Engineering Extracellular Expression Systems in Escherichia coli

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Engineering Extracellular Expression Systems in Escherichia coli Based on Transcriptome Analysis and Cell Growth State Wen Gao, Jun Yin, Lichen Bao, Qun Wang, Shan Hou, Yali Yue, Wenbing Yao, and Xiangdong Gao ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.7b00400 • Publication Date (Web): 18 Apr 2018 Downloaded from http://pubs.acs.org on April 19, 2018

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ACS Synthetic Biology

Engineering Extracellular Expression Systems in Escherichia coli Based on Transcriptome Analysis and Cell Growth State Wen Gao#, Jun Yin#, Lichen Bao, Qun Wang, Shan Hou, Yali Yue, Wenbing Yao*, Xiangdong Gao* Jiangsu Key Laboratory of Druggability of Biopharmaceuticals and State Key Laboratory of Natural Medicines, School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, China *Corresponding authors: Xiangdong Gao, e-mail: [email protected]., telephone: +86-25-83271543, Fax: +86-25-83271249, we designate this one to further communicate with the Editorial and Production offices. Wenbing Yao, e-mail: [email protected]., telephone: +86-25-83271218, Fax: +86-25-83302827. # These authors contributed equally to this work.

ABSTRACT Escherichia coli extracellular expression systems have a number of advantages over other systems, such as lower pyrogen levels and simplifier purification. Various approaches, such as the generation of leaky mutants via chromosomal engineering, have been explored for this expression system. However, extracellular protein yields in leaky mutants are relatively low compared to intracellular expression systems and therefore need to be improved. In this work, we describe the construction, characterization and mechanism of enhanced extracellular expression in Escherichia coli. Based on the localizations, functions and transcription levels of cell envelope proteins, we systematically elucidated the effects of multiple gene deletions on cell growth and extracellular expression using modified CRISPR/Cas9-based genome editing and a FlAsH labeling assay. High extracellular yields of heterologous proteins of different sizes were obtained by screening multiple gene mutations. The enhancement of extracellular secretion was associated with the derepression of translation and translocation. This work utilized universal methods in the design of extracellular expression systems for genes not directly associated with protein synthesis that were used to generate strains with higher protein expression capability. We anticipate that extracellular expression systems may help to shed light on the poorly understood aspects of these secretion processes as well as to further assist in the construction of engineered prokaryotic cells for efficient extracellular production of heterologous proteins.

KEYWORDS extracellular expression, cell growth, membrane permeability, lipoprotein family, genome engineering, apparent molecular size

INTRODUCTION In recent decades, Escherichia coli has remained one of the most widely used hosts to produce heterologous proteins, including nearly 30% of the currently approved recombinant therapeutic proteins. The reason for this widespread use is because of its well-characterized genetics, versatile cloning vectors, various expression systems, rapid growth, low-cost medium and ease of scale-up for

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high protein yield and quality1. Cytoplasmic expression systems are widely used as high-level expression systems, but the intracellular accumulation of heterologous protein can lead to insolubility, cell toxicity, non-native disulfide patterns, and non-native protein folding or aggregation2, 3. Periplasmic expression systems can assist with correct disulfide bond formation due to the oxidizing environment and various chaperones4. The lower concentration of host cell proteins in the periplasmic space also simplifies purification procedures. However, the limited space in the periplasm limits the accumulation of recombinant protein for industrial production. Extracellular expression systems have the advantages of periplasmic expression systems but also have additional attractive features, such as alleviating cell toxicity and the production of less endotoxin for simplifier purification and high yields5. As there is not a dedicated secretion system in E. coli, several approaches have been explored to secrete recombinant proteins into the medium, including engineering secretion pathways6, 7, co-expression of lysis-promoting proteins8, fusion expression of osmotically inducible protein Y9, 10, expression of the carrier protein YebF with unknown translocation mechanisms11, 12, and the use of leaky mutants13. The addition of supplements, such as Triton X-100, glycine or calcium ions, can increase the permeabilization of cell membranes which can lead to the leakage of periplasmic proteins. Most of these approaches influence the production rate and bioactivity of the therapeutics because they require the removal of the fusion protein to expose the authentic N-terminus, complicating the biocatalysis process in the host cells. Therefore, there is a great need to engineer bacteria with a high extracellular secretion capacity. Deletion of lpp significantly increased the outer membrane permeability of E.coli, which was used for the extracellular expression of recombinant proteins14. Chromosomal engineering in host strains has advantages for extracellular protein production, such as the non-specificity of proteins, the use of normal growth conditions without the need for permeabilization supplements and the release of expressed proteins with an authentic N-terminus. Wild-type E. coli cells have a complicated cell envelope, which acts as a barrier to release periplasmic proteins into culture. Cell envelope proteins, which account for approximately 25% of the total bacterial proteins, including lipoproteins, membrane proteins and periplasmic proteins, are necessary for the integrity of the cell membrane15-17. Mutants lacking cell envelope proteins show a periplasmic-leaky phenotype, such as murein lipoprotein (lpp) and outer membrane protein (ompA) mutants18, 19. While extracellular secretion is an attractive option for industry, it remains a challenge because of the relatively low yields achieved compared to cytoplasmic expression20. As high product formation rates are relevant to high growth rates, rapid cell growth is necessary for industrial production6, 21. While the lack of cell envelope proteins caused by gene deletions influences cell growth, such effects on cell growth may not necessarily be limited as long as the gene targeted for deletion is carefully selected. The deletion of different genes leads to different impacts on cell growth. Thus, it is important to determine which gene has the least impact on the final cell density after deletion. The deletion of different genes also leads to different levels of secretory proteins. To determine whether the secreted protein level is relevant to the transcriptional level of the deleted genes, encoded proteins in the same family needs to be investigated. The extracellular secretion of some recombinant proteins with a large apparent molecular size may require a higher leaky capacity compared to small-sized proteins22-24. Whether the leaky capacity of the proteins of different sizes is


