## 4 Discussion  
_P. putida_ LS46 was isolated from wastewater and, like most other _P. putida_ strains, can produce only MCL-PHAs [10]. PHA synthase (PhaC1 and PhaC2) enzymes are highly conserved among different strains of _P. putida_[7]. Some _Pseudomonas_ species, like _Pseudomonas_ sp. 61, _P. mendocina, P. extremaustralis_, and _P. pseudocaligenes_ have been shown to synthesize PHB/PHA co-polymers [41; 42; 43; 44]. Recombinants of _P. putida, P. oleovorans, P. resinocororans, P. entomophila, P. mendocina, P. aeruginosa_, and _P. stutzeri_ that synthesize co-polymers of and MCL-PHAs utilizing low cost carbon sources have been reported in the literature [45; 46]. Even the carbon:nitrogen (C/N) molar ratio has been reported to affect the subunit composition of PHAs in _P. pseudocalisgene_ YS1, with higher C/N molar ratios resulting in greater incorporation of medium chain length monomers in the polymers [47]. A number of PhaC1 proteins of Class I PHA synthases have broad substrate specificity and could produce random co-polymers of 3 HB and 3 HHx. Number of recombinant bacteria that express the _phaC1_ gene of _Aeromonas cavaie_ have been shown to synthesize 3 HB-co-3 HHx polymers [48; 49; 50]. Mutagenesis of PhaC1 of _Pseudomonas_ sp. 61-3 of Glu130 to Asp130 (E130D), Ser325 to Cys325 or Thr325 and Gln481 to Met481, lys481 or Lys481 led to 20%-40% 3 HB in copolymer [51]. In PhaC1\({}_{16}\) only one change identical this to this (Ser325 Thr) mutagenesis was observed. This could be a possible reason for the higher incorporation of 3 HB in copolymer. Figure 11: Comparison of predicted 3D structures of proteins encoded by **(A)**_P. putida_ LS46 PhaC1\({}_{18}\) and **(B)**_PhaC1\({}_{16}\)_. 3D models were constructed with PhaC1 of _P. putida_ LS46 (EMR48251), PhaC1\({}_{16}\) (ALV86417.1) using online Phyre2. Red areas indicate amino acid at putative catalytic sites in the PhaC1 proteins which identical to template. The green area indicates amino acids at the catalytic site, which are different from the template (gastric lipase). The substrate specificity of PHA synthases has been modified by PhaC mutagenesis in _Ralstonia eutropha_ H16, _A. cariae, P. putida_ Gpo1, and _Pseudomonas_ sp. 61-3 [39; 52; 53; 54]. Comparison of the amino sequences of PhaC1\({}_{16}\) protein and other Class I and Class II PHA synthases revealed both low sequence identity and homology with the Class I PHA synthase of _R. eutropha_ H16 (38% identity, 54% homology) and with the Class II PHA synthase of _P. putida_ LS46 (46% identity, 61% homology). Directed evolution of Class II PHA synthase of _Pseudomonas_ sp. 61-3 identified Ser325, Ser447 and Gln481, which are involved in substrate specificity of PHA synthase [55; 56]. Site-directed mutagenesis of _phaC1_ of _P. putida_ KT2440, _P. chlororapalis, P. resinovorans_, and _P. aeuginosa_ at four residues (Glu130, Ser325, Ser477, and Gln481) had a similar effect on substrate specificity [52]. Multiple sequence alignment showed that the six conserved regions have 23 different amino acids that are naturally involved and associated with the substrate specificity. Random mutagenesis of PHA synthase of _P. putida_ led to altered substrate specificity; however, no individual mutation was identified for this change. The PHA synthase of LS46 (PHA\({}_{1\text{S46}}\)) and LS46123 (PHA\({}_{16}\)) had 15 amino acids different but it is difficult to identify the effect of individual amino acids on the substrate specificity of an enzyme. [PERSON] and [PERSON] [40] compared six conserved regions of the PHA synthase of _P. putida_ Gpo1 with mutated PHA synthases, which they showed were able to add a higher mol % of 3 HB to co-polymers. Further comparison of the amino acid sequences of the mutated PHA synthase from _P. putida_ Gpo1 with the PhaC1\({}_{16}\) revealed 18 amino acid substitutions across the six conserved regions. At least 12 amino acids in PhaC1\({}_{16}\) were different to the amino acids of the _P. putida_ LS46 PhaC1.