# Alginate Biosynthesis inAzotobacter vinelandii: Overview of Molecular Mechanisms in Connection with the Oxygen Availability

Authors: Ivette Pacheco-Leyva; Felipe Guevara Pezoa; Alvaro Díaz-Barrera
Journal: International Journal of Polymer Science
 (2016)

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## Abstract

The Gram-negative bacteriumAzotobacter vinelandii can synthetize the biopolymer alginate that has material properties appropriate for plenty of applications in industry as well as in medicine. In order to settle the foundation for improving alginate production without compromising its quality, a better understanding of the polymer biosynthesis and the mechanism of regulation during fermentation processes is necessary. This knowledge is crucial for the development of novel production strategies. Here, we highlight the key aspects of alginate biosynthesis that can lead to producing an alginate with specific material properties with particular focus on the role of oxygen availability linked with the molecular mechanisms involved in the alginate production.


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## Body

## 1. Introduction

Increasing research on the mechanisms of synthesis and biochemical properties of biopolymers, such as polysaccharides, led to improving production process and new applications in diverse areas, mainly in food and pharmaceutical industries [1]. One of the main advantages for the use of biopolymers is its degradability, making them a renewable product option. However, the high costs of biopolymer production are still a major drawback for a widespread industrial application [2].A particular linear polysaccharide with broad growing interest is alginate, which is a structural component of the brown marine algae and the cell wall of bacteria belonging to thePseudomonas andAzotobacter genera [3–5]. The properties of alginates in solution largely depend on four factors: (a) its monomer chemical composition (β-D-mannuronic acid (M-residues) and its epimer, α-L-guluronic acid (G-residues)); (b) the sequence pattern of the monomers; (c) the molecular weight (MW) of the resulting polysaccharide chain; and (d) modifications of the polymer (acetylation degree) [6, 7].However, algal alginates are complex mixtures containing polysaccharides with a wide range of MW and ratios of M : G. Hence alginates with specific defined M : G ratios or a constant range of MW cannot be easily obtained from particular algae species, due to intrinsic environmental culture conditions, thus limiting their use in the pharmaceutical and chemical industries (more details in Section2). For this reason, the bioprocesses research area has become interested in developing strategies to produce alginates with particular molecular characteristics through microbial alginate production. In contrast to algal alginates, microbial alginates present exclusive M-residue acetylation, controlled M : G ratios, and specific MW under specific growth conditions [8–10]. A nonpathogenic bacterium able to produce alginate with high production yields in bioreactors isAzotobacter vinelandii. Yet, the complex regulatory pathways controlling the alginate biosynthesis and material properties in response to external environmental clues remain still unknown, despite some efforts in trying to gain new insights into gene expression patterns under different culturing conditions inA. vinelandii cultures [9, 11–13].In this review, we present an up-to-date biosynthetic overview of microbial alginate biosynthesis fromAzotobacter vinelandii, and the perspectives for production process improvement based on a better understanding on the molecular mechanism underlying polymer biosynthesis in relationship with the oxygen availability during the fermentation process.


