Metabolic engineering of thermophilic Bacillus methanolicus for riboflavin overproduction from methanol.

The growing need of next generation feedstocks for biotechnology spurs an intensification of research on the utilization of methanol as carbon and energy source for biotechnological processes. In this paper, we introduced the methanol-based overproduction of riboflavin into metabolically engineered Bacillus methanolicus MGA3. First, we showed that B. methanolicus naturally produces small amounts of riboflavin. Then, we created B. methanolicus strains overexpressing either homologous or heterologous gene clusters encoding the riboflavin biosynthesis pathway, resulting in riboflavin overproduction. Our results revealed that the supplementation of growth media with sublethal levels of chloramphenicol contributes to a higher plasmid-based riboflavin production titre, presumably due to an increase in plasmid copy number and thus biosynthetic gene dosage. Based on this, we proved that riboflavin production can be increased by exchanging a low copy number plasmid with a high copy number plasmid leading to a final riboflavin titre of about 523 mg L-1 in methanol fed-batch fermentation. The findings of this study showcase the potential of B. methanolicus as a promising host for methanol-based overproduction of extracellular riboflavin and serve as basis for metabolic engineering of next generations of riboflavin overproducing strains.

Efficient cell factories for the production of N-methylated amino acids and for methanol-based amino acid production.

The growing world needs commodity amino acids such as L-glutamate and L-lysine for use as food and feed, and specialty amino acids for dedicated applications. To meet the supply a paradigm shift regarding their production is required. On the one hand, the use of sustainable and cheap raw materials is necessary to sustain low production cost and decrease detrimental effects of sugar-based feedstock on soil health and food security caused by competing uses of crops in the feed and food industries. On the other hand, the biotechnological methods to produce functionalized amino acids need to be developed further, and titres enhanced to become competitive with chemical synthesis methods. In the current review, we present successful strain mutagenesis and rational metabolic engineering examples leading to the construction of recombinant bacterial strains for the production of amino acids such as L-glutamate, L-lysine, L-threonine and their derivatives from methanol as sole carbon source. In addition, the fermentative routes for bioproduction of N-methylated amino acids are highlighted, with focus on three strategies: partial transfer of methylamine catabolism, S-adenosyl-L-methionine dependent alkylation and reductive methylamination of 2-oxoacids.

Unravelling Formaldehyde Metabolism in Bacteria: Road towards Synthetic Methylotrophy.

Formaldehyde metabolism is prevalent in all organisms, where the accumulation of formaldehyde can be prevented through the activity of dissimilation pathways. Furthermore, formaldehyde assimilatory pathways play a fundamental role in many methylotrophs, which are microorganisms able to build biomass and obtain energy from single- and multicarbon compounds with no carbon-carbon bonds. Here, we describe how formaldehyde is formed in the environment, the mechanisms of its toxicity to the cells, and the cell's strategies to circumvent it. While their importance is unquestionable for cell survival in formaldehyde rich environments, we present examples of how the modification of native formaldehyde dissimilation pathways in nonmethylotrophic bacteria can be applied to redirect carbon flux toward heterologous, synthetic formaldehyde assimilation pathways introduced into their metabolism. Attempts to engineer methylotrophy into nonmethylotrophic hosts have gained interest in the past decade, with only limited successes leading to the creation of autonomous synthetic methylotrophy. Here, we discuss how native formaldehyde assimilation pathways can additionally be employed as a premise to achieving synthetic methylotrophy. Lastly, we discuss how emerging knowledge on regulation of formaldehyde metabolism can contribute to creating synthetic regulatory circuits applied in metabolic engineering strategies.

Characterization of two 3-deoxy-d-Arabino-Heptulosonate 7-phosphate synthases from Bacillusmethanolicus.

