Abstract
Escherichia coli, one of the most efficient expression hosts for recombinant proteins (RPs), is widely used in chemical, medical, food and other industries. However, conventional expression strains are unable to effectively express proteins with complex structures or toxicity. The key to solving this problem is to alleviate the host burden associated with protein overproduction and to enhance the ability to accurately fold and modify RPs at high expression levels. Here, we summarize the recently developed optimization strategies for the high-level production of RPs from the two aspects of host burden and protein activity. The aim is to maximize the ability of researchers to quickly select an appropriate optimization strategy for improving the production of RPs.
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Introduction
Since the last century, the emergence of recombinant protein (RP) expression systems has revolutionized biotechnology. Excitingly, with the advancement of biotechnology, the yield of RPs has increased from the gram to the kilogram scale, and the range of applications has expanded from traditional food and chemical industries to biopharmaceuticals [1, 2]. For example, it is projected that the industrial enzyme market will grow from USD 6.6 billion in 2021 to USD 9.1 billion by 2026 [3], illustrating the enormous market value and growth potential of RPs. Similarly, a variety of protein drugs have been successfully marketed, including monoclonal antibodies (mAbs), recombinant vaccines, and hormones, demonstrating that RPs already play a significant role in the biopharmaceutical field [4].
Due to its inexpensive fermentation requirements, rapid proliferation ability and stable high-level expression, Escherichia coli (hereafter E. coli) has become the mainstay of RP expression among prokaryotic expression hosts [5]. As early as the 1970s, E. coli was applied in the production of clinical drugs, such as the hormones somatostatin [6] and insulin [7], which were commercialized early on. As a gold standard for expressing RPs, E. coli BL21(DE3) and the pET expression system are widely used in research and commercial production. This is primarily attributed to the T7 RNA polymerase (RNAP) from λ prophage in the genome of BL21(DE3), which can specifically recognize the T7 promoter (PT7) on the pET plasmid and transcribe at eightfold the speed of the E. coli native RNAP [8, 9]. In recent years, several BL21(DE3)-derived strains have been widely used to produce various types of RPs, including C41/C43(DE3) (for the production of membrane proteins) [10], BL21(DE3)-pLysS (for reduction of T7 RNAP expression intensity) [11], BL21Star(DE3) (for improvement of mRNA stability) [12], and SixPack (for codon bias correction) [13]. Such efficient production capacity has given it an unassailable position in structural research, new enzyme mining and industrial production [14, 15].
Despite the availability of so many alternative expression systems, there is no guarantee that every type of protein will have a high yield or catalytic/functional activity. The occurrence of these phenomena can be attributed to two main aspects: (i) the host burden caused by the massive production of RPs [16] and (ii) the limited post-translational modification (PTM) capacity and generation of inclusion bodies (IBs) [17]. In fact, any production of RPs, especially toxic proteins, will inevitably compete with the host for resources, which are mainly reflected in the additional DNA replication burden, competition for transcription- and translation-related elements (RNAP, ribosomes, tRNA, and amino acids), and the additional energy and substrates consumed by PTMs [18]. For instance, high-level expression of membrane proteins can lead to the saturation of the Sec translocator-dependent transport pathway, affecting electron transport in the respiratory chain and inhibiting the expression of key enzymes of the tricarboxylic acid cycle [19]. Similarly, glucose dehydrogenase (GDH, an industrial enzyme) leads to significant autolysis of the bacterial cell during the later stages of fermentation [20]. To solve this problem, various means of genetic engineering and synthetic biology have been applied to alleviate host burden, including optimization of the expression intensity of T7 RNAP and pET expression systems (Fig. 1A) [21, 22], as well as balancing or decoupling the cell growth and RP production [23,24,25]. These optimization strategies effectively relieve or even remove the metabolic burden and increase the capacity of unit cell production. However, when proteins are synthesized at high rates, limited PTMs and molecular chaperones can lead to protein misfolding and the formation of a large number of IBs, affecting the functional activity and solubility of certain proteins. Therefore, the production of highly active RPs is also an important optimization aim, which can be achieved by strengthening or supplementing PTMs, increasing proteolysis and overexpressing suitable molecular chaperones [26]. This review summarizes different classes of optimization strategies developed in recent years from the two main aspects of alleviating host burden and optimizing protein activity, providing a reference for increasing the production of different RPs and discusses the future development direction of related optimization strategies.
