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Glucaric acid is a value-added dicarboxylic acid that was synthesized mainly through the oxidation of glucose chemically. Due to the problems of the low selectivity, by-products, and highly polluting waste of this process, producing glucaric acid biologically has attracted great attention.

Malonic acid is an important dicarboxylic acid, which can be widely used in the fields of chemical industry, medicine and food. In this study, a recombinant

Adipic Acid (AA) is a valued platform chemical compound, which can be used as a precursor of nylon-6,6. Due to the generation of an enormous amount of nitric oxide metabolites and the growing depletion of oil resources as a result of AA production from a mixture of cyclohexanol and cyclohexanone, the microbial methods for synthesizing AA have attracted significant attention. Of the several AA-producing pathways, the reverse adipate degradation pathway in Thermobifida fusca (Tfu RADP) is reported to be the most efficient, which has been confirmed in Escherichia coli. In this study, the heterologous Tfu RADP was constructed for producing AA in S. cerevisiae by co-expressing genes of Tfu_0875, Tfu_2399, Tfu_0067, Tfu_1647, Tfu_2576, and Tfu_2576. The AA titer combined with biomass, cofactors and other by-products was all determined after fermentation. During batch fermentation in a shake flask, the maximum AA titer was 3.83 mg/L, while the titer increased to 10.09 mg/L during fed-batch fermentation in a 5-L bioreactor after fermentation modification.

To enhance the glucaric acid (GA) production in Saccharomyces cerevisiae, the Vitreoscilla hemoglobin was employed to reinforce cellular oxygen supplement. Additionally, the pH-free fermentation strategy was engaged to lower the cost brought by base feeding during the acid-accumulated and long-period glucaric acid production. Recombinant yeast Bga-4 was constructed harboring Vitreoscilla hemoglobin on the basis of previous Bga-3. Higher glucose uptake rate, growth rate, and ethanol reuse rate were achieved in Bga-4 in shake-flask fermentation than those in Bga-3. Furthermore, the fed-batch fermentation in a 5-L bioreactor was performed without pH control, resulting in a final glucaric acid titer of 6.38 g/L. Both the GA titer and biomass were enhanced along with the efficiency of ethanol re-utilization in the presence of VHb. Moreover, the absence of base feeding for long-period fermentation reduced production cost, which is meaningful for industrial applications.

Pimelic acid is an important seven-carbon dicarboxylic acid, which is broadly applied in various fields. The industrial production of pimelic acid is mainly through a chemical method, which is complicated and environmentally unfriendly. Herein, we found that pimelic acid could be biosynthesized by the reverse adipate-degradation pathway (RADP), a typical Claisen condensation reaction that could be applied to the arrangement of C-C bond. In order to strengthen the supply of glutaryl-CoA precursor, PA5530 protein was used to transport glutaric acid. Subsequently, we discovered that the enzymes in the BIOZ pathway are isoenzyme of the RADP pathway enzymes. By combining the isoenzymes of the two pathways, the titer of pimelic acid reached 36.7 mg ⋅ L

Photothermal therapy (PTT) has received constant attention as efficient cancer therapy method due to locally selective treatment and not affected by tumor microenvironment. In this study, a novel 880 nm near-infrared (NIR) laser triggered photothermal agent (PTA), 3TT-IC-4Cl, was used for PTT of tumor in deep tissue. Folic acid (FA) conjugated amphiphilic block copolymer (folic acid-polyethylene gly-col-poly (β-benzyl-L-aspartate)10, FA-PEG-PBLA10) was employed to encapsulate 3TT-IC-4Cl by nano-precipitation to form stable nanoparticles (TNPs), TNPs exhibit excellent photothermal stability and photothermal conversion efficiency. Furthermore, the in vitro results showed TNPs display excel-lent biocompatibility and significant phototoxicity. These results suggest that 880 nm triggered TNPs has great potential as an effective PTAs for photothermal therapy of tumor in deep tissue.

As an important dicarboxylic acids existing in nature, glucaric acid has been widely used in medical, health, and polymer materials industry, therefore it is considered as one of the "top value-added chemicals from biomass". In this study, using

