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Alginate and PHB synthesis in A. vinelandii
- Molecular and bioengineering strategies to improve alginate and polydydroxyalkanoate production by Azotobacter vinelandii

2. Alginate and PHB synthesis in A. vinelandii

2.1 Alginate biosynthesis and its regulation

Biosynthetic pathway for alginates (Figure 1) is similar in both the Azotobacter and Pseudomonas species and has been the subject of a recent review [9]. Briefly, alginate is synthesized from fructose-6-P, which is isomerized by a bifunctional enzyme phosphomannose isomerase/guanosine diphosphomannose pyrophosphorylase, (PMI-GMP or AlgA) in order to produce mannose-6-P; which is in turn converted by phosphomannomutase (PMM or AlgC) into mannose-1-P; PMI-GMP (AlgA) catalyzes the conversion of mannose-1-P to become GDP-mannose; GDP mannose is oxidized by GDP-mannose dehydrogenase (GMD or AlgD) to GDP-mannuronic acid. Polymerization of GDP-mannuronic acid is carried out by the Alg8 protein, a mannuronate polymerase (MP) [10]. The resulting polymannuronic molecule is then modified by an acetylase complex comprising AlgI, AlgV, AlgF proteins (AlgI, AlgJ and AlgF for P. aeruginosa), and some of the non-acetylated mannuronate residues are epimerized to guluronate by a mannuronate epimerase (ME or AlgG) and then exported through the outer membrane via the pore-forming protein AlgE (AlgJ in A. vinelandii) (Figure 1).

In the case of A. vinelandii but not in that of the Pseudomonas species, the exported polymer is converted to the final alginate by a family of seven homologous secreted mannuronan C-5 extracellular epimerases (AlgE1-7) [11](Figure 1). These epimerases are essential for the formation of mature cysts, as a mutation which inactivates the Type I secretion system responsible for the export of the AlgE1-7 epimerases produced cysts lacking the intine and exine coats and were therefore unable to survive desiccation. This suggests that the guluronic acid residues in alginate are important for the formation of the alginate coat surrounding the cysts [12]. It is thought that the AlgG AlgK, AlgX and AlgL proteins form a scaffold which guides the polymer through the periplasm, to then be secreted across the outer membrane [13,14]. AlgL is also an alginate lyase enzyme [15]. The idea that AlgL played a role in A. vinelandii, concerning the degradion of the alginate capsule during cyst germination was ruled out, as germination was unaffected in an algL mutant [16]. The main role of AlgL in the Pseudomonas species is to degrade alginates which fail to be exported out of the cell and thus remain in the periplasm [14,17,18].

Although Alg44 was originally considered to be a component of the polymerase complex, it was recently proposed to be a part of the periplasmic scaffold and/or to play a role in bridging Alg8 in the cytoplasmic membrane with AlgE (AlgJ in A. vinelandii) [9]. Alg44 protein has a PilZ domain, a putative cyclic diguanosil monophosphate (c-di-GMP) binding domain [19]. c-di-GMP is a novel regulatory molecule identified as a universal secondary messenger in bacteria [20,21]. Thus, the presence of a PilZ domain, suggests a regulatory role for Alg44.

The genes coding for the enzymes of the alginate biosynthesis in A. vinelandii have all been identified (Figure 2). With the exception of algC, they form the algD-8-44-K-J-G-X-L-I-V-F-A cluster [22-28]. Several promoters transcribing this alginate biosynthetic gene cluster have been identified (Figure 2). Three promoters: algDp1, algDp2 and algDp3 located upstream algD [22], alg8p located upstream alg8 [25], and one putative sigma 70 promoter located upstream algG [27]. Two promoters were identified upstream algC: algCp1 and algCp2 [28]. Expression of the alginate biosynthetic genes in A. vinelandii has been shown to be under the control of the algUmucABCD gene cluster, where algU codes for the alternative sigma E factor, required for transcription from the algCp1 and algDp2 promoters [28,29]. MucA and MucB proteins act as antisigma E factors (Figure 2). Therefore, mutational inactivation of algU results in the impairment of alginate synthesis [30], whereas inactivation of mucA leads to alginate overproduction [31]. Expression from the algD promoters is also under the control of the two component global regulatory system GacS-GacA, where GacS acts as a sensor histidine kinase protein which phosphorylates GacA, the response regulator that activates transcription of the target genes in its phosphorylated form. Inactivation of gacS or gacA genes abrogates transcription of algD from its three promoters [32,33]. The rpoS gene encoding the sigma S factor is under the control of GacA. Inactivation of rpoS was shown to impair transcription from the algDp1 promoter [33]. Thus, a regulatory cascade which includes the global regulators GacA and RpoS participates in the control of algD transcription (Figure 2).

