Appl Microbiol Biotechnol DOI 10.1007/s00253-013-5381-0

APPLIED GENETICS AND MOLECULAR BIOTECHNOLOGY

Engineering of EPA/DHA omega-3 fatty acid production by Lactococcus lactis subsp. cremoris MG1363 Mitra Amiri-Jami & Gisele LaPointe & Mansel W. Griffiths

Received: 17 September 2013 / Revised: 31 October 2013 / Accepted: 2 November 2013 # Springer-Verlag Berlin Heidelberg 2014

Abstract Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been shown to be of major importance in human health. Therefore, these essential polyunsaturated fatty acids have received considerable attention in both human and farm animal nutrition. Currently, fish and fish oils are the main dietary sources of EPA/DHA. To generate sustainable novel sources for EPA and DHA, the 35-kb EPA/DHA synthesis gene cluster was isolated from a marine bacterium, Shewanella baltica MAC1. To streamline the introduction of the genes into food-grade microorganisms such as lactic acid bacteria, unnecessary genes located upstream and downstream of the EPA/DHA gene cluster were deleted. Recombinant Escherichia coli harboring the 20-kb gene cluster produced 3.5- to 6.1-fold more EPA than those carrying the 35-kb DNA fragment coding for EPA/DHA synthesis. The 20-kb EPA/DHA gene cluster was cloned into a modified broad-host-range low copy number vector, pIL252m (4.7 kb, Ery) and expressed in Lactococcus lactis subsp. cremoris MG1363. Recombinant L. lactis produced DHA (1.35± 0.5 mg g−1 cell dry weight) and EPA (0.12±0.04 mg g−1 cell dry weight). This is believed to be the first successful cloning and expression of EPA/DHA synthesis gene cluster in lactic M. Amiri-Jami : M. W. Griffiths Department of Food Science, University of Guelph, Guelph, ON, Canada M. Amiri-Jami (*) : M. W. Griffiths Canadian Research Institute for Food Safety, University of Guelph, 43 McGilvray St, Guelph, ON, Canada N1G 2W1 e-mail: [email protected] M. Amiri-Jami e-mail: [email protected] G. LaPointe Department of Food and Nutrition Sciences, University of Laval, Quebec, QC, Canada

acid bacteria. Our findings advance the future use of EPA/ DHA-producing lactic acid bacteria in such applications as dairy starters, silage adjuncts, and animal feed supplements. Keywords Lactic acid bacteria . EPA . DHA . Omega-3 synthesis gene cluster

Introduction Eicosapentaenoic acid (EPA; C20:5n3) and docosahexaenoic acid (DHA; C22:6n3) are long chain polyunsaturated fatty acids (PUFA), known as omega-3 fatty acids. In the past decade, EPA and DHA have become popularized as useful dietary components for their beneficial roles in human health and development (Siriwardhana et al. 2012; Swanson et al. 2012). EPA and DHA have important structural roles in brain and retina tissue (Bradbury 2011; Campoy et al. 2012; Singh 2005). Much has been written in recent years on the remarkably wide-ranging physiological effects of EPA and/or DHA and their role in human health (Fontani et al. 2005; Ruan and So 2012). Furthermore, clinical studies have shown the involvement of EPA/DHA in the prevention of breast cancer, diabetes, inflammation, chronic disorders, and, in particular, prevention, management, and treatment of cardiovascular disease (Pishva et al. 2012; Raatz et al. 2013; Rafraf et al. 2012; Sato et al. 2013; Solanki et al. 2013; Zulyniak et al. 2013). Today, natural dietary sources rich in EPA and DHA are limited to fish and fish oils. In recent years, awareness of the health benefits of EPA/DHA has increased among the general public. Therefore, interest in consuming fish, fish oil, and foods enriched with fish oil has significantly increased. As a result, many food products including dairy products which have been fortified either directly with fish oil or fish meal have been fed to farm animals (Hughes et al. 2012; Nelson and Martini 2009; Zachut et al. 2010). Declining fish

Appl Microbiol Biotechnol

populations and the undesirable fishy flavor of food enriched with fish oil have led to an interest in novel, alternative, sustainable, and convenient sources of EPA/ DHA. A limited number of marine bacteria and marine algae are capable of producing EPA/DHA (Gladyshev et al. 2013). However, commercial production of EPA/ DHA from algae is reported to be limited and costly (Chi et al. 2009; Sijtsma and de Swaaf 2004). As a result, EPA/ DHA extracted from algae is applied mainly to pharmaceutical products and infant formula (Doughman et al. 2007; Kimura et al. 2011). Recent investigations have focused on microorganisms as alternative sources of EPA/DHA; however, despite intensive research in the last decade to generate recombinant EPA/DHA in microorganisms such as Escherichia coli, cyanobacteria, or such higher organisms as plants (Abbadi et al. 2004; Lopez et al. 2013; Orikasa et al. 2009; Orikasa et al. 2004; Takeyama et al. 1997; Yu et al. 2000), currently, there is no recombinant source of these “good fats” available for animal and human consumption. A marine bacterium, Shewanella baltica MAC1, has been isolated from fish intestine that is able to produce EPA and DHA (Amiri-Jami et al. 2006). Conceptually, genes involved in the synthesis of EPA/DHA could be expressed in other microorganisms or in higher organisms. We isolated 16 genes from S. baltica MAC1 of which five (pfaA, pfaB, pfaC, pfaD, and pfaE) were shown to be responsible for both EPA and DHA production (Amiri-Jami and Griffiths 2010). Transformation of this gene cluster to different strains of E. coli resulted in the production of both EPA and DHA by recombinant E. coli (Amiri-Jami and Griffiths 2010). Nevertheless, E. coli is not identified as a food-grade (FG) microorganism, so it cannot be used directly in food products. Lactic acid bacteria (LAB) are recognized as FG microorganisms (Li et al. 2011; Lizier et al. 2010; Peterbauer et al. 2011) for many applications, but do not produce EPA and/or DHA. Lactic acid bacteria carrying EPA/DHA genes will be good candidates as novel and alternative sources for omega-3 fatty acids for use in the fermentation of dairy products and silage. They will have several potential applications in the development of improved dairy products and nutrition. Since 1980, LAB strains have been used as a safe vehicle to deliver vaccine antigens to mucosal tissues. Over 30 studies around the world have used genetically modified LAB in vivo for both mucosal (oral) vaccination and therapy against several bacterial and viral pathogens such as Salmonella enteritidis , Listeria monocytogenes , rotavirus, etc. (Tarahomjoo 2012; Wells 2011). Moreover, there are studies showing the use of LAB for recombinant protein production as an alternative for the production of rennin or chymosin, bacteriocins, chitinase, and antigens (Kumar et al. 2010; Martinez et al. 2013; Nguyen et al. 2012; Wells 2011). These studies show safe uses of GM lactic acid bacteria, and

