Deleting mrdA and mrcB to significantly improve extracellular recombinant protein production in Escherichia coli

Biochemical Engineering Journal 143 (2019) 185–195

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Regular article

Deleting mrdA and mrcB to significantly improve extracellular recombinant protein production in Escherichia coli ⁎

Haiquan Yang , Fuxiang Wang, Haokun Wang, Xiao Lu, Wei Shen, Xianzhong Chen

T

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The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China

H I GH L IG H T S

extracellular protein production was enhanced by deleting genes mrdA and mrcB. • The of deletion mutants was not significantly inhibited. • Growth morphology of deletion mutants was altered. • Cell • Membrane permeability of deletion mutants was improved.

A R T I C LE I N FO

A B S T R A C T

Keywords: Escherichia coli PBP2 PBP1B Double deletion Secretion Cell morphology

In this work, two key genes (mrdA and mrcB) involved in cell wall biosynthesis were deleted (singly and doubly) in Escherichia coli, and the effects on extracellular recombinant protein production were investigated. The mrdA and mrcB genes encode penicillin-binding protein (PBP) 2 and PBP1B, respectively. The growth of deletion mutants was not significantly inhibited compared with control cells, but cell morphology was altered. The concentration of intracellular soluble peptidoglycan was increased in deletion mutants, especially the double deletion mutant. Extracellular protein production was significantly improved in deletion mutants compared with controls, in the order double deletion > single deletion of mrcB > single deletion of mrdA. Extracellular green fluorescent protein production by the double deletion mutant BL21 ΔmrdA/ΔmrcB::pET28a-gfp was most significantly enhanced compared with control cells (2.6-fold). Extracellular production of recombinant fibroblast growth factor receptor 2 and collagen E4 was also improved in deletion mutants compared with control cells. Extracellular amylase activity of the double deletion mutant BL21 ΔmrdA/ΔmrcB::pET28a-amyk was increased 5.2-fold relative to control cells, but activity in BL21 ΔmrdA::pET28a-amyk and BL21 ΔmrcB::pET28a-amyk single deletion mutants was only increased 1.7- and 4.3-fold, respectively. Extracellular α-galactosidase activity of deletion mutants was also improved, especially the double deletion mutant (2.6-fold). Membrane permeability of deletion mutants was improved compared with control cells, which might increase extracellular recombinant protein production.

1. Introduction Bacterial expression systems for recombinant protein production can be rapid, high-yielding and low-cost [1]. Escherichia coli is one of the most widely-used hosts for efficient recombinant protein production [2,3]. However, secretory production of recombinant proteins in E. coli remains problematic [3] because cells lack an efficient extracellular protein secretion mechanism, and most recombinant proteins cannot be directly secreted into the culture medium [4]. Extracellular production of recombinant proteins can be advantageous compared with cytosolic production; it can avoid intracellular proteolysis, simplify protein

⁎

purification, and facilitate correct protein folding [3]. Gram-negative bacteria employ several different types of protein secretion systems including type I–type III systems [5,6], but extracellular secretion of recombinant proteins depends on the characteristics of individuals proteins and signal peptide sequences [3]. In general, E. coli cells can secrete proteins from the periplasm to the culture medium via compromising the integrity of the outer membrane [3]. Interestingly, glycine supplementation might disrupt peptidoglycan cross-linkages and membrane integrity to increase extracellular production of recombinant proteins in E. coli [3,7]. The bacterial cell wall is a cross-linked polymer that is crucial for

Corresponding authors. E-mail addresses: [email protected] (H. Yang), [email protected] (X. Chen).

https://doi.org/10.1016/j.bej.2019.01.003 Received 8 October 2018; Received in revised form 27 December 2018; Accepted 2 January 2019 Available online 03 January 2019 1369-703X/ © 2019 Elsevier B.V. All rights reserved.

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China). Luria-Bertani (LB) medium comprised 5 g/L tryptone, 10 g/L yeast extract and 10 g/L NaCl, and was used for E. coli seed cultures. Terrific broth (TB) medium comprised 12 g/L tryptone, 24 g/L yeast extract, 16.4 g/L K2HPO4, 2.3 g/L KH2PO4 and 5.1 g/L glycerol, and was used for expression of recombinant protein. Kanamycin (100 mg/L) and spectinomycin (100 mg/L) antibiotics (Sangon Biotech Co., Ltd, Shanghai, China) were added where required.

maintaining the morphology and survival of bacterial cells [8]. The stress-bearing peptidoglycan layer is the major constituent of the cell wall, and provides cellular osmotic stability [8–10]. The peptidoglycan layer surrounding bacterial cells in the periplasmic space between the intracellular and extracellular membranes of Gram-negative bacteria maintains cell morphology [11]. Peptidoglycan synthesis includes three stages; (a) activated soluble nucleotide precursors are synthesised in the cytoplasm [12]; (b) lipid-anchored disaccharide-pentapeptide monomers are assembled by attaching nucleotide precursors to undecaprenyl phosphate [13,14]; (c) glycan chains are inserted into the sacculus [15]. The single-layered peptidoglycan sacculus of E. coli (thickness = 3–6 nm) is connected to the outer membrane by interaction with several outer membrane proteins, and cell enlargement is a highly dynamic process [16,17]. Multi-enzyme complexes with dynamic activities facilitate peptidoglycan growth [16]. Glycosyltransferases (GTases) and DD-transpeptidases (DD-TPases) are required during the final step of peptidoglycan synthesis [16]. The peptidoglycan polymer is synthesised by GTases, generating a repeating disaccharide N-acetylglucosamine-Nacetylmuramic acid polymer with stem peptides [8,15]. DD-TPases then cross-link neighbouring strands (peptides) [8]. Peptidoglycan synthases are divided into three types; bifunctional GTase-TPase enzymes known as class A penicillin-binding proteins (PBPs), monofunctional DDTPases known as class B PBPs, and monofunctional GTases [10,16,18,19]. There are three bifunctional synthases (PBP1 A, PBP1B and PBP1C), one GTase (MgtA) and two DD-TPases (PBP2 and PBP3) in E. coli [10]. PBP1B produces glycan chains of approximately 28 disaccharide units and crosslinks ˜40%–50% of peptides, but PBP1A only produces shorter glycan chains (˜28 disaccharide units) and crosslinks ˜20% of peptides [15,20,21]. The DD-TPase activity of PBP2 then attaches newly synthesised PG to sacculi, but PBP3 requires the putative lipid II flippase FtsW for septal localisation (cell division) [10,16,18,19]. In E. coli, proteins without signal peptides can be translocated across both the inner and outer cell membranes into the extracellular environment under conditions of both osmotic stress and translation stress [22]. By modifying the enzymes synthesising the peptidoglycan layer structure, the permeability of the cell wall could be improved. PBPs of E. coli, as peptidoglycan synthases, are divided into high molecular weight (HMW) and low molecular weight (LMW) class [23]. HMW PBPs include PBP1A, PBP1B, PBP2 and PBP3, and LMW PBPs with D-alanylD-alanine carboxypeptidase activity mainly include PBP4, PBP5, PBP6 and PBP6B [23,24]. In our previous work, we deleted genes of PBP4 (dacB) and PBP5 (dacA) to enhance extracellular recombinant protein secretion in E. coli [23]. In the present work, genes encoding PBP2 (mrdA) and PBP1B (mrcB) in E. coli BL21 (DE3) were deleted to investigate their effect on improving the permeability of the cell wall and thereby promoting extracellular protein production (Fig. 1). Recombinant green fluorescent protein (GFP), recombinant fibroblast growth factor receptor 2 (FGFR2), recombinant collagen E4 and recombinant amylase were employed as model proteins to investigate the effects of deleting mrdA and mrcB on extracellular recombinant protein production. We also investigated the cell growth, morphology, accumulation of intracellular soluble peptidoglycan, and membrane permeability of deletion mutants.

