外文翻譯---在酪干乳桿菌bl23的ldh基因分析乳酸生產(chǎn)上的作用 英文

1、J Ind Microbiol Biotechnol (2008) 35:579–586DOI 10.1007/s10295-008-0319-8123ORIGINAL PAPERAnalysis of ldh genes in Lactobacillus casei BL23: role on lactic acid productionJuan Rico · María Jesús Yebra

2、83; Gaspar Pérez-Martínez · Josef Deutscher · Vicente Monedero Received: 21 December 2007 / Accepted: 13 January 2008 / Published online: 30 January 2008 © Society for Industrial Microbiology 2

3、008Abstract Lactobacillus casei is a lactic acid bacteriumthat produces L-lactate as the main product of sugar fermen- tation via L-lactate dehydrogenase (Ldh1) activity. In addi- tion, small amounts of the D-lactate iso

4、mer are produced by the activity of a D-hydroxycaproate dehydrogenase (HicD). Ldh1 is the main L-lactate producing enzyme, but mutation of its gene does not eliminate L-lactate synthesis. A survey of the L. casei BL23 dr

5、aft genome sequence revealed the presence of three additional genes encoding Ldh paralogs. In order to study the contribution of these genes to the glo- bal lactate production in this organism, individual, as well as dou

6、ble mutants (ldh1 ldh2, ldh1 ldh3, ldh1 ldh4 and ldh1 hicD) were constructed and lactic acid production was assessed in culture supernatants. ldh2, ldh3 and ldh4 genes play a minor role in lactate production, as their si

7、ngle muta- tion or a mutation in combination with an ldh1 deletion had a low impact on L-lactate synthesis. A ?ldh1 mutant dis- played an increased production of D-lactate, which was probably synthesized via the activity

8、 of HicD, as it was abolished in a ?ldh1 hicD double mutant. Contrarily to HicD, no Ldh1, Ldh2, Ldh3 or Ldh4 activities could be detected by zymogram assays. In addition, these assays revealed the presence of extra bands

9、 exhibiting D-/L-lactate dehydrogenase activity, which could not be attributed to any of the described genes. These results suggest that L. casei BL23 possesses a complex enzymatic system able to reduce pyruvic to lactic

10、 acid.Keywords Lactobacillus casei · Lactic acid · Lactate dehydrogenase · Hydroxycaproate dehydrogenase · Metabolic engineeringIntroductionThe metabolism of sugars by lactic acid bacteria (LAB) is c

11、haracterized by the production of lactate as the main fer- mentation product via the action of lactate dehydrogenase, which reduces pyruvic acid to lactic acid. Lactic acid pro- duction is important from a biotechnologic

12、al point of view, as it can be produced by LAB fermentation of many natural sources and it can be used in the food, pharmaceutical and biopolymers industries [7]. In Lactobacillus casei BL23, a strain that has been widel

13、y used for genetic, physiological and biochemical studies, two genes encoding proteins with lactate dehydrogenase activity have been described [8, 12, 19]. Gene ldh1 codes for an L-Ldh responsible for the syn- thesis of

14、L-lactate, whilst hicD encodes a D-hydroxyisocap- roate dehydrogenase that provides D-lactate. Mutant strains have been constructed in both genes demonstrating that they were responsible for the main L- and D-lactate for

15、ma- tion in this bacterium [19]. However, an L. casei BL23 ldh1 mutant still produced substantial amounts of L-lactate and the production of D-lactate was increased. A comparable behaviour has also been reported for othe

16、r LAB with deleted ldh genes. In this sense, mutation of the genes encoding L- and D-Ldhs from Lactobacillus plantarum, an organism which produces a mixture of 50% D- and 50% L- lactate, never resulted in a complete lack

17、 of lactate produc- tion [3]. An ldhL mutation in Lactobacillus sakei, a lactic acid bacterium which lacks D-lactate dehydrogenase activ- ity, resulted in a strain with strongly reduced L- and D- lac- tate production (th

18、e D isomer was a consequence of theJ. Rico · M. J. Yebra · G. Pérez-Martínez · V. Monedero (&) Laboratorio de Bacterias Lácticas y Probióticos, IATA-CSIC, P.O. Box 73, 46100 Burjas

19、sot, Valencia, Spain e-mail: btcmon@iata.csic.esJ. Deutscher Laboratorie de Microbiologie et Génétique Moleculaire, AgroParisTech-CNRS-INRA, 78850 Thiverval-Grignon, FranceJ Ind Microbiol Biotechnol (2008) 35:

