Functional dissection and modulation of the BirA protein for improved autotrophic growth of gas‐fermenting Clostridium ljungdahlii

Abstract

Gas-fermenting Clostridium species can convert one-carbon gases (CO 2 /CO) into a variety of chemicals and fuels, showing excellent application prospects in green biological manufacturing. The discovery of crucial genes and proteins with novel functions is important for understanding and further optimization of these autotrophic bacteria. Here, we report that the Clostridium ljungdahlii BirA protein (ClBirA) plays a pleiotropic regulator role, which, together with its biotin protein ligase (BPL) activity, enables an effective control of autotrophic growth of C. ljungdahlii. The structural modulation of ClBirA, combined with the in vivo and in vitro analyses, further reveals the action mechanism of ClBirA's dual roles as well as their interaction in C. ljungdahlii. Importantly, an atypical, flexible architecture of the binding site was found to be employed by ClBirA in the regulation of a lot of essential pathway genes, thereby expanding BirA's target genes to a broader range in clostridia. Based on these findings, molecular modification of ClBirA was performed, and an improved cellular performance of C. ljungdahlii was achieved in gas fermentation. This work reveals a previously unknown potent role of BirA in gasfermenting clostridia, providing new perspective for understanding and engineering these autotrophic bacteria.

A toolbox for genetic manipulation in intestinal Clostridium symbiosum

“…4 C), indicating the impact of birA repression on cell metabolism. This phenotypic change was similar with the previous finding in C. ljungdahlii that the inactivation of birA could impair the cell growth [ 27 ], which was largely due to the damage of BirA's biotin protein ligase activity on the biotinylation of acetyl-CoA carboxylase (ACC) and pyruvate carboxylase (PYC), two crucial enzymes for the basic metabolism in cells [ 27 ]. However, it should be noted that the repression degree on birA based on the above dd Fn Cas12a-crRNA construct was still incomplete, requiring a further promotion ( Fig.…”

Protein acetylation-mediated cross regulation of acetic acid and ethanol synthesis in the gas-fermenting Clostridium ljungdahlii

“…As is shown in Figure 4 A , the acetylation levels of Pta were relatively low at all the three phases but slightly increased with the extension of fermentation; in contrast, AdhE1 exhibited a remarkably enhanced acetylation level at the S phase with the low acetylation levels at E and L stages. It is known that the WT C. ljungdahlii normally produces much less ethanol than acetic acid in gas fermentation ( 20 ); the significantly increased AdhE1 acetylation level and the resulting low AdhE1 activity in the latter fermentation stage (S stage) ( Fig. 4 B ) may lead to the production of less ethanol at this stage than the earlier stages of fermentation.…”

Pooled CRISPR interference screening identifies crucial transcription factors in gas-fermentingClostridium ljungdahlii

“…Thus, to unlock the full potential and guide the improvement of industrial microorganisms, it is crucial to understand the metabolic regulation mechanism and then incorporate the related information into strain design and modification. However, to date, this aspect remains poorly explored in gas-fermenting Clostridium species, with only a few transcription factors that have been identified (Liu et al, 2022b;Zhang et al, 2021;Zhang, 2023;Zhang et al, 2020a). The lack of knowledge regarding crucial regulatory elements as well as the underlying regulatory mechanisms has impeded the improvement of these industrially important bacteria.…”