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Alginate is a linear anionic heteropolysaccharide with a chemical structure consisting of 1,4-linked subunits of β-D-mannuronic acid (M) and its C-5 epimer α-L-guluronic acid (G). It is well known that the monomer composition and molecular weight of alginates affect their properties and influence their use in the food and pharmaceutical industries. Alginate is usually extracted from seaweed for commercial purposes, but can also be produced by bacteria as exopolysaccharide (EPS). Pseudomonas spp. and Azotobacter vinelandii are well-known alginate-producing microorganisms. Their biochemical machinery for alginate biosynthesis is influenced by changing culture conditions and manipulating genes/proteins, making it relatively easy to obtain customized EPS with different molecular weights, M/G compositions, and thus physicochemical properties. Although these two genera have very similar biosynthetic pathways and molecular mechanisms for alginate production, with most of the genes involved being virtually identical, their regulation has been shown to be somewhat different. In this chapter, we present the main steps of alginate biosynthesis in bacteria, including precursor synthesis, polymerization, periplasmic modifications, transport/secretion, and post-secretion modification.
Department of Biochemistry and Molecular Biology, Federal University of Paraná, Curitiba, PR, Brazil
*Address all correspondence to: rvserrato@ufpr.br
1. Introduction
Alginate is a linear anionic biopolymer consisting of β-D-mannuronic acid units (M) and its C5-epimer α-L-guluronic acid (G) linked by 1 → 4 glycosidic bonds. It can be found as a homopolysaccharide (poly-M or poly-G) or as a heteropolysaccharide containing M/G in different ratios and sequences. Alginate has been known to be a major component of brown algae cell walls since the late nineteenth century when scientists were searching for utile products from kelp [1], and the gelatinous material isolated from these seaweeds was then named alginic acid [2]. The molecular backbone of alginate, however, was only described decades later, after the work of Fisher and Dörfel in 1955 [3], and the structural analyses of alginate have been an extensive field of research ever since due to the immense variability of M/G content and sequential organization found throughout the years in many macroalgae [4, 5]. The structural variability presented by this polymer and the ability to form gels with different physicochemical properties is nowadays reflected in its many biotechnological uses in the food sector, cosmetic industry, wastewater treatment, pharmaceutical, and biomedical applications, among others [6, 7, 8, 9].
Even though all commercial alginate produced today is extracted from seaweed, a handful group of specialized microorganisms can also produce this polymer as an exopolysaccharide [10]. Two genera of bacteria, Pseudomonas and Azotobacter, have been extensively studied over the past years on their ability to exudate alginate [11, 12, 13]. The production of alginate by Pseudomonas aeruginosa has been first reported by Linker and Jones in the mid-1960s [14, 15], and most of the knowledge on bacterial alginate production and biosynthesis emerged from the studies with this opportunistic human pathogen for patients afflicted by cystic fibrosis [16, 17], since the exopolymer represents major virulent factors during the lung infection process [18, 19]. Shortly after, alginate was described as an exopolysaccharide produced by Azotobacter vinelandii [20], a nitrogen-fixing soil bacterium found in association with plants. Over the years, other species belonging to these two genera have also been reported to produce alginate, such as P. putida and P. fluorescens [21], P. mendocina [22]; P. syringae [23] and A. chroococcum [24, 25].
The biosynthesis of bacterial alginates exhibits highly comparable mechanisms among the different producing species but differs from the seaweed polysaccharides in one particular characteristic. The microbial polymers are often O-acetylated, submitted to esterification at O-2 and/or O-3 on the D-mannuronate units [26, 27, 28], which affects their physicochemical properties in comparison to the alginate extracted from macroalgae [29, 30, 31, 32]. Furthermore, as well as different M/G compositions and sequences are found on alginates from seaweeds, the gel formation ability and viscosity of bacterial alginates are also affected by the arrangement of M-, G- and MG-blocks along their structures [7, 33]. Some mucoid strains of P. aeruginosa biosynthesize great amounts of alginate containing mainly M- and MG-blocks (Table 1), often O-acetyl-esterified, which leads to an increasing interaction of the polymer with water molecules, polymer extension, and water capacity, thus allowing the development of a thickly natured and highly structured biofilm matrix [34, 35, 36]. Azotobacter spp., on the other hand, produce alginates that are closely associated with the cells and able to capture divalent ions such as Ca2+ that form a hard and brittle gel due to the high content of L-guluronate residues (G-blocks) [37, 38], allowing the formation of desiccation-resistant cysts under adverse environmental conditions [39, 40].
→4)-α-L-GulA-(1 → 4)-β-D-ManA-(1→ or →4)-β-D-ManA-(1 → 4)-α-L-GulA-(1→ G – M or M – G
Seaweed; Azotobacter spp.; Pseudomonas spp.
Table 1.
Most commonly found structural disaccharides (blocks) of alginates produced by seaweeds, Pseudomonas spp. and Azotobacter spp.