The optimization expression strategies for T7 RNAP and pET plasmids. A Illustration of protein expression of recombinant protein genes on pET plasmids. B Optimization of T7 RNAP transcription and translation level, including substitutions of different promoters, and mutations in promoter functional region and RBS sequence. C regulation of T7 RNAP activity. The conventional approach is to utilize lysozyme or light-induction to regulate. D Optimization of pET plasmids based on expression intensity and copy numbers. Among them, the expression intensity was optimized by constructing an ITR library to screen for optimal expression results. The degree of binding of RNA-i to RNA-p determines the replication intensity of the plasmid to control the copy numbers. By constructing a promoter library for RNA-p, replacing the inducible promoter, and using dCas9 to regulate expression intensity, the copy numbers can be controlled
Optimization of target protein expression rate based on the gold standard T7 RNAP platform
When T7 RNAP is sufficiently induced, its powerful transcriptional capacity enables the rapid production of large amounts of mRNA, bringing the yield of RPs to 50% of the total cellular protein in just a few hours [27]. However, a strong production capacity is a double-edged sword, especially in the expression of toxic proteins. Numerous studies have shown that growth inhibition during RP production is mainly attributed to excessively strong gene transcription, and translation further exacerbates the host burden [21, 28, 29]. Therefore, the ability to precisely balance the intensity of RP transcription and translation levels is key to reducing host burden and increasing production. This is usually optimised in two aspects as follows: T7 RNAP and pET plasmid.
Regulation of the target protein expression rate-T7 RNAP
The easiest way to control the expression intensity of RPs is to regulate the amount and activity of T7 RNAP, which is often achieved by optimizing transcription or translation levels. In the BL21(DE3) genome, the T7 RNAP gene is controlled by the lacUV5 promoter (PlacUV5), which is a strongly inducible promoter that ensures rapid expression and accumulation after induction (induced by Isopropyl-beta-d-thiogalactopyranoside (IPTG)) [30]. However, high levels of expression are not compatible with some RPs, especially toxic proteins. Accordingly, many studies increased the production of toxic proteins by reducing the transcript level of T7 RNAP. For example, the membrane protein expression host C41(DE3) was obtained by stress screening, while the autolysin expression host BL21(DE3-lac1G) was constructed by recombining PlacUV5 with Plac sequences [10, 20, 31]. Furthermore, the PlacUV5 is independent of CRP, which makes it leakier than Plac [32]. Replacing the promoter of T7 RNAP with other kinds of inducible promoters is an effective way to regulate transcription levels and reduce leakage (Fig. 1B). Du et al. [32] tested the effects of three inducible promoters (ParaBAD, PrhaBAD and Ptet) on the transcriptional intensity and leaky expression of T7 RNAP, respectively. It was found that all three promoters were suitable for prolonged fermentation of toxic proteins, whereby PrhaBAD and Ptet were able to regulate T7 RNAP transcription more rigorously, providing additional options for the expression of various RPs, especially toxic proteins. Similarly, enhancing the ability to block proteins is also an effective way to reduce leaky expression. In addition to the conversion of PlacUV5 to Plac, the study found that the lac repressor gene (lacI) was also mutated (V192F, referred to as mLacI hereafter) in the membrane protein expression host (C41/C43(DE3)) [33]. Excitingly, mLacI can specifically bind to the lac operator site, but the blocking effect cannot be removed by the addition of IPTG. Based on this phenomenon, Kim et al. [31] developed an anti-leakage expression system for the overproduction of membrane proteins. Among them, mLacI expression is regulated by the rhamnose inducible promoter PrhaBAD. When trace amounts of L-rhamnose were added, T7 RNAP leakage expression could be inhibited during host growth, reducing growth burden. With the increasing concentration of L-rhamnose, mLacI is abundantly produced and thus reduces the transcription intensity of T7 RNAP, even in the presence of IPTG. This approach makes it possible to control the rate of protein production.
