C4-dicarboxylate Transporter Research Articles

Reversal of carbapenem resistance in Pseudomonas aeruginosa by camelid single domain antibody fragment (VHH) against the C4-dicarboxylate transporter.

Antimicrobial resistance is emerging as the new healthcare crisis necessitating the development of newer classes of drugs using non-traditional approaches. Pseudomonas aeruginosa, one of the most common pathogens involved in nosocomial infections, is extremely difficult to treat even with the last resort frontline drug, the carbapenems. As the pathogen has the ability to acquire resistance to new small-molecule antibiotics, being deployed, a novel biological approach has been tried using antibody fragments in combination with carbapenems and β-lactams as adjunct therapy for an enduring solution to the problem. In this study, we developed a camelid antibody fragment (VHH) library against P. aeruginosa and isolated a highly potent hit, PsC23. Mass spectrometry identified the target to be a component of the C4-dicarboxylate transporter that feeds metabolites to the glyoxylate shunt particularly under conditions of oxidative stress. PsC23 is bacteriostatic at a concentration of 1.66 µM (25 µg ml-1) and shows a synergistic effect with both the classes of drugs at an effective concentration of 100-200 nM (1.5-3.0 µg ml-1) when co administered with them. In combination with meropenem the VHH completely cleared the infection from a neutropenic mouse with a carbapenem-resistant P. aeruginosa systemic infection. Blocking the glyoxylate shunt by PsC23 resulted in disruption of energy transduction due to a respiratory shift to the oxygen-depleted TCA cycle causing inhibition of efflux and increased free radical generation from carbapenems and β-lactams exerting a strong bactericidal effect that reversed the resistance to multiple unrelated drugs.

CopG1, a Novel Transcriptional Regulator Affecting Symbiosis in Bradyrhizobium sp. SUTN9-2.

The symbiotic interaction between leguminous and Bradyrhizobium sp. SUTN9-2 mainly relies on the nodulation process through Nod factors (NFs), while the type IV secretion system (T4SS) acts as an alternative pathway in this symbiosis. Two copies of T4SS (T4SS1 and T4SS2) are located on the chromosome of SUTN9-2. ΔT4SS1 reduces both nodule number and nitrogenase activity in all SUTN9-2 nodulating legumes. The functions of three selected genes (copG1, traG1, and virD21) within the region of T4SS1 were examined. We generated deleted mutants and tested them in Vigna radiata cv. SUT4. ΔtraG1 and ΔvirD21 exhibited lower invasion efficiency at the early stages of root infection but could be recently restored. In contrast, ΔcopG1 completely hindered nodule organogenesis and nitrogenase activity in all tested legumes. ΔcopG1 showed low expression of the nodulation gene and ttsI but exhibited high expression levels of the T4SS genes, traG1 and trbE1. The secreted proteins from ΔT4SS1 were down-regulated compared to the wild-type. Although ΔcopG1 secreted several proteins after flavonoid induction, T3SS (nopP and nopX) and the C4-dicarboxylate transporter (dct) were not detected. These results confirm the crucial role of the copG1 gene as a novel key regulator in the symbiotic relationship between SUTN9-2 and legumes.

Metabolic proteins with crucial roles in Edwardsiella tarda antioxidative adaptation and intracellular proliferation.