so, they may be used to safely deliver EPA and DHA in dairy products, silage adjuncts, and/or animal feed supplements. The objective of this research was to clone and express the EPA/DHA gene cluster in lactic acid bacteria in order to generate novel, alternative, and sustainable sources for EPA and DHA. In this study, we transferred a reduced size EPA/ DHA gene cluster resulting in an engineered strain of LAB with the ability to produce both EPA and DHA.

Materials and methods Bacterial strains, plasmids, and culture conditions The bacterial strains and plasmids used in this study are detailed in Tables 1 and 2. The transformants EPI300T1 E. coli strain no. 5 and no. 6 were cultured in Luria Bertani (LB) (Difco, Detroit, USA) broth supplemented with 12.5 μg ml−1 chloramphenicol and incubated at 37 °C overnight for plasmid isolation. Inocula of E. coli DH5α isolates harboring placEPA/ DHA, placBS-PS, E. coli EPI300T1 isolates carrying pfEPA/ DHA, pfBS-PS, or E. coli strains carrying pCC1FOS or pClac shuttle vector (as negative controls) were transferred to 200 ml LB broth supplemented with 50 μg ml−1 kanamycin and 12.5 μg ml−1 chloramphenicol, respectively, and then grown at 15 °C for 3 days for EPA/DHA production. Lactococcus lactis IL 1403+ cells harboring pIL252 and L. lactis subsp. cremoris MG 1363+ carrying pIL252m were grown in M17 broth (Difco) supplemented with 0.5 % glucose (GM17) and 5 μg ml−1 erythromycin (GEM17) at 30 °C for plasmid extraction. L. lactis cremoris MG1363+ carrying pIL252m (negative control) and recombinant L. lactis subsp. cremoris MGED20 carrying pEDSB were cultivated in GEM17 broth at 30 °C overnight. Five milliliters of overnight precultured cells were transferred to 200 ml fresh GEM17 broth and incubated at 10, 15, or 30 °C to examine the effect of temperature on EPA/DHA production. Construction of deletion clone of the 35-kb EPA/DHA gene cluster Plasmid pfEPA/DHA was isolated from EPI300T1 E. coli strain no. 5 with QIAprep Spin Miniprep Kit (Qiagen, Toronto, Canada) as described by the manufacturer. To obtain the 35-kb EPA/DHA gene cluster, isolated pfEPA/DHA plasmid was digested with Not I-HF (New England Biolabs, Whitby, Canada). The NotI-HF-excised 35-kb DNA fragment was purified following gel electrophoresis using gelase enzyme and QIAEX II gel extraction kit (Qiagen) then further digested with BstZ17I and PsiI (Fig 1a). The 20-kb region from the BstZ17I to PsiI sites of the 35-kb DNA fragment was purified from 0.8 % agarose gel and was end repaired to blunt 5′-phoshporylated ends using T4 polymerase and T4

Appl Microbiol Biotechnol Table 1 Bacterial strains used in this study

Strain

Description

Reference/source

F − mcrA Δ(mrr-hsdRMS-mcrBC) Φ80dlac ZΔM15 ΔlacX74recA1 endA1 araD139 Δ(ara, leu)7697 galU galK λ-rpsL nupG trfA tonA EPI 300T1 harboring pfEPA/DHA EPI 300T1 harboring pfBS-PS F-φ80lacZΔM15 Δ(lacZYA-argF) U169 recA1 endA1 hsdR17 (rk-, mk+) gal- phoA supE44 λ-thi-1 gyrA96 relA1 DH5α harboring pClac shuttle vector (7.5 kb) DH5α harboring placEPA/DHA DH5α harboring placBS-PS

Epicentre

E. coli EPI 300T1

EPI 300T1 no. 5 EPI300T1 no. 6 DH5α

a

CRIFS stock strains are deposited in the Canadian Research Institute for Food Safety culture collection

b

Department of food and nutrition Sciences, University of Laval, Quebec, QC, Canada

DH5α+ DH5α no. 2 DH5α no. 10 Lactic acid bacteria L. lactis IL1403 L. lactis IL1403+ L. lactis MG1363 L. lactis MG 1363+ L. lactis MGED20

a

Department of Bioresource Engineering, McGill University, QC, Canada

b Commensal and Probiotics-Host Interactions Laboratory, INRA, MICALIS, Jouy-en-Josas, France

This study This study This study

Plasmid-cured variant of IL594 Harboring pIL252 Plasmid-cured variant of NCDO712 Harboring pIL252m

LaPointe G.b This study LaPointe G. This study

MG 1363 carrying pEDSB

This study

polynucleotide kinase (Epicentre, Madison, USA). The 20 kb blunt-end DNA was ligated to the dephosphorylated pCC1FOS and pClac shuttle vectors (Fig. 1b). The resulting vectors were designated pfBS-PS and placBSPS, respectively (Fig. 1c). The pfBS-PS was packaged in vitro into bacteriophage lambda particles using MaxPlax Lambda packaging extracts (Epicentre) and used to infect EPI 300-T1 E. coli previously grown in maltose-containing broth. Infected cells were plated onto LB plate containing 12.5 μg ml−1 chloramphenicol and incubated at 37 °C overnight. E. coli DH5α was transformed with placBS-PS by electroporation using the GenePulser Xcell Electroporation System (Bio-Rad Laboratories, Mississauga, ON). For electroporation, the placBS-PS was added to 50 μl of the ice-cold cells and then transferred to an ice-cold 0.2-cm electroporation cuvette. A single pulse was applied at a field Table 2 Plasmids used in this study

