Effects of Glucose and Maltose Substrates on the Intracellular Metabolic Flux Distribution of Curdlan Polysaccharides Biosynthesis by <i>Alcaligenes faecalis</i>

doi:10.13523/j.cb.2001008

China Biotechnology

2020, Vol. 40

Issue (5): 30-39 DOI: 10.13523/j.cb.2001008

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-ping^1,^**(

)

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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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

Received: 02 January 2019 Published: 02 June 2020

ZTFLH:

Q815

Corresponding Authors: Ying-ping ZHUANG E-mail: ypzhuang@ecust.edu.cn

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	Ze-jian WANG
	Bo LI
	Ping WANG
	Qin ZHANG
	Hai-feng HANG
	Jian-guang LIANG
	Ying-ping ZHUANG

Cite this article:

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.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2001008 OR https://manu60.magtech.com.cn/biotech/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 + N $H 4 +$ →NADP + Glut + H₂O
r₁₁	Glut + ATP + N $H 4 +$ → 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

Table 1 A. faecalis metabolic network reaction

Table 2 A. faecalis metabolic network equation

Fig.1 The profiles of carbon dioxide emission rate (a), biomass (b), curdlan production (c), sugar consumption (d) during the chemostat cultures of A. faecalis under glucose and maltose as the substrate at the dilution rate of 0.1h^-1

Table 3 Comparison of substrate and product concentration of glucose and maltose fermentation

Table 4 Comparison of substrate and product specific rates of glucose and maltose fermentation

Fig.2 Relative metabolic flux distribution on curdlan production of A. faecalis at dilution rate of 0.1 based on glucose and maltose fermentation at a dilution rate of D=0.1

Fig.3 Absolute relative metabolic flux distribution on curdlan production of A. faecalis at dilution rate of 0.1h^-1 based on glucose and maltose fermentation


[1]	Rinaudo M . Main properties and current applications of some polysaccharides as biomaterials. Polymer International, 2008,57(3):397-430.

[2]	Mckellar R C, Van Geest J, Cui W . Influence of culture and environmental conditions on the composition of exopolysaccharide produced by Agrobacterium radiobacter. Food Hydrocolloids, 2003,17(4):429-437.

[3]	Sutherland I W . Novel and established applications of microbial polysaccharides. Trends in Biotechnology, 1998,16(1):41-46.

[4]	Shih I L, Yu J Y, Hsieh C , et al. Production and characterization of curdlan by Agrobacterium sp. Biochemical Engineering Journal, 2009,43(1):33-40.

[5]	Ding Z, Jia S, Han P , et al. Effects of carbon sources on growth and extracellular polysaccharide production of Nostoc flagelliforme under heterotrophic high-cell-density fed-batch cultures. Journal of Applied Phycology, 2013,25(4):1017-1021.

[6]	Jin L H, Um H J, Yin C J , et al. Proteomic analysis of curdlan-producing Agrobacterium sp. in response to pH downshift. Journal of Biotechnology, 2008,138(3-4):80-87.

[7]	Yu L J, Wu J R, Zheng Z Y , et al. Changes of curdlan biosynthesis and nitrogenous compounds utilization characterized in ntrc mutant of Agrobacterium sp. Atcc 31749. Current Microbiology, 2011,63(1):60-67.

[8]	Yu L J, Wu J R, Zheng Z Y , et al. Changes in gene transcription and protein expression involved in the response of Agrobacterium sp. Atcc 31749 to nitrogen availability during curdlan production. Applied Biochemistry and Microbiology, 2011,47(5):487-493.

[9]	Wu D, Li A, Ma F , et al. Genetic control and regulatory mechanisms of succinoglycan and curdlan biosynthesis in genus Agrobacterium. Applied Microbiology and Biotechnology, 2016,100(14):6183-6192. doi: 10.1007/s00253-016-7650-1

[10]	Zhang Q, Sun J Y, Wang Z J , et al. Kinetic analysis of curdlan production by Alcaligenes faecalis with maltose, sucrose, glucose and fructose as carbon sources. Bioresource Technology, 2018,259(13):319-324.

[11]	王泽建, 张琴, 王萍 , 等. 恒化培养条件下粪产碱杆菌凝胶多糖的发酵动力学研究. 食品工业科技, 2019,40(11):139-146.

[11]	Wang Z J, Zhang Q, Wang P , et al. Study on curdlan fermentation kinetics of Alcaligenes faecalis under chemostat cultivation. Science and Technology of Food Industry, 2019,40(11):139-146.

[12]	Zheng Z Y, Lee J W, Zhan X B , et al. Effect of metabolic structures and energy requirements on curdlan production by Alcaligenes faecalis. Biotechnology and Bioprocess Engineering, 2007,12(4):359-365. doi: 10.1007/BF02931057

[13]	Shi H, Shiraishi M, Shimizu K . Metabolic flux analysis for biosynthesis of poly(β-hydroxybutyric acid) in Alcaligenes eutrophus from various carbon sources. Journal of Fermentation & Bioengineering, 1997,84(6):579-587.

[14]	Kim M K, Ryu K E, Choi WA , et al. Enhanced production of (1,3)-β-d-glucan by a mutant strain of Agrobacterium species. Biochemical Engineering Journal, 2003,16(2):163-168.

[15]	Hongtao Z, Joao Carlos S, Xiaobei Z , et al. Component identification of electron transport chains in curdlan-producing Agrobacterium sp. Atcc 31749 and its genome-specific prediction using comparative genome and phylogenetic trees analysis. Journal of Industrial Microbiology & Biotechnology, 2011,38(6):667-677.

[16]	Welman A D, Maddox I S . Fermentation performance of an exopolysaccharide-producing strain of Lactobacillus delbrueckii subsp. bulgaricus. Journal of Industrial Microbiology and Biotechnology, 2003,30(11):661-668.

[17]	Stephanopoulos G, Aristidou A, Nielsen J . Metabolic engineering: principles and methods. Elsevier: Academic Press, 2003: 433-501.

[18]	Zhang H T, Zhu L, Liu D , et al. Model-based estimation of optimal dissolved oxygen profile in Agrobacterium sp. Fed-batch fermentation for improvement of curdlan production under nitrogen-limited condition. Biochemical Engineering Journal, 2015,103(11):12-21.

[19]	Duan X, Chi Z, Wang L , et al. Influence of different sugars on pullulan production and activities of alpha-phospho glucose mutase, udpg-pyrophosphorylase and glucosyl transferase involved in pullulan synthesis in Aureobasidium pullulans y68. Carbohydr Polym, 2008,73(4):587-593.

[20]	Martin S A, Russell J B . Transport and phosphorylation of disaccharides by the ruminal bacterium Streptococcus bovis. Applied and Environmental Microbiology, 1987,53(10):2388-2393. doi: 10.1128/AEM.53.10.2388-2393.1987

[21]	Velasco S E, Yebra M J, Monedero V , et al. Influence of the carbohydrate source on beta-glucan production and enzyme activities involved in sugar metabolism in Pediococcus parvulus 2.6. International Journal of Food Microbiology, 2007,115(3):325-334.

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