Malonate is a platform chemical that has been utilized to synthesize many valuable chemical compounds. Here, Saccharomyces cerevisiae was metabolically engineered to produce malonate through the malonyl-CoA pathway. To construct the key step of converting malonyl-CoA to malonate, a native mitochondrial 3-hydroxyisobutyryl-CoA hydrolase gene EHD3 was mutated to target the cytoplasm and obtain malonyl-CoA hydrolase activity. The malonyl-CoA hydrolase activity of Ehd3 was achieved by mutating the malonyl-CoA binding site F121 to I121 and the active site E124 to seven amino acids (S/T/H/K/R/N/Q). We identified that the strain with E124S mutation had the highest malonate titer with 13.6 mg/L. Genomic integration of the mutant EHD3 and ACC1** to delta sequence sites was further explored to increase their reliable expression. Accordingly, a screening method with the work flow of fluorescence detection, shake-tube fermentation, and shake-flask fermentation was constructed to screen high copy delta sequences efficiently. The malonate titer was improved to 73.55 mg/L after screening the ∼1500 integrative strains, which was increased 4.4-folds than that of the episomal strain. We further engineered the strain by regulating the expression of key enzyme in the malonyl-CoA pathway to improve the precursor supply and inhibiting its competing pathways, and the final engineered strain LMA-16 produced 187.25 mg/L in the flask, 14-fold compared with the initial episomal expression strain. Finally, the combined efforts increased the malonate titer to 1.62 g/L in fed-batch fermentation.

Dicarboxylic acids are widely used in fine chemical and food industries as well as the monomer for polymerisation of high molecular material. Given the problems of environmental contamination and sustainable development faced by traditional production of dicarboxylic acids based on petrol, new approaches such as bio-based production of dicarboxylic acids drew more attentions. The yeast, Saccharomyces cerevisiae, was regarded as an ideal organism for bio-based production of dicarboxylic acids with high tolerance to acidic and hyperosmotic environments, robust growth using a broad range of substrates, great convenience for genetic manipulation, stable inheritance via sub-cultivation, and food compatibility. In this review, the production of major dicarboxylates via S. cerevisiae was concluded and the challenges and opportunities facing were discussed.Key Points• Summary of current production of major dicarboxylic acids by Saccharomyces cerevisiae.• Discussion of influence factors on four-carbon dicarboxylic acids production by Saccharomyces cerevisiae.• Outlook of potential production of five- and six-carbon dicarboxylic acids by Saccharomyces cerevisiae.

Zinc is essential for almost all living organisms, since it serves as a crucial cofactor for transcription factors and enzymes. However, it is toxic to cell growth when present in excess. The present work aims to investigate the toxicity mechanisms induced by zinc stress in yeast cells. To this end, 108 yeast single-gene deletion mutants were identified sensitive to 6 mM ZnCl2 through a genome-wide screen. These genes were predominantly related to the biological processes of vacuolar acidification and transport, polyphosphate metabolic process, cytosolic transport, the process utilizing autophagic mechanism. A result from the measurement of intracellular zinc content showed that 64 mutants accumulated higher intracellular zinc under zinc stress than the wild-type cells. We further measured the intracellular ROS (reactive oxygen species) levels of 108 zinc-sensitive mutants treated with 3 mM ZnCl2. We showed that the intracellular ROS levels in 51 mutants were increased by high zinc stress, suggesting their possible involvement in regulating ROS homeostasis in response to high zinc. The results also revealed that excess zinc could generate oxidative damage and then activate the expression of several antioxidant defenses genes. Taken together, the data obtained indicated that excess zinc toxicity might be mainly due to the high intracellular zinc levels and ROS levels induced by zinc stress in yeast cells. Our current findings would provide a basis to understand the molecular mechanisms of zinc toxicity in yeast cells.

Adipate is a linear C6 dicarboxylic acid, and is a crucial commercial material mainly used to produce the polymer nylon-6,6. In this study, the pathway producing adipate via a reverse reaction of degradation pathway of adipic acid was ported from Thermobifida fusca to Escherichia coli (E. coli). The pathway contains 6 genes: Tfu_0875, Tfu_2399, Tfu_0067, Tfu_1647, Tfu_2576 and Tfu_2577, which encodes β-ketothiolase, 3-hydroxyacyl-CoA dehydrogenase, 3-hydroxyadipyl-CoA dehydrogenase, 5-carboxy-2-pentenoyl-CoA reductase and adipyl-CoA synthetase, respectively. Of the genes in this pathway, Tfu_1647 is the limited step. Here, we constructed a homology model of 5-carboxy-2-pentenoyl-CoA reductase and found that Lys295 and Glu334 were the active sites. We carried out ten site-directed mutations of these two residues including E334D, K295R, K295Q, K295Y, K295 F, E334R, E334H, E334 K, E334 W, and E334 F. The enzymatic activity of Tfu_1647 in pTrc99A-0067-1647 of E334D, E334 F, and E334R were much higher than that in the control. The Km values of E334D, E334 F, and E334R were significantly reduced compared with the control. The strain with E334D had the highest adipic acid titer (0.23 g/L) with 5.8% of the theoretical yield. The rational reconstruction of 5-carboxy-2-pentenoyl-CoA reductase is a potential approach in improving the enzymatic activity and titer of adipate.