CRIFS stocka This study Invitrogen

strength of 2.5 kV, 200 Ω resistance, and 25 μF capacitance. Immediately after electroporation, the cell suspension was mixed with 0.95 ml of ice-cold S.O.C. medium and incubated at 37 °C for 1.5 h. Appropriate dilutions of the cells were spread on LB plates containing 50 μg ml−1 kanamycin. Randomly chosen antibioticresistant colonies were selected after 24 h growth in order to perform colony PCR for detecting the presence of the pfaA and pfaD genes using forward and reverse primers 5A and 8D as described previously (Amiri-Jami and Griffiths 2010). To determine the insert size in each positive clone for pfaA and pfaD genes, 1 μg of isolated plasmid from each clone was digested with Not IHF. Digested and undigested plasmids were separated in a 1 % agarose gel. In addition, plasmid isolated from control cells which were electroporated in the absence of DNA insert was also digested with Not I-HF.

Plasmid

Description

Reference/source

pCC1FOS pfEPA/DHA pfBS-PS pClac

parABC, Cmr, cloning vector pCC1FOS (8 kb) + 35 kb EPA/DHA gene cluster, Cmr pCC1FOS (8 kb) + 20 kb EPA/DHA gene cluster, Cmr Replication donor plasmid (theta replicating pAMbeta1) Shuttle vector, Emr, APr, Kmr pClac + 35 kb EPA/DHA gene cluster, Kmr pClac + 20 kb EPA/DHA gene cluster, Kmr Low copy number plasmid, Emr Modified pIL252 with NotI site, Emr pIL252m + 20 kb EPA/DHA gene cluster

Epicentre Amiri-Jami and Griffiths (2010) This study Choi, Y. J.a

placEPA/DHA placBS-PS pIL252 pIL252m pEDSB

This study This study Langella, P.b This study This study

Appl Microbiol Biotechnol BstZ17I 1

2

psiI pfaA

pfaE

3

pfaB

pfaC

pfaD

9

35kb

a

10

11

12

13

14 15

16 1kbp

deletion

BstZ17I

psiI

pfaE

pfaA

pfaB

pfaC

pfaD

20kb

b

ligation

T7

cos loxP lacZ parC

ligation Plac pUC ori

Chlr

parB

Apr

RedF pCC1FOS 8139 bp

Kmr NotI 2685 ori V ori 2

parA

Construction of plasmid for cloning EPA/DHA biosynthesis gene cluster in lactic acid bacteria The low copy number pIL252 vector was first modified by replacing the multiple cloning site (MCS) of this vector with a synthetic 160-bp DNA fragment containing a NotI site (Fig. 2). The pIL252 vector and synthetic 160-bp DNA fragment were digested by EcoRI and XhoI at 37 °C for 5 h. Digested fragments were purified using QIAquick PCR purification kit (Qiagen). Cohesive ligation of 160 bp and pIL252 was performed using T4 DNA ligase (Roche, Laval, Canada). The ligation reaction mixture which contained 500 ng plasmid, 100 ng 160 bp DNA fragment, 5 units T4 DNA ligase, 1.5 μl 10× ligation buffer, and sterile H2O in a total volume of 15 μl was incubated at 16 °C overnight. After deactivation of T4 ligase at 65 °C for 10 min, L. lactis MG1363 cells were transformed with 5 μl of reaction using the GenePulser Xcell Electroporation System (Bio-Rad). A single pulse was applied at a field strength of 2.5 kV (peak voltage), 25 μF capacitance, and 400 resistance (Wells et al. 1993). Immediately after transformation, the cell suspension was mixed with 0.95 ml of ice-cold SGM17MC (GM17 medium supplemented with 0.5 mol sucrose, 20 mmol MgCl2, and 2 mmol CaCl2), incubated on ice for 5 min, and then transferred to 30 °C for 2.5 h. Several dilutions of the cells were prepared and 100 μl of each was spread on GM17 plates containing 5 μg ml−1 erythromycin. Plasmid extracts of 10 randomly

pClac

pfBS-PS 28.0 kb

EPA/DHA

c

pCC1FOS

RepE

SacI (7480) 1kbp SpeI (7488) Bam HI (7482) r Em AfeI (868) RepF pClac shuttle 7523 bp

RepD

PT7 RepE f1 ori NotI (3627) XhoI (3633) XbaI (3645) ApaI (3655)

placBS-PS 27.5 kb

EPA/DHA

Fig. 1 Construction of plasmids pfBS-PS and placBS-PS carrying EPA/DHA gene cluster. a Deletion of ORFs located upstream and downstream of EPA/DHA genes (pfaE, pfaA, pfaB, pfaC, pfaD) by BstZ17I and psiI restriction enzymes. b The 20-kb EPA/DHA gene cluster was ligated to vectors pCC1FOS and pClac shuttle vectors as described in “Materials and methods.” c Plasmids pfBS-PS and placBS-PS containing all the genes required for the biosynthesis of EPA/DHA

selected antibiotic-resistant colonies were digested with NotIHF restriction enzyme. The modified plasmid (pIL252m) that was positive for NotI restriction was used for the cloning of 20kb EPA/DHA gene cluster. The plasmid pfBS-PS which contained the 20-kb EPA/DHA gene cluster was digested by NotI-HF at 37 °C overnight. The 20-kb DNA fragment was purified following gel electrophoresis using gelase enzyme and QIAEX II gel extraction kit. Plasmid pIL252m was digested with NotI restriction enzyme, purified, dephosphorylated using Apex Heat-Labile alkaline phosphatase (Epicentre), and ligated to the purified 20-kb EPA/DHA gene cluster using rapid DNA ligation kit (Roche). The ligation reaction mixture which contained 500 ng DNA fragment, 100 ng plasmid, 5 units T4 DNA ligase, 2 μl 10× ligation buffer, and sterile H2O in a total volume of 20 μl was incubated at 16 °C overnight. L. lactis subsp. cremoris MG 1363 was transformed with 5 μl of reaction as described above. Ten random colonies were chosen from the plate and subcultured in 10 ml GM17 supplemented with 5 μg ml−1 erythromycin. Plasmids from 3 ml overnight cultures were extracted using the QIAprep Spin Miniprep kit and digested with NotI-HF restriction enzyme. The plasmid positive for the EPA/DHA gene cluster was designated as pEDSB. Furthermore, PCR was performed for pfaA and pfaD genes using pEDSB as template, using primers and PCR conditions described previously (Amiri-Jami and Griffiths 2010).

