The genomic modification strategies for lysine production in Escherichia coli are primarily designed based on the lysine biosynthesis pathway in E. coli (Figure 1). The synthesis of lysine begins with L-aspartate, which is catalyzed by aspartate aminotransferase (AST) from oxaloacetate. Oxaloacetate is a key intermediate in the tricarboxylic acid (TCA) cycle. Under the catalysis of aspartate kinase (AK), L-aspartate first reacts with ATP to form L-4-asparty phosphate, a crucial initial step in the aspartate pathway. Subsequently, L-4-asparty phosphate undergoes a series of enzymatic reactions to convert into L-aspartate-4-semialdehyde, which is then catalyzed by dihydrodipicolinate synthase (DHDPS) to produce L-2,3-dihydrodipicolinate, a key intermediate in lysine biosynthesis. L-2,3-dihydrodipicolinate is then reduced by dihydrodipicolinate reductase (DHDPR) to tetrahydrodipicolinate. Through a series of enzymatic reactions, tetrahydrodipicolinate is ultimately converted into meso-2,6-diaminopimelate (DAP). This process involves several enzymes, including DAP aminotransferase and DAP decarboxylase. Finally, DAP decarboxylase catalyzes the conversion of meso-2,6-diaminopimelate into L-lysine.

Gene knockout breeding is a method of increasing the lysine production of Escherichia coli through genetic engineering (Theisen and Liao, 2017). This process typically involves targeted knockout or mutation of certain genes in the Escherichia coli genome to optimize metabolic pathways, reduce byproduct production, and increase lysine production. By knocking out metabolic pathway genes that compete with lysine, such as tyrosine and tryptophan synthesis genes, as well as key enzyme genes involved in lysine degradation pathways, the production of by-products can be reduced, lysine consumption can be reduced, and more carbon can be directed toward lysine synthesis and accumulation can be increased (Ohno et al., 2014). By knocking out or mutating feedback inhibition genes (such as dapA) and enhancing key enzymes in the lysine synthesis pathway (such as LysA), the efficiency of lysine synthesis can be further improved. By reducing the generation of by-products such as acetic acid and lactic acid, optimizing the metabolic network, and introducing new metabolic pathways, lysine production can be further optimized (Liu et al., 2023).

Gene editing techniques, including CRISPR/Cas9 and λ—Red homologous recombination, have been applied to modify the genome of Escherichia coli (Zhao et al., 2016). Composite gene editing methods have also been explored, such as combining lambda Red recombination with homologous endonucleases I-SceI or CRISPR/Cas9. Although CRISPR-Cas9 is commonly used for gene knockout, altering multiple genes remains challenging (Su et al., 2020; Feng et al., 2018). Researchers combined CRISPR-Cas9 and MetClo assembly to design a standardized iterative genome editing system for Escherichia coli. This method simplifies the process of introducing multiple gene modifications, making it more efficient (Dong et al., 2021). These techniques have enabled researchers to study gene function and optimize metabolic pathways to produce high-value products.

Targeted mutagenesis refers to the precise mutation of specific genes in the Escherichia coli genome using gene-editing technologies to enhance lysine biosynthesis or reduce lysine degradation pathways (Simon et al., 2018). By mutating key enzyme genes in the pathway, it is possible to decrease by-product formation or increase the activity of critical enzymes. For example, mutating the gene encoding phosphoenolpyruvate carboxylase (PPC) can reduce pyruvate consumption, thereby increasing the supply of lysine precursors (Shimizu and Matsuoka, 2022; Yin et al., 2024). Alternatively, mutating genes encoding regulatory factors can alleviate feedback inhibition in lysine biosynthesis. For instance, in the L-lysine biosynthesis pathway, targeted mutagenesis of the lysC gene, which encodes aspartokinase, and the dapA gene, which encodes dihydrodipicolinate synthase, can relieve feedback inhibition by L-lysine on its biosynthetic pathway (Li et al., 2023).

Promoter replacement is a method used to regulate the expression of target genes by replacing the promoters of genes in Escherichia coli (Marschall et al., 2017). By replacing the original weak promoter with a strong promoter, the transcription level of the target gene can be increased, thereby enhancing lysine synthesis (Jin et al., 2019). For example, replacing the promoter of the dapA gene with the constitutive tac promoter can achieve stable and efficient gene expression (Ying et al., 2014). Through promoter engineering, promoters that are sensitive to environmental conditions (such as temperature, pH, and nutrients) can be designed, enabling dynamic regulation of lysine synthesis (Wang et al., 2020).

