Zaigao Tan

The actin-like cytoskeletal protein MreB controls bacterial cell morphology, yet whether engineered modulation of MreB-associated architecture can improve microbial metabolism remains largely unexplored. Here, we established a chemical-genetic platform in Escherichia coli combining an engineered MreB variant (A53R) that remodels cellular morphology with A22-mediated perturbation of MreB dynamics. MreB(A53R) reshaped cell morphology and engaged an RpoE-Cra-GadE regulatory network associated with cellular adaptation, which remodeled electron transport, redirected carbon flux toward NADPH-generating pathways, and reduced intracellular reactive oxygen species (ROS). Consequently, engineered strains exhibited enhanced formaldehyde tolerance and efficient assimilation of methanol and other C1 substrates. Acute A22 treatment recapitulated the morphological alteration but failed to induce metabolic rewiring, revealing a two-stage adaptive response involving immediate physical adaptation associated with cell envelope reinforcement and long-term transcription-dependent metabolic adaptation to persistent morphological remodeling. The analogous V45R mutation in the native methylotroph Bacillus methanolicus partially recapitulated the improved C1 utilization phenotype, supporting the transferability of MreB-based morphological engineering. Collectively, these findings establish MreB-mediated morphological engineering as a strategy to couple cellular architecture with central carbon and redox metabolism and provide a framework for engineering robust microbial chassis for C1 bioconversion.

The lipase enzyme shows great potential for catalyzing the synthesis of valuable food flavor compounds, such as γ- and δ-lactones, but natural lipases are limited by low conversion rates. In this study, we developed a computational-guided strategy named Synergistic Pairwise Residues Evolution for the efficient directed evolution of Candida antarctica lipase B (CalB). This approach identifies synergistic hotspot residues by combining substrate-contact information with evolutionarily coupled functional sectors. E188Q/I189F, the top-performing mutant with 2.75-fold improved activity, achieved over 99.5% conversion within 5 h at 37 °C, compared with only 49% for wild-type CalB in the synthesis of δ-dodecalactone. This study presents an efficient strategy for building functional enzyme toolkits and demonstrates their high catalytic performance in lactone synthesis.

The research on synthetic methylotrophic bacteria for one-carbon (C1) feedstock assimilation has garnered substantial interest and is regarded as the forefront of biomanufacturing advancements. Nevertheless, the effective utilization of C1 feedstocks faces challenges due to inadequate tolerance toward C1 compounds. This study elucidates that the buildup of formaldehyde causes severe DNA-protein cross-linking (DPC), and thus hampers growth and methanol assimilation in Escherichia coli. To tackle this issue, we exploited a metalloproteinase, SpWss1, from Schizosaccharomyces pombe. By fine-overexpressing SpWss1 in the E. coli genome, we were able to alleviate DPC damage and enhance formaldehyde tolerance. Remarkably, the engineered strain displayed a 10-fold increase in the amount of methanol assimilated (142 mM) compared to that of the control strain lacking SpWss1 (14 mM). Moreover, through iterative substrate feeding of methanol and xylose in shake-flask experiments, the genetically modified strain exhibited improved consumption levels, reaching up to 309 mM (∼10 g/L), making it one of the highest methanol-consuming strains among all E. coli strains without adaptive evolution. Additionally, the modified strain significantly enhanced the sustainable production of valuable products, such as triacetic acid lactone and fatty acids, from methanol. Overall, our findings underscore the significant scientific and biotechnological importance of addressing DPC to optimize C1 assimilation, providing valuable insights for sustainable chemistry, engineering, and industrial biotechnology applications.

Lipid composition represents a significant differentiator across the three domains (eukaryotes, bacteria, and archaea) of cellular life. Eukaryotes possess distinct lipids, such as sterols and sphingolipids, generally, these are not commonly found in typical bacteria and archaea. Sterols play a pivotal role in eukaryotic cellular functions, lanosterol, a key precursor for animal and fungal steroids, has well established functions in eukaryotes, while its potential functions in bacteria remain largely uninvestigated. In this study, we genetically engineered Escherichia coli (E. coli) to reconstruct the biosynthesis of lanosterol, and successfully developed a novel E. coli strain capable of synthesizing lanosterol, although its specific location, such as whether it is incorporated into the cell membrane, remains to be further determined. Comprehensive characterization of the observed phenotypic changes has unveiled that, despite an unaltered growth rate under normal condition, the engineered E. coli strain displayed notably enhanced tolerance to various stresses. Subsequent analysis has indicated that lanosterol plays a role in preserving membrane integrity, fluidity, hydrophobicity, and ATP production, mirroring the functions of sterols in eukaryotes. This study unveils the unexpected capacity of E. coli to synthesize sterols, not only underscores the importance of lanosterol as a precursor for essential cellular lipids but also offers fresh insights into the potential functions of sterols within bacterial systems.

Efficient bioassimilation of one-carbon (C1) feedstocks is often hindered by the toxicity of C1 substrates and/or intermediates. We compared the toxicity of several common C1 substrates/intermediates and found that formaldehyde imposes the highest toxicity on the representative bacterium Escherichia coli. Besides causing chromosomal DNA and protein damage effects, here, we revealed that formaldehyde greatly impairs cell membranes. To this end, here, we sought to remodel the cell membrane of E. coli by introducing a non-native, eukaryote-featured membrane phospholipid composition, phosphatidylcholine (PC). This engineered E. coli strain exhibited significantly increased membrane integrity, resulting in enhanced formaldehyde tolerance. When applied to C1 assimilation, the PC-harboring E. coli consumed up to 4.7 g/L methanol, which is 23-fold higher than that of the control strain (0.2 g/L). In summary, the present study highlights the detrimental impact of formaldehyde-induced membrane damage and thus underscores the significance of membrane remodeling in enhancing formaldehyde tolerance and facilitating the assimilation of C1 substrates.