Biology Reference
In-Depth Information
Besides production of novel derivatives, synthetic biology also offers platforms for
productivity enhancement. Random mutagenesis was applied to a
S. coelicolor
strain
expressing the heterologous DEBS for the production of erythromycin aglycones.
171
Several
strains exhibited two-fold higher productivities and reached
3 g/L of total macrolide
aglycones. By combining classical mutagenesis, recombinant DNA techniques, and process
development, the fermentation achieved 1.3 g/L of 15-methyl-6-dEB, and the productivity
was increased by over 100% in a scalable fermentation process. Furthermore, the
introduction of an engineered mutase-epimerase pathway in
E. coli
, which provides the
extender unit methylmalonyl-CoA to DEBS, enabled the five-fold higher heterologous
production of 6-dEB.
172,173
Also, a deletion strain to increase biosynthetic pathway
carbon flow and an engineered stain which contains an extra copy of a key deoxysugar
glycosyltransferase gene both showed significant improvement in erythronolide B (EB)
and 3-mycarosylerythronolide B (MEB) production, and a seven-fold increase in
erythromycin A titer.
174
.
PRECURSORS FOR ARTEMISININ
The biosynthetic production of artemisinin precursors is the most promising example of
how synthetic biology is revolutionizing natural-product-based drug manufacturing.
Artemisinin is an antimalarial agent naturally produced by sweet wormwood (
Artemisia
annua
). As malaria-causing parasites are becoming increasingly resistant to commonly used
therapies, more focus has been placed on artemisinin-based treatments. An estimated
225 million people are infected with malaria, and over 750 000 people die from it
each year.
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Current artemisinin extraction methods are neither economical nor sustainable
enough to produce the compound for wide use. However, the progress of artemisinin
biosynthesis in
E. coli
and
S. cerevisiae
over the past decade has led to titers that make
fermentative production a practical and viable process.
196
Artemisinin is a sesquiterpene, a C15 molecule consisting of three isoprene building blocks.
Two molecules of isopentenyl pyrophosphate (IPP) and one dimethylallyl pyrophosphate
(DMAPP) react to form farnesyl pyrophosphate (FPP), which can be cyclized to form a
number of sesquiterpenes. Amorpha-4,11-diene synthase (ADS) cyclizes FPP into
amorphadiene, a bicyclic precursor of artemisinin. The gene for ADS was first discovered by
probing an
A. annua
cDNA library with gene fragments derived from other sesquiterpene
synthases.
176
Initial expression of ADS in
E. coli
led to a production of 0.086 mg
caryophyllene equivalent/L/OD
600
(caryophyllene is a sesquiterpene that was used for
standardized measurements when amorphadiene was not commercially available).
177
In
E. coli
, IPP and DMAPP are products of the 1-deoxy-
D
-xylulose 5-phosphate (DXP)
pathway, while in many eukaryotes they are made via the mevalonate pathway. To decouple
amorphadiene production from the native DXP pathway, Martin and coworkers introduced
the
S. cerevisiae
mevalonate pathway into
E. coli
, leading to amorphadiene titers of 6.5 mg
caryophyllene equivalent/L/OD
600
, a 75-fold improvement.
177
The mevalonate pathway was
split into two stages, with the first three enzymes contained on plasmid pMevT and the latter
four along with FPP synthase on pMBIS (
Fig. 10.3
). By optimizing the codons of the pMevT
genes and switching both promoters from lac to the stronger and less regulation-sensitive
lacUV5, amorphadiene production rose to 221 mg/L/OD
600
.
178
Because a considerable
increase was seen when pMBIS genes were placed under lacUV5, it was determined that one
of the latter-stage mevalonate enzymes was rate-limiting. Each of the pMBIS genes was
individually placed on a higher copy number plasmid and coexpressed with pMBIS while
cultures were supplemented with mevalonate, the substrate for the MBIS pathway stage. The
strain overexpressing mevalonate kinase, encoded by
ERG12
, showed considerable
production of amorphadiene, and a codon optimized
ERG12
was placed on a high copy
number plasmid with ADS, leading to amorphadiene titers of 293 mg/L/OD
600
.
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