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relevant to the number of deleted genes or the class of the absent cell envelope protein is still not clear. In this study, we constructed and characterized the enhanced extracellular expression systems in E. coli. Based on a systematic transcriptomic analysis and functional classification25, various gene deletion mutations were constructed using a modified CRISPR/Cas9-based genome editing system. Cell growth and intracellular contents release of mutants were studied to determine the impact of mutations on cell growth. A generic fluorescence detection method using a FlAsH-labeled tetracysteine motif fused to heterologous proteins of different sizes was established to rapidly detect the secretion capacity of the extracellular expression systems. Heterologous proteins of small size, such as interferon alpha, or of large size, such as various PsTag polypeptides, were expressed and detected in the cytoplasm, periplasm and culture medium. PsTag polypeptides of differing lengths (200, 400 and 600 residues) were screened from the libraries of the short peptide segments comprising repeatable sequences to extend the plasma half-life of the peptides and proteins with an increased hydrodynamic radius. These PsTag sequences showed large apparent molecular sizes of 128 kDa for PsTag200, 361 kDa for PsTag400 and 547 kDa for PsTag60026. Various mutants were screened with the goal of establishing a generally applicable extracellular expression system having a minimal impact on cell physiology.

RESULTS AND DISCUSSION Selection of Candidate Genes for Deletion via Transcriptomic Analysis. Because cell envelope proteins, including lipoproteins, membrane proteins and periplasmic proteins, firmly interact with each other by covalent and noncovalent interactions, the lack of these proteins may cause changes to the permeabilization of the outer membrane15. The gene transcription levels of 86 periplasmic lipoprotein genes during the exponential phase (OD600=0.6) were analyzed via transcriptomic analysis to identify targets for deletion. The transcription levels were standardized by reads per kilobase of exon per million mapped sequence reads (RPKM) and were shown in Figure S1. The highest RPKM value (lpp) was over 156,000, which was almost 30-fold more than the second highest value (mepS) and 150,000-fold more than the lowest value (wza). These differences in the transcription level indicated the difference in copy number of the lipoproteins per cell. The deletion of a gene with a higher RPKM value was assumed to have a greater impact on the permeabilization of the membrane. Ten lipoprotein genes with the highest RPKM values were selected as targets for the construction of single- or multi-gene deletion mutants via CRISPR/Cas9 editing to explore the association between the gene expression level and the permeabilization capacity. To systematically study the permeability capacity of the cell envelope-related genes27, genes encoding membrane proteins (ompA, tolA, tolC), periplasmic proteins (oppA, dppA) and peptidoglycan components (mrcA, mrcB) were also selected as candidate genes for deletion. CRISPR/Cas9-Based Iterative Deletion in E. coli. Figure 1 schematically represents the CRISPR/Cas9-based scarless genome editing system used in this study. This system was composed of five elements: cas9 cassette and λ-Red recombineering system, the sgRNA expression plasmid, donor dsDNA, and an inducible plasmid curing system. The expression of cas9 protein was induced by tetracycline. λ-Red recombinase was expressed constitutively on the same plasmid, pCasREDcure, which contained a p15A replication origin and an



inducible p15A-targeting sgRNA cassette. The sgRNA targeting genome was expressed constitutively on pKDG containing a temperature-sensitive replication origin repA101(Ts). Donor dsDNA with upstream and downstream homologous arms to the area of deletion had no selectable marker and were electrotransformed together with pKDG. Table S8 summarized the efficiencies of mutagenesis obtained from all experiments. The curing of the pKDG plasmid was manipulated at 43℃ and the ampicillin-sensitive colonies were ready for the next round of editing. The pCasREDcure plasmid need not be cured until the construction of the desired mutant had been completed. PCasREDcure was eliminated by the expression of the p15A-targeting gRNA. The curing efficiency pCasREDcure cleaved by cas9 could reach 100% (data not shown). This CRISPR/Cas9-based scarless genome editing system saved time for iterative gene editing. For example, if three genome modifications were undertaken, this editing system would take as little as 9 days, while other published methods for scarless genome modifications would take 14 days25, 28. A Fluorescence Detection Method for Evaluating Extracellular Expression Systems. To rapidly measure the extracellular protein expression, a FlAsH labeling system was established using periplasmic expression plasmids. FlAsH-EDT2 is a non-fluorescent biarsenical labeling reagent which became strongly fluorescent upon binding to a tetracysteine motif (Cys-Cys-Pro-Gly-Cys-Cys) with high specificity and high affinity29. To investigate the permeability capacity for different-sized proteins, the periplasmic expression plasmids for IFNα and PsTag polypeptides were constructed with a TC-tag fused to the C-termini. When the TC-tag fusion proteins were secreted into the extracellular medium, the medium sample would produce significant fluorescence after FlAsH-EDT2 binding, indicating the extracellular protein production of the mutants. When the cell pellets were labeled by FlAsH, the fluorescence was due to the intracellular accumulation of proteins. The TC-tagged fusion proteins expressed in the periplasm were separated by osmotic shock for validation in the FlAsH labeling assay. The concentration of FlAsH in the reaction buffer and the concentration of the TC-tag were tested. The TC-tag proteins were diluted 10000, 1000, 500, 100, 40, 20, 10, 5, 4, 3, and 2-fold and reacted with labeling buffer under the same reaction conditions. A positive correlation between the fluorescence intensity and the concentration of TC-tag was observed (Figure S5). These data demonstrated the high sensitivity of detection of the assay and provided a theoretical foundation for the comparison of the permeabilization capacity of mutants. Construction and Growth Characteristics of Single-Gene Deletion Mutants. Single-gene deletion mutants were constructed via CRISPR/Cas9-based gene deletion and plasmid curing procedures. From each transformation, thirty colonies were randomly selected and analyzed by colony PCR to identify correct mutations and to establish the success rate of the mutagenesis30(Figure S6-S7). Table S8 summarized the results of the success rates of different gene deletions, and most of the success rates were over 50%. For a single gene deletion, the mutagenesis efficiency for some target genes, such as tolA, reached 100%, while for other target genes, such as oppA, the efficiency was below 50%. Colonies were still recoverable and represented “escapers”. Under these conditions, cells might escape CRISPR interference from the recombination due to the mutations in the targeted PAM or seed regions31. In this study, the gene deletion efficiency could be improved though optimization of the components, such as gene loci30 and the length or concentration


