Metabolic engineering of Saccharomyces cerevisiae for isoprenoids production by-Regis Rutayisire

Metabolic engineering of Saccharomyces cerevisiae for
isoprenoids production
by Regis Rutayisire, IIT Chicago

Outline
• Background
• Why Saccharomyces cerevisae?
• Biosynthesis of isoprenoid precursors (IPP and DMAPP)
• Approaches to increasing isoprenoids production
• Targeted interventions to increase isoprenoids production
• Examples (artemisinic acid, taxadiene, geraniol, etc.)
• Challenges and advances

Isoprenoid: what is an isoprenoid? Why?
• Any compound derivered from
isopentenyl pyrophosphate (IPP):
C5-Isoprene
• Found in animals, plants, and
microbial species.
• Largest and most diverse class of
natural products in plants.
• Large range of applications.

Isoprenoids
• Present in low quantities in their natural
sources (plants, micro-organisms, etc.)
• Various utilization & high market
demand
Microbial production
Microbes require little and produce the
building blocks of all isoprenoids
Traditional
production
• Plant extraction
• Chemical
synthesis
• In vitro
enzymatic
production, etc.
Impractical
High demand
Low yields/concentrations
Microbial
production
Microbial
production

Outline
• Background
• Why Saccharomyces cerevisae?
• Biosynthesis of isoprenoid precursors (IPP and DMAPP)
• ME Approaches to increasing isoprenoids production
• Targeted interventions to increase isoprenoids production
• Examples (artemisinic acid, taxadiene, geraniol, etc.)
• Challenges and advances

Why Saccharomyces cerevisiae?
First eukaryote whose genome was fully sequenced in the early 1990s
Model organism for research on modern molecular and cell biology,
genetics, biotechnology, drug research, etc.
…rapid growth rate
…easy to modify genetically
…features typical of eukaryotes
…relatively simple (unicellular)
…relatively small genome
…relatively tolerant to harsh conditions (low pH, etc.)
Safest cell factory, classified GRAS (Recognized As Safe by USFDA)
Native isoprenoid pathway

2 distinct pathways for biosynthesis of isoprenoids precursors
The DXP/MEP pathway
• Present in E. coli
• Present in other prokaryotes,
algae and plant plastids
• Condensation of pyruvate and
glyceraldehyde 3-phopshate by
DXS and an additional six steps
transform DXP to IPP and
DMAPP.
The mevalonate (MVA) pathway
• Naturally used for isoprenoids biosynthesis in
S. cerevisae
• Present in archaea, fungi, plant cytoplasm, and
other eukaryotes including mammalian cells
• Condensation of 3 acetyl- CoA to form HMG-
CoA and an additional four steps transform
HMG-CoA to IPP, which is isomerized to
DMAPP by IDI.
• Target of statins: class of cholesterol lowering
drugs inhibiting HMG-CoA Reductase.
IDI: isopentenyl diphosphate isomerase, DXS: DXP synthase, DXP: 1-deoxy-D-xylulose 5-phosphate, MEP: 2-methyl-D-erythritol-4-phosphate,
DMAPP: dimethylallyl diphosphate

The mevalonate pathway of S. cerevisae
SGD-Saccharomyces Genome Database, https://www.yeastgenome.org

Beyond IPP/DMAPP
• IPP monomer: universal building
block for the production of all
isoprenoids, including artemisinin,
carotenoids, and Taxol.
• Geranyl pyrophosphate (GPP):
universal precursor of monoterpenes
obtained by combining two isoprene
(C5) units, further processed by
monoterpene synthases/cyclases
(MTS/C) to produce a vast array of
chemical structures.

Approaches to increasing isoprenoids production
Requires engineering a strain for efficient provision of the universal C5 monomer IPP:
• To manipulate the metabolic flux and regulation of the native MVA pathway
Overexpression of a truncated Hmg1 (tHMG1)
... a transcriptional factor mutant UPC2-1
… a FPP synthase gene-ERG20
Overexpression of all other genes (ERG10, ERG13, tHMGR, ERG12, ERG8 and
IDI1) in the MVA pathway by integrating an additional copy or replacing promoters of
all genes,
Down-regulation of squalene synthase gene-ERG9
Development of a SUE (sterol uptake enhancement) mutant to make ergosterol
biosynthesis nonessential. A k/o mutation of ERG9 was created in the SUE.
Not to ignore the supply of acetyl-CoA, and balancing co-factor usage (NADH-
dependent HMGR)

