Dual genetic level modification engineering accelerate genome evolution of Corynebacterium glutamicum.

High spontaneous mutation rate is crucial for obtaining ideal phenotype and exploring the relationship between genes and phenotype. How to break the genetic stability of organisms and increase the mutation frequency has become a research hotspot. Here, we present a practical and controllable evolutionary tool (oMut-Cgts) based on dual genetic level modification engineering for Corynebacterium glutamicum. Firstly, the modification engineering of transcription and replication levels based on RNA polymerase α subunit and DNA helicase Cgl0854 as the 'dock' of cytidine deaminase (pmCDA1) significantly increased the mutation rate, proving that the localization of pmCDA1 around transient ssDNA is necessary for genome mutation. Then, the combined modification and optimization of engineering at dual genetic level achieved 1.02 × 104-fold increased mutation rate. The genome sequencing revealed that the oMut-Cgts perform uniform and efficient C:G→T:A transitions on a genome-wide scale. Furthermore, oMut-Cgts-mediated rapid evolution of C. glutamicum with stress (acid, oxidative and ethanol) tolerance proved that the tool has powerful functions in multi-dimensional biological engineering (rapid phenotype evolution, gene function mining and protein evolution). The strategies for rapid genome evolution provided in this study are expected to be applicable to a variety of applications in all prokaryotic cells.

Dehydroabietylamine exerts antitumor effects by affecting nucleotide metabolism in gastric cancer.

Nucleotide metabolism is the ultimate and most critical link in the self-replication process of tumors, including gastric cancer (GC). However, in clinical treatment, classic anti-tumor drugs such as 5-fluorouracil (5-FU) are mostly metabolic analogues of purines or pyrimidines, which lack specificity for tumor cells and therefore have significant side effects. It is unclear whether there are other drugs that can target nucleotide metabolism, except for nucleic acid analogues. Here, we found that a natural compound, dehydroabietylamine (DHAA), significantly reduced the viability and proliferation of GC cells and organoids. DHAA disrupts purine and pyrimidine metabolism of GC cells, causing DNA damage and further inducing apoptosis. DHAA treatment decreased transcription and protein levels of key enzymes involved in nucleotide metabolism pathway, with significant reductions in the expression of pyrimidine metabolism key enzymes CAD, DHODH, and purine metabolism key enzymes PAICS. We also found that DHAA directly binds to and reduces the expression of Forkhead box K2 (FOXK2), a common transcription factor for these metabolic enzymes. Ultimately, DHAA was shown to delay tumorigenesis in K19-Wnt1/C2mE transgenic mice model and reduce levels of CAD, DHODH, and PAICS in vivo. We demonstrate that DHAA exerts an anticancer effect on GC by targeting transcription factor FOXK2, reducing transcription of key genes for nucleotide metabolism and impairing nucleotide biosynthesis, thus DHAA is a promising candidate for GC therapy.

The flavohaemoprotein hmp maintains redox homeostasis in response to reactive oxygen and nitrogen species in Corynebacterium glutamicum.

BACKGROUND: During the production of L-arginine through high dissolved oxygen and nitrogen supply fermentation, the industrial workhorse Corynebacterium glutamicum is exposed to oxidative stress. This generates reactive oxygen species (ROS) and reactive nitrogen species (RNS), which are harmful to the bacteria. To address the issue and to maintain redox homeostasis during fermentation, the flavohaemoprotein (Hmp) was employed. RESULTS: The results showed that the overexpression of Hmp led to a decrease in ROS and RNS content by 9.4% and 22.7%, respectively, and improved the survivability of strains. When the strains were treated with H2O2 and NaNO2, the RT-qPCR analysis indicated an up-regulation of ammonium absorption and transporter genes amtB and glnD. Conversely, the deletion of hmp gives rise to the up-regulation of eight oxidative stress-related genes. These findings suggested that hmp is associated with oxidative stress and intracellular nitrogen metabolism genes. Finally, we released the inhibitory effect of ArnR on hmp. The Cc-ΔarnR-hmp strain produced 48.4 g/L L-arginine during batch-feeding fermentation, 34.3% higher than the original strain. CONCLUSIONS: This report revealed the influence of dissolved oxygen and nitrogen concentration on reactive species of Corynebacterium glutamicum and the role of the Hmp in coping with oxidative stress. The Hmp first demonstrates related to redox homeostasis and nitrite metabolism, providing a feasible strategy for improving the robustness of strains.

