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In silico aided metabolic engineering of Streptomyces roseosporus for
daptomycin yield improvement
Di Huanga, Jianping Wena,b,*, Guoying Wanga, Guanghai Yua, Xiaoqiang Jiaa,b, Yunlin
Chenc
a
Department of Biochemical Engineering, School of Chemical Engineering and
Technology, Tianjin University, Tianjin 300072, P. R. China
b
Key Laboratory of Systems Bioengineering (Tianjin University), Ministry of
Education, Tianjin 300072, P. R. China
c
School of Science, Beijing Jiaotong University, Beijing 100044, P. R. China
*Author
for
correspondence:
[email protected]
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Fax:
+86-22-27892061,
E-mail:
Supplementary Material: metabolic network model construction
The network was modified by previous work of our group (data not shown),
which excluded organic acids secretion (except acetate and lactate) and used glucose
as sole carbon source. The reaction network includes Embden-Meyerhof-Parnas
pathway (EMP) and pentose phosphate pathways (PPP), tricarboxylic acid cycle
(TCA), anaplerotic reactions, ammonia and sulfate assimilation, electron transport
reactions, folic acid and thioredoxin reactions. Besides, the Entener–Doudoroff (ED)
pathway and glyoxylate pathway in streptomyces was considered inactive in the
model. The biosynthesis of amino acids, pyrimidine, and purine nucleotides, and the
biosynthesis of macromolecular components of biomass such as RNA, DNA, protein,
fatty acids, phospholipids, carbohydrate, as well as daptomycin production were
added in the network. The anabolic reactions by S. roseosporus for protein, lipid,
RNA, DNA, and biomass synthesis were taken from the reported information for
Streptomyces coelicolor A3(2), Streptomyces avermitilis and Escherichia coli at the
specific growth rate of 0.1 h–1 (Arabolaza et al. 2008; Kaddor et al. 2009; Olukoshi
and Packter 1994; Shahab et al. 1996). Furthermore, the pathway synthesizing
3-methy-glutamate was also added (Mahlert et al. 2007; Milne et al. 2006). The
stoichiometry of daptomycin synthesis was deduced from the structure information
reported by Miao et al. (2005). The detailed stoichiometric coefficient for each
reaction was taken from the KEGG (http://www.genome.jp/kegg/pathway) and
BRENDA (http://www.brenda-enzymes.org/) database.
Appendix A: Metabolic reactions
Embden-Meyerhof-Parnas Pathway
R1. GLC + ATP → G6P + ADP
R2. G6P ↔ F6P
R3. F6P + ATP ↔ G3P + DHAP + ADP
R4. DHAP ↔ G3P
R5. G3P + PI + NAD + ADP ↔ NADH + 3PG + ATP
R6. 3PG ↔ PEP
R7. PEP + ADP → PYR + ATP
Pentose Phosphate Pathway
R8. G6P + 2 NADP ↔ CO2 + RL5P + 2 NADPH
R9. RL5P ↔ R5P
R10. RL5P ↔ X5P
