Levin, D. | Search bioRxiv

bioRxiv2024

Antibiotic-free whole-cell biocatalytic fermentation: Escherichia coli with surface-displayed PETases for sustainable plastic degradation

Plastic pollution has increasingly burdened the environment, driving the need for natural degradation platforms that utilize microbial enzymes to break plastics down into monomers. In this study, we introduce a novel approach using Escherichia coli as a fermentative, antibiotic-free whole-cell biocatalyst with surface-displayed, genomically integrated PETases for efficient plastic degradation. PETases, a class of esterases, catalyze the hydrolysis of polyethylene terephthalate (PET) into mono-2-hydroxyethyl terephthalate (MHET). Surface display of these enzymes was achieved via gene fusions with an N-terminal cysteine (Cys) triacylated anchor, mediated by the Braun lipoprotein (Lpp) signal peptide. To circumvent issues associated with plasmids, - such as genetic instability and reliance on antibiotics - we used a Type I-F CRISPR-associated transposase to insert the genes directly into specific E. coli genome sites. Proper enzyme display and activity on the E. coli surface were confirmed through enzyme activity tests, Western blotting, and flow cytometry, with cells retaining PET degradation ability over multiple generations. High-performance liquid chromatography (HPLC) analysis assessed degradation efficiency, identifying byproducts such as bis (2-hydroxyethyl) terephthalate and terephthalic acid. This study establishes a proof-of-concept for efficient plastic degradation using engineered bacteria as robust, sustainable, and genomically stable whole-cell biocatalysts, providing a promising platform for addressing plastic waste management. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=194 SRC="FIGDIR/small/624590v1_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@1320dd8org.highwire.dtl.DTLVardef@12b025forg.highwire.dtl.DTLVardef@a8b0b3org.highwire.dtl.DTLVardef@e8d2c7_HPS_FORMAT_FIGEXP M_FIG C_FIG One-sentence AbstractEscherichia coli was engineered as a fermentative, antibiotic-free whole-cell biocatalyst, featuring surface-displayed and genomically integrated PETases for efficient plastic degradation. This innovative approach has the potential to transform plastic recycling by enabling sustainable, large-scale degradation of plastic waste through environmentally friendly microbial systems.

synthetic biology↗

bioRxiv2023

In vivo metabolomics identifies CD38 as an emergent vulnerability in LKB1-mutant lung cancer

LKB1/STK11 is a serine/threonine kinase that plays a major role in controlling cell metabolism, resulting in potential therapeutic vulnerabilities in LKB1-mutant cancers. Here, we identify the NAD+ degrading ectoenzyme, CD38, as a new target in LKB1-mutant NSCLC. Metabolic profiling of genetically engineered mouse models (GEMMs) revealed that LKB1 mutant lung cancers have a striking increase in ADP-ribose, a breakdown product of the critical redox co-factor, NAD+. Surprisingly, compared with other genetic subsets, murine and human LKB1-mutant NSCLC show marked overexpression of the NAD+-catabolizing ectoenzyme, CD38 on the surface of tumor cells. Loss of LKB1 or inactivation of Salt-Inducible Kinases (SIKs)--key downstream effectors of LKB1-- induces CD38 transcription induction via a CREB binding site in the CD38 promoter. Treatment with the FDA-approved anti-CD38 antibody, daratumumab, inhibited growth of LKB1-mutant NSCLC xenografts. Together, these results reveal CD38 as a promising therapeutic target in patients with LKB1 mutant lung cancer. SIGNIFICANCELoss-of-function mutations in the LKB1 tumor suppressor of lung adenocarcinoma patients and are associated with resistance to current treatments. Our study identified CD38 as a potential therapeutic target that is highly overexpressed in this specific subtype of cancer, associated with a shift in NAD homeostasis.

cancer biology↗