FEMS Microbiology Letters Advance Access published July 7, 2015

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Title:Isolation of strong constitutive promoters from Lactococcus lactis subsp. lactis N8

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Runing title: Selection of strong promoters

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Author: Duolong Zhua, Fulu Liua, Haijin Xua, Yanling Baia, Xiuming Zhanga , Per Erik Joakim Sarisb

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and Mingqiang Qiaoa *

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Affiliations: a College of Life Sciences, Nankai University, Tianjin, China.

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The Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, Nankai

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University, Tianjin, China.

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b

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* Corresponding author.

Department of Food and Environmental Sciences, University of Helsinki, Finland.

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Address: Room 301, College of Life Sciences, Nankai University, Tianjin, China.

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E-mail address: [email protected]

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Phone: 86-022-2350-3340; Fax: 86-022-2350-3340

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Abstract:

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The synthesis of heterologous proteins in Lactococcus lactis is strongly influenced by the promoter

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selected for the expression. The nisin A promoter is commonly used for induced expression of proteins

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in L. lactis, whereas only few constitutive promoters (P45 and the weaker P32) have been used for

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protein expression studies. In this study, eight different putative strong constitutive promoters were

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identified through transcriptional analysis of L. lactis N8 and were investigated for their capability to

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drive nisZ gene expression with promoters P45 and P32 as control. Four strong promoters (P8, P5, P3

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and P2) were identified for the transcriptional activity higher than P45 through RT-qPCR and Agar-

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Diffusion experiments. In addition, these four promoters were fused to the erythromycin resistant gene

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(ermC) with promoter P45 as control and inserted into the backbone of the pNZ8048 vector. All of the

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transcriptional efficiency of promoters P8, P5, P2, and P3 were higher than promoter P45 based on the

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obtained MIC50 values and all of them showed in different activity levels. In conclusion, four strong

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constitutive promoters with a wide range of promoter activities were identified and are suitable for

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protein production in L. lactis.

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Keywords: Lactococcus lactis; strong promoters; transcriptional analysis; nisZ; MIC50.

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Introduction

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Lactic acid bacteria (LAB) are a group of low G-C content Gram-positive, nonsporulating bacteria and

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have relatively small genome, including Lactococcus, Lactobacillus, Leuconostoc, Pediococcus and

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Streptococcus spp.. LAB are widely found in nature and used in the food industry for production and

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preservation of fermented products. These bacteria are considered to be GRAS (generally regarded as

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safe) organisms. The relationship between human and LAB is very close especially on our surfaces, on

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the mucosa and in the intestine (du Toit et al., 1998). Lactococcus lactis, the model LAB, is a good

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candidate for heterologous protein production and has been extensively used in fermented food for

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several centuries (Yoon et al., 2004). L. lactis is a noncolonizing, nonpathogenic microorganism which

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can also be delivered in vivo with the expression of pharmaceutical protein at a mucosal level, and it

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can be used for the clinical therapeutics in humans (Villatoro-Hernandez et al., 2012).

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Despite the commercial importance of L. lactis there is a lack of efficient constitutive promoters for

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the production of heterologous proteins (Nouaille et al., 2003). The yield of biologically expressed

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proteins depends largely on the transcriptional efficiency of promoters. In previous reports, some

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promoters have been used to express heterologous proteins in L. lactis, including both native and

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synthetic promoters (Rud et al., 2006). Nisin-inducible expression promoter presented in the NICE

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system (Kuipers et al., 1995) is probably the best known example which has been used successfully to

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express many recombinant proteins, including membrane proteins (Mierau & Kleerebezem, 2005;

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Frelet-Barrand et al., 2010) and lysostaphin (Turner et al., 2007). An alternative inducible expression

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system in L. lactis is based on the PZn promoter and the ZitR repressor (Morello et al., 2008). ZitR

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repressor controls the heterologous protein expression by binding to the PZn promoter region when

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zinc is abundant in the medium.