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of dsDNA28. To explore the relationship between cell growth and gene deletions, the cell growth of single-gene deletion mutants was studied and compared to the wild-type strain. Most of the mutants showed similar growth rates compared to the wild-type strain in LB and M9 media (Figure 2 and Table S9-S11). The final cell densities of the △lpp, △pal, △slyB and △ecnB mutants were less than 15% lower than the wild-type strain, and the final cell densities of the △mepS, △arcA, △nlpI, △osmE, △bamD and △cyoA mutants were approximately 40% lower. Except for the △tolC mutant, membrane-associated protein gene mutants had 10% to 15% lower final cell densities compared to the wild-type strain (Figure 2B). The glucose consumption curves of the mutants were above that of the wild-type strain, reflecting a delayed glucose consumption due to differences in cell density (Figure 2C). Lactate dehydrogenase (LDH) is a peripheral membrane respiratory enzyme located on the cytoplasmic side of the inner membrane32. The intracellular contents, such as chromosomal DNA and LDH, are released into the medium when cell lysis occurred33. To investigate whether cell lysis caused the lower final yields for the mutant strains, DNA and LDH release was detected in the culture supernatants for M9 media. The levels of DNA and LDH release were observed throughout all growth phases, including the exponential phase, indicating that cell lysis was not responsible for the lower final yields of the mutant strains (Figure S3B and S3D). Our results also confirmed that secretion of heterologous proteins was not due to lysis. As the genes encoding cell envelope proteins were deleted in the mutants, one possible reason for the relatively low cell density was that the outer membrane or the cytoderm lacked several components, causing defects in the surface structure and the overall cell morphology34. The functions of the proteins encoded by mepS, arcA, nlpI, osmE, bamD, cyoA and tolC are associated with metabolism. The deletion of such genes led to a serious impact on cell growth. The deletion of lipoprotein-encoding genes, such as lpp, pal, slyB, ecnB and membrane-associated protein-encoding genes, such as ompA, tolA, oppA, dppA, mrcA, and mrcB, did not significantly affect cell growth. To investigate the integrity of the cell membranes in single-gene deletion mutants, a double fluorescence staining method was established. Only in viable cells can calcein be generated from the cell membrane permeable Calcein-AM by esterases, resulting in the emission of a strong green fluorescence35. In contrast, Propidium Iodide (PI) can penetrate only bacteria with damaged membranes and cannot pass through an intact membrane. Therefore, only cells with a permeable membrane will be stained by both of Calcein-AM and PI. A staining solution containing 10 µM Calcein-AM and 5 µM PI was established via optimizing the concentrations of Calcein-AM and PI (data not shown). Fluorescence micrographs of the double stained single-gene deletion mutants are shown in Figure 2E. All the single-gene deletion mutants were double-stained by Calcein-AM and PI. These results indicate the deletion of all the targeted genes damaged the integrity of the cell membrane. To investigate the degree of damage to the cell envelope integrity, the fluorescence was measured to compare the differences in PI permeability. The fluorescence of the △lpp, △pal, △ompA and △tolA mutant was higher than other mutants, suggesting a better permeability capacity. Extracellular Expression Systems in Single-Gene Deletion Mutants. IFNα and PsTag polypeptides fused to a TC-tag were expressed in the single-gene deletion