Approaches to increasing isoprenoids production
Requires engineering a strain with high potential of generating IPP:
• To increase the precursor flux by introducing a heterologous pathway to supplement
the native pathway,
Importing DXP/MEP pathway (or its components) from other micro/organisms
(E. coli, etc.) to S. cerevisae
Possible toxicity or inability to functionalize the enzymes/pathway
Higher yields

Terpene biosynthesis in yeast. Figure indicating genes involved and metabolic engineering interventions employed. Upregulated yeast genes indicated in green and
downregulated yeast genes in orange. Enzymes with yield or specificity enhancement or altered by protein engineering are indicated by superscripted asterisks. CPP
copalyl diphosphate, CDS copalyl diphosphate synthase, DTS diterpene synthase, MTS monoterpene synthase, SeACS(L641P) Salmonella enterica acetyl-CoA synthase
mutant L641P

Synopsis of terpene biosynthesis in yeast indicating the genes involved and the metabolic engineering interventions employed. Upregulated yeast genes indicated
indicated in green, downregulated yeast genes in red. Genes whose products have been fused or attached to a synthetic protein scaffold are denoted with supersripted
(f). Enzymes with product yield or specificity improved or altered by protein engineering are indicated by superscripted (e). (CPP, copalyl diphosphate; CDS, copalyl
diphosphate synthase; DTS, diterpene synthase; MTS, monoterpene synthase; SeACS(L641P), Salmonella enterica acetyl-CoA synthase mutant L641P; AtoB-gene
from E. coli, acetoacetyl-CoA synthase/thiolase). Source: Developing a yeast cell factory for the production of terpenoids http://dx.doi.org/10.5936/csbj.201210006

HMGR: HmgIp & Hmg2p
• HMGR-catalyzed reaction produces mevalonic acid from HMG-CoA by reduction
with NADPH.
• Overexpression of a truncated version of HMG1 (tHMG1) improved amorphadiene
levels by >5-fold, taxadiene by 1.5 fold and carotenoid production by 7-fold.
ERG9
• ERG9 codes for squalene synthase (two FPP moieties form squalene) in the
sterol biosynthetic pathway.  major draining route of isoprenoid substrates
• Deletion of ERG9 is lethal, so complete elimination of squalene synthesizing activity
is not feasible. It is replaced by the endogenous promoter PMET3 (methionine
repressible promoter)
• ERG9 dr: 40 g L−1 for the artemisinin precursor amorphadiene, 25 g L−1 of
artemisinic acid, increase in sesquiterpenes cubebol, valencene and patchoulol, etc.

ERG20
• Erg20p enzyme catalyzes the condensation of IPP and DMAPP to form GPP and
subsequently FPP.
• ERG20 overexpression increased amorphadiene and geraniol production.
ACS
The bottleneck in the supply of acetyl-CoA to the mevalonate pathway showed that
overproduction of acetaldehyde dehydrogenase ALD6 and introduction of a
heterologous acetyl-CoA synthase variant from Salmonella enterica (L641P) together
with tHMG1 expression achieved substantial improvements in amorphadiene
production.

UPC2
• Upc2p and Ecm22p are two highly homologous zinc cluster proteins regulating a
number of ERG genes in the yeast ergosterol biosynthetic pathway.
• They positively regulate transcription by binding to sterol response elements in the
promoters of the target genes.
• Overexpression of upc2-1 appeared to exert modest effects on amorphadiene
production which become more pronounced in combination with tHMG1 and
PMET3−ERG9.
IDI1
Encodes for IPP isomerase, catalyzing the conversion of IPP to its isomer
DMAPP. ERG20 adds one molecule of IPP to DMAPP to form GPP. In the case of
monoterpene production, when a rich GPP pool is required, IDI1 overexpression
significantly enhanced monoterpene titers.

BTS1
• BTS1 encodes for geranylgeranyl diphosphate synthase. The enzyme uses FPP and
IPP to synthesize the C20 GGPP substrate.
• Overexpression of BTS1 can provide increases of β‐carotene production between 6.5‐
and 22‐fold. BTS1p has been fused to ERG20p to improve product yields for alcohol
geranylgeraniol (up to 229 mg/L) and miltiradiene.
LPP1, DPP1
• Lpp1p and Dpp1p, two enzymes initially identified as phosphatidic acid
hydrolases were shown to also dephosphorylate isoprenoid phosphates.
• Deletion of DPP1 was reported to result in a modest increase of the sesquiterpene α-
santalene.