3-Deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase catalyzes the condensation of phosphoenolpyruvate (PEP) with d-erythrose 4-phosphate (E4P) and plays an important role in regulating carbon flux toward aromatic amino acid biosynthesis in bacteria and plants. Sequence analysis of the DAHP synthases AroG1 and AroG2 from Bacillus methanolicus MGA3 suggested this thermophilic, methylotrophic bacterium possesses two type Iß DAHP synthases. This study describes production of AroG1 and AroG2 in Escherichia coli as hexa-histidine fused proteins, which were purified by affinity chromatography. Treatment with TEV protease afforded native proteins for characterization and kinetic analysis. AroG1 and AroG2 are, respectively, 30.1 kDa and 40.0 kDa proteins. Both enzymes have maximal activity over a pH range of 6.3-7.2. The apparent kinetic parameters at 50 °C and pH 7.2 for AroG1 are KmPEP 1100 ± 100 µM, KmE4P 530 ± 100 µM, and kcat 10.3 ± 1.2 s-1. The kinetic parameters for AroG2 are KmPEP 90 ± 20 µM, KmE4P 130 ± 40 µM, and kcat 2.0 ± 0.2 s-1. At 50 °C AroG2 retains 50% of its activity after 96 min whereas AroG1 retains less than 5% of its activity after 10 min. AroG2, which contains an N-terminal regulatory domain, is inhibited by chorismate and prephenate but not l-phenylalanine, l-tyrosine, or l-tryptophan. AroG1 is not inhibited by any of the molecules examined. Understanding DAHP synthase regulation in B. methanolicus is a first step toward generating biocatalysts that exploit the target-rich aromatic amino acid biosynthetic pathway for synthesis of chemicals from methanol.

Evaluation of Heterologous Biosynthetic Pathways for Methanol-Based 5-Aminovalerate Production by Thermophilic Bacillus methanolicus.

The use of methanol as carbon source for biotechnological processes has recently attracted great interest due to its relatively low price, high abundance, high purity, and the fact that it is a non-food raw material. In this study, methanol-based production of 5-aminovalerate (5AVA) was established using recombinant Bacillus methanolicus strains. 5AVA is a building block of polyamides and a candidate to become the C5 platform chemical for the production of, among others, δ-valerolactam, 5-hydroxy-valerate, glutarate, and 1,5-pentanediol. In this study, we test five different 5AVA biosynthesis pathways, whereof two directly convert L-lysine to 5AVA and three use cadaverine as an intermediate. The conversion of L-lysine to 5AVA employs lysine 2-monooxygenase (DavB) and 5-aminovaleramidase (DavA), encoded by the well-known Pseudomonas putida cluster davBA, among others, or lysine α-oxidase (RaiP) in the presence of hydrogen peroxide. Cadaverine is converted either to γ-glutamine-cadaverine by glutamine synthetase (SpuI) or to 5-aminopentanal through activity of putrescine oxidase (Puo) or putrescine transaminase (PatA). Our efforts resulted in proof-of-concept 5AVA production from methanol at 50°C, enabled by two pathways out of the five tested with the highest titer of 0.02 g l-1. To our knowledge, this is the first report of 5AVA production from methanol in methylotrophic bacteria, and the recombinant strains and knowledge generated should represent a valuable basis for further improved 5AVA production from methanol.

Developing a Riboswitch-Mediated Regulatory System for Metabolic Flux Control in Thermophilic Bacillus methanolicus.

Genome-wide transcriptomic data obtained in RNA-seq experiments can serve as a reliable source for identification of novel regulatory elements such as riboswitches and promoters. Riboswitches are parts of the 5' untranslated region of mRNA molecules that can specifically bind various metabolites and control gene expression. For that reason, they have become an attractive tool for engineering biological systems, especially for the regulation of metabolic fluxes in industrial microorganisms. Promoters in the genomes of prokaryotes are located upstream of transcription start sites and their sequences are easily identifiable based on the primary transcriptome data. Bacillus methanolicus MGA3 is a candidate for use as an industrial workhorse in methanol-based bioprocesses and its metabolism has been studied in systems biology approaches in recent years, including transcriptome characterization through RNA-seq. Here, we identify a putative lysine riboswitch in B. methanolicus, and test and characterize it. We also select and experimentally verify 10 putative B. methanolicus-derived promoters differing in their predicted strength and present their functionality in combination with the lysine riboswitch. We further explore the potential of a B. subtilis-derived purine riboswitch for regulation of gene expression in the thermophilic B. methanolicus, establishing a novel tool for inducible gene expression in this bacterium.

Methane monooxygenases: central enzymes in methanotrophy with promising biotechnological applications.