## 2. Alginate Structure, Chemical Structure, and Applications

Over the past 40 years, a growing interest in the use of alginate has been observed including different areas, ranging from genetics to pharmaceutics (Figure1).Figure 1
(a) Number of publications indexed in Scopus database (August 2015), using keywordalginate (−) in title, abstract, or keywords. (b) Percentage of the wordalginate distributed in different subject areas.
(a)
(b)Alginate has been placed as the second biopolymer derived from seaweeds with greater demand in the hydrocolloids’ industry [14]. Currently, the only economic way to obtain commercial alginate used for most applications is through its extraction from marine algae, the cost of which ranges between US$ 2 and 20/kg, and with a total market value of around US$ 339 million [14]. Furthermore, alginates of very high purity are used in the pharmaceutical industry where they are sold for up to US$ 3,200/kg.Since alginate is a biodegradable and a biocompatible polysaccharide, it presents a panoply of food, pharmaceutical, and biotechnological applications (Figure1(b)). In the food and pharmaceutical industries, alginate is mainly used as a stabilizing, thickening, or gel-film-forming agent [6, 15–17], Table 1; in medicine it is used as wound healing material [18], as part of medical treatments [19, 20], or as dietary fiber supplements [21, 22]. Alginate showed potential beneficial physiological effects in the gastrointestinal tract [23]. Moreover, hydrogel-alginates are being investigated in biotechnology as drug delivery agents, as cell encapsulation material, and as scaffold material in tissue engineering [24].Table 1
Summary of biotechnological and pharmaceutical applications of alginates based on their molecular weights.
Application of alginate
Molecular weight (kDa)
Reference
Delivery of bioactive compoundsAntioxidantIn vivo tissue scaffoldsAntibacterialDietary supplementCell immobilization
≈15–120
[60–66]
Food stabilizer and preserving agentMicroencapsulation and storage stabilityAntibacterial BioremediationWound healing
≈120–290
[62, 63, 67–76]
Modulation of enzymatic activityExtended-release tablet compound
500–941
[71, 77]Alginate is the main structural component of brown marine algae (Laminaria andMacrocystis) representing about 32% of dry biomass [25], consisting in variable amounts of M-, G-, and MG-residues, linked by 1→4 glycosidic bonds [7]. On the other hand, alginates produced by bacteria are submitted to esterification withO-acetyl groups at theO-2 and/orO-3 of the M-residues [26], where the majority of the M-residues are mono-O-acetylated, and infrequently with 2,3-di-acetylated [27] (Figure 2). Because the monomeric chemical structure of bacterial alginate and the sequence length determine the mechanical properties of the alginates, one of the aims of different investigations is the possibility of manipulating the composition alginates for specific applications have been intensively investigated [28, 29].Figure 2
Representation of the chemical structure from acetylated alginates produced byAzotobacter vinelandii bacterium [28]. Mannuronic (M) and guluronic (G) acid residues are represented in the alginate chain.The obligate aerobe bacteriumAzotobacter vinelandii produces alginate that acts as a diffusion barrier for nutrients and oxygen [30, 31]. It was reported as a bacterium with a highest respiratory rate [32], implying that it adjusts oxygen consumption rates in order to maintain low levels of cytoplasmic oxygen and in this way permitting the oxygen-sensitive enzymes to be active, like nitrogenase, which is responsible for fixing nitrogen [30, 32].A. vinelandii under limitation of carbon source or by induction forms cysts that are more resistant to desiccation and is mainly composed of alginate [33, 34]. It also accumulates the intracellular polyester poly-β-hydroxybutyrate (PHB) as a reserve carbon and energy source [35, 36].Consequently, an increased knowledge about the molecular mechanism involved in alginate biosynthesis will be crucial for the development of novel strategies to improve the production of alginates with defined characteristics tailored for specific applications.


