Mechanism of dimethylenzimidazole (DMB) synthesis and the metabolic engineering of a dietary useful form of cobalamin in Lactobacillus

维生素B12，也被称为钴胺，是人类两种基本酶--甲基丙二酰辅酶A变位酶和蛋氨酸合成酶--所必需的，缺乏B12会阻止这些酶的功能，并导致恶性贫血。虽然这种疾病可能发生在儿童身上，但恶性贫血通常在30岁之前不会出现。确诊时的平均年龄为60岁。这种疾病在所有种族群体中都会发生，但最常见的是斯堪的纳维亚人或北欧后裔。这种疾病可能表现为缺乏维生素B12和/或无法吸收B12，这两种情况都可以通过服用维生素B12来治疗。可以补充维生素B12，但每天需要1000微克。我们通常从饮食中获取维生素B12，特别是从肉类和乳制品中，而不是从水果和蔬菜中，因为它们不包含这种必要的营养。维生素B12缺乏会导致过多的临床表现，包括血液、神经和精神疾病。那些有维生素B12缺乏风险的人包括：1.素食者--植物中缺乏B12会使素食者，尤其是纯素食主义者，容易缺乏维生素B12。2.老年人--70岁以上的人中有高达30%的人缺乏B12，需要补充，在某些情况下，还需要静脉注射B12。3.未出生的婴儿--B12是导致未出生婴儿神经管缺陷的危险因素。因此，有必要提供钴胺水平较高的适当食品。一些国家甚至正在考虑将B12添加到面粉中，就像已经添加了叶酸一样。维生素B12在维生素中是独一无二的，只由原核生物制造。它是通过商业发酵生产的，使用的是特定的菌株，这些菌株已经被选为高B12水平。过去使用了许多菌株和生产方法，但所采用的主要方法包括将维生素的一种成分5，6-二甲基苯并咪唑(DMB)共同喂养到培养基中。在该途径的生物合成中，DMB的合成和附着往往是限速的。有趣的是，任何一次去超市、健康食品店或浏览互联网都会证明，维生素B12可以以一系列形式购买。然而，这些形式中的许多都是无用的，因为它们不含有正确版本的维生素，包括：1.液体培养中出售的某些益生菌2.螺旋藻提取物，作为维生素B12的来源销售，因为人们相信，作为蓝藻，它们可以产生大量的维生素。在这些情况下，被提议的“维生素B12”不是钴胺，而是假钴胺，尽管结构相似，但无法被人类使用。这两个化合物之间的区别在于低配体与含腺嘌呤的伪钴胺和含有5，6-二甲基苯甲酰胺(DMB)的钴胺的相同。肯特和其他地方的专业知识有助于我们了解合成途径，从而确定了负责合成DMB的蛋白质的特征，从而能够生产真正的维生素B12。编码这一关键步骤的基因(Blb)存在于某些细菌菌株中，而在其他细菌菌株中缺失。目前提案中的总体策略是了解DMB是如何由酶blb制造的。随后，BLOB将被转移到一种具有GRAS(通常被认为是安全的)状态的产生假钴胺的益生菌菌株。这是首次尝试创造一种基因工程细菌，用于益生菌制剂，能够在体内提供可持续的维生素B12生产。

Vitamin B12, also known as cobalamin, is required for two essential enzymes in humans, methylmalonyl CoA mutase and methionine synthase and the lack of B12 prevents these enzymes functioning and results in pernicious anaemia. Although a form of the disease can occur in children, pernicious anemia usually does not appear before the age of 30. The average age at diagnosis is 60. The disease occurs in all racial groups, but occurs most often in people of Scandinavian or Northern European descent. The disease may be manifest as a lack of vitamin B12 and / or an inability to absorb B12, both of which may be treated by vitamin B12 administration. Vitamin B12 supplements can be given but 1000 microg / day are required. We ordinarily obtain vitamin B12 from our diet, particularly from meat and dairy products but not from fruit and vegetables, which do not contain this essential nutrient. Vitamin B12 deficiency results in a plethora of clinical manifestations including hematologic, neurologic and psychiatric disorders. Those people at risk of vitamin B12 deficiency include: 1. Vegetarians - the absence of B12 in plants make vegetarians, especially vegans, susceptible to deficiency. 2. Elderly - up to 30% of people over 70 have B12 deficiency and require supplements and, in some cases, intravenous injections of B12 3. Unborn babies - B12 is a contributory risk factor in neural tube defects in unborn babies. There is thus a need to provide suitable foodstuffs with elevated cobalamin levels. Some countries are even contemplating adding B12 to flour in the same way that folate is already added. Vitamin B12 is unique amongst the vitamins, being manufactured only by prokaryotes. It is produced commercially by fermentation using specific strains that have been selected for high B12 levels. Many strains and production methodologies have been used in the past but the principal methodology employed involves the co-feeding of a component of the vitamin called 5,6-dimethyl benzimidazole (DMB) to the culture medium. The synthesis and attachment of DMB is often rate-limiting in the biosynthesis of the pathway. Interestingly, as any trip to supermarket, health food shop or browse of the internet will demonstrate vitamin B12 can be purchased in a range of forms. However, many of these forms are useless as they do not contain the the correct version of the vitamin, and include: 1. Certain probiotic bacteria that are sold in liquid cultures 2. Spirulina extracts, which are marketed as a source of vitamin B12 in the belief that, as cyanobacteria, they produce large amounts of the vitamin. In these cases the proposed 'vitamin B12' is not cobalamin but is pseudocobalamin which, although structurally similar, is unable to be used by humans. The difference between the two compounds is the identity of the lower ligand with pseudocobalamin containing adenine and cobalamin containing 5,6-dimethylbenzamide (DMB). Expertise at Kent and elsewhere has contributed to our understanding of the synthetic pathway, leading to the characterisation of the protein responsible for the synthesis of DMB, enabling the production of true vitamin B12. The gene encoding this critical step (bluB) is present in certain bacterial strains but absent in others. The overall strategy in the current proposal is to understand how DMB is made by the enzyme BluB. Subsequently, bluB will be transferred into a pseudocobalamin-producing probiotic strain that has GRAS (Generally Regarded As Safe) status. This is the first attempt to create a genetically engineered bacterium for use in probiotic formulations with the capability of providing sustainable vitamin B12 production in vivo.