Worldwide, the use of methane is limited to generating power, electricity, heating, and for production of chemicals. We believe this valuable gas can be employed more widely. Here we review the possibility of using methane as a feedstock for biotechnological processes based on the application of synthetic methanotrophs. Methane monooxygenase (MMO) enables aerobic methanotrophs to utilize methane as a sole carbon and energy source, in contrast to industrial microorganisms that grow on carbon sources, such as sugar cane, which directly compete with the food market. However, naturally occurring methanotrophs have proven to be difficult to manipulate genetically and their current industrial use is limited to generating animal feed biomass. Shifting the focus from genetic engineering of methanotrophs, towards introducing metabolic pathways for methane utilization in familiar industrial microorganisms, may lead to construction of efficient and economically feasible microbial cell factories. The applications of a technology for MMO production are not limited to methane-based industrial synthesis of fuels and value-added products, but are also of interest in bioremediation where mitigating anthropogenic pollution is an increasingly relevant issue. Published research on successful functional expression of MMO does not exist, but several attempts provide promising future perspectives and a few recent patents indicate that there is an ongoing research in this field. Combining the knowledge on genetics and metabolism of methanotrophy with tools for functional heterologous expression of MMO-encoding genes in non-methanotrophic bacterial species, is a key step for construction of synthetic methanotrophs that holds a great biotechnological potential.

Establishment of a functional system for recombinant production of secreted proteins at 50 °C in the thermophilic Bacillus methanolicus.

BACKGROUND: The suitability of bacteria as microbial cell factories is dependent on several factors such as price of feedstock, product range, production yield and ease of downstream processing. The facultative methylotroph Bacillus methanolicus is gaining interest as a thermophilic cell factory for production of value-added products from methanol. The aim of this study was to expand the capabilities of B. methanolicus as a microbial cell factory by establishing a system for secretion of recombinant proteins. RESULTS: Native and heterologous signal peptides were tested for secretion of α-amylases and proteases, and we have established the use of the thermostable superfolder green fluorescent protein (sfGFP) as a valuable reporter protein in B. methanolicus. We demonstrated functional production and secretion of recombinant proteases, α-amylases and sfGFP in B. methanolicus MGA3 at 50 °C and showed that the choice of signal peptide for optimal secretion efficiency varies between proteins. In addition, we showed that heterologous production and secretion of α-amylase from Geobacillus stearothermophilus enables B. methanolicus to grow in minimal medium with starch as the sole carbon source. An in silico signal peptide library consisting of 169 predicted peptides from B. methanolicus was generated and will be useful for future studies, but was not experimentally investigated any further here. CONCLUSION: A functional system for recombinant production of secreted proteins at 50 °C has been established in the thermophilic B. methanolicus. In addition, an in silico signal peptide library has been generated, that together with the tools and knowledge presented in this work will be useful for further development of B. methanolicus as a host for recombinant protein production and secretion at 50 °C.

Characterization of D-Arabitol as Newly Discovered Carbon Source of Bacillus methanolicus.

Bacillus methanolicus is a Gram-positive, thermophilic, methanol-utilizing bacterium. As a facultative methylotroph, B. methanolicus is also known to utilize D-mannitol, D-glucose and, as recently discovered, sugar alcohol D-arabitol. While metabolic pathways for utilization of methanol, mannitol and glucose are known, catabolism of arabitol has not yet been characterized in B. methanolicus. In this work we present the elucidation of this hitherto uncharted pathway. In order to confirm our predictions regarding genes coding for arabitol utilization, we performed differential gene expression analysis of B. methanolicus MGA3 cells grown on arabitol as compared to mannitol via transcriptome sequencing (RNA-seq). We identified a gene cluster comprising eight genes that was up-regulated during growth with arabitol as a sole carbon source. The RNA-seq results were subsequently confirmed via qRT-PCR experiments. The transcriptional organization of the gene cluster identified via RNA-seq was analyzed and it was shown that the arabitol utilization genes are co-transcribed in an operon that spans from BMMGA3_RS07325 to BMMGA3_RS07365. Since gene deletion studies are currently not possible in B. methanolicus, two complementation experiments were performed in an arabitol negative Corynebacterium glutamicum strain using the four genes discovered via RNA-seq analysis as coding for a putative PTS for arabitol uptake (BMMGA3_RS07330, BMMGA3_RS07335, and BMMGA3_RS07340 renamed to atlABC) and a putative arabitol phosphate dehydrogenase (BMMGA3_RS07345 renamed to atlD). C. glutamicum is a natural D-arabitol utilizer that requires arabitol dehydrogenase MtlD for arabitol catabolism. The C. glutamicum mtlD deletion mutant was chosen for complementation experiments. Heterologous expression of atlABCD as well as the arabitol phosphate dehydrogenase gene atlD from B. methanolicus alone restored growth of the C. glutamicum ΔmtlD mutant with arabitol. Furthermore, D-arabitol phosphate dehydrogenase activities could be detected in crude extracts of B. methanolicus and these were higher in arabitol-grown cells than in methanol- or mannitol-grown cells. Thus, B. methanolicus possesses an arabitol inducible operon encoding, amongst others, a putative PTS system and an arabitol phosphate dehydrogenase for uptake and activation of arabitol as growth substrate.