[Anti-depression mechanism of Zuojin Pills:based on UHPLC-TOF-MS, network pharmacology, and experimental verification].

This study aims to explore the anti-depression mechanism of Zuojin Pills based on the plasma constituents, network pharmacology, and experimental verification. UHPLC-TOF-MS was used for qualitative analysis of Zuojin Pills-containing serum. Targets of the plasma constituents and the disease were retrieved from PharmMapper and GeneCards. Then the protein-protein interaction(PPI) network was constructed and core targets were screened for GO term enrichment and KEGG pathway enrichment. Cytoscape 3.7.2 was employed construct the "compound-target-pathway" network and the targets and signaling pathways of Zuojin Pills against depression were predicted. CUMS-induced depression mouse model was established to verify the key targets. The results showed that a total of 21 constituents migrating to blood of Zuojin Pills were identified, which were mainly alkaloids. A total of 155 common targets of the constituents and the disease and 67 core targets were screened out. KEGG enrichment and PPI network analysis showed that Zuojin Pills may play a role in the treatment of depression through AMPK/SIRT1, NLRP3, insulin and other targets and pathways. Furthermore, the results of animal experiments showed that Zuojin Pills could significantly improve the depression behaviors of depression, reduce the levels of IL-1ß, IL-6 and TNF-α in hippocampus and serum, activate AMPK/SIRT1 signaling, and reduce the protein expression of NLRP3. In conclusion, Zuojin Pills may play a role in the treatment of depression by activating AMPK/SIRT1 signaling pathway, and inhibiting NLRP3 activation and neuroinflammation in the hippocampus of mice.

Development of a nonauxotrophic L-homoserine hyperproducer in Escherichia coli by systems metabolic engineering.

L-Homoserine is a valuable amino acid as a platform chemical in the synthesis of various important compounds. Development of microbial strains for high-level L-homoserine production is an attractive research direction in recent years. Herein, we converted a wild-type Escherichia coli to a non-auxotrophic and plasmid-free hyperproducer of L-homoserine using systematically metabolic engineer strategies. First, an initial strain was obtained through regulating L-homoserine degradation pathway and enhancing synthetic flow. To facilitate L-homoserine production, flux-control genes were tuned by optimizing the copy numbers in chromosome, and transport system was modified to promote L-homoserine efflux. Subsequently, a strategy of cofactors synergistic utilization was proposed and successfully applied to achieve L-homoserine hyperproduction. The final engineered strain could efficiently produce 85.29 g/L L-homoserine, which was the highest production level ever reported from a plasmid-free, antibiotic-free, inducer-free and nonauxotrophic strain. These strategies used here can be considered for developing microbial cell factory of other L-aspartate derivatives.

Development of a 2-pyrrolidone biosynthetic pathway in Corynebacterium glutamicum by engineering an acetyl-CoA balance route.

2-Pyrrolidone is widely used in the textile and pharmaceutical industries. Here, we established a 2-pyrrolidone biosynthesis pathway in Corynebacterium glutamicum, by expressing glutamate decarboxylase (Gad) mutant and ß-alanine CoA transferase (Act) which activates spontaneous dehydration cyclization of GABA to form 2-pyrrolidone. Also, the 5' untranslated regions (UTR) strategy was used to increase the expression of protein. Furthermore, considering the importance of acetyl-CoA in the 2-pyrrolidone synthesis pathway, the acetyl-CoA synthetase (acsA) gene was introduced to convert acetate into acetyl-CoA thus achieving the recyclability of the economy. Finally, the fed-batch fermentation of the final strain in a 5 L bioreactor produced 10.5 g/L 2-pyrrolidone within 78 h, which increased by 42.5% by altering the level of gene expression. This is the first time to build the basic chemical 2-pyrrolidone from glucose in one step in C. glutamicum.

