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Review

From Gene to Protein: Advances and Challenges in Microbial Production of Immunoglobulins

by
Xinhui Pang
1,
Xin Song
1,
Yongjun Xia
2,
Guangqiang Wang
1,
Xinxin Liu
1,
Zhiqiang Xiong
1,* and
Lianzhong Ai
1,2
1
Shanghai Engineering Research Center of Food Microbiology, School of Health Science and Engineering, University of Shanghai for Science and Technology, Shanghai 200093, China
2
School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China
*
Author to whom correspondence should be addressed.
Fermentation 2026, 12(6), 296; https://doi.org/10.3390/fermentation12060296 (registering DOI)
Submission received: 30 April 2026 / Revised: 18 June 2026 / Accepted: 18 June 2026 / Published: 22 June 2026
(This article belongs to the Section Microbial Metabolism, Physiology & Genetics)
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Abstract

Immunoglobulins exhibit important biological functions, including the neutralization of cytotoxins, enhancement of phagocytic activity, and activation of the complement system, which have driven their widespread application in both the food and pharmaceutical industries. Due to their low cost and short production cycles, microbial expression systems such as bacteria and yeast have been increasingly developed in recent years for immunoglobulin production. However, microbial systems face considerable challenges in ensuring proper protein folding, accurate chain assembly, and the soluble expression of full-length immunoglobulins. Recent optimization strategies have focused on host engineering (e.g., modulating secretion pathways and chaperone proteins), the coordinated regulation of expression elements (e.g., optimizing the light-to-heavy chain ratio), and regulation of fermentation processes. In addition to summarizing the above strategies, this review discusses the progress made in expressing both full-length immunoglobulins and antibody fragments across different microbial hosts, analyzes the advantages and limitations of each system, and explores potential future directions, aiming to provide a reference for the efficient heterologous expression of immunoglobulins.

1. Introduction

Immunoglobulins (Igs), also known as antibodies, are a class of proteins produced by plasma cells differentiated from B lymphocytes upon antigen stimulation, characterized by their ability to specifically bind to the corresponding antigen. To date, more than 140 therapeutic immunoglobulins have been approved by the U.S. Food and Drug Administration (FDA) for the clinical treatment of human diseases [1]. In recent years, full-length immunoglobulin G (IgG) and its fragments (e.g., Fab fragments, single-chain variable fragments [scFv]) have been widely applied in the treatment of immune-related diseases and in the food industry, owing to their specificity and functional activity [2]. However, the complex structures and high molecular weights of these proteins impose stringent demands on heterologous expression host systems. Consequently, achieving efficient, large-scale production of high-quality immunoglobulins has become a central focus of current research.
Currently, the vast majority of therapeutic immunoglobulins are produced using mammalian cell expression systems, primarily due to their superior capacity for proper protein folding and humanized glycosylation modifications. However, this system exhibits several limitations, including operational complexity, prolonged production cycles, high costs, and significant susceptibility to environmental factors during fermentation [3]. For immunoglobulin fragments such as Fab and scFv, or for certain immunoglobulins that do not require complete glycosylation modifications, the excessively long development cycles and high costs associated with mammalian cell systems have become major bottlenecks for heterologous immunoglobulin expression. Therefore, the development of a low-cost, short-cycle, and easily scalable expression system has become the key to overcoming the current obstacles in antibody production.
Microbial systems, particularly those based on E. coli, yeast, Aspergillus, and lactic acid bacteria [4,5,6,7], are increasingly emerging as important complements or alternatives to mammalian cell systems, owing to their advantages including ease of manipulation, short production cycles, low costs, and well-established high-density fermentation processes. Although microbial systems still face challenges such as low folding efficiency, inclusion body formation, and the lack of endogenous humanized glycosylation pathways when handling multimeric proteins with complex disulfide bonds like IgG, gram-level yields of functional Fab and scFv fragments have been successfully achieved through the engineering of microbial secretion pathways, the co-expression of molecular chaperones, or the use of cytoplasmic oxidative folding strategies. Furthermore, recent studies have demonstrated that full-length IgG can also be synthesized in certain engineered bacterial strains. With the advancement of synthetic biology and high-density fermentation technologies, microbial cell factories are expected to emerge as a key platform for the next generation of low-cost, high-performance immunoglobulin manufacturing. This review summarizes the current status of recombinant immunoglobulin expression in various microbial systems, discusses the bottlenecks and challenges limiting large-scale production, and highlights innovative strategies developed to overcome these obstacles.