Appl Microbiol Biotechnol

b

pIL252 4698 bp

(2946)ClaI PstI XhoI XbaI (2910)ClaI HindIII EcoRI

pfBS-PS 28.0 kb

Replacement of MCS with 160 bp synthetic fragment contained a NotI site

EPA/DHA

a pCC1FOS

The EPA/DHA gene cluster (20 kb) was excised from pfBS-PS by NotI digestion NotI

NotI pfaE

pfaA

pfaB

pfaC

pfaD

pIL252m 4707 bp

Ligation of pIL252m and the 20 kb EPA/DHA gene cluster

pIL252m

(2946)ClaI PstI XhoI XbaI (2910)ClaI NotI HindIII EcoRI

Fatty acid analysis The bacterial cells from 200 ml of E. coli transformants, or L. lactis subsp. cremoris MGED20, were harvested by centrifugation at 8,000 rpm for 17 min then freeze dried overnight. Total lipid from freeze-dried cells was extracted as described previously (Amiri-Jami and Griffiths 2010). The fatty acid methyl esters were analyzed using an automated Agilent 6890 Gas Chromatography (GC) system (Agilent, Palo Alto, USA) and a Varian 3800 GC/Saturn 2000 ion trap mass spectrometer in external EI mode (GC-MS) (Varian, Mississauga, Canada) as described previously (Amiri-Jami and Griffiths 2010). Compounds were identified by comparison with relative retention time and mass spectra with that of pure EPA and DHA standards.

Results Elimination of flanking DNA of the EPA/DHA gene cluster resulted in higher production of EPA/DHA by engineered E. coli In the 35-kb gene cluster from S. baltica MAC1, a total of 16 predicted open reading frames (ORFs) were identified. Out of

pEDSB 24.7 kb

c

EPA/DHA

Fig. 2 Construction of plasmid pEDSB. a Multiple cloning site of pIL252 was excised by EcoRI and XhoI digestion, and 160-bp synthetic fragment containing NotI site was ligated to digested pIL252 as described in “Materials and methods.” b Plasmid pfBSPS was digested by NotI to obtain the 20-kb EPA/DHA gene cluster. c Modified pIL252 carrying NotI site was ligated to the EPA/DHA genes as described in “Materials and methods.” Em r erythromycin, Cop copy control region, rep replication region, MCS multiple cloning site

these, five ORFs, namely ORF4 (pfaE), ORF5 (pfaA), ORF6 (pfaB), ORF7 (pfaC), and ORF8 (pfaD), were homologous with EPA/DHA genes (Amiri-Jami and Griffiths 2010). Seven ORFs out of 16 identified ORFs did not possess any significant homology to GenBank sequences and were identified as coding for hypothetical proteins (Amiri-Jami and Griffiths 2010). In this study, we deleted 11 ORFs that were located upstream of pfaE and downstream of pfaD genes (Fig. 1). The 20-kb region from the Bst Z17I to psi I sites of the 35-kb fragment was subcloned into pCC1FOS vector and pClac, which is an E. coli, Lactobacillus, and Lactococcus shuttle vector. The resulting plasmids were designated pfBS-PS and placBS-PS (Fig. 1). E. coli clones containing both the pfaA and pfaD genes were then tested for EPA/DHA production. E. coli EPI300-T1 and DH5α carrying only the plasmid vectors did not produce any EPA or DHA (Table 3). The amount of DHA was not significantly different among E. coli strains with cloned inserts in the two different vector systems (Table 3). However, the level of EPA produced was significantly lower in E. coli DH5α carrying either the long (35 kb) or short (20 kb) inserts in the pClac shuttle vector compared to the EPI300-T1 strain carrying the cloned inserts in the pCC1FOS vector (Table 3). Most interestingly, E. coli transformants harboring the 20-kb gene cluster produced 3.5to 6.1-fold more EPA than those carrying the 35-kb gene

Appl Microbiol Biotechnol Table 3 Percentage of EPA/ DHA in total fatty acids extracted from recombinant E. coli cells

Bacterial strains

Vectors carrying pfa genesa

EPA/DHA produced (% of total FA) EPA

Values are expressed as means from three independent cultivations at 15 °C

S. baltica MAC1

Wild-type chromosomal pfa genes

1.7

0.04

E. coli E. coli E. coli E. coli E. coli E. coli

pfEPA/DHA pfBS-PS placEPA/DHA placBS-PS pCC1FOS pClac

14 30 9.4 22 0 0

0.4 0.3 0.4 0.4 0 0

EPI 300T1 no. 5 EPI 300T1 no. 6 DH5α no. 2 DH5α no. 10 EPI 300T1 DH5α

cluster (Fig. 3). This level of EPA production by E. coli carrying the 20-kb gene cluster was 30–50 times greater than the amount of the fatty acid produced by the original strain, the marine bacterium S. baltica MAC1 (Fig. 3). This suggests that the 20-kb gene cluster codes for all the genes required for the production of EPA/DHA and that the nucleotide sequences upstream to the BstZ17I site and those downstream to the psiI site are not required for EPA/DHA biosynthesis. Sequence analysis of BstZ17I–psiI region showed five ORFs, of which only ORF2 was oriented in the reverse direction (Fig. 1). Domain analysis of these five ORFs indicated several enzyme domains characteristic of functions present in bacterial polyketide synthase-type multienzyme complexes responsible for the biosynthesis of EPA and DHA as described previously for pfaA, pfaB, pfaC, pfaD, and pfaE genes (Amiri-Jami and Griffiths 2010). The modified vector for LAB can maintain the 20-kb EPA/DHA gene cluster To date, there is no report describing the cloning of the large 20-kb EPA/DHA gene cluster to any lactic acid bacteria vector. No L. lactis clones positive for the EPA/DHA genes were obtained when using pClac, pNZ19, pAK80, pGKV210, or pIL253 as cloning vectors (data are not shown). The only positive clones were obtained with the low copy number plasmid pIL252 (4.6 kb). To facilitate the ligation of the 20kb gene cluster to this vector, we modified the pIL252 by replacing its multiple cloning site (140 bp) with a 160-bp synthetic sequence containing a NotI restriction site (Fig. 2). L. lactis MG1363 transformed with the modified plasmid, designated as pIL252m (4.7 kb), gave erythromycin-resistant (Emr) transformants that were screened for the presence of a NotI site, which was absent in pIL252. The isolated plasmids from the 10 selected transformants were digested by NotI. To clone the 20-kb EPA/DHA gene cluster in L. lactis, the constructed pIL252m plasmid was digested with NotI and ligated with the 20-kb EPA/DHA gene cluster cleaved with the same restriction endonucleases. The resulting plasmid was designated as pEDSB (24.7 kb) (Fig. 2). L. lactis MG 1363

