Strain N1 Research Articles

Production of anti-rM14 and anti-rM42 antibodies and their application in the stidy of expression of metalloproteases M14 and M42 of keratinolytic strain of Bacillus paralicheniformis

In the present study, two metalloproteases M14 and M42 from a Kazakh strain of Bacillus paralicheniformis T7 with high proteolytic activity were successfully cloned, expressed and purified. Protease genes were amplified by polymerase chain reaction (PCR) using specific primers selected on the basis of full genome sequencing data of B. paralicheniformis strain N7 (GenBank accession #CP124861). The amplified fragments were cloned in the expression vector pET-28c(+). Recombinant analogs of the metalloproteases M14 and M42 were obtained by plasmid expression in Escherichia coli cells of the strain ArcticExpressRP(DE3). Using metal affinity chromatography, rM14 and rM42 proteins were purified and polyclonal antibodies were produced by immunization of rabbits. Using anti-rM14 and anti-rM42 antibodies, the expression level of native metalloproteases M14 and M42 in cell lysate and culture fluid of B. paralicheniformis T7 strain was examined after culturing the strain on keratin-containing media: chicken feather, horn, hoof, hide and wool of cattle. Substrate-dependent expression of metalloprotease genes of B. paralicheniformis strain T7 was established. The obtained results indicate the prospective use B. paralicheniformis strain T7 as a producer of metalloproteases M14 and M42.

Mixed culture fermentation using Lactobacillus delbrueckii and novel Lactiplantibacillus plantarum strains isolated from traditional chhurpi from Sikkim Himalayan region for potential probiotic and improved functional properties

The aim of the study was to isolate potential probiotic lactic acid bacteria (LAB) strains from the traditional fermented milk products, Chhurpi of the Sikkim Himalayan region and to prepare curd using mixed culture fermentation. A preliminary probiotic assessment of 110 LAB isolates including antimicrobial activity, bile and acid tolerance, surface hydrophobicity, auto-aggregation and co-aggregation, and adhesion to Caco-2 cells, was performed. Lactiplantibacillus plantarum strains N4, CHDE2, CHDE4, and SS22 demonstrated better viability in bile (0.3%) and acid (pH 2.5), adherence with hydrophobicity (13.93–92.85%), auto-aggregation (72.44–89.74%), adhesion to Caco-2 cells (45–73.61%) and antimicrobial activity against food borne pathogens. These strains were used for mixed culture fermentation with a previously screened curd forming strain Lactobacillus delbrueckii WS4. Curd produced using mixed culture fermentation of Lb. delbrueckii WS4 and the selected Lp. plantarum strains had a relatively higher acid production (pH 3.97–4.01), protein hydrolysis, and antioxidant activity. Further, mixed culture fermentation also resulted in the increase of HOCl scavenging activity (12.22–32.96%), myeloperoxidase (MPO) inhibition activity (22.38–42.94%) and angiotensin converting enzyme (ACE) inhibition (15.61–33.03%) as compared to that of WS4 fermentation (P < 0.05). This is the first report on identification of potential probiotic strains from the traditional fermented milk products of Sikkim and application in mixed culture fermentation with enhanced multifunctional potential.

A comprehensive analysis of the WRKY family in soybean and functional analysis of GmWRKY164-GmGSL7c in resistance to soybean mosaic virus

BackgroundSoybean mosaic disease caused by soybean mosaic virus (SMV) is one of the most devastating and widespread diseases in soybean producing areas worldwide. The WRKY transcription factors (TFs) are widely involved in plant development and stress responses. However, the roles of the GmWRKY TFs in resistance to SMV are largely unclear.ResultsHere, 185 GmWRKYs were characterized in soybean (Glycine max), among which 60 GmWRKY genes were differentially expressed during SMV infection according to the transcriptome data. The transcriptome data and RT-qPCR results showed that the expression of GmWRKY164 decreased after imidazole treatment and had higher expression levels in the incompatible combination between soybean cultivar variety Jidou 7 and SMV strain N3. Remarkably, the silencing of GmWRKY164 reduced callose deposition and enhanced virus spread during SMV infection. In addition, the transcript levels of the GmGSL7c were dramatically lower upon the silencing of GmWRKY164. Furthermore, EMSA and ChIP-qPCR revealed that GmWRKY164 can directly bind to the promoter of GmGSL7c, which contains the W-box element.ConclusionOur findings suggest that GmWRKY164 plays a positive role in resistance to SMV infection by regulating the expression of GmGSL7c, resulting in the deposition of callose and the inhibition of viral movement, which provides guidance for future studies in understanding virus-resistance mechanisms in soybean.

