Cadaverine Biosynthesis Research Articles

Fine-Tuning Pyridoxal 5'-Phosphate Synthesis in Escherichia coli for Cadaverine Production in Minimal Culture Media.

Cadaverine is a critical C5 monomer for the production of polyamides. Pyridoxal 5'-phosphate (PLP), as a crucial cofactor for the key enzyme lysine decarboxylase in the cadaverine biosynthesis pathway, has seen a persistent shortage, leading to limitations in cadaverine production. To address this issue, a dual-pathway strategy was implemented, synergistically enhancing both endogenous and heterologous PLP synthesis modules and resulting in improved PLP synthesis. Subsequently, a growth-stage-dependent molecular switch was introduced to balance the precursor competition between PLP synthesis and cell growth. Additionally, a PLP sensor-based negative feedback circuit was constructed by integrating a newly identified PLP-responsive promoter PygjH and an arabinose-regulated system, dynamically regulating the expression of the PLP synthetic genes and preventing excessive intracellular PLP accumulation. The optimal strain, L18, cultivated in the minimal medium AM1, demonstrated cadaverine production with a titer, yield, and productivity of 64.03 g/L, 0.23 g/g glucose, and 1.33 g/L/h, respectively. This represents the highest titer reported to date in engineered Escherichia coli by fed-batch fermentation in a minimal medium.

Combining directed evolution with high cell permeability for high-level cadaverine production in engineered Escherichia coli.

The biosynthesis of cadaverine from lysine is an environmentally promising technology, that could contribute to a more sustainable approach to manufacturing bio-nylon 5X. However, the titer of biosynthesized cadaverine has still not reached a sufficient level for industrial production. A powerful green cell factory was developed to enhance cadaverine production by regulating lipopolysaccharide (LPS) genes and improving membrane permeability. Firstly, 10 LPS mutant strains were constructed and the effect on the growth was investigated. Then, the lysine decarboxylase (CadA) was overexpressed in 10 LPS mutant strains of Escherichia coli MG1655 and the ability to produce cadaverine was compared. Using 20.0gL-1 of L-lysine hydrochloride (L-lysine-HCl) as the substrate for the biotransformation reaction, Cad02 and Cad06 strains exhibited high production levels of cadaverine, with 8.95gL-1 and 7.55gL-1 respectively while the control strain Cad00 only 4.92gL-1 . Directed evolution of CadA was also used to improve its stability under alkaline conditions. The cadaverine production of the Cad02-M mutant stain increased by 1.86 times at pH 8.0. Finally, the production process was scaled up using recombinant whole cells as catalysts, achieving a high titer of 211gL-1 cadaverine (96.8%) by fed-batch bioconversion. This study demonstrates the potential role of LPS in enhancing the efficiency of mass transfer between substrate and enzymes in vivo by increasing cell permeability. The results indicate that the argumentation of cell permeability could not only significantly enhance the biotransformation efficiency of cadaverine, but also provide a universally applicable, straightforward, environment-friendly, and cost-effective method for the biosynthesis of other high-value chemicals.

Deletion of ArmPT, a LamB-like protein, increases cell membrane permeability and antibiotic sensitivity in Vibrio alginolyticus

Vibrio bacterial species are dominant pathogens in mariculture animals. However, the extensive use of antibiotics and other chemicals has increased drug resistance in Vibrio bacteria. Despite rigorous investigative studies, the mechanism of drug resistance in Vibrio remains a mystery. In this study, we found that a gene encoding LamB-like outer membrane protein, named ArmPT, was upregulated in Va under antibiotic stress by RT-qPCR. We speculated that ArmPT might play a role in Va’s drug resistance. Subsequently, using ArmPT gene knockout and gene complementation experiments, we confirmed its role in resistance against a variety of antibiotics, particularly kanamycin (KA). Transcriptomic and proteomic analyses identified 188 and 83 differentially expressed genes in the mutant strain compared with the wild-type (WT) before and after KA stress, respectively. Bioinformatic analysis predicted that ArmPT might control cell membrane permeability by changing cadaverine biosynthesis, thereby influencing the cell entry of antibiotics in Va. The higher levels of intracellular reactive oxygen species and the infused content of KA showed that antibiotics are more likely to enter the Va mutant strain. These results uncover the drug resistance mechanism of Va that can also exist in other similar pathogenic bacteria.