was transformed with a portion of the ligation mixture. Erythromycin-resistant transformants were tested for pfaA and pfaD genes by PCR. Among 10 tested clones, clone no. 3 was positive for both pfaA and pfaD genes. Furthermore, digestion of isolated plasmid DNA from clone no. 3 with NotI resulted in two bands of the correct size, 20 and 4.7 kb corresponding to the EPA/DHA gene cluster and pIL252m, respectively. L. lactis cremoris carrying the 20-kb EPA/DHA gene cluster (clone no. 3) was designated as L. lactis cremoris MGED20. Recombinant production of EPA/DHA in L. lactis GC analysis of FAMEs derived from L. lactis subsp. cremoris MGED20 revealed two extra peaks at retention times corresponding to EPA and DHA standards. No peaks at the EPA/ DHA retention time were identified for the negative control, L. lactis harboring the vector (pIL252m). The EPA/DHA peaks produced by L. lactis MGED20 and identified by GC were further characterized by mass spectrometry. Both 160 140 mg of EPA/ g CDW

a

DHA

120 100 80 60 40 20 0 S. MAC1

E. coli-5

E. coli-6

E. coli-2

E. coli-10

Bacterial Strains Fig. 3 Comparison of EPA production (in milligrams per gram cell dry weight) of S. baltica MAC1 (carrying all pfa genes) with E. coli EPI300T1 no. 5 (harboring pCC1FOS + 35 kb gene cluster), E. coli EPI300T1 no. 6 (harboring pCC1FOS + 20 kb gene cluster), E. coli DH5α no. 2 (harboring pClac + 35 kb gene cluster), and E. coli DH5α no. 10 (harboring pClac + 20 kb gene cluster). S. baltica MAC1 and E. coli strains were grown at 15 °C in triplicates. Total fatty acid was extracted from freeze-dried cells and analyzed as described in “Materials and methods.” Experiment was repeated three times

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electron ionization and chemical ionization modes confirmed that the two extra peaks detected by GC-MS corresponded to the molecular mass of EPA and DHA. Thus, GC and GC-MS results confirmed that the products were EPA and DHA. Furthermore, there was no apparent difference in the fatty acid composition between the wild-type and recombinant L. lactis MGED20, except for the EPA and DHA that were produced. Recombinant L. lactis MGED20 produced 1.35±0.5 mg g−1 dry weight of DHA and 0.12±0.04 mg g−1 dry weight of EPA, while both EPA and DHA were produced at all temperatures tested (Fig. 4). The highest amount of EPA and DHA was produced by L. lactis MGED20 grown at 30 °C.

Discussion In the last 10–15 years, considerable effort has focused on the development of genetic techniques and molecular tools to first characterize and isolate EPA/DHA genes and then transfer the genes to other microorganism or plants in order to create novel sustainable sources for EPA and DHA (Metz et al. 2001;

mg DHA/ g CDW

a

1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 10ºC

b

15ºC Temperature

30ºC

0.16

mg EPA/ g CDW

0.14

0.12 0.1 0.08 0.06 0.04 0.02 0 10°C

15°C Temperature

30°C

Fig. 4 Production of DHA (a) and EPA (b) in L. lactis subsp. cremoris MGED20 containing plasmid pEDSB (pIL252m + 20 kb EPA/DHA gene cluster). L. lactis MGED20 was grown at 10, 15, and 30 °C in triplicate. Total fatty acid was extracted from freeze-dried cells and analyzed as described in “Materials and methods.” Experiment was repeated three times

Okuyama et al. 2007; Orikasa et al. 2009; Sugihara et al. 2008; Zhang et al. 2013). It is reported that several bacterial species of marine origin produce EPA and/or DHA (Gentile et al. 2003; Russell and Nichols 1999). All the genes required for the production of both EPA and DHA have been isolated from a marine bacterium, S. baltica MAC1 (Amiri-Jami and Griffiths 2010). To date, there is no report showing EPA/DHA production by any wild or engineered lactic acid bacterium. In this study for the first time, a plasmid, pEDSB, containing genes responsible for EPA and DHA synthesis were transferred to L. lactis MG. To enable this, the 35-kb EPA/DHA gene cluster originally isolated from S. baltica MAC1 was reduced in size to 20 kb in order to determine if the flanking genes had any impact on omega-3 fatty acid productions. The production of EPA/DHA genes was enhanced when ORFs with unknown functions located upstream and downstream of pfaE (ORF 4) and pfaD (ORF 8) genes were deleted. This suggests that five ORFs spanning 20 kb are essential for the synthesis of EPA and DHA. In addition, it indicates that unknown hypothetical proteins that were identified for ORFs upstream and downstream of pfaE and pfaD genes may possibly downregulate the level of EPA/DHA production. Orikasa et al. (2004) also reported higher production of EPA in E. coli when they downsized their 38 kb EPA biosynthesis gene cluster isolated from Shewanella pneumatophori SCRC2738 to 27 kb nucleotide sequence contained in the XhoI to SpeI region. Our study shows that an additional deletion of 7 kb can be beneficial to EPA and DHA production. Although the basic structure of pfaA-E genes isolated from S. baltica MAC1 showed some similarity to other published pfaA-pfaE genes (Orikasa et al. 2007; Orikasa et al. 2009), the domain structures of each individual gene product are quite different (Amiri-Jami and Griffiths 2010). To introduce the 20-kb EPA/DHA genes to a food-grade microorganism, we investigated several LAB vectors (unpublished data) including pClac, which is an E. coli , Lactobacillus, and Lactococcus shuttle vector. pClac carrying the 20-kb EPA/DHA gene cluster (placBS-PS) was successfully transferred to E. coli DH5α; however, when we transferred placBS-PS to Lactococcus and Lactobacillus strains, no LAB transformants were observed on the plates even after 10 days of incubation. The lack of transformants suggests that the replicon of pClac might be a problem and no replication was obtained when placBS-PS was transferred to LAB strains. In the next step, to test the feasibility of transformation and expression of the large EPA/DHA gene cluster isolated from a gram-negative marine bacterium to LAB strains, the low copy number pIL252 vector was investigated. Several studies have confirmed that this vector can replicate in both L. lactis and Lactobacillus strains and also is able to stably maintain large DNA inserts (D’Angio et al. 1994; Gruzza et al. 1992; O’Sullivan and Klaenhammer 1993; Piard et al. 1997; Simon and Chopin 1988). This vector has a multiple cloning