Unlike the transcriptional level, which is controlled by the promoter and RNAP, the strength of translation is mainly determined by the nucleotide sequence and arrangement of the ribosome binding site (RBS) (Fig. 1B). Liang et al. [34] designed 10 RBS sequences with different expression intensities for expressing T7RNAP using an RBS calculator, which was successfully implemented in five Gram-negative and one Gram-positive bacteria. To further extend the regulatory range, Li et al. [35] constructed a more extensive RBS library of T7 RNAP using CRISPR/Cas9 and cytosine base editor, with expression levels ranging from 28 to 220% of the wild-type strain. Using this library, the authors obtained customized hosts for eight difficult-to-express proteins in just three days. The tested model RPs included an autolytic protein, membrane protein, antimicrobial peptide, and insoluble protein, while the production of the industrial enzyme GDH was increased 298-fold. These results show that optimizing the expression intensity of T7 RNAP can effectively improve the RP production, and regulation of the translational level makes it easier to construct screening libraries and rapidly obtain optimized hosts for individual RPs.
Since it is an enzyme, the catalytic activity of T7 RNAP is also a key factor affecting the rate and efficiency of transcription. Mutations of key amino acid residues in T7 RNAP are one of the most effective methods to tune its activity, whose mechanisms are divided into two categories: weakening the binding ability to PT7 or generating code-shifting mutations to reduce the catalytic activity [36,37,38]. For example, Baumgarten et al. [37] found a single amino acid mutation (A102D) of T7 RNAP in the membrane protein expression host Mt56(DE3), which reduced the ability to bind to the PT7 and decreased the RP production rate. In addition, the addition of T7 RNAP inhibitors is also a way to effectively regulate T7 RNAP activity, and various derivative hosts including BL21(DE3)-pLysS, BL21(DE3)-pLysE, and Lemo21(DE3) have been developed based on this principle [39,40,41] (Fig. 1C). With the development of synthetic biology, researchers hope to change the strength of T7 RNAP activity in logic gates to precisely and dynamically regulate the process of growth and production. A variety of T7 RNAP expression systems regulated by light induction have been developed successively, achieving dynamic regulation of RP production [42,43,44]. For example, the Opto-T7RNAPs system splits the T7RNAP into two fragments and expresses them in tandem with a light-sensitive dimerization domain. When the fragments are expressed and irradiated by the light of a specific wavelength, T7 RNAP can resume its transcriptional activity, with up to 80-fold change in activity between blue light and darkness [43]. Regrettably, these studies have only been validated with fluorescent proteins or lycopene, and have not been applied to RP production.
Regulation of the target protein expression rate-pET plasmid
Another key factor affecting the expression rate of RPs depends on the combination of different elements on the pET plasmid, including sequences of relevant functional regions near PT7 (-35/-10 region, translation initiation region (TIR) and operator sequence) and replicon [45]. As the core region of the pET plasmid, various functional regions near the PT7 determine the rigor of basal expression before induction and the appropriate transcription rate after induction.
To reduce the host burden of leaky expression, several more rigorous inducible systems have been combined with PT7 to increase the yield of toxic or structurally complex proteins, such as the cumate operator [46], inducible translational ON orthogonal riboswitch [47], and temperature-regulated self-induction [48]. After solving the leaky expression problem, an urgent task is to quickly screen the appropriate expression intensity of various RPs. In contrast to complex genomic manipulations, the combination of degenerate primers and MEGAWHOP PCR or enzymatic digestion and ligation allows rapid access to very large libraries of various functional sequences, including promoter mutation and TIR libraries [22, 84, 85]. Among them, Liu et al. [84] utilized phage-assisted continuous evolution technology for rapid optimization of 16S rRNA by screening pressure. After multiple rounds of directed evolution, the mutant o-ribosome achieved faster translation, resulting in 6.3-fold higher RP production than the wild-type. Most importantly, this ribosome can introduce ncAAs into the protein with high efficiency, which is 9.08-fold higher than that of the native ribosome, improving the application of orthogonal translation systems in RP production. In brief, whether it is to inhibit or block the expression of growth-essential genes or to use o-ribosomes to express RPs, the aim is to ensure normal growth of the host during the growth phase (Fig. 2D).
