Engineered microorganisms and methods for improving aldehyde dehydrogenase activity
An aldehyde dehydrogenase, microbial technology, applied in biochemical equipment and methods, botanical equipment and methods, genetic engineering, etc., can solve problems such as undesired, reduced product efficiency or yield, and increased compound cost and complexity
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Embodiment 1
[0153] Example 1. Screening of candidate aldehyde dehydrogenases for activity on adipyl-CoA
[0154] Genes encoding candidate aldehyde dehydrogenases (Ald) were bioinformatically identified in the genomes of multiple species (Table 1). Genes encoding each aldehyde dehydrogenase were synthesized, expressed in E. coli, and Ald activity was assessed.
[0155] The genes encoding the Ald enzyme candidates of Table 1 were cloned into low copy vectors under constitutive promoters and the constructs were transformed into E. coli using standard techniques. The transformants were cultured overnight at 35°C in LB medium in the presence of antibiotics, and then the cells were harvested at room temperature at a speed of 15000 rpm. To prepare lysates, resuspend cells in chemical lysis solution containing lysozyme, nuclease, and 10 mM DTT and incubate at room temperature for at least 30 min. The resulting lysates were used to test for aldehyde dehydrogenase activity.
[0156] The lysate (...
Embodiment 2
[0164] Example 2. Performing an aldehyde dehydrogenase assay to determine substrate specificity
[0165]To determine the substrate preference of several aldehyde dehydrogenases, a substrate-CoA consumption assay was applied using succinyl-CoA and adipyl-CoA substrates. In this assay, the substrate solution contained 0.1M Tris-HCl (pH 7.5), 1 mM adipyl-CoA, 0.2 mM succinyl-CoA, and 0.2 mM acetyl-CoA, and an excess of 1.5 mM NADH or NADPH cofactor . Reactions were initiated by adding lysates to assay buffer and incubated for 2 hours at room temperature. The reaction was quenched with 1% formic acid and then evaluated by LC / MS analytical method to quantify each residual substrate CoA. Ald activity was measured as percent consumption of each CoA substrate. A higher percent consumption of a specific CoA substrate relative to another CoA substrate present in the assay indicates a preference for a specific substrate CoA. figure 2 Peptostreptococcaceae bacterium oral aldehyde deh...
Embodiment 3
[0166] Example 3. In vivo assay of aldehyde dehydrogenase
[0167] Aldehyde dehydrogenases demonstrating an adipyl-CoA substrate preference were also tested in in vivo assays in which a construct encoding a 3-oxoadipyl-CoA thiolase ( Thl), 3-oxoadipyl-CoA dehydrogenase (Hbd) and 3-oxoadipyl-CoA dehydratase (“crotonase” or Crt), 5-carboxy-2-pentenoyl-CoA A E. coli strain with genes for reductase (Ter) and transaminase (TA). Thl, Hbd, Crt, Ter, TA E. coli strains include all pathway enzymes required for the production of 6-aminocaproic acid (6ACA), except the Ald enzyme. Encoding Porphyromonas gingivalisW83Ald (SEQ ID NO:2), Peptostreptococcaceae bacterium oralAld (SEQ ID NO:7), Acidaminococcus massiliensis Ald (SEQ ID NO:28), Collinsella sp.GD7Ald (SEQ IDNO:60) and Romboutsia lituseburensis DSM Ald (SEQ ID NO:60) and Romboutsia lituseburensis DSM Ald (SEQ ID NO:7) ID NO:107) genes were respectively cloned into low copy number plasmid vectors under constitutive promoters. The...
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