## 3. The Biosynthetic-Secretory Route of Alginate Production inAzotobacter vinelandii

Microbial polysaccharides have distinct biological functions, as intracellular storage, as envelope, or as extracellular polymers [37]. Microbial alginate is an extracellular polysaccharide as xanthan, cellulose, and sphingan, among others, and they differ in their biosynthetic pathways routes (recently reviewed in Schmid et al. 2015 [37]). Moreover, alginate is secreted trough a secretion system shared among the Gram-negative bacteria [38].The alginate biosynthesis in bacteriaAzotobacter results from a complex regulatory network of proteins, similar toPseudomonas genera [6, 28, 39].All of the steps involved in the conversion of central sugar metabolites into the alginate precursor inA. vinelandii have been previously identified and characterized [6, 40]. The alginate precursor, GDP-mannuronic acid, is synthesized from fructose-6-phosphate to mannose-6-phosphate by the bifunctional enzyme phosphomannose isomerase (PMI)/guanosine-diphosphomannose pyrophosphorylase (GMP), designated as AlgA, encoded by thealgA gene. A phosphomannomutase (AlgC) directly converts the mannose-6-phosphate into mannose-1-phosphate, which is in turn converted into GDP-mannose by the AlgA enzyme. GDP-mannose is oxidized to GDP-mannuronic acid by GDP-mannose dehydrogenase (AlgD, encoded byalgD gene). Because the intracellular levels of GDP-mannose are high and because it is used in different pathways, it has been proposed as the limiting step of alginate biosynthesis inP. aeruginosa [41].After the production of the polymer precursor GDP-mannuronic acid precursor, its polymerization and transport across the cytoplasmic membrane is carried out by proteins presumably integrating a cytoplasmic membrane complex (polymerase complex). The core of the polymerase complex is composed of the glycosyltransferase Alg8 protein and Alg44 protein [42–44]. Furthermore, the protein AlgK is thought to stabilize the polymerase complex, by interacting with Alg44 [43]. Highlighting the important role of this protein, alginate polymerization does not occur in the absence ofalgK [42, 45].The polymannuronate polysaccharide resulting from polymerization and then translocation to theA. vinelandii periplasm is composed of M-residues, which can then be further modified during its passage across the periplasm [43]. These modifications consist in acetylation, epimerization, and degradation of the M-residues. More specifically, the polymannuronic molecule undergoes anO-acetylase modification, which is catalyzed by an acetylase enzymatic complex composed of AlgI, AlgV (AlgJ inP. aeruginosa), AlgF, and AlgX proteins [46–48]. While M-residueO-acetylation does not occur frequently in alginate, some may be acetylated.O-acetylated M-residues will therefore be protected from epimerization [26], because only nonacetylated M-residues can be epimerized to G-residues by the AlgG epimerase [42], so alginates with a relatively high degree of acetylation display a lower degree of epimerization [27].Alginate depolymerization occurs at the 4-O-glycosidic bond via β-elimination, by alginate lyases which have been the subject of a recent review [28]. TheAzotobacter vinelandii genome encodes six enzymes with alginate lyase activity [31]: the alginate lyase AlgL [49], the bifunctional mannuronan C-5 epimerase and alginate lyase AlgE7 [50], the three AlyA(1–3) lyases [51], and an exolyase, AlyB, that is still uncharacterized [28].Some of the nonacetylated M-residues are then epimerized to G-residues by the bifunctional AlgG epimerase, which converts poly(β-D-mannuronate) to α-L-guluronate. InP. aeruginosa, AlgG is also part of the periplasmic protein complex that serves as a scaffold for leading the newly formed alginate polymer through the periplasmic space to the outer membrane secretin AlgE porin (AlgJ inA. vinelandii) [52]. A scaffold complex helps to transport the recently modified polysaccharide throughout the periplasm towards AlgE before secretion to the extracellular milieu. This complex is thought to be composed of AlgG, AlgK, and AlgX proteins and possibly AlgL [40, 42, 43, 52]. The exported polysaccharide could be then epimerized by seven extracellular Ca2+-dependent epimerases (AlgE1–7) [53]. Based on these evidences, Figure 3 shows a schematic representation of the alginate biosynthetic steps inA.vinelandii.Figure 3
Schematic representation of the alginate biosynthetic steps inAzotobacter vinelandii, from evidence-based protein-protein interaction inP. aeruginosa [28, 42, 43]. The biosynthetic alginate pathway is represented as two complementary stages: on the left, the synthesis of the substrate precursor (GDP-mannuronic acid) and its following polymerization, including transfer from cytoplasm; on the right, the modification (periplasmic and extracellular) of the nascent polymer, as well as the export through the outer membrane of the polymer.