Overexpressing key enzyme genes in the lysine biosynthesis pathway can enhance the activity of these enzymes, thereby increasing lysine production (Li et al., 2017). Aspartokinase is a crucial regulatory enzyme in this pathway, whose activity is subject to feedback inhibition by lysine (Lo et al., 2009). By constructing a feedback-resistant mutant of lysC and overexpressing this gene in Escherichia coli, this feedback inhibition can be alleviated, leading to increased lysine production. Dihydrodipicolinate synthase is the rate-limiting enzyme in lysine biosynthesis, and its overexpression can significantly accelerate lysine synthesis. In addition to lysC and dapA, overexpression of genes such as dapB, dapD, and lysA can also enhance the efficiency of lysine synthesis (Zhang et al., 2023). For instance, Xu J. et al. (2016) studied key enzymes in the L-lysine ester synthesis pathway of a high-yield L-lysine strain, including dihydrodipicolinate synthase (DHDPS) and aspartokinase III (AK III). They modified these enzymes to protect them from feedback inhibition and enhance their activity.

In Escherichia coli, lysine metabolism involves the tricarboxylic acid (TCA) cycle, glycolytic pathway, diaminopimelate pathway, and pentose phosphate pathway (Figure 2). By modulating the carbon flux ratio between the glycolytic and pentose phosphate pathways, the production of NADPH can be increased, which favors lysine biosynthesis (Tsuge and Kondo, 2017; Wendisch, 2017). Additionally, the introduction of a heterologous multifunctional enzyme, which replaces multiple original steps of enzyme-catalyzed reactions, can simplify the lysine biosynthetic pathway, thereby enhancing production efficiency. Furthermore, increasing the concentration of lysine synthesis precursors within the metabolic pathway can effectively boost lysine yield (Banno et al., 2018).

In the lysine biosynthesis pathway, NADH and NADPH are crucial cofactors. Optimizing the balance of these cofactors can significantly enhance lysine production (Wang et al., 2017). By introducing or overexpressing NADH oxidase, the accumulation of NADH can be reduced, thereby increasing the availability of NADPH. Researchers can also optimize the NADPH/NADH ratio by modulating the cellular cofactor regeneration systems (Liu et al., 2018). Alternatively, by knocking out or downregulating metabolic pathways that competitively consume NADPH, more NADPH can be directed toward lysine biosynthesis. For example, knocking out certain genes involved in fatty acid synthesis in Escherichia coli can reduce NADPH consumption. Lysine synthesis requires a substantial amount of ATP. Enhancing ATP generation pathways can also improve lysine production. For instance, overexpressing ATP synthase can increase intracellular ATP levels (Gao et al., 2021). Moreover, through metabolic flux analysis (MFA), researchers can quantitatively analyze the intracellular flow of cofactors and strategically adjust metabolic pathways to optimize lysine production.

In summary, genome editing technologies hold great promise for boosting lysine production in E. coli, benefiting industries such as animal feed and biotechnology. Researchers continue to refine these methods to improve strain performance and yield (Chen et al., 2024a; Eggeling et al., 2015).

The application of biosensors in breeding lysine-producing Escherichia coli offers a novel, rapid, and sensitive method for screening and optimizing high-efficiency strains. Biosensors typically consist of a receptor element that specifically binds to the target product, such as lysine, and a reporter element that generates a measurable signal (Newman et al., 2011). By integrating these sensors into the genome of E. coli, researchers can monitor lysine concentrations in real time and select high-producing strains based on signal intensity. This approach significantly reduces the cumbersome quantitative detection steps in traditional high-throughput screening processes and greatly improves breeding efficiency. For example, Wang et al. (2016) utilized an L-lysine sensor in E. coli to screen mutant strain MU-1 from an atmospheric and room temperature plasma (ARTP) mutagenesis library, achieving a 9.05% increase in conversion rate and a 21% increase in yield compared to the original strain.

In recent years, significant progress has been made in the field of biosensor breeding for lysine-producing E. coli, driven by advancements in synthetic biology and gene editing technologies. Recent research has focused on developing more precise and multifunctional biosensors. For instance, one type of sensor is based on the transcriptional regulator LysG, which responds to high concentrations of L-lysine by promoting the expression of the lysE gene (Steffen et al., 2016). By fusing the lysE gene with a fluorescent protein gene and using fluorescence-activated cell sorting (FACS) to select cells with strong fluorescence signals, high L-lysine-producing strains can be obtained (Ye et al., 2020).

Another type of sensor is based on the interaction between L-lysine and the L-lysine aptamer riboswitch, which results in a regulatory effect (Shi et al., 2018; Wang et al., 2016). For instance, Yang et al. (2013) used an L-lysine riboswitch to regulate the expression of tetA. When the intracellular concentration of L-lysine is high, the expression of tetA is blocked, rendering the cells sensitive to tetracycline. TetA can transport Ni²⁺ into the cytoplasm, leading to cell death. If its expression is blocked, Ni²⁺ intracellular transport is prevented, allowing the cells to grow in a NiCl₂ environment. By selecting an appropriate concentration of NiCl₂ as a selection pressure, strains with high L-lysine production can be screened. They used this biosensor to optimize the promoter of phosphoenolpyruvate carboxylase (encoded by the ppc gene), which plays a key role in the distribution of metabolic flux in L-lysine production, and identified promoters from a constructed ppc promoter library that favored L-lysine ester production (Wang W. et al., 2022).

