Metabolic control analysis enabled the improvement of the L-cysteine production process with Escherichia coli

Abstract

L-cysteine is an amino acid with relevance to the pharmaceutical, food, feed, and cosmetic industry. The environmental and societal impact of its chemical production has led to the development of more sustainable fermentative L-cysteine production processes with engineered E. coli based on glucose and thiosulfate as sulphur source. Still, most of the published processes show low yields. For the identification of further metabolic engineering targets, engineered E. coli cells were withdrawn from a fed-batch production process, followed by in vivo metabolic control analysis (MCA) based on the data of short-term perturbation experiments, metabolomics (LC–MS), and thermodynamic flux analysis (TFA). In vivo MCA indicated that the activities of the L-cysteine synthases of the cells withdrawn from the production process might be limiting, and we hypothesised that the L-cysteine precursor O-acetylserine (OAS) might be exported from the cells faster than it took to transform OAS into L-cysteine. By increasing the expression of the L-cysteine synthases, either sulfocysteine synthase or L-cysteine synthase, which transform OAS into L-cysteine, an improvement of up to 70% in specific L-cysteine productivity and up to 47% in the final L-cysteine concentration was achieved in standardised fed-batch processes thereby increasing the yield on glucose by more than 85 to 9.2% (w/w). Key points • Metabolic control analysis was applied to analyse L-cysteine production with E. coli • OAS export was faster than its transformation to L-cysteine • Overexpression of L-cysteine synthases improved L-cysteine productivity and yield

Challenges and Advances in the Bioproduction of L-Cysteine

“…Most of the methods described above are applied directly on the L-cysteine synthesis pathway, which has been extensively characterized so that the bottlenecks are extensively identified. However, most metabolic pathways are thoroughly interconnected with regulation interactions that have not been yet characterized [ 59 ]. These interactions must then first be mapped out in order to identify further bottlenecks that can be tackled through rational metabolic engineering methods [ 59 ].…”

“…However, most metabolic pathways are thoroughly interconnected with regulation interactions that have not been yet characterized [ 59 ]. These interactions must then first be mapped out in order to identify further bottlenecks that can be tackled through rational metabolic engineering methods [ 59 ].…”

“…However, the economic L-cysteine production by fermentation must also take into account the process engineering viewpoint. Emphasizing energy-efficient sulfur pathways [ 7 , 60 ], selecting economically viable carbon sources [ 61 ], designing scalable fed-batch processes with individual feedings of carbon and sulfur sources [ 59 ], and implementing efficient purification techniques [ 62 ] are important for the industrial-scale production of L-cysteine.…”

“…An in vivo metabolic control analysis (MCA) was applied after rapid media transition [ 68 ] and short-term perturbation experiments [ 69 , 70 ] for the identification of bottlenecks in the L-cysteine synthesis pathway of producer cells withdrawn from this fed-batch process in the production phase. Synthetic biology efforts to increase the expression of OASS-A based on the results of the in vivo MCA led to an increase in the L-cysteine product concentration by 46% in the dual-feeding, fed-batch process, reaching maximal L-cysteine concentrations of 22 g L −1 , which are the highest reported in the literature so far [ 59 ]. The L-cysteine concentration profiles of this process before and after the strain optimization are presented in Figure 3 b.…”

“…This led to an increase in the maximal achieved L-cysteine concentration to 5.1 g L −1 . Caballero et al [59] transferred the shake flask experiments of Daßler et al [51] with E. coli W3110, carrying L-cysteine production plasmids to a stirred-tank bioreactor on a 15 L scale using continuous but independent glucose and ammonium thiosulfate feedings (presented in Figure 3a), thereby increasing the maximal achieved L-cysteine concentration from 72 mg L −1 in shake flasks to 16 g L −1 in the fed-batch process. An in vivo metabolic control analysis (MCA) was applied after rapid media transition [68] and short-term perturbation experiments [69,70] for the identification of bottlenecks in the L-cysteine synthesis pathway of producer cells withdrawn from this fed-batch process in the production phase.…”