2.2. Gene deletion Genes mrdA and mrcB were deleted in the E. coli BL21 (DE3) parent strain using the CRISPR/cas9 system [25] with primers (Table 2) synthesised by Suzhou Hongxun Biotechnology Co., Ltd (Suzhou, China). Four primers, mrdA-FW1/RV1 and mrdA-FW2/RV2, were used to construct the homology regions consisting of upstream/downstream regions of mrdA (mrdA-U, 503 bp) and mrdA (mrdA-D, 528 bp). Using mrdA-U and mrdA-D as model fragments, fusion PCR was performed with primers mrdA-FW1 and mrdA-RV2 to generate the knockout cassette for deletion of mrdA via the CRISPR/cas9 system. Similarly, four primers, mrcB-FW1/RV1 and mrcB-FW2/ RV2, were used to construct the knockout cassette to delete mrcB sing the same approach. The upstream/downstream regions of mrcB were 512 and 521 bp, respectively. The double deletion mutant was obtained by deleting mrcB from the single deletion ΔmrdA strain. 2.3. Construction of recombinant plasmids and expression of recombinant protein in E. coli

2. Materials and methods

The pET28a vector was used to express GFP (GenBank Accession No. U70496.1), FGFR2 (residues 64―309 corresponding to the whole genome sequence, GenBank Accession No. AKA19580.1), amylase AmyK (GenBank Accession No. KF751392) and collagen E4 (MGHHHHHHHLFEVSALEKEVSALEKEVSALEKEVSALEKGGGGGGPKGPKGPKGPKGPKGPKGPKGPKGPKGPKGPKGPKGPKGPKGPKG). The gfp and amyk genes encoding GFP and AmyK were synthesised by Sangon Biotech Co., Ltd. and separately ligated into plasmid pUC57. The resulting pUC57-gfp and pUC57-amyk plasmids served as PCR templates. PCR conditions for gfp were 94 °C for 4 min, followed by 30 cycles at 98 °C for 10 s, 55 °C for 15 s, 72 °C for 48 s, and a final extension at 72 °C for 10 min. PCR conditions for amyk were 94 °C for 4 min, followed by 30 cycles at 98 °C for 10 s, 61 °C for 15 s, 72 °C for 96 s, and a final extension at 72 °C for 10 min. The gfp and amyk amplification products were separately ligated into pET28a between the EcoRI and XhoI restriction enzyme sites. Meanwhile, the fgfr2 and e4 genes encoding Histagged FGFR2 and E4 were synthesised by GENEWIZ Biotech Co. Ltd. (Suzhou, China) and ligated into plasmid pET28a between NcoI and XhoI, and EcoRI and XhoI, respectively. Recombinant plasmids were verified by DNA sequencing and introduced into competent E. coli cells. Recombinant E. coli cells separately harbouring plasmids pET28agfp, pET28a-fgfr2, pET28a-e4 and pET28a-amyk were used to express GFP, FGFR2, E4 and AmyK (Table 1). Cells were cultured at 37 °C in LB medium for 10 h, and 1% (v/v) was transferred into TB medium for recombinant protein expression. Cells were grown at 37 °C in 30 mL TB medium (250 mL shake flasks) containing 30 μg/mL kanamycin. When the absorbance at 600 nm (OD600) reached 0.6, protein expression was induced with 1 mM isopropyl-β-D-thiogalactoside (IPTG) and culturing was continued at 25 °C.

2.1. Strains, plasmids and media

2.4. Determination of dry cell weight (DCW)

Strains and plasmids used in this work are listed in Table 1. E. coli BL21 (DE3) served as the parent strain in this work. PrimeSTAR HS DNA polymerase and T4 DNA Ligase, used to construct the homologous arms and single synthetic guide RNA (sgRNA), were purchased from TaKaRa Biotechnology Co., Ltd (Dalian, China). The inducer L-arabinose was purchased from Merck Life Science Co., Ltd (Shanghai,

Recombinant E. coli culture broth was centrifuged at 1.0 × 104 × g for 10 min at 4 °C. Harvested cells were washed with 10 mL phosphatebuffered saline (PBS; 10 mM Na2HPO4·12H2O, 10 mM NaH2PO4·2H2O and 500 mM NaCl, pH 7.4) and centrifuged at 1.0 × 104 ×g for 10 min at 4 °C. The cell pellet was dried to constant weight at 105 °C for 2 h, and the growth curve of recombinant E. coli cells was drawn based on 186

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Fig. 1. The peptidoglycan synthesised by penicillin-binding protein (PBP) 2 and PBP1B and deletion of their genes in E. coli. A, Reactions catalysed by enzymes involved in peptidoglycan polymerization [42]. DD-TPases, DD-transpeptidases (PBP2, PBP3, PBP1A, PBP1B and PBPC). GTases, glycosyltransferases (PBP1A, PBP1B, PBPC and Mgt). DD-CPase, D,D-carboxypeptidase. LD-TPases, LD-transpeptidases. B, Peptidoglycan chain synthesis with PBP2 and PBP1B.

2.6. Fluorescence-activated cell sorting (FACS) assay

the DCW of recombinant strains cultured in LB medium (20 mL/250 mL shake flask) at 37 °C.

