Rhodococcus opacus PD630 is a non-model Gram-positive bacterium that possesses desirable traits for lignocellulosic biomass conversion. In particular, it has a relatively rapid growth rate, exhibits genetic tractability, produces high quantities of lipids, and can tolerate and consume toxic lignin-derived aromatic compounds. Despite these unique, industrially relevant characteristics, R. opacus has been underutilized because of a lack of reliable genetic parts and engineering tools. In this work, we developed a molecular toolbox for reliable gene expression control and genome modification in R. opacus. To facilitate predictable gene expression, a constitutive promoter library spanning ∼45-fold in output was constructed. To improve the characterization of available plasmids, the copy numbers of four heterologous and nine endogenous plasmids were determined using quantitative PCR. The molecular toolbox was further expanded by screening a previously unreported antibiotic resistance marker (HygR) and constructing a curable plasmid backbone for temporary gene expression (pB264). Furthermore, a system for genome modification was devised, and three neutral integration sites were identified using a novel combination of transcriptomic data, genomic architecture, and growth rate analysis. Finally, the first reported system for targeted, tunable gene repression in Rhodococcus was developed by utilizing CRISPR interference (CRISPRi). Overall, this work greatly expands the ability to manipulate and engineer R. opacus, making it a viable new chassis for bioproduction from renewable feedstocks.

中文翻译：

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用于不透明红球菌PD630基因表达控制和基因组修饰的分子工具包

opacho Rhodococcus opacus PD630是一种非模型革兰氏阳性细菌，具有木质纤维素生物质转化所需的性状。特别是，它具有相对较快的生长速度，具有遗传易加工性，可产生大量脂质，并且可以耐受和食用有毒的木质素衍生的芳香族化合物。尽管具有这些独特的，与工业相关的特征，但由于缺乏可靠的遗传部分和工程工具，欧巴木不被充分利用。在这项工作中，我们开发了一种分子工具箱，用于可靠地进行R. opacus基因表达控制和基因组修饰。为促进可预测的基因表达，构建了构建性启动子文库，其输出跨度约为45倍。为了改善可用质粒的特性，使用定量PCR确定了四个异源和九个内源质粒的拷贝数。通过筛选以前未报道的抗生素抗性标记（HygR）并构建用于临时基因表达的可固化质粒主链（pB264），进一步扩展了分子工具箱。此外，设计了一个用于基因组修饰的系统，并使用转录组数据，基因组结构和生长速率分析的新颖组合鉴定了三个中性整合位点。最后，第一个报道的红球菌靶向，可调基因抑制系统是利用CRISPR干扰（CRISPRi）开发的。总体而言，这项工作极大地扩展了操纵和工程化R. opacus的能力，使其成为可再生原料生物生产的可行的新底盘。