Towards future practical applications, it is necessary to develop the fluorescent sensors with only single fluorophore and single receptor for multiple-analytes detection. In the present work, a novel molecular sensor containing single receptor (the naphthol-azomethine-phenol framework) and single fluorophore (BODIPY), NAP-BODIPY, has been designed and synthesize. Single crystal X-ray diffraction analysis reveals the keto-enamine rather than the enol-imine constitution of the ortho-hydroxy naphthalene azomethine moiety in NAP-BODIPY. As a consequence, along with the conversion between acid and basic conditions in the mixed DMSO/H2O, the enol-keto tautomerization occurs in the ortho-hydroxy naphthalene azomethine moiety of NAP-BODIPY, resulting in a diverse change over the electronic absorption and fluorescence emission spectra. Moreover, addition of Cu2+ into NAP-BODIPY solution leads to an obvious red-shift in the maximum absorption and the naked-eye observed change from pale-orange to magenta turbid due to the metal-induced aggregation interaction. Nevertheless, adding Bi3+ into NAP-BODIPY solution induces a remarkable enhancement over the weak fluorescence emission owing to the inhibition of photo-induced electron transfer process. Subsequent addition of Cu2+ into the NAP-BODIPY-Bi3+ (1:10) system triggers a triple-signal change associated with the metal displacement. This, in combination with the fast responsive time of NAP-BODIPY to acid/base/Cu2+/Bi3+ (within several seconds), endows NAP-BODIPY an ultrafast-detecting sensor nature for multi-analytes. In particular, NAP-BODIPY can selectively stain lysosomes and therefore detect the pH change in live cells upon lysosomal acidification, revealing its great application potential for multiple-analytes.

The calcium/calcineurin signalling pathway is required for cell survival under various environmental stresses. Using Saccharomyces cerevisiae, we explored the mechanism underlying calcium-regulated homeostasis of intracellular reactive oxygen species (ROS). We found that deletion of acyltransferase Akr1 and C-5 sterol desaturase Erg3 increased the intracellular ROS levels and cell death, and this could be inhibited by the addition of calcium. The hexose transporter Hxt1 and the amino acid permease Agp1 play crucial roles in maintaining intracellular ROS levels, and calcium induced the expression of the HXT1 and AGP1 genes. The cytosolic calcium concentration was decreased in both the akr1Δ and erg3Δ mutants relative to wild-type cells, potentially lowering basal expression of HXT1 and AGP1. Moreover, the calcium/calcineurin signalling pathway also induced the expression of AKR1 and ERG3, indicating that Akr1 and Erg3 might perform functions that help yeast cells to survive under high calcium concentrations. Our results provided mechanistic insight into how calcium regulated intracellular ROS levels in yeast.

As a potentially toxic heavy metal, Cadmium (Cd) can cause endoplasmic reticulum and oxidative stress, and thus lead to cell death. To explore the mechanisms of Cd toxicity, we investigated the UPRE-lacZ expression, the intracellular reactive oxygen species (ROS) and cell death in the 151 Cd-sensitive mutants of Saccharomyces cerevisiae in response to Cd stress. We identified 101 genes regulating UPRE-lacZ expression were involved in preventing ROS production and/or cell death from increasing to high levels, while mutants for 72 genes caused both elevated ROS production and cell death, indicating the Cd-induced ROS production and cell death are mediated by UPR. Genes involved in cell wall integrity (CWI) pathway, vacuolar protein sorting (VPS) and vacuolar transport, calcium/calcineurin pathway and PHO pathways were all required for the Cd-induced UPR, intracellular ROS and cell death. To conclude, this study highlights the importance of Cd-induced UPR, intracellular ROS levels and cell death that may play crucial roles in Cd-induced toxicity.

Cadmium is a carcinogen that can induce ER stress, DNA damage, oxidative stress and cell death. The yeast mitogen-activated protein kinase (MAPK) signalling pathways paly crucial roles in response to various stresses. Here, we demonstrate that the unfolded protein response (UPR) pathway, the high osmolarity glycerol (HOG) pathway and the cell wall integrity (CWI) pathway are all essential for yeast cells to defend against the cadmium-induced toxicity, including the elevated ROS and cell death levels induced by cadmium. We show that the UPR pathway is required for the cadmium-induced phosphorylation of HOG_MAPK Hog1 but not for CWI_MAPK Slt2, while Slt2 but not Hog1 is required for the activation of the UPR pathway through the transcription factors of Swi6 and Rlm1. Moreover, deletion of HAC1 and IRE1 could promote the nuclear accumulation of Hog1, and increase the cytosolic and bud neck localisation of Slt2, indicating crucial roles of Hog1 and Slt2 in regulating the cellular process in the absence of UPR pathway. Altogether, our findings highlight the significance of these two MAPK pathways of HOG and CWI and their interrelationship with the UPR pathway in responding to cadmium-induced toxicity in budding yeast.

