【正文】 在這項(xiàng)研究中干酪乳桿菌菌株的使用在表 1 中列出。一個519 bp 的片段從 LDH2 ampliWed PCR 結(jié)合寡核苷酸 5ˊ GATTCCCTACCTTACACG 和 5ˊTCTCAATGATGCCATAAGC 與 EcoRV 分為 pRV300 消化的克隆，給予 pRVldh2。限制性內(nèi)切酶， DNA 的 modiWcation 酶和 T4 連接酶為 New England Biolabs公司和 Roche 公司生產(chǎn)。色譜法在 50176。在第二個用含有 10％的 PAG 電泳進(jìn)行檢測 ％ SDS。對 LDHs 序列的推斷發(fā)現(xiàn)，類似于 LDH1， LDH2 和 LDH3 蛋白質(zhì)進(jìn)行守恒的 NADH 結(jié)合域中含有典型的GX G X X – G 紋路（ X 為任意氨基酸 圖 1 示意圖 4diVerent LDH 基因檢測干酪乳桿菌基本法第二十三條連同他們的基因組范圍內(nèi)。雖然 LDH2 似乎也是 monocistronic 的基因，但 LDH2 擁有在一些二元酸反應(yīng)步驟的脫氫作用證明它是合理的。（ 4）基因編碼的一種應(yīng)激反應(yīng)膜在 ATCC334（ LSEI_1313） GTP 酶是一個 BL23 的假基因（圖 1）。表2 中可以看到， LDH1 突變對主要乳酸生產(chǎn)的影響，以減少乳酸總額的 37％的增幅，對D乳酸進(jìn)行了觀察。乳酸脫氫酶的酶譜檢測活動的乳酸 LDH2， LDH3 和 LDH4 突變的低 eVect，合成促使我們尋找額外的存在 LDH 活性存在于干酪乳桿菌 BL23，我們試圖將粗提取物進(jìn)行酶譜分析檢測。樂隊(duì)相應(yīng)的 HicD 酶被標(biāo)記面板 “基本法”第二十三條（野生型） 。類似結(jié)果也存在于含有或缺乏果糖 1,6二磷酸的過程中，這是 LDH1 激活（數(shù)據(jù)未顯示）。 BL274（ LDH1 hicD） 。在這種情況下，基因組重要的工業(yè)可用性微生物是在發(fā)展至關(guān)重要新戰(zhàn)略旨在改善應(yīng)變。這一假說可以證明，如果三聯(lián)或四聯(lián)的突變體在 LDH 將產(chǎn)生新的基因。此外，酶譜分析表明，微弱的波段與 HicD 活動相關(guān)。然而，酶譜分析 conWrmed 在干酪乳桿菌 BL23 兩個 NADH 的氧化酶誘導(dǎo) LDH1 突變，這說明了細(xì)胞如何響應(yīng)試圖以彌補(bǔ)缺乏通過乳酸脫氫酶 NADH 再生。 致謝 J. Rico 的是關(guān)于 Corporacion AlimentariaPe241。rezMart237。 Gaspar P233。Metabolic engineering Introduction The metabolism of sugars by lactic acid bacteria (LAB) is characterized by the production of lactate as the main fermentation product via the action of lactate dehydrogenase,which reduces pyruvic acid to lactic acid. Lactic acid production is important from a biotechnological 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 widely used for geic, physiological and biochemical studies, two genes encoding proteins with lactate dehydrogenase activity have been described [8, 12,19]. Gene ldh1 codes for an LLdh responsible for the synthesis of Llactate, whilst hicD encodes a Dhydroxyisocaproate dehydrogenase that provides Dlactate. Mutant strains have been constructed in both genes demonstrating that they were responsible for the main L and Dlactate formation in this bacterium [19]. However, an L. casei BL23 ldh1 mutant still produced substantial amounts of Llactate and the production of Dlactate was increased. A parable behaviour has also been reported for other LAB with deleted ldh genes. In this sense, mutation of the genes encoding L and DLdhs from Lactobacillus plantarum, an anism which produces a mixture of 50% D and 50% Llactate, never resulted in a plete lack of lactate production[3]. An ldhL mutation in Lactobacillus sakei, a lactic acid bacterium which lacks Dlactate dehydrogenase activity,resulted in a strain with strongly reduced L and D lactate production (the D isomer was a consequence of the presence of a racemase activity able to transform L into Dlactate)but small amounts of lactate were still produced[15]. Finally, a Lactobacillus fermentum ldhLldhD double knockout was still able to synthesize lactate [1]. EVorts have been made to construct LAB strains aVected in one or several of the identiWed ldh genes, as they can be used in the production through fermentation of nonracemic, optically active lactic acid [1]. In addition, metabolic engineering strategies were applied where oxidation of NADH was coupled to an alternative metabolism of pyruvate, leading to the production of valueadded metabolites [4, 6, 13, 17].However, deletion of known lactate dehydrogenase genes in bination with the expression of enzymatic activities able to reduce pyruvate is not always suYcient to divert a signiWcant pyruvate Xow towards other molecules of interest diVerent from lactic acid. For those reasons, a better study of the enzymes implicated in lactate synthesis bees necessary. In an attempt to discover the genes responsible for residual lactic acid production in an L. casei BL23 ldh1 mutant, we searched its genome for the presence of additional ldh genes and studied the eVects of mutations in the newly found ldhs on lactate production. Material and methods Strains and growth conditions The L. casei strains used in this study are listed in Table were grown in MRS medium (Oxoid) or MRS basal medium, containing per litre: peptone, 10 g。C under static conditions. E. coli DH5_ was used as a cloning host and it was grown in LB medium at 37 176。 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 la