Exchange of the L-cysteine exporter after in-vivo metabolic control analysis improved the L-cysteine production process with engineered Escherichia coli.

BACKGROUND

L-Cysteine is a proteinogenic amino acid of high pharmaceutical and industrial interest. However, the fermentation process for L-cysteine production is faced with multiple obstacles, like the toxicity of L-cysteine for the cells, the low carbon yield of the product, and the low selectivity of the L-cysteine exporter. In previous work, in-vivo metabolic control analysis (MCA) applied to an L-cysteine fed-batch production process with E. coli, followed by the targeted metabolic engineering to reduce an intracellular O-acetylserine (OAS) deficiency, resulted in a significant improvement of the L-cysteine production process with the new producer strain.

RESULTS

In this work, in-vivo MCA was applied to the L-cysteine fed-batch production process with the new producer strain (E. coli W3110 pCysK). The MCA indicated that a simultaneous increase in the exporter's expression and selectivity is required to increase the L-cysteine production further. The exchange of the L-cysteine exporter YdeD present in the plasmid pCysK for the potentially more selective exporter YfiK led to an increase of the maximal L-cysteine concentration by the end of the fed-batch process of 37% to a final concentration of 33.8 g L. The L-cysteine production could also be extended for at least 20 h due to conserved cellular activity as a result of the reduction of carbon loss as OAS.

CONCLUSIONS

It could be shown that the in-vivo MCA methodology can be utilised iteratively with cells from the production process to pinpoint targets for further strain optimisation towards a significant increase in the L-cysteine production with E. coli. The use of this technology in combination with process engineering to adapt the fed-batch process to the modified strain may achieve a further improvement of the process performance.