Manganese is a crucial cofactor for a wide range of enzymes in many living cells. However, excessive manganese can induce cellular toxicity by affecting a number of metabolic reactions and even cause severe neurological diseases in humans. To understand manganese homeostasis fully, a genome-scale screen was performed using the homozygous diploid yeast deletion mutant library. We identified 152 manganese-sensitive and 13 manganese-tolerant gene deletion mutations. We found that 62 of the manganese-sensitive mutants (40% of the total) accumulated higher intracellular manganese compared to wild type. Our results also reinforced the genetic functional link between manganese and calcium, and the addition of 100 mM CaCl2 confirmed that the manganese sensitivities of 103 (67.8 % of the total) strains could be inhibited by calcium. Finally, this study demonstrated that there might be some significant interactions between manganese and calcium regulated by the calcium/calcineurin signaling pathway through the P-type Ca2+- and Mn2+-transporting ATPase, Pmr1. Taken together, our current findings would provide new insights into the molecular causes of manganese toxicity in yeast cells.

Manganese is a crucial cofactor for a wide range of enzymes in many living cells. However, excessive manganese can induce cellular toxicity by affecting a number of metabolic reactions and even cause severe neurological diseases in humans. To understand manganese homeostasis fully, a genome-scale screen was performed using the homozygous diploid yeast deletion mutant library. We identified 152 manganese-sensitive and 13 manganese-tolerant gene deletion mutations. We found that 62 of the manganese-sensitive mutants (40% of the total) accumulated higher intracellular manganese compared to wild type. Our results also reinforced the genetic functional link between manganese and calcium, and the addition of 100 mM CaCl

Glucaric acid has been successfully produced in Escherichia coli and fungus. Here, we first analyzed the effects of different metal ions on glucaric acid production in the engineered Saccharomyces cerevisiae Bga-3 strain harboring the glucaric acid synthesis pathway. We found that magnesium ions could promote the growth rate of yeast cells, and thus, increase the glucaric acid production by elevating the glucose and myo-inositol utilization of Bga-3 strain. RNA-Seq transcriptome analysis results showed that the upregulation of genes involved in the gluconeogenesis pathway, as well as the downregulation of genes associated with the glycolysis pathway and pentose phosphate pathway in response to MgCl2 were all benefit for the enhancement of the glucose-6-phosphate flux, which was the precursor for myo-inositol and glucaric acid. In addition, we found that MgCl2 could also increase the activity of MIOX4, which was also crucial for glucaric acid synthesis. At last, a final glucaric acid titer of 10.6 g/L, the highest reported titer, was achieved in the fed-batch fermentation using a 5-L bioreactor by adding 100 mM MgCl2. Our findings will provide a new way of promoting the production of other chemicals in the engineered yeast cells.

To reduce dependence on petroleum, the biosynthesis of important chemicals from simple substrates using industrial microorganisms has attracted increased attention. Metabolic engineering of Saccharomyces cerevisiae offers a sustainable and flexible alternative for the production of various chemicals. As a key metabolic intermediate, malonyl-CoA is a precursor for many useful compounds. However, the productivity of malonyl-CoA derivatives is restricted by the low cellular level of malonyl-CoA and enzymatic performance. In this review, we focused on how to increase the intracellular malonyl-CoA level and summarize the recent advances in different metabolic engineering strategies for directing intracellular malonyl-CoA to the desired malonyl-CoA derivatives, including strengthening the malonyl-CoA supply, reducing malonyl-CoA consumption, and precisely controlling the intracellular malonyl-CoA level. These strategies provided new insights for further improving the synthesis of malonyl-CoA derivatives in microorganisms.

A cellulase hyperproducing mutant strain, JNDY-13, was obtained using the ARTP mutation system and with Trichoderma reesei RUT-C30 as the parent strain. Whole-genome sequencing of JNDY-13 confirmed that 105 of the 653 SNPs were point mutations, 336 mutations were deletions and 165 were insertions. Moreover, 99 mutations were insertions and duplications. Among all the mutations, the one that occurred in the galactokinase gene might be related to the production of cellulases in T. reesei JNDY-13. Moreover, the up-regulation of cellulase and hemicellulase genes in JNDY-13 might contribute to higher cellulases production. Under optimal conditions, the highest cellulase activity by batch fermentation reached 4.35 U/ml, and the highest activity of fed-batch fermentation achieved was 5.40 U/ml.

