Bhat Etash H, Henard Jessica M, Lee Spencer A, McHalffey Dustin, Ravulapati Mahith S, Rogers Elle V, Yu Logan, Skiles David, Henard Calvin A
Department of Biological Sciences and BioDiscovery Institute, University of North Texas, Denton, TX, USA.
Synth Syst Biotechnol. 2024 Feb 19;9(2):250-258. doi: 10.1016/j.synbio.2024.02.003. eCollection 2024 Jun.
Methanotrophic bacteria are currently used industrially for the bioconversion of methane-rich natural gas and anaerobic digestion-derived biogas to valuable products. These bacteria may also serve to mitigate the negative effects of climate change by capturing atmospheric greenhouse gases. Several genetic tools have previously been developed for genetic and metabolic engineering of methanotrophs. However, the available tools for use in methanotrophs are significantly underdeveloped compared to many other industrially relevant bacteria, which hinders genetic and metabolic engineering of these biocatalysts. As such, expansion of the methanotroph genetic toolbox is needed to further our understanding of methanotrophy and develop biotechnologies that leverage these unique microbes for mitigation and conversion of methane to valuable products. Here, we determined the copy number of three broad-host-range plasmids in Bath and OB3b, representing phylogenetically diverse Gammaproteobacterial and Alphaproteobacterial methanotrophs, respectively. Further, we show that the commonly used synthetic Anderson series promoters are functional and exhibit similar relative activity in and OB3b, but the synthetic series had limited range. Thus, we mutagenized the native particulate methane monooxygenase promoter and identified variants with activity that expand the activity range of synthetic, constitutive promoters functional not only in , but also in . Collectively, the tools developed here advance the methanotroph genetic engineering toolbox and represent additional synthetic genetic parts that may have broad applicability in Pseudomonadota bacteria.
目前，甲烷营养菌在工业上被用于将富含甲烷的天然气和厌氧消化产生的沼气生物转化为有价值的产品。这些细菌还可以通过捕获大气中的温室气体来减轻气候变化的负面影响。此前已经开发了几种用于甲烷营养菌基因和代谢工程的遗传工具。然而，与许多其他工业相关细菌相比，现有的用于甲烷营养菌的工具明显不够完善，这阻碍了这些生物催化剂的基因和代谢工程。因此，需要扩展甲烷营养菌遗传工具箱，以加深我们对甲烷营养作用的理解，并开发利用这些独特微生物来减轻甲烷排放并将其转化为有价值产品的生物技术。在这里，我们确定了三种广泛宿主范围质粒在 Bath 和 OB3b 中的拷贝数，它们分别代表系统发育上不同的γ-变形菌纲和α-变形菌纲甲烷营养菌。此外，我们表明常用的合成安德森系列启动子具有功能，并且在 和 OB3b 中表现出相似的相对活性，但合成系列的范围有限。因此，我们对天然颗粒甲烷单加氧酶启动子进行了诱变，并鉴定出具有活性的变体，这些变体扩展了不仅在 中而且在 中起作用的合成组成型启动子的活性范围。总体而言，这里开发的工具推进了甲烷营养菌基因工程工具箱，并代表了可能在假单胞菌门细菌中具有广泛适用性的其他合成遗传元件。