When the OD600 value of recombinant E. coli cells reached 0.6, 1 mM (w/v, final concentration) IPTG was added and culturing was continued at 25 °C for 8 h. Cells were centrifuged at 1.0 × 104 × g for 10 min at 4 °C, and the cell pellet was resuspended in 10 mM (w/v) PBS buffer to an OD600 (cell density) of 0.5. Resuspended cells were analysed using a FACS Calibur Flow Cytometer (BD Accuri C6, Becton Dickinson, Ashland, USA).

2.5. Determination of glucosamine concentration Glucosamine at different concentrations (0, 5, 10, 15 and 20 μg/mL) was used to prepare a standard curve. A 1 mL volume of acetylacetone solution was mixed with glucosamine solution and boiled for 25 min. The mixture was incubated to complete the reaction, and 1 mL of anhydrous ethanol and 1 mL of p-dimethylaminobenzaldehyde were added to the cooled mixture and incubated at 25 °C for 1 h. The absorbance at 440 nm (A440) was measured with an Epoch2 Microplate Reader (BioTek Instruments, Inc., Winooski, Vermont, USA) and the standard curve was plotted. E. coli cells induced for 8 h were collected and repeatedly frozen and thawed five times. Cells were further lysed using a JXFSTPRP automatic sample rapid grinding machine (Shanghai Jingxin Industrial Development Co., Ltd., Shanghai, China) for 10 min. The cell lysate was centrifuged at 4.5 × 104 × g for 20 min at 4 °C, and the supernatant was freeze-dried to obtain crude soluble peptidoglycan. Crude soluble peptidoglycan obtained was weighed (w1), and a 2 mg sample of which was mixed with 1.5 mL 6 M HCl and incubated for 1 h at 100 °C. The weight (w1) will be used to calculate the glucosamine concentration at last step. Next, 2 mM NaOH solution was added to neutralise the mixture, and deionised water was added to 10 mL (final volume). A 2 mL sample of the reaction solution was mixed with deionised water to 5 mL (final volume), and 1 mL of acetylacetone was added and incubated for 25 min at 100 °C. A 1 mL sample of ethanol and dimethylaminobenzaldehyde was then added to the cooled mixture, incubated for 1 h at 25 °C, and the A440 value was measured to calculate the glucosamine concentration.

2.7. Intracellular and extracellular sample preparation A 1 mL sample of fermentation broth was centrifuged at 1.0 × 104 × g for 10 min at 4 °C. Harvested cells were resuspended in 1 mL PBS buffer (10 mM, pH 7.4) and lysed using a JXFSTPRP automatic sample rapid grinding machine (Shanghai Jingxin Industrial Development Co., Ltd.) for 10 min with 10 s bursts interspersed with 5 s pauses. Lysed cells were used to assay intracellular enzyme activity and protein concentration, soluble peptidoglycan concentration, and membrane permeability, while the supernatant was used to investigate extracellular properties. 2.8. Transmission electron microscopy (TEM) Recombinant E. coli cells were cultured in 20 mL LB medium (250 mL shake flask) at 37 °C to an OD600 of 4.0. Following a 10-fold dilution, cells were cultured on solid LB medium with 1 mM IPTG (w/v) at 4 °C for 24 h, and cell morphology was analysed at 0.5 μm using a Hitachi H7650 TEM (Hitachi Japan Electronics Co., Ltd., Tokyo, Japan). 187

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Table 1 Strains and plasmids used in this work. Factors

Properties and genotype

Sources

Strains E. coli JM109 E. coli BL21 BL21::pET28a BL21::pET28a-amyk BL21::pET28a-gfp BL21::pET28a-fgfr2 BL21::pET28a-e4 BL21 △mrdA BL21 △mrdA::pET28a BL21 △mrdA::pET28a-amyk BL21 △mrdA::pET28a-gfp BL21 △mrdA::pET28a-fgfr2 BL21 △mrdA::pET28a-e4 BL21 △mrcB BL21 △mrcB::pET28a BL21 △mrcB::pET28a-amyk BL21 △mrcB::pET28a-gfp BL21 △mrcB::pET28a-fgfr2 BL21 △mrcB::pET28a-e4 BL21 △mrdA/△mrcB BL21 △mrdA/△mrcB::pET28a BL21 △mrdA/△mrcB::pET28a-amyk BL21 △mrdA/△mrcB::pET28a-gfp BL21 △mrdA/△mrcB::pET28a-fgfr2 BL21 △mrdA/△mrcB::pET28a-e4

Cloning host Wild type E. coli BL21(DE3) E. coli BL21 containing plasmid pET28a E. coli BL21 containing pET28a-amyk E. coli BL21 containing pET28a-gfp E. coli BL21 containing pET28a-fgfr2 E. coli BL21 containing pET28a-e4 E. coli BL21 derivate, deleting mrdA BL21 △mrdA containing pET28a BL21 △mrdA containing pET28a-amyk BL21 △mrdA containing pET28a-gfp BL21 △mrdA containing pET28a-fgfr2 BL21 △mrdA containing pET28a-e4 E. coli BL21 derivate, deleting mrcB BL21 △mrcB containing pET28a BL21 △mrcB containing pET28a-amyk BL21 △mrcB containing pET28a-gfp BL21 △mrcB containing pET28a-fgfr2 BL21 △mrcB containing pET28a-e4 E. coli BL21 derivate, deleting mrdA and mrcB BL21 △mrdA/△mrcB containing pET28a BL21 △mrdA/△mrcB containing pET28a-amyk BL21 △mrdA/△mrcB containing pET28a-gfp BL21 △mrdA/△mrcB containing pET28a-fgfr2 BL21 △mrdA/△mrcB containing pET28a-e4

Novagen Novagen This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work

Plasmidsa pMD19-T vector pCas pTargetF-mrdA pTargetF-mrcB pET28a-amyk pET28a-gfp pET28a-fgfr2 pET28a-e4

TA cloning repA101(Ts) kan Pcas-cas9 ParaB-Red lacIq Ptrc-sgRNA-pMB1 pMB1 aadA sgRNA-mrdA pMB1 aadA sgRNA-mrcB KanR, T7 promoter, pBR322 origin, pET28a containing amyk KanR, T7 promoter, pBR322 origin, pET28a containing gfp KanR, T7 promoter, pBR322 origin, pET28a containing fgfr2 KanR, T7 promoter, pBR322 origin, pET28a containing e4

