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通过代谢工程利用植物乳杆菌高产低热量糖山梨醇。

High-level production of the low-calorie sugar sorbitol by Lactobacillus plantarum through metabolic engineering.

作者信息

Ladero Victor, Ramos Ana, Wiersma Anne, Goffin Philippe, Schanck André, Kleerebezem Michiel, Hugenholtz Jeroen, Smid Eddy J, Hols Pascal

机构信息

Unité de Génétique, Institut des Sciences de la Vie, Université catholique de Louvain, 5 Place Croix du Sud, B-1348 Louvain-La-Neuve, Belgium.

出版信息

Appl Environ Microbiol. 2007 Mar;73(6):1864-72. doi: 10.1128/AEM.02304-06. Epub 2007 Jan 19.

Abstract

Sorbitol is a low-calorie sugar alcohol that is largely used as an ingredient in the food industry, based on its sweetness and its high solubility. Here, we investigated the capacity of Lactobacillus plantarum, a lactic acid bacterium found in many fermented food products and in the gastrointestinal tract of mammals, to produce sorbitol from fructose-6-phosphate by reverting the sorbitol catabolic pathway in a mutant strain deficient for both l- and d-lactate dehydrogenase activities. The two sorbitol-6-phosphate dehydrogenase (Stl6PDH) genes (srlD1 and srlD2) identified in the genome sequence were constitutively expressed at a high level in this mutant strain. Both Stl6PDH enzymes were shown to be active, and high specific activity could be detected in the overexpressing strains. Using resting cells under pH control with glucose as a substrate, both Stl6PDHs were capable of rerouting the glycolytic flux from fructose-6-phosphate toward sorbitol production with a remarkably high efficiency (61 to 65% glucose conversion), which is close to the maximal theoretical value of 67%. Mannitol production was also detected, albeit at a lower level than the control strain (9 to 13% glucose conversion), indicating competition for fructose-6-phosphate rerouting by natively expressed mannitol-1-phosphate dehydrogenase. By analogy, low levels of this enzyme were detected in both the wild-type and the lactate dehydrogenase-deficient strain backgrounds. After optimization, 25% of sugar conversion into sorbitol was achieved with cells grown under pH control. The role of intracellular NADH pools in the determination of the maximal sorbitol production is discussed.

摘要

山梨醇是一种低热量糖醇,因其甜度高且溶解度大,在食品工业中被广泛用作原料。在此,我们研究了植物乳杆菌(一种存在于许多发酵食品及哺乳动物胃肠道中的乳酸菌)通过在缺乏L-和D-乳酸脱氢酶活性的突变菌株中逆转山梨醇分解代谢途径,从6-磷酸果糖生产山梨醇的能力。在基因组序列中鉴定出的两个6-磷酸山梨醇脱氢酶(Stl6PDH)基因(srlD1和srlD2)在该突变菌株中持续高水平表达。两种Stl6PDH酶均显示有活性,且在过表达菌株中可检测到高比活性。以葡萄糖为底物,在pH控制下使用静息细胞时,两种Stl6PDH均能够以极高的效率(61%至65%的葡萄糖转化率)将糖酵解通量从6-磷酸果糖重新导向山梨醇生产,这接近67%的最大理论值。也检测到了甘露醇的产生,尽管其水平低于对照菌株(9%至13%的葡萄糖转化率),这表明天然表达的1-磷酸甘露醇脱氢酶会竞争6-磷酸果糖的重新导向。类似地,在野生型和乳酸脱氢酶缺陷型菌株背景中均检测到该酶的低水平表达。优化后,在pH控制下生长的细胞可实现25%的糖转化为山梨醇。本文还讨论了细胞内NADH库在确定最大山梨醇产量中的作用。

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