Efficient production of 1,2,4-butanetriol from corn cob hydrolysate by metabolically engineered Escherichia coli.

Corn cob is a major waste mass-produced in corn agriculture. Corn cob hydrolysate containing xylose, arabinose, and glucose is the hydrolysis product of corn cob. Herein, a recombinant Escherichia coli strain BT-10 was constructed to transform corn cob hydrolysate into 1,2,4-butanetriol, a platform substance with diversified applications. To eliminate catabolite repression and enhance NADPH supply for alcohol dehydrogenase YqhD catalyzed 1,2,4-butanetriol generation, ptsG encoding glucose transporter EIICBGlc and pgi encoding phosphoglucose isomerase were deleted. With four heterologous enzymes including xylose dehydrogenase, xylonolactonase, xylonate dehydratase, α-ketoacid decarboxylase and endogenous YqhD, E. coli BT-10 can produce 36.63 g/L 1,2,4-butanetriol with a productivity of 1.14 g/[L·h] using xylose as substrate. When corn cob hydrolysate was used as the substrate, 43.4 g/L 1,2,4-butanetriol was generated with a productivity of 1.09 g/[L·h] and a yield of 0.9 mol/mol. With its desirable characteristics, E. coli BT-10 is a promising strain for commercial 1,2,4-butanetriol production.

Use of machine learning-based integration to develop a monocyte differentiation-related signature for improving prognosis in patients with sepsis.

BACKGROUND: Although significant advances have been made in intensive care medicine and antibacterial treatment, sepsis is still a common disease with high mortality. The condition of sepsis patients changes rapidly, and each hour of delay in the administration of appropriate antibiotic treatment can lead to a 4-7% increase in fatality. Therefore, early diagnosis and intervention may help improve the prognosis of patients with sepsis. METHODS: We obtained single-cell sequencing data from 12 patients. This included 14,622 cells from four patients with bacterial infectious sepsis and eight patients with sepsis admitted to the ICU for other various reasons. Monocyte differentiation trajectories were analyzed using the "monocle" software, and differentiation-related genes were identified. Based on the expression of differentiation-related genes, 99 machine-learning combinations of prognostic signatures were obtained, and risk scores were calculated for all patients. The "scissor" software was used to associate high-risk and low-risk patients with individual cells. The "cellchat" software was used to demonstrate the regulatory relationships between high-risk and low-risk cells in a cellular communication network. The diagnostic value and prognostic predictive value of Enah/Vasp-like (EVL) were determined. Clinical validation of the results was performed with 40 samples. The "CBNplot" software based on Bayesian network inference was used to construct EVL regulatory networks. RESULTS: We systematically analyzed three cell states during monocyte differentiation. The differential analysis identified 166 monocyte differentiation-related genes. Among the 99 machine-learning combinations of prognostic signatures constructed, the Lasso + CoxBoost signature with 17 genes showed the best prognostic prediction performance. The highest percentage of high-risk cells was found in state one. Cell communication analysis demonstrated regulatory networks between high-risk and low-risk cell subpopulations and other immune cells. We then determined the diagnostic and prognostic value of EVL stabilization in multiple external datasets. Experiments with clinical samples demonstrated the accuracy of this analysis. Finally, Bayesian network inference revealed potential network mechanisms of EVL regulation. CONCLUSIONS: Monocyte differentiation-related prognostic signatures based on the Lasso + CoxBoost combination were able to accurately predict the prognostic status of patients with sepsis. In addition, low EVL expression was associated with poor prognosis in sepsis.

Activation of the Chemokine Receptor CCR1 and Preferential Recruitment of Gαi Suppress RSV Replication: Implications for Developing Novel Respiratory Syncytial Virus Treatment Strategies.