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Inducible expression system can be suitable in cases where the aim is to overproduce a protein at

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high levels at a specific moment (Eichenbaum et al., 1998). However, it is less suitable for other

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purposes, such as in-situ production in the human body, in which a steady-state gene expression is

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required (Jensen et al., 1993). In these applications, constitutive promoters could be an alternative

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choice. But, studies on isolation of strong constitutive promoters and on constitutive promoters to

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express the heterologous protein in L. lactis have scarcely been described (Koivula et al., 1991) and

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only two constitutive promoters (P45 and P32) are more commonly used to express the recombinant

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proteins (Takala & Saris, 2002; MacCormick et al., 1995). Presently, there are only few choices for 3

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strong constitutive promoters which could be used for expression of heterologous proteins. Some putative promoters were isolated from L. lactis and Bacillus subtilis with a promoter vector

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probe based on the cat gene as described earlier (Koivula et al., 1991). But the selection of putative

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promoters with this method was less of the target purpose and was randomly with unknowing the

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sequence of these putative promoters. Validation of basic tools such as strong constitutive promoters

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with different activities remains to be developed for L. lactis. Nisin is a small, heat-stable and

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biochemically well-characterized natural antibacterial peptide (Mulders et al., 1991) and it could be

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used as a convenient reporter for identifying the transcriptional efficiency of promoters in several

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Gram-positive bacteria because its activity is easily detectable through Agar-Diffusion experiment

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(Qiao et al., 1996). Erythromycin resistant protein (Erm) can also be used as reporter by the detection

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of MIC50 value (Podbielski et al., 1992).

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In our study, eight putative strong promoters were selected through transcriptional analysis. The

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activity of these different promoters was tested in L. lactis NZ9000 with nisZ and ermC as reporter

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genes. Among eight promoters, the transcriptional efficiency of four promoters was higher than

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promoter P45, two higher than P32 and two lower than P32. The constitutive promoter library

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containing promoters with a wide range of promoter activities was constructed which allows for the

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fine-tuning of steady-state gene expression, especially when inducible systems are not applicable.

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Materials and methods

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Bacterial strains, plasmids and culture conditions

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Table S1 reports the L. lactis strains and plasmids used in this work. All strains used in this study

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except L. lactis N8 are L. lactis NZ9000 host harbouring the different plasmids. The L. lactis strains

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were grown at 30°C under static conditions in M17 medium supplemented with 0.5% (w/v) glucose

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and 0.5% (w/v) sucrose (SGM17). Solid medium contained 1.5% agar. The antibiotics

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chloramphenicol and erythromycin were used at a concentration of 5 μg mL-1 for L. lactis.

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DNA manipulations and cloning

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DNA markers, T4 DNA ligase, restriction enzymes were purchased from Takara (Dalian, China) and

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PCR product purification kit was from Thermo Fisher Scientific (Waltham, USA). The commercial

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nisin was purchased from Sigma (St. Louis, USA). L. lactis plasmid DNA, chromosomal DNA and

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total RNA were isolated using a Qiaprep spin kit (small scale) following manufacturer’s instructions. 4

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Polymerase chain reactions (PCR) were performed with the proof-reading DNA polymeras (LA Taq)

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(Takara, Dalian, China). Primers were synthesized by BGI (Beijing, China) and corresponding

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sequences are listed in Table S2 in the supplemental material. PCR products were purified with a High

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Pure PCR product purification kit (Thermo Fisher Scientific, Waltham, USA) according to the protocol

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of the supplier. Recombinant plasmids were introduced into L. lactis NZ9000 by electrotransformation

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using a Bio-Rad Gene Pulser (Bio-Rad Laboratories, Richmond, CA) according to the method

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described earlier (Holo & Nes, 1989).

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Selection and amplification of the putative strong promoters in L. lactis N8 through

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transcriptional analyses

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The total RNA was extracted from L. lactis N8 and sequenced through Illumina HiSeq 2500

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(GENEWIZ service, Hangzhou, China). Eight putative strong transcriptional genes were selected as

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abundances reported in FPKM [FPKM: fragments per kilobase of transcript per million fragments

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sequenced, similar to RPKM: reads per kilobase of gene model exon, per million mapped reads, a

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measure used earlier (Mortazavi et al., 2008)], which represents the quantity of gene transcripts

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(Trapnell et al., 2010). Then the promoters of corresponding genes were analysed by SoftBerry

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software (http://www.softberry.com) and amplified by PCR with the L. lactis N8 chromosomal DNA as

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the template using the primer pairs P1-F/P1-R, P2-F/P2-R, P3-F/P3-R, P4-F/P4-R, P5-F/P5-R, P6-

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F/P6-R, P7-F/P7-R and P8-F/P8-R, respectively. PCRs were performed using the primers listed in

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Table S2. The promoter P32 and P45 were amplified with pMG36e plasmid DNA and pLEB124

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plasmid DNA as the templates using the primer pairs P32-F/P32-R and P45-F/P45-R, respectively.

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Nucleotide sequences were confirmed by nucleotide sequence analysis. The cloned promoters with

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BglII and PstI restriction sites on flanking regions were named by P1-P8.

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Construction of the putative strong promoter plasmids with nisZ reporter gene

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The nisZ gene was amplified by PCR with primers PnisZ-F/PnisZ-R and chromosomal DNA from L.