## 4. Genetic Regulation of Alginate Biosynthesis inAzotobacter vinelandii

InAzotobacter vinelandii the alginate biosynthetic gene cluster is arranged as an operon (Figure 4), containing genes coding for enzymes involved in the synthesis of the alginate precursors, as well as those involved in its polymerization, degradation, acetylation, epimerization, and secretion. The availability of the complete genome sequence ofA. vinelandii [31] also contributes to the better knowledge of this organism.Figure 4
Genetic structure genes involved in alginate biosynthesis and modification inAzotobacter vinelandii. Gene operon for alginate biosynthesisalgD-A, andalgC gene is transcribed separately;alyA1–3 andalyB alginate lyases encoding genes, andalgE1–7 the epimerases genes.Several promoters controlling alginate gene cluster transcription have been described:algDp1 (σD promoter),algDp2 (AlgU σE dependent promoter), andalgDp3 promoters, all located upstream ofalgD [54, 55],alg8p promoter, upstream ofalg8 [44], and a promoter for sigma 70 located upstream ofalgG [49]. In addition, two putative promotersalgCp1 andalgCp2 are situated upstream ofalgC gene (Figure 4) [56].The alginate biosynthetic gene cluster expression is controlled byalgUmucABCD gene cluster, wherealgU encodes the alternative sigma σE factor (AlgU), essential for alginate production [57]. Moreover, AlgU is responsible for transcription driven by thealgCp1 andalgDp2 promoters (Figure 5), but it does not control thealgL or thealgA genes, as described forP. aeruginosa [55].Figure 5
Regulation of alginate biosynthetic genes inA. vinelandii (modified according to reference [40]). Promoters are indicated as banners; mRNAs are indicated as dotted boxes; solid lines indicate the reported mechanism of regulation, and dashed lines indicate unknown mechanism of gene regulation; arrows indicate positive regulation and T-shaped bars indicate negative regulation. OM: outer membrane; PG: peptidoglycan; IM: inner membrane. See text for a more detailed description.The MucA and MucC proteins negatively regulate alginate production, acting as anti-σE factors [54]. MucA represses AlgU protein activity, thus suppressingalgD transcription from thealgDp2 promoter. In contrast,algU gene transcription is autoregulated by AlgU interaction and activation of its σD promoter locus (algUp2) (Figure 5) [54].Additionally, expression of thealgD promoters is controlled by the global two-component system GacS/GacA, which is conserved among Gram-negative bacteria [58]. The GacS/GacA system controls alginate biosynthesis [58], where GacS controls the expression ofalgD from its three promoters [58]. Accordingly, mutations ingacS andgacA significantly reduce thealgD transcript levels [58]. GacA not only is a positive regulator of the biosynthesis of alginate and PHB [58] but also regulates alginate biosynthesis through activation of the small regulatory RNAs, Rsm (rsmZ1 andrsmZ2). These RNAs interact with the rsmA protein, which bindsalgD mRNA and thus acts as a transcriptional repressor [59]. TheA. vinelandii genome encodes nine small RNAs belonging to the Rsm posttranscriptional regulatory system (rsmZ1–7 andrsmY1-2) (Figure 5) [59].Despite the great efforts to understand the alginate biosynthetic gene regulation, little is known about how cultivation conditions could modify gene transcription inA. vinelandii.