ManA units are found O-acetylated on C-2 or C-3 only in bacterial alginates.
GulA (G): guluronic acid; ManA (M): mannuronic acid.
The production of tailor-made alginate in order to obtain polymers with different molecular weights, M/G ratio, and O-acetyl content to suit specific applications has been intensively investigated [41, 42, 43] as it poses as a potential commercial interest for the use-driven design of these polysaccharides in different industrial and medical applications [44, 45, 46]. Altering fermentation parameters and growth conditions in order to induce alginate production and or structural modifications during microbial cultivation has been extensively evaluated, especially for Azotobacter vinelandii [47, 48, 49, 50, 51, 52] due to its non-pathogenic characteristics, which makes this bacterium suitable for biotechnological processes. However, the genome sequencing of both P. aeruginosa [53] and A. vinelandii [54], together with the use of molecular tools for gene cloning, have expanded the possibilities for in vivo fine structural adjustments of alginate, and the isolation of alginate-modifying enzymes is allowing the on-demand chemical tailoring of these bacterial polymers [55, 56, 57, 58], including the suppression or degradation of alginate produced by Pseudomonas spp. for commercial and medical purposes [59, 60, 61, 62].
Understanding the molecular basis involved in bacterial alginate biosynthesis, transport, and metabolism, which are usually under strict regulatory control [63, 64, 65, 66], is paramount in order to develop new strains and mutants guided to the production of the idealized alginate structure in large-scale bioreactors or to be used therapeutically on clinical patients in opposition to alginate-overproducing pathogens [67, 68]. In this chapter, we are going to explore the main steps involved in the biosynthesis of microbial alginate, from the assembly of its precursor units to the polymerization, chemical and structural modifications, and transport through the cytoplasmic and outer membranes until the secretion of the mature polymer by the bacterial cells and the extracellular modifications that may follow. Gene and enzyme regulation will also be briefly addressed when relevant to each of the biosynthetic phases.
2. Overview of the genetics and regulation of bacterial alginate biosynthesis
The genetic organization of alginate-producing bacteria has been extensively analyzed and the regulation of the biosynthetic process is relatively well known, from the biosynthesis of precursor molecules, their polymerization into poly-D-mannuronic acid, chemical modifications/epimerization in the periplasmic space, and secretion to the extracellular environment. All of these steps have been previously identified and characterized in many reviews [42, 69, 70] and the biosynthesis of alginate has been shown to be extremely similar for both Pseudomonas aeruginosa and Azotobacter vinelandii, resulting from a complex regulatory network of proteins [41, 51, 59, 65]. In P. aeruginosa, a 12-gene operon is strictly controlled by algD transcription, the first gene of the alginate biosynthetic operon [53, 71], and two additional internal promoters upstream of algG and algI [72] (Figure 1). Three transcription promoter regions have been described for the algD operon, namely algDp1 (δD promoter, algDp2 (AlgU-δE promoter), and algDp3 [65]. Genes algD and algA, located downstream on the operon, are involved in the biosynthesis of the precursor guanosine diphosphate D-mannuronic acid (GDP-ManA) together with algC, which is found elsewhere in the bacterial chromosome, has its own promoter regions [73], and encodes for a phosphomannomutase [74]. The gene algG encodes for a C5-mannuronate epimerase, which converts D-ManA units into L-GulA in the periplasm [75]. Further structural modification of the nascent alginate polymer is promoted by the products of genes algI, algJ, and algF, all of which are involved in the O-acetylation of ManA units [76, 77]. Periplasmic proteins encoded by genes algXand algK, also present as part of the operon, are supposedly involved in the guiding of alginate through the outer membrane porin (formed by algE) and protection of alginate against degradation of an alginate lyase encoded by algL [77, 78]. The polymerization process to form poly-D-ManA is promoted by two transmembrane proteins (localized on the cytoplasmic membrane) encoded by genes alg8 and alg44 [79, 80].
Figure 1.
Genetic organization of alginate biosynthesis/regulation related genes for Pseudomonas aeruginosa (blue) and Azotobacter vinelandii (green). Gene sizes are not to scale. Numbers on top of each gene indicate bp as available from https://www.ncbi.nlm.nih.gov/ for the genome of P. aeruginosa PAO1 (Ref. NC_002516.2) and A. vinelandii DJ (Ref. NC_012560.1). The direction of transcription and bp for alyB of A. vinelandii is still undetermined.
The alginate biosynthetic genes on Azotobacter vinelandii are also clustered and share high similarity with their previously described P. aeruginosa counterparts. However, despite their essentially identical functions, Pseudomonads algE and algJ genes have been designated, respectively, as algJ and algV in A. vinelandii (Figure 1). Also, A. vinelandii alginate gene cluster has been shown to possess two promoter regions upstream of algD, one of which is an AlgU (δE)-dependent promoter, as in P. aeruginosa, and another RpoS (δs)-dependent [64, 65, 81]. Additionally, other three internal promoters are found on the A. vinelandii operon upstream of alg8 (δs-dependent), algG (δ70-dependent), and algA (regulation unknown) [64, 65]. The levels of algA transcription exclusively from its upstream promoter, however, are not sufficient to sustain alginate production [82]. Only in P. aeruginosa, the transcription of the algA and algL genes has been demonstrated to be controlled by AlgU [51].