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lactis N8 as the template. The PCR product was digested with KpnI and HindIII and cloned into KpnI -

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HindIII-digested pNZ8048, yielding pNZ:nisZ plasmid. The resulting plasmid in which the nisZ gene

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was under the control of the nisin-inducible promoter PnisA, were obtained in L. lactis NZ9000. The

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promoter P32, P45 and P1-P8 fragments were digested with BglII and PstI, respectively, and cloned

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into BglII-PstI-digested pNZ:nisZ, yielding pNZ-32:nisZ, pNZ-45:nisZ and pNZ-(1-8):nisZ plasmids.

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The resulting plasmids were all harboured in L. lactis NZ9000. The hosts of these plasmids were 5

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named as L. lactis NZ9000-PnisA/nisZ, NZ9000-P32/nisZ, NZ9000-P45/nisZ and NZ9000-P(1-8)/nisZ.

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The profile of growth curve of different strains was monitored by measuring the OD600 for 14 h.

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When the OD600 value reached approximately 0.6, the commercial nisin were added to the cultures

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at a concentration of 10 IU/ml (Reunanen & Saris, 2003). The total RNA of L. lactis NZ9000-

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PnisA/nisZ, NZ9000-P32/nisZ, NZ9000-P45/nisZ and NZ9000-P(1-8)/nisZ were isolated from the cells

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cultured for 8 h, 10 h and 12 h, respectively (de Vuyst & Vandamme, 1992). Then the nisZ gene

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expression of these strains were analysed through RT-qPCR with the primer pairs QnisZ-F/R by using

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the comparative CT (2-△△CT) method with L. lactis tufA (Castro et al., 2009), a housekeeping gene

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coding for elongation factor Tu required for the continued translation of mRNA, as a control with the

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primer pairs tufA-F/R. All RT-qPCR reactions were repeated independently four folds.

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For construction of L. lactis nisBTC genes expressing plasmid, nisin-inducible promoter PnisZ and

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nisBTC genes were amplified with L. lactis N8 chromosomal DNA as the template by PCR using the

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primer pairs PP-F/PP-R and Pb-F/Pb-R, respectively. The resulting PnisZ fragment was digested with

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XmaI and BamHI and ligated into the XmaI-BamHI-digested pIL253, yielding pIL253:PnisZ plasmid.

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The resulting plasmid was digested with BamHI and PstI and ligated into the BamH-PstI-digested

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nisBTC fragment, yielding pIL253:PnisZ/nisBTC plasmid. The host was named as L. lactis NZ9000-

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BTC. Then the plasmids pNZ:nisZ, pNZ-32:nisZ, pNZ-45:nisZ and pNZ-(1-8):nisZ were introduced

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into L. lactis NZ9000-BTC. The obtained transformants were named as NZ9000-BTC-PnisA/nisZ,

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NZ9000-BTC-P32/nisZ, NZ9000-BTC-P45/nisZ and NZ9000-BTC-P(1-8)/nisZ and cultured in

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SGM17.

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The nisBTC genes were induced as described above when the OD600 value of these cultures reached

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approximately 0.6. When the cells were cultured for 10 h and 12 h (OD600 ≈1.5), the supernatants were

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collected from 1.5 mL of media by centrifugation at 12000 rpm for 10 min. Then the trypsin was added

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to 1.0 mL supernatants at a concentration of 100 mg mL-1, respectively. The supernatants were

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incubated at 37 °C for 1 h, then shifted to 70 °C for 15 min before they were used in the Agar-Diffusion

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experiment to quantify nisin activity (Wu et al., 2009).

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Construction of the putative strong promoter plasmids with ermC reporter gene

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The ermC gene was amplified with pLEB124 plasmid DNA as the template by PCR using the primer 6

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pairs Perm-F/Perm-R. The PCR product ermC gene fragment was digested with KpnI and HindIII and

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cloned into KpnI-HindIII-digested pNZ8048, yielding pNZ:ermC plasmid. Then the constitutive

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promoter P45 and the selected strong promoters (P8, P5, P3 and P2) with higher transcription

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efficiency than P45 promoter were digested with BglII and PstI, respectively, and cloned into BglII-

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PstI-digested pNZ:ermC, yielding pNZ-45:ermC, pNZ-8:ermC, pNZ-5:ermC, pNZ-3:ermC and pNZ-

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2:ermC plasmids. The resulting plasmids were all harboured in L. lactis NZ9000. The hosts of these

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plasmids were named by L. lactis NZ9000-PnisA/ermC, NZ9000- P45/ermC, NZ9000- P8/ermC,

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NZ9000- P5/ermC, NZ9000- P3/ermC andNZ9000- P2/ermC. The strong promoters (P8, P5, P3 and

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P2) were further identified with ermC reporter gene by the measure of MIC50 value (Cafiso et al.,

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2010).