TaKaRa [25] This work This work This work This work This work This work

a

pTarget was used to express the targeting single synthetic guide RNA (sgRNA) [25]. sgRNA-mrdA, sgRNA with a 20-bp complementary region (N20) sequence for targeting the mrdA region; sgRNA-mrcB, sgRNA with an N20 sequence for targeting the mrcB region. kan, kanamycin resistance gene; aadA, spectinomycin resistance gene; KanR, kanamycin resistance. Table 2 Primers used in this work. Oligonucleotidesa

Sequences (5’→3’)b

Deletion of genesc mrdA-sgRNA-FW mrdA-sgRNA-RV mrdA-FW1 mrdA-RV1 mrdA-FW2 mrdA-RV2 mrcB-sgRNA-FW mrcB-sgRNA-RV mrcB-FW1 mrcB-RV1 mrcB-FW2 mrcB-RV2

GAACGATCCGAATACACCGCGTTTTAGAGCTAGAAATAGCAAGTT ACTAGTATTATACCTAGGACT GGGGATTTTGCTGCTGACCGGCG GTGTCACCCTGATACCACGGTTCGTCATTATTCAGGCGTTCG CGAACGCCTGAATAATGACGAACCGTGGTATCAGGGTGACAC ACATTATGCTGGGTGATAACA GGTTCTGAGCCGCAGTTTGCGTTTTAGAGCTAGAAATAGCAAGTT ACTAGTATTATACCTAGGACT ACTATGAGGATGAAGAACCGATG TGCATGGTCCATAGTGTCAGAGTTTGGCGAAGAGATCATGG CCATGATCTCTTCGCCAAACTCTGACACTATGGACCATGCA GGTTGGATCAAGGATATGTTTG

Plasmid construction GFP-FW GFP-RV AmyK-FW AmyK-RV

CGGAATTCATGAGTAAAGGAGAAGAACTTTTC GAAGATCTTTATTTGTATAGTTCATCCATGC CCGGAATTCATGAGCGAGCTGCCGCAAATC CCGCTCGAGTTAAAAACCGCCATTGAAGGACG

a

FW represents Forward primers. RV represents Reverse primers. Underline letters represent the restriction enzyme sites. Italic letters represent homologous sequences used for gene knockout. c Primers mrdA-sgRNA-FW/RV and mrcB-sgRNA-FW/RV were used to insert the 20-bp complementary region (N20) sequences of mrdA and mrcB into pTargetF to construct recombinant plasmids pTargetF-mrdA and pTargetF-mrcB, respectively. mrdA-FW1/RV1, mrdA-FW2/RV2, mrcB-FW1/RV1 and mrcB-FW2/RV2 were used to construct the homologous arm fragments to delete mrdA and mrcB, respectively. b

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2.9. Determination of GFP fluorescence intensity

2.13. Cell membrane permeability assay

Recombinant E. coli cells harbouring pET28a-gfp were grown to an OD600 of 0.6, induced with 1 mM IPTG to initiate expression of GFP, and culturing was continued at 25 °C. A Synergy H4 Hybrid Multi-Mode Microplate Reader (BioTek Instruments, Inc., Winooski, Vermont, USA) was used to determine the GFP fluorescence intensity at 488 nm (excitation wavelength) and 533 nm (emission wavelength).

The outer membrane permeability of E. coli cells was analysed by determining the uptake of N-phenyl-α-naphthylamine (NPN; Shanghai Aladin Bio-Chem Technology Co., Ltd.) [27]. E. coli cells at an OD600 of 0.6 were induced with IPTG and cultured at 25 °C for a further 4 h. A 200 μL sample of cell suspension (OD600 = 0.5) was mixed with 20 μL NPN (final concentration = 10 mM) and an Epoch2 Multi-Functional Microplate Reader (BioTek Instruments, Inc.) was used to determine the fluorescence intensity at 350 nm (excitation wavelength) and 420 nm (emission wavelength). The permeability of the cell inner membrane was determined by measuring the extent of cytoplasmic access of o-nitrophenyl-β-D-galactopyranoside (ONPG; Shanghai Aladin Bio-Chem Technology Co., Ltd.) [28] using E. coli cells induced with IPTG for 24 h at 25 °C. A 200 μL sample of cell suspension (OD600 = 0.5) was mixed with 20 μL ONPG, incubated for 65 min, and an Epoch2 Multi-Functional Microplate Reader (BioTek Instruments, Inc.) was used to measure the absorbance at 420 nm.

2.10. Determination of amylase activity One unit (U) of amylase was defined as the amount of enzyme producing 1 μmol reducing sugar (glucose) from starch per min under the assay conditions described below. The reaction mixture contained 7.4 g/L (w/v) soluble starch and 30 mM glycine-sodium hydroxide buffer (pH 9.5). The mixture was preheated for 5 min at 50 °C, 100 μL enzyme solution was added, and the mixture (1.35 mL) was incubated for 5 min at 50 °C. Enzyme solution means fermentation supernatant including extracellular enzymes without cells or the supernatant including intracellular enzymes of lysed cells. The reducing sugar content was measured with a modified 3,5-dinitrosalicylic acid (DNS) method [26]. Briefly, the mixture (1 mL) incubated was mixed with 1 mL DNS solution, boiled for 15 min, cooled in ice water, and deionised water was added to 10 mL (total volume). The A540 value was determined using an Epoch2 Microplate Reader (BioTek Instruments, Inc.). Glucose (1 mL) at different concentrations (0, 0.1, 0.2, 0.3, 0.4 and 0.5 g/L) was mixed with 1 mL DNS solution, incubated as described above, and A540 was measured to construct a standard curve.

2.14. Statistical analysis All experiments were independently performed at least three times, and data expressed as mean ± standard deviation (SD). Student’s ttests were used for statistical analysis of differences between mutants and the parent strain following F tests to identify overall significant differences. Following determination of the t value and the degree of freedom, p-values were calculated, and p < 0.05 was considered statistically significant.

2.11. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDSPAGE)

3. Results 3.1. Deletion of mrdA and/or mrcB does not affect the growth of E. coli cells

SDS-PAGE was performed to investigate the effect of deleting mrdA and/or mrcB on extracellular protein production. The difference of extracellular protein production of unit cells between deletion mutants with parent strains was compared in this work. Therefore, the fermentation broth was diluted to the same OD600 value of 1.5, centrifuged, and subjected to SDS-PAGE. A 20 μL sample was mixed with 5 μL of (5×) Sample Loading Buffer (P0015L; Beyotime Biotech Co., Ltd., Shanghai, China) and boiled for 5 min. A 15 μL sample of the mixture was centrifuged at 1.0 × 104 × g for 2 min and separated with a 15% separating gel and a 5% stacking gel using a Mini-PROTEAN Tetra electrophoresis tank (BIO-RAD, California, USA). Gels were stained with 1.2 g/L (w/v) Coomassie Brilliant Blue R250 in 45% (v/v) methanol and 10% (v/v) acetic acid for 2 h at 25 °C, destained with 45% (v/v) methanol and 10% (v/v) acetic acid for 10 h at 25 °C, and visualised with a ChemiDoc XRS + Imaging Systems (BIO-RAD).