2. Immunoglobulin Structure

The overall structure of an immunoglobulin typically consists of two identical light chains (LC) and two identical heavy chains (HC), arranged in a “Y”-shaped configuration, with the chains linked by disulfide bonds (Figure 1) [8]. The LC has a molecular weight of approximately 25 kDa and exists in two functionally identical types, the λ type and the κ type. The HC, with a molecular weight ranging from 50 to 70 kDa, can be classified into five types—α, δ, ε, γ, and μ—based on differences in its constant region, which correspond to distinct antibody classes [9,10]. In terms of functional architecture, immunoglobulins can be divided into the variable region (V region) and the constant region (C region) [11]. The variable region, formed jointly by the N-termini of the LCs and HCs, is responsible for antigen recognition and binding. The constant region extends from the base of the two arms of the “Y”-shaped structure throughout the stem region, and its amino acid sequence is relatively conserved. Following proteolytic cleavage by papain, the immunoglobulin is cleaved into two identical antigen-binding fragments (Fab fragments) and one crystallizable fragment (Fc fragment) [12]. Among these, the Fc region is composed of the constant regions of two HCs. Although it does not directly participate in antigen recognition, it can interact with a variety of cell surface receptors [13]. The Fab region, which consists of the LC, the variable region of the heavy chain (VH), and a portion of the heavy chain constant region, is responsible for the specific binding of antigens [14].

3. Microbial Heterologous Expression System for Immunoglobulin

Microbial expression systems offer advantages such as short production cycles, low costs, and operational convenience, and have thus been widely employed for the expression of immunoglobulins. Among these, the E. coli system is the most extensively utilized due to its well-characterized genetic background and high protein yields. The yeast system possesses relatively robust eukaryotic protein expression capabilities. The Aspergillus system exhibits strong secretory capacity, which helps reduce downstream purification costs. The lactic acid bacteria system, in turn, offers in vivo delivery capabilities with enhanced targeting efficiency [15,16,17,18]. However, the N-glycosylation patterns of immunoglobulins expressed in different microbial hosts vary considerably (Figure 2).

3.1. Yeast Expression System

Yeast, which combines the ease of cultivation characteristic of microorganisms with the post-translational modification capabilities of eukaryotic systems, has emerged as one of the important expression systems for the production of recombinant immunoglobulins [19]. Among them, Pichia pastoris and Saccharomyces cerevisiae are the most widely used hosts at present, owing to their well-defined genetic backgrounds and ease of manipulation [20]. In P. pastoris, recombinant immunoglobulin genes are commonly expressed under the control of the methanol-inducible alcohol oxidase (AOX) promoter; alternatively, some studies employ the constitutive glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter to circumvent the metabolic burden associated with methanol induction [21]. In recent years, through optimization of the AOX1 promoter, screening of signal peptides, and fine-tuning of the expression of the molecular chaperone HAC1, the yield of immunoglobulin VHH in fermenters has reached 2.13 g/L [22].
Although yeast cells possess N-glycosylation pathways similar to those of mammalian cells, the immunoglobulins expressed in yeast typically carry high-mannose-type glycan chains, which may raise immunogenicity concerns [23]. To address this limitation, Parsaie Nasab et al. [24] knocked out the high-mannosyltransferase genes ALG3 and ALG11 to block the synthesis of high-mannose precursors, thereby effectively reducing the level of high-mannose glycosylation. Overall, despite certain limitations, the advantages of the yeast expression system, including short production cycles, low costs, and the capability for high-density fermentation, endow it with considerable application prospects in the production of recombinant immunoglobulins.

3.2. Escherichia coli Expression System

The E. coli expression system, owing to its rapid growth rate and low culture cost, has been widely employed for the production of recombinant immunoglobulins, particularly for immunoglobulin fragments that are relatively simple in structure and do not require post-translational modifications [25,26]. In this system, isopropyl-β-D-thiogalactopyranoside (IPTG) is commonly used as an inducer to direct the secretion of recombinant immunoglobulins into the periplasmic space [27]. The cytoplasm of E. coli is strongly reducing, which is unfavorable for the formation of disulfide bonds in immunoglobulins; consequently, recombinant proteins predominantly accumulate as inclusion bodies and require in vitro renaturation to regain activity. In contrast, the periplasmic space provides an oxidizing environment that facilitates the correct formation of disulfide bonds and the acquisition of soluble, active fragments. However, achieving transmembrane translocation necessitates the fusion of a signal peptide (e.g., pelB, ompA) to the N-terminus of the immunoglobulin gene [28].
Furthermore, the genetically engineered Shuffle® series of strains, through the knockout of glutathione reductase and thioredoxin reductase genes in the cytoplasm, disrupts the reducing capacity of the cytoplasm and converts it into an oxidizing environment. Concurrently, these strains express disulfide bond isomerases, thereby enabling the formation of disulfide bonds in the cytoplasm and the production of soluble, active immunoglobulins [29]. E. coli itself contains endotoxins, posing potential safety concerns. E. coli Nissle 1917 (EcN), a probiotic strain safely used in clinical settings, naturally possesses lipopolysaccharide structures with low immunogenicity. As a “next-generation biomanufacturing platform,” its advantage lies in the low endotoxin content of heterologous protein products, which streamlines the purification workflow and reduces downstream costs. However, wild-type EcN lacks an efficient expression system, making it difficult to meet the large-scale production demands for immunoglobulins. In recent years, researchers have successfully constructed a high-yield strain, EcN: T7ΔompTΔiclRΔarcA, by inserting the T7 RNA polymerase gene into the chromosome and employing CRISPR/Cas9-mediated genome editing to knock out the genes encoding outer membrane protease (ompT), glyoxylate pathway transcriptional repressor (iclR), and aerobic respiration regulatory factor (arcA). The protein yield of this engineered strain reached 89.3% of that of E. coli BL21(DE3) [30]. This advancement positions the E. coli expression system as a promising biomanufacturing platform that combines both economic efficiency and clinical application potential.