Edwardsiella tarda is a severe fish pathogen, featured by its capacity to live inside host phagocytes. For intracellular survival, it is crucial for E. tarda to neutralize the deleterious effect of host reactive oxygen species (ROS). Accumulating evidence suggests that bacterial metabolism is closely connected to oxidative resistance. However, the roles of E. tarda metabolic proteins in antioxidative adaptation and intracellular proliferation remain elusive. In this study, we performed a proteomic analysis on E. tarda and identified 111 proteins responsive to H2O2-mediated oxidative stress. Based on this data, we further obtained eight crucial proteins, including seven metabolic proteins, for E. tarda antioxidation and intracellular infection. Among them, two C4-dicarboxylate transporters were found necessary for E. tarda to disseminate in fish tissues. Furthermore, the substrate of the two transporters was identified as L-aspartate, which was proven to be essential for the full antioxidative capacity of E. tarda. Our results indicate that reprogramming the metabolic flux to the production of pyruvate, a ketoacid capable of neutralizing ROS, was likely a pivotal strategy of E. tarda to survive the oxidative environments inside host cells. Together, the findings of this study highlight the significance of metabolic reprogramming for bacterial redox homeostasis and intracellular infection. IMPORTANCE Edwardsiella tarda is a significant fish pathogen that can live in challenging environments of reactive oxygen species (ROS), such as inside the phagocytes. Metabolic reconfiguration has been increasingly associated with bacterial oxidative tolerance and virulence. However, the metabolic proteins of E. tarda involved in such processes remain elusive. By proteomic analysis and functional characterization of protein null mutants, the present study identified eight crucial proteins for bacterial oxidative resistance and intracellular infection. Seven of them are metabolic proteins dictating the metabolic flux toward the generation of pyruvate, a key metabolite capable of scavenging ROS molecules. Furthermore, L-aspartate uptake, which can fuel the pyruvate generation, was found essential for the full antioxidative capacity of E. tarda. These findings identified seven metabolic proteins involved in bacterial oxidative adaptation and indicate that metabolic reprogramming toward pyruvate was likely a pivotal strategy of bacteria for antioxidative adaptation and intracellular survival.

Metabolic Engineering of Trichoderma reesei for l-Malic Acid Production.

l-Malic acid has various applications in the chemical and food industries. The filamentous fungus Trichoderma reesei is known to be an efficient enzyme producer. Here, through metabolic engineering, T. reesei was constructed for the first time as an excellent cell factory for l-malic acid production. The heterologous overexpression of genes encoding the C4-dicarboxylate transporter from Aspergillus oryzae and Schizosaccharomyces pombe initiated l-malic acid production. The overexpression of pyruvate carboxylase from A. oryzae in the reductive tricarboxylic acid pathway further increased both the titer and yield of l-malic acid, resulting in the highest titer reported in a shake-flask culture. Furthermore, the deletion of malate thiokinase blocked l-malic acid degradation. Finally, the engineered T. reesei strain produced 220.5 g/L of l-malic acid in a 5 L fed-batch culture (productivity of 1.15 g/L/h). A T. reesei cell factory was created for the efficient production of l-malic acid.

Genome-guided comparative in planta transcriptome analyses for identifying cross-species common virulence factors in bacterial phytopathogens.

Plant bacterial disease is a complex outcome achieved through a combination of virulence factors that are activated during infection. However, the common virulence factors across diverse plant pathogens are largely uncharacterized. Here, we established a pan-genome shared across the following plant pathogens: Burkholderia glumae, Ralstonia solanacearum, and Xanthomonas oryzae pv. oryzae. By overlaying in planta transcriptomes onto the pan-genome, we investigated the expression profiles of common genes during infection. We found over 70% of identical patterns for genes commonly expressed by the pathogens in different plant hosts or infection sites. Co-expression patterns revealed the activation of a signal transduction cascade to recognize and respond to external changes within hosts. Using mutagenesis, we uncovered a relationship between bacterial virulence and functions highly conserved and shared in the studied genomes of the bacterial phytopathogens, including flagellar biosynthesis protein, C4-dicarboxylate ABC transporter, 2-methylisocitrate lyase, and protocatechuate 3,4-dioxygenase (PCD). In particular, the disruption of PCD gene led to attenuated virulence in all pathogens and significantly affected phytotoxin production in B. glumae. This PCD gene was ubiquitously distributed in most plant pathogens with high homology. In conclusion, our results provide cross-species in planta models for identifying common virulence factors, which can be useful for the protection of crops against diverse pathogens.

C4-dicarboxylate metabolons: interaction of C4-dicarboxylate transporters of Escherichia coli with cytosolic enzymes.

