Improved production of tryptophan in genetically engineered Escherichia coli with TktA and PpsA overexpression.

Tong Shen,Qingyang Xu,Xixian Xie,Ning Chen,Qing Liu

doi:10.1155/2012/605219

Tong Shen, Qingyang Xu + Show 3 more

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https://doi.org/10.1155/2012/605219

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Abstract

Intracellular precursor supply is a critical factor for amino acid productivity. In the present study, ppsA and tktA genes were overexpressed in genetically engineered Escherichia coli to enhance the availability of two precursor substrates, phosphoenolpyruvate and erythrose-4-phosphate. The engineered strain, TRTH0709 carrying pSV709, produced 35.9 g/L tryptophan from glucose after 40 h in fed-batch cultivation. The two genes were inserted, independently or together, into a low-copy-number expression vector (pSTV28) and transferred to TRTH0709. Fed-batch fermentations at high cell densities of the recombination strains revealed that overexpression of the ppsA gene alone does not significantly increase tryptophan yield. On the other hand, overexpression of the tktA gene, alone or with the ppsA gene, could further improve tryptophan yield to a final tryptophan titer of 37.9 and 40.2 g/L, respectively. These results represent a 5.6% and 11.9% enhancement over the titer achieved by TRTH0709. No evident genetic modifications leading to growth impairment were observed.

Highlights

The aromatic tryptophan is a very important amino acid that is widely used in medicine and as a supplement in animal feeds
Tryptophan can be manufactured through bacterial fermentation by two representative producer organisms, Corynebacterium glutamicum and Escherichia coli
In E. coli, aromatic metabolites are generated from the condensation reaction between phosphoenolpyruvate (PEP) and erythrose 4phosphate (E4P) to form 3-deoxy-D-arabinoheptulosonate 7-phosphate (DAHP)

Summary

Introduction

The aromatic tryptophan is a very important amino acid that is widely used in medicine and as a supplement in animal feeds. Once PEP has been converted to pyruvate by either the phosphotransferase system or the pyruvate kinases, it is less likely to be converted back to PEP because of the high energy cost This stoichiometric limitation may be overcome by overexpression of PEP synthase, which is coded by ppsA, so that more carbon flux will be directed into the aromatic pathway [4]. Another way to increase PEP supply is to recycle PEP from oxaloacetate (OAA). Overexpression of Tal in strains which already overexpress TktA did not show a further increase in production of aromatics This result was attributed to the saturation of E4P supply when TktA was overexpressed [7]

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