 15.47%(Cd5), 16.07%(Cd20) and 7.85%(Cd50), indicating that cadmium stress had a certain effect on the peroxidation of the inner cell membranes in the seedlings that resulted in damage to the cell membrane structure. Superoxide dismutase activity decreased among treatments by 17.05%(Cd5), 10,68%(Cd20), 20.85%(Cd50) and 8.91%(Cd100), while peroxidase activity increased steadily with increasing cadmium concentration; these results suggest that peroxidase is likely the main protective enzyme involved in the reactive oxygen removal system in sassafras seedlings. Upward trends were observed in proline content by 90.76%(Cd5), 74.36%(Cd20), 99.73%(Cd50) and 126.01%(Cd100). The increase in proline content with increasing cadmium concentration indicated that cadmium stress induced proline synthesis to resist osmotic stress in the seedlings. Compared to that in CT, the soluble sugar content declined under the different treatments by 32.84%(Cd5), 5.85%(Cd20), 25.55%(Cd50) and 38.69%(Cd100). Increases were observed in the soluble protein content by 2.34%(Cd5), 21.36%(Cd20), 53.15%(Cd50) and 24.22%(Cd100). At different levels of cadmium stress, the chlorophyll content in the seedlings first increased and then decreased, and it was higher in the Cd5 and Cd20 treatments than that in the CT treatment. These results reflected that cadmium had photosynthesis-promoting effects at low concentrations and photosynthesis-suppressing effects at high concentrations. The photosynthetic gas exchange parameters and photosynthetic light-response parameters showed downward trends with increasing cadmium concentration compared with those in CT; these results reflected the negative effects of cadmium stress on photosynthesis in sassafras seedlings.

Discovery of cyclohexadepsipeptides with anti-Zika virus activities and biosynthesis of the nonproteinogenic building block (3S)-methyl-l-proline.

The fungal cyclohexadepsipeptides destruxins (DTXs), isaridins (ISDs), and isariins (ISRs) are nonribosomal peptides whose structures include a 19-membered ring composed of five amino acid residues and one α- or ß-hydroxy acid residue. These cyclohexadepsipeptides contain unusual nonproteinogenic amino acid-building blocks and possess a range of antiviral, antibacterial, and other activities. The biosynthetic gene clusters for ISDs and ISRs have not been identified, and the biosynthesis of the nonproteinogenic (3S)-methyl-l-proline residue, which is found in DTXs, ISDs, and many other natural products, lacks full characterization. In an ongoing effort to identify compounds that can inhibit the Zika virus (ZIKV), we examined the extract of marine-derived fungus Beauveria felina SX-6-22 and discovered 30 DTXs, ISDs, and ISRs (1-30) including seven new compounds (1-7). The anti-ZIKV assays showed that 9-12 and 16-18 possess inhibitory activities against ZIKV RNA replication and NS5 (nonstructural protein 5) production in ZIKV-infected A549 cells. We sequenced the genome of B. felina SX-6-22 and identified three biosynthetic gene clusters detx, isd and isr, which are responsible for the biosynthesis of DTXs, ISDs, and ISRs, respectively. Comparative analyses of the three gene clusters clarified the biosynthetic relationships among these cyclohexadepsipeptides. Finally, we characterized the entire biosynthesis of nonproteinogenic building block (3S)-methyl-l-proline. The Δ1-pyrroline-5-carboxylate reductases (P5CRs), also used in the biosynthesis of l-proline, were demonstrated to catalyze the final reduction step in (3S)-methyl-l-proline formation, suggesting potential cross talk between primary and secondary metabolisms. These results provide opportunities for biosynthetic pathway engineering to generate new anti-ZIKV cyclohexadepsipeptides.

Abscisic acid-responsive element binding transcription factors contribute to proline synthesis and stress adaptation in Arabidopsis.