Metabolons represent the structural organization of proteins for metabolic or regulatory pathways. Here, the interaction of fumarase FumB, aspartase AspA, and L-tartrate dehydratase TtdAB with the C4-dicarboxylate (C4-DC) transporters DcuA, DcuB, DcuC, and the L-tartrate transporter TtdT of Escherichia coli was tested by a bacterial two-hybrid (BACTH) assay in situ, or by co-chromatography using mSPINE (membrane Streptavidin protein interaction experiment). From the general C4-DC transporters, DcuB interacted with FumB and AspA, DcuA with AspA, whereas DcuC interacted with neither FumB nor AspA. Moreover, TtdT did not interact with TtdAB. The fumB-dcuB, the dcuA-aspA, and the ttdAB-ttdT genes encoding the respective proteins colocalize on the genome and each pair of genes forms cotranscripts, whereas the dcuC gene lies alone. The data suggest the formation of DcuB/FumB and DcuB/AspA metabolons for the uptake of L-malate, or L-aspartate, and their conversion to fumarate for fumarate respiration and excretion of the product succinate. The DcuA/AspA metabolon catalyzes uptake and conversion of L-aspartate to fumarate coupled to succinate excretion. The DcuA/AspA metabolon provides ammonia at the same time for nitrogen assimilation (ammonia shuttle). On the other hand, TtdT and TtdAB are not organized in a metabolon. Reasons for the formation (DcuA/AspA, DcuB/FumB, and DcuB/AspA) or nonformation (DcuC, TtdT, and TtdAB) of metabolons are discussed based on their metabolic roles.

Enterococcus faecalis V583 cell membrane protein expression to alkaline stress.

Enterococcus faecalis is able to adapt to alkaline conditions and is commonly recovered from teeth in which endodontic treatment has failed. The role that E. faecalis membrane proteins play in survival strategies to extreme alkaline conditions is unclear. We grew E. faecalis V583 in a chemostat at pH 8 and 11 at one-tenth the organism’s relative maximum growth rate. Following membrane shaving, isotope-coding protein labels were added at the peptide level to samples and then combined. The relative proportion of membrane proteins were identified using LC-ESI mass spectrometry and MaxQuant analysis. Ratios of membrane proteins were log2 transformed, with proteins deviating by more than 1 SD of the mean considered to be up- or down-regulated. A total of six proteins were up-regulated in pH 11 including: EF0669 (polysaccharide biosynthesis family); EF1927 (glycerol uptake facilitator), and EF0114 (glycosyl hydrolase). A total of five proteins were down-regulated including: EF0108 (C4-dicarboxylate transporter); EF1838 (PTS system IIC component); EF0456 (PTS system IID component); and EF0022 (PTS mannose-specific IID component). In extreme alkaline conditions, the membrane proteins of E. faecalis seem to be involved in a shift of carbohydrate metabolism from the PTS system to glycerol, which supports the formation of a protective capsule protecting the cell.

Genomic rearrangements in the aspA-dcuA locus of Propionibacterium freudenreichii are associated with aspartase activity

Propionibacterium freudenreichii is crucial in Swiss-type cheese manufacture. Classic propionic acid fermentation yields the nutty flavor and the typical eyes. Co-metabolism of aspartate pronounces the flavor of the cheese; however, it also increases the size of the eyes, which can induce splitting and reduce the cheese quality. Aspartase (EC 4.3.1.1) catalyzes the deamination of aspartate, yielding fumarate and ammonia. The aspartase activity varies considerably among P. freudenreichii strains. Here, the correlation between aspartase activity and the locus of aspartase-encoding genes (aspA ) and dcuA encoding the C4-dicarboxylate transporter was investigated in 46 strains to facilitate strain selection for cheese culture. Low aspartase activity was correlated with a particular genomic rearrangement: low in vitro aspartase activity always occurred in strains with gene clusters aspA - dcuA where the dcuA was frameshifted, producing a stop codon or was disrupted by an ISL3-like element. The low aspartase activity could be due to the protein sequence of the aspartase or a dysfunctional DcuA. The highest values of aspartase activity were detected in strains with aspA1 - aspA2-dcuA with a DcuA sequence sharing 99.07 – 100% identity with the DcuA sequence of strain DSM 20271 T and an additional C4-dicarboxylate transporter belonging to the DcuAB family.

Escherichia coli Dcu C4-dicarboxylate transporters dependent proton and potassium fluxes and FOF1-ATPase activity during glucose fermentation at pH 7.5