R11. R5P + X5P ↔ G3P + S7P
R12. X5P + E4P ↔ F6P + G3P
R13. G3P + S7P ↔ E4P + F6P
Branches from Glycolysis Pathway
R14. PYR + NAD + COA → ACCOA + CO2 + NADH
R15. PYR + NAD → AC + CO2 + NADH
R16. PYR + NADH ↔ LAC + NAD
Anaplerotic Reactions
R17. PEP + CO2 + ADP → OA + ATP
R18. PYR + ATP + CO2 → ADP + OA + PI
R19. MAL + NAD → CO2 + NADH + PYR
TCA Cycle
R20. ACCOA + OA → COA + CIT
R21. CIT ↔ ICIT
R22. ICIT + NADP → NADPH + AKG + CO2
R23. AKG + NAD + COA ↔ CO2 + NADH + SUCCOA
R24. GDP + PI + SUCCOA ↔ GTP + SUCC + COA
R25. SUCC + FAD ↔ FUM + FADH2
R26. FUM ↔ MAL
R27. MAL + NAD ↔ NADH + OA
Biosynthesis of Amino Acids
R28. 3PG + NAD + GLU → NADH + AKG + PI + SER
R29. THF + SER ↔ GLY + METTHF
R30. SER + ACCOA + H2S → COA + CYS + AC
R31. PYR + NH3 + NADH ↔ ALA + NAD
R32. 2 PYR + NADPH → NADP + OIVAL + CO2
R33. OIVAL + GLU → AKG + VAL
R34. ACCOA + OIVAL + NAD + GLU → COA + NADH + CO2 + AKG + LEU
R35. R5P + ATP ↔ PRPP + AMP
R36. PRPP + ATP + GLN + 2 NAD → 2 PPI + AKG + AICAR + PI + 2 NADH +
HIS
R37. OA + GLU ↔ ASP + AKG
R38. ASP + ATP + GLN → GLU + ASN + AMP + PPI
R39. ASP + ATP + NADPH → ADP + NADP + PI + ASPSA
R40. ASPSA + NADPH → NADP + HSER
R41. HSER + ATP → ADP + THR + PI
R42. HSER + SUCCOA + CYS ↔ SUCC + HCYS + PYR + NH3 + COA
R43. HCYS + MTHF ↔ THF + MET
R44. THR + PYR + NADPH + GLU → CO2 + NH3 + NADP + AKG + ILE
R45. E4P + 2 PEP + NADPH + ATP → 4 PI + ADP + CHOR
R46. CHOR + GLN → GLU + PYR + AN
R47. AN + PRPP + SER → PPI + CO2 + G3P + TRP
R48. CHOR → PHEN
R49. PHEN + GLU → CO2 + AKG + PHE
R50. PHEN + NAD + GLU → AKG + TYR + CO2 + NADH
R51. AKG + NH3 + NADPH ↔ GLU + NADP
R52. GLU + NH3 + ATP → GLN + ADP + PI
R53. GLU + ATP + 2 NADPH + NADH → ADP + 2 NADP + NAD + PI + PRO
R54. ATP + NADPH + 2 GLU → ADP + NADP + PI + AKG + ORN
R55. GLN + 2 ATP + CO2 → GLU + CAP + 2 ADP + PI
R56. ASP + 2 ATP + CAP + NADPH + 2 GLU → ARG + FUM + AKG + AMP + PPI
+ 2 PI + ADP + NADP
R57. ASPSA + PYR + NADH + NADPH + SUCCOA + GLU → NAD + NADP +
COA + AKG + SUCC + MDAPIM
R58. MDAPIM → LYS + CO2
Biosynthesis of D Family Amino Acids
R59. GLU ↔ DGLU
R60. ALA ↔ DALA
R61. SER ↔ DSER
R62. ASN ↔ DASN
Biosynthesis of Nonprotein Amino Acids
R63. ATP + MET → SAM + PPI + PI
R64. SAM + AKG → MAKG + SAH
R65. SAH → HCYS + ADN
R66. MAKG + VAL → MGLU + OIVAL
R67. TRP + O2 → FKYN
R68. FKYN → FOR + KYN
Biosynthesis of Nucleotides
R69. PRPP + 2 GLN + 2 ATP + GLY + FTHF → PPI + 2 GLU + 2 ADP + 2 PI + THF
+ FGAM
R70. FGAM + 3 ATP + CO2 + ASP → 3 ADP + 3 PI + FUM + AICAR
R71. AICAR + FTHF ↔ THF + IMP
R72. IMP + NAD + 2 ATP + GLN → GLU + AMP + PPI + GDP + NADH + ADP
R73. GDP + ATP ↔ GTP + ADP
R74. GDP + RTHIO + ATP → DGTP + OTHIO + ADP
R75. ADN + ATP ↔ AMP + ADP
R76. AMP + ATP → 2 ADP