## 5. Alginate Production inAzotobacter vinelandii Cultures: The Balance of Alg8 and AlgL by Oxygen Availability

The glycosyltransferase Alg8 protein belongs to the glycosyltransferase type II family and is localized in the inner cell membrane [78]. The glycosyltransferase type II enzyme family catalyzes the transfer of glycosyl residues to an acceptor molecule, during biosynthesis of polysaccharides, such as the cellulose or chitin synthase [79].In bothAzotobacter vinelandii andPseudomonas aeruginosa thealg8 gene encodes the Alg8 protein [44]. InP. aeruginosa it has been demonstrated that by adding additional copies ofalg8 it is possible to increase alginate production by at least 10 times [80], suggesting that this protein might be involved in a rate-limiting step of alginate production. As a consequence, the possibility of manipulating Alg8 protein levels inA. vinelandii may be a valuable approach for increased alginate production, although this has not being done so far. The attempts to reach high Alg8 protein levels were by manipulating thealg8 gene expression via culture conditions. However, it is important to note that alginate production inA. vinelandii is a multienzymatic and complex process.Moreover, the Alg44 protein acts as link between Alg8 and the AlgJ alginate exporter protein [42, 43]. Since Alg44 has a c-di-GMP intracellular binding domain, it was suggested that this protein presents a regulatory role [81]; although the c-di-GMP levels might not have an impact neither on Alg44 stability nor on its localization, it still seems to be required for the activation of Alg8 [42, 43].Interestingly, inA. vinelandii batch, cultures controlling the dissolved oxygen tension (DOT) at 1% present higher levels ofalg8 andalg44 gene expression, when compared with control cultures (5% DOT) [9]; the authors suggested that this behavior can in turn enhance the MW of the alginate produced under low DOT conditions. Moreover, in continuous cultures under non-nitrogen-fixation conditions at different agitation rates (300, 500, and 700 rpm) and different sucrose concentration in the feed medium, the highest alginate MW (obtained at 500 rpm) is correlated with the highestalg8 expression [12], suggesting thatalg8 gene expression can be modulated by not only oxygen availability but also carbon source feed rate, as well. The oxygen availability here is perceived as the amount of oxygen needed for full oxidation of carbon source, taking into account the oxygen transfer rate as well as the DOT level in cultures [82]. Meanwhile, in chemostat cultures under nitrogen-fixation conditions, operated at a dilution rate of 0.07 h−1, expression of bothalg44 andalg8 was affected by changes in agitation rate (400, 500, and 800 rpm), implying that the activity of both genes could be controlled by oxygen availability [13]. Although the highest alginate MW was obtained at 500 rpm, this was not correlated with higheralg8 gene expression, which was obtained at 800 rpm. The differences between the two-chemostat culture conditions might be explained by the activation of the nitrogenase protection machinery (non-nitrogen-fixation versus fixation), where the higher alginate MW have directly linked to thealg8 gene expression under nonfixing conditions. This notion agrees with the fact that nitrogenase activity protects cells from oxygen, thus fostering alginate production [30, 83]. Other possible explanation given is that the culture condition might activate the genes coding for alginate lyases, further discussed in this review. However, more studies are needed, especially those involving gene expression and proteomics profiles duringA. vinelandii cultures in order to have a better insight of alginate polymerization step.A possible link among the low specific oxygen uptake rate (qO2), the MW of the alginate synthesized, andalg8 gene expression was found [11]. This work suggests that when the qO2 value increases by double, the MW of alginate decreases (about 1.6 times), whilealg8 relative expression decreases around sixfold. Moreover, in cultures carried out in continuous mode operated at dilution rate 0.08 h−1, when the qO2 value was 2.2 mmol g−1h−1, both the alginate MW andalg8 gene expression levels were higher than those obtained in cultures in which the qO2 value was double [11]. The same correlation between low qO2 value and highest alginate MW was reported [12], where a slight increment of 1 in the qO2 lead to a reduction in the MW of the alginate produced byA. vinelandii (from 1200 to 500 kDa). Furthermore, in this condition, the lyase-encoding genealgL increased its expression by threefold whilealg8 expression decreased by ninefold. Interestingly, for qO2 values below 2 mmol g−1h−1 [12] or exceeding 5 mmol g−1h−1 [9, 13], the changes in the alginate MW were not correlated withalg8 oralgL gene expression levels. Table 2 summarizes the major changes observed on both the alginate MW and gene expression levels, during the small increment values over the specific oxygen uptake rate ofA. vinelandii cultures.Table 2
