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

维生素B12，也被称为钴胺素，是人体中两种必需酶，甲基丙二酰辅酶A β和甲硫氨酸合成酶所必需的，缺乏B12会阻止这些酶发挥功能并导致恶性贫血。虽然这种疾病的一种形式可以发生在儿童中，但恶性贫血通常不会在30岁之前出现。诊断时的平均年龄为60岁。这种疾病发生在所有种族群体中，但最常发生在斯堪的纳维亚人或北方欧洲人后裔中。该疾病可以表现为缺乏维生素B12和/或不能吸收B12，这两者都可以通过给予维生素B12来治疗。可以补充维生素B12，但需要1000微克/天。我们通常从饮食中获得维生素B12，特别是从肉类和奶制品中，但不从水果和蔬菜中获得，因为它们不含这种必需的营养素。维生素B12缺乏导致过多的临床表现，包括血液学，神经学和精神病学疾病。维生素B12缺乏的风险包括：1。植物中缺乏维生素B12使素食者，特别是纯素食者，容易缺乏维生素B12。2.老年人-高达30%的70岁以上的人有B12缺乏症，需要补充，在某些情况下，静脉注射B12 3。未出生婴儿- B12是未出生婴儿神经管缺陷的一个促成风险因素。因此，需要提供具有升高的钴胺素水平的合适食品。一些国家甚至考虑在面粉中添加维生素B12，就像添加叶酸一样。维生素B12是维生素中唯一的，仅由原核生物生产。它是通过使用特定菌株发酵生产的，这些菌株已被选择用于高B12水平。过去已经使用了许多菌株和生产方法，但所采用的主要方法涉及将称为5，6-二甲基苯并咪唑（DMB）的维生素组分共同进料到培养基中。DMB的合成和连接通常在该途径的生物合成中是限速的。有趣的是，任何去超市、健康食品店或浏览互联网的旅行都会证明维生素B12可以以各种形式购买。然而，许多这些形式是无用的，因为它们不包含正确版本的维生素，包括：1。某些益生菌在液体培养物中出售2.螺旋藻提取物，作为维生素B12的来源销售，相信作为蓝藻，它们产生大量的维生素。在这些情况下，建议的“维生素B12”不是钴胺素，而是假钴胺素，虽然结构相似，但不能被人类使用。这两种化合物之间的区别是下层配体与含有腺嘌呤的假钴胺素和含有5，6-二甲基苯甲酰胺（DMB）的钴胺素的同一性。肯特和其他地方的专业知识有助于我们了解合成途径，从而表征负责合成DMB的蛋白质，从而能够生产真正的维生素B12。编码这一关键步骤的基因（bluB）存在于某些细菌菌株中，但在其他菌株中不存在。当前提案的总体策略是了解酶BluB如何制造DMB。随后，bluB将被转移到具有GRAS（通常被认为是安全的）状态的生产假钴胺素的益生菌菌株中。这是第一次尝试创建用于益生菌制剂的基因工程细菌，具有在体内提供可持续维生素B12生产的能力。

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.