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Supplementary Information

Systems strategies for developing industrial microbial strains

Sang Yup Lee1,2,3,4* & Hyun Uk Kim1,2,4

1Metabolic and Biomolecular Engineering National Research Laboratory, Department of

Chemical and Biomolecular Engineering (BK21 Plus Program), Center for Systems and

Synthetic Biotechnology, Institute for the BioCentury, Korea Advanced Institute of Science

and Technology (KAIST), Daejeon 305-701, Republic of Korea; 2BioInformatics Research

Center, KAIST, Daejeon 305-701, Republic of Korea; 3BioProcess Engineering Research

Center, KAIST, Daejeon 305-701, Republic of Korea; 4The Novo Nordisk Foundation Center

for Biosustainability, Technical University of Denmark, Hørsholm, Denmark.

*e-mail: leesy@kaist.ac.kr

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Supplementary Figure 1. Systems metabolic engineering framework applied to the

production of L-valine using E. coli strains1-3. (a) Systems metabolic engineering framework

tailored to the L-valine production. The whole procedure of the L-valine production using this

systems metabolic engineering framework consists of: first round for the strain improvement

using E. coli K-12 W3110 strain (Steps 1-12)1, second round for the optimization of fed-

batch fermentation (Steps 13-16)2, and third round for the use of E. coli platform strain W to

repeat the systems metabolic engineering procedure applied to the W3110 strain (Step 17 and

underlined steps)3. E. coli W strain secrets less byproducts (i.e., acetic and pyruvic acids), and

has greater L-valine tolerance than the W3110 strain. In the third round, only the underlined

steps were repeated using the E. coli W strain. (b) Metabolic engineering experiments

conducted on E. coli to produce L-valine. Each manipulation shares the same color as the

respective step shown in the framework (a) and their resulting strains (c). Dotted lines with

“X” indicate inactivated reactions. E. coli has three acetohydroxy acid synthase isoenzymes I,

II and III, each encoded by ilvBN, ilvGM, and ilvIH genes, respectively (yellow shadow).

“Dual metabolic system” in Step 16 refers to the use of glucose and acetic acid as nutrients in

order to optimize both cell growth rate and L-valine biosynthesis. Metabolite abbreviations

are: 2kb, 2-ketobutyrate; ac, acetate; accoa, acetyl-CoA; acl, 2-acetolactate; acp, acetyl-

phosphate; dhv, 2,3-dihydroxyisovalerate; f6p, D-fructose 6-phosphate; fbp, D-fructose 1,6-

bisphosphate; g6p, D-glucose 6-phosphate; kiv, 2-ketoisovalerate; leu, L-leucine; mal, malate;

oa, oxaloacetate; pan, pantothenate; pyr, pyruvate; thr, L-threonine; val, L-valine. (c) Titers of

L-valine produced from the middle and final engineered E. coli strains from their batch and

fed-batch cultures. Genotypes of VAL, VAMF, and WLA strains are: W3110 (ΔlacI

attilvG::ptac attilvB::ptac ilvHG41A, C50T ΔilvA ΔpanB ΔleuA); Val (ΔaceF Δmdh ΔpfkA::KmR);

and W (ΔlacI ΔilvA), respectively. Genes shown in the parenthesis next to the strain names

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are the ones overexpressed using plasmids. The ilvBNmut genes are ilvBN genes engineered to

be resistant to feedback inhibition. Because of potential confusions from 3 repeating cycles of

the systems metabolic framework (a) for the studies on L-valine production, experimental

steps corresponding to each strain’s production performance (i.e., L-valine concentration) are

not indicated on the bar graph.

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Supplementary Figure 2. Systems metabolic engineering framework applied to the

production of L-threonine using E. coli strains4, 5. (a) Systems metabolic engineering

framework tailored to the L-threonine production. (b) Metabolic engineering experiments

conducted on E. coli to produce L- threonine. Each manipulation shares the same color as the

respective step shown in the framework (a) and their resulting strains (c). Dotted lines with

“X” indicate inactivated reactions, and those without “X” for downregulated reactions.