During fermentation in Escherichia coli succinate is transported via Dcu transporters, encoded dcuA, dcuB, dcuC and dcuD although the role of DcuD protein has not been elucidated yet. It has been shown contribution of Dcu transporters in the N,N’-dicyclohexylcarbodiimide (DCCD) sensitive proton and potassium transport through the cytoplasmic membrane and membrane-associated ATPase activity. Total H± efflux was decreased ~ 40% while K± uptake was absent in dcuD mutant. DCCD-sensitive H± flux was absent in dcuD nevertheless it was increased ~ 3 fold in dcuACB. K± uptake in dcuACB was stimulated ~ 30% compared to wild type but in DCCD assays K± ions were effluxed with the rate of 0.15 mmol/min per 109 cells/ml. In dcuACB mutant membrane potential (ΔΨ) was ~ 30 mV higher than in wild type. dcuD gene expression was increased in the dcuACB mutant respect to wild type at pH 7.5 (~120%), suggesting that an increment of DcuD activity compensates the lack of DcuA, DcuC and DcuB carriers. It can be concluded that active DcuD is important for H± efflux via the FOF1-ATPase and K± uptake at pH 7.5. In addition, DcuA, DcuB and DcuC transporters are crucial for regulating DCCD-sensitive K± transport and ΔΨ in E. coli.

Evolutionary, genomic, and biogeographic characterization of two novel xenobiotics-degrading strains affiliated with Dechloromonas

Xenobiotics are generally known as man-made refractory organic pollutants widely distributed in various environments. For exploring the bioremediation possibility of xenobiotics, two novel xenobiotics-degrading strains affiliated with Azonexaceae were isolated. We report here the phylogenetics, genome, and geo-distribution of a novel and ubiquitous Azonexaceae species that primarily joins in the cometabolic process of some xenobiotics in natural communities. Strains s22 and t15 could be proposed as a novel species within Dechloromonas based on genomic and multi-phylogenetic analysis. Pan-genome analysis showed that the 63 core genes in Dechloromonas include genes for dozens of metabolisms such as nitrogen fixation protein (nifU), nitrogen regulatory protein (glnK), dCTP deaminase, C4-dicarboxylate transporter, and fructose-bisphosphate aldolase. Strains s22 and t15 have the ability to metabolize nitrogen, including nitrogen fixation, NirS-dependent denitrification, and dissimilatory nitrate reduction. Moreover, the novel species possesses the EnvZ-OmpR two-component system for controlling osmotic stress and QseC-QseB system for quorum sensing to rapidly sense environmental changes. It is intriguing that this new species has a series of genes for the biodegradation of some xenobiotics such as azathioprine, 6-Mercaptopurine, trinitrotoluene, chloroalkane, and chloroalkene. Specifically, glutathione S-transferase (GST) and 4-oxalocrotonate tautomerase (praC) in this novel species play important roles in the detoxification metabolism of some xenobiotics like dioxin, trichloroethene, chloroacetyl chloride, benzo[a]pyrene, and aflatoxin B1. Using data from GenBank, DDBJ and EMBL databases, we also demonstrated that members of this novel species were found globally in plants (e.g. rice), guts (e.g. insect), pristine and contaminated regions. Given these data, Dechloromonas sp. strains s22 and t15 take part in the biodegradation of some xenobiotics through key enzymes.

Salmonella profits from immunometabolism

Host Defense Extensive metabolic rewiring occurs in various immune cells during the course of infection. Whether these changes can be exploited by intracellular pathogens remains an open question. Rosenberg et al. report that infection with Salmonella enterica serovar Typhimurium ( S. Tm) induces the accumulation of the metabolite succinate in macrophages (see the Perspective by Lynch and Lesser). This key intermediate in the citric acid cycle activates virulence genes in S. Tm, leading to microbial resistance. Moreover, the active transport of succinate through the C4-dicarboxylate transporter DcuB is required for S. Tm virulence and survival within macrophages. Sensing of citric acid cycle intermediates may more generally serve as a cue to initiate the resistance programs of intracellular pathogens. Science , this issue p. [400][1]; see also p. [344][2] [1]: /lookup/doi/10.1126/science.aba8026 [2]: /lookup/doi/10.1126/science.abf8414

Metabolic engineering of Aspergillus niger via ribonucleoprotein-based CRISPR\u2013Cas9 system for succinic acid production from renewable biomass