20、579–586 581123Lactate dehydrogenase zymogram assaysLactobacillus casei wild-type and mutant strains were grown in 10 mL of MRS and exponentially growing cells were recovered by centrifugation, washed with Tris–HCl 100 mM

21、 pH 7.4 and resuspended in the same buVer supple- mented with 1 mM dithio-1,4-threitol and 0.5 mM phenylm- ethylsulphonyl Xuoride. Cells were broken by shaking with glass beads (0.1 mm diameter) in a Mini-Beadbeater (Bio

22、- spec) and cellular debris was removed by centrifugation 10 min at 12,000£g and 4 °C. Cellular extracts were loaded onto a 6% non-denaturing PAG and resolved at 90 V. To detect lactate dehydrogenase activity t

23、he gels were incu- bated in 100 mM triethanolamine buVer pH 6.8 containing 0.75 mM NAD+, 0.1 mg/mL nitroblue tetrazolium, 0.02 mg/ mL phenazyne methosulfate and either 200 mM L-lactate or 200 mM of a 50% D-50% L-lactate

24、mixture. In a second assay electrophoresis was carried out with a 10% PAG con- taining 0.05% SDS. The gels were soaked for 60 min in 2% Triton X-100 to eliminate the SDS and transferred to a solu- tion containing 100 mM

25、triethanolamine buVer pH 6.8 with 10 mM fructose-1,6-bisphosphate, 1 mM NADH with or without 25 mM sodium pyruvate. After 60 min of incuba- tion, the gels were rinsed with water for 30 min and incu- bated in a solution c

26、ontaining 0.25 mg/mL nitroblue tetrazolium and 0.02 mg/mL phenazyne methosulfate. Pro- tein bands with NADH oxidase activity were evidenced as clear bands over a dark background.Nucleotide accession numbersThe nucleotide

27、 sequences reported in this paper have been deposited at the EMBL database under accession numbers AM886174, AM886175, AM886176 and AM886177.Resultsldh homologues present in the L. casei BL23 genomeIn addition to the pre

28、viously described Ldh from L. casei BL23 [8, 19], a BLAST search for ldh homologues in the L. casei BL23 draft genome (96% sequence coverage) ren- dered three additional genes encoding putative Ldhs. The three additional

29、 Ldhs (Ldh2, Ldh3 and Ldh4) had a per- centage of identity of 49, 31 and 24%, respectively, when compared to the L. casei BL23 Ldh1 enzyme. Ldh2 had homologies to lactate/malate dehydrogenase enzymes, whereas Ldh3 was mo

30、st similar to L-hydroxyisocaproate dehydrogenases from many bacteria. In Ldh4, sequence homology to other L-Ldhs started at around aminoacid 80, whereas the Wrst N-terminal amino acids only shared a sig- niWcant homology

31、 to the N-terminus of the secondary Ldh from L. lactis (Ldh-2). Sequence analysis of the putative Ldhs revealed that, similar to Ldh1, the Ldh2 and Ldh3 proteins carried conserved NADH-binding domains con- taining the ty

32、pical G-X-G-X-X-G pattern (X is any aminoTable 1 Strains and plasmids used in this study Strain or plasmidCharacteristics Source or referenceLactobacillus casei BL23 Wild-type, genome sequenced at the Université

33、de Caen, CNRSa, INRAb and CSICc B. Chassy, U. IllinoisBL176 BL23 ldh1::pRV300 [19]BL198 BL23 hicD::pRV300 [19]BL249 BL23 ?ldh1 This workBL252 BL23 ?ldh1 ldh2::pRV300 This workBL269 BL23 ldh2::pRV300 This workBL270 BL23 l

34、dh3::pRV300 This workBL271 BL23 ?ldh1 ldh3::pRV300 This workBL272 BL23 ldh4::pRV300 This workBL273 BL23 ?ldh1 ldh4::pRV300 This workBL274 BL23 ?ldh1 hicD::pRV300 This workPlasmidspRV300 Insertional vector for Lactobacil

35、lus, Ampr, Eryr [11]pRV?ldh1 pRV300 with a 1 kb DNA fragment carrying fused 5? and 3? ldh1 regions This workpRVldh2 pRV300 with a 0.5 kb ldh2 fragment cloned at EcoRV site This workpRVldh3 pRV300 with a 0.5 kb ldh3 fragm

36、ent cloned at SmaI site This workpRVldh4 pRV300 with a 0.4 kb ldh4 fragment cloned at EcoRV site This workpVBhic pRV300 with a 0.6 kb hicD fragment cloned at SmaI site [19]a Centre National de la Recher- che ScientiWqueb