Screening of exopolysaccharide-producing Enterobacter aerogenes NJ1023 and its cadaverine biosynthesis promotion.

Enterobacter aerogenes, the gram-negative bacteria belonging to the family Enterobacteriaceae, lacks the ability to synthesize chemicals. However, in this study, a strain of Enterobacter aerogenes NJ1023 screened from the soil containing petrochemicals was found to be capable of producing extracellular polysaccharides (EPSs). After purification of the polysaccharide, the chemical composition and physicochemical properties of the polysaccharide were analyzed by UV-Vis spectra, FTIR spectroscopy and GC-MS, etc. The results showed that: The molecular weight of the polysaccharide produced by this strain was only 2.7×103 Da, which was lower than that reported in other polysaccharides from the same genus. The polysaccharide produced by E. aerogenes NJ1023 mainly comprised xylose, glucose, galactose, and N-acetylglucosamine with a molar ratio of 0.27: 4.52: 1.74: 0.2, which differed from those reported from the same genus. The results demonstrated that lower incubation temperatures and shaking speeds were more favorable for EPSs synthesis, while higher incubation temperatures and shaking speeds favored cell growth. Additionally, the EPSs produced by E. aerogenes NJ1023 significantly protected the Escherichia coli cells against cadaverine stress. Overall, the discovery of EPSs produced by E. aerogenes increased the diversity of bacterial polysaccharides and broadened the potential applications of this species.

Engineering synthetic microbial consortium for cadaverine biosynthesis from glycerol.

1,5-pentanediamine (cadaverine) is a C5 platform chemical, also an important raw material for bio-polyamide PA5X. With increasing concerns about the depletion of fossil resources and global environmental protection, cadaverine bio-production has attracted more attentions. Here, a microbial consortium consisting of Corynebacterium glutamicum cgl-FDK and Escherichia coli BL-ABST-Spy was constructed to de novo synthesize cadaverine utilizing glycerol as the sole carbon resource. The glycerol utilization pathway was initially constructed in C. glutamicum cgl-FDK to produce lysine from glycerol. Then, the pyridoxal 5'-phosphate (PLP) biosynthesis pathway and SpyTag/SpyCatcher protein-ligation system for lysine decarboxylase (CadA) and cadaverine-lysine antiporter protein (CadB) were introduced into E. coli BL-ABST-Spy to synthesize cadaverine from lysine. Furthermore, the fermentation conditions of microbial consortium were optimized and the cadaverine production reached 9.3g/L with glycerol as the sole carbon source. This work provides a promising strategy for efficiently producing cadaverine from glycerol with an artificial microbial consortium.

Cadaverine Biosynthesis in Escherichia сoli Adaptation to Hydrogen Peroxide

Cadaverine Biosynthesis in Escherichia сoli Adaptation to Hydrogen Peroxide

Engineered Halomonas spp. for production of l-Lysine and cadaverine

Cadaverine, a derivative of l-lysine, has been used as a monomer for the synthesis of bio-based nylon-5,6. This study engineered Halomonas bluephagenesis TD1.0 by blocking the feedback inhibition, overexpressing the key l-lysine synthesis genes, strengthening the l-lysine export system and increasing the supply of oxaloacetate for production of l-lysine in the supernatant and PHB in the cells. Subsequently, cadaverine biosynthetic pathway was constructed in H. campaniensis LC-9 to improve the efficiency of de novo cadaverine biosynthesis which combines l-lysine producing H. bluephagenesis TDL8-68–259 and cadaverine producing H. campaniensis LC-9-ldcC-lysP. When H. campaniensis LC-9-ldcC-lysP was used as a whole cell catalysis for cadaverine production, the conversion efficiency of l-lysine to cadaverine reached 100% in the presence of 0.05% Triton X-100 for cell membrane permeability enhancement, resulting in 118 g L-1 cadaverine formed in the fermentor. Thus, Halomonas spp. have been successfully constructed for l-lysine and cadaverine production.

