27
(PLA) whose market is anticipated to reach USD 2091.29 million by 2023, at an
estimated annual growth rate of 20.06% over the forecast period. Its degradability
when exposed to the environment drives the demand for PLA in the environmentally sustainable and advanced packaging segment.
The microbial production of lactate from sugar-based feedstocks has been extensively studied, but it presents the main drawback of subtracting abundant carbon
sources from the global food supplies. Hence, different works started to study processes where the sustainable lactic acid synthesis relies on CO 2 . To this end, in
Niederholtmeyer et al. (2010), Synechococcus elongatus PCC 7942 was engineered
by the expression of the lactate dehydrogenase (encoded by ldhA) and the lactate
transporter (encoded by ldP) from E. coli. Lactate production was further optimized
through the expression of the soluble NADPH/NADH transhydrogenase (encoded
by udhA) from E. coli to balance the lactate dehydrogenase cofactor availability
(NADH). Upon homologous recombination of these genes put under the control of
the IPTG-inducible promoter, the engineered cyanobacterium accumulated an
extracellular lactate concentration of 0.6 mM after 4 days of cultivation.
In Angermayr, Paszota, and Hellingwerf (2012), the lactate dehydrogenases ldhA
from E. coli, ldh from Bacillus subtilis, and ldh from Lactococcus lactis were
expressed in the cyanobacterium Synechocystis sp. PCC 6803. The three genes were
cloned and the constructs placed in separate integration vectors to incorporate the
genes in the neutral docking site slr0168 of Synechocystis sp. PCC6803 genome
under the control of the Ptrc promoter. Among the three lactate dehydrogenases, the
Bacillus subtilis gene resulted in stable L-lactic acid production over prolonged
batch cultivation. Indeed, in 10 days the strain produced lactic acid at a concentration of 0.7 mM, with a maximal rate of 0.0058 mmol lactate/g dry weight/h. Since
increased cofactor availability was expected to stimulate lactic acid production, the
authors co-expressed a soluble transhydrogenase which resulted into increased
intracellular NADH concentration. The transhydrogenase-carrying Synechocystis
strain reached a final concentration of 3.2 mM lactic acid in 2 weeks.
In Angermayr and Hellingwerf (2013), the same authors acknowledged the criticality of addressing the controllability of gene expression in order to optimize the
formation of the product of interest by acting both at the transcriptional and at the
translational levels. In particular, in order to optimize the production of L-lactic acid
from Synechocystis, the exogenous expression of two lactate dehydrogenase
enzymes, from Lactococcus lactis sp. cremoris MG1363 and Bacillus subtilis sp.
168, was subjected to the control of constitutive promoters selected on the basis of
different strength and tested for correlation of the enzyme concentration with
L-lactic acid production rate. This study showed that the strain carrying the lactate
dehydrogenase driven by the strongest promoter resulted into the strongest
Synechocystis lactate producer. The Synechocystis mutant showed a production of
20 mM after 4 weeks under continuous white light irradiation. Gordon et al. (2016)
enhanced the production of L-lactate by engineering Synechococcus sp. PCC 7002
(i) to express an optimized Bacillus subtilis lactate dehydrogenase (LDH) and (ii) to
improve carbon flux to pyruvate. The optimized LDH showed a single amino acid
substitution (Richter et al. 2011) that switches the cofactor requirement of the
enzyme from NADH to NADPH during conversion of pyruvate to lactate. To
1 Use of Carbon Dioxide in Polymer Synthesis
(PLA) whose market is anticipated to reach USD 2091.29 million by 2023, at an
estimated annual growth rate of 20.06% over the forecast period. Its degradability
when exposed to the environment drives the demand for PLA in the environmentally sustainable and advanced packaging segment.
The microbial production of lactate from sugar-based feedstocks has been extensively studied, but it presents the main drawback of subtracting abundant carbon
sources from the global food supplies. Hence, different works started to study processes where the sustainable lactic acid synthesis relies on CO 2 . To this end, in
Niederholtmeyer et al. (2010), Synechococcus elongatus PCC 7942 was engineered
by the expression of the lactate dehydrogenase (encoded by ldhA) and the lactate
transporter (encoded by ldP) from E. coli. Lactate production was further optimized
through the expression of the soluble NADPH/NADH transhydrogenase (encoded
by udhA) from E. coli to balance the lactate dehydrogenase cofactor availability
(NADH). Upon homologous recombination of these genes put under the control of
the IPTG-inducible promoter, the engineered cyanobacterium accumulated an
extracellular lactate concentration of 0.6 mM after 4 days of cultivation.
In Angermayr, Paszota, and Hellingwerf (2012), the lactate dehydrogenases ldhA
from E. coli, ldh from Bacillus subtilis, and ldh from Lactococcus lactis were
expressed in the cyanobacterium Synechocystis sp. PCC 6803. The three genes were
cloned and the constructs placed in separate integration vectors to incorporate the
genes in the neutral docking site slr0168 of Synechocystis sp. PCC6803 genome
under the control of the Ptrc promoter. Among the three lactate dehydrogenases, the
Bacillus subtilis gene resulted in stable L-lactic acid production over prolonged
batch cultivation. Indeed, in 10 days the strain produced lactic acid at a concentration of 0.7 mM, with a maximal rate of 0.0058 mmol lactate/g dry weight/h. Since
increased cofactor availability was expected to stimulate lactic acid production, the
authors co-expressed a soluble transhydrogenase which resulted into increased
intracellular NADH concentration. The transhydrogenase-carrying Synechocystis
strain reached a final concentration of 3.2 mM lactic acid in 2 weeks.
In Angermayr and Hellingwerf (2013), the same authors acknowledged the criticality of addressing the controllability of gene expression in order to optimize the
formation of the product of interest by acting both at the transcriptional and at the
translational levels. In particular, in order to optimize the production of L-lactic acid
from Synechocystis, the exogenous expression of two lactate dehydrogenase
enzymes, from Lactococcus lactis sp. cremoris MG1363 and Bacillus subtilis sp.
168, was subjected to the control of constitutive promoters selected on the basis of
different strength and tested for correlation of the enzyme concentration with
L-lactic acid production rate. This study showed that the strain carrying the lactate
dehydrogenase driven by the strongest promoter resulted into the strongest
Synechocystis lactate producer. The Synechocystis mutant showed a production of
20 mM after 4 weeks under continuous white light irradiation. Gordon et al. (2016)
enhanced the production of L-lactate by engineering Synechococcus sp. PCC 7002
(i) to express an optimized Bacillus subtilis lactate dehydrogenase (LDH) and (ii) to
improve carbon flux to pyruvate. The optimized LDH showed a single amino acid
substitution (Richter et al. 2011) that switches the cofactor requirement of the
enzyme from NADH to NADPH during conversion of pyruvate to lactate. To
1 Use of Carbon Dioxide in Polymer Synthesis