Appl Microbiol Biotechnol

site for both blunt-end and sticky-end generating restriction endonucleases. However, it is noteworthy that several attempts to insert the 20-kb EPA/DHA gene cluster into the polyrestriction site of pIL252 were all unsuccessful. To insert the 20-kb gene cluster to pIL252 MCS, the MCS of pIL252 was replaced with a synthetic sequence, which resulted in the insertion of the large DNA gene cluster into the modified vector. The pIL252m was able to stably support the large DNA fragment. Addition of the NotI site to the MCS, which was absent in pIL252, facilitated the ligation of the large DNA fragment to the pIL252m vector. Transformation of pIL252m carrying the 20-kb gene cluster to L. lactis subsp. cremoris MG 1363 resulted in the production of EPA and DHA. Our unique gene cluster, encoding proteins required for EPA/DHA biosynthesis, was successfully introduced into and expressed in L. lactis subsp. cremoris, which normally produces unsaturated fatty acids up to C18:1 (9c) (oleic acid) (Johnsson et al. 1995; To et al. 2011). To the best of our knowledge, this is the first report of a successful transformation of the 20-kb EPA/ DHA gene cluster to LAB, which resulted in the production of EPA/DHA in L. lactis subsp. cremoris MGED20. The growth of L. lactis subsp. cremoris MGED20 harboring the EPA/DHA gene cluster at different temperatures indicated the higher production of these fatty acids at the optimal growth temperature of 30 °C. The same phenomenon is reported for recombinant production of DHA in E. coli DH5α (Orikasa et al. 2006) and EPA in E. coli EPI300T1 strain no. 5 (Amiri-Jami and Griffiths 2010), when the level of DHA and EPA was increased by increasing the temperature to 15 °C. Likewise, the effect of temperature on EPA production by L. lactis subsp. cremoris MGED20 is the reverse of that seen in S. baltica MAC1, in which the level of EPA and DHA increased when growth temperature was decreased (AmiriJami et al. 2006). In addition, L. lactis subsp. cremoris MGED20 is able to produce both EPA and DHA at 30 °C, while 25 and 20 °C were the highest temperatures for EPA and DHA production, respectively, by recombinant E. coli strains (Amiri-Jami and Griffiths 2010; Orikasa et al. 2006). These results suggest that besides EPA/DHA biosynthesis gene cluster enzymes, some other unknown factor(s) or enzymes may be absent, which would explain the lack of control by temperature on EPA/DHA production in L. lactis subsp. cremoris MGED20. Furthermore, the quantity of EPA and DHA in L. lactis subsp. cremoris harboring pEDSB is lower than that in E. coli harboring pfEPA/DHA, pfBS-PS, or placBS-PS plasmids. One reason might be a lower copy number of the broad-host-range pIL252 vector in the L. lactis subsp. cremoris as compared to E. coli. The copy number of pIL252 is reported to be 6–9 copies/L. lactis cell (el Alami et al. 1992) and that of the ∼25-kb pEDSB might be even lower. Moreover, plasmid replication of vectors carrying large inserts may not closely accompany cell division, and cells without plasmids may have already been enriched when cells were treated for EPA/DHA

extraction. In addition, L. lactis subsp. cremoris has only an inner membrane compared to the E. coli where incorporation in both inner and outer cell membranes may result in higher EPA and DHA production. Further work is necessary to determine if EPA/DHA production can be improved by optimizing the culture medium and incubation conditions. Omega-3 FA-producing E. coli or marine bacteria cannot be used directly in feed/food products as they are not identified as food-grade microorganisms; therefore, lactic acid bacteria, notably those being used for fermentation of dairy products and silage adjuncts, harboring the EPA/DHA gene cluster could be used in a variety of feed and food applications as a novel alternative source for EPA/DHA. Our findings proved the feasibility of using L. lactis for expressing the large EPA/DHA gene cluster. To apply the recombinant LAB on an industrial scale in food, we will use a broad host range low copy number food-grade vector for transformation of EPA/DHA gene cluster to different strains of lactic acid bacteria, which then can be used in dairy products, silage adjuncts, and/or animal feed supplements. Acknowledgments We thank Dr. Young J. Choi and Dr. Philippe Langella for providing us with plasmids. This work was funded by Dairy Farmers of Ontario and the Natural Sciences and Engineering Research Council of Canada.