The glyoxylate shunt is a branch of the tricarboxylic acid (TCA) cycle which directly determines the synthesis of glycolate, and the balance between the glyoxylate shunt and TCA cycle is very important for the growth of Escherichia coli. In order to accumulate glycolate at high yield and titer, strategies for over-expressing glycolate pathway enzymes including isocitrate lyase (AceA), isocitrate dehydrogenase kinase/phosphatase (AceK) and glyoxylate reductase (YcdW) were analyzed. The genes encoding these three enzymes were transcribed under the control of promoter pTrc on pTrc99A, to form pJNU-3, which was harbored by strain Mgly1, resulting in strain Mgly13. Strain Mgly13 produced glycolate with 0.385 g/g-glucose yield (45.2% of the theoretical yield). Citrate synthase (GltA) converted excess acetyl-CoA and oxaloacetate to citrate and was over-expressed by pJNU-4 (pCDFDuet-1 backbone). Thus, the resulting strain Mgly134 produced glycolate with a 0.504 g/g-glucose yield (59.3% of the theoretical yield). We then eliminated the pathways involved in the degradation of glycolate, resulting in strain Mgly434, which produced glycolate with 92.9% of the theoretical yield. Following optimization of fermentation, the maximum glycolate titer from strain Mgly434 was 65.5 g/L.

Background: Sodium dodecyl sulfate (SDS) is one of the most widely used anionic alkyl sulfate surfactants. Toxicological information on SDS is accumulating, however, mechanisms of SDS toxicity regulation remain poorly understood. In this study, the relationship between the SDS-sensitive mutants and their intracellular ROS levels has been investigated. Results: Through a genome-scale screen, we have identified 108 yeast single-gene deletion mutants that are sensitive to 0.03% SDS. These genes were predominantly related to the cellular processes of metabolism, cell cycle and DNA processing, cellular transport, transport facilities and transport routes, transcription and the protein with binding function or cofactor requirement (structural or catalytic). Measurement of the intracellular ROS (reactive oxygen species) levels of these SDS-sensitive mutants showed that about 79% of SDS-sensitive mutants accumulated significantly higher intracellular ROS levels than the wild-type cells under SDS stress. Moreover, SDS could generate oxidative damage and up-regulate several antioxidant defenses genes, and some of the SDS-sensitive genes were involved in this process. Conclusion: This study provides insight on yeast genes involved in SDS tolerance and the elevated intracellular ROS caused by SDS stress. Our findings provide a basis to understand molecular mechanisms underlying the detoxification of SDS by yeast cells.

Additional file 1 : Table S1. Primers used in this study.

Adipic acid is an important dicarboxylic acid, which is an essential building block to synthesize nylon 6-6 fiber. Adipic acid is primarily synthesized from chemical plant, however, this process is associated with a number of environmental concerns including heavy pollution, toxic catalyst and harsh reaction conditions. A decent amount of adipic acid was produced by reconstructing the reversed adipate-degradation pathway (RADP) from Thermobifida fusca in Escherichia coli. However, IPTG was used in the previous study, which was not feasible in the fermentation industry. In this study, strong promoter-5'-UTR complexes (PUTR) were chosen to construct a highly efficient induction-free system to produce adipic acid. First, comparisons of various exogenous 5'-UTR Complexes, as well as a series of E. coli host strains, demonstrated that those genes using E. coli K12 MG1655 as the host strain produced the highest titer of adipic acid. Subsequently, optimizations were applied to enhance the titer of adipate biosynthesizing strains. The highest titer of adipate of 57.6 g L-1 was achieved by fed-batch fermentation. This work offers a better way to enhance the industrial titer of adipate.

Background: Sodium dodecyl sulfate (SDS) is one of the most widely used anionic alkyl sulfate surfactants. Toxicological information on SDS is accumulating, however, mechanisms of SDS toxicity regulation remain poorly understood. In this study, the relationship between the SDS-sensitive mutants and their intracellular ROS levels has been investigated. Results : Through a genome-scale screen, we have identified 108 yeast single-gene deletion mutants that are sensitive to 0.03% SDS. These genes were predominantly related to the cellular processes of metabolism, cell cycle and DNA processing, cellular transport, transport facilities and transport routes, transcription and the protein with binding function or cofactor requirement (structural or catalytic). We further measured the intracellular ROS (reactive oxygen species) levels of 108 SDS-sensitive mutants treated with 0.015% SDS. The results showed that 85 SDS-sensitive mutants accumulated significantly higher intracellular ROS levels under SDS stress than the wild-type cells. Moreover, SDS could generate oxidative damage and up-regulate several antioxidant defenses genes, and some of the SDS-sensitive genes were involved in this process. Conclusion: This study provides insight on yeast genes involved in SDS tolerance and the elevated intracellular ROS caused by SDS stress. Our findings provide a basis to understand molecular mechanisms underlying the detoxification of SDS by yeast cells.