R77. ATP + RTHIO → DATP + OTHIO
R78. CAP + ASP + NAD + PRPP → NADH + PPI + UMP + CO2 + PI
R79. ATP + UMP ↔ ADP + UMP
R80. ATP + UDP ↔ ADP + UTP
R81. ATP + UTP + NH3 → ADP + PI + CTP
R82. CDP + ATP ↔ CTP + ADP
R83. CDP + RTHIO + ATP → DCTP + OTHIO + ADP
R84. DCTP + METTHF → DHF + NH3 + DTTP
Folate biosynthesis and Interconversion of One-Carbon Units
R85. DHF + NADPH → NADP + THF
R86. THF + FOR + ATP → FTHF + PI + ADP
R87. FTHF ↔ METHF
R88. METHF + NADPH ↔ METTHF + NADP
R89. METTHF + FADH2 → MTHF + FAD
R90. OTHIO + NADPH → RTHIO + NADP
Biosynthesis of Carbohydrate
R91. F6P + GLN + ACCOA + UTP → GLU + COA + UDPNAG + PPI
R92. G6P + UTP → PPI + UDPGAL
Biosynthesis of Peptidoglycan
R93. UDPNAG + PEP + NADPH → UDPNAM + PI + NADP
R94. 2 DALA + ATP → ALAALA + ADP + PI
Biosynthesis of Triacylglycerol
R95. DHAP + NADH ↔ GL3P + NAD
R96. ACCOA + ATP + CO2 ↔ MALCOA + ADP + PI
R97. MALCOA + ACP → MALACP + COA
R98. ACCOA + ACP → ACACP + COA
R99. ACACP + 6 MALACP + 12 NADPH → 12 NADP + C140ACP + 6 CO2 + 6
ACP
R100. ACACP + 6.5 MALACP + 13 NADPH → 13 NADP + C150ACP + 6.5 CO2 +
6.5 ACP
R101. ACACP + 7 MALACP + 14 NADPH → 14 NADP + C160ACP + 7 CO2 + 7
ACP
R102. ACACP + 7.5 MALACP + 15 NADPH → 15 NADP + C170ACP + 7.5 CO2 +
7.5 ACP
R103. ACACP + 8 MALACP + 15 NADPH → 15 NADP + C181ACP + 8 CO2 + 8
ACP
Biosynthesis of Phospholipid
R104. GL3P + 0.094 C140ACP + 0.294 C150ACP + 0.262 C160ACP + 0.293
C170ACP + 0.057 C181ACP → AGL3P + ACP
R105. AGL3P + 0.094 C140ACP + 0.294 C150ACP + 0.262 C160ACP + 0.293
C170ACP + 0.057 C181ACP → PA + ACP
R106. PA + CTP ↔ CDPDG + PPI
R107. CDPDG + SER ↔ CMP + PS
R108. PS → PE + CO2
R109. CDPDG + GL3P ↔ CMP + PGP
R110. PGP → PI + PG
R111. CDPDG + PG → CMP + CL
Biosynthesis of Teichoic acid
R112. GL3P + CTP → PPI + CDPGL
R113. 12 CDPGL → 12 CMP + POLYGP
Maintenance Energy and Other reactions
R114. ATP → ADP + PI
R115. PPI → 2 PI
R116. NADH + 2 ADP + 2 PI + 0.5 O2 → 2 ATP + NAD
R117. FADH2 + ADP + PI + 0.5 O2 → ATP + FAD
R118. NADPH + NAD → NADP + NADH
R119. SO4 + 2 ATP + 4 NADPH → 2 ADP + H2S + 2 PI + 4 NADP
Biomass Synthesis
R120. 0.469 DATP + 1.149 DCTP + 0.469 DTTP + 1.149 DGTP + 4.4 ATP → 4.4
ADP + 4.4 PI + 3.236 PPI + DNA
R121. 0.518 POLYGP + 0.129 LYS + 0.129 UDPNAG + 0.129 ATP → TEICH +
0.129 UDP + 0.129 ADP + 0.129 PI
R122. 0.600 ATP + 0.826 GTP + 1.031 CTP + 0.662 UTP + 1.25 ATP → 1.25 ADP +
1.25 PI + RNA + 3.119 PPI
R123. 1.007 UDPNAM + 1.197 UDPNAG + 1.900 ALAALA + 0.950 ALA + 1.140
MDAPIM + 1.014 DGLU + 0.973 GLY + 5.026 ATP → PEPTIDOGLYCAN + 0.950
DALA + 1.197 UDP + 1.007 UMP + 5.026 ADP + 5.026 PI
R124. 1.089 PE + 0.250 PG + 0.052 CL → PHOSPHOLIPID
R125. 1.244 GL3P + 0.050 C140ACP + 1.677 C150ACP + 0.421 C160ACP + 1.570