High-level production of the agmatine in engineered Corynebacterium crenatum with the inhibition-releasing arginine decarboxylase.

BACKGROUND: Agmatine is a member of biogenic amines and is an important medicine which is widely used to regulate body balance and neuroprotective effects. At present, the industrial production of agmatine mainly depends on the chemical method, but it is often accompanied by problems including cumbersome processes, harsh reaction conditions, toxic substances production and heavy environmental pollution. Therefore, to tackle the above issues, arginine decarboxylase was overexpressed heterologously and rationally designed in Corynebacterium crenatum to produce agmatine from glucose by one-step fermentation. RESULTS: In this study, we report the development in the Generally Regarded as Safe (GRAS) L-arginine-overproducing C. crenatum for high-titer agmatine biosynthesis through overexpressing arginine decarboxylase based on metabolic engineering. Then, arginine decarboxylase was mutated to release feedback inhibition and improve catalytic activity. Subsequently, the specific enzyme activity and half-inhibitory concentration of I534D mutant were increased 35.7% and 48.1%, respectively. The agmatine production of the whole-cell bioconversion with AGM3 was increased by 19.3% than the AGM2. Finally, 45.26 g/L agmatine with the yield of 0.31 g/g glucose was achieved by one-step fermentation of the engineered C. crenatum with overexpression of speAI534D. CONCLUSIONS: The engineered C. crenatum strain AGM3 in this work was proved as an efficient microbial cell factory for the industrial fermentative production of agmatine. Based on the insights from this work, further producing other valuable biochemicals derived from L-arginine by Corynebacterium crenatum is feasible.

Efficient D-allulose synthesis under acidic conditions by auto-inducing expression of the tandem D-allulose 3-epimerase genes in Bacillus subtilis.

BACKGROUND: D-allulose, a hexulose monosaccharide with low calorie content and high sweetness, is commonly used as a functional sugar in food and nutrition. However, enzyme preparation of D-allulose from D-frutose was severely hindered by the non-enzymatic browning under alkaline and high-temperature, and the unnecessary by-products further increased the difficulties in separation and extraction for industrial applications. Here, to address the above issue during the production process, a tandem D-allulose 3-epimerase (DPEases) isomerase synergistic expression strategy and an auto-inducible promoter engineering were levered in Bacillus subtilis 168 (Bs168) for efficient synthesis of D-allulose under the acidic conditions without browning. RESULTS: First, based on the dicistron expression system, two DPEases with complementary functional characteristics from Dorea sp. CAG:317 (DSdpe) and Clostridium cellulolyticum H10 (RCdpe) were expressed in tandem under the promoter HpaII in one cell. A better potential strain Bs168/pMA5-DSdpe-RCdpe increases enzyme activity to 18.9 U/mL at acidic conditions (pH 6.5), much higher than 17.2 and 16.7 U/mL of Bs168/pMA5-DSdpe and Bs168/pMA5-RCdpe, respectively. Subsequently, six recombinant strains based on four constitutive promoters were constructed in variable expression cassettes for improving the expression level of protein. Among those engineered strains, Bs168/pMA5-PspoVG-DSdpe-PsrfA-RCdpe exhibited the highest enzyme activity with 480.1 U/mL on fed-batch fermentation process in a 5 L fermenter at pH 6.5, about 2.1-times higher than the 228.5 U/mL of flask fermentation. Finally, the maximum yield of D-allulose reached as high as 163.5 g/L at the fructose concentration (50% w/v) by whole-cell biocatalyst. CONCLUSION: In this work, the engineered recombinant strain Bs168/pMA5-PspoVG-DSdpe-PsrfA-RCdpe was demonstrated as an effective microbial cell factory for the high-efficient synthesis of D-allulose without browning under acidic conditions. Based on the perspectives from this research, this strategy presented here also made it possible to meet the requirements of the industrial hyper-production of other rare sugars under more acidic conditions in theory.