Respiratory syncytial virus (RSV) is a major pathogen that can cause acute respiratory infectious diseases of the upper and lower respiratory tract, especially in children, elderly individuals, and immunocompromised people. Generally, following viral infection, respiratory epithelial cells secrete cytokines and chemokines to recruit immune cells and initiate innate and/or adaptive immune responses. However, whether chemokines affect viral replication in nonimmune cells is rarely clear. In this study, we detected that chemokine CCL5 was highly expressed, while expression of its receptor, CCR1, was downregulated in respiratory epithelial cells after RSV infection. When we overexpressed CCR1 on respiratory epithelial cells in vivo or in vitro, viral load was significantly suppressed, which can be restored by the neutralizing antibody for CCR1. Interestingly, the antiviral effect of CCR1 was not related to type I interferon (IFN-I), apoptosis induction, or viral adhesion or entry inhibition. In contrast, it was related to the preferential recruitment and activation of the adaptor Gαi, which promoted inositol 1,4,5-triphosphate receptor type 3 (ITPR3) expression, leading to inhibited STAT3 phosphorylation; explicitly, phosphorylated STAT3 (p-STAT3) was verified to be among the important factors regulating the activity of HSP90, which has been previously reported to be a chaperone of RSV RNA polymerase. In summary, we are the first to reveal that CCR1 on the surface of nonimmune cells regulates RSV replication through a previously unknown mechanism that does not involve IFN-I induction. IMPORTANCE Our results revealed a novel mechanism by which RSV escapes innate immunity. That is, although it induces high CCL5 expression, RSV might attenuate the binding of CCL5 by downregulating the expression of CCR1 in respiratory epithelial cells to weaken the inhibitory effect of CCR1 on HSP90 activity and thereby facilitate RSV replication in nonimmune cells. This study provides a new target for the development of co-antiviral inhibitors against other components of the host and co-molecular chaperone/HSP90 and provides a scientific basis for the search for effective broad-spectrum antiviral drugs.

Sertraline Is an Effective SARS-CoV-2 Entry Inhibitor Targeting the Spike Protein.

The global spread of the novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the continuously emerging new variants underscore an urgent need for effective therapeutics for the treatment of coronavirus disease 2019 (COVID-19). Here, we screened several FDA-approved amphiphilic drugs and determined that sertraline (SRT) exhibits potent antiviral activity against infection of SARS-CoV-2 pseudovirus (PsV) and authentic virus in vitro. It effectively inhibits SARS-CoV-2 spike (S)-mediated cell-cell fusion. SRT targets the early stage of viral entry. It can bind to the S1 subunit of the S protein, especially the receptor binding domain (RBD), thus blocking S-hACE2 interaction and interfering with the proteolysis process of S protein. SRT is also effective against infection with SARS-CoV-2 PsV variants, including the newly emerging Omicron. The combination of SRT and other antivirals exhibits a strong synergistic effect against infection of SARS-CoV-2 PsV. The antiviral activity of SRT is independent of serotonin transporter expression. Moreover, SRT effectively inhibits infection of SARS-CoV-2 PsV and alleviates the inflammation process and lung pathological alterations in transduced mice in vivo. Therefore, SRT shows promise as a treatment option for COVID-19. IMPORTANCE The study shows SRT is an effective entry inhibitor against infection of SARS-CoV-2, which is currently prevalent globally. SRT targets the S protein of SARS-CoV-2 and is effective against a panel of SARS-CoV-2 variants. It also could be used in combination to prevent SARS-CoV-2 infection. More importantly, with long history of clinical use and proven safety, SRT might be particularly suitable to treat infection of SARS-CoV-2 in the central nervous system and optimized for treatment in older people, pregnant women, and COVID-19 patients with heart complications, which are associated with severity and mortality of COVID-19.

Structural basis for the arsenite binding and translocation of Acr3 antiporter with NhaA folding pattern.

The arsenical resistance-3 (ACR3) family constitutes the most common pathway that confers high-level resistance to toxic metalloids in various microorganisms and lower plants. Based on the structural model constructed by AlphaFold2, the Acr3 antiporter from Bacillus subtilis (Acr3Bs ) exhibits a typical NhaA structure fold, with two discontinuous helices of transmembrane (TM) segments, TM4 and TM9, interacting with each other and forming an X-shaped structure. As the structural information available for these important arsenite-efflux pumps is limited, we investigated the evolutionary conservation among 300 homolog sequences and identified three conserved motifs in both the discontinuous helices and TM5. Through site-directed mutagenesis, microscale thermophoresis (MST), and fluorescence resonance energy transfer (FRET) analyses, the identified Motif C in TM9 was found to be a critical element for substrate binding, in which N292 and E295 are involved in substrate coordination, while R118 in TM4 and E322 in TM10 is responsible for structural stabilization. In addition, the highly conserved residues on Motif B of TM5 are potentially key factors in the protonation/deprotonation process. These consensus motifs and residues are essential for metalloid compound translocation of Acr3 antiporters, by framing the core domain and the typical X-shaped of NhaA fold.