3.3. Bacillus Subtilis Expression System

B. subtilis is the model organism of Gram-positive bacteria and is recognized by the FDA as a Generally Recognized as Safe (GRAS) strain. It is endotoxin-free, possesses a strong secretion capacity that simplifies downstream purification, and has well-established molecular biology tools that facilitate genetic engineering. In addition, it can be cultivated to high cell densities, making it a highly advantageous expression host [31]. To enhance immunoglobulin expression levels and reduce the metabolic burden imposed by non-essential genes, researchers have streamlined the B. subtilis genome by eliminating 20% of its non-essential genes. On this basis, they constructed protease-deficient strains by knocking out major protease genes such as aprE and nprE, which significantly increased the immunoglobulin content in the fermentation supernatant [32]. Neither protease deletion nor chaperone overexpression alone was sufficient to increase immunoglobulin yield; only the combination of both strategies improved expression efficiency. During the expression of single-chain antibody fragments (SCA), low expression levels of the chaperones GroES/GroEL and DnaK/DnaJ/GrpE, deficiency of PrsA, and high activity of the WprA protease were identified as the primary reasons for low protein production. By enhancing chaperone co-expression while simultaneously reducing protease activity, the secreted yield of SCA was increased to 10–15 mg/L [33]. Currently, only immunoglobulin fragments have been successfully expressed in B. subtilis; therefore, the expression of full-length immunoglobulins and the lack of a suitable glycosylation machinery remain limitations of this expression system.

3.4. Aspergillus Expression System

Aspergillus species are recognized as “superfactories” for protein secretion and are designated as GRAS strains by the U.S. FDA. As eukaryotes, Aspergillus possesses post-translational modification capabilities including protein folding, disulfide bond formation, and glycosylation, which are essential for the proper assembly and functional activity of antibodies requiring complex folding and modifications, particularly full-length IgG [17]. However, the glycosylation pattern of Aspergillus is of the high-mannose type, which differs from human-type glycan chains.
To modify the glycosylation pattern of Aspergillus, Huynh et al. [34] fused the LCs and HCs of an immunoglobulin with endogenous secretory proteins from Aspergillus oryzae and employed the CRISPR/Cas9 gene editing system to knock out the Golgi α-1,6-mannosyltransferase Aooch1 gene in a protease-deficient A. oryzae strain. As a result, a yield of 39.7 mg/L of full-length immunoglobulin was obtained from the culture supernatant, and the purified immunoglobulin exhibited antigen-binding and neutralizing activities comparable to those of immunoglobulins produced in mammalian cells. In addition to full-length immunoglobulins, nanobodies, characterized by their small molecular size and simple structure, have gradually become a research focus. Some researchers have enhanced the secretory yield of nanobodies in A. oryzae by fusing them with the Taka-amylase A signal sequence and the N-terminal 28 amino acids of Rhizopus oryzae lipase [35]. The robust protein secretion capacity, post-translational modification capabilities, and generally recognized safety of Aspergillus species confer significant advantages for immunoglobulin production. However, differences in glycosylation patterns and the issue of protease degradation remain major limitations at present.