Proline accumulation is one of the most common adaptive responses of higher plants against abiotic stresses like drought. It plays multiple roles in osmotic adjustment, cell homeostasis and stress recovery. Genetic regulation of proline accumulation under drought is complex, and transcriptional cascades modulating proline is poorly understood. Here, we employed quadruple mutant (abf1 abf2 abf3 abf4) to dissect the role of ABA-responsive elements (ABREs) binding transcription factors (ABFs) in modulating proline accumulation across varying stress scenarios. ABREs are present across the promoter of the P5CS1 gene, whose upregulation is considered a hallmark for drought inducible proline accumulation. Upon ABA treatment, P5CS1 mRNA expression and proline content in the shoot were significantly higher in Col-0 compared to the quadruple mutant. Similar results were found at 2 h and 3 h after acute dehydration. We quantified proline at different time points after drought stress treatment. The proline content was higher in wild type (Col-0) than the quadruple mutant at the early stage of drought. Notably, the proline accumulation in wild type increased at a slower rate than the quadruple mutant 7 d after drought stress. Besides, the quadruple mutant displayed significant oxidative damage, low tissue turgidity and higher membrane damage under terminal drought stress. Both terminal drought stress and long-term constant water stress revealed substantial differences in growth rate between wild type and quadruple mutant. The study provides evidence that ABFs are involved in drought stress response, such as proline biosynthesis in Arabidopsis.

Mitochondrial NADP(H) generation is essential for proline biosynthesis.

The coenzyme nicotinamide adenine dinucleotide phosphate (NADP+) and its reduced form (NADPH) regulate reductive metabolism in a subcellularly compartmentalized manner. Mitochondrial NADP(H) production depends on the phosphorylation of NAD(H) by NAD kinase 2 (NADK2). Deletion of NADK2 in human cell lines did not alter mitochondrial folate pathway activity, tricarboxylic acid cycle activity, or mitochondrial oxidative stress, but rather led to impaired cell proliferation in minimal medium. This growth defect was rescued by proline supplementation. NADK2-mediated mitochondrial NADP(H) generation was required for the reduction of glutamate and hence proline biosynthesis. Furthermore, mitochondrial NADP(H) availability determined the production of collagen proteins by cells of mesenchymal lineage. Thus, a primary function of the mitochondrial NADP(H) pool is to support proline biosynthesis for use in cytosolic protein synthesis.

Overexpression of an Agave Phosphoenolpyruvate Carboxylase Improves Plant Growth and Stress Tolerance.

It has been challenging to simultaneously improve photosynthesis and stress tolerance in plants. Crassulacean acid metabolism (CAM) is a CO2-concentrating mechanism that facilitates plant adaptation to water-limited environments. We hypothesized that the ectopic expression of a CAM-specific phosphoenolpyruvate carboxylase (PEPC), an enzyme that catalyzes primary CO2 fixation in CAM plants, would enhance both photosynthesis and abiotic stress tolerance. To test this hypothesis, we engineered a CAM-specific PEPC gene (named AaPEPC1) from Agave americana into tobacco. In comparison with wild-type and empty vector controls, transgenic tobacco plants constitutively expressing AaPEPC1 showed a higher photosynthetic rate and biomass production under normal conditions, along with significant carbon metabolism changes in malate accumulation, the carbon isotope ratio δ13C, and the expression of multiple orthologs of CAM-related genes. Furthermore, AaPEPC1 overexpression enhanced proline biosynthesis, and improved salt and drought tolerance in the transgenic plants. Under salt and drought stress conditions, the dry weight of transgenic tobacco plants overexpressing AaPEPC1 was increased by up to 81.8% and 37.2%, respectively, in comparison with wild-type plants. Our findings open a new door to the simultaneous improvement of photosynthesis and stress tolerance in plants.

Mitochondrial NADP+ is essential for proline biosynthesis during cell growth.

Nicotinamide adenine dinucleotide phosphate (NADP+) is vital to produce NADPH, a principal supplier of reducing power for biosynthesis of macromolecules and protection against oxidative stress. NADPH exists in separate pools, in both the cytosol and mitochondria; however, the cellular functions of mitochondrial NADPH are incompletely described. Here, we find that decreasing mitochondrial NADP(H) levels through depletion of NAD kinase 2 (NADK2), an enzyme responsible for production of mitochondrial NADP+, renders cells uniquely proline auxotrophic. Cells with NADK2 deletion fail to synthesize proline, due to mitochondrial NADPH deficiency. We uncover the requirement of mitochondrial NADPH and NADK2 activity for the generation of the pyrroline-5-carboxylate metabolite intermediate as the bottleneck step in the proline biosynthesis pathway. Notably, after NADK2 deletion, proline is required to support nucleotide and protein synthesis, making proline essential for the growth and proliferation of NADK2-deficient cells. Thus, we highlight proline auxotrophy in mammalian cells and discover that mitochondrial NADPH is essential to enable proline biosynthesis.