Molecular weight of alginate and relative gene expression ofalg8 and algL with respect to the qO2 variations.
Specific oxygen uptake rate (mmol g−1 h−1)
1–5
5–10
Alginate molecular weight (kDa)
500–1350
480–870
algL gene expression (fold change)
Until 15
0.5–1.5
alg8 gene expression (fold change)
Until 9.0
1.0–2.0
Source: [9, 11–13].Furthermore, theAzotobacter vinelandii genome encodes six enzymes with alginate lyase activity [31]: the alginate lyase AlgL [49], the bifunctional mannuronan C-5 epimerase and alginate lyase AlgE7 [50], and the three AlyA(1–3) lyases [51].The AlyA1, AlyA2, and AlyA3 belong to the PL7 polysaccharide lyase family, containing an alginate lyase module, linked to three calcium-binding modules [28, 51]. AlyA1 and AlyA2 are more likely to be periplasmic (AlyA1, UniProtKB-M9YEJ6; AlyA2, UniProtKB-C1DHI8) whereas the AlyA3 protein has secreted signal C-terminal domain (AlyA3, UniProtKB-C1DQS5), which is needed for efficient germination inA. vinelandii [51]. In chemostat cultures, conducted at dilution rate of 0.07 h−1 with agitation of 500 rpm, highest alginate MW was reported [13]. In this condition, an increment in the agitation rate (from 400 to 600 rpm) leads to an increment in the lyase-encoding genesalyA1,algL, andalyA2 by twofold.ThealgGXLIVFA operon encodes the AlgL protein responsible for the periplasmic alginate lyase activity inA. vinelandii. Disruption of thealgL gene generated a strain that overproduces alginate, suggesting that this enzyme is important for alginate biosynthesis [84]. Furthermore, the increase inalgL expression was not correlated with a decrease in alginate MW in chemostat cultures [12]. However,algL gene expression pattern could also be affected by the qO2 (manipulated by changes in the agitation rate) in chemostat. Supporting this observation, chemostat cultures also showed an increase inalgL gene expression (around eightfold) together with higher MW alginate production [11, 12]. By using anA. vinelandii mutant strain carrying algL::WGm nonpolar mutation [84] and culturing under 3% of DOT, no alterations were found in alginate lyase activity in culture broth comparing with the wild-type strain. However, alginates with a high MW were obtained [85], suggesting that the lower MW of the alginate correlates with the higher alginate lyase AlgL activity.InA. vinelandii ATCC 9046 strain cultures carried out at 1 and 5% DOT, the expression of higher alginate lyase genes (algL,alyA1,alyA2,alyA3, andalgE7) correlated with the lower DOT and with the higher MW alginate production [9]. In these conditions (1% DOT), the intracellular and extracellular lyase activities were lower, comparing with cultures grown at 5% DOT, suggesting that dissolved oxygen affected the activity of the alginate lyases and/or their gene expression. However, the alginate lyase activity (intracellular and extracellular) seemed to be associated with the exponential phase of the cultures, where, in the ATCC strain cultured, the maximum of alginate lyase activity was found in the prestationary phase and dropping in the stationary phase [9, 85].As stated previously (Table2), in cultures with qO2 between 2 mmol g−1h−1 and 5 mmol g−1 h−1 [9, 11–13], the activity of intracellular lyases, namely, AlgL, presented a basal level which was not correlated with a rise in their gene transcriptional levels [9]. This behaviorper se may explain the observed rise in alginate MW (Table 2). Even though these observations indicate that dissolved oxygen affects intracellular as well as extracellular alginate lyase activities, it is possible that different alginate lyases could be expressed at different physiological states, as suggested by the study of AlyE3, which is essential for the efficient cyst germination inA. vinelandii [51].It is important to note that although the AlgL is localized in the periplasm, it has an N-terminal secretion signal (AlgL, UniProtKB-O5219), suggesting that AlgL secretion can occur in response to diverse environmental stimuli (i.e., oxygen concentration). This notion is supported by the observation that AlgL extracellular activity is highly dependent on the dissolved oxygen and that the role of alginate lyase is restricted to a postpolymerization step [9, 85]. Similarly, the alginate lyase AlyA3 also presents extracellular activity, whereas AlyA1 and AlyA2 appear to be periplasmic [51]. These data strongly suggest that alginate lyase expression and extracellular activity occur in response to dissolved oxygen concentrations. Therefore, a detailed analysis of dynamic variations in expression levels and in enzymatic activity throughout the culture is warranted to understand more deeply the alginate polymerization process.In summary, current evidence indicates that when values ofqO2 vary between 2 and 5 mmol g−1h−1 in cultures ofA. vinelandii, a rise in expression ofalgL together with a decrease in expression ofalg8 correlates with a decrease in alginate MW (Table 2). As such, this range of qO2 could be a target in the development of strategies to manipulate the characteristics of alginates.