Optimizing protein activity—another key to the production
In addition to ensuring the quantity of RPs, the functional activity of the protein at high yields is also a key focus of RP production. When the expression rate or quantity of RPs exceeds the capacity of the host cell, it will result in a large number of proteins that misfold and aggregate, eventually producing IBs [17]. This phenomenon has greatly hindered the use of E. coli in various fields, especially the expression of protein-based drugs. The key reason for the generation of IBs is the limited PTM capacity and folding efficiency, which are the top priorities for optimizing the functional activity of RPs.
Enhancement of post-translational modifications
Most proteins with complex structures contain multiple disulfide bonds (DSBs) that maintain their normal conformation, including insulin [7] and epidermal growth factor [86]. As an oxidative process, the natural DSB formation is completed in the periplasmic space of E. coli and not in the reductive environment of the cytoplasm, which requires the protein to be localized and translocated to the appropriate location for modification [87]. The common protein translocation pathways are divided into three main categories: SecB-dependent, SRP-mediated and TAT translocation pathways [88]. Among them, SecB-dependent and SRP-mediated pathways both complete the translocation process by binding to SecA, and genetic fusion of signal peptides to RPs can enable them to utilize these pathways to translocate. Commonly used signal peptides include pelB, OmpA and DsbA [89, 90], but each signal peptide triggers a different mechanism that greatly affects the effectiveness of RP transport. In contrast to SRP-mediated DsbA, SecB-dependent OmpA drives the synthesis of endogenous secreted and membrane proteins, preventing Sec translocator saturation [89]. In recent years, the TAT translocation pathway has attracted the interest of researchers due to its natural "quality control" system, which can prioritize the output of correctly folded proteins [91]. The "TatExpress" strain was successfully developed and applied for the gram-level production of human growth hormone, proving its great potential [92]. In addition to the above translocation pathways, a signal peptide based on the N-terminal sequence of penicillin-binding protein 2 (PBP2) was shown to anchor the fusion protein to the cytoplasmic membrane. Interestingly, the high expression of PBP2 affects morphological changes in E. coli (rods to spheres) and interacts with lysis transglycosylase leading to host lysis [93]. This phenomenon has the potential to be developed into a self-cleaving transport system for rapidly accumulating RPs production.
Compared to the narrow periplasmic space, the cytoplasm has enough space to accomplish more protein folding and increase productivity. By blocking the natural reduction pathway in a Δgor/ΔtrxB strain, the reductive cytoplasmic environment becomes oxidative, which facilitates the formation of DSBs [94]. The earliest commercial DSB-forming E. coli strain, Origami from Novagen, was developed based on this principle. By overexpressing sulfhydryl oxidase from the yeast mitochondria and disulfide bond isomerase from human cells, a host called CyDisCo was developed for the production of RPs with high DSB content, and was able to produce even perlecan with 44 DSBs (Fig. 3A) [95, 96]. Apart from the above, other means of optimization, including replacement of sulfhydryl oxidases from other sources [97], inversion or development of a periplasmic transmembrane disulfide bond-forming enzyme DsbB [98, 99], were also used to improve the efficiency and capacity of DSB formation.