Genetic analysis of Pycr1 and Pycr2 in mice.

The final step in proline biosynthesis is catalyzed by three pyrroline-5-carboxylate reductases, PYCR1, PYCR2, and PYCR3, which convert pyrroline-5-carboxylate (P5C) to proline. Mutations in human PYCR1 and ALDH18A1 (P5C Synthetase) cause Cutis Laxa (CL), whereas mutations in PYCR2 cause hypomyelinating leukodystrophy 10 (HLD10). Here, we investigated the genetics of Pycr1 and Pycr2 in mice. A null allele of Pycr1 did not show integument or CL-related phenotypes. We also studied a novel chemically-induced mutation in Pycr2. Mice with recessive loss-of-function mutations in Pycr2 showed phenotypes consistent with neurological and neuromuscular disorders, including weight loss, kyphosis, and hind-limb clasping. The peripheral nervous system was largely unaffected, with only mild axonal atrophy in peripheral nerves. A severe loss of subcutaneous fat in Pycr2 mutant mice is reminiscent of a CL-like phenotype, but primary features such as elastin abnormalities were not observed. Aged Pycr2 mutant mice had reduced white blood cell counts and altered lipid metabolism, suggesting a generalized metabolic disorder. PYCR1 and -2 have similar enzymatic and cellular activities, and consistent with previous studies, both were localized in the mitochondria in fibroblasts. Both PYCR1 and -2 were able to complement the loss of Pro3, the yeast enzyme that converts P5C to proline, confirming their activity as P5C reductases. In mice, Pycr1; Pycr2 double mutants were sub-viable and unhealthy compared to either single mutant, indicating the genes are largely functionally redundant. Proline levels were not reduced, and precursors were not increased in serum from Pycr2 mutant mice or in lysates from skin fibroblast cultures, but placing Pycr2 mutant mice on a proline-free diet worsened the phenotype. Thus, Pycr1 and -2 have redundant functions in proline biosynthesis, and their loss makes proline a semi-essential amino acid. These findings have implications for understanding the genetics of CL and HLD10, and for modeling these disorders in mice.

Activation of proline biosynthesis is critical to maintain glutamate homeostasis during acute methamphetamine exposure.

Methamphetamine (METH) is a highly addictive psychostimulant that causes long-lasting effects in the brain and increases the risk of developing neurodegenerative diseases. The cellular and molecular effects of METH in the brain are functionally linked to alterations in glutamate levels. Despite the well-documented effects of METH on glutamate neurotransmission, the underlying mechanism by which METH alters glutamate levels is not clearly understood. In this study, we report an essential role of proline biosynthesis in maintaining METH-induced glutamate homeostasis. We observed that acute METH exposure resulted in the induction of proline biosynthetic enzymes in both undifferentiated and differentiated neuronal cells. Proline level was also increased in these cells after METH exposure. Surprisingly, METH treatment did not increase glutamate levels nor caused neuronal excitotoxicity. However, METH exposure resulted in a significant upregulation of pyrroline-5-carboxylate synthase (P5CS), the key enzyme that catalyzes synthesis of proline from glutamate. Interestingly, depletion of P5CS by CRISPR/Cas9 resulted in a significant increase in glutamate levels upon METH exposure. METH exposure also increased glutamate levels in P5CS-deficient proline-auxotropic cells. Conversely, restoration of P5CS expression in P5CS-deficient cells abrogated the effect of METH on glutamate levels. Consistent with these findings, P5CS expression was significantly enhanced in the cortical brain region of mice administered with METH and in the slices of cortical brain tissues treated with METH. Collectively, these results uncover a key role of P5CS for the molecular effects of METH and highlight that excess glutamate can be sequestered for proline biosynthesis as a protective mechanism to maintain glutamate homeostasis during drug exposure.

Pyrroline-5-carboxylate synthase senses cellular stress and modulates metabolism by regulating mitochondrial respiration.

