Enhanced Extracellular Expression of Gene-Optimized Thermobifida Fusca
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Process Biochemistry 50 (2015) 1039–1046 Contents lists available at ScienceDirect Process Biochemistry jo urnal homepage: www.elsevier.com/locate/procbio Enhanced extracellular expression of gene-optimized Thermobifida fusca cutinase in Escherichia coli by optimization of induction strategy a,b a,b a,b,∗ Lingqia Su , Ruoyu Hong , Jing Wu a State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, China b School of Biotechnology and Key Laboratory of Industrial Biotechnology Ministry of Education, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, China a r t i c l e i n f o a b s t r a c t Article history: Our previous study demonstrated that when Thermobifida fusca cutinase was expressed in Escherichia coli Received 13 November 2014 without mediation of a signal peptide, it could release to the culture medium via the enhanced membrane Received in revised form 26 March 2015 permeability, which was based on its limited phospholipid hydrolysis activity. The goal of the present Accepted 29 March 2015 work was to achieve highly efficient extracellular production of the recombinant signal peptide-free Available online 10 April 2015 cutinase in E. coli. The codons of the T. fusca cutinase gene were optimized for expression in E. coli, and a recombinant expression system was constructed using this optimized gene. After that, the induction strat- Keywords: egy was optimized using high-cell-density cultivation in a 3-L fermentor. Results showed that the optimal Cutinase −1 induction condition was at a dry cell weight (DCW) of 13 g L , and an IPTG/lactose combination induction Escherichia coli strategy is proposed, in which IPTG is added once in a final concentration of 25.0 M, and lactose is fed Extracellular expression −1 −1 −1 −1 Codon optimization at a rate of 0.5 g L h . In this condition, an extracellular cutinase activity of 2258.5 U mL (5.1 g L ) Induction strategy was achieved, which represented the highest cutinase production ever reported, and demonstrated the potential of this system for the industrial production of cutinase. © 2015 Elsevier Ltd. All rights reserved. 1. Introduction obtained from these systems to meet the needs of laboratory research, but the disadvantages including low productivity and Cutinase is a multi-functional enzyme that hydrolyses a variety long fermentation period have made it difficult to meet the needs of substrates, including polyesters, water-insoluble triglycerides of industrial applications. and soluble esters. It is also capable of catalyzing esterification and With the development of recombinant DNA technology, a sub- transesterification [1–3]. Therefore, it is of great potential for uses stantial number of genetically engineered microorganisms have in the textile, detergent, ester synthesis and environmental pro- been constructed to express high levels of cutinase in homolo- tection industries [3–7]. In order to satisfy the demand of these gous or heterologous hosts. Subsequent optimization of regulatory industrial applications, the high-yield preparation of cutinase has elements and fermentation strategies further increased cutinase drawn extensive attention in recent years. production [3,13–16]. Since all of the cutinases isolated from Many cutinase-producing fungi and bacteria have been isolated wild-type microorganisms so far have been found to be secreted and screened. The optimization of cutinase production from these enzymes, and the extracellular expression of recombinant proteins wild-type strains has been widely studied in the past decades. has the advantages of improved product quality and simplified Reports have shown that the yield of cutinase is influenced by downstream isolation process [17], signal peptides are usually both the composition of the medium and culture conditions [8–11]. employed to mediate the extracellular expression of recombinant Certain additives, including cutin, cutin hydrolysis monomers, and cutinase in these reports. To date, the highest extracellular cuti- −1 some lipids can be used as inducers to improve the production nase yield reported, 3.8 g L , was from Glomerella cingulata, which of cutinase [9,12]. Sufficient quantities of cutinases have been was obtained from dense culture of Pichia pastoris grown under fed-batch conditions [13]. In our previous work, the gene encoding Thermobifida fusca cuti- ∗ nase (Tfu 0883, NCBI accession number AAZ54921) was identified Corresponding author at: State Key Laboratory of Food Science and Technol- and extracellularly expressed in Escherichia coli using a variety ogy, Jiangnan University, Wuxi, Jiangsu 214122, China. Tel.: +86 510 85327802; of strategies, including utilizing different secretion systems, co- fax: +86 510 85326653. E-mail address: [email protected] (J. Wu). expression of key secretion transport proteins, and optimization of http://dx.doi.org/10.1016/j.procbio.2015.03.023 1359-5113/© 2015 Elsevier Ltd. All rights reserved. 