4 Metabolic Engineering in Acetogens
Metabolic engineering offers unique opportunities to eliminate unwanted
side-products, synthesize novel, high-valuable compounds and increase the yield of
natural products. A prerequisite for metabolic engineering is the knowledge of the
biosynthetic pathway and its bioenergetics. The biochemistry and the bioenergetics
have extensively been studied in the past for acetogenic bacteria such as A. woodii
[4, 33], C. ljungdahlii [34] and C. autothenagoenum [19, 83]. The availability of
genome sequences has further developed the use of acetogenic bacteria as a production platform. However, the use of acetogens as biocatalysts for biotechnological applications requires strategies for genetic manipulation. The major
challenge was to establish a genetic toolbox for acetogenic bacteria. Problems that
had to be overcome in establishing a system for genetic manipulation in acetogenic
bacteria included a strong restriction-modification system, efficient degradation of
external DNA by nucleases and the thick outer layer of these mostly Gram-positive
bacteria [99, 100]. The development of strategies to overcome these problems has
drastically increased over the past decade which led to an advanced genetic toolbox
for acetogens. So far, genetic systems have been reported for A. woodii [62, 101],
C. aceticum [102], C. autoethanogenum, C. ljungdahlii [103], E. limosum [104] and
T. kivui [105]. These acetogenic bacteria have been genetically modified to produce
highly valuable chemicals such as acetone, butanol, butyrate, 2,3-BD and
poly-3-hydroxybutyrate (PHB).
Strategies to enhance acetone production on a genetic level by using engineered
acetogens have come into focus recently. Acetone is mainly used as an industrial
solvent and serves as precursor for the generation of plastics. With a global market
of estimated 4.04 billion USD in 2019 and more than 6 million tons produced, the
interest of acetone is immense. These strategies are based of the implementation
from the genes of the acetone pathway in C. acetobutylicum (thlA, ctfA, ctfB, and
adc) [102, 106, 107]. In the natural acetone pathway of C. acetobutylicum, two
molecules of acetyl-coA are converted to acetoacetyl-CoA by a thiolase A (thlA).
Next, the CoA moiety from acetoacetyl-CoA is then transferred to acetate and
acetoacetate as well as acetyl-CoA is formed. In a final step, the formed acetoacetate is converted into acetone and CO 2 . This reaction is catalyzed by an acetoacetate decarboxylase (Adc) (Fig. 4). The successful expression of these genes in
a plasmid-based approach was first shown in 2012 with a recombinant C. aceticum
strain. In a closed batch experiment with H 2 + CO 2 as a sole carbon and energy
source, acetone was produced up to 8 mg/l by the recombinant strain. In 2014, the
successfully expression of the genes involved in the acetone pathway from
C. acetobutylicum under control of a lactose-inducible promoter could also be
shown for C. ljungdahlii in closed batched experiments using CO as substrate
[106]. However, it was shown that C. ljungdahlii is converting the produced acetone to isopropanol by a NADPH-dependent primary-secondary alcohol dehydrogenase [87]. To solve this problem, the respective gene was deleted, and acetone
production was further enhanced by exchanging the thiolase gene thlA from
122
D. Litty and V. Müller
Metabolic engineering offers unique opportunities to eliminate unwanted
side-products, synthesize novel, high-valuable compounds and increase the yield of
natural products. A prerequisite for metabolic engineering is the knowledge of the
biosynthetic pathway and its bioenergetics. The biochemistry and the bioenergetics
have extensively been studied in the past for acetogenic bacteria such as A. woodii
[4, 33], C. ljungdahlii [34] and C. autothenagoenum [19, 83]. The availability of
genome sequences has further developed the use of acetogenic bacteria as a production platform. However, the use of acetogens as biocatalysts for biotechnological applications requires strategies for genetic manipulation. The major
challenge was to establish a genetic toolbox for acetogenic bacteria. Problems that
had to be overcome in establishing a system for genetic manipulation in acetogenic
bacteria included a strong restriction-modification system, efficient degradation of
external DNA by nucleases and the thick outer layer of these mostly Gram-positive
bacteria [99, 100]. The development of strategies to overcome these problems has
drastically increased over the past decade which led to an advanced genetic toolbox
for acetogens. So far, genetic systems have been reported for A. woodii [62, 101],
C. aceticum [102], C. autoethanogenum, C. ljungdahlii [103], E. limosum [104] and
T. kivui [105]. These acetogenic bacteria have been genetically modified to produce
highly valuable chemicals such as acetone, butanol, butyrate, 2,3-BD and
poly-3-hydroxybutyrate (PHB).
Strategies to enhance acetone production on a genetic level by using engineered
acetogens have come into focus recently. Acetone is mainly used as an industrial
solvent and serves as precursor for the generation of plastics. With a global market
of estimated 4.04 billion USD in 2019 and more than 6 million tons produced, the
interest of acetone is immense. These strategies are based of the implementation
from the genes of the acetone pathway in C. acetobutylicum (thlA, ctfA, ctfB, and
adc) [102, 106, 107]. In the natural acetone pathway of C. acetobutylicum, two
molecules of acetyl-coA are converted to acetoacetyl-CoA by a thiolase A (thlA).
Next, the CoA moiety from acetoacetyl-CoA is then transferred to acetate and
acetoacetate as well as acetyl-CoA is formed. In a final step, the formed acetoacetate is converted into acetone and CO 2 . This reaction is catalyzed by an acetoacetate decarboxylase (Adc) (Fig. 4). The successful expression of these genes in
a plasmid-based approach was first shown in 2012 with a recombinant C. aceticum
strain. In a closed batch experiment with H 2 + CO 2 as a sole carbon and energy
source, acetone was produced up to 8 mg/l by the recombinant strain. In 2014, the
successfully expression of the genes involved in the acetone pathway from
C. acetobutylicum under control of a lactose-inducible promoter could also be
shown for C. ljungdahlii in closed batched experiments using CO as substrate
[106]. However, it was shown that C. ljungdahlii is converting the produced acetone to isopropanol by a NADPH-dependent primary-secondary alcohol dehydrogenase [87]. To solve this problem, the respective gene was deleted, and acetone
production was further enhanced by exchanging the thiolase gene thlA from
122
D. Litty and V. Müller