Bioaugmentation is an effective treatment method to reduce nitrogenous pollutants from wastewater. A strain of DYTN-1, which could effectively remove TN from sewage, was isolated from the sludge of a wastewater treatment plant and was identified as Paracoccus denitrificans. The TN in wastewater reduced to

Background Cobalt is an important metal cofactor of many living cells. However, excessive cobalt is toxic and can cause cell death and even several diseases in humans. Saccharomyces cerevisiae is a useful tool for studying metal homeostasis and many of the genes and pathways are highly conserved in higher eukaryotes including humans. Methods The intracellular cobalt and reactive oxygen species (ROS) levels were measured by an atomic absorption spectrometer and DHE staining method, respectively. The expression of genes involved in scavenging oxidative stress was tested by qPCR method, while the expression of UPRE-lacZ report gene was analyzed via β-galactosidase activity assay. Results Using a genome-scale genetic screen, 153 cobalt-sensitive and 37 cobalt-tolerant gene deletion mutants were identified from Saccharomyces cerevisiae. We showed that 101 of the cobalt-sensitive mutants accumulated higher intracellular cobalt compared to wild-type. The intracellular ROS levels in 112 of the mutants were induced by cobalt, which might be caused by the decreased expression of genes involved in scavenging oxidative stress in response to cobalt. Moreover, more than one-third of the cobalt-sensitive mutants were also sensitive to tunicamycin, and cobalt stress might induce the unfolded protein response (UPR) through serine/threonine kinase and endoribonuclease Ire1. Conclusions This study reinforced the fact that cobalt toxicity might be due to the high intracellular cobalt and ROS levels, and the endoplasmic reticulum stress responses induced by cobalt. General significance Elucidating the toxicity mechanisms of cobalt stress response will help reveal new routes for the treatment of the diseases induced by cobalt.

BackgroundSodium dodecyl sulfate (SDS) is one of the most widely used anionic alkyl sulfate surfactants. Toxicological information on SDS is accumulating, however, mechanisms of SDS toxicity regulation remain poorly understood. In this study, the relationship between the SDS-sensitive mutants and their intracellular ROS levels has been investigated.ResultsThrough a genome-scale screen, we have identified 108 yeast single-gene deletion mutants that are sensitive to 0.03% SDS. These genes were predominantly related to the cellular processes of metabolism, cell cycle and DNA processing, cellular transport, transport facilities and transport routes, transcription and the protein with binding function or cofactor requirement (structural or catalytic). Measurement of the intracellular ROS (reactive oxygen species) levels of these SDS-sensitive mutants showed that about 79% of SDS-sensitive mutants accumulated significantly higher intracellular ROS levels than the wild-type cells under SDS stress. Moreover, SDS could generate oxidative damage and up-regulate several antioxidant defenses genes, and some of the SDS-sensitive genes were involved in this process.ConclusionThis study provides insight on yeast genes involved in SDS tolerance and the elevated intracellular ROS caused by SDS stress, which is a potential way to understand the detoxification mechanisms of SDS by yeast cells.

Figure S1. Shake flask fermentation of Mgly624 in different concentrations of organic nitrogen source. Figure S2. GO histogram of DEGs. Figure S3. Shake flask fermentation of Mgly624 in different concentrations of glutamate. Figure S4. Growth curve following Fe3+ addition to Mgly624 cultures. Table S1. The stoichiometry of producing glycolate from glucose in Mgly624. Table S2. Significantly altered genes in the Group B vs. C comparison. Table S3. Description of significantly altered genes related to N-regulation and amino acids metabolism in the Group B vs. C comparison. (DOCX 207 kb)

A cellulase hyperproducing mutant strain, JNDY-13, was obtained using the ARTP mutation system and with

Saccharomyces cerevisiae Rch1 is structurally similar to both the vertebrate solute carrier SLC10A7 and Candida albicans Rch1. We show here that ScRCH1 is a functional homolog of CaRCH1. In S. cerevisiae, overexpression of ScRCH1 suppresses, but deletion of ScRCH1 does not affect, the lithium and rapamycin tolerance of pmr1 cells. Overexpression of ScRCH1 reduces expression of ENA1, prevents sustained accumulation of cytosolic calcium and reduces the activation level of calcium/calcineurin signaling in pmr1 cells. Therefore, similar to the situation in the pathogen C. albicans, ScRch1 negatively regulates the cytosolic homeostasis in response to high levels of extracellular calcium. ScRch1 proteins distribute as multiple foci in the plasma membrane prior to cell division, move toward and concentrate at the bud neck as the bud grows in size, and disperse again along the plasma membrane immediately prior to cytokinesis. Furthermore, our genetic and biochemical data also demonstrate that transcriptional expression of RCH1 is positively regulated by calcium/calcineurin signaling through the sole CDRE element in its promoter.