BackgroundSuccinic acid has great potential to be a new bio-based building block for deriving a number of value-added chemicals in industry. Bio-based succinic acid production from renewable biomass can provide a feasible approach to partially alleviate the dependence of global manufacturing on petroleum refinery. To improve the economics of biological processes, we attempted to explore possible solutions with a fungal cell platform. In this study, Aspergillus niger, a well-known industrial production organism for bio-based organic acids, was exploited for its potential for succinic acid production.ResultsWith a ribonucleoprotein (RNP)-based CRISPR–Cas9 system, consecutive genetic manipulations were realized in engineering of the citric acid-producing strain A. niger ATCC 1015. Two genes involved in production of two byproducts, gluconic acid and oxalic acid, were disrupted. In addition, an efficient C4-dicarboxylate transporter and a soluble NADH-dependent fumarate reductase were overexpressed. The resulting strain SAP-3 produced 17 g/L succinic acid while there was no succinic acid detected at a measurable level in the wild-type strain using a synthetic substrate. Furthermore, two cultivation parameters, temperature and pH, were investigated for their effects on succinic acid production. The highest amount of succinic acid was obtained at 35 °C after 3 days, and low culture pH had inhibitory effects on succinic acid production. Two types of renewable biomass were explored as substrates for succinic acid production. After 6 days, the SAP-3 strain was capable of producing 23 g/L and 9 g/L succinic acid from sugar beet molasses and wheat straw hydrolysate, respectively.ConclusionsIn this study, we have successfully applied the RNP-based CRISPR–Cas9 system in genetic engineering of A. niger and significantly improved the succinic acid production in the engineered strain. The studies on cultivation parameters revealed the impacts of pH and temperature on succinic acid production and the future challenges in strain development. The feasibility of using renewable biomass for succinic acid production by A. niger has been demonstrated with molasses and wheat straw hydrolysate.

Solvent accessibility changes in a Na+-dependent C4-dicarboxylate transporter suggest differential substrate effects in a multistep mechanism

The divalent anion sodium symporter (DASS) family (SLC13) plays critical roles in metabolic homeostasis, influencing many processes, including fatty acid synthesis, insulin resistance, and adiposity. DASS transporters catalyze the Na+-driven concentrative uptake of Krebs cycle intermediates and sulfate into cells; disrupting their function can protect against age-related metabolic diseases and can extend lifespan. An inward-facing crystal structure and an outward-facing model of a bacterial DASS family member, VcINDY from Vibrio cholerae, predict an elevator-like transport mechanism involving a large rigid body movement of the substrate-binding site. How substrate binding influences the conformational state of VcINDY is currently unknown. Here, we probe the interaction between substrate binding and protein conformation by monitoring substrate-induced solvent accessibility changes of broadly distributed positions in VcINDY using a site-specific alkylation strategy. Our findings reveal that accessibility to all positions tested is modulated by the presence of substrates, with the majority becoming less accessible in the presence of saturating concentrations of both Na+ and succinate. We also observe separable effects of Na+ and succinate binding at several positions suggesting distinct effects of the two substrates. Furthermore, accessibility changes to a solely succinate-sensitive position suggests that substrate binding is a low-affinity, ordered process. Mapping these accessibility changes onto the structures of VcINDY suggests that Na+ binding drives the transporter into an as-yet-unidentified conformational state, involving rearrangement of the substrate-binding site–associated re-entrant hairpin loops. These findings provide insight into the mechanism of VcINDY, which is currently the only structurally characterized representative of the entire DASS family.

L-Aspartate as a high-quality nitrogen source in Escherichia coli: Regulation of L-aspartase by the nitrogen regulatory system and interaction of L-aspartase with GlnB.

Escherichia coli uses the C4-dicarboxylate transporter DcuA for L-aspartate/fumarate antiport, which results in the exploitation of L-aspartate for fumarate respiration under anaerobic conditions and for nitrogen assimilation under aerobic and anaerobic conditions. L-Aspartate represents a high-quality nitrogen source for assimilation. Nitrogen assimilation from L-aspartate required DcuA, and aspartase AspA to release ammonia. Ammonia is able to provide by established pathways the complete set of intracellular precursors (ammonia, L-aspartate, L-glutamate, and L-glutamine) for synthesizing amino acids, nucleotides, and amino sugars. AspA was regulated by a central regulator of nitrogen metabolism, GlnB. GlnB interacted with AspA and stimulated its L-aspartate deaminase activity (NH3 -forming), but not the reverse amination reaction. GlnB stimulation required 2-oxoglutarate and ATP, or uridylylated GlnB-UMP, consistent with the activation of nitrogen assimilation under nitrogen limitation. Binding to AspA was lost in the GlnB(Y51F) mutant of the uridylylation site. AspA, therefore, represents a new type of GlnB target that binds GlnB (with ATP and 2-oxoglutarate), or GlnB-UMP (with or without effectors), and both situations stimulate AspA deamination activity. Thus, AspA represents the central enzyme for nitrogen assimilation from L-aspartate, and AspA is integrated into the nitrogen assimilation network by the regulator GlnB.

