Department of Bioscience and Biotechnology, Faculty of Agriculture, Shinshu University, Nagano, Japan.
Appl Environ Microbiol. 2013 Aug;79(15):4586-94. doi: 10.1128/AEM.00828-13. Epub 2013 May 24.
To develop the infrastructure for biotin production through naturally biotin-auxotrophic Corynebacterium glutamicum, we attempted to engineer the organism into a biotin prototroph and a biotin hyperauxotroph. To confer biotin prototrophy on the organism, the cotranscribed bioBF genes of Escherichia coli were introduced into the C. glutamicum genome, which originally lacked the bioF gene. The resulting strain still required biotin for growth, but it could be replaced by exogenous pimelic acid, a source of the biotin precursor pimelate thioester linked to either coenzyme A (CoA) or acyl carrier protein (ACP). To bridge the gap between the pimelate thioester and its dedicated precursor acyl-CoA (or -ACP), the bioI gene of Bacillus subtilis, which encoded a P450 protein that cleaves a carbon-carbon bond of an acyl-ACP to generate pimeloyl-ACP, was further expressed in the engineered strain by using a plasmid system. This resulted in a biotin prototroph that is capable of the de novo synthesis of biotin. On the other hand, the bioY gene responsible for biotin uptake was disrupted in wild-type C. glutamicum. Whereas the wild-type strain required approximately 1 μg of biotin per liter for normal growth, the bioY disruptant (ΔbioY) required approximately 1 mg of biotin per liter, almost 3 orders of magnitude higher than the wild-type level. The ΔbioY strain showed a similar high requirement for the precursor dethiobiotin, a substrate for bioB-encoded biotin synthase. To eliminate the dependency on dethiobiotin, the bioB gene was further disrupted in both the wild-type strain and the ΔbioY strain. By selectively using the resulting two strains (ΔbioB and ΔbioBY) as indicator strains, we developed a practical biotin bioassay system that can quantify biotin in the seven-digit range, from approximately 0.1 μg to 1 g per liter. This bioassay proved that the engineered biotin prototroph of C. glutamicum produced biotin directly from glucose, albeit at a marginally detectable level (approximately 0.3 μg per liter).
为了通过天然生物素缺陷型谷氨酸棒杆菌来开发生物素生产的基础设施，我们尝试将该生物体工程改造为生物素原养型和生物素超营养型。为了使生物体具有生物素原养型，我们将大肠杆菌的 cotranscribed bioBF 基因引入到原本缺乏 bioF 基因的谷氨酸棒杆菌基因组中。该菌株仍然需要生物素才能生长，但可以被外源的吡咯烷二羧酸（pimelic acid）取代，吡咯烷二羧酸是生物素前体吡咯烷二羧酸硫酯的来源，该硫酯与辅酶 A（CoA）或酰基载体蛋白（ACP）相连。为了在吡咯烷二羧酸硫酯与其专用前体酰基辅酶 A（或 -ACP）之间架起桥梁，我们进一步在工程菌株中表达了枯草芽孢杆菌的 bioI 基因，该基因编码一种 P450 蛋白，可裂解酰基-ACP 中的碳-碳键，生成吡咯烷酰-ACP。这导致生物素原养型能够从头合成生物素。另一方面，野生型谷氨酸棒杆菌中的生物素摄取基因 bioY 被破坏。虽然野生型菌株每升需要大约 1 μg 的生物素才能正常生长，但 bioY 缺陷型（ΔbioY）菌株每升需要大约 1 mg 的生物素，几乎是野生型水平的 3 个数量级。ΔbioY 菌株对前体脱硫生物素也有类似的高需求，脱硫生物素是 bioB 编码的生物素合酶的底物。为了消除对脱硫生物素的依赖，我们进一步在野生型菌株和 ΔbioY 菌株中破坏了 bioB 基因。通过选择性地使用这两个菌株（ΔbioB 和 ΔbioBY）作为指示菌株，我们开发了一种实用的生物素生物测定系统，可以定量测量 7 位数范围内的生物素，从每升约 0.1 μg 到 1 g。该生物测定法证明，谷氨酸棒杆菌的工程生物素原养型可以直接从葡萄糖中产生生物素，尽管检测水平较低（每升约 0.3 μg）。
