3.2.3.2 Genetically Engineered Pathways for PHA Biosynthesis
Genetic engineering has garnered research interest in PHA biosynthesis because it is
useful to biosynthesize PHA with targeted monomer composition and desired
properties and at a higher yield. Furthermore, genetic approaches also open a
possibility in the production of 3HB-based copolymers using various cheap and
renewable carbon substrates such as waste materials and carbon dioxide, ensuring
PHA production to be more economical for replacing PBP. Concerning PHA
biosynthesis, genetic or metabolic engineering can be used either to upgrade the
existing PHA biosynthetic pathway in a natural PHA producer to increase the flux
for PHA monomer synthesis or to modify a non-PHA producer by incorporating
essential genes for PHA production. A number of essential PHA synthetic genes
such as phaC, phaA, phaB, phaG, and phaJ from PHA producers could be cloned
into E. coli or other nonnatural PHA producers. For example, mcl-PHA
homopolymers are generally not naturally and widely occurring PHAs, unlike
scl-PHA. Therefore, gene modification is required to construct an artificial biosynthetic pathway in bacteria to biosynthesize near mcl homopolymers using renewable
resources. Two mcl-PHA homopolymers, poly(3-hydroxydecanoate) [P(3HD)] and
poly(3-hydroxydodecanoate) [P(3HDD)], have biosynthesized from metabolically
engineered E. coli LSBJ using pure fatty acids (Hiroe et al. 2016). The production of
this mcl-PHA was further enhanced to 1.47 g/L and 0.42 g/L, respectively, when
metal components in the culture medium were increased (Hiroe et al. 2016). PHAs
with controlled monomer composition can be produced by recombinant bacteria
encompassing a combination of the substrate specificities of PHA synthase and
monomer-supplying enzymes. The use of (R)-specific enoyl-CoA hydratase (PhaJ)
in PHA biosynthesis from fatty acid ß-oxidation cycle in recombinant bacteria has
been determined (Tsuge et al. 2003). The presence of either PhaJ1 Pa or PhaJ4 Pa in
recombinant E. coli led to the high levels of PHA accumulation of 36–41 wt.% in dry
cells consisting of mainly scl- and mcl-3HA units, respectively. In addition, phaJ1 Pa
and phaJ2 Pa , homologous to the A. caviae PhaJ (phaJ Ac ), were cloned in E. coli
DH5α strain to investigate the monomer-supplying ability for PHA synthesis from
β-oxidation cycle. The recombinants harboring phaJ1 Pa or phaJ2 Pa showed high (R)specific enoyl-CoA hydratase activity with different substrate specificities, that is,
specific for scl- and mcl-enoyl-CoAs, respectively. Meanwhile, co-expression of
these two hydratase genes with phaC in E. coli LS5218 resulted in the accumulation
of PHA up to 14–29 wt% of cell dry weight from dodecanoate as a sole carbon
source (Tsuge et al. 2000).
72
R. Sivashankari and T. Tsuge
Genetic engineering has garnered research interest in PHA biosynthesis because it is
useful to biosynthesize PHA with targeted monomer composition and desired
properties and at a higher yield. Furthermore, genetic approaches also open a
possibility in the production of 3HB-based copolymers using various cheap and
renewable carbon substrates such as waste materials and carbon dioxide, ensuring
PHA production to be more economical for replacing PBP. Concerning PHA
biosynthesis, genetic or metabolic engineering can be used either to upgrade the
existing PHA biosynthetic pathway in a natural PHA producer to increase the flux
for PHA monomer synthesis or to modify a non-PHA producer by incorporating
essential genes for PHA production. A number of essential PHA synthetic genes
such as phaC, phaA, phaB, phaG, and phaJ from PHA producers could be cloned
into E. coli or other nonnatural PHA producers. For example, mcl-PHA
homopolymers are generally not naturally and widely occurring PHAs, unlike
scl-PHA. Therefore, gene modification is required to construct an artificial biosynthetic pathway in bacteria to biosynthesize near mcl homopolymers using renewable
resources. Two mcl-PHA homopolymers, poly(3-hydroxydecanoate) [P(3HD)] and
poly(3-hydroxydodecanoate) [P(3HDD)], have biosynthesized from metabolically
engineered E. coli LSBJ using pure fatty acids (Hiroe et al. 2016). The production of
this mcl-PHA was further enhanced to 1.47 g/L and 0.42 g/L, respectively, when
metal components in the culture medium were increased (Hiroe et al. 2016). PHAs
with controlled monomer composition can be produced by recombinant bacteria
encompassing a combination of the substrate specificities of PHA synthase and
monomer-supplying enzymes. The use of (R)-specific enoyl-CoA hydratase (PhaJ)
in PHA biosynthesis from fatty acid ß-oxidation cycle in recombinant bacteria has
been determined (Tsuge et al. 2003). The presence of either PhaJ1 Pa or PhaJ4 Pa in
recombinant E. coli led to the high levels of PHA accumulation of 36–41 wt.% in dry
cells consisting of mainly scl- and mcl-3HA units, respectively. In addition, phaJ1 Pa
and phaJ2 Pa , homologous to the A. caviae PhaJ (phaJ Ac ), were cloned in E. coli
DH5α strain to investigate the monomer-supplying ability for PHA synthesis from
β-oxidation cycle. The recombinants harboring phaJ1 Pa or phaJ2 Pa showed high (R)specific enoyl-CoA hydratase activity with different substrate specificities, that is,
specific for scl- and mcl-enoyl-CoAs, respectively. Meanwhile, co-expression of
these two hydratase genes with phaC in E. coli LS5218 resulted in the accumulation
of PHA up to 14–29 wt% of cell dry weight from dodecanoate as a sole carbon
source (Tsuge et al. 2000).
72
R. Sivashankari and T. Tsuge