Construction and characterization of broad-host-range reporter plasmid suitable for on-line analysis of bacterial host responses related to recombinant protein production.

BACKGROUND: Bacteria are widely used as hosts for recombinant protein production due to their rapid growth, simple media requirement and ability to produce high yields of correctly folded proteins. Overproduction of recombinant proteins may impose metabolic burden to host cells, triggering various stress responses, and the ability of the cells to cope with such stresses is an important factor affecting both cell growth and product yield. RESULTS: Here, we present a versatile plasmid-based reporter system for efficient analysis of metabolic responses associated with availability of cellular resources utilized for recombinant protein production and host capacity to synthesize correctly folded proteins. The reporter plasmid is based on the broad-host range RK2 minimal replicon and harbors the strong and inducible XylS/Pm regulator/promoter system, the ppGpp-regulated ribosomal protein promoter PrpsJ, and the σ32-dependent synthetic tandem promoter Pibpfxs, each controlling expression of one distinguishable fluorescent protein. We characterized the responsiveness of all three reporters in Escherichia coli by quantitative fluorescence measurements in cell cultures cultivated under different growth and stress conditions. We also validated the broad-host range application potential of the reporter plasmid by using Pseudomonas putida and Azotobacter vinelandii as hosts. CONCLUSIONS: The plasmid-based reporter system can be used for analysis of the total inducible recombinant protein production, the translational capacity measured as transcription level of ribosomal protein genes and the heat shock-like response revealing aberrant protein folding in all studied Gram-negative bacterial strains.

Corrigendum: Genome-Based Genetic Tool Development for Bacillus methanolicus: Theta- and Rolling Circle-Replicating Plasmids for Inducible Gene Expression and Application to Methanol-Based Cadaverine Production.

[This corrects the article DOI: 10.3389/fmicb.2016.01481.].

Genetic Tools and Techniques for Recombinant Expression in Thermophilic Bacillaceae.

Although Escherichia coli and Bacillus subtilis are the most prominent bacterial hosts for recombinant protein production by far, additional species are being explored as alternatives for production of difficult-to-express proteins. In particular, for thermostable proteins, there is a need for hosts able to properly synthesize, fold, and excrete these in high yields, and thermophilic Bacillaceae represent one potentially interesting group of microorganisms for such purposes. A number of thermophilic Bacillaceae including B.methanolicus, B.coagulans, B.smithii, B.licheniformis, Geobacillus thermoglucosidasius, G. kaustophilus, and G. stearothermophilus are investigated concerning physiology, genomics, genetic tools, and technologies, altogether paving the way for their utilization as hosts for recombinant production of thermostable and other difficult-to-express proteins. Moreover, recent successful deployments of CRISPR/Cas9 in several of these species have accelerated the progress in their metabolic engineering, which should increase their attractiveness for future industrial-scale production of proteins. This review describes the biology of thermophilic Bacillaceae and in particular focuses on genetic tools and methods enabling use of these organisms as hosts for recombinant protein production.

Detailed transcriptome analysis of the plant growth promoting Paenibacillus riograndensis SBR5 by using RNA-seq technology.

