Submitted to: Journal of Industrial Microbiology & Biotechnology

Supplementary Material3

A novel aceE mutation leading to a bettergrowth profile and a higher L-serine production in a high-yield L-serine-producing Corynebacterium glutamicumstrain

Wen Guo1, 3, Ziwei Chen 1, 3, Xiaomei Zhang1, 3, Guoqiang Xu1, 2, Xiaojuan Zhang1, Jinsong Shi1, 3, Zhenghong Xu1, 3,*

1Laboratory of Pharmaceutical Engineering, School of Pharmaceutics Science, Jiangnan University, Wuxi 214122, People’s Republic of China

2National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi 214122, People’s Republic of China

3The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, People’s Republic of China

*Corresponding author

Zhenghong Xu,

Mailing address:Laboratory of Pharmaceutical Engineering, School of Pharmaceutics Science,Jiangnan University, Wuxi 214122, People’s Republic of China

Phone: +86-510-85918206; Fax: +86-510-85918206

E-mail address:

Metabolic flux analysis

The model was based on utilizing sucrose as the carbon source. The reactions involved in the metabolic network and metabolites depicted by reactions in the metabolic flux analysiswere listed in Table S1 and Table S2. The metabolite uptake and output rates were measured to estimate intracellular fluxes by a pseudo-steady-state approximation. Elementary mode analysis was constructed using MATLAB software version 7.0. Stoichiometric coefficients of the metabolites presented in the modes were written in the form of a matrix equation (eq. (1)):

Ar=0 (1)

Where A is an n×k matrix of stoichiometric coefficients, n represents the metabolic node in the mode and k represents the total number of the reaction numbers. The stoichiometric model in this study consists of 28 reactions and 19 compounds; the degree of freedom (DF) is 28-19=9, which means that the rate of 9 compounds should be measured to determine all of the reaction rates in the metabolic network. External metabolites measured for quantification, including sucrose, serine, glycine, alanine, valine, threonine, lysine, proline and biomass. The result of the MFA was shown in Fig S1.

Table S1 Equations for reactions involved in the metabolic flux analysis (MFA) ofC.glutamicum

r1: Suc+2PEP=2G6P+2PYR
r2: G6P+H2O+2NADP=Ru5P+CO2+2NADPH
r3: Ru5P=R5P
r4: Ru5P=X5P
r5: X5P+E4P=F6P+GAP
r6: X5P+R5P=S7P+GAP
r7: GAP+S7P=F6P+E4P
r8: G6P=F6P
r9: F6P+ATP=FBP+ADP
r10: FBP=2GAP
r11: GAP+ADP+NAD=G3P+NADH+ATP
r12: G3P+NAD+Glu=Ser(i)+NADH+αKG
r13: Ser(i)=Ser(ex)
r14: Ser(i)+THF=Gly(ex)+METHF
r15: G3P=PEP+H20
r16: PEP+ADP=ATP+PYR
r17: PEP+CO2+GDP=OAA+GTP
r18: PYR+Glu+NADPH=αKG +NADP+Val(ex)
r19: PYR+Glu=Ala(ex)+αKG
r20: PYR+CoA+NAD=AcCoA+CO2+NADH
r21: AcCoA+OAA+H2O =αKG+CoA+CO2
r22: NH3+αKG+NADPH=Glu +2H20+NADP
r23 Glu+2NADPH=Pro(ex)+2NADP
r24: αKG+H20+FAD+2NAD+ADP=OAA+FADH2+2NADH+CO2+ATP
r25: OAA+Glu=ASP+αKG
r26: ASP+2NADPH=Thr(ex)+2NADP
r27:ASP+Glu+PYR+2NADPH=Lys(ex)+αKG
r28:0.205G6P+0.308F6P+0.879R5P+0.268E4P+0.129GAP+1.295G3P+0.652PEP+
2.604PYR+3.177AcCoA+1.68OAA+1.244αKG+16.429NADPH+17.002ATP+3.111NAD
=Biomass+16.429NADP+3.177CoA+17.002ADP+3.111NADH+1.227CO2

Table S2Metabolites depicted by reactions in the metabolic flux analysis (MFA) ofC.glutamicum

G6P: / 2r1-r2-r8-0.205r28=0
F6P: / r5+r7+r8-r9-0.308r28=0
FBP: / r9-r10=0
GAP: / r5+r6-r7+2r10-r11-0.129r28=0
G3P: / r11-r12-r15-1.295r28=0
Ser: / r12-r13-r14=0
PEP: / -2r1+r15-r16-r17-0.625r28=0
Pyr: / 2r1+r16-r18-r19-r20-r27-2.604r28=0
AcCoA: / r20-r21-3.177r28=0
αKG: / r12+r18+r19+r21-r22-r24+r25+r27-1.244r28=0
Glu: / -r12-r18-r19+r22-r23-r25-r27=0
OAA: / -r21+r24-r25-1.68r28=0
Asp: / r25-r26-r27=0
Ru5P: / r2-r3-r4=0
X5P: / r4-r5-r6=0
R5P: / r3-r6-0.879r28=0
S7P: / r6-r7=0
E4P: / -r5+r7-0.268r28=0
NADPH: / 2r2-r18-r22-2r23-2r26-2r27-16.429r28=0

Fig S1Carbon flux distribution of SYPS-062-33a (upper numbers of each pair) and 33a(aceE594) (lowernumbers of each pair) during growth on sucrose. All fluxes are expressed as the molar percentage of the mean specific sucrose uptake rate (0.95 mmolg-1h-1for SYPS-062-33a, 0.89 mmolg-1h-1 for 33a(aceE594)), which is defined as 100%. A replicate experiment showed consistent resultswith a standard deviation of 5%