L-Cysteine and L-methionine stand out as the sole two sulfur-containing amino acids among the canonical 20 amino acids that are used in protein synthesis (Brosnan and Brosnan, 2006; Yamazaki et al., 2020). The remaining canonical amino acids are constructed exclusively from carbon, hydrogen, oxygen and nitrogen atoms. Both sulfur and oxygen are the members of Group 6 in the periodic table, enabling them to form similar covalent bonds. However, sulfur's lower electronegativity distinguishes it, contributing to the unique properties of the sulfur-containing amino acids (Brosnan and Brosnan, 2006).

L-Cysteine, characterized by its thiol group, serves as a catalyst for biological enzymes, and is particularly significant in maintaining redox conditions. L-Cysteine also plays a crucial role in folding, assembly and stability of proteins through the formation of disulfide bonds (Takagi and Ohtsu, 2017; Colovic et al., 2018; Kawano et al., 2018). Beyond its vital role in metabolism, L-cysteine has broad applications in the agricultural, food, pharmaceutical and cosmetic sectors, with the market for products containing L-cysteine experiencing significant growth (Takagi and Ohtsu, 2017; Kallscheuer, 2018; Heieck et al., 2023). L-Methionine, as an essential amino acid, is necessary for humans and other animals (Sanderson et al., 2019). It is employed in protein synthesis and acts as a precursor for various metabolites like glutathione and S-adenosylmethionine (SAM) (Park et al., 2007; Willke, 2014). Consequently, L-methionine is extensively used in the pharmaceutical sector, the food industry and as feed additives (Willke, 2014).

Until recently, the most common and cost-effective method of L-cysteine production depends on keratin hydrolysis derived from animals. However, this process raises significant environmental and health issues, including concerns related to bovine spongiform encephalopathy (Joo et al., 2017). To circumvent the drawbacks, alternative technologies such as enzymatic conversion and fermentation have been explored. The asymmetrical hydrolysis of DL-2-amino-∆2-thiazoline-4-carboxylic acid (DL-ATC) to L-cysteine using enzymes from Pseudomonas spp. is restricted by product inhibition (Shiba et al., 2002). Currently, microbial fermentation is considered a promising solution due to its cost-efficiency, scalability and eco-friendly nature (Joo et al., 2017). Several microorganisms like Escherichia coli, Corynebacterium glutamicum and Pantoea ananatis have been genetically engineered to produce L-cysteine (Takumi et al., 2017; Wei et al., 2019; Heieck et al., 2023). Among these organisms, E. coli stands out as a promising candidate due to its rapid growth in cost-effective media, robustness for industrial processes, well-characterized metabolism and available molecular tools for genetic engineering (Kawano et al., 2018). In contrast, C. glutamicum has relatively limited genetic tools and shown relatively lower productivity compared to E. coli, while the metabolism of P. ananatis is not as well-understood as that of E. coli. Notably, E. coli has achieved the highest reported titers of L-cysteine, with up to 14.16 g/L, surpassing the levels achieved in C. glutamicum (8.45 g/L) and P. ananatis (2.2 g/L) (Takagi and Ohtsu, 2017; Usuda et al., 2022; Du et al., 2023; Zhang et al., 2023b). Thus, the review mainly concentrates on E. coli for L-cysteine production.

L-Methionine, on the other hand, is primarily synthesized chemically, a process that involves hazardous substances and results in a racemic mixture of D,L-methionine, necessitating further chiral resolution by aminoacylase (Mohany et al., 2021). Microbial fermentation could overcome this hurdle, providing an alternative route for L-methionine production. Although microorganisms can synthesize L-methionine through the aspartate metabolic pathway, the production of L-methionine is often limited due to the multibranched and multilevel regulated biosynthetic pathway (Huang et al., 2018a; François, 2023). Persistent efforts have been made to develop satisfactory strains for large-scale or even industrial fermentation through systematic genetic modification and metabolic engineering. Most of these works focus on E. coli and the highest titer of L-methionine was 21.28 g/L produced by E. coli (Cai et al., 2023). A coupled fermentation-enzymatic process to produce L-methionine based on the intermediate O-succinyl-L-homoserine (OSH) or O-acetyl-L-homoserine (OAH) of the L-methionine biosynthetic pathway has been developed. In this process, the fermentation product OSH/OAH reacts with methyl mercaptan, catalyzed by OSH/OAH sulfhydrylase, to produce L-methionine (Shim et al., 2017; Zhu et al., 2021). Although this fermentation-enzymatic coupling route has been industrialized, the production scale is small compared with that of chemical synthesis. As such, microbial fermentation represents a viable method for the production of the sulfur-containing amino acids L-cysteine and L-methionine. Particularly, E. coli has been extensively researched as a preferred host strain for the fermentative production of L-cysteine and L-methionine.

This article provides an updated overview of the genetic and molecular mechanisms involved in the production of L-cysteine and L-methionine in E. coli. It covers the key metabolic pathways of these amino acids, as well as the genetic regulations mediated by feedback inhibition and transcriptional regulators. Additionally, the article explores the transport mechanisms of these desired compounds. Furthermore, the article systematically summarizes the various metabolic engineering strategies that have been employed to enhance the production of L-cysteine and L-methionine. These strategies include the identification of new targets, modulation of metabolic fluxes, modification of transport systems, dynamic regulation strategies and optimization of fermentation conditions. The provided strategies and design principles aim to facilitate the development of strain and process engineering for the direct fermentation of sulfur-containing amino acids.