Pyrroline-5-carboxylate synthase (P5CS) catalyzes the synthesis of pyrroline-5-carboxylate (P5C), a key precursor for the synthesis of proline and ornithine. P5CS malfunction leads to multiple human diseases; however, the molecular mechanism underlying these diseases is unknown. We found that P5CS localizes in mitochondria in rod- and ring-like patterns but diffuses inside the mitochondria upon cellular starvation or exposure to oxidizing agents. Some of the human disease-related mutant forms of P5CS also exhibit diffused distribution. Multimerization (but not the catalytic activity) of P5CS regulates its localization. P5CS mutant cells have a reduced proliferation rate and are sensitive to cellular stresses. Flies lacking P5CS have reduced eclosion rates. Lipid droplets accumulate in the eyes of the newly eclosed P5CS mutant flies, which degenerate with aging. The loss of P5CS in cells leads to abnormal purine metabolism and lipid-droplet accumulation. The reduced lipid-droplet consumption is likely due to decreased expression of the fatty acid transporter, CPT1, and few ß-oxidation-related genes following P5CS knockdown. Surprisingly, we found that P5CS is required for mitochondrial respiratory complex organization and that the respiration defects in P5CS knockout cells likely contribute to the metabolic defects in purine synthesis and lipid consumption. This study links amino acid synthesis with mitochondrial respiration and other key metabolic processes, whose imbalance might contribute to P5CS-related disease conditions.

PINCH-1 regulates mitochondrial dynamics to promote proline synthesis and tumor growth.

Reprograming of proline metabolism is critical for tumor growth. Here we show that PINCH-1 is highly expressed in lung adenocarcinoma and promotes proline synthesis through regulation of mitochondrial dynamics. Knockout (KO) of PINCH-1 increases dynamin-related protein 1 (DRP1) expression and mitochondrial fragmentation, which suppresses kindlin-2 mitochondrial translocation and interaction with pyrroline-5-carboxylate reductase 1 (PYCR1), resulting in inhibition of proline synthesis and cell proliferation. Depletion of DRP1 reverses PINCH-1 deficiency-induced defects on mitochondrial dynamics, proline synthesis and cell proliferation. Furthermore, overexpression of PYCR1 in PINCH-1 KO cells restores proline synthesis and cell proliferation, and suppresses DRP1 expression and mitochondrial fragmentation. Finally, ablation of PINCH-1 from lung adenocarcinoma in mouse increases DRP1 expression and inhibits PYCR1 expression, proline synthesis, fibrosis and tumor growth. Our results identify a signaling axis consisting of PINCH-1, DRP1 and PYCR1 that regulates mitochondrial dynamics and proline synthesis, and suggest an attractive strategy for alleviation of tumor growth.

Comparative physiological and root proteome analyses of two sorghum varieties responding to water limitation.

When exposed to drought stress many plants reprogram their gene expression to activate adaptive biochemical and physiological responses for survival. However, most of the well-studied adaptive responses are common between drought-sensitive and drought-tolerant species, making it difficult to identify the key mechanisms underpinning successful drought tolerance in crops. We developed a sorghum experimental system that compares between drought-sensitive (ICSB338) and enhanced drought-tolerant (SA1441) varieties. We show that sorghum activates a swift and robust stomatal shutdown to preserve leaf water content when water stress has been sensed. Water uptake is enhanced via increasing root cell water potential through the rapid biosynthesis of predominantly glycine betaine and an increased root-to-shoot ratio to explore more soil volume for water. In addition to stomatal responses, there is a prompt accumulation of proline in leaves and effective protection of chlorophyll during periods of water limitation. Root and stomatal functions rapidly recover from water limitation (within 24 h of re-watering) in the drought-tolerant variety, but recovery is impaired in the drought-sensitive sorghum variety. Analysis of the root proteome revealed complex protein networks that possibly underpin sorghum responses to water limitation. Common and unique protein changes between the two sorghum varieties provide new targets for future use in investigating sorghum drought tolerance.