The effects of deleting mrdA and/or mrcB on extracellular protein production in E. coli were investigated using the CRISPR/cas9 system. Positive colonies were verified by colony PCR using mrdA-S1 and mrdAA2, and mrcB-S1 and mrcB-A2, respectively (Fig. 1), and both genes were successfully deleted. Analysis of the effects of the deletions on cell growth (Fig. 2) showed that the maximum DCW of mutant strains BL21 ΔmrdA, BL21 ΔmrcB and BL21 ΔmrdA/ΔmrcB was decreased by 5.5%, 9.7% and 17.4%, respectively, compared with control cells (Fig. 2A). The maximum specific growth rates of mutant strains BL21 ΔmrdA, BL21 ΔmrcB and BL21 ΔmrdA/ΔmrcB were decreased from 1.8 h−1 for control cell to 1.5, 1.4 and 1.3 h−1, respectively (Fig. 2B). These results indicate that deletion of mrdA (PBP2) or mrcB (PBP1B) did not significantly inhibit the growth of E. coli cells. 3.2. Deletion of mrdA and/or mrcB affects E. coli cell morphology

2.12. Determination of α-galactosidase activity FACS can be used to quantitatively analyse the cell morphology of microorganisms [29]. Herein, the effects of deleting mrdA and/or mrcB on E. coli cell morphology were analysed by FACS. Forward-scattered and side-scattered light was plotted to investigate the cell population distribution of deletion mutants and the parent strain (Fig. 3A–D). As shown in Fig. 3E–H, E. coli cell morphology was significantly affected by deletion of genes mrdA and mrcB. The distribution of forward-scattered light was graphed to visualise differences in E. coli cell shape [29]. FACS gates were used to provide a relative comparison of cell distribution among gates. The amount of BL21 ΔmrdA::pET28a, BL21 ΔmrcB::pET28a and BL21 ΔmrdA/ΔmrcB::pET28a in gates was decreased by 2.9%, 8.3% and 12.5%, respectively, compared with control cells (Fig. 3E–H). These results indicate that the cell morphology of deletion mutants was altered, especially the double deletion mutant. Deletion of mrcB had a more pronounced effect on cell morphology than deleting mrdA. TEM was then applied to visualise the changes in cell

Under the conditions described below, the amount of enzyme required to produce 1 μmol p-nitrophenol per min was defined as one unit (U) of α-galactosidase activity. Paranitrophenol-α-D-galactopyranose (pNPG) was used as substrate to measure α-galactosidase activity, and the reaction product was monitored by measuring the A400 value. The reaction mixture contained 10 mM (w/v) p-NPG, 10 mM (w/v) citrate buffer (pH 5.8) and 100 μL enzyme solution. Enzyme solution means fermentation supernatant including extracellular enzymes without cells. The mixture was incubated at 45 °C for 15 min, and the reaction was terminated by adding 3 mL 0.25 M (w/v) sodium carbonate. An Epoch2 Multi-Functional Microplate Reader (BioTek Instruments, Inc.) was used to measure the A400 corresponding to the p-nitrophenol produced. The p-nitrophenol samples at different concentrations (0, 0.2, 0.4, 0.6, 0.8 and 1.0 mM) served as standards for plotting a standard curve. 189

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Fig. 2. The effect of deleting mrdA and mrcB on the growth of E. coli. A, Dry cell weight (DCW). The p value indicates significant difference compared with control cell. 1.5 h: ΔmrdA, p < 0.05; ΔmrcB, p < 0.05; ΔmrdA/ΔmrcB, p > 0.05. 3.0 h: ΔmrdA, p > 0.05; ΔmrcB, p < 0.05; ΔmrdA/ΔmrcB, p < 0.01. 4.5 h: ΔmrdA, p > 0.05; ΔmrcB, p < 0.05; ΔmrdA/ΔmrcB, p < 0.05. 6.0 h: ΔmrdA, p < 0.05; ΔmrcB, p < 0.01; ΔmrdA/ΔmrcB, p < 0.01. 8.0 h: ΔmrdA, p < 0.05; ΔmrcB, p < 0.01; ΔmrdA/ΔmrcB, p < 0.01. 10.0 h: ΔmrdA, p < 0.01; ΔmrcB, p < 0.01; ΔmrdA/ ΔmrcB, p < 0.01. B, The specific growth rate (SGR). It is also known as continuous growth rate, the data of which were calculated based on the mean of dry cell weights using an equation (SGR = (lnW2-lnW1)/(t2-t1)) from Hoffmann and Poorter [43]. Control, Wild type E. coli BL21. ΔmrdA, BL21 ΔmrdA. ΔmrcB, BL21 ΔmrcB. ΔmrdA/ΔmrcB, BL21 ΔmrdA/ΔmrcB.

the double deletion.

morphology in more detail (Fig. 3I–L). The cell shape of deletion mutants was more irregular than control cells, with perforated defects evident, especially for the double deletion mutant. These results indicate that deletion of mrdA and mrcB inhibited the biosynthesis of the cell wall peptidoglycan network of E. coli, and this was more acute for

Fig. 3. The effects of deleting mrdA and mrcB on the cell morphology of E. coli. A–H, Fluorescence-activated cell sorting (FACS) analysis of E. coli cells. A–D, Dot plot of E. colicells with forward-scattered light (x-axis) and side-scattered light (y-axis). E–H, Numbers of E. coli cells according to the amount of forward-scattered light. FACS gates were drawn around control cell and superimposed on other deletion mutants to give a relative comparison of cell distribution among gates. I–L, Transmission electron microscopy (TEM) assay on cell morphology. A, E and I, BL21::pET28a (control cell). B, F and J, BL21 ΔmrdA::pET28a. C, G and K, BL21 ΔmrcB::pET28a. D, H and L, BL21 ΔmrdA/ΔmrcB::pET28a. 190

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Fig. 4. Analysis of glucosamine concentrations. The y-axis represents the glucosamine concentration of soluble peptidoglycan, indicating different concentration of the intracellular soluble peptidoglycan in E. coli strains. Control, BL21::pET28a. ΔmrdA, BL21 ΔmrdA::pET28a. ΔmrcB, BL21 ΔmrcB::pET28a. ΔmrdA/ΔmrcB, BL21 ΔmrdA/ΔmrcB::pET28a. Asterisks indicate significant difference compared with control cell (**p < 0.01).