1040 L. Su et al. / Process Biochemistry 50 (2015) 1039–1046 environmental conditions [18–21]. When cutinase was expressed 2.3. Construction of recombinant E. coli for expression of by the mediation of signal peptides pelB and HlyAs, the extracellu- codon-optimized cutinase lar cutinase activities were 149.2 U/mL and 334 U/mL, respectively, under optimized cultivation conditions in shake flasks [19,20]. The plasmid with the codon-optimized T. fusca cutinase gene With further exploration of the latter one in a 3 L fermenter, the was digested with restriction enzymes NdeI and HindIII. The cutinase yield reached 725 U/mL [21]. In these experiments, an target fragment was then ligated into similarly restricted expres- anomalous phenomenon was discovered, in which T. fusca cuti- sion vector pET-24a(+), resulting in the plasmid pET-24a(+)/cut. nase without mediation of a signal peptide could be transferred This plasmid was used to transform chemically competent E. coli from the cytoplasm to the culture medium in percentages as high BL21(DE3) for expression of cutinase. as 92.5%. Further study about the underlying mechanism of this remarkable phenomenon showed that the enzyme has phospho- lipid hydrolysis activity. After synthesized in the cytoplasm, the 2.4. Media and feeding solutions cutinase without signal peptide could fold into an active form and −1 partially hydrolyze the phospholipids of the membrane, leading to Luria-Bertani (LB) medium, which contained (g L ) NaCl 10.0, the enhanced membrane permeability, which eventually results in tryptone 10.0, yeast extract 5.0, was used for seed cultivation. A the release of cutinase from the cytoplasm to the culture medium. shake-flask culture was grown in Terrific Broth (TB) medium that −1 Moreover, it should be noted that although the membrane was consisted of (g L ) glycerol 5.0, tryptone 12.0, yeast extract 24.0, destroyed to some extent, no obvious cell lysis occurred [21]. K2HPO4 16.4, and KH2PO4 2.3. Cutinase production in a 3-L fermen- In the present study, the extracellular production of recom- tor was performed using a modified semisynthetic medium [22]. −1 binant, gene-optimized, signal peptide-free T. fusca cutinase was The initial batch medium contained (g L ) tryptone 30.0, yeast · performed under high-cell-density cultivation in a 3-L fermentor. extract 20.0, MgSO4 7H2O 2.0, K2HPO4 14.6, glycerol 8.0, (NH4)2-H- −1 In the T7 expression system used in this study, the protein expres- citrate 1.0, and trace metal solution 1.0 mL L , pH 7.0. The feeding −1 sion relies on the strong promoter of the T7 phage, which was only solution was (g L ) tryptone 50.0, yeast extract 50.0, MgSO4 7H2O recognized by the RNA polymerase of the same phage. Both lac- 3.4, and glycerol 500.0. All of the media were supplemented with −1 tose and IPTG could induce the synthesis of the T7 RNA polymerase 30 g mL kanamycin. under control of the inducible lacUV5 promoter in the chromosom- ally integrated cassette. Then the T7 RNA polymerase recognizes the T7 promoter, leading to the production of RNA and the expres- 2.5. Cultivation conditions sion of the target protein. Due to the difference that lactose and IPTG causes a mild and drastic induction, respectively, the type and 2.5.1. Shake-flask concentration of the inducer usually played a critical role in cell Seed cultures were started by inoculating 50 mL of LB medium growth and protein production [22–26], therefore, the induction in a 250 mL shake flask with 100 L sample of a frozen (kept at − ◦ process was optimized in detail. 80 C) glycerol stock of the E. coli expression strain described above, and shaking the resulting culture at 200 rpm for 8 h at ◦ 37 C. A sample (5%, v/v) of this culture was used to inoculate 2. Methods 50 mL of TB medium in a 250 mL shake flask. The resulting culture ◦ was then shaken at 200 rpm and 37 C. When the culture reached 2.1. Bacterial strains, plasmid and materials an OD600 of 1.5, lactose was added to induce cutinase expres- sion. After induction, incubation was continued for an additional The E. coli strain JM109 was used for construction of plasmids 26 h. and E. coli strain BL21(DE3) was used for recombinant cutinase production. The pET-24a(+) vector from Novagen was used as the expression vector. Restriction enzymes, alkaline phosphatase, T4 2.5.2. Bioreactor DNA ligase, and the agarose gel DNA purification kit were pur- Seed cultures were prepared as described above, and then used chased from TakaRa (Dalian, China). The 4-nitrophenyl butyrate to inoculate semisynthetic medium (1.2 L) for fed-batch cultiva- (pNPB) was obtained from Sigma (Shanghai, China). Other chem- tion in a 3-L fermentor (BioFlo 110, New Brunswick Scientific Co., ◦ icals were obtained from Sinopharm Chemical Reagent Co., Ltd New Jersey, USA) kept at 37 C and pH 7.0.