2.2. Polyhydroxybutyrate synthesis and its regulation

PHB in A. vinelandii is synthesized in three steps from acetyl-CoA [34]. A β-Ketothiolase catalyses the first reaction i.e. the condensation of two molecules of acetyl-CoA to form acetoacetyl-CoA, which is reduced by the NADPH dependent acetoacetyl-CoA reductase to produce β-hydroxybutyryl-CoA. PHB synthase catalyses the final reaction: the polymerization of β-hydroxybutyryl-CoA (Figure 1). A PHB biosynthetic gene cluster phbBAC, coding for the enzymatic activities β-ketothiolase, acetoacetyl-CoA reductase and PHB synthase respectively has been described in A. vinelandii [7,35,36] and Azotobacter sp. strain FA8 (Figure 2) [37]. In the same DNA region where phbBAC genes are found, other genes related to PHB synthesis were also found: phbR, which codes for a member of the AraC family of transcriptional activators; phbP, coding for a putative granule-associated protein, and phbF, a putative regulator of the phbP [7,36]

The regulation of polyhydroxybutyrate synthesis in A. vinelandii seems to be complex (Figure 2). In addition to the allosteric control of the first biosynthetic enzyme, β-ketothiolase, by the CoA/acetyl-CoA ratio which was described several years ago [38], other regulatory systems are involved. Transcription of the phbBAC biosynthetic operon is initiated from two overlapping promoters, pB1 and pB2. PhbR, encoded by phbR, activates transcription of the PHB biosynthetic operon from the pB1 promoter, whereas transcription from pB2 is dependent on the sigma factor RpoS and increases during the stationary phase of growth. Transcription of phbR itself also starts from two promoters, pR1 and pR2. Transcription from pR2 is also induced during the stationary phase and is dependent on RpoS, although probably in an indirect manner [36]. Thus, the following regulation model was proposed: in exponentially growing cells, the balanced growth conditions inhibit the β-ketothiolase activity which is present, and there is also a low transcription of phbBAC caused by both the lack of RpoS, which affects transcription from one of the promoters of the PHB biosynthetic operon, and by the low concentraction of PhbR, whose transcription is also partially dependent on this sigma factor [36]. On entering into the stationary phase, the increase in transcription of rpoS and consequently of phbR, stimulates transcription of the phbBAC operon. In addition, the tricarboxylic acid cycle activity may slow down during the stationary phase, allowing for an increase in the acetyl-CoA/CoA ratio, which relieves the inhibitory effect on β-ketothiolase.

The two-component global regulatory system formed by the sensor kinase GacS [32] and its corresponding response regulator GacA [33] is also involved in the control of PHB production in A. vinelandii (Figure 2). Either gacS or gacA mutations diminish PHB production. The model proposes that GacA plays a role as a positive regulator of PHB synthesis in its phosphorylated form. GacA is required for transcription of rpoS [33]. Hence, at least part of the control that this system exerts on PHB production can be explained by its effect on the expression of the sigma factor RpoS.

The nitrogen-related phosphotransferase system (PTSNtr; Figure 2), a homolog of the phosphoenol pyruvate-sugar phosphotransferase system (PTS) which mediates the uptake and concomitant phosphorylation of glucose and other carbohydrates in a number of bacterial genera [39] is also involved in the control of PHB accumulation in A. vinelandii. A mutation on ptsP, encoding enzyme INtr, lowers the accumulation of PHB [6]. This regulation is probably exerted through a phosphate relay, where enzyme INtr autophosphorylates using phosphoenolpyruvate, and IIANtr protein appears to be the terminal phosphoryl acceptor (and acts as a negative regulator of PHB synthesis (G. Espín, unpublished data).

It has also been argued that the Fnr-like regulatory protein called CydR may control PHB synthesis in A. vinelandii [40]. CydR acts as a repressor in the transcription of cydAB, the genes coding for the cytochrome bd terminal oxidase required for aerotolerant nitrogen fixation. The DNA binding capacity of CydR is diminished in the presence of oxygen, and transcription of cydAB is derepressed [41]. While looking for CydR regulated genes, Wu et al. [40] found that a cydR mutant overexpresses β-ketothiolase and acetoacetyl-CoA reductase, and accumulates PHB throughout the exponential growth rate. It is probable that the role of CydR in the control of PHB synthesis is related to the redox state of the cell. However, the mechanism used and its relationship with other regulatory systems is unclear.

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