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The production of PHA in Gram-positive organisms such as C. glutamicum and L. lactis

is relativity poor (22% and 6 %, CDW respectively) in comparison to their Gram- negative counterparts (Jo et al., 2006; Mifune et al., 2009). However, in a recent publication by Mifune et al. (2009), it was suggested PHB yield in L. lactis could be improved by looking at the organism‟s metabolic flux, in particular the pyruvate pool/flux in LDH deficient mutant strains of L. lactis NZ9000. To assess this hypothesis, two currently available mutant strains NZ9010 and NZ9020 were employed in this study. NZ9010 in a derivative of NZ9000 with a single ldh gene knockout, while NZ9020 is a derivative of NZ9010 with an additional lactate dehydrogenase gene (ldhB) knockout (Bongers et al., 2003; Hoefnagel et al., 2002). PHB production in the non- native host L. lactis was mediated by the transformation of plasmid pNZ-CAB, harbouring codon optimised genes (PHA operon) from C. necator (Figure 36).

Several parameters were assessed to characterise L. lactis mutant strains when growth under PHB accumulating growth condition over a 24 hour period, these include optical density, media pH, lactate and acetate. No differences were seen in the growth characteristics or media pH when L. lactis NZ strains (NZ9010, and NZ9020) grown in GM17 media harbouring plasmid pNZ-CAB or pNZ-8148 (Figure 43). This suggests PHB production does not impact on growth or pH. It was observed that both LDH deficient mutants NZ9010 and NZ9020 independent of plasmid expressed reached high cell densities in comparison to control NZ9000. Although, NZ9020 tends to have a much longer lag phase in comparison to both NZ9000 and NZ9010. This increase growth to high cell densities seen in mutant strains is most likely corresponding to the pH in the media, as lower pH seen in NZ9000 may be inhibitory to growth (Mifune et al., 2009). Change in the media pH is primarily due to the synthesis of lactate acid as a product of fermentation (Hoefnagel et al., 2002). pH levels measured in growth culture show LDH mutants NZ9010 and NZ9020 produce very little lactate in comparison to wildtype strain NZ9000 (Figure 43). Lactate analysis of culture medium confirms the LDH knockout phenotype for both NZ9010 and NZ9020, as insignificant levels of lactate (< 5mM) was produced in comparison to NZ9000 (34 -36 mM) in the 24 hour sample of the culture medium (Figure 44). The level of lactate produced correlates with what was observed with the change in pH in the culture medium (Figure 43).

After confirming the L. lactis LDH knockout phenotype in NZ9010 and NZ9020, a redistribution of the carbon flux from pyruvate which is normally directed to lactate production towards other products such as acetate or PHB biogenesis indirectly via increase in acetyl-CoA (Figure 5). Acetate analysis of the growth medium shows increase in the level of acetate produced in LDH deficient strains which is significantly higher than the control (NZ9000) (Figure 45), confirming what was expected and whats shown in literature (Hoefnagel et al., 2002). However, it is interesting to note the level of acetate in strains harbouring plasmid pNZ-CAB under PHB accumulating condition is slightly higher than its corresponding strain harbouring plasmid pNZ-8148 (negative control). The formation of PHB was expected to decrease the level of available acetyl- CoA for acetate formation due to the increased distribution of acetyl-CoA towards PHB biogenesis.

GC/MS analysis showed that under aerobic PHB accumulating growth conditions, positive control NZ9000 harbouring pNZ-CAB showed an average yield of 3.57 % PHB

per mg/dry weight, which is in similar to that previously published by Mifune et al.

(2009) for PHB accumulation under aerobic condition, with glucose as the carbon source and additive L-Arginine (Table 12). All negative controls were negative. However, very little to no PHB is present in NZ9010 and NZ9020 in comparison to NZ9000 harbouring plasmid pNZ-CAB. This result was unexpected and in addition, the higher level of acetate found in NZ9010 and NZ9020 harbouring plasmid pNZ-CAB

may suggest the LDH knockout phenotype may have a negative impact on PHB biogenesis. However, under anaerobic PHB accumulating growth conditions, showed a considerable increase in levels of PHB produced in LDH deficient mutants in comparison to cultivation under aerobic conditions. L. lactis is a facultative aerobe, meaning the main metabolism is through the anaerobic pathway and this could explain the increase in PHB in L. lactis mutants grown under anerobic condition. Under these conditions an increase in lactate formation in normal wildtype L. lactis is expected when compared to growth under aerobic conditions. Since L. lactis LDH mutants are unavailable to produce significant amounts of lactate, we would see this correlating to an increase in PHB yield due to the indirect increase in available acetyl-CoA under anaerobic growth conditions. This hypothesis could be tested by quantifiying levels of lactate and acetate produced during anaerobic fermentation with wildtype and LDH mutants and comparing results to cultivation under aerobic conditions.

Acetate formation is seen to be dominant over PHB biogenesis and results in increase acetyl-CoA being directed towards acetate formation and not PHB. This may suggests why NZ9020 has much higher production of PHB in comparison to NZ9010. This is illustrated in Figure 45, where the production of acetate in NZ9020 is much slower than NZ9010 and has allowed more time for acetyl-CoA to be directed towards PHB biogenesis.

PHB production using LDH deficient mutants NZ9010 and NZ9020 did not mediate significant increases in PHB in comparison to NZ9000. Other means of increasing PHB yield could include knocking out the acetate pathway in the LDH deficient strains, therefore increasing levels of available acetyl-CoA. Another possibility mentioned by Mifune et al. (2009), involves the modification to gene expression of the pha genes. For example, typical modifications include gene dosage e.g. by increasing plasmid-copy- numbers; looking at maintaining plasmid stability; transcriptional elements e.g.