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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2020, Vol. 40 Issue (5): 30-39    DOI: 10.13523/j.cb.2001008
研究报告     
葡萄糖和麦芽糖碳源底物对粪产碱杆菌合成凝胶多糖的胞内代谢流影响*
王泽建1,栗波2,王萍1,张琴1,杭海峰1,梁剑光2,庄英萍1,**()
1 华东理工大学生物反应器工程国家重点实验室 上海 200237
2 苏州大学医学部药学院 苏州 215123
Effects of Glucose and Maltose Substrates on the Intracellular Metabolic Flux Distribution of Curdlan Polysaccharides Biosynthesis by Alcaligenes faecalis
WANG Ze-jian1,LI Bo2,WANG Ping1,ZHANG Qin1,HANG Hai-feng1,LIANG Jian-guang2,ZHUANG Ying-ping1,**()
1 State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai Institute of Biomanufacturing Technology & Collaborative Innovation Center, Shanghai 200237, China
2 College of Pharmaceutical Science,Soochow University, Suzhou 215123, China
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摘要：

麦芽糖和葡萄糖对粪产碱杆菌发酵合成凝胶多糖有着显著的影响,为了详细分析两种底物对凝胶多糖合成的影响机制,利用恒化培养实验及稳态碳平衡代谢分析,研究发现在稀释速率为0.1h-1时,利用麦芽糖和葡萄糖为碳源底物的条件下粪产碱杆菌的微观代谢途径通量有较大的差异。以麦芽糖为底物时凝胶多糖的摩尔得率为53.8%,比葡萄糖为碳源时的摩尔得率(36.9%)高出了45.8%以上。同时以麦芽糖为碳源时HMP途径的绝对代谢通量比葡萄糖时的通量提升了40%以上。这条途径通量的增加,提升了NADPH还原力供给速率,促进了依赖于还原力NADPH的凝胶多糖合成途径通量,提升了碳源底物向产物的摩尔转化速率。而且代谢流分析结果显示ED途径通量和能量提供也是影响粪产碱杆菌凝胶多糖合成效率的关键因素。麦芽糖作为碳源底物过程中维持的较低的残留葡萄糖浓度解除了高葡萄糖浓度条件下对凝胶多糖合成的抑制,能够实现更高通量的ATP能量提供效率,更加促进了凝胶多糖合成通量。

关键词： 凝胶多糖;  代谢途径通量分析;  粪产碱杆菌;  恒化发酵    
Abstract:

Maltose and glucose have significant effects on the production of curdlan by fermentation of Alcaligenes faecalis. The chemostat culture and steady-state carbon balanced metabolic flux analysis were applied to evaluate the effect of the substrates on curdlan biosynthesis in detail. Results demonstrated that the intracellular metabolism of A. faecalis were significantly different under the substrates of maltose and glucose as the carbon substrate at the dilution rate of 0.1h -1. The relative metabolic flux analysis showed the curdlan yield reached 53.8% under maltose source, which was more than 45.8% higher than that of glucose (36.9%). At the same time, the absolute metabolic flux of the HMP pathway increased more than 40% than that of glucose, and enhanced the supply rate of NADPH. The higher NADPH supply level promotes the flux ratio of curdlan biosynthesis, which depends on NADPH cofactors, and increases the molar conversion rate of curdlan from carbon source substrate. Moreover, the metabolic flux distribution results also showed that the ED pathway distribution and energy supply are also the key factors affecting the curdlan biosynthesis efficiency of A. faecalis. The lower residual glucose concentration with maltose as carbon source substrate could relieve the inhibition on curdlan synthesis, and could achieve higher flux ratio of ATP supply for promoting the curdlan biosynthesis efficiency.

Key words: Curdlan    Metabolic flux analysis    Alcaligenes faecalis    Chemostat culture
收稿日期: 2019-01-02 出版日期: 2020-06-02
ZTFLH:  Q815  
基金资助: * 国家科技重大专项(2017YFF0204602)
通讯作者: 庄英萍     E-mail: ypzhuang@ecust.edu.cn
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王泽建
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梁剑光
庄英萍

引用本文:

王泽建,栗波,王萍,张琴,杭海峰,梁剑光,庄英萍. 葡萄糖和麦芽糖碳源底物对粪产碱杆菌合成凝胶多糖的胞内代谢流影响*[J]. 中国生物工程杂志, 2020, 40(5): 30-39.

WANG Ze-jian,LI Bo,WANG Ping,ZHANG Qin,HANG Hai-feng,LIANG Jian-guang,ZHUANG Ying-ping. Effects of Glucose and Maltose Substrates on the Intracellular Metabolic Flux Distribution of Curdlan Polysaccharides Biosynthesis by Alcaligenes faecalis. China Biotechnology, 2020, 40(5): 30-39.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2001008    或    https://manu60.magtech.com.cn/biotech/CN/Y2020/V40/I5/30