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Results and Discussion

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Selection of the putative strong constitutive promoters in L. lactis N8 through transcriptional

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analysis

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Eight putative strong promoters were selected by FPKM value through transcriptional analyses with

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nisZ gene as control (Table 1) and analysed by SoftBerry software (Fig. 1) (http://www.softberry.com).

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Eight putative strong promoters were named by P1-P8 on the basis of FPKM value. Putative promoter -

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35 and -10 regions and the putative ribosome-binding site (RBS) were marked with different colors by

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SoftBerry software.

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Identification of the putative strong constitutive promoters through transcriptional analysis of

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nisZ reporter gene

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Before the total RNA was extracted, the profile of growth curve of the corresponding hosts were

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monitored for 14 h. The results showed that there was no significant difference between the control

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strain NZ9000 and the studied strains (Fig. S1). Then the total RNA of NZ9000-PnisA/nisZ, NZ9000-

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P32/nisZ, NZ9000-P45/nisZ and NZ9000-P(1-8)/nisZ were extracted from the cells cultured for 8 h, 10

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h and 12 h and were converted to cDNA, respectively. The nisZ gene expression of these strains was

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analysed through RT-qPCR using the primers QnisZ-F/R with the tufA used as reference gene. All RT-

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qPCR reactions were repeated independently four folds. The transcriptional level of different promoters

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was compared to P45 promoter at 8 h, 10 h and 12 h, respectively (Fig. 2). The results indicated that the

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transcriptional efficiency of the promoter P8, P5, P3, PnisA, P2, P45, P1, P6, P32, P7 and P4 was 7

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decreasing in turn and the difference reached the maximum at 10 h. The transcriptional efficiency of P8

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promoter was about 4.5 folds of P45, P5 was about 3.1 folds of P45, P3 was about 2.2 folds of P45 and

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P2 was about 1.7 folds of P45.

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Identification of the putative strong constitutive promoters with nisin antibacterial activity

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through Agar-Diffusion experiment.

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Antibacterial activity of nisin of supernatants of L. lactis NZ9000-BTC-PnisA/nisZ, NZ9000-BTC-

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P32/nisZ, NZ9000-BTC-P45/nisZ and NZ9000-BTC-P(1-8)/nisZ strains was monitored after

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fermentation of 10 h and 12 h (Fig. 3). The diameter of inhibition zone was measured when the

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indicator bacteria were cultured for 24 h (Table 2). Three independent experiments were performed,

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and result of one was presented in Fig. 3, the two other results were in Fig. S2 and Fig. S3. L. lactis

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NZ9000 harbouring empty pNZ8048 and pIL253 plasmids were used as controls. The results showed

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that the diameter of inhibition zone of the promoter P8, P5, P3, PnisA, P2, P45, P1, P6, P32, P7 and P4

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decreased gradually in order. This indicated the transcriptional efficiency was decreasing gradually as

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well.

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Assessment of the transcriptional efficiency of the ermC reporter expressed under the control of

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The promoter P8, P5, P3 and P2 were selected and used to transcribe ermC gene. The MIC50 of L.

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lactis NZ9000-P45/ermC, NZ9000-P8/ermC, NZ9000-P5/ermC, NZ9000-P3/ermC and NZ9000-

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P2/ermC were measured (Table 3). The results indicated that the transcriptional efficiency of promoters

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decreased gradually from P8 to P45 (P8>P5>P2>P3>P45) similarly as in the Agar-Diffusion

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experiment results described above except for P2 and P3 promoter. The promoter P3 was more

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efficient than P2 based on RT-qPCR and Agar-Diffusion experiment analysis while the P2 promoter

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was more efficient than P3 judged from this result. This difference may be the result of differences in

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translational activities of the two mRNAs with different reporters.

the selected strong promoters

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In this study, we established a targeted procedure to obtain constitutive strong promoters in L.

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lactis, constructed a constitutive promoter library and identified the four strongest constitutive

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promoters. The constructed vectors with the selected strong promoters were successfully used to

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express nisZ and ermC genes in L. lactis strains, showing the effectiveness of selected constitutive

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strong promoters to produce heterologous proteins in L. lactis strains. These promoters could be an

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effective alternative to nisin-inducible expression vectors (Bryan et al., 2000; Sorvig et al., 2005) for 8

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production of non-toxic proteins. The wide range of promoter activities in the constitutive promoter

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library allows for the fine-tuning of gene expression, which is preferable for certain research

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applications such as metabolic control analysis and metabolic optimization (Jensen & Hammer, 1998).