Exclusively in Azotobacter spp., seven genes encoding C5-ManA epimerases have been identified, namely, algE1-algE7 [58, 83, 84]. The translational products of these (AlgE1-AlgE7) are exported to the cell surface and released into the extracellular environment. AlgE1–7 catalyze the Ca2+-dependent epimerization of D-ManA units into L-GulA units in the alginate polymer after its secretion through the outer membrane by the AlgJ porin [51, 83]. These extracellular epimerases are structurally unrelated to AlgG, a Ca2+-independent C5-mannuronate epimerase found in the periplasm, and all seven algE1–7 genes have been reported to be regulated by the sigma factor RpoS [85]. AlgE7 has been reported to also have lyase activity, which leads to depolymerization of alginate via β-elimination at the 4-O-glycosidic bonds [86]. In addition, four other alginate lyase genes (alyA1–3 and alyB) different from algL, which is part of the biosynthetic operon, have been described for A. vinelandii [42].
AlgU, previously called AlgT, δE, or δ22 [87], is homologous to the alternative sigma factor of the stress response regulator RpoE from Escherichia coli [88] and plays a key role as the main regulator of bacterial alginate biosynthesis. AlgU ist is encoded together with the mucABCD operon, which contains four genes (mucA, mucB, mucC, and mucD). In both, A. vinelandii and Pseudomonas spp., the muc genes are located downstream of the algU gene, forming the algUmucABCD regulatory gene cluster [64, 65, 89]. Transcription of the biosynthetic algD operon is activated by the presence of AlgU, which in turn requires the activation of two other promoters located upstream of algC (algCp1) and an AlgU (δE)-dependent promoter (algDp2) [65, 90]. In A. vinelandii, however, AlgU does not participate in the regulation of algL and algA genes as it has been reported for P. aeruginosa [51, 91]. Together, algU and the mucABCD genes form the principal regulatory switch that controls the conversion between mucoid and non-mucoid phenotypes of P. aeruginosa [64, 65, 89], and in other Pseudomonads, such as P. fluorescens and P. syringae, the mucC gene is absent and the transcription mucD is not dependent on AlgU [92, 93].
The regulatory proteins encoded by the mucABCD operon are found either embedded in the inner cell membrane or soluble in the periplasmic space. MucA is a transmembrane protein that has anti-δE activity and acts as a negative regulator of AlgU and is required for maintaining cell viability in P. aeruginosa [94]. MucB has a hydrophobic cavity that interacts with MucA forming a stable complex (MucA-MucB) and serving as a fine-tune controlling mechanism that protects MucA from cleavage of its periplasmic domains by proteases [95, 96]. The role of MucC still remains to be fully determined, however, it has been hypothesized that this is an inner membrane protein that might act synergistically with MucA and MucB to negatively regulate AlgU in A. vinelandii, but not in P. aeruginosa [89, 97]. On the other hand, the role of MucD has been thoroughly determined as being a serine protease/chaperone-like protein with an important regulatory function that acts on the alginate-biosynthetic complex through AlgX [10], and also plays a central role in AlgU activation [89] functioning as a negative regulator localized in the periplasm (Figure 2).
Figure 2.
Schematic representation for the genetic regulation of bacterial alginate biosynthesis. Transcription regulation (full lines) or protein-protein modulation (dashed lines) are indicated in blue (positive regulation/modulation) or red (negative modulation).
Another set of genes and their respective translational products are part of a two-component signal transduction system and directly participate in the regulatory control network of alginate biosynthesis [89]. Four out of five chromosomal regulatory genes (algB, algP, algQ, algR, and algZ/amrZ) found in Pseudomonads and A. vinelandii transcribe in the opposite direction from the previously described algD- and algUmucABCD-operons, except for algB, which has the same transcriptional orientation of the biosynthetic cluster [82]. The promoter regions of algR and algB have significant homology to other promoters recognized by δE, and these genes are positively regulated by AlgU-(δE) [89]. Their translational products, AlgR and AlgB, act directly on different sites of the algD-operon promoter in P. aeruginosa [98, 99]. In contrast, AlgB and AlgR are not required for activation of algD transcription in A. vinelandii [82], and mutations in algB did not affect alginate production for this bacterium, as recently reported [100]. The kinase AlgQ transcribed from algQ undergoes autophosphorylation using ATP or GTP, and transfers a phosphate group to algR [82]. Since AlgR binds to specific sequences of the algD-promoter region, it up-regulates the transcription of the biosynthesis operon in P. aeruginosa, together with AlgU. Although algC transcription is independent of other alginate genes, it has been demonstrated that algC is up-regulated by the presence of functional algR [73]. A ribbon-helix-helix DNA binding protein, AlgZ (also called AmrZ – alginate and motility regulator) is the product of gene algZ and is a strictly δ22-dependent kinase that binds to sequences localized upstream of the algD promoter, activating algD transcription [82]. It has been reported that algZ− mutants of A. vinelandii are unable to produce alginate, thus, it is proposed that AlgZ is required for the transcription of the biosynthetic algD operon [100], however, the molecular mechanism of the algD regulation by AlgZ is yet to be elucidated. AlgP was determined to be a histone-like protein that binds to the algD promoter and aids DNA looping to positively regulate this gene and activate translation [89].