Simultaneous determination of nicotinamide and N1 -methylnicotinamide in human serum by LC-MS/MS to associate their serum concentrations with obesity.

A rapid and sensitive LC-MS/MS method was developed and validated for the simultaneous determination of nicotinamide and its metabolite N1 -methylnicotinamide in human serum. Serum samples were prepared by protein precipitation with acetonitrile. The chromatographic separation was performed on a Waters Spherisorb S5 CN microbore column (2.0 × 100 mm, 5 µm) with gradient elution within 7 min. Acetonitrile and 5 mm ammonium formate aqueous solution (containing 0.1% formic acid) were used as mobile phases. Nicotinamide, N1 -methylnicotinamide and N'-methylnicotinamide (internal standard) were detected with a triple-quadrupole tandem mass spectrometer in the positive ion mode. Multiple reaction monitoring was used to monitor precursor to product ion transitions of m/z 123.1 → 80.1 for nicotinamide, m/z 137.1 → 94.1 for N1 -methylnicotinamide and m/z 137.1 → 80.1 for the internal standard. The linear ranges of nicotinamide and N1 -methylnicotinamide were 5.000-160.0 and 2.500-80.00 ng/ml, respectively. The intra- and inter-day precisions (RSD) of both analytes were within 6.90%. The recoveries were >88%. The analytes were proven to be stable during all sample storage, preparation and analytic procedures. The method was successfully applied to determine the concentrations of nicotinamide and N1 -methylnicotinamide in human serum to investigate the association between their concentrations and obesity in 1160 Chinese subjects.

Microbial production of riboflavin: Biotechnological advances and perspectives.

Riboflavin is an essential nutrient for humans and animals, and its derivatives flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) are cofactors in the cells. Therefore, riboflavin and its derivatives are widely used in the food, pharmaceutical, nutraceutical and cosmetic industries. Advances in biotechnology have led to a complete shift in the commercial production of riboflavin from chemical synthesis to microbial fermentation. In this review, we provide a comprehensive review of biotechnologies that enhance riboflavin production in microorganisms, as well as representative examples. Firstly, the synthesis pathways and metabolic regulatory processes of riboflavin in microorganisms; and the current strategies and methods of metabolic engineering for riboflavin production are systematically summarized and compared. Secondly, the using of systematic metabolic engineering strategies to enhance riboflavin production is discussed, including laboratory evolution, histological analysis and high-throughput screening. Finally, the challenges for efficient microbial production of riboflavin and the strategies to overcome these challenges are prospected.

Engineering of microbial cells for L-valine production: challenges and opportunities.

L-valine is an essential amino acid that has wide and expanding applications with a suspected growing market demand. Its applicability ranges from animal feed additive, ingredient in cosmetic and special nutrients in pharmaceutical and agriculture fields. Currently, fermentation with the aid of model organisms, is a major method for the production of L-valine. However, achieving the optimal production has often been limited because of the metabolic imbalance in recombinant strains. In this review, the constrains in L-valine biosynthesis are discussed first. Then, we summarize the current advances in engineering of microbial cell factories that have been developed to address and overcome major challenges in the L-valine production process. Future prospects for enhancing the current L-valine production strategies are also discussed.

Enhanced production of L-arginine by improving carbamoyl phosphate supply in metabolically engineered Corynebacterium crenatum.

