Enhanced extracellular production of L-asparaginase from Bacillus subtilis 168 by B. subtilis WB600 through a combined strategy

Abstract

L-asparaginase (EC 3.5.1.1, ASN) exhibits great commercial value due to its uses in the food and medicine industry. In this study, we reported the enhanced expression of type II ASN from Bacillus subtilis 168 in B. subtilis WB600 through a combined strategy. First, eight signal peptides (the signal peptide of the ASN, ywbN, yvgO, amyE, oppA, vpr, lipA, and wapA) were used for ASN secretion in B. subtilis by using Hpa II promoter, respectively. The signal peptide wapA achieved the highest extracellular ASN activity (28.91 U/mL). Second, Hpa II promoter was replaced by a strong promoter, P43 promoter, resulting in 38.1 % enhanced ASN activity. By two rounds of error-prone PCR mutation, the P43 promoter variants with remarkably enhanced strength (D7, E2, H6, B2, and F3) were identified. B2 (-28: A → G, -13: A → G) achieved ASN activity up to 51.13 U/mL. Third, after deletion of the N-terminal 25-residues, ASN activity reached 102.41 U/mL, which was 100 % higher than that of the intact ASN. At last, the extracellular ASN of the B. subtilis arrived at 407.6 U/mL (2.5 g/L of ASN protein) in a 3-L bioreactor by using a fed-batch strategy. The purified ASN showed maximal activity at 65 °C and its half-life at 65 °C was 61 min. The K and k of the ASN were 5.29 mM and 54.4 s, respectively. To the best of our knowledge, we obtained the highest yield of ASN in a food-grade host ever reported, which may benefit the industrial production and application of ASN.

Design of a high‐efficiency synthetic system for l‐asparaginase production in Bacillus subtilis

“…In this study, P 43 showed the best effect on improving the transcription level of L-asparaginase gene throughout the exponential phase and the early stationary phase (Figures 3 and 4), and the transcription level and protein expression level are not directly correlated (Figure 1). These results are in accordance with the previous reports [12,49]. Further, we found that the secondary structures in RBS regions, which were caused by mismatch between the promoter and RBS, were the reason of the inconsistent enzyme production with transcription levels, so an appropriate RBS sequence that improved the rate of translation was key to further enhance enzyme production.…”

“…However, the low yield of L-asparaginase has restricted the application of this enzyme in the food and pharmaceutical industries. A series of strategies have been used to improve L-asparaginase production, such as optimization of fermentation medium and process [9][10][11], signal peptide screening, promoter mutation, and N-terminal deletion [12]. To date, the highest yield of L-asparaginase obtained was 2168 U/mL, which was reported in our previous work [13].…”

Combinatorial Fine-Tuning of GNA1 and GlmS Expression by 5’-Terminus Fusion Engineering Leads to Overproduction of N-Acetylglucosamine inBacillus subtilis

“…Transcriptional analysis of ecglmS was conducted as described by Feng et al Total RNA was extracted using the RNAisoPlus kit (TaKaRa, Dalian, China), and used to synthesize the cDNA using the PrimeScript RT reagent Kit Perfect Real Time (TaKaRa Bio‐Inc, Otsu, Shiga, Japan). Quantitative PCR (qPCR) was conducted by a LightCycler 480 II Real‐time PCR instrument (Roche Diagnostics, Mannheim, Germany) using a SYBR Premix Ex Taq Kit (TaKaRa).…”

Chasing bacterial chassis for metabolic engineering: a perspective review from classical to non‐traditional microorganisms

“…Numerous examples on the optimization of heterologous enzyme production in B . subtilis have been published – which attempt to improve the overall process efficiency, finding new interesting enzymes and to develop more suitable chassis for the industry (Chen et al ., ; Feng et al ., ).…”

“…Some examples of industrially relevant enzymes produced in B. subtilis chassis are subtilisin (an alkaline serine protease), aand b-amylases, b-glucanases and laccases (Schallmey et al, 2004). Numerous examples on the optimization of heterologous enzyme production in B. subtilis have been publishedwhich attempt to improve the overall process efficiency, finding new interesting enzymes and to develop more suitable chassis for the industry Feng et al, 2017). Apart from its prominent role in protein production, B. subtilis is also used for industrial processes aimed at the synthesis of nucleotides, vitamins, surfactants and antibiotics, for example bacitracin and subtilin.…”