3. Use of mutants for modifying alginate characteristics or for improving alginate production
Genetic strategies for the construction of A. vinelandii mutants have proven to be useful for the elucidation of regulatory aspects in the biosynthesis of alginate and therefore for the construction of A. vinelandii mutants exhibiting higher specific alginate production. Moreover, it has been useful for generating strains which produce alginates with specific chemical characteristics. The rationale for the construction of such mutants is discussed in the following section and the main results in terms of the alginate concentration and its molecular characteristics are also summarized in Table 1 for cultures in shake flasks, and in Table 2 for cultures carried out in fermentors.
3.1 Alginate production by A. vinelandii mutants with increased algD transcription
As previously discussed, an extensive work has been carried out, in order to unveil the regulatory network which controls alginate synthesis in A. vinelandii [24,28-33,42,43]. The global regulators GacA/GacS and the sigma factor AlgU seem to represent key elements in this regulation, and the algD promoters are targets in A. vinelandii. A fact which upholds this observation is that transcription of the A. vinelandii algD gene correlates with the production of alginate [22,31,42]
mucA and mucABCD mutations increased algD transcription two and four-fold, respectively [31]. When transferred to the wild type AEIV, a non-highly mucoid strain, these mutations increased alginate levels up to six-fold. However, in the background of the highly mucoid strain ATCC 9046, only a minor increment of 39 % (for the mucA mutation) and 85 % (for the mucABCD mutation) was observed. To our knowledge, the A. vinelandii ATCC 9046 mucABCD mutant derivative named JRA4, showed the highest level of specific alginate production (8.9 mg of alginate/mg of protein), ever reported for this bacterium. However, spontaneous derivatives with reduced levels of alginate production (2.5 mg/mg of protein) appeared at a high frequency, implying that elevated AlgU activity and/or increased alginate production are deleterious to A. vinelandii [31].
Random Tn5 mutagenesis of WI12 strain, an ATCC 9046 derivative carrying an algD::lacZ transcriptional fusion was carried out [42]. Two mutants called AC28 [42] and RC26 [4], which showed a 3 and 1.9-fold increase in levels of algD transcription respectively were identified. The Tn5 mutations in strains AC28 and RC26 were shown to reside within the ampDE operon (muc28 mutation) which participates in the intracellular recycling of the cell wall, and within a gene named muc26, which encodes a conserved hypothetical protein (Gene Object ID 400259050) of the A. vinelandii genome project [44], respectively. The double ampDE mutation, but not the single ampD or ampE mutations exhibited a three-fold increase in algD transcription. Transfer of the ampDE::Tn5 mutation to the ATCC 9046 strain was unsuccessful. However, when transferred to the AEIV strain, this mutation was viable and increased alginate production eight-fold.
The muc26 mutation was transferred to wild type strain ATCC 9046 in order to produce mutant CN26. Although a high alginate concentration (7 g/L) was obtained in the culture of this mutant in shake flasks (Table 1), under controlled conditions of pH (7.2), agitation rate (300 rpm) and dissolved oxygen tension (DOT) (3% DOT), this muc26 mutant did not improve the volumetric production of alginate when compared to the wild type strain ATCC 9046 (Table 2) [4].
Since alginate and PHB are two polymers which compete for the carbon source, we hypothesized that phbR mutation would improve alginate yield in the muc26 mutant background. Therefore, a double mutant (muc26 phbR) was constructed, and named DM [4]. No increase in alginate production was apparent; however, a significant increment in molecular weight (from 0.8 × 106 to 4.0 × 106 Da) of alginate produced by strain DM was observed, when the cells were cultured under controlled DOT conditions (Table 2). This is the highest which has been reported for a bacterial alginate. This value is higher than that of a commercial alginate from Macrosystis pyrifera (1.1 × 106 Da) and that of the alginate produced by the A. vinelandii parental strain in shake flasks (1.9 × 106 Da) [45]. The polymer produced by any of the single mutants, muc26 (CN26 strain) or phbR (AT268 strain) showed wild type characteristics (Table 2). Since the mechanism determining the molecular mass of alginate is poorly understood and since the muc26 mutation interrupted a gene encoding a hypothetical protein, it is difficult to offer an explanation for the high molecular weight exhibited by the alginate produced by the DM mutant.
3.2 A phbBAC mutant increases alginate production
A. vinelandii converts carbon substrates to alginate and PHB, thus the synthesis of PHB is undesirable when optimizing alginate production. Strain AT6, a derivative of ATCC 9046, carrying a phbBAC mutation which abrogates PHB synthesis, was shown to increase the alginate yield up to a value of 3.02 g alg/g protein (Table 1), presumably as a consequence of the greater availability of carbon source. However, the volumetric yield of alginate was not significantly increased due to a deleterious effect of this mutation upon cell growth [46].