mutants, and the supernatant fractions were measured using a FlAsH labeling assay to evaluate the extracellular accumulation of proteins. Among the lipoprotein gene deletion mutants, the fluorescence of extracellular IFNα was significantly higher in the △lpp, △mepS, △arcA, △nlpI and △pal mutants, while the fluorescence observed for the △slyB, △osmE, △bamD, △cyoA and △ecnB strains was slightly enhanced compared to the wild-type strain (Figure 3A, 3B). The differences in the PsTag200 and PsTag400 extracellular protein levels in the single mutants were similar to the expression level of IFNα, which was consistent with the SDS-PAGE results (Figure 3A, 3C, 3D). The lpp, mepS, arcA, nlpI and pal genes had higher RPKM values and higher extracellular protein secretion levels in mutations, respectively, than those observed slyB, osmE, bamD, cyoA and ecnB. Coupled with the cell growth state, the △lpp and △pal mutants were more suitable for the extracellular protein expression. In addition, the fluorescence of IFNα, PsTag200 and PsTag400 extracellular proteins was significantly higher in the △ompA, △tolA and △tolC mutants, while the fluorescence in the △oppA, △dppA, △mrcA and △mrcB mutants was slightly enhanced (Figure 3A). The fluorescence from the extracellular secretion of the PsTag600 fusion proteins in all these single-gene deletion mutants was similar to that observed in the wild-type strain (Figure 3A), which indicated that proteins such as PsTag600 of up to 547 kDa cannot be secreted into the medium when expressed in single-gene deletion mutants. To explore the reasons for the extracellular expression difference, the amounts of PsTag200 in the cytoplasm, periplasm and extracellular medium were detected in the wild-type strain and single-gene deletion mutants (Figure 4A). The mutants with higher extracellular protein secretions, such as the △lpp and △pal mutants, had a lower accumulation of PsTag200 in the cytoplasm and periplasm. The total expression of PsTag200 in these mutants was also elevated when calculating the total amounts in cytoplasm, periplasm and extracellular medium. For a preliminary study on the cellular mechanism of enhanced extracellular secretion of recombinant proteins, we analyzed the mRNA levels of PsTag200 in the wild-type strain and in single-gene deletion mutants by RT-qPCR. As shown in Figure 4B, no significant differences in mRNA levels of PsTag200 in different strains, indicating that the enhanced extracellular protein expression was associated with the derepression of translation. Furthermore, the transcription levels of secB, secA, secY encoding functional proteins in Sec protein translocation pathway were detected by RT-qPCR. SecB protein is a chaperone interacted with newly synthesized recombinant protein precursors in posttranslational translocation, while SecA level is regulated by the secretion needs of the cell36, 37. The synthesis of secB mRNA in mutants with higher extracellular secretions was increased (Figure 4C), which indicated the enhancement of recombinant protein translocation levels. Interestingly, the secA and secY mRNA levels did not significantly change between the wild-type and mutant strains (Figure 4D,4E). However, we cannot rule out the possibility that SecA protein synthesis was elevated during the derepression of secretion block in these mutants according to the notion that secA derepression might occur at a posttranscriptional level36, 38. Construction and Growth Characteristics of Multi-Gene Deletion Mutants. To secrete proteins with large apparent sizes and to obtain an enhanced yield of proteins with small apparent sizes, multi-gene deletion mutants may be required. We selected the mutants with the highest secretion and exhibited the least impact on cell growth, such as the △lpp, △pal, △ompA and △tolA mutants, for use in iterative engineering. Double-gene and triple-gene deletion mutants were


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constructed. From each transformation, thirty colonies were randomly selected for analysis by colony PCR. As the number of deleted genes increased, the success rate of the represented deletion was decreased (Table S8). To explore the relationship between cell growth and multi-gene deletions, the cell growth of multi-gene deletion mutants was studied and compared to the wild-type strain. As shown in Figure 2D, multi-gene deletion mutants exhibited slightly lower growth rates compared to the wild-type strain. The final cell densities of the △lpp△mrcA, △lpp△mrcB, △pal△mrcA, △pal△mrcB, △pal△ompA, △pal△tolA, △lpp△oppA, and △lpp△dppA mutants were 20% lower than the wild-type strain, and the final cell densities of the △ompA△dppA and △tolA△dppA mutants were 40% lower than the wild-type strain. The deletion of the lipoprotein-encoding gene combined with the membrane protein-encoding genes, periplasmic protein-encoding genes or peptidoglycan component-encoding genes had less impact on cell growth among these double-gene deletion mutants. The double-gene deletion mutants exhibited the least impact on cell growth were engineered in the next round of deletion. As the success rate of the deletion decreased, only the △pal△mrcA△tolA and △pal△mrcA△ompA mutants were obtained. The final cell density of the △pal△mrcA△ompA mutant was 50% lower than the wild-type strain, and the final cell density of △pal△mrcA△tolA mutant was 30% lower than the wild-type strain. These results indicated that the deletion of the lipoprotein-encoding gene pal, the peptidoglycan component-encoding gene mrcA and the membrane protein-encoding gene tolA had a low impact on cell growth according to the data. Extracellular Expression Systems in Multi-Gene Deletion Mutants. All of the multi-gene deletion mutants showed similar fluorescence of the extracellular IFNα-TC tag proteins compared to the single-gene deletion mutants (Figure 5A, 5B). This result indicated that multi-gene deletion had little influence on the extracellular expression of small-molecule proteins. Thus, single-gene deletion mutants were more suitable for extracellular production of small-molecule proteins. The fluorescence of PsTag200 and PsTag400 extracellular proteins in all the double-gene deletions was remarkably enhanced compared to in the single-gene deletion mutants, and the △lpp△mrcA, △pal△mrcA, △pal△ompA and △pal△tolA mutants had the highest fluorescence, which is consistent with the SDS-PAGE results (Figure 5A, 5C, 5D). The differences in the extracellular yields in the double-gene deletion mutants were similar to those in the single-gene deletion mutants. For example, the PsTag200 extracellular expression in the △pal△tolA mutant was higher than that observed in the △pal△mrcA mutant, while the PsTag200 extracellular protein expression in the △tolA mutant was higher than that observed in the △mrcA mutant. However, for the extracellular secretion of PsTag600, only the △pal△ompA and △pal△tolA mutants had significantly enhanced fluorescence compared to the wild-type strain. These results indicated that the deletion of the lipoprotein- and membrane protein-encoding genes increased the permeabilization capacity in the both extracellular yields and the larger apparent molecular size of the protein (up to 547 kDa), while others only increased the extracellular yields. The extracellular secretion of PsTag200, PsTag400 and PsTag600 in the triple-gene deletion mutants was significantly enhanced compared to those in the double-gene deletion mutants, but the differences in yields between the two triple-gene deletion mutants were not remarkable (Figure 5A,