Cofactor self-sufficient by co-immobilization of pyridoxal 5′-phosphate and lysine decarboxylase for cadaverine production

Cadaverine (1,5-diaminopentane) has widespread applications in industry, agriculture and medicine. Cadaverine can be biotransformed from l-lysine by lysine decarboxylasey which is a pyridoxal 5′-phosphate (PLP)-dependent enzyme. Herein, barium alginate microspheres were fabricated by electro-dripping and employed as immobilized metal ion affinity chromatography matrix to purify and immobilize engineered lysine decarboxylase CadA-EK. Besides, polyethyleneimine (PEI) was employed for co-immobilizing PLP and enzymes (cCadA-EK-PEI/PLP). The cCadA-EK-PEI/PLP can produced 184.53 g/L cadaverine with yield of 90.03% in 2 mol/L lysine solution. And the stability of co-immobilized CadA-EK was 1–4 times higher than that of iCadA-EK + PLP in environmental factors, and the catalytic efficiency kcat/Km was increased by 3.4-fold. Furthermore, a continuous flow microreactor with the co-immobilized enzymes and cofactors was developed to catalyze lysine, with 14.28 g/(L·h) productivity of cadaverine, which displayed great potential for cadaverine biosynthesis. This work provides alternative strategy for the one-step purification and co-immobilization of enzyme and cofactor.

Cadaverine biosynthesis contributes to decreased Escherichia coli susceptibility to antibiotics

Some bacterial stress responses are involved in survival under antibiotic treatment and contribute to less susceptible microbial forms selection. Here, we tested the role of cadaverine, one of the biogenic polyamines considered as universal adaptogens, in the processes. The expression of ldcC and cadA genes, encoding cadaverine-producing lysine decarboxylase, increased in Escherichia coli cells exposed to β-lactams and fluoroquinolones but not aminoglycosides. The transcriptional regulators RpoS and SoxS controlled the expression of ldcC and cadA, respectively, in response to antibiotics. Exogenous cadaverine had little effect on E. coli antibiotic susceptibility, whereas non-antibiotic-induced endogenous cadaverine contributed to its tolerance to β-lactams, fluoroquinolones, and aminoglycosides. Antibiotic-induced cadaverine synthesis promoted bacterial survival under fluoroquinolone exposure, as well as could contribute to low-resistant bacterial forms development. Selection under the fluoroquinolone levofloxacin exposure toward bacteria with an increased ability to synthesize cadaverine and negative correlation between LdcC activity and fluoroquinolone susceptibility in the selected forms were demonstrated. The same correlation in a special group of low-level resistant clinical E. coli isolates was revealed. So, cadaverine biosynthesis appeared to be a significant player in decreased E. coli antibiotic susceptibility development.

Green chemical and biological synthesis of cadaverine: recent development and challenges.

Cadaverine has great potential to be used as an important monomer for the development of a series of high value-added products with market prospects. The most promising strategies for cadaverine synthesis involve using green chemical and bioconversion technologies. Herein, the review focuses on the progress and strategies towards the green chemical synthesis and biosynthesis of cadaverine. Specifically, we address the specific biosynthetic pathways of cadaverine from different substrates as well as extensively discussing the origination, structure and catalytic mechanism of the key lysine decarboxylases. The advanced strategies for process intensification, the separation and purification of cadaverine have been summarized. Furthermore, the challenging issues of the environmental, economic, and applicable impact for cadaverine production are also highlighted. This review concludes with the promising outlooks of state-of-the-art applications of cadaverine along with some insights toward their challenges and potential improvements.

