26
widely ranging from 25 to 100 kDa, acts as a temporary nitrogen reserve material.
Arginine is a limiting factor for the accumulation of cyanophycin (Maheswaran
et al. 2006). The amount of cyanophycin per dry cell weight (g CP per g CDW) can
vary from roughly 1% during their exponential growth phase to 18% during stationary phase (Trautmann et al. 2016). In the last decades, isolated cyanophycin has
acquired increasing interest for the synthesis of biodegradable equivalents to the
polyacrylates (Simon 1971; Trautmann et al. 2016). Different studies highlighted
that the titer of this polymer can be increased by growing cyanobacteria under stationary phase or under conditions of unbalanced growth such as sulfate or phosphate limitations or in the presence of the protein synthesis inhibitors such as
chloramphenicol. For instance, in Simon (1976), CP production in the cyanobacterium Anabaena cylindrica was enhanced from 0.53% to 7.2% per CDW by adding
chloramphenicol to the culture during the logarithmic phase. Conversely, Allen,
Hutchison, and Weathers (1980) increased the CP production from 1.8% per CDW
to 5.7% growing the cyanobacterium Aphanocapsa 6308 under phosphate limitation. More recently, due to the expansion of the available genetic toolbox, metabolic
engineering approaches have been undertaken for increasing the production of cyanophycin in different bacteria species. In Lütte et al. (2012), the Gram-negative
hydrogen-oxidizing bacterium Cupriavidus necator was engineered to express a
plasmid carrying the Synechocystis sp. PCC 6308 cyanophycin synthetase gene
(cphA) under the control of the strong promoter of the C. necator operon containing
the genes encoding most of the enzymes of the Calvin–Benson–Bassham cycle, by
which C. necator fixes CO 2 during its lithoautotrophic growth. The cells, grown in
a 10 L fermenter in an atmosphere consisting of H 2 , CO 2 , and O 2 (80%, 10%, and
10%, respectively), led to an accumulation of CP equal to 7% per cell dry weight.
Alternative engineering approaches leveraged on the role of the signal processor
P II in the control of cyanophycin synthesis. In response to the nitrogen status, P II was
found to regulate N-acetyl-l-glutamate kinase (NAGK), which catalyzes the initial
reaction of the cyclic arginine synthesis pathway. Indeed, the formation of a complex between non-phosphorylated P II and NAGK results in the NAGK activation.
More precisely, in the nonactivated state, NAGK activity provides sufficient arginine for the synthesis of proteins; in the P II -activated state, nitrogen in surplus can
be stored under the form of cyanophycin (Maheswaran et al. 2006). Watzer et al.
(2015) demonstrated that a single amino acid replacement in the P II protein could
increase the CP accumulation in the cyanobacterium up to 47.4% per CDW through
constitutive activation of NAGK under phosphate limitation. Additional studies
explored the performance of these mutants analyzing the specific phosphor quota
necessary to trigger cyanophycin accumulation (Trautmann et al. 2016).
1.5.2 Lactic Acid
Lactic acid exists in nature in two enantiomeric (L- and D-) forms and is utilized in
a variety of industries including cosmetics, chemicals, food and beverages, pharmaceutical, and medical. Lactic acid supplies the building blocks to polylactic acid
A. A. Azim et al.
widely ranging from 25 to 100 kDa, acts as a temporary nitrogen reserve material.
Arginine is a limiting factor for the accumulation of cyanophycin (Maheswaran
et al. 2006). The amount of cyanophycin per dry cell weight (g CP per g CDW) can
vary from roughly 1% during their exponential growth phase to 18% during stationary phase (Trautmann et al. 2016). In the last decades, isolated cyanophycin has
acquired increasing interest for the synthesis of biodegradable equivalents to the
polyacrylates (Simon 1971; Trautmann et al. 2016). Different studies highlighted
that the titer of this polymer can be increased by growing cyanobacteria under stationary phase or under conditions of unbalanced growth such as sulfate or phosphate limitations or in the presence of the protein synthesis inhibitors such as
chloramphenicol. For instance, in Simon (1976), CP production in the cyanobacterium Anabaena cylindrica was enhanced from 0.53% to 7.2% per CDW by adding
chloramphenicol to the culture during the logarithmic phase. Conversely, Allen,
Hutchison, and Weathers (1980) increased the CP production from 1.8% per CDW
to 5.7% growing the cyanobacterium Aphanocapsa 6308 under phosphate limitation. More recently, due to the expansion of the available genetic toolbox, metabolic
engineering approaches have been undertaken for increasing the production of cyanophycin in different bacteria species. In Lütte et al. (2012), the Gram-negative
hydrogen-oxidizing bacterium Cupriavidus necator was engineered to express a
plasmid carrying the Synechocystis sp. PCC 6308 cyanophycin synthetase gene
(cphA) under the control of the strong promoter of the C. necator operon containing
the genes encoding most of the enzymes of the Calvin–Benson–Bassham cycle, by
which C. necator fixes CO 2 during its lithoautotrophic growth. The cells, grown in
a 10 L fermenter in an atmosphere consisting of H 2 , CO 2 , and O 2 (80%, 10%, and
10%, respectively), led to an accumulation of CP equal to 7% per cell dry weight.
Alternative engineering approaches leveraged on the role of the signal processor
P II in the control of cyanophycin synthesis. In response to the nitrogen status, P II was
found to regulate N-acetyl-l-glutamate kinase (NAGK), which catalyzes the initial
reaction of the cyclic arginine synthesis pathway. Indeed, the formation of a complex between non-phosphorylated P II and NAGK results in the NAGK activation.
More precisely, in the nonactivated state, NAGK activity provides sufficient arginine for the synthesis of proteins; in the P II -activated state, nitrogen in surplus can
be stored under the form of cyanophycin (Maheswaran et al. 2006). Watzer et al.
(2015) demonstrated that a single amino acid replacement in the P II protein could
increase the CP accumulation in the cyanobacterium up to 47.4% per CDW through
constitutive activation of NAGK under phosphate limitation. Additional studies
explored the performance of these mutants analyzing the specific phosphor quota
necessary to trigger cyanophycin accumulation (Trautmann et al. 2016).
1.5.2 Lactic Acid
Lactic acid exists in nature in two enantiomeric (L- and D-) forms and is utilized in
a variety of industries including cosmetics, chemicals, food and beverages, pharmaceutical, and medical. Lactic acid supplies the building blocks to polylactic acid
A. A. Azim et al.