Development and application of bioparts for carbon dioxide fixing Acetogen, Eubacterium limosum ATCC8486 via integration of multi-omics data

Abstract

Bioconversion of sustainable substrates into value-added compounds has received much attention to replace petrochemical process. Of the bioconversion platforms, acetogens contains the Wood-Ljungdahl pathway (WLP) that condensate carbon monoxide (CO) and carbon dioxide ($CO_2$) into acetyl-CoA, one of important central metabolites. The acetogenic metabolism via the WLP was reported to be energetically the most efficient pathway, compared to the other six natural $CO_2$ fixation pathways without losing ATP molecules under the autotrophic growth condition. Recently, to systemically understand the advantages, studies on the genome and transcription of acetogens at the genome wide level have been actively pursed. Despite the obtained systematic understandings via omics studies, developing acetogens to industrial strains requires genetic modification tools, which severely limited. In this study, omics data sets composed of genome sequencing, RNA sequencing, ribosome profiling, and differential RNA sequencing were utilized to construct synthetic biology tools for Eubacterium limosum ATCC8486. For the construction of the tools, one hundred different bioparts were developed by using 18 promoters and 18 5' UTRs that obtained from the genome, then the DNA delivery efficiency was increased by investigating optimal cell density, field strength, cell wall-weakening agents, and cell membrane-disturbing agents. Through the investigation, the efficiency increased by 3,247-folds higher from $7.7 \times 10^1$ to $2.5 \times 10^5$ colony-forming $unit/\mu g$ of DNA/optical density at 600 nm. Using the optimized approach, the synthesized bioparts were introduced and expression levels of the bioparts were characterized using enhanced green fluorescent protein. The characterized bioparts were integrated to acetolactate synthase and acetolactate decarboxylase, which responsible for synthesizing acetoin, resulting production of 1.36 mM acetoin under autotrophic growth condition in the airlift bioreactor.