References Abbadi A, Domergue F, Bauer J, Napier JA, Welti R, Zahringer U, Cirpus P, Heinz E (2004) Biosynthesis of very-long-chain polyunsaturated fatty acids in transgenic oilseeds: constraints on their accumulation. Plant Cell 16:2734–2748 Amiri-Jami M, Griffiths MW (2010) Recombinant production of omega3 fatty acids in Escherichia coli using a gene cluster isolated from Shewanella baltica MAC1. J Appl Microbiol 109:1897–1905 Amiri-Jami M, Wang H, Kakuda Y, Griffiths MW (2006) Enhancement of polyunsaturated fatty acid production by Tn5 transposon in Shewanella baltica. Biotechnol Lett 28:1187–1192 Bradbury J (2011) Docosahexaenoic acid (DHA): an ancient nutrient for the modern human brain. Nutrients 3:529–554 Campoy C, Escolano-Margarit MV, Anjos T, Szajewska H, Uauy R (2012) Omega 3 fatty acids on child growth, visual acuity and neurodevelopment. Br J Nutr 2:85–106 Chi Z, Liu Y, Frear C, Chen S (2009) Study of a two-stage growth of DHA-producing marine algae Schizochytrium limacinum SR21 with shifting dissolved oxygen level. Appl Microbiol Biotechnol 81:1141–1148 D’Angio C, Beal C, Boquien CY, Corrieu G (1994) Influence of dilution rate and cell immobilization on plasmid stability during continuous cultures of recombinant strains of Lactococcus lactis subsp. lactis. J Biotechnol 34:87–95 Doughman SD, Krupanidhi S, Sanjeevi CB (2007) Omega-3 fatty acids for nutrition and medicine: considering microalgae oil as a vegetarian source of EPA and DHA. Curr Diabetes Rev 3:198–203 el Alami N, Boquien CY, Corrieu G (1992) Batch cultures of recombinant Lactococcus lactis subsp. lactis in a stirred fermentor. I. Effect of plasmid content on bacterial growth and on genetic stability in pure cultures. Appl Microbiol Biotechnol 37:358–363

Appl Microbiol Biotechnol Fontani G, Corradeschi F, Felici A, Alfatti F, Migliorini S, Lodi L (2005) Cognitive and physiological effects of omega-3 polyunsaturated fatty acid supplementation in healthy subjects. Eur J Clin Invest 35:691–699 Gentile G, Bonasera V, Amico C, Giuliano L, Yakimov MM (2003) Shewanella sp. GA-22, a psychrophilic hydrocarbonoclastic Antarctic bacterium producing polyunsaturated fatty acids. J Appl Microbiol 95:1124–1133 Gladyshev MI, Sushchik NN, Makhutova ON (2013) Production of EPA and DHA in aquatic ecosystems and their transfer to the land. Prostaglandins Other Lipid Mediat http://dx.doi.org/10.1016/j. prostaglandins2013.03.002 Gruzza M, Duval-Iflah Y, Ducluzeau R (1992) Colonization of the digestive tract of germ-free mice by genetically engineered strains of Lactococcus lactis: study of recombinant DNA stability. Microb Releases 1:165–171 Hughes BH, Brian Perkins L, Calder BL, Skonberg DI (2012) Fish oil fortification of soft goat cheese. J Food Sci 77:S128–S133 Johnsson T, Nikkila P, Toivonen L, Rosenqvist H, Laakso S (1995) Cellular fatty acid profiles of Lactobacillus and Lactococcus strains in relation to the oleic acid content of the cultivation medium. Appl Environ Microbiol 61:4497–4499 Kimura F, Ito S, Endo Y, Doisaki N, Koriyama T, Miyazawa T, Fujimoto K (2011) Supplementation of DHA-rich microalgal oil or fish oil during the suckling period in mildly n-3 fatty acid-deficient rat pups. Lipids 46:1101–1110 Kumar A, Grover S, Sharma J, Batish VK (2010) Chymosin and other milk coagulants: sources and biotechnological interventions. Crit Rev Biotechnol 30:243–258 Li R, Takala TM, Qiao M, Xu H, Saris PE (2011) Nisin-selectable foodgrade secretion vector for Lactococcus lactis. Biotechnol Lett 33: 797–803 Lizier M, Sarra PG, Cauda R, Lucchini F (2010) Comparison of expression vectors in Lactobacillus reuteri strains. FEMS Microbiol Lett 308:8–15 Lopez NR, Haslam RP, Usher SL, Napier JA, Sayanova O (2013) Reconstitution of EPA and DHA biosynthesis in Arabidopsis: iterative metabolic engineering for the synthesis of n-3 LC-PUFAs in transgenic plants. Metab Eng 17:30–41 Martinez FA, Balciunas EM, Converti A, Cotter PD, de Souza Oliveira RP (2013) Bacteriocin production by Bifidobacterium spp. A review. Biotechnol Adv 31:482–488 Metz JG, Roessler P, Facciotti D, Levering C, Dittrich F, Lassner M, Valentinem R, Lardizabal K, Domergue F, Yamada A, Yazawa K, Knauf V, Browse J (2001) Production of polyunsaturated fatty acids by polyketide synthases in both prokaryotes and eukaryotes. Science 293:290–293 Nelson KA, Martini S (2009) Increasing omega fatty acid content in cow’s milk through diet manipulation: effect on milk flavor. J Dairy Sci 92:1378–1386 Nguyen HA, Nguyen TH, Nguyen TT, Peterbauer CK, Mathiesen G, Haltrich D (2012) Chitinase from Bacillus licheniformis DSM13: expression in Lactobacillus plantarum WCFS1 and biochemical characterisation. Protein Express Purif 81:166– 174 O’Sullivan DJ, Klaenhammer TR (1993) High- and low-copy-number Lactococcus shuttle cloning vectors with features for clone screening. Gene 137:227–231 Okuyama H, Orikasa Y, Nishida T, Watanabe K, Morita N (2007) Bacterial genes responsible for the biosynthesis of eicosapentaenoic and docosahexaenoic acids and their heterologous expression. Appl Environ Microbiol 73:665–670 Orikasa Y, Ito Y, Nishida T, Watanabe K, Morita N, Ohwada T, Yumoto I, Okuyama H (2007) Enhanced heterologous production of eicosapentaenoic acid in Escherichia coli cells that co-express eicosapentaenoic acid biosynthesis pfa genes and foreign DNA