Moreover, researchers have developed multiplex biosensor systems capable of detecting multiple metabolites or intermediate products simultaneously, providing a more comprehensive assessment of metabolic pathway optimization (Chu et al., 2022). Another important advancement is the integration of machine learning with biosensors, allowing for the analysis of large datasets to predict and design more efficient sensor systems. The combination of these technologies has greatly enhanced the potential applications of biosensors in the breeding of lysine-producing strains, further advancing the production efficiency of lysine in E. coli.

In recent years, the combination of lysine biosensors and fluorescence-activated cell sorting (FACS) has established a classical high-throughput screening strategy (Della Corte et al., 2020). This system enables the screening of cells containing plasmid libraries as well as cells with genomic mutations. For instance, the study by Wang Yan et al. introduced a novel approach for evolving industrial lysine-producing strains using an L-lysine-inducible promoter. Based on intracellular enhanced green fluorescent protein (EGFP) levels controlled by the promoter, fluorescence-activated cell sorting was initially used to screen 186 strains from a library of 10 million mutants derived from a high-lysine-producing Escherichia coli strain. A multiparameter evaluation of lysine concentration, yield, and cell-specific productivity in these strains identified two mutants, MU-1 and MU-2, which achieved lysine yields of 136.51 ± 1.55 g/L and 133.29 ± 1.42 g/L, respectively, in a 5-liter fermenter. Compared to the original strain, the lysine concentration and yield of the MU-1 strain increased by 21.00 and 9.05%, respectively, while those of the MU-2 strain increased by 18.14 and 10.41%, respectively. The mutant selection and evaluation system developed in this study can be applied to continuously enhance lysine strains for industrial production (Fang et al., 2024; Schendzielorz et al., 2014).

Additionally, droplet microfluidics technology allows for high-throughput screening in tiny droplets. Using Mass-Activated Droplet Sorting (MADS), cell variants with high lysine production can be screened based on signal intensity measured by electrospray ionization mass spectrometry (ESI-MS) (Payne et al., 2023). Strategies based on tRNA modification can be used to effectively identify and screen strains with high amino acid yields (Blay et al., 2020). For example, a research group at Beijing Institute of Technology proposed a strategy called AMINO, which screens high-producing amino acid strains based on synthetic tRNA CUA. This strategy utilizes aminoacylation reactions to reduce the affinity of tRNA for aminoacyl-tRNA synthetase by modifying the anticodon of tRNA, thereby screening for strains with high yields of the target amino acid (Guo et al., 2023). Other studies have employed large-scale activated droplet sorting (MADS) technology to screen E. coli mutants growing in microdroplets in a high-throughput manner. This method selects droplets based on signal intensity measured by electrospray ionization mass spectrometry (ESI-MS), enabling screening at a rate of 0.5 samples per second, thus rapidly identifying high-lysine-producing mutants (Payne et al., 2023).

Directed evolution simulates the natural selection pressures in vitro, allowing Escherichia coli to gradually adapt to high-lysine production environments, thereby selecting mutant strains capable of maintaining high growth and production levels under elevated lysine concentrations (Matsui and Asano, 2015). Recently, Xurong Yao and colleagues designed a synthetic acid-tolerance module to finely regulate the expression of multifactorial gene blocks. Through directed evolution, they developed an acid-responsive asr promoter library, from which four variants were selected for constructing the synthetic module. The laboratory strain Escherichia coli MG1655 was first selected for growth under mildly acidic conditions (pH 5–6), followed by an evaluation of the lysine production performance of the industrial lysine-producing E. coli strain SCEcL3. The results demonstrated that this synthetic biology strategy significantly enhanced the robustness and productivity of both strains in mildly acidic environments (Yao et al., 2022).

Significant progress has been made in breeding Escherichia coli for lysine production using microbial consortia design. Researchers have combined genetically engineered lysine-assimilating E. coli with glutamate-assimilating Corynebacterium glutamicum to form a synthetic symbiotic consortium. In this alliance, E. coli secretes fructose as a carbon source, which C. glutamicum utilizes for growth and metabolism. Meanwhile, C. glutamicum synthesizes and secretes L-lysine, which is then utilized by E. coli. This mutualistic relationship effectively converts sucrose into L-lysine (Jia et al., 2016). This approach not only increases L-lysine production and reduces costs but also provides environmental sustainability benefits. By leveraging the metabolic capabilities of each organism, the consortium optimizes resource utilization and enhances overall process efficiency. Future research may further refine these microbial communities to expand their applications in biotechnology and industrial processes (Figure 3).

