【導(dǎo)讀】LDH1是主要的L-乳酸生產(chǎn)酶，但突變它的基因并沒(méi)有消除L-乳酸的。一項(xiàng)稱作“干酪乳桿菌基本法第二十三條草案的基因組序列”的調(diào)查揭示了相。關(guān)的另外三個(gè)基因編碼乳酸脫氫酶旁系。為了研究這些基因?qū)θ虻倪@種微生物的貢。獻(xiàn)，個(gè)體乳酸以及雙突變體的生。產(chǎn)，構(gòu)建了乳酸生產(chǎn)的上清培養(yǎng)評(píng)估。LDH2，LDH3和LDH4基因乳酸生產(chǎn)中發(fā)揮的作。用不大，因?yàn)樗麄兊膯我煌蛔兓蛟诮M合與LDH1缺失突變對(duì)L-乳酸合成的影響很小。相反，可能是HicD，在沒(méi)有LDH1，LDH2，LDH3或LDH4活動(dòng)的情況下。通過(guò)酶譜分析檢測(cè)。活性，這不能歸咎于任何所描述的基因。統(tǒng)，能夠減少乳酸丙酮酸。從而降低丙酮酸，乳酸。和D-乳酸的形成表明突變株已在這兩個(gè)基因上建成。突變體仍然產(chǎn)生大量的L-乳酸D-乳酸。一個(gè)與其他實(shí)驗(yàn)室刪除LDH基因相似的行為也。由于這些原因，一個(gè)更好的涉及乳酸合成酶的研究將成為必要。identiWed限制分析，并命名為PRVΔLDH1。構(gòu)建LDH1缺陷菌株，干酪乳桿菌BL23通過(guò)克隆選

　　

【正文】 NADH with or without 25 mM sodium pyruvate. After 60 min of incubation, the gels were rinsed with water for 30 min and incubated in a solution containing mg/mL nitroblue tetrazolium and mg/mL phenazyne methosulfate. Protein bands with NADH oxidase activity were evidenced as clear bands over a dark background. Nucleotide accession numbers The nucleotide sequences reported in this paper have been deposited at the EMBL database under accession numbersAM886174, AM886175, AM886176 and AM886177. Results ldh homologues present in the L. casei BL23 genome In addition to the previously described Ldh from L. casei BL23 [8, 19], a BLAST search for ldh homologues in the BL23 draft genome (96% sequence coverage) rendered three additional genes encoding putative Ldhs. The three additional Ldhs (Ldh2, Ldh3 and Ldh4) had a percentage of identity of 49, 31 and 24%, respectively, when pared to the L. casei BL23 Ldh1 enzyme. Ldh2 had homologies to lactate/malate dehydrogenase enzymes,whereas Ldh3 was most similar to Lhydroxyisocaproate dehydrogenases from many bacteria. In Ldh4, sequence homology to other LLdhs started at around aminoacid 80,whereas the Wrst Nterminal amino acids only shared a signiWcant homology to the Nterminus of the secondary Ldh from L. lactis (Ldh2). Sequence analysis of the putative Ldhs revealed that, similar to Ldh1, the Ldh2 and Ldh3 proteins carried conserved NADHbinding domains containing the typical GXGXXG pattern (X is any amino acid). Furthermore, the catalytic MGEHG[D/E][S/T]site, where H is the catalytic histidine, was also present in Ldh2 and Ldh3, supporting thus the hypothesis that they encode potential Ldh enzymes. Ldh4 was the enzyme exhibiting the lowest similarity to Ldh1 and conservation of the above sequence motifs was poor. In order to assign putative roles to the new proteins, the geic contexts of their corresponding genes were studied (Fig. 1). As previously described, ldh1 is a monocistronic gene and no genes related to carbon metabolism are located next to it. ldh2 is Xanked at its 5_end by the divergently transcribed gdh gene, encoding a NADPdependent glutamate dehydrogenase and at its 3_ end by genes encoding putative amino acid and dicarboxylic acid transporters. Although it appears that ldh2 is also a monocistronic gene, it is plausible that ldh2 carries a function in the dehydrogenation step of some dicarboxylic acids. ldh3 is Xanked by genes encoding a putative deacetylase, a membrane protein and an ADPribose pyrophosphatase. ldh4 is located at the 3_end of a gene cluster encoding the diVerent subunits of the pyruvate dehydrogenase plex and seems to form an operon with a gene encoding a conserved hypothetical protein and a putative archaealtype fructose1,6bisphosphatase of the inositolmonophosphatase family. While the pyruvate dehydrogenase plex is involved in pyruvate metabolism as it converts this pound into acetylCoA under aerobic conditions, fructose1,6bisphosphatase takes part in the gluconeogenic pathway and therefore no clear link with Ldh could be recognized. In conclusion, genome context of the new ldh genes did not allow establishing likely hypotheses about their cellular roles. A similar search carried out by using the plete genome sequence of L. casei ATCC334[14], the newly proposed L. casei typestrain, revealed the occurrence of the same additional ldh genes. The geic structure of the DNA regions carrying the ldhs was identicalin both strains except that: (1) in ATCC334, the gdh gene(LSEI_0635) adjacent to ldh2 and the putative proline iminopeptidase gene (LSEI_2604) close to ldh3 are pseudogenes。(2) an IS3related insertion element is present in ATCC 334 between lpdA and ldh4。 (3) the gene encoding a putative PadRlike regulator in BL23 is absent from ATCC 334。 and (4) the gene encoding a stressresponse membrane GTPase in ATCC334 (LSEI_1313) is a pseudogene in BL23 (Fig. 1).Construction of diVerent ldh mutants and their eVect on lactate production In a previous work, an ldh1 mutant was constructed by insertion of a nonreplicative plasmid carrying an internal fragment of the gene [19]. In order to produce a stable mutant and to avoid the presence of an antibiotic resistance marker, L. casei BL23 was transformed with plasmid pRV ldh1, which carried the 5 and 3 Xanking regions of ldh1. After a Wrst singlecrossover integration, a stable mutant was selected which underwent a second rebination event, thus leading to a plete deletion of ldh1 in the pared to the wildtype, the new deletion mutant (strain BL249) exhibited a behaviour similar to that of the previous insertional mutant BL176: culture supernatants always reached a higher Wnal pH, it produced CO2 from glucose and LLdh activity in crude extracts was reduced by 95% (data not shown). Subsequently, the ldh2, ldh3, ldh4 and hicD genes were inactivated in the ldh1 background giving four double mutants (Table 1). In a similar way, the ldh2, ldh3 and ldh4 genes were inactivated in the L. casei wildtype, giving three diVerent single mutants (Table 1). In order to study the eVect of the diVerent mutations on lactic acid production, all strains were grown in MRS basal medium containing % glucose and lactic acid concentrations were measured after plete glucose can be seen in Table 2, a _ldh1 mutation had a major impact on lactate production, with a reduction of 37% in total lactic acid, whereas an increase in the percentage of Dlactate was observed. The ldh2, ldh3 and ldh4 single mutants behaved like the wildtype (data not shown). Furthermore, bination of ldh2, ldh3 or ldh4 mutations with an ldh1 deletion did not produce a clear eVect on total lactate production, although the D/Llactate ratio increased more than twofold in the ldh1 ldh2 and ldh1 ldh4 double knockouts pared to the single ldh1 mutant. These results suggested tha