Background Glucaric acid is a high-value-added chemical that can be used in various fields. Because chemical oxidation of glucose to produce glucaric acid is not environmentally friendly, microbial production has attracted increasing interest recently. Biological pathways to synthesize glucaric acid from glucose in both Escherichia coli and Saccharomyces cerevisiae by co-expression of genes encoding myo-inositol-1-phosphate synthase (Ino1), myo-inositol oxygenase (MIOX), and uronate dehydrogenase (Udh) have been constructed. However, low activity and instability of MIOX from Mus musculus was proved to be the bottleneck in this pathway. Results A more stable miox4 from Arabidopsis thaliana was chosen in the present study. In addition, high copy delta-sequence integration of miox4 into the S. cerevisiae genome was performed to increase its expression level further. Enzymatic assay and quantitative real-time PCR analysis revealed that delta-sequence-based integrative expression increased MIOX4 activity and stability, thus increasing glucaric acid titer about eight times over that of episomal expression. By fed-batch fermentation supplemented with 60 mM (10.8 g/L) inositol, the multi-copy integrative expression S. cerevisiae strain produced 6 g/L (28.6 mM) glucaric acid from myo-inositol, the highest titer that had been ever reported in S. cerevisiae. Conclusions In this study, glucaric acid titer was increased to 6 g/L in S. cerevisiae by integrating the miox4 gene from A. thaliana and the udh gene from Pseudomonas syringae into the delta sequence of genomes. Delta-sequence-based integrative expression increased both the number of target gene copies and their stabilities. This approach could be used for a wide range of metabolic pathway engineering applications with S. cerevisiae. Electronic supplementary material The online version of this article (10.1186/s12934-018-0914-y) contains supplementary material, which is available to authorized users.

Additional file 1: Table S1. Primers used in this study. Figure S1. MIOX4 activity of the episomal expression plasmid in the wild-type strain and opi1 mutant strain with myo-inositol. All experiments were performed in triplicates and the error bar represented mean Âą standard deviation. Figure S2. myo-inositol residue in shake flask (A) and fed-batch (B) cultures when fed 60 mM (10.8 g/L) myo-inositol to the culture. All experiments were performed in triplicates and the error bar represented mean Âą standard deviation.

ESCRT components function to form multivesicular bodies for sorting of proteins destined to the yeast vacuole. The calcium hypersensitivity of ESCRT mutants is mainly due to repressed expression of PMR1 through the Rim101/Nrg1 pathway in budding yeast. Here, we show that overexpression of PMC1 and its negative regulator gene NYV1 suppresses and increases calcium hypersensitivity of ESCRT mutants, respectively. Consistently, deletion of NYV1 suppresses their calcium hypersensitivity. Expression of NYV1 is dramatically reduced in ESCRT mutants. Promoter analysis demonstrates that both Nrg1 and Mig1 repress NYV1 expression. Deletion of ESCRTs increases Nrg1 binding, but not Mig1-binding, to the NYV1 promoter. Deletion of MIG1 increases calcium sensitivity of ESCRT mutants due to derepression of NYV1 expression.

The activated retrograde (RTG) pathway controls transcription of target genes through a heterodimer of transcription factors, Rtg1 and Rtg3, in Saccharomyces cerevisiae . Here, we have identified the sole homologous gene CaRTG3 that encodes a protein of 520 amino acids with characteristics of the basic helix–loop–helix/leucine zipper (bHLH/Zip) family in Candida albicans . Deletion of CaRTG3 results in C. albicans cells being sensitive to high concentrations of calcium and lithium cations as well as sodium dodecyl sulfate and activates the calcium/calcineurin signaling pathway in C. albicans cells. CaRTG3 is also involved in the tolerance of C. albicans cells to the antifungal drugs azoles and terbinafine, but not to the antifungal drugs casponfungin and amphotericin B as well as the cell-wall-damaging reagents Calcoflour White and Congo red. In contrast to ScRtg3, CaRtg3 is not involved in the osmolar response and is constitutively localized in the nucleus. However, deletion of CaRTG3 results in a delay in serum-induced filamentation of C. albicans cells. Therefore, CaRtg3 plays a role in tolerance to cations and antifungal drugs as well as serum-induced filamentation in C. albicans .

