Enhanced extracellular production of maltotetraose amylase from Pseudomonas saccharophila in Bacillus subtilis through regulatory element optimization

Maltotetraose amylase (Mta) catalyzes the hydrolysis of amylaceous polysaccharides into maltotetraose, which is an important functional sugar used in the food industry. However, the lack of efficient expression systems for recombinant Mta has hindered its scale-up production and application. In this study, a codon-optimized mta gene from Pseudomonas saccharophila was efficiently produced in Bacillus subtilis by optimizing the regulatory elements. First, a plasmid library containing 173 different signal peptide sequences placed upstream of mta gene was constructed, and transformed into B. subtilis strain WB800N( amyE Δ1) for high-throughput screening. The signal peptide yhcR was found to significantly enhance the secretion of Mta, reaching an activity of 75.4 U/mL in the culture medium. After optimization of the promoters, the Mta activity was further increased to 100.3 U/mL using a dual-promoter P HpaII P amyE . Finally, the carbon sources and nitrogen sources for recombinant Mta production were optimized, yielding a highest Mta activity of 288.9 U/mL under the optimal culture conditions. The crude enzyme solution containing recombinant Mta produced a highest maltotetraose yield of 70.3% with 200 g/L of maltodextrin as the substrate. Therefore, the present study have demonstrated a high yield of Mta produced in B. subtilis , laying the foundation for large-scale Mta production and application.


Introduction
Maltotetraose is a straight-chain maltooligosaccharide which is composed of four α-D-glucopyranosyl units linked by α-1,4 glycosidic bonds [1,2].Maltotetraose offers various advantages in food processing, including low sweetness, high moisture retention, and reduced production of Maillard reaction products in the course of food processing [3].In addition, maltotetraose can inhibit the growth of intestinal putrefactive bacteria, potentially improving colonic health without disrupting the balance of the intestinal microflora [1].Currently, maltotetraose is increasingly used as an ingredient in the food industry.
Maltotetraose is commercially produced by enzymatic treatment of amylaceous polysaccharides.Maltotetraose amylase (Mta), a member of the glycoside hydrolase family 13 (GH13), releases maltotetraose by cleaving the fourth α-1,4 glycosidic bond from the non-reducing ends of starch or maltodextrin [1,3,4].Mta is primarily found in Pseudomonas bacterial strains, with the enzyme from Pseudomonas saccharophila being the most extensively studied.However, the low yield of Mta produced in the wild bacterial strains has impeded its industrial production and application.The extracellular activity of Mta produced by Pseudomonas sp.IMD353 reached 29 U/ mL in the shake flask cultivation [5].Through the optimization of fermentation conditions, a Mta activity of 6.8 U/mL was achieved in Pseudomonas stutzeri AS22 [6].Although Escherichia coli (E.coli), the most commonly used host for expressing heterologous proteins, has been used for heterologous expression of Mta from Pseudomonas saccharophila, the yield of soluble recombinant Mta in E. coli is typically insufficient, and the recombinant enzyme has been detected primarily in inclusion bodies [7].In addition, E. coli is unsuitable for producing Mta in the food industry due to safety concerns related to endotoxin synthesis.
Mta is extensively used in the food industry for maltotetraose production and has been approved for use in many countries.Following a comprehensive safety assessment by the European Food Safety Authority (EFSA), Mta is considered not to cause safety concerns, and dietary exposure to this enzyme is not expected to induce allergenic problems under the intended conditions of use [8].The production of recombinant Mta in food-grade microbial strains would facilitate its widespread application in the food industry.Bacillus subtilis, a well-known grampositive bacterium, has been extensively used in the food industry for enzyme production [9].Using B. subtilis as a host for enzyme production offers a number of advantages, including superior heterologous proteins secretion, non-pathogenicity, and ease of large-scale cultivation [9,10].Food enzymes such as pullulanase, subtilisin and asparaginase were expressed by recombinant B. subtilis strains that have been approved by the US Food and Drug Administration (FDA) as the generally recognized as safe (GRAS) status [11][12][13].Mta has been expressed in Bacillus subtilis strains.The Mta from Pseudomonas saccharophila STB07 has been expressed in Bacillus subtilis WB600, resulting in an extracellular activity of 230 U/mL in the shake flask cultivation using TB medium [4,14].The Mta from Pseudomonas saccharophila has been expressed and secreted in Bacillus subtilis WS11, and an activity level of 236 U/mL was achieved in the shake flask after optimizing the culture conditions [15].Given the growing demand for maltotetraose, it is essential to develop an efficient expression system for production of maltotetraose amylase using food-grade bacteria such as B. subtilis.Nevertheless, the yield of recombinant Mta produced by B. subtilis needs to be increased, and the effects of regulatory elements such as signal peptide (SP) sequences and promoters on Mta expression have not been systematically studied and optimized.
The signal peptide, a polypeptide chain located at the N-terminus of the secretory protein precursor, influences the expression and secretion of heterologous proteins in B. subtilis [16,17].The prediction of the optimal signal peptide for a specific protein remains challenging.The construction of a signal peptide library followed by high-throughput screening has proven to be an efficient strategy for signal peptide optimization in the B. subtilis expression system [9,17].After high-throughput screening of a signal peptide library, a recombinant strain harboring CitH signal peptide was found to yield the highest secretion for a thermostable glycosidase in B. subtilis, with an activity of 5.20 U/mL in the culture medium, compared to the original activity of 1.00 U/mL produced with the aprE signal peptide [18].Through the construction and screening of a signal peptide library, the production of a raw strach-degrading α-amylase AmyZ1 in B. subtilis was enhanced with an optimal YpuA signal peptide, which exhibited a 1.28-fold higher AmyZ1 activity compared to the original amyQ signal peptide [19].
To efficiently produce heterologous proteins in B. subtilis, a strong promoter is essential.Several constitutive promoters have been identified for directing the expression of recombinant proteins in B. subtilis, such as the B. subtilis constitutive promoter P 43 , the Staphylococcus aureus constitutive promoter P HpaII , and the B. subtilis α-amylase promoter P amyE [16,20,21].Using a constitutive promoter for the production of heterologous proteins eliminates the need for commonly used inducer such as IPTG and xylose [16,22], and allows gene transcription during cell growth, thereby providing a more simple, cost-effective and safe expression process.Recent studies have demonstrated that combining different constitutive promoters in tandem resulted in a higher yield of multiple heterologous proteins than a single promoter [23].The productivity of the thermostable 4-α-glucanotransferase from Thermus scotoductus in B. subtilis was increased more than ten-fold using a dualpromoter compared to the single promoter P HpaII [20].A higher level of β-cyclodextrin glycosyltransferase was produced in B. subtilis using the optimized dual-promoter P HpaII P amyQ ' in comparison to the original single promoter P HpaII and P amyQ ' [24].In addition, the dualpromoter P HpaII P gsiB produced greater aminopeptidase activity in B. subtilis than the individual promoters P HpaII and P gsiB [25].
In this study, the regulatory elements in the recombinant plasmid were optimized to enhance the expression of Mta in recombinant B. subtilis strains.First, a signal peptide library was constructed, followed by high-throughput screening, yielding the optimal signal peptide.Next, the expression of Mta was improved through the optimization of the different types of dualpromoters.Finally, the culture medium for the resulting recombinant B. subtilis strain was optimized during the shake flask cultivation.The production of maltotetraose using the crude solution containing the recombinant enzymes was also evaluated.The findings presented in this study establish a highly efficient system for the expression of recombinant Mta, therefore facilitating the scale-up production and application of Mta.