The optimization strategies to enhance PTMs. A Principle of disulfide bond formation in the cytoplasm using the CyDisCo system. B Modification process of phosphorylation and acetylation. P: phosphonate; AC: acetyl. C Modification process of glycosylation by overexpression of a heterologous N/O-glycosylase. D Introduction of PTMs via ncAA. The figure shows the principle of phosphoserine introduction
In addition to the formation of DSBs, the efficiency of other PTMs also affects the functional activity of RPs, such as phosphorylation, acetylation (Fig. 3B), glycosylation and many other modifications that are often found in mAbs and functional proteins [100,101,102]. Among them, glycosylation is one of the most abundant and complex PTMs [103]. By linking monosaccharides, oligosaccharides or polysaccharides to proteins, the variety of protein functional activities is greatly expanded. Currently, over 70% of therapeutic proteins are modified by glycosylation, and precision glycosylation can effectively enhance the use of glycoproteins in the medical industry [102]. Compared to eukaryotes, E. coli does not have a natural mechanism for glycosylation of encoded proteins. Therefore, it can be used as a suitable chassis cell to develop bottom-up glycoengineering for different types of glycoproteins [104]. The first N-glycosylation expression system was successfully developed in E. coli by introducing genes related to N-glycosylation of Campylobacter jejuni, opening the curtain on the glycoprotein synthesis in E. coli [105] (Fig. 3C). Over the last two decades, many efforts have conferred the potential to produce a wide range of N/O-glycoproteins from E. coli or cell-free extracts, including optimization of glycosyltransferase substrate identification and orthogonality [102, 106,107,108], exploration of glycosylase function from multiple sources [107,108,109] and optimization of host environment, metabolic pathways and culture conditions [110,111,112,113]. Based on these studies, a variety of medically relevant products are in production and in the clinical phase, such as recombinant vaccine exotoxin A [114], therapeutic protein O-glycosylated interferon-α2b [115] and N-glycosylated mannose3-N-acetylglucosamine2 [116]. In a similar way to DSB, the glycosylation process in the above systems is mostly completed in the periplasmic space. In recent years, several studies have identified cytoplasmic glycosylation systems in various bacteria, laying the foundation for the development of novel glycosylation systems in E. coli [117,118,119]. Among them, the asparagine (N)-glucosyltransferase from Actinobacillus pleuropneumoniae (ApNGT) can be actively expressed in the E. coli cytoplasm and transfer glucose residues to the naturally N-terminal glycosylation site of the protein (e.g. recombinant human EPO) [117]. Based on this discovery, Tytgat et al. [120] developed an N-glycosylation system in E. coli cytoplasm. Using ApNGT in combination with various oligosaccharide synthesis pathways (e.g. human milk oligosaccharides and glycosphingolipids), glycosylation modifications of various glycoproteins (glycoconjugate vaccines and multivalent glycopolymers) have been achieved. Surprisingly, the system can complete the glycosylation of megadalton protein assemblies, which can be used as customized carriers for delivery of drugs and vaccines.
It is worth mentioning that the orthogonality of ncAAs with specific codons can be used to introduce various types of modified amino acids more directly and precisely. Park et al. [121] successfully introduced phosphorylated serine residues into RPs at specific sites by orthogonal pairing of SepRS/tRNASep (Fig. 3D). Similarly, phosphor-threonine [122] and phospho-tyrosine [123] were utilized for RP modification. In addition to phosphorylation, acetylation, methylation and ubiquitination have been successfully introduced into various RPs [124]. In conclusion, the introduction of PTMs using ncAAs has the potential to once again make E. coli a "star host" for biopharmaceuticals.
Elimination of inclusion bodies
In addition to limited PTMs, a variety of factors such as misfolding, low solubility, and host burden also contribute to IB formation. Three strategies are usually used to solve the problems: (i) enhancing solubility; (ii) improving correct folding efficiency; (iii) optimizing the appropriate expression intensity. Among them, the relevant aspects of (iii) have been described above.