DIAZOTROPHIC ENDOPHYTE Klebsiella sp. N5 FROM SORGHUM SHOWS CROSS-COLONIZATION AND PLANT GROWTH PROMOTION IN RICE

Rice necessitates the highest levels of N fertilizers during its cultivation increasing demand for nitrogen fertilizers as well as negatively affecting the environment. Endophytic diazotrophic bacteria were isolated from sorghum, out of 35 isolates Klebsiella sp. N5 showed maximum potential for biological nitrogen fixation. Primary screening of strain N5 was done with acetylene reduction assay indicating dry root activity of 638.55 nmol C2H4 h-1 g-1. That was further confirmed by nifH gene amplification. The strain was identified morphologically, biochemically and 16S rDNA sequencing analysis and identified as Klebsiella sp. strain N5. This strain exhibited other plant growth-promoting (PGP) traits, such as phosphate solubilization, production of indole acetic acid, siderophore activity, ACC deaminase activity, and anti-fungal activity. PGP endophyte N5 was inoculated into rice seedlings to investigate its interaction under axenic conditions and to characterize its ability to colonize non-native host plant rice. Extensive colonization of rice roots was corroborated by Scanning electron microscopy. In-planta experiments with rice seeds inoculated with Klebsiella sp. N5 showed significant improvements in various agronomic traits such as increase in shoot length, root length, fresh and dry weight of root and shoot, chlorophyll content, and nitrogen content of the plant and soil compared to the untreated plants. Klebsiella sp. N5, a cross colonizer from sorghum (C4 plant) to rice (C3 plant) plays a crucial role in facilitating the acquisition of essential resources such as nitrogen, phosphorus, and iron through direct mechanisms. This study expands the horizon for its potential as a valuable bioinoculant for sustainable agricultural practices, particularly in non-native host rice, paving the way for future field trials.

Preparation of anti-staphylococcal packaging material using antimicrobial metabolite from Lactiplantibacillus plantarum strain N1 isolated from fermented sausage from Egypt

Preparation of anti-staphylococcal packaging material using antimicrobial metabolite from Lactiplantibacillus plantarum strain N1 isolated from fermented sausage from Egypt

Unravelling the Mechanisms of Heavy Metal Tolerance: Enhancement in Hydrophilic Antioxidants and Major Antioxidant Enzymes Is Not Crucial for Long-Term Adaptation to Copper in Chlamydomonas reinhardtii.

Understanding of the mechanisms of heavy metal tolerance in algae is important for obtaining strains that can be applied in wastewater treatment. Cu is a redox-active metal directly inducing oxidative stress in exposed cells. The Cu-tolerant Chlamydomonas reinhardtii strain Cu2, obtained via long-term adaptation, displayed increased guaiacol peroxidase activity and contained more lipophilic antioxidants, i.e., α-tocopherol and plastoquinol, than did non-tolerant strain N1. In the present article, we measured oxidative stress markers; the content of ascorbate, soluble thiols, and proline; and the activity of superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) in N1 and Cu2 strains grown in the absence or presence of excessive Cu. The Cu2 strain displayed less pronounced lipid peroxidation and increased APX activity compared to N1. The amount of antioxidants was similar in both strains, while SOD and CAT activity was lower in the Cu2 strain. Exposure to excessive Cu led to a similar increase in proline content in both strains and a decrease in ascorbate and thiols, which was more pronounced in the N1 strain. The Cu2 strain was less tolerant to another redox-active heavy metal, namely chromium. Apparently other mechanisms, probably connected to Cu transport, partitioning, and chelation, are more important for Cu tolerance in Cu2 strain.

Characterization of Probiotic Properties and Whole-Genome Analysis of Lactobacillus johnsonii N5 and N7 Isolated from Swine.

