ondly, expression of the plant-derived amorphadiene synthase had to be optimized, and,
finally, cytochromes and dehydrogenases
involved in the oxidation of amorphadiene to
artemisinic acid had to be optimized. With
these modifications 25 g/L of the desired compound can be accumulated, which led to the
industrialization of the process (Kung et al.
2018). The time from initial inception of the
project to market entrance was about 14 years,
this included an increase of the amorphadiene
titer greater than ten million-fold from the first
proof-of-concept to industry-readiness (Hale
et al. 2007).
D. Opioids
Sixteen different opiate alkaloids are currently
approved by the US Food and Drug Administration (USFDA) for medical use. Currently,
the sole commercial source of opiate alkaloids
is opium poppy cultivation, which leads to
fluctuations in supply and quality of the
pharmaceutically important substances. The
core structure of opioids is pentacyclic with
multiple chiral centers, which makes chemical
synthesis on commercial scale impossible. This
prompted efforts to reconstitute the opiate
alkaloid production in microbial systems.
Expression of 21 enzymes in baker’s yeast
allowed for the first time the production of
thebaine from sugar in 2015 (Galanie et al.
2015). Thebaine is an opioid structure, from
which various pharmaceutically important opiates can be chemically or microbially synthesized. This work was the result of a decade of
work and provided the proof-of-principle that
heterologous microbial opiate production is
possible. This work is not only important
from an industrial point of view, but it is a
prime example for how such an applied aim
helps to solve groundbreaking questions
about biology itself. Many crucial, hitherto
unknown enzymes have been identified. The
key enzyme of the pathway catalyzes the epimerization of S-reticuline to R-reticuline. It was
identified in the course of such projects from
three groups at the same time. Only its identification allowed the proof-of-principle of opiate
production yeasts. However, the insights go
much deeper: the final step of thebaine synthesis was long time thought to occur spontaneously in nature. Recently, this hypothesis could
be falsified and an enzyme for this reaction
has been identified. The initially produced
amounts of thebaine were in a scale of mg/L,
which differs about six orders of magnitude
from the commercially useful concentration of
5 g/L (Galanie et al. 2015). Expression of this
enzyme catalyzing the last step of thebaine synthesis together with the remaining pathway
dramatically increases thebaine production to
about 700 mg/L (Chen et al. 2018).
The construction of the first proof-of-concept strain is quite interesting also from a
synthetic biology point of view: the pathway
has been split into seven functional modules
(Ho ¨hne and Kabisch 2016). One module
ensures sufficient supply of the primary metabolites, four modules comprise the main pathway of the opiate production—split at
branching points in the biosynthetic pathway.
One module encodes the enzymes for tetrahydrobiopeterin production—a required enzymatic co-factor, which is naturally not present
in yeast, and finally, one module comprises
enzymes catalyzing rate-limiting steps, which
have been identified by metabolic flux analyses.
Starting from these modules other related alkaloids are accessible. Li et al., for example, report
the successful production of noscapine—an
antitussive, with anticancer properties, in
baker’s yeast. Expression of 31 enzymes was
required to obtain this substance, of which 25
are heterologous from plants, mammals, and
bacteria (Li et al. 2018).
In the meantime many other alkaloids have
been successfully synthesized in yeasts. Interestingly, there are only very few examples of
other production hosts than baker’s yeast. For
the assembly of very complicated heterologous
pathways, this traditional workhorse seems still
to be the preferred choice. It can be noted
also that most of the endeavors are at a proofof-principle level. It remains to be seen which of
the substances ever reach the markets as microbial products.
13 Yeast Cell Factories
327
finally, cytochromes and dehydrogenases
involved in the oxidation of amorphadiene to
artemisinic acid had to be optimized. With
these modifications 25 g/L of the desired compound can be accumulated, which led to the
industrialization of the process (Kung et al.
2018). The time from initial inception of the
project to market entrance was about 14 years,
this included an increase of the amorphadiene
titer greater than ten million-fold from the first
proof-of-concept to industry-readiness (Hale
et al. 2007).
D. Opioids
Sixteen different opiate alkaloids are currently
approved by the US Food and Drug Administration (USFDA) for medical use. Currently,
the sole commercial source of opiate alkaloids
is opium poppy cultivation, which leads to
fluctuations in supply and quality of the
pharmaceutically important substances. The
core structure of opioids is pentacyclic with
multiple chiral centers, which makes chemical
synthesis on commercial scale impossible. This
prompted efforts to reconstitute the opiate
alkaloid production in microbial systems.
Expression of 21 enzymes in baker’s yeast
allowed for the first time the production of
thebaine from sugar in 2015 (Galanie et al.
2015). Thebaine is an opioid structure, from
which various pharmaceutically important opiates can be chemically or microbially synthesized. This work was the result of a decade of
work and provided the proof-of-principle that
heterologous microbial opiate production is
possible. This work is not only important
from an industrial point of view, but it is a
prime example for how such an applied aim
helps to solve groundbreaking questions
about biology itself. Many crucial, hitherto
unknown enzymes have been identified. The
key enzyme of the pathway catalyzes the epimerization of S-reticuline to R-reticuline. It was
identified in the course of such projects from
three groups at the same time. Only its identification allowed the proof-of-principle of opiate
production yeasts. However, the insights go
much deeper: the final step of thebaine synthesis was long time thought to occur spontaneously in nature. Recently, this hypothesis could
be falsified and an enzyme for this reaction
has been identified. The initially produced
amounts of thebaine were in a scale of mg/L,
which differs about six orders of magnitude
from the commercially useful concentration of
5 g/L (Galanie et al. 2015). Expression of this
enzyme catalyzing the last step of thebaine synthesis together with the remaining pathway
dramatically increases thebaine production to
about 700 mg/L (Chen et al. 2018).
The construction of the first proof-of-concept strain is quite interesting also from a
synthetic biology point of view: the pathway
has been split into seven functional modules
(Ho ¨hne and Kabisch 2016). One module
ensures sufficient supply of the primary metabolites, four modules comprise the main pathway of the opiate production—split at
branching points in the biosynthetic pathway.
One module encodes the enzymes for tetrahydrobiopeterin production—a required enzymatic co-factor, which is naturally not present
in yeast, and finally, one module comprises
enzymes catalyzing rate-limiting steps, which
have been identified by metabolic flux analyses.
Starting from these modules other related alkaloids are accessible. Li et al., for example, report
the successful production of noscapine—an
antitussive, with anticancer properties, in
baker’s yeast. Expression of 31 enzymes was
required to obtain this substance, of which 25
are heterologous from plants, mammals, and
bacteria (Li et al. 2018).
In the meantime many other alkaloids have
been successfully synthesized in yeasts. Interestingly, there are only very few examples of
other production hosts than baker’s yeast. For
the assembly of very complicated heterologous
pathways, this traditional workhorse seems still
to be the preferred choice. It can be noted
also that most of the endeavors are at a proofof-principle level. It remains to be seen which of
the substances ever reach the markets as microbial products.
13 Yeast Cell Factories
327