5C, 5D, 5E). Despite a slightly lower cell density of the triple-gene deletion mutants, the total yields of extracellular proteins in the triple-gene deletion mutants from an equal volume of fermentation supernatants were higher than those observed in the single-gene or double-gene deletion mutants. Therefore, triple-gene deletion mutants may be more suitable for extracellular production of large-molecule proteins. When monitoring the extracellular and intracellular accumulations of PsTag600 in the △pal△mrcA△tolA mutant, the protein accumulation in the cytoplasm and periplasm was stable and reached a maximal accumulation at 4 and 8 h, respectively, after induction, while the extracellular accumulation kept rising during the entire incubation (Figure 5F). This result indicated that the extracellular secretion continuously promoted the expression of the recombinant protein during the stationary phase. Taking the results of the single- and the multi-gene deletions together, the permeability capacity for the apparent molecular size of the recombinant protein and the extracellular yields were correlated with the number and combination of deletions for the cell envelope genes. Extracellular secretion of recombinant proteins in E. coli exhibits advantages in simplifying the purification process and higher product quality. However, it has not been widely used in the industry due to the relatively low yields and growth conditions compared to the cytoplasmic expression system39. The genes targeted for engineering need to be carefully selected to overcome these limitations. Lpp (Braun’s lipoprotein) is present at as many as 750,000 copies per cell, making it the most numerically abundant protein in E. coli40. There are over 80 different kinds of lipoproteins experimentally verified in E. coli, which account for 2.0% of the total proteome41, 42. The data for the single-gene deletion mutants in the lipoprotein-encoding genes exhibited relevance between the expression level of the target gene and the membrane permeability of the deletion mutant. Most of the lipoproteins are abundant in the periplasmic space, have a small size and are not attached or covalently attached via periplasmic proteins or membrane proteins43, 44. As the copy number of the lipoproteins is typically higher than other periplasmic proteins, lipoproteins occupy a specific space in the periplasmic space and the outer membrane45, 46. The deletion of these genes may disrupt reactions relevant to the integrity of the cell envelope. The extracellular yields of the double-gene deletion mutants had relevance to the yields of the single-gene deletion mutants of each gene. The results of the secretion of PsTag200 in the △pal△tolA and △pal△mrcA strains indicated that the permeability capacity of multi-gene deletion mutants might be equal to the total permeability capacity of each gene. Interestingly, the △pal△tolA mutant had an increased permeability capacity for both yields and a larger apparent molecular size of proteins compared to the △pal and △tolA mutants, while the △pal△mrcA mutant only increased the permeability capacity for the yields compared to the △pal and △mrcA mutants. This result may be relevant to the functional properties of the proteins encoded by tolA and mrcA47-49. Pal-Tal is an important transport pathway, and the lack of two compounds in the pathway may create a permeable space due to their adjacent localization and the fact that they are covalently or non-covalently bound34, 50, 51 . Therefore, the increased extracellular yields in the △pal and △mrcA mutants resulted from the increased permeability capacity of these two genes deleted independently. However, it is still unclear whether metabolism changes caused by the gene deletion might influence the expression level of other proteins with similar or relevant functions, which needs to be further elucidated.


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We have previously constructed a novel recombinant polypeptide, PsTag, which should be useful in the development of biological drugs with properties comparable to those achievable by PEGylation, but with potentially less side effects24, 26. Although at an early stage, a number of recombinant polypeptides possess the potential to deliver superior biopharmaceuticals with an improved safety profile and increased patient compliance. Such a large hydrodynamic volume of PsTag polypeptide fusion proteins may not achieve a high yield expressed in the cytoplasm in E. coli, which limits the application of this technology24. The recombinant polypeptides that all share this common problem are due to their similar compositions52, 53. Extracellular expression systems may be a better option for the production of PsTag polypeptides as there is no space limit in the medium. After the third iteration of engineering E. coli JM109, PsTag polypeptides in different apparent molecular sizes were secreted into the medium with significantly enhanced extracellular productions. That greatly simplifies bioproduction and processing because PsTag fusion proteins were purified directly from the medium. These mutants were expected to be applicable in large-scale production for therapeutic proteins of varying size. Conclusion Our work has established extracellular expression systems to achieve high productions of heterologous proteins in Escherichia coli. The results demonstrated the effects and mechanisms of cell envelope-related gene editing on cell growth and extracellular expression. The extracellular expression systems provided genome engineering guidelines for high extracellular production of recombinant proteins. Single-gene deletion mutants were more suitable for the high extracellular expression of small-molecule proteins. Multi-gene deletion mutants should be chosen via the comprehensive selection of the number and species of deleted genes for the high extracellular expression of large-molecule proteins. The extracellular expression systems should help to shed light on the poorly understood aspects of these secretion processes as well as to further assist in the construction of engineering prokaryotic cells for efficient secretion of heterologous proteins.