Aspartokinase isozymes I and III in E. coli are encoded by thrA and lysC, respectively

(yellow shadow). Metabolite abbreviations are: ac, acetate; accoa, acetyl-CoA; asp, L-

aspartate; aspp, L-4-aspartyl phosphate; aspsa, L-aspartate semialdehyde; g6p, D-glucose 6-

phosphate; gly, glycine; hser, homoserine; hserp, homoserine phosphate; icit, isocitrate; ile,

L-isoleucine; lys, L-lysine; met, L-methionine; oa, oxaloacetate; pep, phosphoenolpyruvate;

pyr, pyruvate; thr, L-threonine. (c) Titers of L-threonine produced from the middle and final

engineered E. coli strains from their batch and fed-batch cultures. Genes shown in the

parenthesis next to the strain names are the ones overexpressed using plasmids. The “R3” in

the parentheses indicates rhtABC genes.

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Supplementary Figure 3. Heterologous metabolic pathways constructed in microbial hosts.

(a) Production of adipic acid in E. coli6. Genes in red were expressed in the final strain. The

selected genes are: paaH1, 3-hydroxyacyl-CoA reductase from Ralstonia eutropha; ter,

trans-enoyl-CoA reductase from Euglena gracilis; ech, putative enoyl-CoA hydratase from R.

eutropha; ptb, phosphate butyryltransferase from Clostridium acetobutylicum; buk1, butyryl

kinase from C. acetobutylicum. (b) Production of 3-hydroxypropionic acid in S. cerevisiae7.

Multiple candidate genes were examined for each reaction step, and genes in red were

incorporated into the final strain. The selected genes are: TcPAND, aspartate decarboxylase

from Tribolium castaneum; BcBAPAT, β-alanine-pyruvate aminotransferase from Bacillus

cereus; EcYDFG, 3-hydroxypropanoate dehydrogenase from E. coli. (c) Production of

artemisinic acid in S. cerevisiae8. All the five genes are from the plant Artemisia annua, and

they all function together to oxidize amorphadiene to artemisinic acid. (d) Production of

erythromycin A and its derivatives in E. coli9. In addition to the erythromycin biosynthetic

gene cluster from Saccharopolyspora erythraea, following heterologous genes were

expressed in order to concentrate precursor pools: pcc, propionyl-CoA carboxylase from

Streptomyces coelicolor; mce, methylmalonyl-CoA epimerase from S. coelicolor; matB,

malonyl-CoA ligase from Rhizobium trifolii. Different combinations of genes were examined

for the citramalate pathway, and the shown in red gave the greatest product titer in this

pathway.

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References

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2. Park, J.H., Kim, T.Y., Lee, K.H. & Lee, S.Y. Fed-batch culture of Escherichia coli forL-valine production based on in silico flux response analysis. Biotechnol. Bioeng. 108,934-946 (2011).

3. Park, J.H., Jang, Y.S., Lee, J.W. & Lee, S.Y. Escherichia coli W as a new platformstrain for the enhanced production of L-valine by systems metabolic engineering.Biotechnol. Bioeng. 108, 1140-1147 (2011).

4. Lee, K.H., Park, J.H., Kim, T.Y., Kim, H.U. & Lee, S.Y. Systems metabolicengineering of Escherichia coli for L-threonine production. Mol. Syst. Biol. 3, 149(2007).

5. Lee, J.W. et al. Development of sucrose-utilizing Escherichia coli K-12 strain bycloning beta-fructofuranosidases and its application for L-threonine production. Appl.Microbiol. Biotechnol. 88, 905-913 (2010).

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7. Borodina, I. et al. Establishing a synthetic pathway for high-level production of 3-hydroxypropionic acid in Saccharomyces cerevisiae via beta-alanine. Metab. Eng. 27,57-64 (2015).

8. Paddon, C.J. et al. High-level semi-synthetic production of the potent antimalarialartemisinin. Nature 496, 528-532 (2013).

9. Jiang, M. & Pfeifer, B.A. Metabolic and pathway engineering to influence native andaltered erythromycin production through E. coli. Metab. Eng. 19, 42-49 (2013).

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