Development of modular expression across phylogenetically distinct diazotrophs

Kulakowski, Shawn; Rivier, Alex; Kuo, Rita; Mengel, Sonya; Eng, Thomas

doi:10.1093/jimb/kuae033

Title: Development of modular expression across phylogenetically distinct diazotrophs

Journal Article · 09 September 2024 · Journal of Industrial Microbiology and Biotechnology

DOI: https://doi.org/10.1093/jimb/kuae033 · OSTI ID:2476437

Kulakowski, Shawn ^[1];

^[1];

^[1]; Mengel, Sonya ^[1];

^[1]

Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)

Diazotrophic bacteria can reduce atmospheric nitrogen into ammonia enabling bioavailability of the essential element. Many diazotrophs closely associate with plant roots increasing nitrogen availability, acting as plant growth promoters. These associations have the potential to reduce the need for costly synthetic fertilizers if they could be engineered for agricultural applications. However, despite the importance of diazotrophic bacteria, genetic tools are poorly developed in a limited number of species, in turn narrowing the crops and root microbiomes that can be targeted. Here, we report optimized protocols and plasmids to manipulate phylogenetically diverse diazotrophs with the goal of enabling synthetic biology and genetic engineering. Three broad-host-range plasmids can be used across multiple diazotrophs, with the identification of one specific plasmid (containing origin of replication RK2 and a kanamycin resistance marker) showing the highest degree of compatibility across bacteria tested. We then demonstrated modular expression by testing seven promoters and eleven ribosomal binding sites using proxy fluorescent proteins. Finally, we tested four small molecule inducible systems to report expression in three diazotrophs and demonstrated genome editing in Klebsiella michiganensis M5al.

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Research Organization:: Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)

Sponsoring Organization:: USDOE; USDOE Laboratory Directed Research and Development (LDRD) Program; USDOE Office of Science (SC), Biological and Environmental Research (BER); USDOE Office of Science (SC), Office of Workforce Development for Teachers & Scientists (WDTS)

Grant/Contract Number:: AC02-05CH11231

OSTI ID:: 2476437

Journal Information:: Journal of Industrial Microbiology and Biotechnology, Journal Name: Journal of Industrial Microbiology and Biotechnology Vol. 51; ISSN 1367-5435

Publisher:: SpringerCopyright Statement

Country of Publication:: United States

Language:: English

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59 BASIC BIOLOGICAL SCIENCES
biological nitrogen fixation
diazotroph
rhizosphere
synthetic biology

Title: Development of modular expression across phylogenetically distinct diazotrophs

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