In our previous microbiome profiling analysis, Lactobacillus (L.) johnsonii was suggested to contribute to resistance against chronic heat stress-induced diarrhea in weaned piglets. Forty-nine L. johnsonii strains were isolated from these heat stress-resistant piglets, and their probiotic properties were assessed. Strains N5 and N7 exhibited a high survival rate in acidic and bile environments, along with an antagonistic effect against Salmonella. To identify genes potentially involved in these observed probiotic properties, the complete genome sequences of N5 and N7 were determined using a combination of Illumina and nanopore sequencing. The genomes of strains N5 and N7 were found to be highly conserved, with two N5-specific and four N7-specific genes identified. Multiple genes involved in gastrointestinal environment adaptation and probiotic properties, including acidic and bile stress tolerance, anti-inflammation, CAZymes, and utilization and biosynthesis of carbohydrate compounds, were identified in both genomes. Comparative genome analysis of the two genomes and 17 available complete L. johnsonii genomes revealed 101 genes specifically harbored by strains N5 and N7, several of which were implicated in potential probiotic properties. Overall, this study provides novel insights into the genetic basis of niche adaptation and probiotic properties, as well as the genome diversity of L. johnsonii.

Mechanism of phenanthrene degradation by the halophilic Pelagerythrobacter sp. N7

PAHs has shown worldwide accumulation and causes a significant environmental problem especially in saline and hypersaline environments. Moderately halophilic bacteria could be useful for the bioremediation of PAH pollution in hypersaline environments. Pelagerythrobacter sp. N7 was isolated from the PAH-degrading consortium 5H, which was enriched from mixed saline soil samples collected in Shanxi Province, China. 16S rRNA in the genomic DNA revealed that strain N7 belonged to Pelagerythrobacter. Strain N7 exhibited a high tolerance to a wide range of salinities (1–10%) and was highly efficient under neutral to weak alkaline conditions (pH 6–9). The whole genome of strain N7 was sequenced and analyzed, revealing an abundance of catabolic genes. Using the whole genome information, we conducted preliminary research on key enzymes and gene clusters involved in the upstream and downstream PAH degradation pathways of strain N7, thereby inferring its degradation pathway for phenanthrene and naphthalene. This study adds to our understanding of PAH degradation in hypersaline environments and, for the first time, identifies a Pelagerythrobacter with PAH-degrading capability. Strain N7, with its high efficiency in phenanthrene degradation, represents a promising resource for the bioremediation of PAHs in hypersaline environments.

Protease production and molecular characterization of a protease dipeptidyl-aminopeptidase gene from different strains of Sordaria fimicola.

The current research was designed to reach extracellular protease production potential in different strains of Sordaria fimicola which were previously obtained from Dr. Lamb (Imperial College, London) from North Facing Slope and South Facing Slope of Evolution Canyon. After initial and secondary screening, two hyper-producers strains S2 and N6 were selected for submerged fermentation and cultural conditions including temperature, pH, incubation period, inoculum size, substrate concentration, and different carbon and nitrogen sources were optimized for enzyme production. S2 strain showed maximum protease production of 3.291 U/mL after 14 days of incubation at 30 °C with 7 pH, 1% substrate concentration and 1 mL inoculum, While N6 strain showed maximum protease production of 1.929 U/mL under fermentation optimized conditions. Another aim of the present research was to underpin the biodiversity of genetics and post-translational modifications (PTMs) of protease DPAP (peptidyl-aminopeptidase) in Sordaria fimicola. Five polymorphic sites were observed in amino acid sequence of S. fimicola strains with reference to Neurospora crassa. PTMs prediction from bioinformatics tools predicted 38 phosphorylation sites on serine residues for protease peptidyl-aminopeptidase in S1 strain of S. fimicola while 45 phosphorylation sites on serine in N7 strain and 47 serine phosphorylation modifications were predicted in N. crassa. Current research gave an insight that change in genetic makeup effected PTMs which ultimately affected the production of protease enzyme in different strains of same organism (S. fimicola). The production and molecular data of the research revealed that environmental stress has strong effects on the specific genes through mutations which may cause genetic diversity. S. fimicola is non- pathogenic fungus and has a short life cycle. This fungus can be chosen to produce protease enzyme on a commercial scale.