METHODS Bacterial Strains and Growth Conditions. E. coli JM109, which was bought form Takara Biomedical Technology (Beijing, China), was used as a cloning host and the parental strain for the genome engineering procedures. The bacterial strains used in this study were given in Table S1. All strains were routinely grown aerobically in LB media at 30 ℃ or 37 ℃, and M9 media (6.8 g Na2HPO4,3.0 g KH2PO4,0.5 g NaCl,1.0 g NH4Cl,3.5 g glucose, 0.50 g MgSO47H2O, 0.015 g CaCl2 2H2O) was used for a cell growth and metabolism assay. When needed, appropriate concentrations of antibiotics (100 µg/ml ampicillin, 50 µg/ml kanamycin and 25 µg/ml chloramphenicol) were added to the medium. IPTG (isopropyl-β-D-thiogalactopyranoside, 200 µM) and tetracycline (100 ng/ml) were added to the medium to induce gene expression under the control of the PTAC promoter and PTET promoter respectively. Transcriptomic Analysis. A fresh single colony of E. coli JM109 was inoculated into 3 ml of LB media and cultured overnight at 200 rpm. Overnight cultures were diluted 1:100 into 20 ml LB media and cultured at 200 rpm for approximately 4 h. When the value of OD600 was approximately 0.6, the cells were harvested during the



exponential phase using centrifugation at 3500 g for 5 min. Total RNA extraction and transcriptomic analysis were conducted by GENEWIZ Suzhou, China. Plasmid Construction. The plasmids and primers used in this study are listed in Table S2-S7. The two-plasmid system, including pCasREDcure and pKDG series, which carried the cas9 gene and sgRNA separately, was used for iterative genome engineering. The pCasREDcure plasmid, for whose construction the pCas9 plasmid (ADDGENE #4287631) was used as a template, consisted of the cas9 gene under the control of the PTET promoter, the constitutive expression of λ-Red cassette and the sgRNA with a PTAC promoter guiding the p15A replicon. The λ-Red recombinase gene was amplified from pKD46 using the RED-F1/R1 and RED-F2/R2 primers. The PTAC-gRNA fragment was constructed by fusing the PTAC promoter amplified from pGEX-4T-2 by primers TAC-F1/R1 and the overlapping PCR product of the g-p15A-1 to g-p15A-6 primers together. pCasRED was constructed via a one-step cloning kit (Vazyme Biotech Co., Ltd) by recombining the pCas vector digested with Bsu36 I and the λ-Red cassette. PCasREDcure was constructed by recombining the vector pCasRED digested with Xba I and the overlap PCR product of PTAC and gRNA-p15A. The pKDG series possessed sgRNA with a constitutive promoter guiding the genome and a temperature-sensitive replicon. pKDG was constructed by ligating the AmpR-repA101(Ts) fragment digested from pKD46 using Ava I/Nco I with the Ava I/Nco I digested overlap PCR product of the primers gRNA-1 to gRNA-654. Donor dsDNA in this study only had two homologous arms and no selectable marker. To construct donor dsDNA, upstream and downstream homologous arms were separately amplified from the E. coli JM109 genome using the donor-1/2 and donor-3/4 primers and then fused together by overlap PCR. The fusion proteins containing a tetracysteine motif for FlAsH labeling were encoded from pBtac-cgt-PsTag(n) -TC series. The PTAC promoter fragment was amplified from pGEX-4T-2 by the TAC-F2/R2 primers and ligated into pBAD/Myc-HisA after digestion with Afl II/Nco I to create the vector pBtac. The pBtac-PsTag(n) series was constructed by insertion of DNA encoding the PsTag(n) cassette digested from pET28a-PsTag(n)-FGF2126 between the Nco I and Hind III sites of pBtac. pBtac-cgt-PsTag(n) was constructed by ligating the vector pBtac-PsTag(n) digested with Nco I/BamH I with the BspH I/BamH I-digested overlap PCR product of the CGTase signal peptide gene. Multiple restriction sites were cloned between the EcoR I and Hind III sites of pBtac-cgt-PsTag(n) to create pBtac-cgt-PsTag(n) -XX with the primers XX-F/R. A tetracysteine motif (-GGGGSFLNCCPGCCMEP-)29 gene amplified by primers TC-F/R was inserted between the Xba I and Xho I sites of pBtac-cgt-PsTag(n) -XX to create pBtac-cgt-PsTag(n) -TC series. The gene encoding interferon alpha 2b (GenBank accession no. AAP20099.1) was PCR-amplified from a previously synthesized IFN-containing pUC57 vector (GenScript, Nanjing, China) and inserted between the BamH I and Xba I sites of pBtac-cgt-PsTag(n)-TC to create pBtac-cgt-IFN-TC. Gene Deletion via CRISPR-Cas9. E coli JM109 competent cells harboring pCasREDcure were prepared as previously described25. Tetracycline (a final concentration of 100 ng/ml) was added to the culture for cas9 induction at an OD600 of approximately 0.6, while λ-Red was constitutively expressed during the incubation. After induction for over 30 minutes, the cells were made electrocompetent by cold-sterile ddH2O/glycerol as described previously and were concentrated into 50 µl for each reaction. For electrotransformation, 50 µl of cells were mixed with 100 ng of donor dsDNA and 50 ng of pKDG plasmid and then transferred to a 2-mm electroporation