### 5.1. Oxygen Sensing Mechanisms inAzotobacter vinelandii

Current evidences demonstrate that the oxygen transfer rate, the dissolved oxygen tension levels, and the oxygen uptake rate affect alginate biosynthesis inA. vinelandii cultures [8, 9, 12, 13, 36, 40, 86–89]. Despite the importance of the oxygen and the intrinsic relationship with it, no strong evidence of the molecular mechanism involved in sensing it duringA. vinelandii culturing is available, as well as its further downstream mechanism still being lacking. In this section we discuss that oxygen availability duringA. vinelandii culturing is a key factor and we suggest a possible mechanism of action.InA. vinelandii the mechanism involved in sensing oxygen availability remains to be fully investigated. In bacteria, several oxygen sensing mechanisms exist. However they can be clustered in two groups based on how the signal is perceived. One category can interact with external environment while, on the other hand, the second category senses physiological changes resulting from variations in the external environment. Nevertheless, both sensing mechanisms operating together control directly the switch between aerobic and anaerobic metabolism [90]. Among the oxygen sensing mechanism, the FNR, ArcA/B, and ubiquinone-8 (Q8) are well characterized inE. coli [90].InA. vinelandii the absence of an Fnr-like protein, CydR, overexpressing the β-ketothiolase and acetoacetyl-coA reductase [91], both enzymes catalyze the production of β-hydroxybutyryl-CoA, which is the PHB precursor [40]. It has been demonstrated that low aeration culture conditions inA. vinelandii cultures enhanced the metabolic flux from pyruvate towards acetyl-CoA. This had an influence on the increment on the metabolic flux towards PHB production, concomitantly with the higher alginate production [8], suggesting that the aeration conditions could affect the alginate production, by regulating possible gene targets of CydR. Supporting this observation, batch cultures ofA. vinelandii OP mutant strain carried out at 600 rpm showed lowest qO2 compared with wild-type strain (ATCC 9046) [92]. TheA. vinelandii OP strain contains an insertion element in thealgU gene, which in turn represses alginate synthesis [93] and it has been suggested that AlgU is required forcydR gene expression [94].CydR controls the expression ofcydAB operon that encodes a cytochromebd terminal oxidase, andcydAB gene expression correlates with the NADH:ubiquinone oxidoreductase activity (NDHII) [91]. InA. vinelandii, the Na+-translocating NADH:ubiquinone oxidoreductases (Na+-NQR) are encoded in thenqr operon, and it had been linked to regulating negatively alginate production [95]. Additionally,A. vinelandii genome contains genes linked to NADH:ubiquinone oxidoreductases (NDH), the NDH-II type, and 13 genes encoding subunits of NDH-I type [95]. The NADH oxidation inA. vinelandii is mediated by two NADH:ubiquinone oxidoreductases [96], and the fast NADH oxidation is linked to a fast quinone reduction. TheubiC-A operon inA. vinelandii is responsible for the transcription of the genes necessaries for Q8 biosynthesis [95]. A mutation in the intragenic regionubiA correlates with lower Q8 protein levels, accompanied with an improvement in the alginate production, but all the more, with a higher expression of biosynthetic alginate genes,algD,algC, andalgA. Moreover, the Q8 protein seems to be responsible for at least 8% of the respiratory capacity inA. vinelandii, during low and high aeration cultures [95].Interestingly, in other bacteria asE. coli, the role of quinones as a redox signal for the pathways involved in sensing oxygen and regulation of expression of genes involved in oxidative and fermentative catabolism is well known, specifically the ArcB/A two-component system [97–99].Figure6 summarizes the plausible regulation ofalg genes inA. vinelandii, via a signaling cascade activated by oxygen availability. On one hand, the Na+NQR protein regulates negativelyalgD andalgC gene targets, while the ArcB/A two-component system regulatesalgD andalg8 gene expression under oxygen availability. When oxygen is limiting, the sensor kinase ArcB autophosphorylates and then transphosphorylates the regulator ArcA, which activatesalgD,alg8, andalg44 gene expression. The autophosphorylation of ArcB is inhibited at higher oxygen concentrations, by the accumulation of Q8 (oxidized form). In this sense, inA. vinelandii, a tight control ofalg genes via a signaling cascade activated by oxygen availability may exist (Figure 6).Figure 6
Schematic representation of the possible gene regulation mechanism by oxygen inAzotobacter vinelandii. Oxygen availability is depicted in the figure as low O2 (left side) and high O2 (right side). Light red dotted boxes indicate the Na+-translocating NADH:ubiquinone oxidoreductase (Na+NQR) that regulates negativelyalgD andalgC gene targets, although the exact mechanism ofalgD andalgC gene regulation at high O2 by Na+NQR is still unknown. Gray slashed boxes represent the ArcB/A two-component redox sensor: under high oxygen availability, the autophosphorylation of ArcB (B blocks) is inhibited by oxidized quinones (Q8). ArcA (A blocks) in the nonphosphorylated state is unable to bind specifically toalgD,alg8, andalg44 gene targets. Low oxygen causes a decrease in the level of oxidized quinones (Q8H2), allowing the autophosphorylation of ArcA. ArcA-P binds specifically to its target sites and coordinates the cellular response to oxygen availability. Arrows indicate positive regulation and T-shaped bars indicate negative regulation. Flag-type boxes indicate genes described in the figure. Question mark indicates unknown gene regulation mechanism. OM: outer membrane; PG: peptidoglycan; IM: inner membrane.Although recently Flores et al., 2015 [36], discussed mainly the influence of the oxygen on production of alginate duringA.vinelandii cultures, not much attention is paid to which molecular pathways are involved during alginate biosynthesis. In our work, we propose a possible mechanism of action of the oxygen availability duringA.vinelandii culturing, offering a new path to look at and in this way contributing to the better knowledge of controlling bacterial alginates production.Despite the enormous efforts in understanding the microbial alginate biosynthesis under defined culture conditions, there is still a way to go. The decoding of theA. vinelandii genome has open the possibility to getting access to new information; however no wide genetic screen studies during alginate production have been reported yet. So, it will be necessarily an improvement in the knowledge ofA. vinelandii alginate biosynthesis gene regulation in alginate production processes, in order to generate a tailored and affordable alginate product.


