do not polymerize C4 monomers. However, PhaC from Pseudomonas sp. 61-3 was
shown to be able to polymerize scl-HA prior to the expression of respective genes in
R. eutropha PHA-negative mutants (Matsusaki et al. 1998, 2000; Tsuge et al. 2005).
Besides, PhaC from Bacillus megaterium is interestingly dissimilar to other PhaCs
despite composing two subunits of PhaC and PhaR (Tomizawa et al. 2011) such as
of class III synthase, and therefore, it has been classified into a new category, class
IV. At present PhaC class IV category encompasses only PhaCs from Bacillus
(Tsuge et al. 2015). Generally, with the supply of precursors, PhaCs from Bacillus
were able to polymerize copolymers. For instance, P(3HB-co-3HV) could be
biosynthesized using Bacillus with the supply of propionate and valerate precursors.
However, it has shown that some Bacillus sp. are able to polymerize P(3HB-co3HV) with up to 2 mol% 3HV fraction using glucose as a sole carbon source
(Mizuno et al. 2010). A similar situation was observed in another study, where it
was then known that P(3HB-co-3HV) with 2–12 mol% 3HV can be produced in
some Bacillus sp. using glucose alone (Masood et al. 2013).
Pursuing the current trends, PHA copolymer compositions can be altered using
mutated PHA synthase genes (Taguchi et al. 2003). Change in monomer composition in PHA copolymer using PHA synthase can be linked to the change in substrate
specificities toward target substrate monomers; thus, noticeable changes happen in
related metabolic pathways. Point mutagenesis of Ala510 in PhaC Re has changed the
polymerization activity of PhaC and hinted the magnificent influence of amino acid
at the position 510 in PHA biosynthesis (Tsuge et al. 2004a). In addition, PhaC from
Aeromonas caviae (PhaC Ac ) with two mutations, N149S/D171G (NSDG), has
expressed it in PHA-negative strain and obtains higher 3HHx fraction than of the
wild-type PhaC (Tsuge et al. 2007). Besides, the PhaC gene from Pseudomonas
putida KT2440 (phaC1 Pp ) was randomly mutated to achieve a high yield of poly
(3-hydroxydodecanoate), P(3HDD) (Hiroe et al. 2018). From the random mutant
library, PhaC1 Pp mutants with increased activity and specificity for the polymerization of 3HDD were selected for the further enzyme optimization to enhance the
production of P(3HDD). Therefore, it is evident that causing a change in PhaC using
molecular techniques such as point mutation or random mutation indeed affects the
overall biosynthesis results.
3.2.2 Potential Carbon Sources for PHA Biosynthesis
The high production cost of PHA, unlike PBP, is one of the major factors that hinder
it from getting commercialized. One of the plausible ways to bring down the
production cost of PHA is by using cheaper carbon sources. A good carbon source
is required for microbes to grow well before entering to PHA production phase prior
to stress induced through nutrient (nitrogen/phosphorus) limitation. A variety of
cheap structurally related and unrelated carbon sources were/are being evaluated to
be used in large-scale PHA production using wild-type or recombinant strains.
Typically, using wild-type PHA producers, structurally unrelated carbon sources
such as glucose are used to produce P(3HB) homopolymer, whereas structurally
3 Development of Polyhydroxyalkanoate (PHA) and Its Copolymers as a Possible. . .
63
shown to be able to polymerize scl-HA prior to the expression of respective genes in
R. eutropha PHA-negative mutants (Matsusaki et al. 1998, 2000; Tsuge et al. 2005).
Besides, PhaC from Bacillus megaterium is interestingly dissimilar to other PhaCs
despite composing two subunits of PhaC and PhaR (Tomizawa et al. 2011) such as
of class III synthase, and therefore, it has been classified into a new category, class
IV. At present PhaC class IV category encompasses only PhaCs from Bacillus
(Tsuge et al. 2015). Generally, with the supply of precursors, PhaCs from Bacillus
were able to polymerize copolymers. For instance, P(3HB-co-3HV) could be
biosynthesized using Bacillus with the supply of propionate and valerate precursors.
However, it has shown that some Bacillus sp. are able to polymerize P(3HB-co3HV) with up to 2 mol% 3HV fraction using glucose as a sole carbon source
(Mizuno et al. 2010). A similar situation was observed in another study, where it
was then known that P(3HB-co-3HV) with 2–12 mol% 3HV can be produced in
some Bacillus sp. using glucose alone (Masood et al. 2013).
Pursuing the current trends, PHA copolymer compositions can be altered using
mutated PHA synthase genes (Taguchi et al. 2003). Change in monomer composition in PHA copolymer using PHA synthase can be linked to the change in substrate
specificities toward target substrate monomers; thus, noticeable changes happen in
related metabolic pathways. Point mutagenesis of Ala510 in PhaC Re has changed the
polymerization activity of PhaC and hinted the magnificent influence of amino acid
at the position 510 in PHA biosynthesis (Tsuge et al. 2004a). In addition, PhaC from
Aeromonas caviae (PhaC Ac ) with two mutations, N149S/D171G (NSDG), has
expressed it in PHA-negative strain and obtains higher 3HHx fraction than of the
wild-type PhaC (Tsuge et al. 2007). Besides, the PhaC gene from Pseudomonas
putida KT2440 (phaC1 Pp ) was randomly mutated to achieve a high yield of poly
(3-hydroxydodecanoate), P(3HDD) (Hiroe et al. 2018). From the random mutant
library, PhaC1 Pp mutants with increased activity and specificity for the polymerization of 3HDD were selected for the further enzyme optimization to enhance the
production of P(3HDD). Therefore, it is evident that causing a change in PhaC using
molecular techniques such as point mutation or random mutation indeed affects the
overall biosynthesis results.
3.2.2 Potential Carbon Sources for PHA Biosynthesis
The high production cost of PHA, unlike PBP, is one of the major factors that hinder
it from getting commercialized. One of the plausible ways to bring down the
production cost of PHA is by using cheaper carbon sources. A good carbon source
is required for microbes to grow well before entering to PHA production phase prior
to stress induced through nutrient (nitrogen/phosphorus) limitation. A variety of
cheap structurally related and unrelated carbon sources were/are being evaluated to
be used in large-scale PHA production using wild-type or recombinant strains.
Typically, using wild-type PHA producers, structurally unrelated carbon sources
such as glucose are used to produce P(3HB) homopolymer, whereas structurally
3 Development of Polyhydroxyalkanoate (PHA) and Its Copolymers as a Possible. . .
63