3.5. Lactic Acid Bacteria Expression System

Lactic acid bacteria (primarily Lactococcus lactis and Lactobacillus species) are probiotics commonly used in the food industry. They produce no endotoxins and possess a high level of biosafety. These bacteria can tolerate the gastrointestinal environment and colonize the intestinal mucosa. Following genetic engineering, they are capable of targeted, sustained production and release of immunoglobulin fragments. Consequently, the research focus on lactic acid bacteria is gradually expanding from their role as mere production tools toward their development as live biotherapeutic products [36].
Murakami et al. [37] found that subcutaneous injection of anti-interleukin-31 (IL-31) immunoglobulin exhibited adverse effects and posed difficulties in achieving targeted delivery to local lesions; to address this issue, the team designed an IL-31 single-chain variable fragment (scFv) based on an approved therapeutic antibody as a template and employed L. lactis as the expression host, under the control of the inducible Nisin promoter. They successfully expressed the IL-31 scFv fragment with antigen-binding activity. IL-31 is involved in the pathogenesis of pulmonary and respiratory diseases, and this study provides a new approach for the mucosal delivery of immunoglobulin fragment-based therapies using lactic acid bacteria. In response to the limitations of traditional IL-23/Th17 axis antibody therapies for intestinal inflammation, such as high costs and significant systemic immune side effects, researchers have constructed a food-grade L. lactis strain capable of secreting a REX protein blocker targeting the IL-23 receptor. The specific binding capability of this protein to the IL-23 receptor was validated through in vitro experiments [38]. An in vivo expression system (LIVE) in lactic acid bacteria has also been constructed to produce the anti-inflammatory cytokine IL-10. In a mouse model of colitis, the IL-10 expressed by this system exhibited significant anti-inflammatory effects at the mucosal surface [39]. The expression of immunoglobulins using lactic acid bacteria as a vehicle to construct novel local delivery systems provides a potential low-cost, locally acting therapeutic strategy for achieving mucosal localized delivery of biologics to modulate disease.

3.6. Comparative Summary of Microbial Expression Systems

The five microbial expression systems described above each possess distinct advantages and limitations, and the selection of an appropriate system is instrumental in improving the synthesis efficiency of different immunoglobulins (Table 1). For instance, the E. coli system, which lacks glycosylation capacity, is primarily used for the production of Fab fragments and non-glycosylated full-length IgG. The yeast expression system, in addition to producing immunoglobulin fragments, enables the expression of full-length IgG following glycosylation engineering. The Aspergillus system is capable of glycosylating full-length IgG; however, the glycan pattern is of the high-mannose type, necessitating further modification. The lactic acid bacteria system is mainly employed for the production of immunoglobulin fragments.

4. Bottlenecks in the Heterologous Expression of Immunoglobulins in Microbial Systems

The relatively low yield of immunoglobulins in stable expression systems represents a common and multifaceted challenge, the causes of which span multiple levels of cellular physiology. Immunoglobulins themselves may exert a certain degree of cytotoxicity on microbial expression hosts, thereby interfering with normal cell growth and proliferation [53]. Figure 3 provides an overview of the major molecular engineering strategies currently employed to enhance the microbial production of immunoglobulins. Within the cell, failure of proper immunoglobulin folding and assembly impedes efficient secretion, while complex post-translational modification processes can also act as bottlenecks in expression efficiency [54]. Therefore, addressing the issue of low immunoglobulin expression remains a considerable challenge.

4.1. Protein Folding and Assembly

The most fundamental challenge in the heterologous expression of immunoglobulins lies in their complex molecular architecture. Immunoglobulins are tetrameric proteins assembled from two HCs and two LCs through precise intra-chain and inter-chain disulfide bond pairing [55]. However, the cytoplasmic environment of prokaryotic expression systems such as E. coli is reducing, which is unfavorable for the correct formation of disulfide bonds. Consequently, recombinant proteins often accumulate as misfolded inclusion bodies. Furthermore, the LCs and HCs must be synthesized, translocated, and assembled in an appropriate stoichiometric ratio. Studies have shown that co-expression of the molecular chaperones GroEL and GroES can facilitate the correct folding of polypeptide chains, thereby enhancing the solubility of immunoglobulins [5]. In addition to co-expressing molecular chaperones, the use of signal peptides to direct immunoglobulins to the periplasmic space also promotes correct folding. A nanobody is a single-domain antibody fragment derived from naturally occurring heavy-chain-only antibodies found in certain organisms, such as camelids, and consists solely of the variable domain of the heavy chain (VHH) while retaining antigen-binding capacity [56]. When comparing the efficiencies of three signal peptides OmpA, PelB, and L-AsPsII, immunoglobulins fused with PelB or OmpA achieved a soluble nanobody yield of approximately 0.4 mg/L in shake flask cultures [27], because the periplasmic space provides an oxidizing environment. Distinct from traditional optimization strategies, researchers have recently proposed a translation pausing strategy, which involves redesigning the coding DNA sequence to introduce mutations that alter translation pause sites without changing the amino acid sequence, thereby promoting correct protein folding and improving the solubility of heterologous proteins in E. coli [57].