3.3. Deletion of mrdA and/or mrcB increases intracellular soluble peptidoglycan accumulation The E. coli cell wall peptidoglycan is a polymer consisting of alternating β(1→4)-linked N-acetyl-glucosamine and N-acetylmuramic acid resides attached to peptide side chains [30]. Herein, we measured the glucosamine concentration of soluble peptidoglycan to analyse changes in intracellular soluble peptidoglycan concentration in the deletion mutants and the parent strain. As shown in Fig. 4, the glucosamine concentrations in deletion mutants BL21 ΔmrdA::pET28a, BL21 ΔmrcB::pET28a and BL21 ΔmrdA/ΔmrcB::pET28a were 41.5, 48.7 and 54.1 mg/g, respectively, compared with only 37.0 mg/g in control cells. These results indicate that deletion of mrdA and/or mrcB increased intracellular soluble peptidoglycan accumulation, especially in the double deletion mutant. Interestingly, single deleting mrcB had a more pronounced effect on intracellular soluble peptidoglycan accumulation than single deletion of mrdA.

Fig. 5. The effects of deleting mrdA and mrcB on the extracellular production of recombinant green fluorescent protein (GFP), fibroblast growth factor receptor 2 (FGFR2) and collagen E4 in E. coli. A, Extracellular specific fluorescence intensity. Asterisks indicate significant difference compared with control cell (**p < 0.01). The inner, B and C, Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of GFP, FGFR2 and E4, respectively. The inner, GFP. Control, BL21::pET28a-gfp. N-Control, BL21::pET28a (negative control). ΔmrdA, BL21 ΔmrdA::pET28a-gfp. ΔmrcB, BL21 ΔmrcB::pET28a-gfp. ΔmrdA/ΔmrcB, BL21 ΔmrdA/ΔmrcB::pET28a-gfp. Arrow, Target proteins. B, FGFR2. Control, BL21::pET28a-fgfr2. N-Control, BL21::pET28a (negative control). ΔmrdA, BL21 ΔmrdA::pET28a-fgfr2. ΔmrcB, BL21 ΔmrcB::pET28a-fgfr2. ΔmrdA/ΔmrcB, BL21 ΔmrdA/ΔmrcB::pET28a-fgfr2. Arrow, Target proteins (the band above). C, E4. Control, BL21::pET28a-e4. N-Control, BL21::pET28a (negative control). ΔmrdA, BL21 ΔmrdA::pET28a-e4. ΔmrcB, BL21 ΔmrcB::pET28ae4. ΔmrdA/ΔmrcB, BL21 ΔmrdA/ΔmrcB::pET28a-e4. Arrow, Target proteins.

3.4. Deleting mrdA and/or mrcB affects extracellular production of recombinant GFP, FGFR2 and E4

of deleting mrdA and/or mrcB on extracellular protein production in E. coli. As shown in Fig. 5B and C, extracellular production of recombinant FGFR2 and E4 was improved in the deletion mutants compared with control cells.

In order to verify the effects of deleting mrdA and/or mrcB on the extracellular secretion of recombinant GFP, recombinant plasmid pET28a-gfp harbouring the GFP gene was transformed into E. coli BL21 (DE3), and deletion mutants BL21 ΔmrdA, BL21 ΔmrcB and BL21 ΔmrdA/ΔmrcB. As shown in Fig. 5A, the extracellular specific fluorescence intensity of deletion mutants BL21 ΔmrdA::pET28a-gfp, BL21 ΔmrcB::pET28a-gfp and BL21 ΔmrdA/ΔmrcB::pET28a-gfp was increased from 1.8 × 105 A.U./g for control cells to 2.1 × 105, 3.2 × 105 and 4.7 × 105 A.U./g, respectively. The extracellular specific fluorescence intensity of the double deletion mutant BL21 ΔmrdA/ΔmrcB::pET28agfp was significantly higher than both single deletion mutants and control cells. The extracellular specific fluorescence intensity of single deletion mutant BL21 ΔmrcB::pET28a-gfp was higher than that of BL21 ΔmrdA::pET28a-gfp, and both were increased relative to the parent strain. SDS-PAGE was performed to assess the extracellular recombinant GFP concentration of the deletion mutants. As shown in Fig. 5A, the extracellular recombinant GFP concentration of deletion mutants was higher than control cells, suggesting that deletion of mrdA and/or mrcB increased the extracellular production of recombinant GFP in E. coli, especially double deletion. Additionally, recombinant proteins FGFR2 and E4 were also employed as model proteins to investigate the effects

3.5. Deleting mrdA and/or mrcB affects extracellular production of recombinant amylase Recombinant amylase AmyK (˜60 kDa) was employed as a model protein to further verify the effect of deleting mrdA and/or mrcB on the production of extracellular proteins in E. coli. The recombinant plasmid pET28a-amyk harbouring the amylase gene was transformed into E. coli BL21, BL21 ΔmrdA, BL21 ΔmrcB and BL21 ΔmrdA/ΔmrcB. The extracellular amylase specific activity of BL21 ΔmrdA::pET28a-amyk, BL21 ΔmrcB::pET28a-amyk and BL21 ΔmrdA/ΔmrcB::pET28a-amyk was 9.6 × 102, 2.4 × 103 and 2.9 × 103 U/g at 20 h, respectively (Fig. 6A), compared with only 5.6 × 102 U/g for the parent strain. Thus, extracellular production of recombinant amylase was significantly enhanced by deleting mrdA and/or mrcB, especially double deletion. Extracellular amylase specific activity of the double deletion mutant BL21 ΔmrdA/ ΔmrcB::pET28a-amyk was 5.2-fold greater than control cells at 20 h. The effects of deleting mrdA and mrcB on the extracellular distribution of amylase were further investigated (Fig. 6B). The proportion of 191

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Fig. 6. The effects of deleting mrdA and mrcB on the extracellular production of recombinant amylase in E. coli. A, Extracellular amylase specific activity. B, Percentage of extracellular activity of total amylase activity. Asterisks indicate significant difference compared with control cell (none, p > 0.05; *p < 0.05; **p < 0.01). C, SDS-PAGE analysis of extracellular amylase. Arrow, amylase (20 h). M, the standard molecular weight protein markers. Control, BL21-pET28aamyk. N-Control, BL21-pET28a (negative control). ΔmrdA, BL21 ΔmrdA::pET28a-amyk. ΔmrcB, BL21 ΔmrcB::pET28aamyk. ΔmrdA/ΔmrcB, BL21 ΔmrdA/ΔmrcB::pET28a-amyk.

