Combinatorial gene inactivation of aldehyde dehydrogenases mitigates aldehyde oxidation catalyzed by E. coli resting cells

Butler, Neil D.; Anderson, Shelby R.; Dickey, Roman M.; Nain, Priyanka; Kunjapur, Aditya M.

doi:10.1016/j.ymben.2023.04.014

Cited by 10 publications

(2 citation statements)

References 50 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The copyright holder for this preprint (which this version posted May 2, 2023. ; https://doi.org/10.1101/2023.05.02.539072 doi: bioRxiv preprint in the previously engineered RARE.Δ6 strain under aerobic growth. Although we recently verified that the RARE.Δ6 strain reliably stabilizes a broad range of aromatic aldehydes under these conditions 40 , we were surprised to observe TPAL reduction at our first time point of 4 h with greater reduction seen at 24 h (Fig. 1C).…”

Section: Tpal Is Reduced In Mg1655 and Rareδ6 Under Aerobic Conditionsmentioning

confidence: 61%

Genome engineering allows selective conversions of terephthalaldehyde to multiple valorized products in bacterial cells

Dickey

Jones

Butler

et al. 2023

Preprint

View full text Add to dashboard Cite

Deconstruction of polyethylene terephthalate (PET) plastic waste generates opportunities for valorization to alternative products. We recently designed an enzymatic cascade that could produce terephthalaldehyde (TPAL) from terephthalic acid. Here, we showed that the addition of TPAL to growing cultures of Escherichia coli wild-type strain MG1655 and an engineered strain for reduced aromatic aldehyde rection (RARE) strain resulted in substantial reduction. We then investigated if we could mitigate this reduction using multiplex automatable genome engineering (MAGE) to create an E. coli strain with 10 additional knockouts in RARE. Encouragingly, we found this newly engineered strain enabled a 2.5-fold higher retention of TPAL over RARE after 24h. We applied this new strain for the production of para-xylylenediamine (pXYL) and observed a 6.8-fold increase in pXYL titer compared to RARE. Overall, our study demonstrates the potential of TPAL as a versatile intermediate in microbial biosynthesis of chemicals that derived from waste PET.

show abstract

Section: Tpal Is Reduced In Mg1655 and Rareδ6 Under Aerobic Conditionsmentioning

confidence: 61%

Genome engineering allows selective conversions of terephthalaldehyde to multiple valorized products in bacterial cells

Dickey

Jones

Butler

et al. 2023

Preprint

View full text Add to dashboard Cite

show abstract

“…To investigate whether the knockout of genes that encode aldehyde reductases would result in improved yields of β-OH-nsAA, we transformed the plasmid that harbors our TTA expression cassette into another E. coli strain that was engineered to stabilize aromatic aldehydes, the RARE strain 46 . The RARE strain has been shown to stabilize many aryl aldehydes, including 1 , 9 , and 12 , by eliminating potential reduction pathways 46 , 47 . We then repeated the experiment in the RARE strain and once again found that PbTTA produced the highest titer with 0.61 ± 0.04 mM produced with 100 mM l -Thr and 0.13 ± 0.01 mM produced with natural l -Thr levels (Fig.…”

Section: Resultsmentioning

confidence: 99%

Discovery of l-threonine transaldolases for enhanced biosynthesis of beta-hydroxylated amino acids

Jones,

Butler,

Anderson

et al. 2023

Commun Biol

Self Cite

View full text Add to dashboard Cite

Beta-hydroxy non-standard amino acids (β-OH-nsAAs) have utility as small molecule drugs, precursors for beta-lactone antibiotics, and building blocks for polypeptides. While the L-threonine transaldolase (TTA), ObiH, is a promising enzyme for β-OH-nsAA biosynthesis, little is known about other natural TTA sequences. We ascertained the specificity of the TTA enzyme class more comprehensively by characterizing 12 candidate TTA gene products across a wide range (20-80%) of sequence identities. We found that addition of a solubility tag substantially enhanced the soluble protein expression level within this difficult-to-express enzyme family. Using an optimized coupled enzyme assay, we identified six TTAs, including one with less than 30% sequence identity to ObiH that exhibits broader substrate scope, two-fold higher L-Threonine (L-Thr) affinity, and five-fold faster initial reaction rates under conditions tested. We harnessed these TTAs for first-time bioproduction of β-OH-nsAAs with handles for bio-orthogonal conjugation from supplemented precursors during aerobic fermentation of engineered Escherichia coli, where we observed that higher affinity of the TTA for L-Thr increased titer. Overall, our work reveals an unexpectedly high level of sequence diversity and broad substrate specificity in an enzyme family whose members play key roles in the biosynthesis of therapeutic natural products that could benefit from chemical diversification.

show abstract

Genome engineering allows selective conversions of terephthalaldehyde to multiple valorized products in bacterial cells

Dickey,

Jones,

Butler

et al. 2023

AIChE Journal

Self Cite

View full text Add to dashboard Cite

Deconstruction of polyethylene terephthalate (PET) plastic waste generates opportunities for valorization to alternative products. We recently designed an enzymatic cascade that could produce terephthalaldehyde (TPAL) from terephthalic acid. Here, we showed that the addition of TPAL to growing cultures of Escherichia coli wild‐type strain MG1655 and an engineered strain for reduced aromatic aldehyde reduction (RARE) strain resulted in substantial reduction. We then investigated if we could mitigate this reduction using multiplex automatable genome engineering (MAGE) to create an E. coli strain with 10 additional knockouts in RARE. Encouragingly, we found this newly engineered strain enabled a 2.5‐fold higher retention of TPAL over RARE after 24 h. We applied this new strain for the production of para‐xylylenediamine (pXYL) and observed a 6.8‐fold increase in pXYL titer compared with RARE. Overall, our study demonstrates the potential of TPAL as a versatile intermediate in microbial biosynthesis of chemicals that derived from waste PET.

show abstract

Combinatorial gene inactivation of aldehyde dehydrogenases mitigates aldehyde oxidation catalyzed by E. coli resting cells

Cited by 10 publications

References 50 publications

Genome engineering allows selective conversions of terephthalaldehyde to multiple valorized products in bacterial cells

Genome engineering allows selective conversions of terephthalaldehyde to multiple valorized products in bacterial cells

Discovery of l-threonine transaldolases for enhanced biosynthesis of beta-hydroxylated amino acids

Genome engineering allows selective conversions of terephthalaldehyde to multiple valorized products in bacterial cells

Contact Info

Product

Resources

About