Abstract
Acetaldehyde dehydrogenases are potential enzyme preparations that can be used to detoxify acetaldehyde and other exogenous aldehydes from pharmaceuticals, food, and biofuel production. In this study, we enhanced the expression of acetaldehyde dehydrogenase sourced from Issatchenkia terricola (istALDH) in Bacillus subtilis using a combinatorial strategy for the optimization of signal peptides, promoters, and growth conditions. First, a library of various signal peptides was constructed to identify the optimal signal peptides for efficient istALDH secretion. The signal peptide yqzG achieved the highest extracellular istALDH activity (204.85 ± 3.31 U/mL). Second, the aprE promoter was replaced by a constitutive promoter (i.e., P43) and an inducible promoter (i.e., Pglv), resulting in 12.40% and 19.97% enhanced istALDH, respectively. Furthermore, the tandem promoter P43-Pglv provided a better performance, resulting in 30.96% enhanced istALDH activity. Third, the production of istALDH was optimized by testing one factor at a time. Physical parameters were optimized including the inducer (e.g., maltose) concentrations, incubation temperatures, and inoculation amounts, and the results were 2.0%, 35 °C, and 2.0%, respectively. The optimized medium results were 2.0% glucose, 1.5% peptone, 2.5% yeast extract, 1% NaCl, and 0.5% (NH4)2SO4. The extracellular istALDH activity was 331.19 ± 4.19 U/mL, yielding the highest production reported in the literature to date.
Highlights
The results showed that both Pglv and P43 could increase the expression of the Issatchenkia terricola acetaldehyde dehydrogenase (istALDH)
To the best of our knowledge, this study was the first to report on the optimization of the secretion for istALDH in B. subtilis via signal peptides (SPs) screening and promoter replacement
YpzG and tandem promoter P43 -Pglv were the best candidates for istALDH secretion
Summary
Bacillus subtilis is well known as a “generally regarded as safe”(GRAS) strain in the food industry and can be used as a microbial factory for large-scale modern industrial fermentation and production [10]. Attempts to produce foreign proteins using Bacillus strains have failed or led to disappointing yields [15], and the expression of ALDHs in B. subtilis has not yet been reported. Expression components, such as signal peptides (SPs) and promoters optimized for homologous proteins, can improve their expression levels. ALDH production level reported in the B. subtilis expression system, to date
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