fragments including a high-performance catalase gene, vktA. Biotechnol Lett 29:803–809 Orikasa Y, Nishida T, Yamada A, Yu R, Watanabe K, Hase A, Morita N, Okuyama H (2006) Recombinant production of docosahexaenoic acid in a polyketide biosynthesis mode in Escherichia coli . Biotechnol Lett 28:1841–1847 Orikasa Y, Tanaka M, Sugihara S, Hori R, Nishida T, Ueno A, Morita N, Yano Y, Yamamoto K, Shibahara A, Hayashi H, Yamada Y, Yamada A, Yu R, Watanabe K, Okuyama H (2009) pfaB products determine the molecular species produced in bacterial polyunsaturated fatty acid biosynthesis. FEMS Microbiol Lett 295:170–176 Orikasa Y, Yamada A, Yu R, Ito Y, Nishida T, Yumoto I, Watanabe K, Okuyama H (2004) Characterization of the eicosapentaenoic acid biosynthesis gene cluster from Shewanella sp. strain SCRC-2738. Cell Mol Bio 50:625–630 Peterbauer C, Maischberger T, Haltrich D (2011) Food-grade gene expression in lactic acid bacteria. Biotechnol J 6:1147–1161 Piard JC, Hautefort I, Fischetti VA, Ehrlich SD, Fons M, Gruss A (1997) Cell wall anchoring of the Streptococcus pyogenes M6 protein in various lactic acid bacteria. J Bacteriol 179:3068–3072 Pishva H, Amini M, Eshraghian MR, Hosseini S, Mahboob SA (2012) Effects of EPA supplementation on plasma fatty acids composition in hypertriglyceridemic subjects with FABP2 and PPARalpha genotypes. J Diabetes Metab Disord. doi: 10.1186/2251-6581-11-25 Raatz SK, Rosenberger TA, Johnson LK, Wolters WW, Burr GS, Picklo MJ (2013) Dose-dependent consumption of farmed Atlantic salmon (Salmo salar) increases plasma phospholipid n-3 fatty acids differentially. J Acad Nutr Diet 113:282–287 Rafraf M, Mohammadi E, Asghari-Jafarabadi M, Farzadi L (2012) Omega-3 fatty acids improve glucose metabolism without effects on obesity values and serum visfatin levels in women with polycystic ovary syndrome. J Am Coll Nutr 31:361–368 Ruan D, So SP (2012) Screening and identification of dietary oils and unsaturated fatty acids in inhibiting inflammatory prostaglandin E2 signaling in fat stromal cells. BMC Complement Altern Med 12:143 Russell NJ, Nichols DS (1999) Polyunsaturated fatty acids in marine bacteria—a dogma rewritten. Microbiology 145:767–779 Sato SB, Park J, Kawamoto J, Sato S, Kurihara T (2013) Inhibition of constitutive Akt (PKB) phosphorylation by docosahexaenoic acid in the human breast cancer cell line MDA-MB-453. Biochim Biophys Acta 1831:306–313 Sijtsma L, de Swaaf ME (2004) Biotechnological production and applications of the omega-3 polyunsaturated fatty acid docosahexaenoic acid. Appl Microbiol Biotechnol 64:146–153 Simon D, Chopin A (1988) Construction of a vector plasmid family and its use for molecular cloning in Streptococcus lactis. Biochimie 70: 559–566 Singh M (2005) Essential fatty acids, DHA and human brain. Indian J Pediatr 72:239–242 Siriwardhana N, Kalupahana NS, Moustaid-Moussa N (2012) Health benefits of n-3 polyunsaturated fatty acids: eicosapentaenoic acid and docosahexaenoic acid. Adv Food Nutr Res 65:211–222 Solanki P, Aminoshariae A, Jin G, Montagnese TA, Mickel A (2013) The effect of docosahexaenoic acid (DHA) on expression of IL-1β, IL-6, IL-8, and TNF-alpha in normal and lipopolysaccharide (LPS)-stimulated macrophages. Quintessence Int 44:163 Sugihara S, Orikasa Y, Okuyama H (2008) An EntD-like phosphopantetheinyl transferase gene from Photobacterium profundum SS9 complements pfa genes of Moritella marina strain MP-1 involved in biosynthesis of docosahexaenoic acid. Biotechnol Lett 30:411–414 Swanson D, Block R, Mousa SA (2012) Omega-3 fatty acids EPA and DHA: health benefits throughout life. Adv Nutr 3:1–7 Takeyama H, Takeda D, Yazawa K, Yamada A, Matsunaga T (1997) Expression of the eicosapentaenoic acid synthesis gene cluster from

Appl Microbiol Biotechnol Shewanella sp. in a transgenic marine cyanobacterium, Synechococcus sp. Microbiology 143:2725–2731 Tarahomjoo S (2012) Development of vaccine delivery vehicles based on lactic acid bacteria. Mol Biotechnol 51:183–199 To TM, Grandvalet C, Tourdot-Marechal R (2011) Cyclopropanation of membrane unsaturated fatty acids is not essential to the acid stress response of Lactococcus lactis subsp. cremoris. Appl Environ Microbiol 77:3327–3334 Wells J (2011) Mucosal vaccination and therapy with genetically modified lactic acid bacteria. Annu Rev Food Sci Technol 2:423–445 Wells JM, Wilson PW, Le Page RW (1993) Improved cloning vectors and transformation procedure for Lactococcus lactis. J Appl Bacteriol 74:629–636

Yu R, Yamada A, Watanabe K, Yazawa K, Takeyama H, Matsunaga T, Kurane R (2000) Production of eicosapentaenoic acid by a recombinant marine cyanobacterium, Synechococcus sp. Lipids 35:1061–1064 Zachut M, Arieli A, Lehrer H, Livshitz L, Yakoby S, Moallem U (2010) Effects of increased supplementation of n-3 fatty acids to transition dairy cows on performance and fatty acid profile in plasma, adipose tissue, and milk fat. J Dairy Sci 93:5877–5889 Zhang R, Zhu Y, Ren L, Zhou P, Hu J, Yu L (2013) Identification of a fatty acid (6)-desaturase gene from the eicosapentaenoic acid-producing fungus Pythium splendens RBB-5. Biotechnol Lett 35:431–438 Zulyniak MA, Perreault M, Gerling C, Spriet LL, Mutch DM (2013) Fish oil supplementation alters circulating eicosanoid concentrations in young healthy men. Metabolism 62:1107–1113