Cis-Element Engineering Promotes the Expression of Bacillus subtilis Type I L-Asparaginase and Its Application in Food

Abstract

Type I L-asparaginase from Bacillus licheniformis Z-1 (BlAase) was efficiently produced and secreted in Bacillus subtilis RIK 1285, but its low yield made it unsuitable for industrial use. Thus, a combined method was used in this study to boost BlAase synthesis in B. subtilis. First, fifteen single strong promoters were chosen to replace the original promoter P43, with PyvyD achieving the greatest BlAase activity (436.28 U/mL). Second, dual-promoter systems were built using four promoters (PyvyD, P43, PaprE, and PspoVG) with relatively high BlAase expression levels to boost BlAase output, with the engine of promoter PaprE-PyvyD reaching 502.11 U/mL. The activity of BlAase was also increased (568.59 U/mL) by modifying key portions of the PaprE-PyvyD promoter. Third, when the ribosome binding site (RBS) sequence of promoter PyvyD was replaced, BlAase activity reached 790.1 U/mL, which was 2.27 times greater than the original promoter P43 strain. After 36 h of cultivation, the BlAase expression level in a 10 L fermenter reached 2163.09 U/mL, which was 6.2 times greater than the initial strain using promoter P43. Moreover, the application potential of BlAase on acrylamide migration in potato chips was evaluated. Results showed that 89.50% of acrylamide in fried potato chips could be removed when combined with blanching and BlAase treatment. These findings revealed that combining transcription and translation techniques are effective strategies to boost recombinant protein output, and BlAase can be a great candidate for controlling acrylamide in food processing.

Enhanced Thermostability and Molecular Insights for l-Asparaginase from Bacillus licheniformis via Structure- and Computation-Based Rational Design

“…l -Asparaginase ( l -ASNase, amidohydrolase enzyme, EC3.5.1.1) can catalyze the production of non-essential amino acid l -asparagine (L-ASN) to l -aspartic acid (L-ASP) and ammonia. , This enzyme displays great potential for a wide range of applications in the food and pharmaceutical industries, such as reducing the formation of carcinogenic acrylamide in fried foods and treating acute lymphoblastic leukemia (ALL) . Due to the short half-life, commercial l -ASNase coming from Escherichia coli (EcA) and Erwinia chrysanthemi (ErA) have a high-dose-dependent disadvantage for ALL treatment, causing severe toxic effects on patients .…”

Current state of molecular and metabolic strategies for the improvement of L-asparaginase expression in heterologous systems

“…subtilis hosts, and the optimization of these conserved regions was considered one of the important strategies to increase the yield of recombinant proteins. Niu et al (2022), studying the production of L-ASNase using a dual promoter system, modified the -35 and -10 sequences of these promoters, obtaining three mutations, achieving an improvement of 6.6; 7.3 and 13.3% in the expression levels and the transcription intensity was 4.37, 4.15 and 4.86 times greater than that of the P43 promoter. After 36 h of culture, the L-ASNase expression level in a 10 L fermenter reached 2163.09 U/mL, which was 6.2 times greater than the initial strain that was using the P43 promoter.…”

“…Their work improved the transcription levels of the gene of the enzyme 2.09 times more than the original strain by using the P 43 promoter and an optimized ribosomal binding site (RBS). Niu et al (2022) developed an approach similar to the one used by Li et al (2019), but with the difference that they established a dual-promoter system and optimized the its core regions (-35 and -10 boxes). The dual-promoter systems performed ideally when nine of the sixteen dual-promoter systems were used (P aprE -P 43 , P yvyD -P 43 , P spoVG -P 43 , P aprE -P aprE , P yvyD -P aprE , P yvyD -P yvyD , P 43 -P yvyD , P aprE -P yvyD , and P spoVG -P yvyD ) obtaining greater yields than the original P 43 promoter.…”

“…Of 300 sequences that were designed, the mutant RBS that achieved the greatest yield was chosen, having managed to produce a total activity of 5278 U/mL. Niu et al (2022), using the online tool "RBS Calculator v2.0" (SalisLab, 2022) and analyzing 22 RBS sequences, managed to select a sequence capable of improving expression by 1.39 times.…”

“…The expression system used was pET14b-SUMO, improving catalytic efficiency up to 6 times more than the wild enzyme (Karamitros & Konrad, 2014;Karamitros & Konrad, 2016). There are also other N-Terminal fusion proteins that have been employed for the expression of L-ASNase, such as GST (glutathione S-transferase); and the affinity tags used to facilitate purification, like Poly-His, which are often used in the L-ASNase expression hosts (de Almeida Parizotto et al, 2021;Effer et al, 2019;Feng et al, 2017;Niu et al, 2022). These tags are summarized in Table 2.…”