_Applied_  
_sciences_  
_Article_  
**Synthesis and Physical Properties of**  
**Polyhydroxyalkanoate Polymers with Different**  
**Monomer Compositions by Recombinant**  
_Pseudomonas putida_ **LS46 Expressing a Novel PHA**  
**SYNTHASE (PaaC16) Enzyme**  
**[PERSON] 1, [PERSON] 1, [PERSON] 1, [PERSON] 1, [PERSON] 2, [PERSON] 2 and [PERSON] 1,* 1**  
Footnote 11: foot  
PHA polymers are thermoplastic and differences in their physical and thermal properties are a function of their subunit composition. PHA polymers may be homopolymers containing only one type of 3-hydroxy fatty acid monomer, they may be copolymers containing two types of 3-hydroxy fatty acid monomers, or they may be heteropolymers containing 3-hydroxy fatty acids of several different chain lengths. Homopolymers, random copolymers, and block copolymers of PHA may be produced depending on the bacterial species and growth conditions [4]. Poly-(3-hydroxybutyrate), PHB, is a homopolymer containing four carbon subunits of 3-hydroxybutyrate (3 HB), and the most widespread and best characterized member of the polyhydroxyalkanoate family [5; 6]. PHB is highly crystalline (>50%), and consequently is relatively brittle and stiff, with a low elongation to break ratio. For these reasons, efforts in compounding PHB are mainly focused on the search of plasticizers and nucleating agents capable of reducing the crystallization process and improving flexibility. Copolymers containing a mixture of four and five carbon chain length subunits may be produced by culturing bacteria that synthesize these polymers with valeric acid, which results in the formation of PHAs containing 3-hydroxyvalerate (3 HV) or 4-hydroxybutyrate (4 HB) monomers. The incorporation of HV into PHB polymers results in a poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3 HB/3 HV)], which is less stiff and brittle than pure P(3 HB) [7]. Medium chain length PHAs are flexible and elastic, having low crystallinity with low tensile strength and high elongation-to-break ratios. In comparison to SCL-PHA, MCL-PHA polymers have low melting temperatures, low glass transition temperatures, and higher elongation-to-break ratios [8]. Copolymers of PHB and PHV with different mol % of 3 HV (up to 71 mol % 3 HV) were produced by _Alcaligenes eutropha_ using different substrates [9; 10]. Nodax(tm) class PHA copolymers produced by Meridian (Bainbridge, GA, USA) are also copolymers of 3 HB and 3-hydroxyhexanoate (3 HHx) fatty acid acids in varying proportions [11]. Recombinants of _P. putida_ GPp104 carrying PHA synthase of _Aeronomas hydrophulia_ could add only 14 mol % 3 HHx into copolymer [12]. The combination of 3-hydroxyhexanoate (3 HHx) and 3-hydroxyocatanoate (3 HO) subunits with 3 HB subunits imparts significant changes in the thermal and physical properties of these copolymers. _Pseudomonas putida_ LS46 synthesizes MCL-PHAs consisting of 3 HHx, 3 HO, 3 HD), and/or 3-hydroxydodecanoate (3 HDD) monomers in different proportions [10]. The _P. putida_ LS46 genome was sequenced and regulatory genes and proteins associated with PHA synthesis were identified using transcriptomics and proteomic analyses [13; 14; 15; 16]. The Type II PHA synthase enzyme (PhaC1, encoded by _phaC1_) expressed by _P. putida_ is unable to add 3-hydroxybutyrate (3 HB) monomers to MCL-PHA polymers. Substrate specificity of PHA synthase is responsible for monomer composition. Few PHA synthases with wide substrate specificity have been identified in _Aeronomas caviae_ and _Pseudomonas stutzeri_ and recombinant expressing these PHA synthases in PHA negative mutant of _Ralstonia eutropha_, produced copolymer of SCL- and MCL-PHA copolymer [17]. Combinatorial mutations in _P. putida_, _P. oleovorans_, and _P. aeruginosa_ with the ability to synthesize PHAs with 3 HB, 3 HHx, 3 HO, 3 HD, or 3 HDD monomers have been described [18]. Two natural isolates of _Pseudomonas_ sp. 61-3 and _Pseudomonas_ sp. MBEL 6-19 were able to produce copolymers of 3 HB with HHx, HO, and HD [19; 20; 21].