BACKGROUND: The plant growth promoting rhizobacterium Paenibacillus riograndensis SBR5 is a promising candidate to serve as crop inoculant. Despite its potential in providing environmental and economic benefits, the species P. riograndensis is poorly characterized. Here, we performed for the first time a detailed transcriptome analysis of P. riograndensis SBR5 using RNA-seq technology. RESULTS: RNA was isolated from P. riograndensis SBR5 cultivated under 15 different growth conditions and combined together in order to analyze an RNA pool representing a large set of expressed genes. The resultant total RNA was used to generate 2 different libraries, one enriched in 5'-ends of the primary transcripts and the other representing the whole transcriptome. Both libraries were sequenced and analyzed to identify the conserved sequences of ribosome biding sites and translation start motifs, and to elucidate operon structures present in the transcriptome of P. riograndensis. Sequence analysis of the library enriched in 5'-ends of the primary transcripts was used to identify 1082 transcription start sites (TSS) belonging to novel transcripts and allowed us to determine a promoter consensus sequence and regulatory sequences in 5' untranslated regions including riboswitches. A putative thiamine pyrophosphate dependent riboswitch upstream of the thiamine biosynthesis gene thiC was characterized by translational fusion to a fluorescent reporter gene and shown to function in P. riograndensis SBR5. CONCLUSIONS: Our RNA-seq analysis provides insight into the P. riograndensis SBR5 transcriptome at the systems level and will be a valuable basis for differential RNA-seq analysis of this bacterium.

l-lysine production by Bacillus methanolicus: Genome-based mutational analysis and l-lysine secretion engineering.

Bacillus methanolicus is a methylotrophic bacterium with an increasing interest in academic research and for biotechnological applications. This bacterium was previously applied for methanol-based production of l-glutamate, l-lysine and the five-carbon diamine cadaverine by wild type, classical mutant and recombinant strains. The genomes of two different l-lysine secreting B. methanolicus classical mutant strains, NOA2#13A52-8A66 and M168-20, were sequenced. We focused on mutational mapping in genes present in l-lysine and other relevant amino acid biosynthetic pathways, as well as in the primary cell metabolism important for precursor supply. In addition to mutations in the aspartate pathway genes dapG, lysA and hom-1, new mutational target genes like alr, proA, proB1, leuC, odhA and pdhD were identified. Surprisingly, no mutations were found in the putative l-lysine transporter gene lysEMGA3. Inspection of the wild type B. methanolicus strain PB1 genome sequence identified two homologous putative l-lysine transporter genes, lysEPB1 and lysE2PB1. The biological role of these putative l-lysine transporter genes, together with the heterologous l-lysine exporter gene lysECg from Corynebacterium glutamicum, were therefore investigated. Our results demonstrated that the titer of secreted l-lysine in B. methanolicus was significantly increased by overexpression of lysECg while overexpression of lysEMGA3, lysEPB1 and lysE2PB1 had no measurable effect.

Magnesium aminoclay-based transformation of Paenibacillus riograndensis and Paenibacillus polymyxa and development of tools for gene expression.

Members of the genus Paenibacillus are widespread facultative anaerobic, endospore-forming bacteria. Some species such as Paenibacillus riograndensis or Paenibacillus polymyxa fix nitrogen and may play an important role in agriculture to reduce mineral nitrogen fertilization in particular for non-legume plants. The genetic manipulation of Paenibacillus is an imperative for the functional characterization, e.g., of its plant growth-promoting activities and metabolism. This study showed that P. riograndensis and P. polymyxa can be readily transformed using physical permeation by magnesium aminoclays. By means of the fluorescent reporter genes gfpUV, mcherry, and crimson, a two-plasmid system consisting of a theta-replicating plasmid and a rolling circle-replicating plasmid was shown to operate in both species. Xylose-inducible and mannitol-inducible fluorescent reporter gene expression was demonstrated in the compatible two-plasmid system by fluorescence-activated cell scanning. As a metabolic engineering application, the biotin requiring P. riograndensis was converted to a biotin-prototrophic strain based on mannitol-inducible expression of the biotin biosynthesis operon bioWAFDBI from Bacillus subtilis.

Genome-Based Genetic Tool Development for Bacillus methanolicus: Theta- and Rolling Circle-Replicating Plasmids for Inducible Gene Expression and Application to Methanol-Based Cadaverine Production.

Bacillus methanolicus is a thermophilic methylotroph able to overproduce amino acids from methanol, a substrate not used for human or animal nutrition. Based on our previous RNA-seq analysis a mannitol inducible promoter and a putative mannitol activator gene mtlR were identified. The mannitol inducible promoter was applied for controlled gene expression using fluorescent reporter proteins and a flow cytometry analysis, and improved by changing the -35 promoter region and by co-expression of the mtlR regulator gene. For independent complementary gene expression control, the heterologous xylose-inducible system from B. megaterium was employed and a two-plasmid gene expression system was developed. Four different replicons for expression vectors were compared with respect to their copy number and stability. As an application example, methanol-based production of cadaverine was shown to be improved from 11.3 to 17.5 g/L when a heterologous lysine decarboxylase gene cadA was expressed from a theta-replicating rather than a rolling-circle replicating vector. The current work on inducible promoter systems and compatible theta- or rolling circle-replicating vectors is an important extension of the poorly developed B. methanolicus genetic toolbox, valuable for genetic engineering and further exploration of this bacterium.