Integrated gene engineering synergistically improved substrate-product transport, cofactor generation and gene translation for cadaverine biosynthesis in E. coli

Several approaches for efficient production of cadaverine, a bio-based diamine with broad industrial applications have been explored. Here, Serratia marcescens lysine decarboxylase (SmcadA) was expressed in E. coli; mild surfactants added in biotransformation reactions; the E. coli native lysine/cadaverine antiporter cadB, E. coli pyridoxal kinases pdxK and pdxY overexpressed and synthetic RBS libraries screened. Addition of mild surfactants and overexpression of antiporter cadB increased cadaverine biosynthesis of SmcadA. Moreover, expression of pdxY gene yielded 19.82 g/L in a reaction mixture containing added cofactor precursor pyridoxal (PL), without adding exogenous PLP. The screened synthetic RBS1, applied to fully exploit pdxY gene expression, ultimately resulted in PLP self-sufficiency, producing 27.02 g/L cadaverine using strain T7R1_PL. To boost SmcadA catalytic activity, the designed mutants Arg595Lys and Ser512Ala had significantly improved cumulative cadaverine production of 219.54 and 201.79 g/L respectively compared to the wild-type WT (181.62 g/L), after 20 h reaction. Finally, molecular dynamics simulations for WT and variants indicated that increased flexibility at the binding sites of the protein enhanced residue-ligand interactions, contributing to high cadaverine synthesis. This work demonstrates potential of harnessing different pull factors through integrated gene engineering of efficient biocatalysts and gaining insight into the mechanisms involved through MD simulations.

Cadaverine, a metabolite of the microbiome, reduces breast cancer aggressiveness through trace amino acid receptors

Recent studies showed that changes to the gut microbiome alters the microbiome-derived metabolome, potentially promoting carcinogenesis in organs that are distal to the gut. In this study, we assessed the relationship between breast cancer and cadaverine biosynthesis. Cadaverine treatment of Balb/c female mice (500 nmol/kg p.o. q.d.) grafted with 4T1 breast cancer cells ameliorated the disease (lower mass and infiltration of the primary tumor, fewer metastases, and lower grade tumors). Cadaverine treatment of breast cancer cell lines corresponding to its serum reference range (100–800 nM) reverted endothelial-to-mesenchymal transition, inhibited cellular movement and invasion, moreover, rendered cells less stem cell-like through reducing mitochondrial oxidation. Trace amino acid receptors (TAARs), namely, TAAR1, TAAR8 and TAAR9 were instrumental in provoking the cadaverine-evoked effects. Early stage breast cancer patients, versus control women, had reduced abundance of the CadA and LdcC genes in fecal DNA, both responsible for bacterial cadaverine production. Moreover, we found low protein expression of E. coli LdcC in the feces of stage 1 breast cancer patients. In addition, higher expression of lysine decarboxylase resulted in a prolonged survival among early-stage breast cancer patients. Taken together, cadaverine production seems to be a regulator of early breast cancer.

Crystal Structure and Pyridoxal 5-Phosphate Binding Property of Lysine Decarboxylase from Selenomonas ruminantium.

Lysine decarboxylase (LDC) is a crucial enzyme for acid stress resistance and is also utilized for the biosynthesis of cadaverine, a promising building block for bio-based polyamides. We determined the crystal structure of LDC from Selenomonas ruminantium (SrLDC). SrLDC functions as a dimer and each monomer consists of two distinct domains; a PLP-binding barrel domain and a sheet domain. We also determined the structure of SrLDC in complex with PLP and cadaverine and elucidated the binding mode of cofactor and substrate. Interestingly, compared with the apo-form of SrLDC, the SrLDC in complex with PLP and cadaverine showed a remarkable structural change at the PLP binding site. The PLP binding site of SrLDC contains the highly flexible loops with high b-factors and showed an open-closed conformational change upon the binding of PLP. In fact, SrLDC showed no LDC activity without PLP supplement, and we suggest that highly flexible PLP binding site results in low PLP affinity of SrLDC. In addition, other structurally homologous enzymes also contain the flexible PLP binding site, which indicates that high flexibility at the PLP binding site and low PLP affinity seems to be a common feature of these enzyme family.

