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Development of Biotin-Prototrophic and -Hyperauxotrophic Corynebacterium glutamicum Strains

机译：谷氨酸棒状杆菌生物素原养和超营养养株的研制

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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 (Δ bioY ) required approximately 1 mg of biotin per liter, almost 3 orders of magnitude higher than the wild-type level. The Δ bioY 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 Δ bioY strain. By selectively using the resulting two strains (Δ bioB and Δ bioBY ) 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).
机译：为了开发通过自然生物素营养缺陷型谷氨酸棒状杆菌生产生物素的基础设施，我们尝试将生物体改造为生物素营养缺陷型和生物素营养缺陷型。为了使生物素具有原养性，将大肠杆菌的共转录的bioBF基因引入到最初缺乏bioF基因的谷氨酸棒杆菌基因组中。所得菌株仍需要生物素才能生长，但可用外源庚二酸代替，后者是与辅酶A（CoA）或酰基载体蛋白（ACP）连接的生物素前体庚二酸硫酯的来源。为了弥合庚二酸硫酯与其专用的前体酰基辅酶A（或-ACP）之间的鸿沟，枯草芽孢杆菌的bioI基因编码了一个P450蛋白，该蛋白可切割酰基ACP的碳-碳键以生成庚二酰ACP通过使用质粒系统在工程菌株中进一步表达α1，β1，β2，β1，β2，β2，β2，β2，β2，β2，β2，β2，β2，β2，β2，β2。这产生了能够从头合成生物素的生物素原养生物。另一方面，在野生型谷氨酸棒杆菌中破坏负责生物素摄取的bioY基因。为了正常生长，野生型菌株每升需要约1μg生物素，而bioY破坏剂（ΔbioY）每升需要约1 mg生物素，比野生型水平高出近3个数量级。 ΔbioY菌株对前体脱硫生物素（bioB编码的生物素合酶的底物）显示出相似的高要求。为了消除对脱硫生物素的依赖性，在野生型菌株和ΔbioY菌株中都进一步破坏了bioB基因。通过选择性地使用所得的两种菌株（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，该系统可以量化生物素，范围为每升约0.1μg至1 g的七位数。该生物测定法证明，谷氨酸棒状杆菌的工程化生物素原养生物直接从葡萄糖产生了生物素，尽管可检测到的水平很小（每升约0.3μg）。

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