Taken together, these results suggest that the use of A. vinelandii ATCC 9046 mutant derivatives with higher levels of alginate, due either to an increase in AlgU activity or to an increase in the available carbon source seems not to be feasible for industrial purposes and suggests that a different approach must be taken in order to improve volumetric alginate yield in this A. vinelandii strain. This fact is further supported by a previous suggestion that strain ATCC 9046 contains a muc-1 mutation which increases AlgU activity and consequently alginate production [29]. Therefore, it is likely that the metabolic limits for alginate production have already been reached for the ATCC 9046 strain and that any other mutation increasing alginate production will prove either to be unviable, or to be viable, but at the expense of cell growth.
3.3 Alginate lyase and its role in the molecular weight of alginate
The rheological and gel-forming properties of alginate are largely dependent on the molecular mass distribution (MMD) and the relative content of D-mannuronic and L-guluronic monomers [1]. Oxygen concentration has been shown to influence the molecular mass of alginate produced by A. vinelandii [47]. A high molecular mass alginate can be produced at a DOT of 3 %. However, a drop in the mean molecular mass of alginate was observed towards the end of the incubation period, presumably as a consequence of a de-polymerization activity carried out by an alginate lyase [47]. To investigate the effect of A. vinelandii AlgL on the molecular weight of alginate, a non-polar algL::Gm mutant was constructed, and was named SML2. No alginate lyase activity was detected in the SML2 strain. Since AlgE7 epimerase exhibits alginate lyase activiy in vitro [48], it is possible that this activity is either very low or is only expressed under specific conditions. The specific production of alginate by the SML2 strain was reduced 35 %, when the cells were cultured in a fermenter at 3 % of DOT and 300 rpm, perhaps as a consequence of the gentamycin cassette insertion which might have had some polar effect on genes downstream algL. In contrast to the wild type strain, in A. vinelandii SML2 cultures, no drop in the MMM was observed (Table 2), indicating that AlgL was responsible for the de-polymerization of alginate [16].
3.4 Alginate acetylation
Bacterial alginates differ from algal alginates because of the presence of O-acetylated mannuronate residues; the majority of these residues are mono-O-acetylated but a few are 2,3-di-O-acetylated [49]. In an attempt to obtain an A. vinelandii mutant producing non-acetylated alginate strain, an algF::Tc non-polar mutant was constructed and was named AJ34. This mutant produced a non-acetylated alginate which appeared to confer a rough phenotype to the colony and a reduction in the formation of cysts resistant to desiccation [27]. Alginate production by AJ34 strain mutant was reduced by 50 %, probably due to a polar effect on algA transcription from the tetracyclin cassette promoter [27]. Further work is needed in order to assess the rheological characteristics of this polymer and to investigate whether these non-acetylated alginates constitute better substrates for the A. vinelandii AlgE1-7 epimerases, which only modify non-acetylated M residues.
3.5 A. vinelandii epimerases
As pointed out previously, alginates are composed of variable amounts of (1–4)-β-D-mannuronic acid (M residues) and its epimer, α-L-guluronic acid (G residues). Their relative content and sequence distribution vary widely and have profound effects on the physicochemical properties of the polymer. In A. vinelandii, a family of seven secreted and Ca++ dependent C-5 epimerases (designated AlgE1-AlgE7) have been identified and these are responsible for generating a variety of epimerization patterns, including G blocks of various lengths [50]. Each enzyme is composed of two structurally distinct modules, designated A and R. The A modules (about 385 amino acids) are present in one or two copies in each enzyme, while the R modules (about 155 amino acids) are present in between one and seven copies. It was shown that the A module alone is sufficient for both the epimerization, as well as the determination of sequence distribution [51]. G blocks are of great biological and biotechnological significance as they are a prerequisite for the formation of strong polymer gels in the presence of divalent cations like Ca2+; however the composition of the commercially available alginates vary, depending on the source of the polymer. Hence extensive work has been carried out in order to investigate the epimerization properties for each A. vinelandii C-5 epimerase, with the aim of increasing the G content of commercially available alginates, containing a wide range of initial G residues, or in order to produce alginates with specialized properties [50,52,53]. While algE4 activity introduces alternating M and G residues into substrate, the remaining six enzymes introduce a mixture of continuous stretches of G residues and alternating sequences. Furthermore, Bjerkan et al. [52] constructed hybrid mannuronan C-5 epimerases, by exchanging parts of the sequences encoding the A module of AlgE2 (which generates consecutive stretches of G residues) and AlgE4 (which generates alternating structures). These hybrid enzymes introduce a variety of new monomer-sequence patterns into their substrate. Those authors also identified some regions of the A module, important for the specificity or processivity of the enzymes. These studies, besides helping to elucidate the structure-function relationship for each enzyme, open up new possibilities for biotechnological applications.
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