Improving the secretion of cadaverine in Corynebacterium glutamicum by cadaverine–lysine antiporter

Cadaverine (1,5-pentanediamine, diaminopentane), the desired raw material of bio-polyamides, is an important industrial chemical with a wide range of applications. Biosynthesis of cadaverine in Corynebacterium glutamicum has been a competitive way in place of petroleum-based chemical synthesismethod. To date, the cadaverine exporter has not been found in C. glutamicum. In order to improve cadaverine secretion, the cadaverine-lysine antiporter CadB from Escherichia coli was studied in C. glutamicum. Fusion expression of cadB and green fluorescent protein (GFP) gene confirmed that CadB could express in the cell membrane of C. glutamicum. Co-expression of cadB and ldc from Hafnia alvei in C. glutamicum showed that the cadaverine secretion rate increased by 22% and the yield of total cadaverine and extracellular cadaverine increased by 30 and 73%, respectively. Moreover, the recombinant strain cultured at acid and neutral pH separately hardly had any difference in cadaverine concentrations. These results suggested that CadB could be expressed in the cell membrane of C. glutamicum and that recombinant CadB could improve cadaverine secretion and the yield of cadaverine. Moreover, the pH value did not affect the function of recombinant CadB. These results may be a promising metabolic engineering strategy for improving the yield of the desired product by enhancing its export out of the cell.

Molecular Basis for the Maintenance of Envelope Integrity in Selenomonas ruminantium: Cadaverine Biosynthesis and Covalent Modification into the Peptidoglycan Play a Major Role

Polyamine is a small organic polycation composed of a hydrocarbon backbone with multiple amino groups which ubiquitously exists in all living organisms from bacteria to higher animals. The critical step of polyamine biosynthesis generally includes the amino acid-decarboxylating reaction to produce the primary diamines, such as a synthesis of putrescine (NH(3)(+)·(CH(2))(4)·NH(3)(+)) from ornithine, and cadaverine (NH(3)(+)·(CH(2))(5)·NH(3)(+)) from lysine, which are catalyzed by pyridoxal-5'-phosphate (PLP; vitamin B(6))-dependent decarboxylases. Synthesized polyamines are implicated in a wide variety of cytoplasmic reactions such as DNA replication and protein synthesis, and are essential for proper growth of the organisms. However, in Selenomonas ruminantium, a strictly anaerobic Gram-negative bacterium dominant in sheep rumen, cadaverine displays its function in a quite distinctive scheme compared to the general bacteria reported. It serves as an essential constituent of the peptidoglycan for the maintenance of envelope integrity through an interaction with the periplasm-exposed SLH domain of Mep45, the outer membrane protein of this bacterium. Furthermore, cytoplasmic biosynthesis of cadaverine occurs totally in a eukaryotic-like manner rather than in a conventional way of bacteria. Lysine/ornithine decarboxylase (LDC/ODC), a PLP-dependent enzyme responsible for cadaverine synthesis in this bacterium, displays significant homology to the eukaryotic ODC but not to the general bacterial LDC nor ODC, and its activity is tightly regulated by antizyme-mediated proteolysis, a regulatory process generally found in eukaryotes. These findings represent the biological diversity of this bacterium beyond the preexisting knowledge related to the polyamine-physiology, cell envelope-architecture, and the regulatory system for the enzyme. In this review we will describe (i) the cadaverine-containing peptidoglycan of S. ruminantium: its chemical structure, biosynthesis, and biological function, and (ii) cellular biosynthesis of cadaverine by LDC/ODC and its antizyme-mediated regulation. In addition, we will briefly refer to (iii) the phylogenetic position and characteristics of S. ruminantium and its unique cadaverine-physiology.

Molecular dissection of the Selenomonas ruminantium cell envelope and lysine decarboxylase involved in the biosynthesis of a polyamine covalently linked to the cell wall peptidoglycan layer.