Identification and engineering a C4-dicarboxylate transporter for improvement of malic acid production in Aspergillus niger.

Modification of C4-dicarboxylate transport processes is an important strategy for the development of efficient malic acid producing cell factory in Aspergillus niger. However, there is a lack of identification and functional research of malic acid transport proteins, which seriously hinders the construction of high-yield malic acid metabolic engineering strains. A C4-dicarboxylate transport protein (DCT) DCT1 is identified as major malic acid transport protein and exhibits significant elevation in malic acid production when overexpressed. DCT1 is found by homology searches and domain analyses with SpMAE1 from Schizosaccharomyces pombe as the template. Phylogenetic and domain analyses show that DCTs belong to voltage-dependent slow-anion channel transporter (SLAC1) family and are members of Tellurite-resistance/Dicarboxylate Transporter (TDT) Family. DCT1 disruption dramatically decreases malic acid titer by about 85.6% and 96.2% at 3days and 5days compared with the parent strain, respectively. Meanwhile, the citric acid titers increase by 36.4% and 13.7% at 3days and 5days upon DCT1 deficiency. These results suggest that DCT1 is the major malic acid transporter in A. niger. Overexpression of dct1 with its native promoter significantly improves malic acid production yielding up to 13.86g/L and 30.79g/L at 3days and 5days, respectively, which is 36.8% and 22.8% higher than those in the parent strain. However, the citric acid has no significant change during the 5-day fermentation. These results demonstrate the importance of C4-dicarboxylate transporters for the efficient production of malic acid. Furthermore, enhancement of malic acid transport process is a feasible approach of efficient malic acid production in this citric acid producing A. niger strain. KEY POINTS: • A dicarboxylate transporter DCT1 is identified as a major malic acid transporter. • DCT1 deficiency results in significant decrease of malic acid. • DCT1 overexpression leads to increased titers of malic acid. • Enhancement of malic acid transport is vital for malic acid production in A. niger.

Engineering the transmission efficiency of the noncyclic glyoxylate pathway for fumarate production in Escherichia coli

BackgroundFumarate is a multifunctional dicarboxylic acid in the tricarboxylic acid cycle, but microbial engineering for fumarate production is limited by the transmission efficiency of its biosynthetic pathway.ResultsHere, pathway engineering was used to construct the noncyclic glyoxylate pathway for fumarate production. To improve the transmission efficiency of intermediate metabolites, pathway optimization was conducted by fluctuating gene expression levels to identify potential bottlenecks and then remove them, resulting in a large increase in fumarate production from 8.7 to 16.2 g/L. To further enhance its transmission efficiency of targeted metabolites, transporter engineering was used by screening the C4-dicarboxylate transporters and then strengthening the capacity of fumarate export, leading to fumarate production up to 18.9 g/L. Finally, the engineered strain E. coli W3110△4-P(H)CAI(H)SC produced 22.4 g/L fumarate in a 5-L fed-batch bioreactor.ConclusionsIn this study, we offered rational metabolic engineering and flux optimization strategies for efficient production of fumarate. These strategies have great potential in developing efficient microbial cell factories for production of high-value added chemicals.

A Novel Nitrite-Base Aerobic Denitrifying Bacterium Acinetobacter sp. YT03 and Its Transcriptome Analysis

Nitrite in a water environment is very harmful to humans and aquatic animals. A novel aerobic denitrifying bacterium able to utilize -N as the only nitrogen source was isolated for the purpose of removing nitrite from water, which was identified as Acinetobacter sp. and named as YT03. The growth and denitrification activity of strain YT03 was assessed comprehensively. Results showed that the nitrite in water with an initial concentration of 10 mg L–1 could be completely removed within 6 h by strain YT03, and the optimal conditions for strain YT03 to remove nitrite were as follows: sodium succinate as the carbon source, C/N ratio of 16, pH of 6.5, temperature of 30°C, and shaking speed of 250 rpm. An RNA-Seq transcriptome analysis was used to find genes associated with nitrite removal. Compared with the removal of ammonia nitrogen, 47 genes were significantly differentially expressed, including 20 up-regulated and 27 down-regulated genes, mainly involved in the transport process, biosynthetic process, and so on. And among the differentially expressed genes, C4-dicarboxylate transporter (DctA) and nitrate/nitrite transporter (Nrt) might be of importance for the efficient utilization of carbon and nitrogen sources in aerobic nitrite denitrification with sodium succinate by strain YT03.