## 5.1. Oxygen Sensing Mechanisms inAzotobacter vinelandii

Current evidences demonstrate that the oxygen transfer rate, the dissolved oxygen tension levels, and the oxygen uptake rate affect alginate biosynthesis inA. vinelandii cultures [8, 9, 12, 13, 36, 40, 86–89]. Despite the importance of the oxygen and the intrinsic relationship with it, no strong evidence of the molecular mechanism involved in sensing it duringA. vinelandii culturing is available, as well as its further downstream mechanism still being lacking. In this section we discuss that oxygen availability duringA. vinelandii culturing is a key factor and we suggest a possible mechanism of action.InA. vinelandii the mechanism involved in sensing oxygen availability remains to be fully investigated. In bacteria, several oxygen sensing mechanisms exist. However they can be clustered in two groups based on how the signal is perceived. One category can interact with external environment while, on the other hand, the second category senses physiological changes resulting from variations in the external environment. Nevertheless, both sensing mechanisms operating together control directly the switch between aerobic and anaerobic metabolism [90]. Among the oxygen sensing mechanism, the FNR, ArcA/B, and ubiquinone-8 (Q8) are well characterized inE. coli [90].InA. vinelandii the absence of an Fnr-like protein, CydR, overexpressing the β-ketothiolase and acetoacetyl-coA reductase [91], both enzymes catalyze the production of β-hydroxybutyryl-CoA, which is the PHB precursor [40]. It has been demonstrated that low aeration culture conditions inA. vinelandii cultures enhanced the metabolic flux from pyruvate towards acetyl-CoA. This had an influence on the increment on the metabolic flux towards PHB production, concomitantly with the higher alginate production [8], suggesting that the aeration conditions could affect the alginate production, by regulating possible gene targets of CydR. Supporting this observation, batch cultures ofA. vinelandii OP mutant strain carried out at 600 rpm showed lowest qO2 compared with wild-type strain (ATCC 9046) [92]. TheA. vinelandii OP strain contains an insertion element in thealgU gene, which in turn represses alginate synthesis [93] and it has been suggested that AlgU is required forcydR gene expression [94].CydR controls the expression ofcydAB operon that encodes a cytochromebd terminal oxidase, andcydAB gene expression correlates with the NADH:ubiquinone oxidoreductase activity (NDHII) [91]. InA. vinelandii, the Na+-translocating NADH:ubiquinone oxidoreductases (Na+-NQR) are encoded in thenqr operon, and it had been linked to regulating negatively alginate production [95]. Additionally,A. vinelandii genome contains genes linked to NADH:ubiquinone oxidoreductases (NDH), the NDH-II type, and 13 genes encoding subunits of NDH-I type [95]. The NADH oxidation inA. vinelandii is mediated by two NADH:ubiquinone oxidoreductases [96], and the fast NADH oxidation is linked to a fast quinone reduction. TheubiC-A operon inA. vinelandii is responsible for the transcription of the genes necessaries for Q8 biosynthesis [95]. A mutation in the intragenic regionubiA correlates with lower Q8 protein levels, accompanied with an improvement in the alginate production, but all the more, with a higher expression of biosynthetic alginate genes,algD,algC, andalgA. Moreover, the Q8 protein seems to be responsible for at least 8% of the respiratory capacity inA. vinelandii, during low and high aeration cultures [95].Interestingly, in other bacteria asE. coli, the role of quinones as a redox signal for the pathways involved in sensing oxygen and regulation of expression of genes involved in oxidative and fermentative catabolism is well known, specifically the ArcB/A two-component system [97–99].Figure6 summarizes the plausible regulation ofalg genes inA. vinelandii, via a signaling cascade activated by oxygen availability. On one hand, the Na+NQR protein regulates negativelyalgD andalgC gene targets, while the ArcB/A two-component system regulatesalgD andalg8 gene expression under oxygen availability. When oxygen is limiting, the sensor kinase ArcB autophosphorylates and then transphosphorylates the regulator ArcA, which activatesalgD,alg8, andalg44 gene expression. The autophosphorylation of ArcB is inhibited at higher oxygen concentrations, by the accumulation of Q8 (oxidized form). In this sense, inA. vinelandii, a tight control ofalg genes via a signaling cascade activated by oxygen availability may exist (Figure 6).Figure 6
Schematic representation of the possible gene regulation mechanism by oxygen inAzotobacter vinelandii. Oxygen availability is depicted in the figure as low O2 (left side) and high O2 (right side). Light red dotted boxes indicate the Na+-translocating NADH:ubiquinone oxidoreductase (Na+NQR) that regulates negativelyalgD andalgC gene targets, although the exact mechanism ofalgD andalgC gene regulation at high O2 by Na+NQR is still unknown. Gray slashed boxes represent the ArcB/A two-component redox sensor: under high oxygen availability, the autophosphorylation of ArcB (B blocks) is inhibited by oxidized quinones (Q8). ArcA (A blocks) in the nonphosphorylated state is unable to bind specifically toalgD,alg8, andalg44 gene targets. Low oxygen causes a decrease in the level of oxidized quinones (Q8H2), allowing the autophosphorylation of ArcA. ArcA-P binds specifically to its target sites and coordinates the cellular response to oxygen availability. Arrows indicate positive regulation and T-shaped bars indicate negative regulation. Flag-type boxes indicate genes described in the figure. Question mark indicates unknown gene regulation mechanism. OM: outer membrane; PG: peptidoglycan; IM: inner membrane.Although recently Flores et al., 2015 [36], discussed mainly the influence of the oxygen on production of alginate duringA.vinelandii cultures, not much attention is paid to which molecular pathways are involved during alginate biosynthesis. In our work, we propose a possible mechanism of action of the oxygen availability duringA.vinelandii culturing, offering a new path to look at and in this way contributing to the better knowledge of controlling bacterial alginates production.Despite the enormous efforts in understanding the microbial alginate biosynthesis under defined culture conditions, there is still a way to go. The decoding of theA. vinelandii genome has open the possibility to getting access to new information; however no wide genetic screen studies during alginate production have been reported yet. So, it will be necessarily an improvement in the knowledge ofA. vinelandii alginate biosynthesis gene regulation in alginate production processes, in order to generate a tailored and affordable alginate product.


## 6. Conclusion

In the present review we discuss that oxygen availability duringAzotobacter vinelandii cultures might exert a tight control over the expression of alginate-related genes, which will impact the quality of the polysaccharide or will regulate enzymatic activities that modified the nascent alginate chain. Current evidence indicates a prevailing equilibrium inalg8 andalgL gene expression, which is being regulated by oxygen availability. This equilibrium will further impact the alginate molecular weight. Accordingly, more information regarding oxygen sensing, transportation, and signaling pathways during specific culture conditions ofA. vinelandii will be needed in order to obtain alginates with specific characteristics.



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*Source: 2062360-2016-02-29.xml*