ß-Galactosidase: a traditional enzyme given multiple roles through protein engineering.

ß-Galactosidases are crucial carbohydrate-active enzymes that naturally catalyze the hydrolysis of galactoside bonds in oligo- and disaccharides. These enzymes are commonly used to degrade lactose and produce low-lactose and lactose-free dairy products that are beneficial for lactose-intolerant people. ß-galactosidases exhibit transgalactosylation activity, and they have been employed in the synthesis of galactose-containing compounds such as galactooligosaccharides. However, most ß-galactosidases have intrinsic limitations, such as low transglycosylation efficiency, significant product inhibition effects, weak thermal stability, and a narrow substrate spectrum, which greatly hinder their applications. Enzyme engineering offers a solution for optimizing their catalytic performance. The study of the enzyme's structure paves the way toward explaining catalytic mechanisms and increasing the efficiency of enzyme engineering. In this review, the structure features of ß-galactosidases from different glycosyl hydrolase families and the catalytic mechanisms are summarized in detail to offer guidance for protein engineering. The properties and applications of ß-galactosidases are discussed. Additionally, the latest progress in ß-galactosidase engineering and the strategies employed are highlighted. Based on the combined analysis of structure information and catalytic mechanisms, the ultimate goal of this review is to furnish a thorough direction for ß-galactosidases engineering and promote their application in the food and dairy industries.

Evolutionary conservative analysis revealed novel functional sites in the efflux pump NorA of Staphylococcus aureus.

OBJECTIVES: The NorA antiporter of Staphylococcus aureus belongs to the major facilitator superfamily (MFS) and extrudes various kinds of drugs. With no structure available for this drug efflux pump, the aim of this study was to explore its important structural elements that contribute to substrate binding and drug transport. METHODS: Evolutionary conservative analyses were conducted on different compilations of NorA homologues to identify conservative motifs and residues. Site-directed mutations were constructed to verify the functional changes in NorA efflux capacities and the conformational changes were further measured by fluorescence resonance energy transfer (FRET) and microscale thermophoresis (MST) analysis. RESULTS: Besides Motif-A, Motif-B and Motif-C that were reported previously in MFS proteins, two other motifs, Motif-1 and Motif-2, were identified in NorA. Site-directed mutations of Motif-1 and Motif-2 as well as 11 predicted binding sites all caused remarkable reductions in drug resistance and efflux activity. Among these, mutant F16A/E222A/F303A/D307A showed an altered binding affinity for tetraphenylphosphonium chloride when measured by MST and Motif-1 mutant G114D/A117E/D118G/V119I and Motif-2 mutant Q325E/G326E/A328E/G330E displayed obvious conformational alterations when compared with the wild-type NorA in the FRET signal spectra. CONCLUSIONS: The NorA structure agrees well with the typical structures of MFS proteins, with two newly identified motifs (Motif-1 and Motif-2) that are critical to the structural stability of NorA, and sites F16, E222, F303 and D307 are involved in substrate binding.

Improving the ability of CAR-T cells to hit solid tumors: Challenges and strategies.

Chimeric antigen receptor T cell (CAR-T) therapy is a late-model of immune cell therapy that has been shown to be effective in refractory/recurrent B-cell leukemia and lymphoma. Compared with the traditional anti-tumor methods, CAR-T cell therapy has the advantages of higher specificity, stronger lethality and longer-lasting efficacy. Although CAR-T cells have made significant progress in the treatment of hematologic malignancies, diverse difficulties remain in the treatment of solid tumors, including immune escape due to tumor antigen heterogeneity, preventing entry or limiting the persistence of CAR-T cells by physical or cytokine barriers and along with other immunosuppressive molecule and cells in the tumor microenvironment (TME). Otherwise, the intracellular signaling of CAR also impact on CAR-T cells persistence. Appropriate modification of intracellular costimulatory molecular signal in the structure of CAR or coexpression of CAR and cytokines can provide a way to enhance CAR-T cells activity. Additionally, CAR-T cells dysfunction due to T cell exhaustion is associated with multi-factors, especially transcription factors, such as c-Jun, NR4A. Engineering CAR-T cells to coexpress or knockout transcription factors in favor of TCM memory CAR-T cells differentiation was proved to prolonged the survival of CAR-T cells. Finally, combination of CAR-T cells with oncolytic viruses, nanoparticles or immune checkpoint inhibitors provides an effective measure to improve CAR-T cells function. Here, we discuss all of these advances and challenges and review promising strategies for treating solid tumors. In particular, we also highlight that CAR-T cells have enormous potential to be used in combination with other immunotherapies.