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cuvette. Electroporation was performed on a Gene Pulser cuvette (Bio-Rad) at 1.8 kV and cells were immediately resuspended in 1 ml of room temperature SOC medium for 2 hours before being spread onto LB agar containing 25 µg/ml chloramphenicol and 100 µg/ml ampicillin and incubated overnight at 30 ℃. Transformants were identified by colony PCR and DNA sequencing and then the correct mutant colonies were inoculated for plasmid curing for iterative engineering. Plasmid Curing. For the curing of the pKDG series, the correct mutant colonies harboring both pCasREDcure and pKDG were spread onto LB agar containing 25 µg/ml of chloramphenicol overnight at 43 ℃ and the colonies were tested for ampicillin sensitivity. Colonies were inoculated for the next gene editing before testing the ampicillin sensitivity because of its high curing efficiency. For the curing of the pCasREDcure, the colonies were inoculated for 12 h in LB with 100 ng/ml tetracycline and 200 µM IPTG at 37 ℃ and then spread onto LB agar. It was confirmed that the pCasREDcure plasmids were eliminated from the colonies, which showed no resistance to chloramphenicol. Integrity Assay of Cell Membranes. Overnight cultures of wild-type E. coli JM109 and mutants were diluted 100-fold in fresh LB medium and cultured at 200 rpm for approximately 4 h. The cells were harvested at an OD600 of approximately 0.8 using centrifugation at 3500 g for 5 min. The cells were washed gently with sterile PBS buffer and resuspended to a final concentration of 1×106~1×107 cells/ml. Then, 100 µl of the suspension was mixed with an equal volume of 1X staining solution containing 10 µM Calcein-AM and 5 µM PI (propidium iodide)55. After 30 minutes of incubation at room temperature in the dark, the stained cells were washed gently with sterile PBS buffer and concentrated to 10 µl. The stained cells were spotted on microscope slides and observed with an Olympus IX 73 fluorescence microscope (Olympus Inc., Japan) with 400X magnification and the fluorescence emission spectrum was measured by 490/545 nm excitation and 515/617 nm emission in a fluorescence spectrophotometer, and each value was normalized by A600. Cell Viability Assays of Deletion Mutants. LB and M9 media were used for cultivations. Overnight cultures were diluted into 200 ml of LB medium at a starting OD600 of 0.01 and were shaken at 200 rpm at 37℃. Samples were taken from each culture at regular intervals for measuring the optical density at 600 nm. The supernatants were separated by centrifugation at 3500 g for 5 minutes for cell lysis assays. Lactate dehydrogenase assay kit (Nanjing Jiancheng Bioengineering Institute, China) was used for the determination of lactate dehydrogenase release. DsDNA HS Assay Kit for Qubit (Yeasen, China) was used for quantitating double-stranded DNA in the supernatants. Overnight cultures were diluted into 200 ml of M9 media (containing 3.5 g/L glucose) at a starting OD600 of 0.01 to determine the glucose consumption. The glucose consumptions in M9 media were measured by a Glucose Assay Kit (Nanjing Jiancheng Bioengineering Institute, China). Proteins Expression and FlAsH Labeling Assay. Overnight cultures of wild-type E. coli JM109 and mutants harboring pBtac-cgt-PsTag(n)-TC series were transferred into 200 ml of fresh LB medium with 100 mg/ml ampicillin and incubated at 37 ℃ before adding 200 µM IPTG to induce protein expression at an OD600 of approximately 1.0. After protein expression for 12 h at 30 ℃, the culture samples were harvested at indicated time points, and the supernatant and cell pellets were separated by centrifugation at 3500 g for 20 minutes at 22 ℃ to avoid cold shock. Periplasm and cytoplasm proteins were separated by osmotic shock method and resuspended in PBS



buffer to a final volume equal to the original culture volume. After filtrating the supernatant through a 0.22 µm membrane, 20 µl of labeling solution containing 20 µM FlAsH-EDT2 and 10 mM DTT were added to each well in a 96-well plate following addition of 180 µl of proteins samples (extracellular, periplasmic, cytoplasmic and total intracellular proteins)56. After incubation in the dark for 1 h at 37 ℃, fluorescence was measured by 508 nm excitation and 528 nm emission, and each value was normalized by the A600. Data were expressed as the mean ± SEM of biological triplicates. Real-time PCR analysis. For quantitative RT-qPCR analyses, total RNA of the wild-type strain and single-gene deletion strains was extracted from the same cell culture which was utilized to analyze transcription levels after 10 h induction. Total RNA was isolated from the cell pellet by using EasyPureTM RNA Kit (Transgen, China) and cDNA was synthesized using GoTaq® 2-Step RT qPCR System (Promega, USA). The quantitative RT-PCR was performed by using SYB green (Applied Biosystem, USA) according to the manufacturer’s instructions (Stepone plus, Applied biosystem). The mRNA expression was normalized to the level of 16s rRNA mRNA. Primers were synthesized by GenScript and were listed in Supporting Information Table S7.

ASSOCIATED CONTENT Supporting Information

Supporting Information Available: Plasmids, strains and primers used in this study, mutagenesis efficiency, the FlAsH assay for TC-fusion proteins, the fluorescence assay of double-stained mutant cells, and PCR analysis of mutants. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]., telephone: +86-25-83271543, Fax: +86-25-83271249. We designate this

one to further communicate with the Editorial and Production offices. *E-mail: [email protected]., telephone: +86-25-83271218, Fax: +86-25-83302827. #

These authors contributed equally to this work. Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS This research was supported by the National Natural Science Foundation of China (No. 81430082, 81703402), the National Science and Technology Major Project(2018ZX09201001-003-002), the Project funded by China Postdoctoral Science Foundation (2017M611957), the Fundamental Research Funds for the Central Universities (2015XPT02), A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions, and the “111 Project” from the Ministry of Education of China and the State Administration of Foreign Expert Affairs of China (No. 111-2-07).

