94
Utilization of such pH-resistant fungi can reduce the cost of alkalization required to
back titrate the produced acids and minimize the waste formation. Guevarra and
Tabuchi first described the production of IA and 2-hydroxyparaconic in unbuffered
media from glucose by U. cynodontis [37]. In this study, the growth of cells was
investigated at different temperatures. Low temperature (25 °C) was found to be the
best for cell growth and resulted in the highest organic acid titers. In 2014, Geiser
et al. screened 68 strains of the Ustilaginaceae family for the synthesis of carboxylic acids, polyols, and glycolipids from glucose [36]. Among the tested fungi,
U. cynodontis was identified as an efficient producer of IA with 3.3 g L
−1
titer under
relatively low pH. In a most recent pioneer work, Hosseinpour et al. applied
morphological and metabolic engineering to generate an efficient pH-tolerant host
to produce IA under biotechnologically relevant environments [38]. The yeast-like
morphology of U. cynodontis was preserved by disturbing the signal transduction
route and by deletions of ras2, fuz7, and ubc3 genes under stress-inducing conditions. This metabolically engineered strain produced 21 g L
−1
IA in a fed-batch
fermentation process with pulse feeding at pH 3.8.
Aspergillus niger Aspergillus niger (A. niger) was identified as an emerging alternative for the synthesis of IA. A. niger is presently employed to synthesize citric
acid on an industrial scale [39]. The biotechnological route to produce citric acid is
similar to IA. It is beneficial because the current fermentation setup of citric acid
can be employed to produce IA on a commercial scale from this fungus. A. niger
cannot produce IA naturally because it lacks the essential enzyme cis-aconitate
decarboxylase (cadA) [40, 41]. Sole expression of the enzyme is not adequate
because such genetically modified strain produces very low concentration of IA
(0.05 g L
−1
). Li et al. demonstrated that 2.5 g L
−1
IA could be achieved by overexpression of a mitochondrial carrier or a plasma membrane carrier [42]. In 2013,
Maassen et al. showed that co-overexpression of cadA (encoding a cis-aconitate
decarboxylase) and acoA (encoding a aconitase decarboxylase) genes in the cytosol
and mitochondria doubles the productivity of IA from glucose [43]. Interestingly,
expression of acoA, mttA (encoding a putative mitochondrial transporter), and mfsA
genes (encoding a plasma membrane transporter) in the A. niger strain led to 24
times higher IA yield (4 g g
−1
) compared to CadA [39].
In 2016, Hossain et al. genetically modified wild A. niger by incorporating a
cytosolic citrate synthase (citB) gene. The resulting strains produced about
26.2 g L
−1
of IA with a production rate of 0.35 g L
−1
h
−1
in batch cultivations without
yielding any by-products [44]. They proposed that by-product formation was suppressed by the overexpression of citrate synthase that also improved the biosynthesis pathway toward IA. For improved yield of IA, the same research group recently
expanded the cytosolic acetyl-CoA pool by introducing acl1 and acl2 genes that
encode together for ATP-citrate lyase (ACL). Using fused A. niger cells, very high
IA titer volume, up to 42.7 g L
−1
, from glucose was reported [45]. They found that
metabolic engineering of ACL increases glycolytic flux that leads to an enhanced IA
K. Avasthi et al.
Utilization of such pH-resistant fungi can reduce the cost of alkalization required to
back titrate the produced acids and minimize the waste formation. Guevarra and
Tabuchi first described the production of IA and 2-hydroxyparaconic in unbuffered
media from glucose by U. cynodontis [37]. In this study, the growth of cells was
investigated at different temperatures. Low temperature (25 °C) was found to be the
best for cell growth and resulted in the highest organic acid titers. In 2014, Geiser
et al. screened 68 strains of the Ustilaginaceae family for the synthesis of carboxylic acids, polyols, and glycolipids from glucose [36]. Among the tested fungi,
U. cynodontis was identified as an efficient producer of IA with 3.3 g L
−1
titer under
relatively low pH. In a most recent pioneer work, Hosseinpour et al. applied
morphological and metabolic engineering to generate an efficient pH-tolerant host
to produce IA under biotechnologically relevant environments [38]. The yeast-like
morphology of U. cynodontis was preserved by disturbing the signal transduction
route and by deletions of ras2, fuz7, and ubc3 genes under stress-inducing conditions. This metabolically engineered strain produced 21 g L
−1
IA in a fed-batch
fermentation process with pulse feeding at pH 3.8.
Aspergillus niger Aspergillus niger (A. niger) was identified as an emerging alternative for the synthesis of IA. A. niger is presently employed to synthesize citric
acid on an industrial scale [39]. The biotechnological route to produce citric acid is
similar to IA. It is beneficial because the current fermentation setup of citric acid
can be employed to produce IA on a commercial scale from this fungus. A. niger
cannot produce IA naturally because it lacks the essential enzyme cis-aconitate
decarboxylase (cadA) [40, 41]. Sole expression of the enzyme is not adequate
because such genetically modified strain produces very low concentration of IA
(0.05 g L
−1
). Li et al. demonstrated that 2.5 g L
−1
IA could be achieved by overexpression of a mitochondrial carrier or a plasma membrane carrier [42]. In 2013,
Maassen et al. showed that co-overexpression of cadA (encoding a cis-aconitate
decarboxylase) and acoA (encoding a aconitase decarboxylase) genes in the cytosol
and mitochondria doubles the productivity of IA from glucose [43]. Interestingly,
expression of acoA, mttA (encoding a putative mitochondrial transporter), and mfsA
genes (encoding a plasma membrane transporter) in the A. niger strain led to 24
times higher IA yield (4 g g
−1
) compared to CadA [39].
In 2016, Hossain et al. genetically modified wild A. niger by incorporating a
cytosolic citrate synthase (citB) gene. The resulting strains produced about
26.2 g L
−1
of IA with a production rate of 0.35 g L
−1
h
−1
in batch cultivations without
yielding any by-products [44]. They proposed that by-product formation was suppressed by the overexpression of citrate synthase that also improved the biosynthesis pathway toward IA. For improved yield of IA, the same research group recently
expanded the cytosolic acetyl-CoA pool by introducing acl1 and acl2 genes that
encode together for ATP-citrate lyase (ACL). Using fused A. niger cells, very high
IA titer volume, up to 42.7 g L
−1
, from glucose was reported [45]. They found that
metabolic engineering of ACL increases glycolytic flux that leads to an enhanced IA
K. Avasthi et al.