4.2. Glycosylation Modification

Asparagine-linked glycosylation (N-glycosylation) of the Fc region of immunoglobulin molecules is critically important for their effector functions. Prokaryotic systems (such as E. coli) lack glycosylation capacity. Although non-glycosylated immunoglobulins produced in such systems retain antigen-binding ability, they cannot effectively mediate downstream immune effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) [4]. Yeast systems (such as P. pastoris) possess glycosylation capacity but typically produce high-mannose-type glycan chains, making them unsuitable for the direct production of therapeutic immunoglobulins [58]. Bacteria such as E. coli lack oligosaccharyltransferase (OST) enzymes capable of installing N-glycans at the conserved N297 site of the Fc region. Engineered bacterial strains with N-glycosylation capabilities have now been developed. Sotomayor et al. [59] discovered an OST enzyme from Desulfovibrio marinus (DmPglB) that utilizes bacterial-derived N-glycans to glycosylate the Fc fragment. The Fc expressed using this method was able to bind key receptors involved in ADCC, providing a new strategy for producing fully functional immunoglobulins in bacterial systems. Immunoglobulins expressed by wild-type P. pastoris possess high-mannose-type N-glycans and phosphomannose modifications, which can affect protein function. In a study on the receptor-binding domain (RBD), researchers found that phosphomannose modification increases the surface charge of RBD and enhances its adsorption to aluminum hydroxide adjuvants, thereby boosting the immune response [60]. Although that study used the RBD antigen as a model, its glycosylation strategy offers valuable insights for the production of near-humanized glycosylated immunoglobulins in yeast.

4.3. Secretion and Production of Intact Antibodies

Efficient expression of full-length immunoglobulins in microorganisms has remained a persistent challenge. E. coli, due to its lack of glycosylation capacity, is primarily used for the production of smaller antibody fragments such as Fab and scFv. Although yeast systems can produce full-length immunoglobulins, their yield and production cycle still require further optimization [61]. The LCs and HCs of immunoglobulins can be efficiently expressed in P. pastoris, but the assembly efficiency between them remains relatively low. This is attributed to the retention of the HC in the endoplasmic reticulum (ER), which triggers protein degradation mechanisms. To address this issue, ER-associated degradation (ERAD)-related genes were knocked out; however, this did not improve the LC and HC assembly efficiency. Notably, the retention of the HC in the ER upregulates the expression of the BiP (binding immunoglobulin protein) chaperone. Consequently, it is hypothesized that the HC binds to BiP, thereby spatially hindering its assembly with the LCs [62]. Therefore, the rational design of molecular chaperones to favor the correct assembly of immunoglobulin LCs and HCs represents a promising strategy.

4.4. Effects of Protein Expression on Host Cells

Extensive misfolding and aggregation of immunoglobulins within the cell can impose a “toxic” stress on the host. The use of low-copy plasmids instead of high-copy plasmids reduces the leaky expression of the target protein prior to induction, thereby alleviating the toxic stress on the host strain [63]. Immunoglobulin fragments contain disulfide bonds and require proper folding within the oxidative environment of the periplasmic space. When expression levels exceed the folding capacity of the periplasm, unfolded or misfolded intermediates aggregate to form inclusion bodies, exerting a toxic effect on the cell [64]. The substantial retention of immunoglobulins within the ER triggers ER stress. In response to this condition, the cell initiates the unfolded protein response (UPR) as a defense mechanism [65]. Studies have found that the expression of single-chain antibody (scAb) can lead to cell lysis, whereas co-expression of the molecular chaperone SKP with the target protein effectively prevents this phenomenon [66]. However, if ER stress persists and cannot be alleviated, the UPR shifts from a “self-rescue” mode to a “suicide” mode, causing irreversible damage to the cell. Aberrant activation of the melanoma differentiation-associated gene 5 (MDA5) protein can directly trigger sustained activation of ER stress signaling, disrupt intracellular protein homeostasis, and exert destructive effects on cells by driving UPR-related signaling pathways [67]. A thorough understanding of the impact of protein toxicity on the expression efficiency of immunoglobulins can help elucidate the intrinsic mechanisms underlying low expression levels and provide a theoretical basis for the development of corresponding optimization strategies. Furthermore, carbon source allocation significantly impacts protein expression, as cells must reroute resources such as amino acids and glucose toward heterologous protein synthesis, frequently at the cost of reduced growth [68].

4.5. Translation Barriers Caused by Codon Preferences

Codon preference exerts a decisive influence on the expression efficiency of immunoglobulins. As shown in Figure 4, the traditional view holds that codon optimization is primarily aimed at ensuring smoothness of the translation process [69]. If the immunoglobulin gene contains a large number of “rare codons”—codons that are used with extremely low frequency by the host cell—ribosomes may pause or even dissociate at these positions [70]; this not only slows down the translation rate but may also lead to the failure of protein synthesis. However, a 2024 study by Giguère and colleagues at the Massachusetts Institute of Technology, published in Science, proposed a different perspective, pointing out that immunoglobulin genes have evolved a conserved codon usage pattern characterized by the frequent utilization of “rare codons.” In antibody-secreting cells (such as plasma cells), the cell upregulates the inosine (I34) modification level of tRNAs, thereby enhancing the “wobble” decoding capacity of tRNAs and enabling a single tRNA to recognize multiple otherwise mismatched codons [71]. This discovery reveals that the codon usage pattern of immunoglobulin genes has co-evolved a precisely adapted relationship with the tRNA pool within B cells. Therefore, the impact of codons on immunoglobulin expression efficiency no longer depends solely on simple codon optimization but is closely related to the tRNA abundance of the host cell. The synergistic action of these two factors ensures efficient immunoglobulin synthesis while maintaining proper folding and avoiding ER stress and cellular toxicity.

