Genetic engineering was also shown to be crucial to increase the metabolic
capacity of S. oneidensis MR-1. The heterologous incorporation of metabolic
pathways allowed S. oneidensis MR-1 to use glucose, xylose or glycerol as the sole
carbon and energy source for electricity production in MFC [164–166]. Furthermore, the heterologous expression of proteorhodopsin, a light-dependent proton
pump, led Shewanella to consume lactate at an increased rate when it is illuminated
which was reflected by the increase in current generation when compared with
wild-type organism [167]. Recently, genetic manipulation of S. oneidensis MR-1
allowed the modification of this organism to use electrons from a cathode to drive
reduction of acetoin to 2,3-butanediol, demonstrating the capacity to genetically
engineer a microbial electrosynthesis pathway [168].
Another approach used to enhance the rate of EET in S. oneidensis MR-1 was
the increase of the intracellular electron pool, by engineering and driving the
metabolic flux toward the enhancement of intracellular NADH regeneration [169].
In this work three different modules (the de novo pathway, the salvage pathway and
the universal biosynthesis pathway) were over-expressed, and the capacity for
electricity production of mutated S. oneidensis MR-1 was evaluated. The increase
in electricity generation and Coulombic efficiency showed that an increase in the
NAD(H+) pool results in the transfer of more electrons from increased oxidation of
the electron donor to the EET pathway, enhancing intracellular electron flux and
EET rate [169].
Mediated electron transfer has been demonstrated to be one of the most
important mechanism for S. oneidensis MR-1 in performing EET. Promoting redox
shuttle biosynthesis in this organism also enhances EET efficiency in BES [163,
170]. The heterologous expression of the flavin biosynthesis pathway from Bacillus
subtilis enhanced EET rate of S. oneidensis MR-1, with an increase of 13,2 times of
the maximum power output when compared with wild-type strain [170]. Likewise,
the homologous expression of the flavin biosynthesis gene cluster ribA-E in S.
oneidensis MR-1 increased the maximum current density by approximate 110%
[163]. Furthermore, overexpression of the gene ydeH from E. coli in S. oneidensis
MR-1, responsible for the biosynthesis of c-di-GMP enhanced biofilm formation
and bioelectricity generation [171]. The high levels of intracellular c-di-GMP
promote the expression of adhesive matrix components, which facilitates bacterial
biofilm formation. The maximum power density obtained with the engineered strain
was *2.8 times higher than that achieved by the wild-type strain [171].
The recent work on the CRISPR/Cas9 approach to manipulate S. oneidensis
MR-1 enables the precise site-directed mutagenesis of the bacterial chromosome
[172]. This allows the modification of several different genes, and the introduction
of various types of mutations, including individual base changes and net gene
deletion in this model strain [172]. This approach will simplify the genetic
manipulation of this electroactive organism facilitating the implementation of
high-throughput genomic engineering technologies, contributing to the improvement of this type of organisms towards the practical implementation of BES.
232
B. M. Fonseca et al.
capacity of S. oneidensis MR-1. The heterologous incorporation of metabolic
pathways allowed S. oneidensis MR-1 to use glucose, xylose or glycerol as the sole
carbon and energy source for electricity production in MFC [164–166]. Furthermore, the heterologous expression of proteorhodopsin, a light-dependent proton
pump, led Shewanella to consume lactate at an increased rate when it is illuminated
which was reflected by the increase in current generation when compared with
wild-type organism [167]. Recently, genetic manipulation of S. oneidensis MR-1
allowed the modification of this organism to use electrons from a cathode to drive
reduction of acetoin to 2,3-butanediol, demonstrating the capacity to genetically
engineer a microbial electrosynthesis pathway [168].
Another approach used to enhance the rate of EET in S. oneidensis MR-1 was
the increase of the intracellular electron pool, by engineering and driving the
metabolic flux toward the enhancement of intracellular NADH regeneration [169].
In this work three different modules (the de novo pathway, the salvage pathway and
the universal biosynthesis pathway) were over-expressed, and the capacity for
electricity production of mutated S. oneidensis MR-1 was evaluated. The increase
in electricity generation and Coulombic efficiency showed that an increase in the
NAD(H+) pool results in the transfer of more electrons from increased oxidation of
the electron donor to the EET pathway, enhancing intracellular electron flux and
EET rate [169].
Mediated electron transfer has been demonstrated to be one of the most
important mechanism for S. oneidensis MR-1 in performing EET. Promoting redox
shuttle biosynthesis in this organism also enhances EET efficiency in BES [163,
170]. The heterologous expression of the flavin biosynthesis pathway from Bacillus
subtilis enhanced EET rate of S. oneidensis MR-1, with an increase of 13,2 times of
the maximum power output when compared with wild-type strain [170]. Likewise,
the homologous expression of the flavin biosynthesis gene cluster ribA-E in S.
oneidensis MR-1 increased the maximum current density by approximate 110%
[163]. Furthermore, overexpression of the gene ydeH from E. coli in S. oneidensis
MR-1, responsible for the biosynthesis of c-di-GMP enhanced biofilm formation
and bioelectricity generation [171]. The high levels of intracellular c-di-GMP
promote the expression of adhesive matrix components, which facilitates bacterial
biofilm formation. The maximum power density obtained with the engineered strain
was *2.8 times higher than that achieved by the wild-type strain [171].
The recent work on the CRISPR/Cas9 approach to manipulate S. oneidensis
MR-1 enables the precise site-directed mutagenesis of the bacterial chromosome
[172]. This allows the modification of several different genes, and the introduction
of various types of mutations, including individual base changes and net gene
deletion in this model strain [172]. This approach will simplify the genetic
manipulation of this electroactive organism facilitating the implementation of
high-throughput genomic engineering technologies, contributing to the improvement of this type of organisms towards the practical implementation of BES.
232
B. M. Fonseca et al.