Carbamoyl phosphate is an important precursor for L-arginine and pyrimidines biosynthesis. In view of this importance, the cell factory should enhance carbamoyl phosphate synthesis to improve related compound production. In this work, we verified that carbamoyl phosphate is essential for L-arginine production in Corynebacterium sp., followed by engineering of carbamoyl phosphate synthesis for further strain improvement. First, carAB encoding carbamoyl phosphate synthetase II was overexpressed to improve the synthesis of carbamoyl phosphate. Second, the regulation of glutamine synthetase increases the supply of L-glutamine, providing an effective substrate for carbamoyl phosphate synthetase II. Third, carbamate kinase, which catalyzes inorganic ammonia synthesis carbamoyl phosphate, was screened and selected to assist in carbamoyl phosphate supply. Finally, we disrupted ldh (encoding lactate dehydrogenase) to decrease by-production formation and save NADH to regenerate ATP through the electron transport chain. Subsequently, the resulting strain allowed a dramatically increased L-arginine production of 68.6 ± 1.2 g∙L-1, with an overall productivity of 0.71 ± 0.01 g∙L-1∙h-1 in 5-L bioreactor. Stepwise rational metabolic engineering based on an increase in the supply of carbamoyl phosphate resulted in a gradual increase in L-arginine production. The strategy described here can also be implemented to improve L-arginine and pyrimidine derivatives. KEY POINTS: • The L-arginine production strongly depended on the supply of carbamoyl phosphate. • The novel carbamoyl phosphate synthesis pathway for C. crenatum based on carbamate kinase was first applied to L-arginine synthesis. • ATP was regenerated followed with the disruption of lactate formation.

Absolute protein assay for the simultaneous quantification of two epoxide hydrolases in rats by mass spectrometry-based targeted proteomics.

Epoxide hydrolases catalyze the hydrolysis of both exogenous and endogenous epoxides to the corresponding vicinal diols by adding water. Microsomal and soluble epoxide hydrolase are two main mammalian enzymes that have been intensely characterized. The purpose of this investigation was to develop and validate a proteomics assay allowing the simultaneous quantification of microsomal and soluble epoxide hydrolase in rats. Protein quantification was realized through targeted proteomics using liquid chromatography with tandem mass spectrometry for the determination of trypsin-specific surrogate peptides after digestion. Stable isotope-labeled peptides were used as the internal standards. The chromatography of the surrogate peptides was performed on an Agilent SB C18 column (100 mm × 4.6 mm, 1.8 µm) with gradient elution. Acetonitrile containing 0.1% formic acid and 0.1% formic acid aqueous solution were used as mobile phases. A multiple reaction monitoring method in a positive ionization mode was used for the simultaneous detection of the peptides. The method was validated concerning the specificity, linearity, within-day and between-day accuracy and precision, matrix effect, stability, and digestion efficiency. The developed assay was successfully used to quantify the protein levels of microsomal and soluble epoxide hydrolase in rat liver, kidney, and heart S9 samples.

A nomogram to predict stricture-free survival in patients with ureteral stricture after balloon dilation.

BACKGROUND: Balloon dilation is a commonly used minimally invasive endourological treatment of ureteral stricture, but the postoperative recurrence rate is relatively high. And factors contributing to recurrence after treatment are poorly understood. Herein, we sought to develop a novel clinical nomogram to predict ureteral stricture-free survival in patients suffering from ureter stricture and performed balloon dilation. METHODS: The nomogram was established based on a retrospective analysis of 321 patients who received endoscopic balloon dilation alone for ureter strictures from January 2016 to January 2020 in Sun Yat-sen Memorial Hospital using the Cox regression model. Perioperative clinical data and disease outcomes were analyzed. The primary endpoint was the onset of ureteral re-stricture after ureter balloon dilation. Discrimination of the nomogram was assessed by the concordance index (C-index) and the calibration curve. The results were internally validated using bootstrap resampling. RESULTS: Overall, 321 patients with a median follow-up of 590 days were enrolled in the study, among which 97 patients (30.2%) developed recurrence of ureteral stricture during follow-up. Five variables remained significant predictors of ureteral re-stricture after multivariable analyses: stricture nature (P < 0.001), urinary nitrite (P = 0.041), CKD (P = 0.005), stent retention time (P < 0.001), and balloon size (P = 0.029). The calibration craves for the probability of 1-, 3-, and 5-years stricture-free survival (SFS) presented satisfied with the consistency of nomogram prediction and actual observation. The C-index of the model was 0.74 (95% CI 0.70-0.79). CONCLUSIONS: Our study developed the first nomogram to effectively predict stricture-free survival in patients suffering from ureter stricture after balloon dilation. It is helpful to identify the optimal patients with ureter stricture for balloon dilation and improve treatment outcomes. However, further external validation of the nomogram is warranted.

