Large-Scale Synthesis of Acyl-ACPs

doi:10.13523/j.cb.20180409

China Biotechnology

2018, Vol. 38

Issue (4): 63-69 DOI: 10.13523/j.cb.20180409

Large-Scale Synthesis of Acyl-ACPs

Wei DING^1,²,Yan-bin FENG¹,Xu-peng CAO¹,Song XUE¹(

)

1 Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China
2 University of Chinese Academy of Sciences, Beijing 100049, China

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Abstract

Acyl-acyl carrier proteins (acyl-ACPs) are substrates for many biosynthesis pathways. However, acyl-ACP is substituted by acyl-CoA for studies in vitro as a result of supply restriction, which causes many questions in enzymatic analysis. Thus, obtaining large scale of acyl-ACP steadily is very important to study the related enzymes and metabolic pathways in vitro. acyl-ACP synthetase catalyze the conversion of holo-ACP using fatty acid as acyl donor in vitro while no productivity has been reported before. Here an acyl-ACP synthetase from Vibrio harveyi was used to catalyze the synthesis of (C4~C18) with holo-ACP, and the yield of acyl-ACP was confirmed by high performance liquid chromatography (HPLC). The results indicated that the yields of medium chains (C4~C14) acyl-ACPs were more than 90.0% while the long chain yields of 16:0-ACP and 18:1-ACP were 85.9% and 25.7%, respectively. Via introducing Li ⁺ to the reaction system, the yield of long chain acyl-ACPs were elevated above 90.0%。Then the reaction parameters were optimized in the enlarged reaction system, and more than 20mg acyl-ACPs were steadily obtained. Additionally, two species of holo-ACP were used to validate the versatility of the reaction system. Finally the acyl-ACP activity was conformed using a glycerol-3-phosphate acyltransferase. The synthesis of different chain acyl-ACPs are of great significance for research of the catalysis mechanism of related enzymes.

Key words： acyl-ACP synthetase acyl-ACP HPLC

Received: 30 January 2018 Published: 08 May 2018

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	Wei DING
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	Song XUE

Cite this article:

Wei DING,Yan-bin FENG,Xu-peng CAO,Song XUE. Large-Scale Synthesis of Acyl-ACPs. China Biotechnology, 2018, 38(4): 63-69.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20180409 OR https://manu60.magtech.com.cn/biotech/Y2018/V38/I4/63

Table 1 Enzymes using acyl-ACP as substrates

Fig.1 Synthesis of acyl-ACP using acyl-ACP synthetase

Table 2 Gradient condition of mobile phase for HPLC

Table 3 The synthesis systems of the acyl-ACP

Fig.2 HPLC analysis of apo- and holo-ACP

Table 4 The retention time and yield of different chain length acyl-ACPs

Table 5 The synthetic efficiency of acyl-ACP in different reaction

Fig.3 HPLC chromatograms of Sp6803 ACPs
(a) holo-ACP and apo-ACP (b) 18:1-ACP

Table 6 Optimization of 18:1-ACP synthesis in large-scale

Fig.4 The bioactivity assays of acyl-ACPs with glycerol-3- phosphate acyltransferase in vitro
Lane 1: Standard LPA (17:0); Lane 2: Negative control; Lane 3: E.coli 16:0-ACP as substrate; Lane 4: E.coli 18:1-ACP as substrate; Lane 5: Sp6803 18:1-ACP as substrate


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