Other regulatory systems are also present during alginate biosynthesis, including post-translational and post-transcriptional regulations [99]. A two-component system (GacS/GacA) acts as a global regulator affecting the transcription of algD and indirectly controlling alginate biosynthesis in A. vinelandii through a signaling cascade involving the post-translational system RsmA/RsmB [65]. It has been described that GacS controls the expression of algD from its three promoters and that gacS− and gacA− mutants of A. vinelandii have significantly lower levels of algD transcripts [51, 81]. Despite being a well-conserved regulatory system among Gram-negative bacteria, the GacS-RsmA pathway has not been reported to regulate alginate biosynthesis in P. aeruginosa [64, 99, 101]. It has been recently announced that the secondary messenger bis-(3′-5′) cyclic dimeric-GMP (c-di-GMP) directly regulates the transcription of the alginate operon in P. aeruginosa [59]. c-di-GMP also acts as a post-translational regulator binding to PilZ domains on proteins such as the one located at the C-terminal end of Alg44 [102, 103]. The binding of c-di-GMP to the Alg44-PilZ domain is essential to activate the alginate polymerase complex Alg8-Alg44 [104].
Although the genetic organization and regulation of alginate biosynthesis seem to be similar in Pseudomonas spp. and Azotobacter vinelandii, these two genera of bacteria have strikingly different ecologies and are present in very specific habitats, which, not surprisingly, reflect the different roles alginate plays in these microorganisms. As shown above, some marked differences between these bacteria regarding alginate biosynthesis-genes distribution and regulation are well reported in the scientific literature [51, 65, 82, 99]. However, there is still much to be unveiled on the genetics and enzymatic regulatory complex for these bacteria, especially for A. vinelandii, which is considered a generally recognized as safe (GRAS) microorganism, with much potential for the biotechnological industry. Therefore, a broad understanding of all the biochemical and regulatory aspects of alginate biosynthesis, combined with the testing of culture conditions and molecular biology tools is paramount for the development of optimal growth methods and strains able to produce this exopolysaccharide in vivo or with post-production in vitro modifications to achieve defined G/M composition, molecular weight and ideal tailor-made physicochemical properties [46, 105, 106, 107, 108].
It is known that bacterial alginate is initially synthesized as a linear poly-D-mannuronic acid homopolysaccharide before undergoing chemical modifications and epimerization in the periplasmic space, which implies in mannuronic acid (ManA) being the main precursor molecule specifically involved in the biosynthesis of bacterial alginate. However, prior to its utilization to form the poly-D-ManA chain that initiates alginate biosynthesis, ManA must first be synthesized from fructose-6-phosphate (F6P), which is derived from the cell’s central carbon metabolism, and then activated with a high-energy bond to yield the sugar nucleotide GDP-ManA [109]. All steps leading to the conversion of F6P to GDP-ManA, the basic building block of alginate, occur in the cytosol and have been extensively analyzed and reported in the past, and a summary of this process is shown in Figure 3.
Three enzymes are directly involved in GDP-ManA biosynthesis, namely, AlgA (isomerase/pyrophosphorylase, AlgC (phosphomutase), and AlgD (dehydrogenase). GDP-ManA biosynthesis begins with the conversion of F6P to mannose-6-phosphate (M6P). This reaction is catalyzed by the bifunctional enzyme AlgA through its phosphomannose isomerase (PMI) activity [110]. Through the formation of M6P, F6P-derived carbon molecules are committed to the alginate biosynthetic pathway, and M6P is rapidly converted to mannose-1-phosphate (M1P) by the Mg2+-dependent phosphomannomutase AlgC [111], a crucial enzyme not only for the biosynthesis of alginate but also required for the production other exopolysaccharides [112], lipopolysaccharides, and rhamnolipids [113, 114]. AlgA is required once again in the next reaction step, however, it will now employ its guanosine-diphosphomannose pyrophosphorylase (GMP) activity to convert M1P to GDP-mannose, simultaneously hydrolyzing GTP and pyrophosphate (PPi). GDP-ManA is finally obtained by the irreversible oxidation of GDP-mannose by AlgD, an NAD+-dependent guanosine-diphospho-D-mannose dehydrogenase that catalyzes the limiting step and major control point of alginate biosynthesis [41, 115, 116].
