A cell engineering approach to enzyme-based fed-batch fermentation.
A cell engineering approach to enzyme-based fed-batch fermentation.
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DOI：
10.1186/s12934-021-01634-y
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发表时间：
2021-07-24
影响因子：
6.4
通讯作者：
Ward JM
中科院分区：
文献类型：
--
作者：
Sibley M;Ward JM
与大肠杆菌发酵相关的一个基本问题是在分批培养中实现高细胞密度的困难，这在很大程度上归因于当提供足够的葡萄糖以满足所需的细胞密度时，通过一种称为溢出代谢的现象产生和积累醋酸盐。虽然补料批次配置是减少此类问题的标准方法，但传统的补料批次系统需要在较小规模应用时出现问题的部件。一种替代方案是开发一种系统，利用淀粉的酶降解以受控的速度释放葡萄糖。然而，到目前为止，淀粉酶还只应用于外源培养，而我们的目标是设计和构建一种能够自我调节的基于酶的补料分批发酵的自我分泌的淀粉酶底盘。克隆并在大肠杆菌BL21(DE3)和W3110中表达了一个可能的葡萄糖淀粉酶，该酶具有显著的葡萄糖释放淀粉酶活性。在用DsbA信号序列取代酶的天然信号肽后，胞外淀粉分解活性得到提高，从而形成了一株能够在限定的培养基中利用淀粉作为唯一碳源的糖淀粉酶分泌菌株。在大肠杆菌W3110中引入PcstA，一种葡萄糖敏感的K12亲和启动子，并将其与Vioraceum葡萄糖淀粉酶一起整合到E.ColiW3110中，与同等数量的葡萄糖培养物(OD600 ∼ 15)相比，在淀粉(OD600 ∼ 30)上生长的细胞密度得到了增加。最后，通过同时表达酵母菌糖化酶和重组目的蛋白(EGFP)，建立了一种新型的基于自分泌酶的补料分批发酵系统，使在含有淀粉的培养基中的发酵产量比葡萄糖当量提高了四倍。这项研究通过分泌一种以前没有特征的细菌糖淀粉酶，开发了一种新的能够将淀粉直接转化为葡萄糖的淀粉分解大肠杆菌菌株。与同等数量的葡萄糖相比，该菌株在淀粉上生长时能够实现更高的细胞密度以及相关的重组蛋白产量的增加，这首次展示了一种基于酶的补料分批发酵的细胞工程方法。网上版载有补充材料，可在10.1186/s12934-021-01634-y查阅。
A fundamental problem associated with E. coli fermentations is the difficulty in achieving high cell densities in batch cultures, attributed in large part to the production and accumulation of acetate through a phenomenon known as overflow metabolism when supplying enough glucose for the cell density desired. Although a fed-batch configuration is the standard method for reducing such issues, traditional fed-batch systems require components which become problematic when applying them at smaller scale. One alternative has been the development of a system whereby the enzymatic degradation of starch is used to release glucose at a controlled rate. However, to date, amylolytic enzymes have only been applied to the culture exogenously, whereas our goal is to design and construct a self-secreting amylolytic chassis capable of self-regulated enzyme-based fed-batch fermentation. A putative glucoamylase from C. violaceum has been cloned and expressed in E. coli BL21(DE3) and W3110, which exhibits significant glucose releasing amylolytic activity. Extracellular amylolytic activity was enhanced following a replacement of the enzymes native signal peptide with the DsbA signal sequence, contributing to a glucoamylase secreting strain capable of utilising starch as a sole carbon source in defined media. Introduction of PcstA, a glucose sensitive K12 compatible promoter, and the incorporation of this alongside C. violaceum glucoamylase in E. coli W3110, gave rise to increased cell densities in cultures grown on starch (OD600 ∼ 30) compared to those grown on an equivalent amount of glucose (OD600 ∼ 15). Lastly, a novel self-secreting enzyme-based fed-batch fermentation system was demonstrated via the simultaneous expression of the C. violaceum glucoamylase and a recombinant protein of interest (eGFP), resulting in a fourfold increase in yield when grown in media containing starch compared with the glucose equivalent. This study has developed, through the secretion of a previously uncharacterised bacterial glucoamylase, a novel amylolytic E. coli strain capable of direct starch to glucose conversion. The ability of this strain to achieve increased cell densities as well as an associated increase in recombinant protein yield when grown on starch compared with an equivalent amount of glucose, demonstrates for the first time a cell engineering approach to enzyme-based fed-batch fermentation. The online version contains supplementary material available at 10.1186/s12934-021-01634-y.
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DOI：
10.1093/protein/2.6.473
发表时间：
1989-03-01
期刊：
PROTEIN ENGINEERING
影响因子：
--
作者：
BLANCHINROLAND, S;MASSON, JM
通讯作者：
MASSON, JM
影响因子：
4.4
作者：
Phue, Je-Nie;Kedem, Benjamin;Shiloach, Joseph
通讯作者：
Shiloach, Joseph
影响因子：
3.2
作者：
Schierle, CF;Berkmen, M;Beckwith, J
通讯作者：
Beckwith, J
DOI：
10.1002/path.1700750102
发表时间：
1958-01-01
期刊：
JOURNAL OF PATHOLOGY AND BACTERIOLOGY
影响因子：
--
作者：
DIBLE, JH
通讯作者：
DIBLE, JH
影响因子：
3.8
作者：
Jeude, M.;Dittrich, B.;Buechs, J.
通讯作者：
Buechs, J.