5. Impact of Bioreactors on the Heterologous Expression of Immunoglobulins in Microorganisms

5.1. Effects of Fermentation Process Parameters on Immunoglobulin Yield

In microbial cell factories (e.g., E. coli, P. pastoris), the expression of immunoglobulins (IgG, Fab, scFv) depends not only on genetic construction but is also significantly influenced by fermentation process parameters [72]. Due to the complex molecular structure of immunoglobulins characterized by multiple disulfide bonds, high folding requirements, and glycosylation modifications, even minor fluctuations during fermentation can directly affect product yield, quality, and biological activity. The promoting effect of high-density fermentation on immunoglobulin expression has been validated across multiple microbial hosts. In the P. pastoris expression system, the yield of nanobodies has reached 2.13 g/L, representing a 49.8-fold increase compared to the initial shake flask culture strain [22]. Furthermore, in glycoengineered P. pastoris strains cultivated under scaled-up conditions, the yield of monoclonal antibodies has also stably exceeded 1 g/L [73].
However, during fermentation, variations in process parameters also affect the expression level and quality of immunoglobulins. In P. pastoris, the AOX1 promoter-driven expression system is strictly induced by methanol. Studies have shown that even the presence of extremely low residual glycerol concentrations (below 6 mg·g−1·h−1) can inhibit the transcriptional activity of the AOX1 promoter, thereby reducing antibody expression levels. To overcome this challenge, the use of an online methanol sensor to achieve precise methanol feeding control can effectively avoid substrate inhibition and optimize induction performance [74]. In high-density fermentation, in addition to the factors mentioned above, parameters such as temperature, pH, and dissolved oxygen concentration must be maintained within specific ranges. Under the conditions of pH 5.5–7.5, temperature 16–24 °C, dissolved oxygen concentration 0.85–3.40 mg/L, and a methanol feed rate of 9–19 mg·g−1·h−1, glycoengineered P. pastoris produces over 1 g/L of functional immunoglobulin [75]. Fluorescence-activated cell sorting (FACS) based on cell surface markers can enrich high-producing P. pastoris strains approximately 5000-fold from a low-frequency mixture of 1:100,000. Following sorting, the immunoglobulin yield of the isolated strains can be increased by 30–300% [76].

5.2. Bottlenecks and Challenges of High Density Fermentation

Although high-density fermentation significantly increases productivity, multiple challenges remain during the expression of immunoglobulins. First, there is the pressure of protein folding and secretion. High-level expression of recombinant proteins often leads to ER stress, triggering the UPR, which may result in protein degradation or growth arrest of the cells. Second, the overexpression of heterologous proteins competes for the cell’s own energy and amino acid resources, leading to reduced competitiveness of the production strain within the fermenter. Third, in the high-cell-density fermentation environment, heterologous proteins are susceptible to proteolytic hydrolysis by host proteases, thereby affecting product integrity and recovery yield.
To mitigate the aforementioned issues, optimization of both the expression system and fermentation process is required. Studies have shown that overexpression of the molecular chaperones immunoglobulin-binding protein (BiP) or PDI in S. cerevisiae can increase the secretion titer of single-chain immunoglobulins by 2- to 8-fold [77]. In P. pastoris, overexpression of the core regulatory factor MSN4 or its synthetic variant synMSN4 has been shown to increase recombinant protein secretion titers by up to 4-fold, enabling scFv yields exceeding 2.5 g/L and VHH titers surpassing 8 g/L [78]. In the process of heterologous protein production in E. coli, it has been observed that when anabolic capacity does not match catabolic capacity, E. coli growth is inhibited. This phenomenon occurs because excessive carbon uptake leads to the secretion of byproducts such as pyruvate, acetate, and glutamate, prompting the cell to reduce glucose uptake and consequently suppress cell growth [79].
The two-stage temperature-shift strategy has been widely applied in the production of immunoglobulins. The initial stage, which involves biomass accumulation, requires the maximum specific growth rate to accelerate cell division. In the subsequent stage, the metabolic rate is reduced to alleviate ER stress and inhibit protease activity, thereby enabling the stable expression of immunoglobulin [41,80]. Kasli et al. [81] demonstrated in their study that induction at 20–25 °C is superior to induction at 37 °C. Low-temperature induction slows down the translation rate of ribosomes, allowing proper disulfide bond pairing and folding of immunoglobulin molecules, thereby increasing the solubility of scFv by 90%. High-density cultivation of P. pastoris induces the expression of the Pep4p protease, which is secreted into the extracellular environment and attacks the hinge region of antibodies, leading to antibody fragmentation. Reducing the temperature to approximately 20 °C significantly lowers the enzymatic reaction rate, thereby protecting immunoglobulin integrity and improving immunoglobulin yield [82]. Through precise regulation of carbon source feeding strategies, environmental parameters, and co-expression of molecular chaperones, antibody yields exceeding the gram-per-liter level can be achieved in hosts such as E. coli and yeast, thereby providing a new pathway for low-cost, high-efficiency immunoglobulin production.