3.7. Deleting mrdA and/or mrcB affects E. coli membrane permeability

extracellular activity relative to total activity was significantly enhanced in deletion mutants compared with control cells, especially in the double deletion mutant. The proportion of extracellular activity for BL21 ΔmrdA::pET28a-amyk, BL21 ΔmrcB::pET28a-amyk and BL21 ΔmrdA/ΔmrcB::pET28a-amyk was increased from 18.6% for control cells to 26.7%, 40.9% and 50.0%, respectively. The extracellular amylase distribution of the double deletion mutant BL21 ΔmrdA/ ΔmrcB::pET28a-amyk was higher than the single deletion mutants and control cells, by 1.2-, 1.9- and 2.7-fold relative to BL21 ΔmrdA::pET28aamyk, BL21 ΔmrcB::pET28a-amyk and control cells, respectively. The effects of deleting mrdA and/or mrcB on extracellular recombinant amylase production were also investigated by SDS-PAGE (Fig. 6C). Protein bands corresponding to amylase in deletion mutants was more obvious than in control cells. Thus, deletion of mrdA and/or mrcB increased extracellular production of amylase, especially in the double deletion mutant BL21 ΔmrdA/ΔmrcB::pET28a-amyk.

As shown in Fig. 7B, the fluorescence intensity of deletion mutant E. coli cells bound to NPN was enhanced compared with control cells, especially for the double mutant. The NPN fluorescence intensity of deletion mutants BL21 ΔmrdA::pET28a, BL21 ΔmrcB::pET28a and BL21 ΔmrdA/ΔmrcB::pET28a was increased from 4.8 × 104 A.U. for control cells to 6.3 × 104, 6.5 × 104 and 6.7 × 104 A.U., respectively. We therefore presumed that deletion of mrdA and/or mrcB disrupted the peptidoglycan network to increase outer membrane permeability. Inner membrane permeability was also investigated (Fig. 7C). Cytoplasmic β-galactosidase in E. coli cells with cytoplasmic membrane damage can hydrolyse ONPG, resulting in a yellow colour. The results showed that the absorbance at 420 nm was ordered double deletion mutant BL21 ΔmrdA/ΔmrcB::pET28a > single deletion mutants BL21 ΔmrdA::pET28a and BL21 ΔmrcB::pET28a > control cells (Fig. 7C). These results suggest that double deletion of mrdA and mrcB increased the inner membrane permeability to a greater extent than single deletions.

3.6. Deleting mrdA and/or mrcB affects the extracellular distribution of αgalactosidase The intracellular enzyme α-galactosidase was used to investigate the effect of deleting mrdA and/or mrcB on extracellular protein production and cell permeability. Deleting mrdA and/or mrcB significantly improved extracellular α-galactosidase specific activity, especially double deletion. As shown in Fig. 7A, the extracellular α-galactosidase specific activity of deletion mutants BL21 ΔmrdA::pET28a, BL21 ΔmrcB::pET28a and BL21 ΔmrdA/ΔmrcB::pET28a was improved from 66.8 U/g for control cells to 117.9, 153.6 and 173.0 U/g, respectively. The extracellular α-galactosidase specific activity of BL21 ΔmrdA::pET28a, BL21 ΔmrcB::pET28a and BL21 ΔmrdA/ΔmrcB::pET28a was increased by 1.8-, 2.3- and 2.6-fold relative to control cells, respectively. This indicates that deletion of mrdA and/or mrcB might improve the permeability of the E. coli cell membrane to promote extracellular production of α-galactosidase.

4. Discussion PBP2 and PBP1B play critical roles in the formation of peptidoglycan linkages during the synthesis of peptidoglycan layers in E. coli [31]. Herein, we singly and doubly deleted PBP2 and PBP1B genes (mrdA and mrcB) in E. coli BL21 (DE3) to improve extracellular protein production. Three deletion mutants, BL21 ΔmrdA, BL21 ΔmrcB and BL21 ΔmrdA/ΔmrcB, were successfully constructed using the CRISPR/ cas9 system. The growth of deletion mutants was not significantly inhibited compared with control cells. E. coli cells possess three types of peptidoglycan synthases; bifunctional GTase-Tpases (PBP1A, PBP1B and PBP1C), two DD-TPases (PBP2 and PBP3) and a GTase (MgtA) [10,18,19]. PBP1B and PBP1 A are partially redundant, one of which is needed for viability of cells [10,32]. It was concluded that deletion of 192

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Fig. 7. The effect of deleting mrdA and mrcB on membrane permeability of E. coli. A, The effects of deleting mrdA and mrcB on the extracellular distribution of α-galactosidase in E. coli. Asterisks indicate significant difference compared with control cell (none, p > 0.05; *p < 0.05; **p < 0.01). B, The effect of deleting mrdA and mrcB on the outer membrane permeability of E. coli. Asterisks indicate significant difference compared with control cell (**p < 0.01). C, The effect of deleting mrdA and mrcB on the inner membrane permeability of E. coli. p < 0.01 (significant difference). Control, BL21::pET28a. ΔmrdA, BL21 ΔmrdA::pET28a. ΔmrcB, BL21 ΔmrcB::pET28a. ΔmrdA/ΔmrcB, BL21 ΔmrdA/ΔmrcB::pET28a.