The use of peptide tags is the most direct and effective means to enhance the solubility of RPs. Common tags include maltose binding protein (MBP), glutathione-S-transferase (GST), carbohydrate-binding module (CBM), thioredoxin, and NusA, which have been reviewed by Ki et al. [125]. Notably, a novel CBM (CBM66) was shown to have a pro-solubilizing effect on several types of RPs and to increase production titer [126]. For example, the combination of poly (ethylene terephthalate) hydrolase and CBM resulted in a 3.7-fold improvement compared to the other commercial labels (MBP and GST), without affecting protein bioactivity. However, if the molecular weight of the peptide tag is close to or larger than that of the RP, it will override the solubility of the RP itself. Furthermore, the subsequent label removal can negatively affect the solubility and stability of RPs. Conversely, the use of peptide tags with smaller molecular weights allows more reliable evaluation and optimization of the solubility of RPs. In recent years, a variety of low-molecular-weight protein tags have contributed to the solubilization and yield enhancement of various RPs, including the NEXT tag [127], low-molecular-weight protamine [128], and 6HFh8 [129]. Kim et al. utilized 6HFh8 [129] to express a variety of growth factor proteins. Among them, 6HFh8-aFGF and 6HFh8-VEGF165 obtained high respective yields of 9.7 and 3.4 g/L in a 5-L batch supplement fermentation, with a purity of more than 99%. The removal of the small peptide tags does not significantly affect the solubility and functional activity, which is suitable for the purification of small RPs.
Molecular chaperones are a class of auxiliary proteins that facilitate the folding and assembly of peptide structures, ensuring proper folding and preventing the aggregation of newly translated peptides [130]. E. coli possesses several molecular chaperone systems, such as GroES/EL and DnaK-DnaJ-GrpE, all with different functions [131]. Among them, DnaK-DnaJ-GrpE not only helps correctly fold newly translated peptides, but also functions during co- and post-translational modification. By contrast, the GroES/EL system associates with peptides only post-translationally, powering the repair of misfolded proteins [127]. It is easy to understand that the folding efficiency can be effectively enhanced by overexpression of molecular chaperones, which is usually done in three combinations: GroES/GroEL, DnaK-DnaJ-GrpE, and co-expression. However, co-expression is usually not better than expressing a single factor, and only some chaperones can have a beneficial effect on protein folding [132]. Huang et al. [133] expressed distinct combinations of molecular chaperones to enhance the solubility and activity of polyunsaturated fatty acid isomerase (PAI). The results showed that overexpression of GroES/EL increased the solubility of PAI from 29 to 97% and improved its specific activity by 57.8%. By contrast, the co-expression of DnaK-DnaJ-GrpE or GroES/EL had a weakening effect, resulting in only an 11.9% increase in activity.
Conclusion and outlook
Different types of RPs from different origins have highly specific characteristics, and there can be no single optimization strategy that applies to all proteins. This review summarizes the recently developed optimization strategies from the two major aspects of alleviating the host burden and optimizing functional activity, which helps researchers quickly select an appropriate expression strategy for their protein of interest (Table 1, Fig. 4). Encouragingly, with the continued development of synthetic biology, systems biology, and various gene editing tools, it is becoming less difficult to rapidly develop a customized host. Multiple in vivo mutagenesis strategies facilitate adaptive laboratory evolution for rapid screening of strongly tolerant expression hosts, including DNA replication proteins, RNAP and T7 RNAP fused with base deaminases [134,135,136,137]. Construction of artificial organelles allows for E. coli compartmentalization, which has the potential to accomplish precise PTMs [138, 139]. In addition, researchers are updating the BL21(DE3) genome annotation, as well as combining mathematical modeling, statistical analysis, and computer aided design to achieve precise optimization [140, 141]. In conclusion, we have reason to believe that E. coli will remain one of the brightest stars among RP production hosts.
The routine workflow for expression optimization based on protein properties
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This work was supported by the Nature Science Foundation of Jiangsu Province (No. BK20202002).
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Zhang, ZX., Nong, FT., Wang, YZ. et al. Strategies for efficient production of recombinant proteins in Escherichia coli: alleviating the host burden and enhancing protein activity. Microb Cell Fact 21, 191 (2022). https://doi.org/10.1186/s12934-022-01917-y
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DOI: https://doi.org/10.1186/s12934-022-01917-y