The wild type of Selenomonas ruminantium subsp. lactilytica, which is a strictly anaerobic, Gram-negative bacterium isolated from sheep rumen, requires one of the normal saturated volatile fatty acids with 3 to 10 carbon atoms for its growth in a glucose medium; however, no such obligate requirement of fatty acid is observed when the cells are grown in a lactate medium. This bacterium is characterized by a unique structure of the cell envelope and a novel lysine decarboxylase and its regulatory protein. In the first part of this article, we will refer to the chemical structure of phospholipid and lipopolysaccharide in the cell membranes of this bacterium compared with that from the general Gram-negative bacteria for understanding their biological functions. S. ruminantium has neither free nor bound forms of Braun lipoprotein which plays an important role of the maintenance of the structural integrity of the cell surface in general Gram-negative bacteria. However, S. ruminantium has cadaverine, which links covalently to the peptidoglycan as a pivotal constituent for the cell division. In the second part of this article, we will refer to the chemical structure of the cadaverine-containing peptidoglycan, its biosynthesis, and the biological function. In the third part of this article, we will depict the molecular cloning of the genes encoding S. ruminanitum lysine decarboxylase (LDC) and its regulatory protein of 22-kDa (22-kDa protein; P22) which has similar characteristics to that of antizyme of ornithine decarboxylase in eukaryotic cells, and the molecular dissection of these proteins for understanding the regulation of cadaverine biosynthesis. Finally, we will illustrate a proposed structure of the cell envelope, a processes of biosynthesis of the cadaverine-containing peptidoglycan layer, and the LDC degradation mechanism in S. ruminantium, on the basis of the analyses of the cell envelope components, the results from the in vitro experiments on the biosynthesis of the peptidoglycan layer, and the current status of the knowledge on LDC and P22 in this organism.

Chromosomal localization of osmotic and salt stress-induced differential alterations in polyamine content in wheat

Osmotic and salinity-induced polyamine accumulation were compared in callus cultures of drought and salt tolerant wheat ( Triticus aestivum L) cultivars and in disomic substitution lines. Putrescine, spermidine and spermine occurred in all cultures. Mannitol-induced osmotic stress increased putrescine in all, and cadaverine in two varieties, while salt stress increased spermidine titer, the accumulation rate being higher in sensitive than in tolerant varieties. Specific chromosome (5A and 7A) involvement in osmotic stress induced spermidine accumulation revealed that mannitol was the most effective stress agent and only spermidine titer of Chinese Spring was significantly changed as a consequence of chromosome substitution. The A genome of Cappelle Desprez (donor) substituted into Chinese Spring (recipient) appears to carry genes involved in the control of osmotic stress induced spermidine accumulation, and the genes controlling cadaverine biosynthesis may be localized in chromosome 5B.

Construction of an Escherichia coli strain unable to synthesize putrescine, spermidine, or cadaverine: characterization of two genes controlling lysine decarboxylase.

We have previously described a polyamine-deficient strain of Escherichia coli that contained deletions in speA (arginine decarboxylase), speB (agmatine ureohydrolase), speC (ornithine decarboxylase), and speD (adenosylmethionine decarboxylase). Although this strain completely lacked putrescine and spermidine, it was still able to grow at a slow rate indefinitely on amine-deficient media. However, these cells contained some cadaverine (1,5-diaminopentane). To rule out the possibility that the presence of cadaverine permitted the growth of this strain, we isolated a mutant (cadA) that is deficient in cadaverine biosynthesis, namely, a mutant lacking lysine decarboxylase, and transduced this cadA gene into the delta (speA-speB) delta speC delta D strain. The resultant strain had essentially no cadaverine but showed the same phenotypic characteristics as the parent. Thus, these results confirm our previous findings that the polyamines are not essential for the growth of E. coli or for the replication of bacteriophages T4 and T7. We have mapped the cadA gene at 92 min; the gene order is mel cadA groE ampA purA. A regulatory gene for lysine decarboxylase (cadR) was also obtained and mapped at 46 min; the gene order is his cdd cadR fpk gyrA.

Biosynthesis of Cadaverine in Mice under the Influence of an Anabolic Steroid

The content of cadaverine (1,5-diaminopentane) in the kidney and urine was investigated in mice treated with the anabolic steroid Durabolin (nondrolone phenpropionate). After administration of this steroid cadaverine was found in the kidneys, whereas this amine could not be detected in the kidney of controls. The urinary excretion of cadaverine was elevated 50 times after Durabolin administration. An enzyme catalyzing the formation of cadaverine from lysine was shown for the first time to be present in mammalian tissue, namely in the kidney of mice after Durabolin administration.