Transformation of Folate-Consuming Lactobacillus gasseri into a Folate Producer

Five genes essential for folate biosynthesis in Lactococcuslactis were cloned on a broad-host-range lactococcal vectorand were transferred to the folate auxotroph Lactobacillus gasseri.As a result L. gasseri changed from a folate consumer to a folateproducer. This principle can be used to increase folate levelsin many fermented food products.

INTRODUCTION

Top

Introduction

Folate is an essential component in the human diet, and adequateintake of folate may prevent the occurrence of diseases andsyndromes like neural tube defect, coronary heart disease, anemia,and certain types of cancer (9). Food products like green vegetables,meat, and fermented dairy products contain significant folatelevels. Despite this, folate deficiency occurs throughout theworld, including several well-developed countries. Recently,it has been shown that metabolic engineering can be used toincrease folate levels in fermented foods (12).

Lactic acid bacteria such as Lactococcus lactis and Lactobacillusplantarum have the ability to synthesize folate, which is abiological cofactor involved in their amino acid and nucleotidemetabolism (7, 11). The genes for folate biosynthesis have beenidentified (6, 12). The biosynthetic pathway includes sevenconsecutive steps, in which the precursor guanosine triphosphateis converted into tetrahydrofolate (10). However, some lacticacid bacteria, such as Lactobacillus gasseri strain ATCC 33323,cannot synthesize folate, because the genes involved in folatebiosynthesis are lacking in the genome except for the two genes,folA and folC, involved in regeneration and retention of reducedfolates taken up from the medium (http://genome.jgi-psf.org/draft_microbes/lacga/lacga.draft.html).

The folate biosynthetic genes of L. lactis MG1363 are organizedin a folate gene cluster, consisting of six genes (folA, folB,folKE, folP, ylgG, and folC) (Fig. 1) (12). In the present workwe describe the transformation of the folate-consuming L. gasseriinto a folate producer by the transfer of a broad-host-rangeplasmid containing the folate gene cluster from L. lactis.

View larger version (18K):
[in this window]
[in a new window]
FIG. 1. Construction of pNZ7019 by restriction of pNZ7017 and the amplification of the folate gene cluster from chromosomal DNA of L. lactis MG1363.

Cloning and transformation of the folate gene cluster of L. lactis into L. gasseri.

Top

Cloning and Transformation of...

The plasmid pNZ7017 (12) was digested by using XbaI and SphI(both Invitrogen, Paisley, United Kingdom) as restriction enzymes.The 3.1-kb DNA fragment that was obtained from the digestionconsisted of the constitutive pepN promoter (14), a part ofthe multiple cloning site, chloroamphenicol resistance marker,and replication genes that originated from pNZ12 (1). The folategene cluster (folB, folKE, folP, ylgG, and folC) of L. lactiswas amplified by PCR by using high-fidelity Pwo polymerase (Invitrogen).The forward primer SphfolB-F (5'-AGGAAGCATGCCTTACAAAATAAAACTTAATAATATG-3')was extended at the 5' end, creating an SphI restriction siteoverlapping the start codon of folB. The reverse primer folCXba-R(5'-TCTCTAGACTACTTTTCTTTTTTCAAAAATTCACG-3') was extended atthe 5' end, creating an XbaI restriction site that overlappedthe stop codon of folC (Fig. 1). The amplified PCR fragmentwas restricted with XbaI and SphI. Subsequently, the two fragmentswere ligated by using T4 ligase (Invitrogen), generating a translationalfusion between the constitutive promoter of the pepN gene (14)and the folate gene cluster (Fig. 1). The resulting plasmidwas designated pNZ7019. After transformation to L. lactis NZ9000and subsequent cultivation of the strain, the plasmid was harvestedas described previously (2).