Fermentation temperature is one of the critical factors affecting lysine production efficiency in Escherichia coli. Temperature directly influences the metabolic activity, enzyme functionality, and growth rate of the cells. During lysine fermentation, a temperature of approximately 37°C is typically chosen, as it is the optimal growth temperature for E. coli, maximizing biomass accumulation and lysine yield. However, excessively high temperatures can intensify cellular stress responses and reduce enzyme activity, leading to a decrease in lysine production. Conversely, lower temperatures may slow metabolic rates but can help reduce by-product formation and extend the fermentation period. Therefore, precise temperature control is crucial for optimizing lysine production (Ma et al., 2021).

Recent advancements have shown that dynamic regulation of fermentation temperature can further enhance lysine production efficiency. For instance, maintaining a higher temperature during the initial phase of fermentation can promote rapid E. coli growth and early product accumulation, while lowering the temperature in the later stages can reduce by-product formation and prolong cell viability, thereby improving the final lysine yield. Additionally, some studies have explored the application of temperature-sensitive regulatory elements, enabling E. coli to automatically adjust its metabolic pathways in response to temperature changes, maximizing lysine synthesis. By utilizing computer simulations and systems biology approaches, researchers can predict cellular metabolic behavior under various temperature conditions, optimizing the fermentation process. The application of these dynamic temperature control strategies and intelligent regulatory methods holds significant promise for advancing the industrial-scale production of lysine.

Dissolved oxygen (DO) plays a crucial role in the fermentation process of L-lysine production by Escherichia coli, influencing cell growth, metabolic activities, and the final yield of L-lysine. E. coli is an aerobic microorganism, and during fermentation, an adequate oxygen supply can promote efficient carbon source metabolism, thereby increasing cell growth rates and product accumulation. However, excessively high DO levels may induce oxidative stress, inhibiting L-lysine synthesis, while insufficient DO can lead to the accumulation of metabolic byproducts and reduced fermentation efficiency. Therefore, maintaining an optimal DO level is essential for the efficient production of L-lysine by E. coli.

The intelligent monitoring and dynamic control of DO during fermentation have become focal points of research. Advanced sensor technology is utilized to monitor DO levels in real time, and in combination with computer control systems, enables dynamic adjustment of DO to meet the changing oxygen demands at different stages of the fermentation process (Zhou et al., 2011). Additionally, research has explored methods such as optimizing agitation speed, aeration rates, and employing microbubble oxygenation techniques to improve oxygen transfer efficiency, ensuring that E. coli obtains sufficient oxygen supply under high-density culture conditions.

pH directly influences the physiological state of cells, enzyme activity, and the balance of metabolic pathways. During the initial stages of fermentation, a lower pH favors rapid growth of Escherichia coli; however, as fermentation progresses, the accumulation of L-lysine can lead to acidification of the culture medium. If not controlled, the resulting pH drop may inhibit cellular activity and reduce L-lysine yield. Therefore, maintaining an optimal pH is crucial for achieving efficient L-lysine production. Buffer systems can be employed to stabilize the pH during fermentation and minimize its impact on L-lysine production. For example, adding pH buffers like phosphate or adjusting the initial pH of the medium can provide high buffering capacity and consistent pH levels (Ferreira et al., 2015). The addition of weak bases such as calcium carbonate to neutralize fermentation acids can also help regulate pH. Additionally, the use of ammonia for neutralization is also another popular approach.

The composition of the fermentation medium is crucial for enhancing lysine production in Escherichia coli. Traditional fermentation media typically consist of carbon sources, nitrogen sources, inorganic salts, trace elements, and vitamins. Common carbon sources include glucose, sucrose, and corn steep liquor, where the choice and concentration of the carbon source directly affect the growth rate of the microbial cells and lysine accumulation. Nitrogen sources usually involve yeast extract, soybean meal hydrolysate, or inorganic nitrogen (such as ammonium salts) to provide essential amino acids and other nitrogenous compounds necessary for cell growth. Additionally, inorganic salts and trace elements like phosphates, sulfates, calcium, magnesium, and zinc play vital roles in metabolic activities, while vitamins such as biotin and niacin are important for maintaining enzyme activity and promoting metabolism (Wang et al., 2023). Through optimization methods like response surface analysis, the optimal conditions for the medium composition can be systematically determined. In recent years, attention has also been given to non-traditional carbon sources, such as glycerol and cellulosic hydrolysates, as potential medium alternatives. These novel carbon sources not only enhance lysine yield but also offer advantages like reducing production costs and minimizing waste discharge. Furthermore, the development of E. coli specific media that can adapt to high-density fermentation, as well as multifunctional media formulations that integrate pH adjustment and foam control, has become a current research focus.