Abstract
BackgroundA 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.ResultsA 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.ConclusionsThis 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.
Highlights
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
To achieve the cell densities required for many purposes batch growth, where all the glucose is added to the media at the start, will lead to overflow metabolism and acetate build up
Identification and characterisation of novel bacterial glucoamylases Glucoamylases are able to successively hydrolyse the terminal α-1,4 glycosidic links from the non-reducing ends of polysaccharides [12], predominantly generating glucose as opposed to other maltodextrins. They are the enzyme of choice for current enzyme-based fed-batch fermentation systems, the majority of previously characterised glucoamylases, and those which are primarily used for industrial purposes, are of fungal origin, largely from Aspergillus niger and Rhizopus oryzae [13]
Summary
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. By limiting the availability of glucose, the specific growth rate of cells within the culture can be reduced below the threshold required to initiate overflow metabolism, minimising the production and accumulation of acetate This concept of substrate limited fed-batch fermentation is a commonly used method to minimise the detrimental effects of overflow metabolism and increase cell densities and recombinant protein yields. Regardless of the mechanism, traditional fed-batch fermentation is often an impractical solution for smaller scale fermentations such as those in shake flasks, owing to the sophistication of the control systems required, as well as other technical difficulties including unsatisfactory flow and mixing of the small, concentrated feed volumes Many of these issues are being challenged through the development and increased availability of miniature bioreactors, for example the ambr® 250 from Sartorius or the DASbox® from Eppendorf, shake flasks still provide an inexpensive and effective way of reproducibly performing many types of industrially-relevant cell cultivations for process development, and are still widely used across industry and academia [5]
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