C170ACP + 0.014 C181ACP → TAG + 3.732 ACP + 1.244 PI
R126. 1.350 ALA + 0.352 ARG + 0.394 ASN + 0.391 ASP + 0.165 CYS + 0.382
GLN + 0.379 GLU + 2.015 GLY + 0.131 HIS + 0.477 ILE + 0.742 LEU + 0.499 LYS
+ 0.221 MET + 0.238 PHE + 0.422 PRO + 0.459 SER + 0.465 THR + 0.059 TRP +
0.159 TYR + 0.797 VAL + 40.0 ATP → 40.0 ADP + 40.0 PI + PROTEIN
R127. 1.897 UDPNAG + 3.794 UDPGAL → 5.691 UDP + CARBOHYDRATE
R128. 0.412 PROTEIN + 0.167 RNA + 0.036 DNA + 0.042 PHOSPHOLIPID +
0.033 TAG + 0.110 PEPTIDOGLYCAN + 0.044 CARBOHYDRATE + 0.066 TEICH
+ 47 ATP → BIOMASS + 47 ADP + 47 PI
Daptomycin Synthesis
R129. DEC + DALA + 2 GLY + 3 ASP + DASN + ORN + MGLU + DSER + THR +
TRP + KYN + 14 ATP → DAPTOMYCIN + 14 ADP
Membrane Transport
R130. GLC_e → GLC
R131. CO2 → CO2_e
R132. NH3_e → NH3
R133. O2_e → O2
R134. PI_e + ATP → 2 PI + ADP
R135. SO4_e + ATP → SO4 + ADP + PI
R136. LAC → LAC_e
R137. AC → AC_e
R138. DAPTOMYCIN → DAPTOMYCIN_e
Appendix B: Abbreviations used in metabolic reactions
3PG
3-Phospho-D-glycerate
AC
Acetate
ACACP
Acyl-[acyl-carrier protein]
ACCOA
Acetyl-CoA
ACP
Acyl-carrier protein
ADN
Adenosine
ADP
Adenosine diphosphate
AGL3P
Acyl-sn-glycerol 3-phosphate
AICAR
1-(5'-Phosphoribosyl)-5-amino-4-imidazolecarboxamide
AKG
α-Ketoglutarate
ALA
L-Alanine
ALAALA
D-alanyl-D-alanine
AMP
Adenosine monophosphate
AN
Anthranilate
ARG
L-Arginine
ASN
L-Asparagine
ASP
L-Aspartate
ASPSA
L-Aspartate 4-semialdehyde
ATP
Adenosine triphosphate
BIOMASS
Biomass
C140ACP
Myristoyl-[acyl-carrier protein]
C150ACP
Pentadecanoyl-[acyl-carrier protein]
C160ACP
Hexadecanoyl-[acyl-carrier protein]
C170ACP
Heptadecanoyl-[acyl-carrier protein]
C181ACP
Oleoyl-[acyl-carrier protein]
CAP
Carbamoyl phosphate
CARBOHYDRATE Carbohydrates (biomass component)
CDP
Cytidine diphosphate
CDPDG
CDP-diacylglycerol
CDPGL
CDP-glycerol
CHOR
Chorismate
CIT
Citrate
CL
Cardiolipin (biomass component)
CMP
Cytidine monophosphate
CO2
Carbon dioxide
COA
Coenzyme A
CTP
Cytidine triphosphate
CYS
L-Cysteine
DALA
D-alanine
DAPTOMYCIN
Daptomycin
DASN
D-Asparagine
DEC
Decanoic acid
DGLU
D-Glutamate
DHAP
Glycerone phosphate
DHF
Dihydrofolate
DNA
Deoxyribonucleic acid
DSER
D-Serine
E4P
D-Erythrose 4-phosphate
F6P
beta-D-Fructose 6-phosphate
FAD
Flavin adenine dinucleotide oxidized
FADH2
Flavin adenine dinucleotide reduced
FGAM
2-(Formamido)-N1-(5'-phosphoribosyl)acetamidine
FKYN
L-Formylkynurenine
FOR
Formate
FTHF
10-Formyltetrahydrofolate
FUM
Fumarate
G3P
D-Glyceraldehyde 3-phosphate
G6P
alpha-D-Glucose 6-phosphate
GDP
Guanosine diphosphate
GL3P
sn-Glycerol-3-phosphate
GLC
alpha-D-Glucose
GLN
L-Glutamine
GLU
L-Glutamate
GLY
Glycine
GTP
Guanosine triphosphate
H2S
Hydrogen sulfide
HCYS
Homocysteine
HIS