L. gasseri (ATCC 33323) was transformed with purified pNZ7019by using an established procedure (8) and was plated on MRSmedium (Merck, Darmstadt, Germany) containing 10 µg ofchloramphenicol/ml. After incubation for 40 h at 37°C, chloramphenicol-resistantcolonies were examined for the presence of pNZ7019 by usingrestriction analyses. An L. gasseri colony harboring pNZ7019was used for renewed cultivation by using the same growth conditionsas previously described. Random amplified polymorphic DNA fingerprintanalysis was used to confirm the identity of the transformantharboring pNZ7019 as L. gasseri ATCC 33323 (results not shown).

Conversion of folate consumer into folate producer.

Top

Conversion of folate consumer...

A modified Folic Acid Casei Medium (FACM) (Difco, Becton Dickinsonand Co., Sparks, Md.) was developed for growth and subsequentfolate analysis of the L. gasseri wild-type strain and the L.gasseri strain harboring pNZ7019. The FACM was enriched with1 mg of vitamin B₁₂ (Sigma-Aldrich Chemie, Gmbh, Steinheim,Germany)/liter and 1 ml of Tween 80 (Merck, Darmstadt, Germany)/liter.The wild-type strain could not grow at 37°C unless folatewas added (1.0 mg/liter), whereas the strain harboring pNZ7019showed folate-independent growth.

Folate levels were quantified by using the Lactobacillus caseimicrobiological assay, including enzymatic deconjugation ofthe polyglutamate tail (5, 11, 12). The L. gasseri strain harboringpNZ7019 produced significant intracellular and extracellularfolate levels (Fig. 2). As expected, the wild-type strain consumedfolate from the medium and intracellular folate concentrationsremained below the detection limit (Fig. 2).

View larger version (26K):
[in this window]
[in a new window]
FIG. 2. Folate production of the L. gasseri wild-type strain (side marked 1) and the L. gasseri strain harboring pNZ7019 (side marked 2), grown on modified FACM. White bar, extracellular folate levels; gray bar, intracellular folate levels; and black bar, total folate levels. The L. gasseri wild-type strain could not grow without supplementation of folate. Therefore, folate levels depicted in the figure are corrected for folate added to the medium. Error bars indicate the standard deviation of the folate microbiological assay over two independent measurements.

Stability of the folate production in the L. gasseri strain.

Top

Stability of the folate...

L. gasseri strain ATCC 33323 harboring pNZ7019 was cultivatedfor approximately 30 generations on MRS medium supplementedwith 10 µg of chloramphenicol/ml at 37°C. The culturewas plated on MRS agar plates supplemented with 10 µgof chloramphenicol/ml. Subsequently, 100 colonies were transferredto folate-free FACM plates containing 10 µg of chloramphenicol/ml.Since all colonies grew on these plates, it appears that thefolate biosynthesis is stably maintained in the pNZ7019 vectorfor more than 30 generations of growth in the presence of folate.Sequential cultivation in folate-rich medium resulted in decreasedfolate production by the transformant (data not shown). Thisis presumably a result of instability of the folate gene cluster.

Conclusion.

Top

Conclusion.

The five genes, i.e., folB, folKE, folP, ylgG, and folC, directingfolate biosynthesis in L. lactis were transferred to L. gasseriby using a derivative of the broad-host-range vector pNZ12 (1).These genes are sufficient to introduce a folate biosynthesispathway in this folate auxotroph lactic acid bacterium, therebytransforming a folate consumer into a folate producer. L. gasseriis currently marketed as a probiotic (4), and when the describedstrategy is used, this lactic acid bacterium can be used toenrich (fermented) foods with the essential B vitamin, folate,in addition to conferring its health-promoting effect on theconsumer (3, 13).

FOOTNOTES

* Corresponding author. Mailing address: NIZO food research, P.O. Box 20, 6710 BA Ede, The Netherlands. Phone: 31-318-659540. Fax: 31-318-650400. E-mail: jeroen.hugenholtz{at}nizo.nl.

REFERENCES

Top