Transcriptome analysis of thermophilic methylotrophic Bacillus methanolicus MGA3 using RNA-sequencing provides detailed insights into its previously uncharted transcriptional landscape.

BACKGROUND: Bacillus methanolicus MGA3 is a thermophilic, facultative ribulose monophosphate (RuMP) cycle methylotroph. Together with its ability to produce high yields of amino acids, the relevance of this microorganism as a promising candidate for biotechnological applications is evident. The B. methanolicus MGA3 genome consists of a 3,337,035 nucleotides (nt) circular chromosome, the 19,174 nt plasmid pBM19 and the 68,999 nt plasmid pBM69. 3,218 protein-coding regions were annotated on the chromosome, 22 on pBM19 and 82 on pBM69. In the present study, the RNA-seq approach was used to comprehensively investigate the transcriptome of B. methanolicus MGA3 in order to improve the genome annotation, identify novel transcripts, analyze conserved sequence motifs involved in gene expression and reveal operon structures. For this aim, two different cDNA library preparation methods were applied: one which allows characterization of the whole transcriptome and another which includes enrichment of primary transcript 5'-ends. RESULTS: Analysis of the primary transcriptome data enabled the detection of 2,167 putative transcription start sites (TSSs) which were categorized into 1,642 TSSs located in the upstream region (5'-UTR) of known protein-coding genes and 525 TSSs of novel antisense, intragenic, or intergenic transcripts. Firstly, 14 wrongly annotated translation start sites (TLSs) were corrected based on primary transcriptome data. Further investigation of the identified 5'-UTRs resulted in the detailed characterization of their length distribution and the detection of 75 hitherto unknown cis-regulatory RNA elements. Moreover, the exact TSSs positions were utilized to define conserved sequence motifs for translation start sites, ribosome binding sites and promoters in B. methanolicus MGA3. Based on the whole transcriptome data set, novel transcripts, operon structures and mRNA abundances were determined. The analysis of the operon structures revealed that almost half of the genes are transcribed monocistronically (940), whereas 1,164 genes are organized in 381 operons. Several of the genes related to methylotrophy had highly abundant transcripts. CONCLUSION: The extensive insights into the transcriptional landscape of B. methanolicus MGA3, gained in this study, represent a valuable foundation for further comparative quantitative transcriptome analyses and possibly also for the development of molecular biology tools which at present are very limited for this organism.

Complete genome sequence of Bacillus methanolicus MGA3, a thermotolerant amino acid producing methylotroph.

Bacillus methanolicus MGA3 was isolated from freshwater marsh soil and characterised as a thermotolerant and methylotrophic L-glutamate producer. The complete genome consists of a circular chromosome and the two plasmids pBM19 and pBM69. It includes genomic information about C1 metabolism and amino acid biosynthetic pathways.

[Health-promoting properties of pectin]. / Prozdrowotne wlasciwosci pektyn.

Pectin, a heteropolysaccharide commercially derived from the cell wall of higher plants, is mainly used in food as a gelling agent in jams and jellies as well as a stabilizer in fruit juice and milk drinks. It has also received great interest as a source of dietary fiber. Furthermore, pectin is proved to have diverse biological activities including lipid and cholesterol level lowering effects, serum glucose and insulin content lowering effects, gastric emptying delay, and anti-cancer activities. Pectin and pectic oligosaccharides have been shown to induce apoptosis in human colonic adenocarcinoma cells and to have anti-metastatic properties. Dietary pectin can bind metal ions, particularly lead ions, thus reducing their retention in the body and diminishing their toxic effects. On the other hand, pectin enhances intestinal solubility and absorption of ferric iron. Pectin with a low degree of esterification or having a large volume of linear oligogalacturonide segments shows significant mucoadhesion capacity in the gastrointestinal tract. In this way pectin forms a physical barrier protecting epithelium against opportunistic microbial invasion during stress.