L-Histidine
HSER
L-Homoserine
ICIT
Isocitrate
ILE
L-Isoleucine
IMP
Inosine monophosphate
KYN
L-Kynurenine
LAC
(R)-Lactate, D-Lactate
LEU
L-Leucine
LYS
L-Lysine
MAKG
3-methyl-2-oxoglutarate
MAL
Malate
MALACP
Malonyl-[acyl-carrier protein]
MALCOA
Malonyl-CoA
MDAPIM
meso-2, 6-Diaminopimelate
MET
L-Methionine
METHF
5, 10-Methenyltetrahydrofolate
METTHF
5, 10-Methylenetetrahydrofolate
MGLU
3-Methyl Glutamate
MTHF
5-Methyltetrahydrofolate
NAD
Nicotinamide adenine dinucleotide oxidized
NADH
Nicotinamide adenine dinucleotide reduced
NADP
Nicotinamide adenine dinucleotide phosphate oxidized
NADPH
Nicotinamide adenine dinucleotide phosphate reduced
NH3
Ammonia
O2
Oxygen
OA
Oxaloacetate
OIVAL
(R)-2-Oxoisovalerate
ORN
L-Ornithine
OTHIO
Oxidized thioredoxin
PA
Phosphatidate
PE
Phosphatidylethanolamine
PEP
Phosphoenolpyruvate
PEPTIDOGLYCAN Peptidoglycan (biomass component)
PG
Phosphatidylglycerol
PGP
Phosphatidylglycerophosphate
PHE
L-Phenylalanine
PHEN
Prephenate
PHOSPHOLIPID
Phospholipids (biomass component)
PI
Orthophosphate
POLYGP
12-residues chain (teichoic acid component)
PPI
Pyrophosphate
PRO
L-Proline
PROTEIN
Proteins (biomass component)
PRPP
5-Phospho-alpha-D-ribose 1-diphosphate
PS
Phosphatidylserine
PYR
Pyruvate
R5P
D-Ribose 5-phosphate
RL5P
D-Ribulose 5-phosphate
RNA
Ribonucleic acid
RTHIO
Reduced thioredoxin
S7P
Sedoheptulose 7-phosphate
SAH
S-Adenosyl-L-homocysteine
SAM
S-Adenosyl-L-methionine
SER
L-Serine
SO4
Sulfate
SUCC
Succinate
SUCCOA
Succinyl-CoA
TAG
Triacylglycerols (biomass component)
TEICH
Teichoic acid (biomass component)
THF
Tetrahydrofolate
THR
L-Threonine
TRP
L-Tryptophan
TYR
L-Tyrosine
UDP
Uridine diphosphate
UDPGAL
UDP-D-galactose
UDPNAG
UDP-N-acetyl-D-glucosamine
UDPNAM
UDP-N-acetylmuramate
UMP
Uridine monophosphate
UTP
Uridine triphosphate
VAL
L-Valine
X5P
D-Xylulose 5-phosphate
References
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Table S1 Metabolic characterization of the parental strain S. roseosporus LC-511 and overexpression recombinants of zwf2, dptI and dptJ during
exponential growth period cultivated in defined medium
Paramatera S. roseosporus LC-511 HP-Z2
HP-I
HP-J
HP-IJ
HP-Z2-IJ
qGLC
0.425±0.111
0.488±0.133 0.432±0.141 0.440±0.177 0.466±0.123 0.439±0.117
µ
0.035±0.007
0.038±0.008 0.033±0.010 0.035±0.011 0.036±0.008 0.038±0.009
CDAP
245.4±13.3
279.1±11.2 270.2±10.9 294.1±11.4 302.5±12.2 322.5±9.9
YDPT/X
20.71±1.11
20.69±1.97 23.56±2.12 25.09±1.55 27.74±0.98 30.86±1.13
a
qGLC, specific glucose consumption rate, mmol/g DCW/h; µ, specific growth rate, h-1; CDAP, daptomycin concentration, mg/L; YDPT/X, datomycin
content, mg/g DCW
Results are represented as mean±SD of three independent observations