Biochemical Characterization and Structural Insight into Interaction and Conformation Mechanisms of Serratia marcescens Lysine Decarboxylase (SmcadA).

Inducible lysine decarboxylases (LDCs) are essential in various cellular processes of microorganisms and plants, especially under acid stress, which induces the expression of genes encoding LDCs. In this study, a novel Serratia marcesenes LDC (SmcadA) was successfully expressed in E. coli, purified and characterized. The protein had an optimal pH of 6 and a temperature of 40 °C and phylogenetic analysis to determine the evolution of SmcadA, which revealed a close relation to Enterobacteriaceae, Klebsiella sp., among others. The molecular weight of SmcadA was approximately 75 kDa after observation on SDS-PAGE and structural modeling showed the protein as a decamer, comprised of five interlinked dimers. The biocatalytic activity of the purified wild-type SmcadA (WT) was improved through site directed mutations and the results showed that the Arg595Lys mutant had the highest specific activity of 286.55 U/mg, while the Ser512Ala variant and wild-type SmcadA had 215.72 and 179.01 U/mg, respectively. Furthermore, molecular dynamics simulations revealed that interactions through hydrogen bonds between the protein residues and cofactor pyridoxal-5-phosphate (PLP) are vital for biocatalysis. Molecular Dynamics (MD) simulations also indicated that mutations conferred structural changes on protein residues and PLP hence altered the interacting residues with the cofactor, subsequently influencing substrate bioconversion. Moreover, the temperature also induced changes in orientation of cofactor PLP and amino acid residues. This work therefore demonstrates the successful expression and characterization of the purified novel lysine decarboxylase from Serratia marcesenes and provided insight into the mechanism of protein-cofactor interactions, highlighting the role of protein-ligand interactions in altering cofactor and binding site residue conformations, thus contributing to improved biocatalysis.

PII Signal Transduction Protein GlnK Alleviates Feedback Inhibition of N-Acetyl-l-Glutamate Kinase by l-Arginine in Corynebacterium glutamicum.

PII signal transduction proteins are ubiquitous and highly conserved in bacteria, archaea, and plants and play key roles in controlling nitrogen metabolism. However, research on biological functions and regulatory targets of PII proteins remains limited. Here, we illustrated experimentally that the PII protein Corynebacterium glutamicum GlnK (CgGlnK) increased l-arginine yield when glnK was overexpressed in Corynebacterium glutamicum Data showed that CgGlnK regulated l-arginine biosynthesis by upregulating the expression of genes of the l-arginine metabolic pathway and interacting with N-acetyl-l-glutamate kinase (CgNAGK), the rate-limiting enzyme in l-arginine biosynthesis. Further assays indicated that CgGlnK contributed to alleviation of the feedback inhibition of CgNAGK caused by l-arginine. In silico analysis of the binding interface of CgGlnK-CgNAGK suggested that the B and T loops of CgGlnK mainly interacted with C and N domains of CgNAGK. Moreover, F11, R47, and K85 of CgGlnK were identified as crucial binding sites that interact with CgNAGK via hydrophobic interaction and H bonds, and these interactions probably had a positive effect on maintaining the stability of the complex. Collectively, this study reveals PII-NAGK interaction in nonphotosynthetic microorganisms and further provides insights into the regulatory mechanism of PII on amino acid biosynthesis in corynebacteria.IMPORTANCE Corynebacteria are safe industrial producers of diverse amino acids, including l-glutamic acid and l-arginine. In this study, we showed that PII protein GlnK played an important role in l-glutamic acid and l-arginine biosynthesis in C. glutamicum Through clarifying the molecular mechanism of CgGlnK in l-arginine biosynthesis, the novel interaction between CgGlnK and CgNAGK was revealed. The alleviation of l-arginine inhibition of CgNAGK reached approximately 48.21% by CgGlnK addition, and the semi-inhibition constant of CgNAGK increased 1.4-fold. Furthermore, overexpression of glnK in a high-yield l-arginine-producing strain and fermentation of the recombinant strain in a 5-liter bioreactor led to a remarkably increased production of l-arginine, 49.978 g/liter, which was about 22.61% higher than that of the initial strain. In conclusion, this study provides a new strategy for modifying amino acid biosynthesis in C. glutamicum.