Long Noncoding RNA NRAV Promotes Respiratory Syncytial Virus Replication by Targeting the MicroRNA miR-509-3p/Rab5c Axis To Regulate Vesicle Transportation.

Respiratory syncytial virus (RSV) is an enveloped RNA virus which is responsible for approximately 80% of lower respiratory tract infections in children. Current lines of evidence have supported the functional involvement of long noncoding RNA (lncRNA) in many viral infectious diseases. However, the overall biological effect and clinical role of lncRNAs in RSV infection remain unclear. In this study, lncRNAs related to respiratory virus infection were obtained from the lncRNA database, and we collected 144 clinical sputum specimens to identify lncRNAs related to RSV infection. Quantitative PCR (qPCR) detection indicated that the expression of lncRNA negative regulator of antiviral response (NRAV) in RSV-positive patients was significantly lower than that in uninfected patients, but lncRNA psoriasis-associated non-protein coding RNA induced by stress (PRINS), nuclear paraspeckle assembly transcript 1 (NEAT1), and Nettoie Salmonella pas Theiler's (NeST) showed no difference in vivo and in vitro Meanwhile, overexpression of NRAV promoted RSV proliferation in A549 and BEAS-2B cells, and vice versa, indicating that the downregulation of NRAV was part of the host antiviral defense. RNA fluorescent in situ hybridization (FISH) confirmed that NRAV was mainly located in the cytoplasm. Through RNA sequencing, we found that Rab5c, which is a vesicle transporting protein, showed the same change trend as NRAV. Subsequent investigation revealed that NRAV was able to favor RSV production indirectly by sponging microRNA miR-509-3p so as to release Rab5c and facilitate vesicle transportation. The study provides a new insight into virus-host interaction through noncoding RNA, which may contribute to exploring potential antivirus targets for respiratory virus.IMPORTANCE The mechanism of interaction between RSV and host noncoding RNAs is not fully understood. In this study, we found that the expression of long noncoding RNA (lncRNA) negative regulator of antiviral response (NRAV) was reduced in RSV-infected patients, and overexpression of NRAV facilitated RSV production in vitro, suggesting that the reduction of NRAV in RSV infection was part of the host antiviral response. We also found that NRAV competed with vesicle protein Rab5c for microRNA miR509-3p in cytoplasm to promote RSV vesicle transport and accelerate RSV proliferation, thereby improving our understanding of the pathogenic mechanism of RSV infection.

Efficient 2,3-butanediol production from whey powder using metabolically engineered Klebsiella oxytoca.

BACKGROUND: Whey is a major pollutant generated by the dairy industry. To decrease environmental pollution caused by the industrial release of whey, new prospects for its utilization need to be urgently explored. Here, we investigated the possibility of using whey powder to produce 2,3-butanediol (BDO), an important platform chemical. RESULTS: Klebsiella oxytoca strain PDL-0 was selected because of its ability to efficiently produce BDO from lactose, the major fermentable sugar in whey. After deleting genes pox, pta, frdA, ldhD, and pflB responding for the production of by-products acetate, succinate, lactate, and formate, a recombinant strain K. oxytoca PDL-K5 was constructed. Fed-batch fermentation using K. oxytoca PDL-K5 produced 74.9 g/L BDO with a productivity of 2.27 g/L/h and a yield of 0.43 g/g from lactose. In addition, when whey powder was used as the substrate, 65.5 g/L BDO was produced within 24 h with a productivity of 2.73 g/L/h and a yield of 0.44 g/g. CONCLUSION: This study demonstrated the efficiency of K. oxytoca PDL-0 for BDO production from whey. Due to its non-pathogenicity and efficient lactose utilization, K. oxytoca PDL-0 might also be used in the production of other important chemicals using whey as the substrate.