3.2 Biosynthesis of the poly-D-ManA linear chain
Polymerization of GDP-ManA precursors to polymannuronate is carried out by two enzymes located at the inner membrane of bacteria, interfacing the cytosolic material with the other periplasmatic proteins in the alginate biosynthetic complex. Both enzymes, namely Alg8 and Alg44, are indispensable for alginate production [65, 69, 117]. Alg8 is believed to be the catalytic subunit of alginate polymerase since it shares homologies with class II glycosyltransferases [118], which are responsible for the transfer of sugar units from an activated monosaccharide donor to an acceptor molecule. Similarities in the polymerization mechanism of alginate with the biosynthesis of other polysaccharides have been suggested [119], and since the catalytic mechanism of class II glycosyltransferases requires an inversion of the anomeric configuration at the reaction center, in order to yield β-linked products such as the nascent alginate polymer (poly-β-D-ManA) the donor substrates must be and α-linked sugar nucleotide (i.e.: GDP-α-D-ManA) [69] (Figure 4). Membrane topology analysis of Alg8 has shown that this protein has at least four transmembrane helices, a short periplasmic loop, and an extended cytoplasmic loop containing the glycosyltransferase domain [118, 119, 120]. It has been reported that alg8 overexpression in both A. vinelandii and P. aeruginosa greatly increases alginate production in comparison to wild-type strains [80]. The molecular weight of alginate polymers has also been shown to be higher when alg8 expression increases in A. vinelandii [121].
Figure 4.
Proposed mechanism reaction for the polymer-level epimerization of β-D-ManA into α-L-GulA by the periplasmic C5-mannuronan epimerase AlgG.
The copolymerase Alg44 is the most controversial protein in the entire alginate biosynthesis complex. It is mostly accepted as being a transmembrane protein localized in the bacterial inner membrane and bears a c-di-GMP-binding PilZ domain at its cytosolic N-terminal portion [79, 103, 104]. However, because its complete structure has not yet been determined, and due to its homology with MexA, a periplasmic membrane fusion protein involved in the multi-drug efflux system [122], some reports have proposed that Alg44 lacks transmembrane domains and instead participates in the multi-protein scaffold formation of the alginate biosynthetic complex bridging Alg8 in the inner membrane to the porin (AlgE or AlgJ) in the outer membrane [79, 112, 117]. As previously stated, however, Alg44 is required for the polymerization of ManA together with Alg8, and its regulatory function via the second messenger c-di-GMP in the Alg8-Alg44 complex has been demonstrated [45, 123]. One particular c-di-GMP synthesizing protein, namely MucR, has been shown to specifically influence the alginate biosynthesis in both P. aeruginosa [124] and A. vinelandii [125], and mutations in the PilZ domain of Alg44 has resulted in the loss of alginate production [112].
3.3 Periplasmic chemical modifications of the polymannuronic acid
After polymerization by the Alg8-Alg44 complex, the nascent poly-D-ManA chain is directed across a multi-enzymatic scaffold structure situated in the periplasmic space, where ManA units may suffer epimerization or O-acetylation [10, 65, 80]. The enzymes involved in these processes are the best-analyzed proteins in the biosynthesis process of alginate [126, 127], and the understanding of their reaction mechanisms is an important factor to enable the production of tailored polymers [10, 128]. Although neither epimerization nor O-acetyl-esterification is essential for the bacterial production of alginate, these chemical modifications significantly alter the physicochemical properties of the polymer [31, 32, 38].
The O-acetylation of alginate is a process naturally occurring only in bacteria. During its transit through the periplasm, some ManA units of the polymannuronate chain are O-acetyl-esterified at positions C-2 and/or C-3 by the combined activity of four enzymes: AlgX, AlgF, AlgI, and AlgJ/AlgV [10, 65, 69, 77, 110]. The acetyl groups from the donor molecules (probably acetyl-CoA, but still undetermined), are transported from the cytosol to the periplasm by AlgI, a 7-helical transmembrane protein with homology to DltB (D-alanyltransferase) in Bacillus subtilis [76], to AlgJ (in P. aeruginosa) or AlgV (in A. vinelandii). AlgJ/AlgV is found on the periplasm, closely associated with AlgI, and also anchored in the cytoplasmic membrane presumably by its hydrophobic signal peptide [129]. It shows high homology with AlgX, another protein present in the alginate biosynthesis complex. The structure of AlgX has been shown to have an N-terminal hydrolase domain involved in the acetylation of alginate, and a C-terminal portion where a sugar-binding domain is possibly involved in the binding and orientation of the polymannuronate chain [78, 130]. AlgJ/AlgV and AlgX show about 70% analogy [76, 77], very similar topology [129], and are both required for the esterification of alginate. AlgF has no sequence homology with any other proteins involved in the O-acetylation [10], and algF− mutants of A. vinelandii have been shown to produce non-acetylated alginate [131]. The order of transfer of the acetyl donor to each of the acetylation-related enzymes is uncertain, but the fact is that AlgJ/V performs is directly involved in the O-acetylation of ManA residues and that AlgF serves presumably an accessory role due to the lack of identifiable catalytic residues [129]. Moreover, it is clear that AlgX is also able to catalyze the direct O-acetylation of alginate, receiving the acetate or acetyl donor molecules either from AlgJ/V or AlgF [129]. AlgX has also been shown to interact with MucD in some sort of undetermined regulatory mechanism, which affects the formation of alginate [65, 69, 132].