De Novo Engineering of Corynebacterium glutamicum for l-Proline Production.

l-Proline is an important amino acid that has various industrial applications. Industrial l-proline-producing strains are obtained by the mutagenesis of Corynebacterium glutamicum. In this study, the optimized C. glutamicum genome-editing tools were further applied in the de novo construction of a hyper-l-proline-producing strain. Overexpression of a feedback inhibition-resistant γ-glutamic kinase mutant ProBG149K, deletion of a proline dehydrogenase to block l-proline degradation, overexpression of glutamate dehydrogenase to increase glutamate synthesis flux, the mutation of 6-phosphate gluconate dehydrogenase and glucose-6-phosphate-dehydrogenase in the pentose phosphate pathway to enhance NADPH supply, the deletion of pyruvate aminotransferase to decrease the byproduct l-alanine synthesis, and weakening of α-ketoglutarate dehydrogenase to regulate the TCA cycle were combined to obtain ZQJY-9. ZQJY-9 produced 19.68 ± 0.22 g/L of l-proline in flask fermentation and was also demonstrated at the 3 L bioreactor level by fed-batch fermentation producing 120.18 g/L of l-proline at 76 h with the highest productivity of 1.581 g/L/h.

Directed Evolution of Ornithine Cyclodeaminase Using an EvolvR-Based Growth-Coupling Strategy for Efficient Biosynthesis of l-Proline.

l-Proline takes a significant role in the pharmaceutical and chemical industries as well as graziery. Typical biosynthesis of l-proline is from l-glutamate, involving three enzyme reactions as well as a spontaneous cyclization. Alternatively, l-proline can be also synthesized in l-ornithine and/or l-arginine producing strains by an ornithine aminotransferase (OCD). In this study, a strategy of directed evolution combining rare codon selection and pEvolvR was developed to screen OCD with high catalytic efficiency, improving l-proline production from l-arginine chassis cells. The mutations were generated by CRISPR-assisted DNA polymerases and were screened by growth-coupled rare codon selection system. OCDK205G/M86K/T162A from Pseudomonas putida was identified with 2.85-fold increase in catalytic efficiency for the synthesis of l-proline. Furthermore, we designed and optimized RBS for the BaargI and Ppocd coupling cascade using RedLibs, as well as sRNA inhibition of argF to moderate l-proline biosynthesis in l-arginine overproducing Corynebacterium crenatum. The strain PS6 with best performance reached 15.3 g/L l-proline in the shake flask and showed a titer of 38.4 g/L in a 5 L fermenter with relatively low concentration of residual l-ornithine and/or l-arginine.

Abscisic acid mediated proline biosynthesis and antioxidant ability in roots of two different rice genotypes under hypoxic stress.

BACKGROUND: Abscisic acid (ABA) and proline play important roles in rice acclimation to different stress conditions. To study whether cross-talk exists between ABA and proline, their roles in rice acclimation to hypoxia, rice growth, root oxidative damage and endogenous ABA and proline accumulation were investigated in two different rice genotypes ('Nipponbare' (Nip) and 'Upland 502' (U502)). RESULTS: Compared with U502 seedlings, Nip seedlings were highly tolerant to hypoxic stress, with increased plant biomass and leaf photosynthesis and decreased root oxidative damage. Hypoxia significantly stimulated the accumulation of proline and ABA in the roots of both cultivars, with a higher ABA level observed in Nip than in U502, whereas the proline levels showed no significant difference in the two cultivars. The time course variation showed that the root ABA and proline contents under hypoxia increased 1.5- and 1.2-fold in Nip, and 2.2- and 0.7-fold in U502, respectively, within the 1 d of hypoxic stress, but peak ABA production (1 d) occurred before proline accumulation (5 d) in both cultivars. Treatment with an ABA synthesis inhibitor (norflurazon, Norf) inhibited proline synthesis and simultaneously aggravated hypoxia-induced oxidative damage in the roots of both cultivars, but these effects were reversed by exogenous ABA application. Hypoxia plus Norf treatment also induced an increase in glutamate (the main precursor of proline). This indicates that proline accumulation is regulated by ABA-dependent signals under hypoxic stress. Moreover, genes involved in proline metabolism were differentially expressed between the two genotypes, with expression mediated by ABA under hypoxic stress. In Nip, hypoxia-induced proline accumulation in roots was attributed to the upregulation of OsP5CS2 and downregulation of OsProDH, whereas upregulation of OsP5CS1 combined with downregulation of OsProDH enhanced the proline level in U502. CONCLUSION: These results suggest that the high tolerance of the Nip cultivar is related to the high ABA level and ABA-mediated antioxidant capacity in roots. ABA acts upstream of proline accumulation by regulating the expression of genes encoding the key enzymes in proline biosynthesis, which also partly improves rice acclimation to hypoxic stress. However, other signaling pathways enhancing tolerance to hypoxia in the Nip cultivar still need to be elucidated.