Coupling between d-3-phosphoglycerate dehydrogenase and d-2-hydroxyglutarate dehydrogenase drives bacterial l-serine synthesis.

l-Serine biosynthesis, a crucial metabolic process in most domains of life, is initiated by d-3-phosphoglycerate (d-3-PG) dehydrogenation, a thermodynamically unfavorable reaction catalyzed by d-3-PG dehydrogenase (SerA). d-2-Hydroxyglutarate (d-2-HG) is traditionally viewed as an abnormal metabolite associated with cancer and neurometabolic disorders. Here, we reveal that bacterial anabolism and catabolism of d-2-HG are involved in l-serine biosynthesis in Pseudomonas stutzeri A1501 and Pseudomonas aeruginosa PAO1. SerA catalyzes the stereospecific reduction of 2-ketoglutarate (2-KG) to d-2-HG, responsible for the major production of d-2-HG in vivo. SerA combines the energetically favorable reaction of d-2-HG production to overcome the thermodynamic barrier of d-3-PG dehydrogenation. We identified a bacterial d-2-HG dehydrogenase (D2HGDH), a flavin adenine dinucleotide (FAD)-dependent enzyme, that converts d-2-HG back to 2-KG. Electron transfer flavoprotein (ETF) and ETF-ubiquinone oxidoreductase (ETFQO) are also essential in d-2-HG metabolism through their capacity to transfer electrons from D2HGDH. Furthermore, while the mutant with D2HGDH deletion displayed decreased growth, the defect was rescued by adding l-serine, suggesting that the D2HGDH is functionally tied to l-serine synthesis. Substantial flux flows through d-2-HG, being produced by SerA and removed by D2HGDH, ETF, and ETFQO, maintaining d-2-HG homeostasis. Overall, our results uncover that d-2-HG-mediated coupling between SerA and D2HGDH drives bacterial l-serine synthesis.

d-2-Hydroxyglutarate dehydrogenase plays a dual role in l-serine biosynthesis and d-malate utilization in the bacterium Pseudomonas stutzeri.

Pseudomonas is a very large bacterial genus in which several species can use d-malate for growth. However, the enzymes that can metabolize d-malate, such as d-malate dehydrogenase, appear to be absent in most Pseudomonas species. d-3-Phosphoglycerate dehydrogenase (SerA) can catalyze the production of d-2-hydroxyglutarate (d-2-HG) from 2-ketoglutarate to support d-3-phosphoglycerate dehydrogenation, which is the initial reaction in bacterial l-serine biosynthesis. In this study, we show that SerA of the Pseudomonas stutzeri strain A1501 reduces oxaloacetate to d-malate and that d-2-HG dehydrogenase (D2HGDH) from P. stutzeri displays d-malate-oxidizing activity. Of note, D2HGDH participates in converting a trace amount of d-malate to oxaloacetate during bacterial l-serine biosynthesis. Moreover, D2HGDH is crucial for the utilization of d-malate as the sole carbon source for growth of P. stutzeri A1501. We also found that the D2HGDH expression is induced by the exogenously added d-2-HG or d-malate and that a flavoprotein functions as a soluble electron carrier between D2HGDH and electron transport chains to support d-malate utilization by P. stutzeri These results support the idea that D2HGDH evolves as an enzyme for both d-malate and d-2-HG dehydrogenation in P. stutzeri In summary, D2HGDH from P. stutzeri A1501 participates in both a core metabolic pathway for l-serine biosynthesis and utilization of extracellular d-malate.

Potassium resistance of halotolerant and alkaliphilic Halomonas sp. Y2 by a Na+-induced K+ extrusion mechanism.

In most halophiles, K+ generally acts as a major osmotic solute for osmotic adjustment and pH homeostasis. However, strains also need to extrude excessive intracellular K+ to avoid its toxicity. In the halotolerant and alkaliphilic Halomonas sp. Y2, an Na+-induced K+ extrusion process was observed when the cells were confronted with high extracellular K+ pressure and supplementation by millimolar Na+ ions. Among three mechanosensitive channels (KefA) and two K+/H+ antiporters founded in the genome of the strain, ke1 displayed around 3-5-fold upregulation to ion stress at pH 8.0, while much higher upregulation of Ha-mrp was observed at pH 10.0. Compared to the growth of wild-type Halomonas sp. Y2, deletion of these genes from the strain resulted in different growth phenotypes in response to the osmotic pressure of potassium. In combination with the transcriptional response of these genes, we proposed that the KefA channel of Ke1 is the main contributor to the K+-extrusion process under weak alkalinity, while the Mrp system plays critical roles in alleviating K+ contents at high pH. The combination of these strategies allows Halomonas sp. Y2 to grow over a range of extracellular pH and ion concentrations, and thus protect cells under high osmotic stress conditions.