6. Summary and Outlook

In the fields of food and medicine, the use of microorganisms as cell factories for immunoglobulin production holds significant importance. Considerable progress has been made in the production of immunoglobulins (IgG, scFv, etc.) using microbial systems. However, the correct formation of disulfide bonds, accurate assembly of LCs and HCs, glycosylation modifications, and high-titer, high-stability industrial production remain major challenges for achieving efficient immunoglobulin expression. The impact of heterologous protein expression on host cells, translation obstacles caused by codon bias, and severe ER stress induced by high-intensity induction are also important factors restricting the stable expression efficiency of immunoglobulins. In the food industry, applications such as oral passive immunization and food safety testing require cost-effective production, food-grade hosts (e.g., lactic acid bacteria), and stability under processing and gastrointestinal conditions [83,84]. Therefore, integrated strategies including host strain engineering, optimization of expression vector elements, and regulation of fermentation conditions are essential to address these challenges and enhance immunoglobulin production in both fields.
Although protein expression efficiency has been improved through modification of target gene sequences and expression strains, achieving glycosylated full-length immunoglobulin production and resolving the problem of low expression levels for certain proteins remain the most prominent limitations of microbial expression systems. In the future, the design of target gene sequences using artificial intelligence and computational language models, combined with machine learning-assisted fermentation strategies, holds promise for overcoming the bottlenecks of non-expression or low expression levels, thereby enabling more stable and higher-yield immunoglobulin production in microbial systems.

Author Contributions

Writing—original draft preparation, X.P.; writing—review and editing, Z.X. and L.A.; supervision, Z.X., G.W. and X.L.; project administration, Z.X., X.S. and Y.X.; funding acquisition, Z.X. and L.A. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Key Research and Development Program of China (No. 2023YFF1103400), National Center of Technology Innovation for Dairy (No. 2024-KFKT-020), Key Special Project of Synthetic Biology in Shanghai “Science and Technology Innovation Action Plan” (grant No. 23HC1400900 and 25HC2830500), National Science Fund for Distinguished Young Scholars (grant No. 32025029), and Shanghai Engineering Research Center of Food Microbiology (grant No. 19DZ2281100).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

No new data were created or analyzed in this study. Data sharing is not applicable to this article.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
IgsImmunoglobulins
FDAFood and Drug Administration
IgGImmunoglobulin G
LCLight chains
HCHeavy chains
GRASGenerally Recognized as Safe
ADCCAntibody-dependent cell-mediated cytotoxicity
OSTOligosaccharyltransferase

