Enhanced extracellular raw starch-degrading α-amylase production in Bacillus subtilis by promoter engineering and translation initiation efficiency optimization

Abstract

Background A raw starch-degrading α-amylase from Pontibacillus sp. ZY (AmyZ1), previously screened by our laboratory, showed a promising application potential for starch-processing industries. However, the AmyZ1 secretory production still under investigation, which seriously restricts its application in the starch-processing industry. On the other hand, Bacillus subtilis is widely used to achieve the extracellular expression of target proteins. Results AmyZ1 secretory production was achieved in B. subtilis and was enhanced by promoter engineering and translation initiation efficiency optimization. First, based on the different phase-dependent promoters, the dual-promoter PspoVG–PspoVG142 was constructed by combining dual-promoter engineering and promoter modification. The corresponding strain BZd34 showed an extracellular AmyZ1 activity of 1437.6 U/mL during shake flask cultivation, which was 3.11-fold higher than that of the original strain BZ1 (PgroE). Then, based on translation initiation efficiency optimization, the best strain BZd343 containing optimized 5'-proximal coding sequence (opt3) produced the highest extracellular α-amylase activity of 1691.1 U/mL, which was 3.65-fold higher than that of the strain BZ1. Finally, cultivation of BZd343 in 3-L fermenter exhibited an extracellular AmyZ1 activity of 14,012 U/mL at 48 h, with productivity of 291.9 U/mL·h. Conclusions This is the first report of recombinant expression of AmyZ1 in B. subtilis and the expression level of AmyZ1 represents the highest raw starch-degrading α-amylase level in B. subtilis to date. The high-level expression of AmyZ1 in this work provides a foundation for its industrial production. The strategies used in this study also provide a strategic reference for improving the secretory expression of other enzymes in B. subtilis.

Enhanced extracellular production of raw starch-degrading α-amylase in Bacillus subtilis through expression regulatory element modification and fermentation optimization

“…Luria − Bertani (LB) medium was used for E. coli growth and seed fermentation of B. subtilis . The original fermentation medium used for shake flask fermentation is 2 × YT medium containing 10 mM calcium ions [ 2 ]. For 3-L fermenter fermentation, the original basic medium (w/v) contained 1% glycerol, 2.4% tryptone, 1.5% yeast extract, 0.75% NaCl, and 3 mL/L trace element solution [ 11 ].…”

“…In starch processing, α-amylase is often used to hydrolyze the internal α-1,4 glycosidic bonds and degrade them into low-polymerization dextrins as well as maltose and glucose [ 1 ]. Traditional starch processing requires thermostable α-amylase, but there are problems such as high energy consumption and complicated process due to its action temperature of 95–105 ℃ [ 2 ]. On the contrary, using raw starch-degrading α-amylases (RSDA) can effectively solve the above problems, because it can efficiently hydrolyze raw starch below the gelatinization temperature (40 °C), so receiving increased attention.…”

Using glycerol-inducible expression system to overexpressed maltooligosaccharide-forming α-amylase in Bacillus subtilis

“…For obtaining more BcMFAse, we focused on the promoter. As part of expression system, the promoter plays an important role in the protein production [10]. Currently, many strategies about promoters have been developed to improve the recombinant expression level of target proteins in B. subtilis [11][12][13][14][15][16][17][18].…”

Purification and characterization of moderately thermostable raw-starch digesting α-amylase from endophytic <i>Streptomyces mobaraensis </i>DB13<i> </i>associated with <i>Costus speciosus</i>

“…It hydrolyzes the α-1,4-glucosidic bonds of starch, releasing reducing sugars. The enzyme has great significance in food, medicinal, textile, paper, and chemical industries (Li et al, 2022;Sajedi et al, 2005). It is usually produced by plants, animals, and microbes, of which α-amylase obtained from microbes, provides a significant contribution.…”

“…Alpha-amylase with enviable properties, like raw starch-degrading are of great importance. Starch gelatinization is an important step in starch processing industries, where starch is liquefied by thermostable amylase on heating to approximately to 95-105°C (Li et al, 2022). The method augments the consumption of energy and costs; in contrast, the application of raw starch digesting amylases (RSDA) that directly hydrolyze raw starch without gelatinization, has generated worldwide importance.…”