Respiratory Syncytial Virus Replication Is Promoted by Autophagy-Mediated Inhibition of Apoptosis.

Respiratory syncytial virus (RSV) is the main cause of acute lower respiratory tract infection (ALRI) in children worldwide. Virus-host interactions affect the progression and prognosis of the infection. Autophagy plays important roles in virus-host interactions. Respiratory epithelial cells serve as the front line of host defense during RSV infection, However, it is still unclear how they interact with RSV. In this study, we found that RSV induced autophagy that favored RSV replication and exacerbated lung pathology in vivo Mechanistically, RSV induced complete autophagy flux through reactive oxygen species (ROS) generation and activation of the AMP-activated protein kinase/mammalian target of rapamycin (AMPK-MTOR) signaling pathway in HEp-2 cells. Furthermore, we evaluated the functions of autophagy in RSV replication and found that RSV replication was increased in HEp-2 cells treated with rapamycin but decreased remarkably in cells treated with 3-methylademine (3-MA) or wortmannin. Knockdown key molecules in the autophagy pathway with short hairpinp RNA (shRNA) against autophagy-related gene 5 (ATG5), autophagy-related gene 7 (ATG7), or BECN1/Beclin 1 or treatment with ROS scavenger N-acetyl-l-cysteine (NAC) and AMPK inhibitor (compound C) suppressed RSV replication. 3-MA or shATG5/BECN1 significantly decreased cell viability and increased cell apoptosis at 48 hours postinfection (hpi). Blocking apoptosis with Z-VAD-FMK partially restored virus replication at 48 hpi. Those results provide strong evidence that autophagy may function as a proviral mechanism in a cell-intrinsic manner during RSV infection.IMPORTANCE An understanding of the mechanisms that respiratory syncytial virus utilizes to interact with respiratory epithelial cells is critical to the development of novel antiviral strategies. In this study, we found that RSV induces autophagy through a ROS-AMPK signaling axis, which in turn promotes viral infection. Autophagy favors RSV replication through blocking cell apoptosis at 48 hpi. Mechanistically, RSV induces mitophagy, which maintains mitochondrial homeostasis and therefore decreases cytochrome c release and apoptosis induction. This study provides a novel insight into this virus-host interaction, which may help to exploit new antiviral treatments targeting autophagy processes.

High ectoine production by an engineered Halomonas hydrothermalis Y2 in a reduced salinity medium.

BACKGROUND: As an attracted compatible solute, 1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine) showed great potentials in various field. However, lower productivity and high saline medium seriously hinder its wide applications. RESULTS: The entire ectoine metabolism, including pathways for ectoine synthesis and catabolism, was identified in the genome of an ectoine-excreting strain Halomonas hydrothermalis Y2. By in-frame deletion of genes encoding ectoine hydroxylase (EctD) and (or) ectoine hydrolase (DoeA) that responsible for ectoine catabolism, the pathways for ectoine utilization were disrupted and resulted in an obviously enhanced productivity. Using an optimized medium containing 100 g L-1 NaCl in a 500-mL flask, the double mutant of Y2/ΔectD/ΔdoeA synthesized 3.13 g L-1 ectoine after 30 h cultivation. This is much higher than that of the wild type strain (1.91 g L-1), and also exceeds the production of Y2/ΔectD (2.21 g L-1). The remarkably enhanced accumulation of ectoine by Y2/ΔectD/ΔdoeA implied a critical function of Doe pathway in the ectoine catabolism. Furthermore, to reduce the salinity of fermentation medium and overcome the wastewater treatment difficulty, mutants that lacking key Na+/H+ antiporter, Mrp and (or) NhaD2, were constructed based on strain Y2/ΔectD/ΔdoeA. As a result, the Mrp-deficient strain could synthesize equal amount of ectoine (around 7 g L-1 or 500 mg (g DCW) -1) in the medium containing lower concentration of NaCl. During a fed-batch fermentation process with 60 g L-1 NaCl stress, a maximum 10.5 g L-1 ectoine was accumulated by the Mrp-deficient strain, with a specific production of 765 mg (g DCW)-1 and a yield of 0.21 g g-1 monosodium glutamate. CONCLUSION: The remarkably enhanced production of ectoine by Y2/ΔectD/ΔdoeA implied the critical function of Doe pathway in the ectoine catabolism. Moreover, the reduced salinity requirement of Mrp-deficient strain implied a feasible protocol for many compatible solute biosynthesis, i.e., by silencing some Na+/H+ antiporters in their halophilic producers and thus lowering the medium salinity.