LysR-Type Transcriptional Regulator MetR Controls Prodigiosin Production, Methionine Biosynthesis, Cell Motility, H2O2 Tolerance, Heat Tolerance, and Exopolysaccharide Synthesis in Serratia marcescens.

Prodigiosin, a secondary metabolite produced by Serratia marcescens, has attracted attention due to its immunosuppressive, antimicrobial, and anticancer properties. However, information on the regulatory mechanism behind prodigiosin biosynthesis in S. marcescens remains limited. In this work, a prodigiosin-hyperproducing strain with the BVG90_22495 gene disrupted (ZK66) was selected from a collection of Tn5G transposon insertion mutants. Using real-time quantitative PCR (RT-qPCR) analysis, ß-galactosidase assays, transcriptomics analysis, and electrophoretic mobility shift assays (EMSAs), the LysR-type regulator MetR encoded by the BVG90_22495 gene was found to affect prodigiosin synthesis, and this correlated with MetR directly binding to the promoter region of the prodigiosin-synthesis positive regulator PigP and hence negatively regulated the expression of the prodigiosin-associated pig operon. More analyses revealed that MetR regulated some other important cellular processes, including methionine biosynthesis, cell motility, H2O2 tolerance, heat tolerance, exopolysaccharide synthesis, and biofilm formation in S. marcescens Although MetR protein is highly conserved in many bacteria, we report here on the LysR-type regulator MetR exhibiting novel roles in negatively regulating prodigiosin synthesis and positively regulating heat tolerance, exopolysaccharide synthesis, and biofilm formation.IMPORTANCESerratia marcescens, a Gram-negative bacterium, is found in a wide range of ecological niches and can produce several secondary metabolites, including prodigiosin, althiomycin, and serratamolide. Among them, prodigiosin shows diverse functions as an immunosuppressant, antimicrobial, and anticancer agent. However, the regulatory mechanisms behind prodigiosin synthesis in S. marcescens are not completely understood. Here, we adapted a transposon mutant library to identify the genes related to prodigiosin synthesis, and the BVG90_22495 gene encoding the LysR-type regulator MetR was found to negatively regulate prodigiosin synthesis. The molecular mechanism of the metR mutant hyperproducing prodigiosin was investigated. Additionally, we provided evidence supporting new roles for MetR in regulating methionine biosynthesis, cell motility, heat tolerance, H2O2 tolerance, and exopolysaccharide synthesis in S. marcescens Collectively, this work provides novel insight into regulatory mechanisms of prodigiosin synthesis and uncovers novel roles for the highly conserved MetR protein in regulating prodigiosin synthesis, heat tolerance, exopolysaccharide (EPS) synthesis, and biofilm formation.

Optimization of l-arginine purification from Corynebacterium crenatum fermentation broth.

l-Arginine has many special physiological and biochemical functions, with wide applications in the food and pharmaceutical industry. Few studies on the purification of l-arginine from fermentation broth have been conducted; however, none of them were systematic enough for industrial scale-up. Therefore, it is necessary to develop a highly efficient and systematic process for the purification of l-arginine from fermentation broth. In this study, we screened out a cation exchange resin, D155, having high exchange capacity, high selectivity, and easy elution capacity, and analyzed its adsorption isotherm, thermodynamics, and kinetics using different models. Further, the process parameters of fixed-bed ion exchange adsorption and elution were optimized, and the penetration curve during the operation was modeled. Based on the fixed-bed ion-exchange parameters, a 30-column continuous ion-exchange system was designed, and the flow velocity in each zone was optimized. Finally, to obtain a high purity of l-arginine, the purification tests were conducted using anion exchange resin 711, and an l-arginine yield of 99.1% and purity of 98.5% was obtained. This effective and economical method also provides a promising strategy for separation of other amino acids from the fermentation broth, which is of great significance to the l-arginine fermentation industry.