Schematic representation of the engineered artemisinic acid biosynthetic pathway in S. cerevisiae
strain EPY224 expressing CYP71AV1 and CPR.
Blue: directly upregulated, Purple: indirectly upregulated: Red: Repression, Green: FPP to AA from
A. Annua.
The engineered taxadiene biosynthetic pathway in S. cerevisiae

He: Heterologous expression, Oe: Overexpression of self-cloning gene(s), Dr: down regulation, De: Deletion, Mu: Mutation
Adapted from: Hara, Kiyotaka Y., Michihiro Araki, Naoko Okai, Satoshi Wakai, Tomohisa Hasunuma and Akihiko Kondo. “Development of bio-based fine
chemical production through synthetic bioengineering.” Microbial cell factories (2014).

Challenges
• Achieving high titers: most common
• Identification of novel downstream genes
• Building a functional MEP pathway in S. cerevisae (genes, etc)
• Heterologous pathways may lead to the accumulation of toxic intermediates.
• Vast amount of data generated by new technologies
• Finding the best mutant from millions of possibilities

Advancements
1. Genome editing and transcriptional regulation
• Yeast Oligo-Mediated Genome Engineering (YOGE)
• Transcription activator like (TAL) effector nucleases
• Multiplex CRISPR-based techniques introduced combinations of recombinant
pathway genes Seamless, marker-less, multi-loci genome editing: simultaneous
manipulation of genes, synthetic genome construction, library generation, etc.
• CRISPR/Cas-homology directed repair (HDR): k/o of genes with 100% efficiency
• eMAGE (eukaryotic multiplex automated genome engineering)
• Pathway enzyme and transporter engineering (directed evolution, etc).
• Plasmid copy number, promoter and terminator engineering
Genes expression and proteins engineering

Advancements (cont’d)
2. Substrate range
Engineering a strain that can use more sustainable, cheaper and generally available
carbon sources (lignocellulose, xylose, etc.)
4. Adaptive evolution and reverse metabolic engineering
Development of a non Saccharomyces cell factory mutant, tolerance level engineering
(high temperature, inhibitors, etc.),
5. Mathematical models
To characterize the performance of mutant strains at various levels of biological
processes

Multiplexed Genetic Engineering in S. cerevisiae for Lycopene production Diagram of Yeast Oligo-mediated Genome Engineering (YOGE)
applications. Synthetically assembled foreign DNA or natural genomes can
be modified with YOGE.

References
[1] D. R. Kutyna and A. R. Borneman, “Heterologous production of flavour and aroma compounds in
Saccharomyces cerevisiae,” Genes (Basel)., vol. 9, no. 7, 2018.
[2] B. Huang, J. Guo, B. Yi, X. Yu, L. Sun, and W. Chen, “Heterologous production of secondary
metabolites as pharmaceuticals in Saccharomyces cerevisiae,” Biotechnol. Lett., 2008.
[3] J. A. Chemler, Y. Yan, and M. A. G. Koffas, “Biosynthesis of isoprenoids, polyunsaturated fatty acids
and flavonoids in Saccharomyces cerevisiae,” Microb. Cell Fact., vol. 5, no. February, 2006.
[4] M. S. Siddiqui, K. Thodey, I. Trenchard, and C. D. Smolke, “Advancing secondary metabolite
biosynthesis in yeast with synthetic biology tools,” FEMS Yeast Res., vol. 12, no. 2, pp. 144–170,
2012.
[5] W. Schwab, B. M. Lange, and M. Wüst, Biotechnology of natural products. 2017.
[6] K. Y. Hara, M. Araki, N. Okai, S. Wakai, T. Hasunuma, and A. Kondo, “Development of bio-based
fine chemical production through synthetic bioengineering,” Microb. Cell Fact., vol. 13, no. 1, 2014.
[7] S. C. Kampranis and A. M. Makris, “Developing a Yeast Cell Factory for the Production of
Terpenoids,” Comput. Struct. Biotechnol. J., vol. 3, no. 4, p. e201210006, 2012.
[8] J. Lian, S. Mishra, and H. Zhao, “Recent advances in metabolic engineering of Saccharomyces
cerevisiae: New tools and their applications,” Metab. Eng., no. April, pp. 0–1, 2018.

Thank you!!! Comments and questions?

Metabolic engineering of Saccharomyces cerevisiae for isoprenoids production by-Regis Rutayisire

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