2,3-Butanediol catabolism in Pseudomonas aeruginosa PAO1.

2,3-Butanediol (2,3-BD) is a primary microbial metabolite that enhances the virulence of Pseudomonas aeruginosa and alters the lung microbiome. 2,3-BD exists in three stereoisomeric forms: (2R,3R)-2,3-BD, meso-2,3-BD and (2S,3S)-2,3-BD. In this study, we investigated whether and how P. aeruginosa PAO1 utilizes these 2,3-BD stereoisomers and showed that all three stereoisomers were transformed into acetoin by (2R,3R)-2,3-butanediol dehydrogenase (BDH) or (2S,3S)-2,3-BDH. Acetoin was cleaved to form acetyl-CoA and acetaldehyde by acetoin dehydrogenase enzyme system (AoDH ES). Genes encoding (2R,3R)-2,3-BDH, (2S,3S)-2,3-BDH and the E1 and E2 components of AoDH ES were identified as part of a new 2,3-BD utilization operon. In addition, the regulatory protein AcoR promoted the expression of this operon using acetaldehyde, a cleavage product of acetoin, as its direct effector. The results of this study elucidate the integrated catabolic role of 2,3-BD and may provide new insights in P. aeruginosa-related infections.

Engineering of glycerol utilization in Gluconobacter oxydans 621H for biocatalyst preparation in a low-cost way.

BACKGROUND: Whole cells of Gluconobacter oxydans are widely used in various biocatalytic processes. Sorbitol at high concentrations is commonly used in complex media to prepare biocatalysts. Exploiting an alternative process for preparation of biocatalysts with low cost substrates is of importance for industrial applications. RESULTS: G. oxydans 621H was confirmed to have the ability to grow in mineral salts medium with glycerol, an inevitable waste generated from industry of biofuels, as the sole carbon source. Based on the glycerol utilization mechanism elucidated in this study, the major polyol dehydrogenase (GOX0854) and the membrane-bound alcohol dehydrogenase (GOX1068) can competitively utilize glycerol but play no obvious roles in the biocatalyst preparation. Thus, the genes related to these two enzymes were deleted. Whole cells of G. oxydans ∆GOX1068∆GOX0854 can be prepared from glycerol with a 2.4-fold higher biomass yield than that of G. oxydans 621H. Using whole cells of G. oxydans ∆GOX1068∆GOX0854 as the biocatalyst, 61.6 g L-1 xylonate was produced from 58.4 g L-1 xylose at a yield of 1.05 g g-1. CONCLUSION: This process is an example of efficient preparation of whole cells of G. oxydans with reduced cost. Besides xylonate production from xylose, other biocatalytic processes might also be developed using whole cells of metabolic engineered G. oxydans prepared from glycerol.

A Bacterial Multidomain NAD-Independent d-Lactate Dehydrogenase Utilizes Flavin Adenine Dinucleotide and Fe-S Clusters as Cofactors and Quinone as an Electron Acceptor for d-Lactate Oxidization.

