Enhanced Extracellular Expression of Gene-Optimized Thermobifida Fusca

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

State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, China

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.

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

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. After the initial glyc-

(Shanghai, China). erol was completely consumed, which was detected by a sudden

increase in both dissolved oxygen and pH, the feeding phase of

the fed-batch cultivation was carried out using a two-stage feed-

2.2. Codon optimization and synthesis of the optimized gene

ing strategy as previously described [22] with some modiﬁcations.

The feeding rate was ﬁrstly increased according to exponential

The codon usage in the gene encoding T. fusca cutinase was ana-

feeding method, and the cell growth was controlled at a speciﬁc

−

lyzed using a graphical codon usage analyzer (http://gcua.schoedl. 1

growth rate () of 0.23 h . When the dry cell weight (DCW)

de/), and optimized by replacing the rarely used codons with

of the culture reached a certain level, the feeding rates depend-

codons preferred by E. coli using DNA works software (http://

ing on the cell growth and glycerol consumption under different

mcl1.ncifcrf.gov/dnaworks/). The encoded amino acid sequence

fermentation conditions were shifted to a gradient-decreasing

was preserved during this process. The free energy of mRNA fold-

method, which would neither make obvious accumulation of glyc-

ing was analyzed using RNA Structure 5.2 software. Optimization

erol nor be deﬁcient for cell growth or cutinase expression. During

of the DNA sequence and synthesis of the codon-optimized gene

the entire process, the dissolved oxygen level was maintained at

were performed by Shanghai Generay Biotech Co., Ltd. (Shanghai,

approximately 30% air saturation by cascading the agitation speed

−

China). In order to facilitate the ligation of the optimized gene 1

(300–900 rpm), adjusting the air-ﬂow rate (2.0–4.0 L min ) and

and the pET-24a(+) vector in the following study, the recogni-

supplementing the air with pure oxygen. Antifoam was added

tion sequences for the restriction enzymes, NdeI and HindIII, were

manually when necessary. At various times, aliquots of the cul-

added in the forward and reverse ends of the optimized gene,

ture were removed and assayed for bacterial biomass and cutinase respectively. activity. Download English Version: https://daneshyari.com/en/article/34349

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