Abstract

The creation of microbial cell factories for sustainable production of natural products is important for medical and industrial applications. This requires stable expression of biosynthetic pathways in a host organism with favorable fermentation properties such as Bacillus subtilis. The aim of this study is to construct B. subtilis strains that produce valuable terpenoid compounds by overexpressing the innate methylerythritol phosphate (MEP) pathway. A synthetic operon allowing the concerted and regulated expression of multiple genes was developed. Up to 8 genes have been combined in this operon and a stably inherited plasmid-based vector was constructed resulting in a high production of C₃₀ carotenoids. For this, two vectors were examined, one with rolling circle replication and another with theta replication. Theta-replication constructs were clearly superior in structural and segregational stability compared to rolling circle constructs. A strain overexpressing all eight genes of the MEP pathway on a theta-replicating plasmid clearly produced the highest level of carotenoids. The level of transcription for each gene in the operon was similar as RT-qPCR analysis indicated. Hence, that corresponding strain can be used as a stable cell factory for production of terpenoids. This is the first report of merging and stably expressing this large-size operon (eight genes) from a plasmid-based system in B. subtilis enabling high C₃₀ carotenoid production.

中文翻译：

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调节的合成操纵子促进枯草芽孢杆菌中多基因萜类途径的稳定过表达。

摘要

建立用于可持续生产天然产物的微生物细胞工厂对于医疗和工业应用至关重要。这需要在具有良好发酵特性的宿主生物（如枯草芽孢杆菌）中稳定表达生物合成途径。这项研究的目的是构建通过过度表达先天性甲基赤藓糖醇磷酸（MEP）途径产生有价值的萜类化合物的枯草芽孢杆菌菌株。开发了一种合成操纵子，该操纵子允许多个基因协调一致地表达。该操纵子中已整合了多达8个基因，并构建了稳定遗传的基于质粒的载体，从而导致了C ₃₀的高产量类胡萝卜素。为此，检查了两个载体，一个具有滚环复制，另一个具有theta复制。与滚环构建体相比，Theta复制构建体在结构和偏析稳定性方面明显优越。在theta复制质粒上过表达MEP途径的所有八个基因的菌株显然产生了最高水平的类胡萝卜素。操纵子中每个基因的转录水平与RT-qPCR分析表明的相似。因此，该相应的菌株可以用作稳定的细胞工厂以生产萜类化合物。这是在枯草芽孢杆菌中基于质粒的系统融合并稳定表达这种大操纵子（八个基因）的第一份报告，可实现高C ₃₀类胡萝卜素的生产。