Evaluation of the growth of \(\textit{Vibrio natriegens}\) strains in a medium containing chitin derivatives and shrimp shell hydrolysate

Chitin from crustacean waste can be the future substrate for bioindustry towards the circular economy concept, especially for countries having large production of sea products like Vietnam. However, the chemical conversion of chitin into monomers implies high amounts of NaCl in a mixture with glucosamine, N-acetyl glucosamine, and acetate. Thus, a bacterial strain that can tolerate high salt concentration, and use chitin monomers as the sole carbon source such as Vibrio natriegens holds great potential in producing bioproducts from chitin derivatives. In this study, V. natriegens strains 10.3 and N5.3 isolated from Vietnam were compared with the reference strain V. natriegens DSM 759 for their growth performances in medium containing separately glucose and chitin monomers. Strain N5.3 showed the best growth rate among the 3 tested strains, exceptionally in medium containing glucosamine with nearly 1.5 times faster than strains 10.3 and DSM 759. Strain N5.3 cultured in bioreactor at 30 g/L NaCl indicated growth rate ranging from 0.614 to 0.881 h-1 in medium containing glucose, glucosamine, N-acetyl glucosamine, and shrimp shell hydrolysate. The formation of acetate was observed during the exponential growth of strain N5.3 in medium with glucose and N-acetyl glucosamine but not in medium with glucosamine or shrimp shell hydrolysate. The faster growth of all tested strains on N-acetyl glucosamine compared to glucosamine suggested metabolism of these substrates of V. natriegens similar to Eescherichia coli.

Uranium bioprecipitation mediated by a phosphate-solubilizing Enterobacter sp. N1-10 and remediation of uranium-contaminated soil

Uranium (U) pollution in soils is prevalent worldwide and poses a significant health risk that will require remediation approaches. However, traditional U bioreduction by sulfate reducing bacteria (SRB) are sensitive to oxygen and are not suitable for treating aerobic topsoil. Bioprecipitation of U into uranyl phosphate (UP) mediated by phosphate-solubilizing microorganism (PSM) is not affected by oxygen. In this study, PSM strains were isolated and used for U-contaminated soil remediation. Microbial metabolites and the mechanism of PSM bioprecipitation were revealed. The results showed that strain Enterobacter sp. N1-10 had the highest phosphate-solubilizing capacity (dissolved P was 409.51 ± 8.48 mg/L). Uranium bioprecipitation was investigated by culturing the bacterium in the presence of 50 mg/L U and in the cell-free culture supernatant. The results showed that strain N1–10 had a high U removal rate (99.45 ± 0.43 %) after adding 50 mg/L U to the culture medium. A yellow precipitate was immediately formed when uranyl nitrate solution was added to the cell-free culture supernatant. The analysis indicated that bacterium produced lactic acid (37.58 mg/L), citric acid (4.76 mg/L), succinic acid (2.03 mg/L), and D-glucuronic acid (1.94 mg/L); the four organic acids solubilized Ca3(PO4)2 to form stable uranyl phosphate precipitate. The application of strain N1-10 and Ca3(PO4)2 significantly decreased the bioavailability of soil U (43.54 ± 0.52 %). In addition, pot experiments showed that PSM N1-10 and Ca3(PO4)2 promoted plant growth and markedly reduced U accumulation by pakchoi. These results demonstrate that PSM N1-10 and Ca3(PO4)2 exhibit a great potential for U bioremediation.

Polyamine-producing bacteria inhibit the absorption of Cd by spinach and alter the bacterial community composition of rhizosphere soil

Polyamines (PAs) are small aliphatic nitrogenous bases with strong biological activity that participate in plant stress response signaling and the alleviation of damage from stress. Herein, the effects of the PA-producing bacterium Bacillus megaterium N3 and PAs on the immobilization of Cd and inhibition of Cd absorption by spinach and the underlying mechanisms were studied. A solution test showed that strain N3 secreted spermine and spermidine in the presence of Cd. Both strain N3 and the PAs (spermine+spermidine) immobilized Cd and increased the pH of the solution. Untargeted metabolomics results showed that strain N3 secreted PAs, N1-acetylspermidine, 3-indolepropionic acid, indole-3-acetaldehyde, cysteinyl-gamma-glutamate, and choline, which correlated with plant growth promotion and Cd immobilization. A pot experiment showed that rhizosphere soil inoculation with strain N3 and PAs improved spinach dry weight and reduced spinach Cd absorption compared with the control. These positive effects were likely due to the increase in rhizosphere soil pH and NH4+-N and PA contents, which can be attributed primarily to Cd immobilization. Moreover, inoculation with strain N3 more effectively inhibited the absorption of Cd by spinach than spraying PAs, mainly because strain N3 enabled a better relative abundance of bacteria (Microvirga, Pedobacter, Bacillus, Brevundimonas, Pseudomonas, Serratia, Devosid, and Aminobacter), that have been reported to have the ability to resist heavy metals and produce PAs. Strain N3 regulated the structure of rhizosphere functional bacterial communities and inhibited Cd uptake by spinach. These results provide a theoretical basis for the prevention of heavy metal absorption by vegetables using PA-producing bacteria.