The esterification of ManA units on hydroxyl groups at C-2 and/or C-3 prevents degradation of the growing alginate polymer by AlgL, an alginate lyase present in the periplasm which will not be discussed in detail herein. O-acetylation also hampers the epimerization of esterified ManA units by AlgG, which is a C5-mannuronan epimerase of the alginate biosynthesis complex and catalyzes the epimerization of β-D-mannuronate (M-blocks) to α-L-guluronate (G-blocks) at polymer level [92]. Non-acetylated ManA units may undergo epimerization by AlgG, which causes the monosaccharide unit to suffer a dramatic change in both its anomeric configuration (from β to α) and pyranose chair conformation (from 4C1 to 1C4) (Figure 4). Such considerable structural changes have very important effects on the physicochemical properties of the mature alginate depending on the proportion and sequence of M-blocks that go through epimerization [69, 112, 133]. AlgG adopts a right-handed parallel β-helix fold, and the catalytic mechanism proposed for this epimerase is based on the β-elimination (not hydrolysis) reaction of polysaccharide lyases [134], which involves the neutralization of the carboxylate group of the uronic acid, removal of the proton at the C-5 position and cleavage of the glycosidic bond with the formation of a double bond between C-4 and C-5 for the formation of a transient glycal intermediate. For the epimerization reaction, however, glycosidic linkage remains intact, and a proton is added to the opposite side of the C-5 to form the epimer [135]. Functional analysis of AlgG mutants suggests that His319 is the catalytic base and that Arg345 is responsible for neutralizing the carboxylic groups during the epimerase reaction. It has also been proposed that water is the likely catalytic acid [134].
Polymer-level epimerization of monosaccharide units is rarely found in nature, and thus far has only been described for the biosynthesis of alginate (both microbial and algal) and glycosaminoglycans (animal origin) [136]. Hence, AlgG is a very exclusive and extraordinary enzyme found in both P. aeruginosa and A. vinelandii. These enzymes show an optimum pH for activity between 6.0 and 7.5 and share about 60% sequence identity [134, 135]. However, differently from Pseudomonads, the A. vinelandii genome also encodes a family of seven extracellular Ca2+-dependent epimerases (AlgE1–AlgE7), which will be discussed later in this chapter.
3.4 Secretion of the mature alginate chain
The two remaining proteins that are part of the alginate biosynthesis complex are AlgK and AlgE/AlgJ (Pseudomonas/Azotobacter). AlgK is a lipoprotein of unclear function found anchored into the inner leaflet of the bacterial outer membrane [137]. It is hypothesized that AlgK may have a protective role, guiding the nascent alginate polymer through the biosynthetic multi-protein complex in the periplasmic space, and preventing it from being degraded by lyases, since the lack of AlgK has been shown to result in the secretion of free uronic acids and/or short-chain alginate products [78, 132], showing that AlgK an essential element for the biosynthesis of full-length alginate and the development of the mucoid phenotype in P. aeruginosa [138]. Additionally, AlgK is proposed to interact with both Alg44, stabilizing the Alg8-Alg44 polymerase complex, and the porin protein AlgE/AlgJ [112, 137]. There is some evidence to suggest that AlgK is involved in the localization of AlgE/AlgJ to the outer membrane [139]. Also, it has been shown that AlgK may form complexes with AlgX and MucD in the periplasmic space, possibly halting the regulatory role of MucD due to its sequestration by an AlgK/AlgX complex in the biosynthetic multi-protein scaffold [65]. The large number of interactions of AlgK with other proteins may be due to its structure containing several tetratricopeptide-like helical motifs which allow unspecific protein-protein interactions [139].
AlgE was first described in the early 1990s as an integral outer membrane protein strongly associated with the mucoid phenotype of P. aeruginosa [140, 141]. Its counterpart in A. vinelandii, AlgJ, shares a high degree of similarity and is thought to perform the same function as in Pseudomonads [142]. Although AlgE is classified as a member of the general diffusion porin family [143], biochemical and electrophysiological analyses suggest that this protein is specifically responsible for the passage of alginate into the extracellular environment through the outer membrane, rather than allowing the passage of small molecules and ions, and that it forms a strongly positively charged anion-selective pore that can be partially blocked by GDP-ManA [144, 145]. AlgE/AlgJ is a monomeric 18-stranded β-barrel protein homologous to the OprD family of substrate-specific porins. In addition to its alginate transport function, it has been shown to play an important role in the assembly of the multiprotein complex involved in alginate biosynthesis [145, 146].