Development of a Cre-loxP-based genetic system in Aspergillus niger ATCC1015 and its application to construction of efficient organic acid-producing cell factories

The filamentous fungus Aspergillus niger is widely used in the biotechnology industry for the production of chemicals and enzymes. Engineering of this valuable organism to improve its productivity is currently hampered by the lack of efficient genetic tools. Here, a Cre-loxP-based system for gene editing in A. niger was developed and its application in construction of A. niger cell factories to produce various organic acids was explored. Two established inducible systems, the xylanase A gene promoter Pxln and Tet-on system, were examined for driving cre expression and thus selection marker hyh deletion. Under inducing conditions, the efficiency of loxP site-specific recombination in the strain with cre driven by Pxln is about 2%, while cre driven by Tet-on system is about 34% which was used as the platform strain for further genetic engineering. As a proof of application of this system, strains containing different copies of oxaloacetate acetylhydrolase-encoding gene (oahA) were constructed, and the resultant strain S428 showed as high as 3.1-fold increase in oxalic acid production. Furthermore, an efficient malate-producing strain was generated through four-step genetic manipulation (oahA deletion, pyc, mdh3 and C4-dicarboxylate transporter gene c4t318 insertion). The resultant strain S575 achieved a titer 120.38 g/L malic acid with the flask culture, and a titer 201.24 g/L malic acid in fed-batch fermentation. These results demonstrated that this modified Cre-loxP system is a powerful tool for genetic engineering in A. niger, which has the potential to be genetically modified as a viable aciduric platform strain to produce high levels of various organic acids.

Gill transcriptomic analysis in fast- and slow-growing individuals of Mytilus galloprovincialis

The molecular basis underlying the mechanisms at the origin of growth variation in bivalves is still poorly understood, although several genes have been described as upregulated in fast-growing individuals. In the present study, we reared mussel spat of the species Mytilus galloprovincialis under diets below the pseudofaeces threshold (BP) and above the pseudofaeces threshold (AP). After 3 months, F and S mussels from each condition were selected to obtain 4 experimental groups: FBP, SBP, FAP and SAP. We hypothesized that the nurturing conditions during the growing period would modify the molecular basis of their growth rate differences.To decipher the molecular mechanisms underlying the growth variation, the gill transcriptomes for the four mussel groups were analysed. Gene expression analysis revealed i) a low number (12) of genes differentially expressed in association with diet and ii) 117 genes differentially expressed by the fast- and slow-growing mussels. According to Biological Process GO term analysis transcriptomic differences between the F and S mussels were mainly based on the upregulation of: response to the stimulus, growth and cell activity. Regarding the KEGG terms, carbohydrate metabolism and the Krebs cycle were upregulated in F mussels, whereas biosynthetic processes were upregulated in S mussels. In accordance with their larger gill surface area and higher rates of feeding and growth, the F individuals overexpressed genes in their gill tissues, and these were involved in i) growth (insulin-like growth factors and myostatin); ii) maintenance of the structure and functioning of extracellular matrix (collagen, laminin, fibulin and decorin); iii) filtration and ciliary activity (mucin, fibrocystin, dynein and tilB homolog protein genes); iv) aerobic metabolism (citrate synthase and carbonic anhydrase); and v) the immune-system, probably in association with haemocytes. In contrast, S individuals overexpressed a different series of genes pertaining to immune system (leucine-rich repeat protein and galectin), along with genes involved in the response to cellular stress (Heat shock protein (HSP24) and metalloendopeptidase) as well as anaerobic metabolism (C4-dicarboxylate transporter). These results might suggest that S individuals would have a greater prevalence of pathogens/diseases or a higher susceptibility to the pathogens.

Role of dcu C4-dicarboxylate transporters in H2 production during fermentation of glucose or glycerol

Role of dcu C4-dicarboxylate transporters in H2 production during fermentation of glucose or glycerol