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Fermentation 12 00296 g001
Figure 1. Schematic diagram of immunoglobulin structure. (a) Mammalian immunoglobulin G; (b) Avian immunoglobulin Y. The light chain of mammalian immunoglobulin G (IgG) consists of a variable domain (VL) and a constant domain (CL), whereas the heavy chain is composed of a variable domain (VH) and three constant domains (CH1, CH2, and CH3). The antigen-binding fragment (Fab) comprises VH, VL, CH1, and CL, while the crystallizable fragment (Fc) is formed by the CH2 and CH3 domains of the heavy chain, with an intervening hinge region, disulfide bonds are represented by red lines. In avian immunoglobulin Y (IgY), the light chain comprises VL and CL, whereas the heavy chain consists of a VH and four constant domains (CH1, CH2, CH3, and CH4). The Fab portion includes VH, VL, CH1, and CL, while the Fc region is formed by the CH2, CH3, and CH4 domains of the heavy chain, lacking a hinge region, disulfide bonds are indicated by red lines.
Figure 1. Schematic diagram of immunoglobulin structure. (a) Mammalian immunoglobulin G; (b) Avian immunoglobulin Y. The light chain of mammalian immunoglobulin G (IgG) consists of a variable domain (VL) and a constant domain (CL), whereas the heavy chain is composed of a variable domain (VH) and three constant domains (CH1, CH2, and CH3). The antigen-binding fragment (Fab) comprises VH, VL, CH1, and CL, while the crystallizable fragment (Fc) is formed by the CH2 and CH3 domains of the heavy chain, with an intervening hinge region, disulfide bonds are represented by red lines. In avian immunoglobulin Y (IgY), the light chain comprises VL and CL, whereas the heavy chain consists of a VH and four constant domains (CH1, CH2, CH3, and CH4). The Fab portion includes VH, VL, CH1, and CL, while the Fc region is formed by the CH2, CH3, and CH4 domains of the heavy chain, lacking a hinge region, disulfide bonds are indicated by red lines.
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Figure 2. Comparative N-glycosylation patterns of immunoglobulin expressed in different microbial hosts.
Figure 2. Comparative N-glycosylation patterns of immunoglobulin expressed in different microbial hosts.
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Figure 3. Key challenges in recombinant protein production and their impact on host cell physiology.
Figure 3. Key challenges in recombinant protein production and their impact on host cell physiology.
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Figure 4. Impact of codon usage bias on the expression of immunoglobulins [71].
Figure 4. Impact of codon usage bias on the expression of immunoglobulins [71].
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Table 1. Progress in immunoglobulin production by different microbial expression systems.
Table 1. Progress in immunoglobulin production by different microbial expression systems.
Expression SystemAntibody FormatEngineering
Strategies		Production
(mg/L)		Reference
E. coliIgGSelection of an appropriate expression host; 5′ UTR engineering to optimize translation initiation efficiency; co-expression of molecular chaperones (e.g., DsbC).362[40]
IgGRandom mutagenesis of the translation initiation region (TIR) sequence to select for optimal protein synthesis efficiency; co-expression of molecular chaperones (DsbC and DsbA).1050[41]
FabFusion of signal peptides (OmpA, PelB) to the N-terminus; optimization of culture temperature and induction conditions.1000[42]
scFvStrain selection (BL21(DE3) pLysS, ArcticExpress(DE3)); Plackett-Burman design for optimization of induction conditions (IPTG, 2xYT, lactose combination).34[43]
scFvEngineered strain RV04 (BW25113 Δpka ΔarcA); knockout of pka and arcA to eliminate acetate accumulation and promote growth.462[44]
P. pastorisIgGHigh-throughput screening to obtain glycoengineered strains; combination with oxygen-limited culture strategies to enhance antibody yield.1900[45]
scFvUtilization of the eukaryotic modification system of yeast to obtain soluble, correctly folded products.-[46]
scFvOverexpression of endoplasmic reticulum chaperones; upregulation of endogenous protein disulfide isomerase (PDI) expression.4000[47]
S. cerevisiaeIgGDirected evolution of the α-mating factor leader peptide to screen for mutants that significantly enhance secretion efficiency.0.1[48]
VHH-FcKnockout of protease genes (VPS30, PEP1, ALG3); medium optimization (arginine, 4-phenylbutyric acid, Tween-20).2.5[49]
B. subtilisscFvFusion of LipA signal peptide sequence of B. megaterium before scFv; using fermentation tanks for high-density cultivation.130[50]
scFvGenome streamlining of B. subtilis combined with knockout of the protease genes aprE and nprE.6[32]
AspergillusIgGFusion of A. oryzae secretory proteins to the LCs and HCs of the immunoglobulin, respectively; knockout of the Aooch1 gene in A. oryzae.39.7[34]
VHHFusion of nanobodies with two secretory guiding elements; cultivation using Sakaguchi flasks.73.8[35]
L. lactisscFvCodon optimization of the immunoglobulin gene sequence; optimization of nisin induction concentration.-[51]
scFvCodon optimization of the immunoglobulin gene sequence; fusion of an appropriate signal peptide; optimization of nisin induction concentration and culture conditions.-[52]
Note: “-” indicates not reported.
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Pang, X.; Song, X.; Xia, Y.; Wang, G.; Liu, X.; Xiong, Z.; Ai, L. From Gene to Protein: Advances and Challenges in Microbial Production of Immunoglobulins. Fermentation 2026, 12, 296. https://doi.org/10.3390/fermentation12060296

AMA Style

Pang X, Song X, Xia Y, Wang G, Liu X, Xiong Z, Ai L. From Gene to Protein: Advances and Challenges in Microbial Production of Immunoglobulins. Fermentation. 2026; 12(6):296. https://doi.org/10.3390/fermentation12060296

Chicago/Turabian Style

Pang, Xinhui, Xin Song, Yongjun Xia, Guangqiang Wang, Xinxin Liu, Zhiqiang Xiong, and Lianzhong Ai. 2026. "From Gene to Protein: Advances and Challenges in Microbial Production of Immunoglobulins" Fermentation 12, no. 6: 296. https://doi.org/10.3390/fermentation12060296

APA Style

Pang, X., Song, X., Xia, Y., Wang, G., Liu, X., Xiong, Z., & Ai, L. (2026). From Gene to Protein: Advances and Challenges in Microbial Production of Immunoglobulins. Fermentation, 12(6), 296. https://doi.org/10.3390/fermentation12060296

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