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The constitutive promoter library is useful for achieving stable protein production, especially when

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inducible systems are not applicable. In addition, fermentation is simplified as no inducer needs to be

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added and the consequent production cost will be reduced for the addition of inducer compounds will

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not be used. Furthermore, with the development of synthetic biology, the constitutive promoters are

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required when many metabolic pathways are to be modified in the L. lactis strains in which some

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specific exogenous genes need to be inserted in the target chromosomal DNA (Kleerebezem et al.,

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2000; Song et al., 2014).

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Table 1. The FPKM value of eight putative strong transcriptional genes Promoter Gene ID Protein characteristics PnisZ

FPKM value*

NisZ protein

703.82

P1

N8GL000144

hypothetical protein L84477

38584.25

P2

N8GL000508

ClpB protein

36456.87

P3

N8GL001051

linoleate isomerase

3231.64

P4

N8GL001519

osmotically inducible protein C

11080.47

P5

N8GL001528

hypothetical protein L1010

7477.33

P6

N8GL001575

hypothetical protein L141485

3517.06

P7

N8GL000762

hypothetical protein L106755

4968.22

P8

N8GL001078

phosphopentomutase

4823.52

381 382

*FPKM value: fragments per kilobase of transcript per million fragments sequenced, represented the

383

formula:

quantity

of

gene

transcript.

384 385

12

FPKM

calculation

385 386

387 388 389 390 391

392 393 394

Table 2. Inhibition zone of L. lactis 9000/pIL253:PnisZ/nisBTC having different promoters Promoter Inhibition zone (mm) Foldsa Inhibition zone (mm) Folds 10 h 12 h P45 12.00±0.06 1 12.24±0.12 1 P8

16.24±0.11*

2.06

17.48±0.06*

2.24

P5

15.12±0.08*

1.78

15.72±0.13*

1.82

P3

14.22±0.14*

1.56

14.60±0.17*

1.56

PnisA

13.86±0.04*

1.47

14.12±0.04*

1.53

P2

13.28±0.23*

1.32

13.84±0.12*

1.38

P1

11.58±0.10

0.90

12.16±0.03

0.98

P6

11.24±0.13*

0.81

11.92±0.14

0.92

P32

10.64±0.07*

0.66

11.66±0.16*

0.86

P7

10.56±0.08*

0.64

10.86±0.13*

0.67

P4

9.62±0.12*

0.41

9.74±0.04*

0.41

a

Folds= [(P1-8 or P32 or PnisA)-8 mm]/(P45-8 mm)

* indicates the difference is significant at the 0.05 level between P45 promoter and selected promoter.

Table 3. MIC50 of the four putative strong promoters (N=3, X±SD) Strains NZ9000NZ9000NZ9000NZ9000P45/ermC P8/ermC P5/ermC P3/ermC

NZ9000P2/ermC

MIC50 1200±50 2800±34* 2400±43* 1300±44 1500±52* (µg mL-1) * indicates the difference is significant at the 0.05 level between P45 promoter and selected promoter.

13

394

395 396 397 398 399 400

Fig. 1. Putative strong promoters and structure of the promoter region. The putative -35 regions are marked with blue color, putative -10 regions are marked with orange color and the putative ribosomebinding site (RBS) are marked with red color.

14

400

401 402 403 404 405 406 407

Fig. 2. Transcriptional analysis using RT-qPCR. Total RNA of different time (A: 8 h; B: 10 h; C: 12 h) were extracted from the cultures of the corresponding hosts. RNA was converted to cDNA, and PCRs were performed using the primers listed in Table S2. All PCRs were performed four times. * indicates the difference is significant at the 0.05 level between P45 promoter and selected promoter.

15

407 408

409 410 411 412 413 414 415

Fig. 3. Results of Agar-Diffusion experiment with L. lactis 9000/pIL253:PnisZ/nisBTC having the promoters under study connected to nisZ. Three independent experiments were done, and this picture represents one. A: cells were cultured for 10 h; B: cells were cultured for 12 h; C: control, L. lactis NZ9000 harbouring empty pNZ8048 and pIL253 plasmids.

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Isolation of strong constitutive promoters from Lactococcus lactis subsp. lactis N8.

The synthesis of heterologous proteins in Lactococcus lactis is strongly influenced by the promoter selected for the expression. The nisin A promoter ...
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