Bøe Cathrine Arnason, Holo Helge
Laboratory of Microbial Gene Technology, Faculty of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, Ås, Norway.
Tine SA, Oslo, Norway.
Front Bioeng Biotechnol. 2020 Mar 18;8:191. doi: 10.3389/fbioe.2020.00191. eCollection 2020.
Cheese produced with is the main source of vitamin K2 in the Western diet. Subclinical vitamin K2 deficiency is common, calling for foods with enhanced vitamin K2 content. In this study we describe analyses of vitamin K2 (menaquinone) production in the lactic acid bacterium ssp. strain MG1363. By cloning and expression from strong promoters we have identified genes and bottlenecks in the biosynthetic pathways leading to the long-chained menaquinones, MK-8 and MK-9. Key genes of the biosynthetic menaquinone pathway were overexpressed, singly or combined, to examine how vitamin K2 production can be enhanced. We observed that the production of the long menaquinone polyprenyl side chain, rather than production of the napthoate ring (1,4-dihydroxy-2-naphtoic acid), limits total menaquinone synthesis. Overexpression of genes causing increased ring formation ( and ) led to overproduction of short chained MK-3, while overexpression of other key genes ( and ) resulted in enhanced full-length MK-9 production. Of two putatively annotated prenyl diphosphate synthases we pinpoint () to be important for menaquinone production in . The genes , and were found to be important contributors to menaquinone levels as single overexpression of these genes double and more than triple the total menaquinone content in culture. Combined overexpression of , and increased menaquinone levels to a higher level than obtained individually. When the overproducing strains were applied for milk fermentations vitamin K2 content was effectively increased 3-fold compared to the wild type. The results provide a foundation for development of strains to ferment foods with increased functional value i.e., higher vitamin K2 content.
用[具体物质]生产的奶酪是西方饮食中维生素K2的主要来源。亚临床维生素K2缺乏很常见，因此需要增加维生素K2含量的食物。在本研究中，我们描述了对乳酸杆菌[具体菌种]亚种MG1363中维生素K2（甲萘醌）产生的分析。通过从强启动子进行克隆和表达，我们确定了导致长链甲萘醌MK-8和MK-9的生物合成途径中的基因和瓶颈。生物合成甲萘醌途径的关键基因被单独或组合过表达，以研究如何提高维生素K2的产量。我们观察到，长链甲萘醌聚异戊二烯侧链的产生，而非萘酸盐环（1,4-二羟基-2-萘甲酸）的产生，限制了总甲萘醌的合成。导致环形成增加的基因（[具体基因1]和[具体基因2]）过表达导致短链MK-3过量产生，而其他关键基因（[具体基因3]和[具体基因4]）过表达则导致全长MK-9产量增加。在两个假定注释的异戊二烯二磷酸合酶中，我们确定[具体基因5]对[具体菌种]中甲萘醌的产生很重要。发现基因[具体基因6]、[具体基因7]和[具体基因8]是甲萘醌水平的重要贡献者，因为这些基因的单一过表达使培养物中的总甲萘醌含量增加了两倍和三倍以上。[具体基因6]、[具体基因7]和[具体基因8]的组合过表达使甲萘醌水平提高到比单独过表达更高的水平。当将过量生产菌株用于牛奶发酵时，与野生型相比，维生素K2含量有效地增加了3倍。这些结果为开发具有更高功能价值（即更高维生素K2含量）的发酵食品菌株奠定了基础。
