Improved Membrane Permeability via Hypervesiculation for In Situ Recovery of Lycopene in Escherichia coli

Abstract

Lycopene biosynthesis is frequently hampered by downstream processing hugely due to its inability to be secreted out from the producing chassis. Engineering cell factories can resolve this issue by secreting this hydrophobic compound. A highly permeable E. coli strain was developed for a better release rate of lycopene. Specifically, the heterologous mevalonate pathway and crtEBI genes from Corynebacterium glutamicum were overexpressed in Escherichia coli BL21 (DE3) for lycopene synthesis. To ensure in situ lycopene production, murein lipoprotein, lipoprotein NlpI, inner membrane permease protein, and membrane-anchored protein in TolA-TolQ-TolR were deleted for improved membrane permeability. The final strain, LYC-8, produced 438.44 ± 8.11 and 136.94 ± 1.94 mg/L of extracellular and intracellular lycopene in fed-batch fermentation. Both proteomics and lipidomics analyses of secreted outer membrane vesicles were perfect indicators of hypervesiculation. Changes in the ratio of saturated fatty acids, unsaturated fatty acids, and cyclopropane fatty acids coupled with the branching and acyl chain lengths altered the membrane fatty acid composition. This ensured membrane fluidity and permeability for in situ lycopene release. The combinatorial deletion of these genes altered the cellular morphology. The structural and morphological changes in cell shape, size, and length were associated with changes in the mechanical strength of the cell envelope. The enhanced lycopene production and secretion mediated by improved membrane permeability established a cell lysis-free system for an efficient releasing rate and downstream processing, demonstrating the importance of vesicle-associated membrane permeability in efficient lycopene production.

Transcending membrane barriers: advances in membrane engineering to enhance the production capacity of microbial cell factories

“…This strategy resulted in a 61% increase in yield for CAR025. Similarly, Fordjour et al [ 82 ], by knocking out genes such as TolA-TolQ TolR, successfully constructed a vesicle system in E. coli , thereby improving cell membrane permeability and increasing lycopene production. In application cases aimed at enhancing the PHB production of cell factories, Wang et al through genomic and transcriptomic analysis, studied flagella assembly in the wild-type E. coli W3110 strain and mutant strains DwaaF, DwaaC, and DwaaG, which only synthesize lipopolysaccharides of varying lengths, concluding that flagella assembly in E. coli depends on the length of lipopolysaccharides; by disrupting all gene clusters related to the polysaccharide part of lipopolysaccharides (LPS), colanic acid (CA), flagella, and/or fimbriae, the outer membrane of E. coli W3110 was successfully modified, creating favorable conditions for PHB production [ 53 , 83 ].…”

Advances in the Discovery and Engineering of Gene Targets for Carotenoid Biosynthesis in Recombinant Strains

“…Furthermore, researchers engineered E. coli with a novel transport system utilizing artificial membrane vesicles to effectively secrete hydrophobic molecules, resulting in a notable 61% increase in β-carotene production [98]. To enhance the release rate of lycopene, a highly permeable E. coli strain was created by deleting the lpp, nlpI, mlaE, and tolA genes [99]. The presence of lipopolysaccharides, which are the primary constituents of the outer membrane in E. coli, The toxicity of carotenoid products poses a significant obstacle to microbial carotenoid production, and the implementation of transporter-mediated carotenoid secretion presents a promising solution to this issue [94].…”