比速率 反应式
r1 Glc + ATP →G-6-P+ADP
r2 G-6-P ? F-6-P
r3 ATP + F-6-P → ADP + 2GAP
r4 GAP + ADP + NAD ? ATP + NADH + G-3-P + H+
r5 G-3-P ? PEP + H2O
r6 ADP + PEP → ATP + PYR
r7 PYR + NAD + COA → Ac-COA + CO2 + NADH
r8 OAA + Ac-COA + NAD →CO2+ NADH + α-KG +COA
r9 α-KG + ADP + FAD + 2NAD → ATP + CO2 + FADH2 + 2 NADH + OAA
r10 NADPH + α-KG + NH4+→NADP + Glut + H2O
r11 Glut + ATP + NH4+ → ADP + Glum
r12 G-6-P + NADP → 6-P-G + NADPH
r13 6-P-G + NADP → Ru-5-P + NADPH
r14 3P5P → 2 F6P + G3P
r15 Ru-5-P → Xu-5-P
r16 Ri-5-P + Xu-5-P →E-4-P + F-6-P
r17 F-6-P + GAP ? Ri-5-P + E-4-P
r18 6-P-G → GAP + PYR
r19 2 ADP + NADH + 0.5 O2 → 2 ATP + NAD + H2O
r20 ATP → ADP
r21 FADH2+ ADP → ATP + H2O + FAD
r22
 0.374 Ac-COA + 4.11 ATP + 0.036 E-4-P + 0.007 F-6-P + 0.15 G-3-P + 0.021 G-6-P+0.013 GAP+0.025 Glum + 0.832 Glut + 0.179 OAA + 0.052 PEP + 0.283 PYR + 0.09 Ri-5-P + 0.826 NADPH+ 0.312 NAD → 4.11 ADP + 0.261 CO2 + 0.751 α-KG + BIOMASS + 0.826 NADP + 0.312NADH
r23 G-6-P + UTP → [C6H10O5] + UDP
r24 UDP + ATP →UTP + ADP
表1  A.faecalis代谢网络方程式
序号 节点 平衡方程
1 Glc -r1=V底物消耗速率
2 G-6-P G: r1-r2-r12-0.0021r24-r21=0
M: 2r1-r2-r12-0.021r24-r21=0
3 F-6-P r2-r3+r14+r15-0.007r24=0
4 GAP 2r3-r4+r15+r17-0.013r24=0
5 G-3-P r4-r5-0.15r24=0
6 PEP r5-r6-0.052r24=0
7 PYR r6-r7+r17-r18-0.032r24=V丙酮酸生成速率
8 Ac-COA r7-r8-0.074r24=0
9 OAA -r8+r9+r18-0.192r24=VOAA生成速率
10 α-KG r8-r9-r10+0.082r24=0
11 Glut r10-r11-0.145r24=0
12 Glum r11-0.025r24=0
13 6-P-G r12-r13-r16=0
14 R-5-P r13-2r14-r15-0.011r24=0
15 E-4-P r14-r15-0.036r24=0
17 KDPG r16-r17=0
18 NADPH -r10+r12+r13-r19-0.826r24=0
19 NADH r4+r7+r8+2r9-r21+0.312r24=0
20 ATP G: -r1-r3+r4+r6+r9-r11+2r19-r20-4.11r24-r22=0
M: -2r1-r3+r4+r6+r9-r11+2r19-r20-4.11r24-r22=0
21 UTP -r22+r21=0
22 UDPG r22-r23=0
23 Curdlan r23=Vcurdlan生成速率
24 Biomass r24=μ菌体比增长速率
25 CO2 r7+r8+r9-r16+r22=0
表2  A.faecalis代谢网络方程式
图1  恒化培养过程中(稀释速率D为0.1h-1)麦芽糖和葡萄糖为碳源条件下粪产碱杆菌的二氧化碳释放速率(CER)(a)、菌浓(b)、凝胶多糖产量(c)和残糖浓度(d)的变化
碳源 葡萄糖
(g/L)		 麦芽糖
(g/L)		 PYR
(g/L)		 OAA
(g/L)		 O2
[mmol/(L·h)]		 CO2
[mmol/(L·h)]		 生物量
(g/L)		 凝胶多糖
(g/L)
葡萄糖 16.52 - 0.30 - 24.65 25.86 5.37 7.81
麦芽糖 0.81 8.79 - 0.61 25.34 27.06 5.48 15.90
表3  葡萄糖和麦芽糖发酵底物和产物浓度比较
碳源底物 葡萄糖 麦芽糖
比葡萄糖消耗速率qs[mmol/(gDCW·h)] 2.42 -
比麦芽糖消耗速率qs[mmol/(gDCW·h)] - 1.67
比PYR合成速率[mmol/(gDCW·h)] 0.063 -
比OAA合成速率[mmol/(gDCW·h)] - 0.084
比CO2生成速率[mmol/(gDCW·h)] 4.81 4.94
比菌体生长速率[mmol/(gDCW·h)] 1.06 1.08
比凝胶多糖合成速率qp[mmol/(gDCW·h)] 0.89 1.79
碳回收率(%) 100.03 100.25
表4  葡萄糖和麦芽糖发酵底物和产物比速率比较
图2  稀释率 D=0.1条件下麦芽糖(左)和葡萄糖(右)为底物时A. faecalis胞内的相对代谢流分布(mmol/100mmol)
图3  稀释率 D=0.1h-1条件下麦芽糖(左)和葡萄糖(右)为底物的A. faecalis 产凝胶多糖绝对代谢流分布 [mmol/(g DCW·h)]
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[1] 柯为. 固定化酶技术生产天冬素[J]. 中国生物工程杂志, 1985, 5(4): 69-69.
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