Bacterial membrane-associated NAD-independent d-lactate dehydrogenase (Fe-S d-iLDH) oxidizes d-lactate into pyruvate. A sequence analysis of the enzyme reveals that it contains an Fe-S oxidoreductase domain in addition to a flavin adenine dinucleotide (FAD)-containing dehydrogenase domain, which differs from other typical d-iLDHs. Fe-S d-iLDH from Pseudomonas putida KT2440 was purified as a His-tagged protein and characterized in detail. This monomeric enzyme exhibited activities with l-lactate and several d-2-hydroxyacids. Quinone was shown to be the preferred electron acceptor of the enzyme. The two domains of the enzyme were then heterologously expressed and purified separately. The Fe-S cluster-binding motifs predicted by sequence alignment were preliminarily verified by site-directed mutagenesis of the Fe-S oxidoreductase domain. The FAD-containing dehydrogenase domain retained 2-hydroxyacid-oxidizing activity, although it decreased compared to the full Fe-S d-iLDH. Compared to the intact enzyme, the FAD-containing dehydrogenase domain showed increased catalytic efficiency with cytochrome c as the electron acceptor, but it completely lost the ability to use coenzyme Q10 Additionally, the FAD-containing dehydrogenase domain was no longer associated with the cell membrane, and it could not support the utilization of d-lactate as a carbon source. Based on the results obtained, we conclude that the Fe-S oxidoreductase domain functions as an electron transfer component to facilitate the utilization of quinone as an electron acceptor by Fe-S d-iLDH, and it helps the enzyme associate with the cell membrane. These functions make the Fe-S oxidoreductase domain crucial for the in vivo d-lactate utilization function of Fe-S d-iLDH.IMPORTANCE Lactate metabolism plays versatile roles in most domains of life. Lactate utilization processes depend on certain enzymes to oxidize lactate to pyruvate. In recent years, novel bacterial lactate-oxidizing enzymes have been continually reported, including the unique NAD-independent d-lactate dehydrogenase that contains an Fe-S oxidoreductase domain besides the typical flavin-containing domain (Fe-S d-iLDH). Although Fe-S d-iLDH is widely distributed among bacterial species, the investigation of it is insufficient. Fe-S d-iLDH from Pseudomonas putida KT2440, which is the major d-lactate-oxidizing enzyme for the strain, might be a representative of this type of enzyme. A study of it will be helpful in understanding the detailed mechanisms underlying the lactate utilization processes.

Coordination of metabolic pathways: Enhanced carbon conservation in 1,3-propanediol production by coupling with optically pure lactate biosynthesis.

Metabolic engineering has emerged as a powerful tool for bioproduction of both fine and bulk chemicals. The natural coordination among different metabolic pathways contributes to the complexity of metabolic modification, which hampers the development of biorefineries. Herein, the coordination between the oxidative and reductive branches of glycerol metabolism was rearranged in Klebsiella oxytoca to improve the 1,3-propanediol production. After deliberating on the product value, carbon conservation, redox balance, biological compatibility and downstream processing, the lactate-producing pathway was chosen for coupling with the 1,3-propanediol-producing pathway. Then, the other pathways of 2,3-butanediol, ethanol, acetate, and succinate were blocked in sequence, leading to improved d-lactate biosynthesis, which as return drove the 1,3-propanediol production. Meanwhile, efficient co-production of 1,3-propanediol and l-lactate was also achieved by replacing ldhD with ldhL from Bacillus coagulans. The engineered strains PDL-5 and PLL co-produced over 70g/L 1,3-propanediol and over 100g/L optically pure d-lactate and l-lactate, respectively, with high conversion yields of over 0.95mol/mol from glycerol.

Receptor usage and cell entry of bat coronavirus HKU4 provide insight into bat-to-human transmission of MERS coronavirus.

Middle East respiratory syndrome coronavirus (MERS-CoV) currently spreads in humans and causes ∼ 36% fatality in infected patients. Believed to have originated from bats, MERS-CoV is genetically related to bat coronaviruses HKU4 and HKU5. To understand how bat coronaviruses transmit to humans, we investigated the receptor usage and cell entry activity of the virus-surface spike proteins of HKU4 and HKU5. We found that dipeptidyl peptidase 4 (DPP4), the receptor for MERS-CoV, is also the receptor for HKU4, but not HKU5. Despite sharing a common receptor, MERS-CoV and HKU4 spikes demonstrated functional differences. First, whereas MERS-CoV prefers human DPP4 over bat DPP4 as its receptor, HKU4 shows the opposite trend. Second, in the absence of exogenous proteases, both MERS-CoV and HKU4 spikes mediate pseudovirus entry into bat cells, whereas only MERS-CoV spike, but not HKU4 spike, mediates pseudovirus entry into human cells. Thus, MERS-CoV, but not HKU4, has adapted to use human DPP4 and human cellular proteases for efficient human cell entry, contributing to the enhanced pathogenesis of MERS-CoV in humans. These results establish DPP4 as a functional receptor for HKU4 and host cellular proteases as a host range determinant for HKU4. They also suggest that DPP4-recognizing bat coronaviruses threaten human health because of their spikes' capability to adapt to human cells for cross-species transmissions.