The amino acid L-cysteine, harbouring a thiol group, provides a high redox activity in cell metabolism, plays a crucial role in protein folding, functions as a catalytic residue of several enzymes and serves as a building block of 5-L-glutamyl-L-cysteinylglycine (GSH) and as a donor compound of sulphur, which is required for the synthesis of Fe/S clusters, biotin, coenzyme A and thiamine (1, 2).
Besides the essential function in metabolism, L-cysteine is also of considerable industrial importance, with applications ranging from pharmaceutical products and cosmetics over food production to feed additives in livestock farming.
Its market size is projected to reach a value of 683.5 million US-Dollars by 2028 at a compound annual growth rate (CAGR) of 6.4% during 2022–2028 with an annual global production volume of 14.000 t in 2015 (3). To date, the cheapest and thereby most prevalent means of L-cysteine production involves chemical hydrolysis of - and extraction from - keratinous biomass, such as feathers, pig bristles and animal hair by means of electrolysis (4). Up to 27 tons of hydrochloric acid are required to obtain 100 kg of a racemic mixture of cysteine from 1.000 kg raw material (4, 5). In order to circumvent negative impacts upon the environment associated with hydrochloric waste disposal, alternative technologies such as fermentation and enzymatic conversion have been explored and rapidly gained significance since their implementation. In 2004, 12% of the globally manufactured L-cysteine global originated from fermentation (6).
The enzymatic conversion of DL-2-amino-∆2-thiazoline-4-carboxylic acid (D-ATC) to L-cysteine with Pseudomonas spp. derived enzymes is limited by product inhibition (7, 8). For biotechnological L-cysteine production, the bacteria C. glutamicum, and E. coli harbouring optimised plasmids represent the dominant expression organisms. Since yields from C. glutamicum are low (approx. 950 mg/L), E. coli is the preferred host for L-cysteine production by fermentation (9).
However, there are still major obstacles in upscaling fermentation processes with engineered microorganisms. The stability of strains with synthetic production is highly fragile and presents a challenge when implementing bioprocesses on a large scale (10, 11). Declining productivity affects the economic feasibility of fermentations over extended time periods.
Cells possess a finite pool of resources that are required for growth and homeostasis such as replication, transcription, translation, and numerous enzymatic reactions. Depending on signals from the environment and growth conditions, cells must economise on these resources to streamline their vitality and survival (12, 13). The introduction of designed plasmid constructs and the upregulation of the genetic elements for recombinant L-cysteine production pose a defiance to the tightly regulated homeostasis within host cells (14, 15). This metabolic load hinders the expression of other genes, thereby negatively affecting growth rate and promoting evolutionary pressure (16–18). In microorganisms, several concepts are reported that can lead to a selection advantage and thus to both phenotypic and genotypic variation within populations (19, 20). In bacteria, activation of mobile genetic elements, such as insertion sequences (IS) and corresponding transposons can lead to mutagenesis-based inactivation of synthetic constructs (21). In addition, expression of regulatory elements of the SOS response has been shown to increase the expression of error-prone DNA polymerases, which can indirectly induce further mutations in recombinant gene elements, such as plasmids (22, 23). These effects have a negative impact on the time dependent-productivity (space-time yield) and consequently the total yield of the biomolecule to be produced.
Fitness and productivity of a producing organism can be improved by several means, including rational metabolic engineering, adaptive laboratory evolution (ALE) (24, 25), the use of reduced and minimal genomes (26, 27), and the improvement of fermentation conditions (28). Metabolic engineering of E. coli strains optimising L-cysteine production, targets the overexpression of specific bottleneck genes (Fig. 1). In order to capture 3-P-glycerate from glycolysis and feed it into the synthesis of the precursor amino acid L-serine to finally convert it to L-cysteine, the two feedback-resistant genes serA and cysE are overexpressed (29, 30). An L-cysteine production pathway uncoupled from glycolysis involves the assimilatory reduction of sulphate. With the expression of cysM, assimilated thiosulphate is converted to L-cysteine via an intermediate step. Since large amounts of L-cysteine have an inhibitory effect on E. coli cells or are even toxic, the overexpression of an L-cysteine efflux gene (eamA) is essential (31).
With the advent of high-resolution omics technologies, system-wide characterization of metabolic stress during recombinant production was facilitated. Despite the necessity of establishing these technologies, high costs, methodological effort as well as the required bioinformatic knowhow still constrains the wide-spread applicability of these technologies (28).
This study investigates the genotypic and phenotypic characteristics of engineered L-cysteine producing E. coli strains by simulating an industrial fermentation process. Within 60 generations, L-cysteine productivity was observed to collapse by up to 85%. Hence, parallelly to the E. coli K-12 production strain W3110, a reduced genome strain almost free of any IS (MDS42), was selected as a control which showed stable L-cysteine productivity and growth fitness throughout the whole simulation. For the first time, using both comparative RNA- and plasmid deep sequencing on early and late populations, strong differences in L-cysteine and sulphur metabolism were uncovered. Moreover, predominantly IS3 and 5 family transposases appear to induce rapid plasmid rearrangements, which we investigated in an industrially relevant E. coli L-cysteine production system. Both observations, presumably in combination, disrupt L-cysteine production in late W3110 populations.