Selection of rhizobial strains differing in their nodulation kinetics under low temperature in four temperate legume species

AbstractBackgroundWinter climate change including frequent freeze‐thaw episodes and shallow snow cover will have major impacts on the spring regrowth of perennial crops. Non‐bloating perennial forage legume species including sainfoin, birdsfoot trefoil, red clover, and alsike clover have been bred for their adaptation to harsh winter conditions. In parallel, the selection of cold‐tolerant rhizobial strains could allow earlier symbiotic nitrogen (N) fixation to hasten spring regrowth of legumes.MethodsTo identify strains forming nodules rapidly and showing high N‐fixing potential, 60 rhizobial strains in association with four temperate legume species were evaluated over 11 weeks under spring soil temperatures for kinetics of nodule formation, nitrogenase activity, and host yield.ResultsStrains differed in their capacity to form efficient nodules on legume hosts over time. Strains showing higher nitrogenase activity were arctic strain N10 with sainfoin and strain L2 with birdsfoot trefoil. For clovers, nitrogenase activity was similar for control and inoculated plants, likely due to formation of effective nodules in controls by endophyte rhizobia present in seeds.ConclusionsSelection based on nodulation kinetics at low temperature, nitrogenase activity, and yield was effective to identify performant rhizobial strains for legume crops. The use of cold‐tolerant strains could help mitigate winter climatic changes.

Bioremediation of metribuzin-contaminated soil by corn straw biochar-immobilized Bacillus cereus N1

The bioremediation effects of metribuzin-degrading bacterium Bacillus cereus N1 immobilized with corn straw biochar (BC) were studied. The results showed that 72.15% of strain N1 and 15.31% of metribuzin was adsorbed by BC, with the highest absorption peak intensity obtained by the immobilized bacterial microspheres (PSBN1). The Mechanism of PSBN1 removal of metribuzin was investigated using scanning electron microscopy, distribution and mass transfer pathways of metribuzin in PSBN1, and adsorption kinetics and isotherms of BC. A total of 62.26% of metribuzin was degraded by PSBN1 in 120 d of storage at 4 °C, which could be reused for 7 times with the degradation efficiency maintained above 75%. The optimal degradation conditions of metribuzin were determined to obtain the degradation rate of 92.48% in 5 d. The degradation rates of metribuzin reached 84.12% and 83.73% in sterilized and non-sterilized soils, respectively, in 28 d. The bioremediation effect of PSBN1 was verified by the maximum recovery rate (183%) of fresh plant weight in metribuzin sensitive crop and the greatly restored soil enzymatic activities. To conclude, PSBN1 was suitable for extended storage time with high degradation efficiency maintained, providing strong experimental evidence to support the commercialization and long-term preservation of metribuzin-degrading microbial agents.

GAR-transferase contributes to purine synthesis and mitochondrion function to maintain fungal development and full virulence of Penicillium digitatum

Penicillium digitatum is one of the most critical phytopathogens during the citrus postharvest period. However, the molecular mechanism of pathogenesis remains to be further explored. Purine is a multiple functional substance in organisms. To verify the role of the de novo purine biosynthesis (DNPB) pathway in P. digitatum, we investigated the third gene Pdgart, glycinamide ribonucleotide (GAR)-transferase, of this pathway in this study. The deletion mutant ΔPdgart was generated in the principle of homologous recombination via Agrobacterium tumefaciens-mediated transformation (ATMT). The phenotypic assay indicated that the ΔPdgart mutant displayed severe defects in hyphae growth, conidiation and germination, which can be rescued by the addition of exogenous ATP and AMP. Compared with wild-type strain N1, the ATP level of strain ΔPdgart was detected to be sharply declined during conidial germination, and this was resulted from the damage to purine synthesis and aerobic respiration. The pathogenicity assay suggested that mutant ΔPdgart infected citrus fruit but attenuated disease, which was owing to its reduced production of organic acids and activities of cell wall degradation enzymes. Additionally, the ΔPdgart mutant showed altered sensitivity to stress agents and fungicides. Taken together, the present study provides insights into the essential functions of Pdgart, and paves the way for further study and novel fungicide development.