peptidoglycan. Deleting mrdA and mrcB significantly improved the extracellular production of recombinant proteins in E. coli. Extracellular GFP production levels of the double deletion mutant BL21 ΔmrdA/ ΔmrcB::pET28a-gfp were significantly improved compared with those of single deletion strains. The extracellular specific fluorescence intensity of BL21 ΔmrdA::pET28a-gfp, BL21 ΔmrcB::pET28a-gfp and BL21 ΔmrdA/ΔmrcB::pET28a-gfp was increased by 1.2-, 1.7- and 2.6-fold, respectively, compared with control cells. Deletion of mrdA and mrcB also increased extracellular production of recombinant FGFR2, E4, and amylase. The extracellular amylase specific activity of double deletion mutant BL21 ΔmrdA/ΔmrcB::pET28a-amyk was increased by 5.2-fold relative to control cells, while the extracellular specific amylase activity of single deletion mutants BL21 ΔmrdA::pET28a-amyk and BL21 ΔmrcB::pET28a-amyk was only increased 1.7- and 4.3-fold, respectively. Extracellular production by the double deletion mutant BL21 ΔmrdA/ ΔmrcB::pET28a-amyk was more significantly enhanced than that of the single deletion mutants, indicating that deletion of both mrdA and mrcB is important for extracellular production of recombinant proteins in E. coli. However, in our previous work, we found that double deletion of dacA and dacB did not increase extracellular production of recombinant amylase compared with that of control cells [23]. It was indicated that deleting genes (mrdA and mrcB) of PBP2 with DD-TPase activity and PBP1B with both GTase activity and DD-TPase activity were important for enhancement of recombinant protein extracellular production compared with deletion of PBP4 and PBP5 genes with D-alanyl-D-alanine carboxypeptidase activity. The intracellular enzyme α-galactosidase was also used to verify the effects of gene deletion on cell integrity to explain the enhanced extracellular production by the deletion mutation. The extracellular αgalactosidase specific activity of deletion mutants was significantly enhanced compared with control cells, especially in the case of the double deletion mutant. These results indicate that deletion of mrdA and mrcB destroyed the cell integrity to improve the extracellular production of proteins in E. coli, especially double deletion. Peptidoglycan forms a mesh-like sacculus around the cytoplasmic membrane that is critical for protecting against various stresses such as osmotic pressure [16,31]. PBP 1B, a class A PBP, is a bifunctional GTase-TPase, while PBP2, a class B PBP, is a monofunctional DD-TPase [10,40]. Peptidoglycan synthesis requires both GTases and DD-TPases, since GTases polymerise the glycan chains and DD-TPases cross-link the attached peptides [10,41]. It has been demonstrated that recombinant proteins can be translocated across both the inner and outer cell membranes of E. coli into the culture medium under osmotic stress and translation stress [22]. Herein, deletion of mrdA and mrcB (especially double deletion) disturbed the synthesis of the peptidoglycan layer to promote recombinant protein translocation across both the inner and outer membranes into the extracellular medium. Deleting mrdA and mrcB significantly enhanced the permeability of

PBP1B and PBP2 does not significantly inhibit cell growth. PBP1B and PBP2 play an important role during the initial stages of peptidoglycan synthesis, cell division, elongation, and maintaining cell robustness [33], and their deletion might affect E. coli cell morphology. Herein, FACS analysis was used to investigate the effects of deleting mrdA and mrcB on cell morphology. Based on quantitative FACS analysis, E. coli cell morphology was significantly affected by deletion of mrdA and mrcB, especially in the case of the double deletion mutant. TEM can also be used to analyse cell morphological changes [34]. In the present work, TEM was used to further study the effects of deleting mrdA and mrcB genes on cell morphology. The cell shape of deletion mutants was irregular compared with that of control cells, especially for the double deletion mutant. The cell wall surface structure of single deletion mutants BL21 ΔmrdA::pET28a and BL21 ΔmrcB::pET28a included perforated defects not present in the parent strain, and these defects were more pronounced in the double deletion mutant. This indicates that deletion of mrdA and mrcB inhibited the synthesis of cell wall peptidoglycan, especially double deletion. Consistently, deletion of PBP1B and PBP2 genes is known to disrupt cell wall synthesis and destroy the cell wall structure [35]. An intact peptidoglycan sacculus, with the ability to bear stress is critical for maintaining the shape of bacterial cells [17,36], and this is synthesised and modified by PBPs [36]. The DD-TPase activity of PBP1A and PBP1B is important for assembling the cell wall and forming the normal rod-shaped morphology of E. coli cells [22]. Cross-linking between peptidoglycan chains is catalysed by PBP2, which plays a critical role in maintaining the strength and shape of the cell structure [37,38]. In this work, we found that deletion of mrdA and mrcB (especially double deletion) significantly promoted the accumulation of soluble peptidoglycan, and the results further suggested that the deletions affected the synthesis of cell wall peptidoglycan, especially double deletion. Some peptidoglycan synthases are important for peptidoglycan synthesis and precursor accumulation, as demonstrated for the peptidoglycan synthesis enzyme MurF that decreases the amount of UDPMurNAc-pentapeptide [39]. The peptidoglycan sacculus of E. coli is a single-layer mesh-like structure surrounding the cytoplasmic membrane made from glycan strands connected by short peptides [16,40]. These short peptides include L- and D-amino acids, which are liked to Nacetylmuramic acid (MurNAc) residues, and the glycan strands of peptidoglycan consist of alternating N-acetylglucosamine (GlcNAc) and MurNAc residues linked by β-1,4 glyosidic bonds [40]. In the process of peptidoglycan sacculus growth, GTase activity is needed to polymerise undecaprenyl-pyrophosphoryl-MurNAc(pentapeptide)-GlcNAc (the precursor of lipid II) to form glycan chains, and DD-TPase activity is also required to form the peptide cross-links [10,41]. PBP1B is a bifunctional class A PBP with GTase-TPase activity, and PBP2 is a class B PBP with DD-TPase activity [10]. We presumed that deletion of PBP2 gene mrdA and PBP1B gene mrcB would affect the synthesis of cell wall peptidoglycan and promote the accumulation of soluble 193

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the outer and inner membrane of E. coli in this work, presumably by disrupting the peptidoglycan network. Analysis of cell morphology and the accumulation of soluble peptidoglycan confirmed that peptidoglycan synthesis and structure were disrupted by deleting mrdA and mrcB. Peptidoglycan is an important component in the E. coli cell wall that surrounds the cytoplasmic membrane to provide mechanical strength and protect cells against osmotic stress [10,16]. Peptidoglycan synthases include bifunctional class A PBPs, monofunctional class B PBPs, and monofunctional GTases [10,16,19,41]. PBP2 as a class B PBP and PBP1B is a class A PBP, and both are important for peptidoglycan synthesis, since their deletion disrupts the peptidoglycan network and increases the permeability of the membrane. Enhanced membrane permeability is the main reason that extracellular recombinant protein production in deletion mutants was significantly improved compared with control cells. Double deletion of mrdA and mrcB increased membrane permeability to a greater extent than single deletion, and deleting mrcB ad a more pronounced effect on membrane permeability than deleting mrdA. PBP1B is a bifunctional enzyme with GTase-TPase activity, but PBP2 is a monofunctional DD-TPase [10,16,19,41]. The effects on peptidoglycan network disruption were ordered double deletion > single deletion of mrcB > single deletion of mrdA, as verified by analysis of cell morphology and accumulation of soluble peptidoglycan. These results are also consistent with extracellular recombinant protein production, which was ordered double deletion > single deletion of mrcB > single deletion of mrdA. It was presumed that the deletion mutants might be susceptible to osmotic stresses and be easily damaged by shear forces during cultivation, especially in a fermentor with high stirring speed.

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