4. Mutations which increase PHB production
4.1 Blocking alginate synthesis, promote PHB accumulation
Alginate synthesis may constitute a waste of substrate when seeking to optimize PHB production. The effects on PHB accumulation of two mutations causing different blockades in the alginate biosynthetic pathway have been described. Martínez et al. [54] evaluated the effect of a mutation in the algK gene on PHB production (Figure 1). This gene encodes a protein which is probably involved in guiding alginate for secretion, and protecting it from AlgL degradation [17,55] and whose inactivation impairs alginate production [26]. When compared to that of the wild strain, PHB accumulation of the algK mutant, increased by 50 %, measured as milligrams per milligram of protein. The algK mutant also showed a 50 % higher yield of PHB per sucrose consumed. However, the possibility that this strain might still have the potential to drain carbon for the synthesis of alginate precursors was raised. Evidence supporting this theory is derived from the description of a P. aeruginosa algK mutant| which is also unable to produce alginate and has been shown to secrete uronic acids [56]. These acids seem to be the products of alginate degradation by AlgL [17].
A more recent work [46] studied the effect of a blockade on the first enzymatic step of the alginate biosynthetic pathway, the phosphomannose isomerase (Figure 1), on PHB production. This enzyme, together with the guanosine diphospho-D-mannose pyrophosphorylase (third step of this pathway) is encoded by the algA gene as a bifunctional enzyme. The algA mutation (strain AT41, Figure 3) impaired alginate production and increased PHB accumulation (in grams of PHB per gram of protein) between 75 % and 500 %, depending on the medium used, with a 61 % higher yield (gram of PHB per gram of glucose consumed). It is interesting to note that the algA mutation not only increased the capacity of the bacterium to produce more PHB per biomass unit, but also permitted better growth of the mutant, influencing the volumetric production of PHB and improving it up to 10-fold.
4.2 The lack of the anaplerotic enzyme pyruvate carboxylase promotes PHB accumulation
An article published recently [57] describes another interesting mutation found to positively affect PHB accumulation capacity. A strain accumulating 3.5 times more PHB (grams of PHB/gram of protein) than its parental strain, a non-alginate producing strain was identified, by mini Tn5 mutagenesis. The Tn5 insertion knocked out a gene (pycA) coding for a subunit of the pyruvate carboxylase. This enzyme catalyzes the ATP-dependent carboxylation of pyruvate, to generate oxaloacetate which replenishes the tricarboxylic acid cycle, during cell-material biosynthesis (Figure 1). The authors hypothesized that the knocking out of pycA results in a lower oxaloacetate pool, and as the flux of acetyl-CoA in the TCA cycle depends on this being condensated with oxaloacetate to produce citrate, the result is that the acetyl-CoA remains available for PHB synthesis, inducing a high PHB accumulation. A low carbon flux throughout the TCA cycle would not only favor PHB accumulation due to a higher acetyl-CoA availability, but would also diminish the CoASH pool, favoring β-ketothiolase activity.
4.3 A defect in the respiratory NADH oxidase improves PHB synthesis
Several years ago another mutation affecting central metabolism was reported as positively affecting the PHB accumulation capacity in A. vinelandii. Page and Knosp [58] reported the isolation of A. vinelandii UWD, a mutant which produces PHB during exponential growth, without requiring nutrient limitation. The reason for this phenotype was apparently a defect in the respiratory NADH oxidase, which increases NADH concentration, lowering the citrate synthase activity. The PHB production increased 2.6 to 3.4 fold, measured as a percentage of the dry weight basis, and the PHB yield (grams of PHB/gram of glucose consumed) increased 5 – 6.6 fold.
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