Here we have identified 120 gene deletion mutants that are sensitive to 0.4M calcium in Saccharomyces cerevisiae. Twenty-seven of these mutants are of genes involved in the vacuolar protein sorting pathway, including those encoding the seven components of the ESCRT complexes, and ten of them encode the components and assembly factors of the vacuolar H+-ATPase. Both Mediator and Paf1 complexes modulating the activity of the general transcription machinery are involved in the calcium sensitivity of yeast cells. Most of these mutants show elevated intracellular calcium contents in response to calcium stress. The calcium sensitivity of 106 mutants can be completely suppressed by 10mM Mg2+, 56 of which can also be suppressed by the inhibitor of calcineurin, cyclosporine A. Therefore, the calcium sensitivity of nearly a half of these 120 mutations is at least partially due to the activation of calcineurin and can be modulated by magnesium ion.

The Vcx1-M1 mutant is known to confer calcineurin-dependent Mn2+tolerance in budding yeast. Here, we demonstrate that another Vcx1 mutant, Vcx1-D1 with calcineurin-independent vacuolar Ca2+/H+exchanger activity, confers calcineurin-independent Mn2+tolerance. Unlike Vcx1-M1, the Mn2+tolerance conferred by Vcx1-D1 is dependent on the presence of Pmr1 or Pmc1. The Pmr1-dependent Mn2+tolerance of Vcx1-D1 requires the presence of calcineurin but not the functioning of the Ca2+/calcineurin signaling pathway. Similar to the wild-type Vcx1, C-terminally green fluorescent protein tagged Vcx1-D1 and Vcx1-M1 mutants localize to the endoplasmic reticulum instead of its normal vacuolar destination, but they remain functional in Ca2+sensitivity and Mn2+tolerance.

Type 2C protein phosphatases (PP2C) are monomeric enzymes that require magnesium or manganese ions for their activities. There are seven PP2C genes in Candida albicans . Here, we demonstrate that CaPTC5 encodes a mitochondrial PP2C enzyme. Expression of CaPTC5 transcripts remains constant during the serum-induced hyphal development. Deletion of CaPTC5 does not affect the in vitro filamentation but renders C. albicans cells sensitive to terbinafine and cadmium, and this sensitivity is complemented by the Saccharomyces cerevisiae ScPTC5 . Deletion of CaPTC6 does not have any additive effect on, but deletion of CaPTC7 blocks, the terbinafine sensitivity owing to deletion of CaPTC5 . In addition, we have shown that deletion of CaPTC6 also renders C. albicans cells sensitive to cadmium, while deletion of CaPTC7 leads to a high cadmium tolerance, and this tolerance is abolished by further deletion of CaPTC5 or CaPTC6 . Furthermore, double deletion of CaPTC6 and CaPTC7 renders C. albicans cells more tolerant to azoles, but deletion of CaPTC5 and CaPTC7 slightly increases the azole sensitivity of C. albicans cells. Our results demonstrate that three mitochondrial PP2C genes CaPTC5 , CaPTC6 and CaPTC7 interact differentially in the response of C. albicans cells to antifungal drugs and cadmium.

The endosomal sorting complex required for transport (ESCRT) complexes function to form multivesicular bodies for sorting of proteins destined for the yeast vacuole or the mammalian lysosome. ESCRT components are well conserved in eukaryotes, and their mutations cause neurodegenerative diseases and other cellular pathologies in humans. PMR1 is the orthologous gene of two human genes for calcium pumps secretory pathway Ca(2+)-ATPase (SPCA1, ATP2C1) and sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA, ATP2A2), which are mutated in Hailey-Hailey and Darier genetic diseases, respectively. Here we show that deletion mutation of ESCRT components Snf7, Snf8, Stp22, Vps20, Vps25, Vps28, or Vps36 activates the calcium/calcineurin signaling in yeast cells, but surprisingly leads to a nearly 50% reduction in expression of the ER/Golgi calcium pump gene PMR1 independent of calcium stress. These ESCRT mutants are known to have a defect in Rim101 activation. Ectopic expression of a constitutively active form of Rim101 or further deletion of NRG1 in these mutants partially suppresses their calcium hypersensitivity. Deletion of NRG1 also completely rescues the expression of PMR1 in these mutants to the level of the wild type. Promoter mutagenesis, gel electrophoretic mobility shift assay, and chromatin immunoprecipitation analysis demonstrate that Nrg1 binds to two motifs in the PMR1 promoter. In addition, expression of PMR1 under the control of its promoters with mutated Nrg1-binding motifs suppresses the calcium hypersensitivity of these ESCRT mutants. Collectively, these data have uncovered a function of ESCRT components in regulating PMR1 expression through the Nrg1/Rim101 pathway. Our findings provide important clues for understanding human diseases related to calcium homeostasis.