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Figure 1. CRISPR/Cas9-based scarless system for iterative two-gene deletions in E. coli."On day 1 the pCasREDcure plasmid was transformed into wild type E.coli, followed by plating on Cm+ LB at 37℃. On day 2 the resulting strain was electrotransformed with pKDG-A plasmid and donor-A together, and plated on Tet+, Cm+, Amp+ LB at 30℃. On day 3 colonies were screened by PCR and grown at 43℃ for the elimation of pKDG-A overnight. On day 4 Amp-sensitivie colonies were identified. The resulting strain was electrotransformed with pKDG-B plasimd and donor-B together. On day 5 and day 6, double-gene deletion mutants were identified and were incubated in Cm+ LB with IPTG after the elimation of pCasREDcure. On day 7 double-gene deletion strain with no plamids was identified. 59x43mm (600 x 600 DPI)


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Figure 2. Comparison of growth curve and glucose consumption of wild-type strain and mutants. (A) Growth curves in LB media of lipoptrotein-encoding gene deletion mutants. (B) Growth curves in LB media of membrane-associated protein-encoding gene deletion mutants. (C) Glucose consumption in M9 media of the single-gene deletion mutants. The △mepS, △arcA, △nlpI, △osmE, △bamD, △cyoA and △tolC mutants were not tested as their poor cell growth states. (D) Growth curves in LB media of the multi-gene deletion mutants. Error bars represented standard deviations from the mean. Data presented were averages from three separate experiments. (E) Microsopy imaging of single-gene deletion cells after double stained with Calcein-AM and PI (bar=20µm). Cells were harvested at OD600=0.8 (“exponential phase”). Green fluorescence represented cells stained by Calcein. Red fluorescence represented cells stained by PI. The △mepS, △arcA, △nlpI, △osmE, △bamD, △cyoA and △tolC mutants were not tested as their poor cell growth states. 162x262mm (600 x 600 DPI)




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Figure 3. FlAsH assay for extracellular protein expression in single-gene deletion mutants. (A) FlAsH labeling of wild-type strian and single-gene deletion mutants expressing IFN-TC tag and PsTag200/400/600TC tag. Data were expressed as the mean ± SEM of biological triplicates. (B) SDS-PAGE analysis for IFN-TC tag in the supernatant fraction from the same strain as in panel A. (C) SDS-PAGE analysis for PsTag200-TC tag in the supernatant fraction from the same strain as in panel A. (D) SDS-PAGE analysis for PsTag400-TC tag in the supernatant fraction from the same strain as in panel A. Lane 1~18 represesnted the extracelluar protein expression in the wild-type strain and the △lpp, △mepS, △arcA, △nlpI, △pal, △slyB, △osmE, △bamD, △cyoA, △ecnB, △ompA, △tolA, △tolC, △oppA, △dppA, △mrcA, △mrcB mutants respectively. 174x171mm (300 x 300 DPI)



Figure 4. In-depth analysis of PsTag200 expression in the wild-type strain and the single-gene deletion mutants via Sec pathway. (A) PsTag200-TC tag localization in the cytoplasm, periplasm and extracellular medium in different strains. (B) Comparison of the transcript levels of PsTag200 in the wild-type strain and mutants. (C, D and E) Comparison of the transcript levels of Sec components in the wild-type strain and mutants. The expression of mRNA encoding SecB, SecA and SecY was analyzed via quantitative RT-PCR respectively. Values are normalized to their average 16s rRNA values. **P＜0.01,*P＜0.05(N=3, paired twotailed t-test). 133x99mm (300 x 300 DPI)


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Figure 5. FlAsH assay for extracellular protein expression in the multi-gene deletion mutants."(A) FlAsH labeling of the wild-type strian and the multi-gene deletion mutants expressing IFN-TC tag and PsTag200/400/600-TC tag. Data were expressed as the mean ± SEM of biological triplicates. (B) SDS-PAGE analysis for PsTag200-TC tag in the supernatant fraction from the same strain as in panel A. (C) SDS-PAGE analysis for PsTag400-TC tag in the supernatant fraction from the same strain as in panel A. (D) SDS-PAGE analysis for PsTag600-TC tag in the supernatant fraction from the same strain as in panel A. Lane 1~13 represented the extracelluar protein expression in the wild-type strain and the △lpp△mrcA, △lpp△mrcB, △pal△mrcA, △pal△mrcB, △lpp△oppA, △lpp△dppA, △pal△ompA, △pal△tolA, △ompA△dppA, △tolA△dppA, △pal△mrcA△tolA, △pal△mrcA△ompA mutants respectively. (E) Time profiles of PsTag600-TC tag production in the △pal△mrcA△tolA mutant in shake flask cultivation. The origin of coordinates represented the time point of IPTG induction. (F) Time profiles of the cytoplasmic accumulations(●), periplamic accumulations(■), and extracellular accumulations(▲) of PsTag600 in the △ pal△mrcA△tolA mutant. 76x41mm (300 x 300 DPI)



For Table of Contents Use Only Title: Engineering Extracellular Expression Systems in Escherichia coli Based on Transcriptome Analysis and Cell Growth State Authors: Wen Gao#, Jun Yin#, Lichen Bao, Qun Wang, Shan Hou, Yali Yue, Wenbing Yao*, Xiangdong Gao*

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Engineering Extracellular Expression Systems in Escherichia coli

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