Secretion usually indicates the end of the bacterial alginate biosynthetic pathway, especially in Pseudomonads, and a schematic representation of the entire biosynthetic/regulatory process can be found in Figure 5. After release into the environment, alginate may undergo further non-enzymatic structural modifications such as de-O-acetylation and/or depolymerization, albeit to a very modest extent, depending on the chemical conditions in the medium surrounding the cell. However, the alginate produced by Azotobacter vinelandii can undergo very significant enzymatic processing extracellularly due to the presence of C5-mannuronan epimerases and alginate lyases, which are also secreted by this bacterium.
Figure 5.
Schematic representation of bacterial alginate biosynthesis, regulation, periplasmic transportation/modification, secretion, and post-secretion modification. Protein structures are merely illustrative (https://pdb101.rcsb.org/) and the nomenclature used is that established for P. aeruginosa encoded genes. (*A. vinelandii counterparts or enzymes that are exclusively found in the Azotobacter genus are marked with an asterisk).
3.5 Post-secretion chemical modifications of alginate
3.5.1 Extracellular epimerases
As previously described, Azotobacter vinelandii encodes seven different C5-ManA epimerases (AlgE1–AlgE7) that are exported to the extracellular environment and are able to promote polymer-level epimerization of D-ManA units into L-GulA on alginate. Although the epimerization mechanism for these enzymes is very similar to the mechanism proposed for AlgG, extracellular epimerases have been found to be Ca2+-dependent, in contrast to the periplasmic counterpart. It has been considered that extracellular alginate epimerases are necessary for A. vinelandii cells for the creation of a diffusion barrier against O2 [84] as a protective mechanism for its oxygen-sensitive nitrogenase complex and that enzymes of the AlgE family play an important role in the encystment process induced by adverse environmental conditions [147]. Also, only alginate-producing cells may produce cysts that can germinate after storage [65]. Alginates produced by Pseudomonads show lower L-GulA content in comparison to the A. vinelandii polymers [38, 65, 70]. Furthermore, alginates isolated from Azotobacter spp. contain G-blocks (consecutive L-GulA residues), while in Pseudomonas spp. alginates have been found to contain only single L-GulA units on their backbone structure [70]. It is proposed that post-secretion epimerization of alginate by AlgE1–7 is the reason for the higher amounts of L-GulA found in the alginate produced by A. vinelandii [10, 65, 118]. As the G-block amounts increase, so does the affinity of this polymer for divalent ions, which are responsible for the formation of cross-links between alginate chains creating a more rigid gel surrounding A. vinelandii cells, being physicochemically much different from the lower viscosity alginate produced by bacteria of the genus Pseudomonas [10]. Even though consecutive GulA units have not been found in alginates produced by Pseudomonads to date, it has been reported that P. syringae pv glycinea encodes a C5-mannuronan epimerase designated PsmE, which was found to be able to introduce G-blocks on alginate in vitro [65, 148]. Differently from the A. vinelandii extracellular epimerases, PsmE is able to epimerize acetylated D-ManA units after removing O-acetyl groups, thus showing a bifunctional deacetylase/epimerase characteristic [148].
Isomerases of the AlgE family have been shown to be differentially expressed during the life cycle of A. vinelandii and to generate different patterns of G-blocks on alginate [147]. Five of the algE genes of A. vinelandii are found clustered in the genome, while algE5 and algE7 are found isolated elsewhere (see Figure 1), and each correspondingly encoded enzyme appears to have different specificities in terms of substrate preferences and non-random formation of G- and GM-blocks on the alginate backbone [149]. These extracellular alginate epimerases from A. vinelandii are about 70% identical but share less than 10% sequence identity with AlgG [134]. The characterization of AlgEs from A. vinelandii has been made at certain levels after cloning, heterogeneous expression, and purification, and three putative AlgE-like isomerases have also been described and characterized in A. chroococcum [58]. All of the AlgE isomerases are structurally organized into two types of alternating modules, each with different roles for these enzymes, namely: A-module and R-module (Table 2). It is known that the catalytic site is located in the A-modules, each comprising about 385 amino acids (with a primary amino acid sequence homology of ~85%). These are always followed by 1–4 R-modules (~155 amino acids each), which, in turn, are thought to modulate the epimerization rate by stabilizing the process with an extended alginate binding site [150, 151], and have also been shown to play a role in the epimerization pattern of the final alginate product [152]. A-modules have been calculated to bind 11 uronic acid units, while each of the R-modules can bind five units with different binding strengths, which also affects the processivity of a particular AlgE enzyme [151]. Four to seven copies of a nine amino acid long motif are located in the N-terminal region of the R-modules and are known to be Ca2+-binding sites [153]. Calcium ions are important not only for the structural integrity of the protein but also for neutralizing the charges of ManA during the epimerization reaction, in a similar manner Arg345 functions in AlgG [149, 150]. In addition, the last R-module of each extracellular AlgE usually contains an unstructured peptide involved in the secretion of the enzyme [151]. Despite the large sequence homology they share, different AlgE epimerases catalyze distinct residue sequences in the alginate product. It has been proposed that the different epimerization patterns depend on the concerted action of both the A- and R-modules, rather than only the catalytic activity of A-modules [150].