The proline synthesis enzyme P5CS forms cytoophidia in Drosophila.

Compartmentation of enzymes via filamentation has arisen as a mechanism for the regulation of metabolism. In 2010, three groups independently reported that CTP synthase (CTPS) can assemble into a filamentous structure termed the cytoophidium. In searching for CTPS-interacting proteins, here we perform a yeast two-hybrid screening of Drosophila proteins and identify a putative CTPS-interacting protein, △1-pyrroline-5-carboxylate synthase (P5CS). Using the Drosophila follicle cell as the in vivo model, we confirm that P5CS forms cytoophidia, which are associated with CTPS cytoophidia. Overexpression of P5CS increases the length of CTPS cytoophidia. Conversely, filamentation of CTPS affects the morphology of P5CS cytoophidia. Finally, in vitro analyses confirm the filament-forming property of P5CS. Our work links CTPS with P5CS, two enzymes involved in the rate-limiting steps in pyrimidine and proline biosynthesis, respectively.

The combined effect of Cr(III) and NaCl determines changes in metal uptake, nutrient content, and gene expression in quinoa (Chenopodium quinoa Willd.).

Many areas of the world are affected simultaneously by salinity and heavy metal pollution. Halophytes are considered as useful candidates in remediation of such soils due to their ability to withstand both osmotic stress and ion toxicity deriving from high salt concentrations. Quinoa (Chenopodium quinoa Willd) is a halophyte with a high resistance to abiotic stresses (drought, salinity, frost), but its capacity to cope with heavy metals has not yet been fully investigated. In this pot experiment, we investigated phytoextraction capacity, effects on nutrient levels (P and Fe), and changes in gene expression in response to application of Cr(III) in quinoa plants grown on saline or non-saline soil. Plants were exposed for three weeks to 500 mg kg-1 soil of Cr(NO3)3·9H2O either in the presence or absence of 150 mM NaCl. Results show that plants were able tolerate this soil concentration of Cr(III); the metal was mainly accumulated in roots where it reached the highest concentration (ca. 2.6 mg g-1 DW) in the presence of NaCl. On saline soil, foliar Na concentration was significantly reduced by Cr(III). Phosphorus translocation to leaves was reduced in the presence of Cr(III), while Fe accumulation was enhanced by treatment with NaCl alone. A real-time RT-qPCR analysis was conducted on genes encoding for sulfate, iron, and phosphate transporters, a phytochelatin, a metallothionein, glutathione synthetase, a dehydrin, Hsp70, and enzymes responsible for the biosynthesis of proline (P5CS), glycine betaine (BADH), tocopherols (TAT), and phenolic compounds (PAL). Cr(III), and especially Cr(III)+NaCl, affected transcript levels of most of the investigated genes, indicating that tolerance to Cr is associated with changes in phosphorus and sulfur allocation, and activation of stress-protective molecules. Moderately saline conditions, in most cases, enhanced this response, suggesting that the halophytism of quinoa could contribute to prime the plants to respond to chromium stress.

Inhibiting both proline biosynthesis and lipogenesis synergistically suppresses tumor growth.

Cancer cells often proliferate under hypoxia and reprogram their metabolism. However, how to find targets to effectively block the hypoxia-associated metabolic pathways remains unclear. Here, we developed a tool to conveniently calculate electrons dissipated in metabolic transformations. Based on the law of conservation of electrons in chemical reactions, we further built up an electron balance model for central carbon metabolism, and it can accurately outline metabolic plasticity under hypoxia. Our model specifies that glutamine metabolism reprogrammed for biosynthesis of lipid and/or proline actually acts as the alternative electron bin to enable electron transfer in proliferating cells under hypoxia. Inhibition of both proline biosynthesis and lipogenesis can synergistically suppress cancer cell growth under hypoxia and in vivo tumor onset. Therefore, our model helps to reveal combinations of potential targets to inhibit tumor growth by blocking hypoxia-rewired metabolism and provides a useful tool for future studies on cancer metabolism.