## 3 Results
### Comparison between PhaC1 Proteins of P. putida LS46 and P. putida LS46123  
The _phaC1\({}_{16}\)_ gene was isolated from a metagenomic library prepared from soil and cloned [26] and subsequently expressed in _P. putida_ LS46123. Analyses of the nucleotide and amino acidsequences of _phaC1\({}_{16}\)_ revealed that it was closely related to the _phaC1_ gene of _Pseudomonas_ sp. F15. PhaC1\({}_{16}\) protein had a 48% amino acid sequence identity to PhaC1 protein of _Pseudomonas_ sp. TTU2014-080 ASC, and an 84% sequence identity to a PhaC1 protein from an uncultured clone 50 (sequence ID ALV86626). _P. putida_ LS46 PhaC1 protein had an amino acid sequence identity of 46% with the PhaC1\({}_{16}\) protein (EMR48251). Other PhaC1 proteins from _P. mendocina, P. alcaligenes, P. stutzeri, P. aeruginosa,_ and _Pseudomonas_ sp. 61-3 had 45% to 47% similarity to the PhaC1\({}_{16}\) protein. These Pseudomonas are also known to produce SCL- and MCL-PHAs.  
The amino acid sequences of the _P. putida_ LS46 PhaC1\({}_{8}\) protein were aligned with those of the _P. putida_ KT2440 PhaC1\({}_{\rm pp}\), _P. mendocina_ symp, _P. olevovorans_ PhaC1\({}_{\rm po}\), and PhaC1\({}_{16}\) proteins (Figure 10). The amino acid position numbers in Table 4 are derived from the alignment of PhaC1\({}_{\rm po}\) with wild-type and mutant PhaC1 proteins from _P. putida_ Gpo1 that resulted in high incorporation of 3-hydroxybutyrate in co-polymers (Table 4) as described earlier [39], whereas the amino acid position numbers in Figure 10 are based on the alignment of the PhaC1 protein of _P. putida_ LS46 with PhaC1\({}_{16}\)[39]. The amino acid sequences of the four PhaC1 proteins showed three gaps when aligned with the Clone 16 PhaC1\({}_{16}\). Consequently, the amino acid positions indicated in Table 4 and Figure 10 do not correspond.  
Figure 8: Tan delta (G\({}^{\prime\prime}\)/G\({}^{\prime}\) ratio) of PHA polymers synthesized by _P. putida_ LS46123 grown on: C6, Hexanoic acid; C8, Octanoic acid; C9, Nonanoic acid; and FFA, biodiesel-derived free fatty acids (REG-FFA).  
Figure 9: Complex viscosity of PHA polymers synthesized by _P. putida_ LS46123 grown on: C6, Hexanoic acid; C8, Octanoic acid; C9, Nonanoic acid; and FFA, biodiesel-derived free fatty acids (REG-FFA).  
Six conserved regions have been earlier identified in Class I and Class II PhaC1 proteins and mutation in these regions affect substrate specificity of PHA synthase. The amino acid sequence of conserved regions of PhaC1\({}_{\text{ls}}\) and PhaC1\({}_{\text{16}}\) showed some similarities and differences (Table 4). All PhaC1 have a Lipase Box sequence required for PhaC activity (Figure 10). In the first conserved region of the _P. putida_ LS46 PhaC1 protein, two phenylalanine (F229 and F232) were replaced by tyrosine (Y229) and leucine (L232) in the PhaC1\({}_{\text{16}}\) (Table 4; Figure 10).  
Differences between the PhaC1\({}_{\text{16}}\) and the PhaC1\({}_{\text{po}}\) proteins were also detected in the F2, F3, F4, F5, and F6 conserved regions of the protein (Table 4). Four amino acids in F2, two in F4, three in F5, and four in F6 were different in PhaC1\({}_{\text{16}}\) compared with PhaC1\({}_{\text{ls}}\). Some of the changes in amino acid corresponded to changes in the mutated PhaC1\({}_{\text{po}}\), which have been shown to be responsible for the addition of greater mol % of 3 HB in polymers. Ser 325 is a critical amino acid in Class II PHA synthases that synthesize MCL-PHAs, and, with three other key amino acid residues--Glu130, Ser477, and Gln481--has been shown to be associated with specificity of the PHA synthase enzyme (PhaC1) in _P. putida_ KT2440. However, no differences in in key amino acids associated with PHA Synthase catalytic site (Cys296, Asp451, and His479) were observed between the endogenous PhaC1\({}_{\text{ls}}\) of _P. putida_ LS46 and PhaC1\({}_{\text{16}}\) expressed in _P. putida_ LS46123.