Structural organization, substrate specificity, and products formed by extracellular C5-ManA epimerases of Azotobacter vinelandii.
Bifunctional extracellular epimerase/lyase enzymes. A-modules: Location of the catalytic site; R-modules: Ca2+-binding sites and substrate-binding/stabilization extended sites.
Although the extracellular C5-epimerases AlgE1–7 play an important role in the addition of L-GulA units to the alginate produced by A. vinelandii, these enzymes have also been cloned and isolated to be used for in vitro modification of macroalgal-derived alginate [128, 149]. AlgE1 and AlgE3 are the only epimerases that contain two catalytic A-modules, one of which introduces single G units that form alternating GM sequences, while the other can generate G-blocks with consecutive GulA units [65, 154]. AlgE2 and AlgE5 both consist of an A-module followed by four R-modules, and they primarily introduce short G-blocks on the alginate chain [149]. The same module organization is found in AlgE6, which, however, is able to introduce longer G-blocks on polymannuronic acid [147, 153]. AlgE4 consists of only one A-module and one R-module and is the smallest enzyme among the extracellular C5-epimerases. Although it inserts a high proportion of GulA units into the alginate chain, this enzyme is unable to attach successive G-blocks to the polymer [65, 149]. AlgE7 generally produces the same GM alternating products as AlgE1, AlgE3, and AlgE4. However, AlgE7 and AlgE2 have also been shown to have present lyase activity (breaks G-MM and G-GM bonds), which is thought to share the same active site with epimerase activity [86]. These bifunctional enzymes can degrade the alginate chain after the epimerization reaction, but at different rates. AlgE2 is thought to have low lyase activity and has been shown to perform 1 to 3 elimination reactions per thousand epimerized units [149, 155]. In contrast, AlgE7 has a much more pronounced lyase activity, with a predicted number of 3–4 glycosyl bond breaks per 26 conversions of D-ManA to L-GulA units [86].
Regarding the reaction mechanisms of Ca2+-dependent AlgE epimerases, two reaction modes have been proposed: (1) the preferred attack mode, in which the enzyme preferentially attacks M-units with G residues as the nearest neighbor and detaches from the substrate after each epimerization reaction [58, 149], and (2) the processive mode, in which there is a relative displacement of the enzyme and the alginate chain that allows the conversion of the nearest M-unit without dissociation of the enzyme-substrate complex [149, 153]. AlgE4 has been reported to have a processive mode of action by sliding along the substrate chain and epimerizing every other M residue [156], and this is thought to be due to the 180°-turn between the successive β-(1 → 4)-linked ManA units in the alginate polymer [153]. AlgE2 follows the mechanism of preferred attack mode and is related to the Ca2+ concentration and substrate type of the blocks found in alginate [149, 155]. The remaining AlgE epimerases appear to behave according to either a preferred attack mode or a combination of the two mechanisms [149].
Despite being in general a relatively well-known process, as shown throughout this chapter, several steps of the bacterial alginate biosynthesis need to be further explored, especially those involving regulatory genes and proteins, to determine their full role during the biosynthetic process and, more importantly, how this knowledge can be useful in the production of tailored alginate polymers for various industrial and commercial uses. In addition to the alginate-producing Pseudomonads and Azotobacter spp. that have been the focus herein, a vast number of organisms bear in their biochemical machinery enzymes capable of degrading alginate (alginate lyases), including algae, marine invertebrates, and fungi, some very specific bacteriophages, and several bacteria such as Bacillus circulans, Klebsiella pneumoniae, Sphingomonas spp., and Vibrio alginolyticus, among others. The role of lyases in organisms that do not produce alginate can be explained by their metabolic ability to use alginate as a sole or secondary carbon source. Interest in these alginate-degrading enzymes has increased in recent years, as it has been shown that alginate-derived oligosaccharides have interesting biotechnological potential, as widely reported in the scientific literature. However, this is a topic that would require an entirely new chapter to adequately address.
The author thanks the Department of Biochemistry and Molecular Biology of the Federal University of Paraná, Curitiba- PR, Brazil, and its collaborators who supported this initiative. This work was not funded by any scientific agency or representative of the Brazilian government.
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Written By
Rodrigo Vassoler Serrato
Submitted: 23 November 2022Reviewed: 01 December 2022Published: 23 December 2022
Open access peer-reviewed chapter - ONLINE FIRST