Asp305Gly mutation improved the activity and stability of the styrene monooxygenase for efficient epoxide production in Pseudomonas putida KT2440.

BACKGROUND: Styrene monooxygenase (SMO) catalyzes the first step of aromatic alkene degradation yielding the corresponding epoxides. Because of its broad spectrum of substrates, the enzyme harbors a great potential for an application in medicine and chemical industries. RESULTS: In this study, we achieved higher enzymatic activity and better stability towards styrene by enlarging the ligand entrance tunnel and improving the hydrophobicity through error-prone PCR and site-saturation mutagenesis. It was found that Asp305 (D305) hindered the entrance of the FAD cofactor according to the model analysis. Therefore, substitution with amino acids possessing shorter side chains, like glycine, opened the entrance tunnel and resulted in up to 2.7 times higher activity compared to the wild-type enzyme. The half-lives of thermal inactivation for the variant D305G at 60 °C was 28.9 h compared to only 3.2 h of the wild type SMO. Moreover, overexpression of SMO in Pseudomonas putida KT2440 with NADH regeneration was carried out in order to improve biotransformation efficiency for epoxide production. A hexadecane/buffer (v/v) biphasic system was applied in order to minimize the inactivation effect of high substrate concentrations on the SMO enzyme. Finally, SMO activities of 190 U/g CDW were measured and a total amount of 20.5 mM (S)-styrene oxide were obtained after 8 h. CONCLUSIONS: This study offers an alternative strategy for improved SMO expression and provides an efficient biocatalytic system for epoxide production via engineering the entrance tunnel of the enzyme's active site.

Synthetic engineering of Corynebacterium crenatum to selectively produce acetoin or 2,3-butanediol by one step bioconversion method.

BACKGROUND: Acetoin (AC) and 2,3-butanediol (2,3-BD) as highly promising bio-based platform chemicals have received more attentions due to their wide range of applications. However, the non-efficient substrate conversion and mutually transition between AC and 2,3-BD in their natural producing strains not only led to a low selectivity but also increase the difficulty of downstream purification. Therefore, synthetic engineering of more suitable strains should be a reliable strategy to selectively produce AC and 2,3-BD, respectively. RESULTS: In this study, the respective AC (alsS and alsD) and 2,3-BD biosynthesis pathway genes (alsS, alsD, and bdhA) derived from Bacillus subtilis 168 were successfully expressed in non-natural AC and 2,3-BD producing Corynebacterium crenatum, and generated recombinant strains, C. crenatum SD and C. crenatum SDA, were proved to produce 9.86 g L-1 of AC and 17.08 g L-1 of 2,3-BD, respectively. To further increase AC and 2,3-BD selectivity, the AC reducing gene (butA) and lactic acid dehydrogenase gene (ldh) in C. crenatum were then deleted. Finally, C. crenatumΔbutAΔldh SD produced 76.93 g L-1 AC in one-step biocatalysis with the yield of 0.67 mol mol-1. Meanwhile, after eliminating the lactic acid production and enhancing 2,3-butanediol dehydrogenase activity, C. crenatumΔldh SDA synthesized 88.83 g L-1 of 2,3-BD with the yield of 0.80 mol mol-1. CONCLUSIONS: The synthetically engineered C. crenatumΔbutAΔldh SD and C. crenatumΔldh SDA in this study were proved as an efficient microbial cell factory for selective AC and 2,3-BD production. Based on the insights from this study, further synthetic engineering of C. crenatum for AC and 2,3-BD production is suggested.