Draft Genome Sequence of Bacillus anthracis N1, Isolated from a Recreational Freshwater Kettle Lake in Ontario, Canada.

Bacillus anthracis is widespread in soil and a causative agent of anthrax, primarily in herbivores. Here, we report the draft genome sequence of Bacillus anthracis strain N1, which was isolated from a recreational freshwater lake and found to carry multiple antibiotic resistance genes and biosynthetic gene clusters.

Draft Genome Sequence of Exiguobacterium sp. Strain N5, Isolated from a Recreational Freshwater Kettle Lake in Ontario

Exiguobacterium spp. are facultative anaerobic, Gram-positive, non-spore-forming bacilli, reported to tolerate extreme environments. Here, we report the draft genome sequence of Exiguobacterium sp. strain N5, isolated from a recreational freshwater lake.

High-Quality Genome Resource of Bacillus subtilis N4, a Biological Control Agent Isolated from Phoebe bournei.

Bacillus subtilis strain N4, an endophyte isolated from the healthy leaves of Phoebe bournei, possesses an excellent biocontrol effect against stem canker of P. bournei caused by Lasiodiplodia theobromae. To understand its biocontrol mechanism, we assembled a high-quality genome of N4 using a combination of second- and third-generation sequencing methods. The N4 genome contained one circular DNA chromosome of 4,218,183 bp length with 43.5% GC content and 11 secondary metabolite biosynthesis gene clusters, including genes for subtilomycin synthesis. This high-quality genome assembly and gene annotation resource will provide better insights into the biocontrol potential of B. subtilis strain N4 against stem canker of P. bournei.

Movement Protein Mediates Systemic Necrosis in Tomato Plants with Infection of Tomato Mosaic Virus.

The necrogenic strain N5 of tomato mosaic virus (ToMV-N5) causes systemic necrosis in tomato cultivar Hezuo903. In this work, we mapped the viral determinant responsible for the induction of systemic necrosis. By exchanging viral genes between N5 and a non-necrogenic strain S1, we found that movement protein (MP) was the determinant for the differential symptoms caused by both strains. Compared with S1 MP, N5 MP had an additional ability to increase virus accumulation, which was not due to its functions in viral cell-to-cell movement. Actually, N5 MP, but not S1 MP, was a weak RNA silencing suppressor, which assisted viral accumulation. Sequence alignment showed that both MPs differed by only three amino acid residues. Experiments with viruses having mutated MPs indicated that the residue isoleucine at position 170 in MP was the key site for MP to increase virus accumulation, but also was required for MP to induce systemic necrosis in virus-infected tomato plants. Collectively, the lethal necrosis caused by N5 is dependent on its MP protein that enhances virus accumulation via its RNA silencing suppressor activity, probably leading to systemic necrosis responses in tomato plants.

Carotenoids production and genome analysis of a novel carotenoid producing Rhodococcus aetherivorans N1

Carotenoids are a series of natural pigments with unique structure and physiological functions. In this study, a novel Rhodococcus aetherivorans strain N1 was discovered, which can produce 6.4 mg/g carotenoids including β-carotene, zeaxanthin and isorenieratene from glucose. Moreover, strain N1 can directly produce 3.0 mg/g carotenoids from the undetoxified straw hydrolysate, representing the highest carotenoids production from the undetoxified lignocellulosic hydrolysate. The crude carotenoid extracts of strain N1 showed efficient free radical scavenging activity and stability. Strain N1 has complete methylerythritol 4-phosphate (MEP) pathway and related genes for carotenoid synthesis, especially the rare aromatic carotenoid of isorenieratene. Genomic comparison between strain N1 and other carotenoid producing Rhodococcus sp. strains showed the conservatism and universality of carotenoids synthesis gene. These results proved that R. aetherivorans strain N1 can serve as a promising producer for the industrialization of carotenoid production.