Strains, plasmids and primers
The bacterial strains, plasmids, and primers used in this study are listed in Table 1.The codon-optimized gene encoding Mta (GenBank accession number E02567.1) from Pseudomonas saccharophila was synthesized by Synbio Technologies (Suzhou, China).The PrimeSTAR HS DNA polymerase, In-Fusion HD Cloning Kit, E. coli HST08 Premium Competent Cells, DNA and protein molecular weight markers were purchased from Takara (Dalian, China).Maltotetraose standard (HPLC ≥ 98%) was purchased from Yuanye Bio-Technology Co., Ltd (Shanghai, China).Maltodextrin (DE 7-9) was purchased from Baolingbao Biotech Corporation (Shandong, China).All other chemical reagents used in this study were of analytical grade unless otherwise stated.
Luria-Bertani (LB) medium (10 g/L tryptone, 5 g/L yeast extract, and 10 g/L sodium chloride) was used to cultivate E. coli and B. subtilis strains.LBG medium (LB medium containing 10 g/L glucose) was used in the experiment of the construction of B. subtilis WB800N(amyEΔ1) strain.2×Yeast Extract and Tryptone (YT) medium (16 g/L tryptone, 10 g/L yeast extract, and 5 g/L sodium chloride) was used to culture B. subtilis strains for protein expression.

Construction of B. subtilis WB800N(amyEΔ1) strain
The amyE gene in the B. subtilis WB800N genome was inactivated using a previously reported method [26].In brief, the pDGIEF plasmid was first digested with the restriction enzyme Sac I and then purified.The linearized pDGIEF was transformed into the B. subtilis WB800N competent cells, which were then spread on LBG plates containing 100 µg/mL of spectinomycin and incubated for 16 h at 37 ℃.To confirm the disruption of the amyE gene, the resulting spectinomycin-resistant colonies were subjected to the starch-plate assay as described previously [26].The successful integration of the spectinomycin resistance gene and mazF cassette into the amyE locus via a double-crossover event disrupted the amyE gene, resulting in amylase-deficient transformants and unable to form transparent plaques in the starchplate assay.The spectinomycin-resistant colonies were also streaked on LB plates supplemented with 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG), but no colonies formed due to the mazF expression, which was controlled by an IPTG-inducible P spac promoter, inhibiting the growth of the strains.After that, the spectinomycin-resistant colonies were inoculated into antibiotic-free LBG medium and incubated for 24 h at 37 ℃, and then spread on LB plates supplemented with 1 mM IPTG.This process resulted in the deletion of the mazF cassette and spectinomycin resistance gene, leaving a scar of directlyrepeated (DR) sequence at the amyE locus, which replaces partial amyE sequence and disrupts the amyE open reading frame via a single-crossover event.The colonies formed were selected and tested for spectinomycin susceptibility.The spectinomycin-sensitive colonies were reverified using a starch-plate assay and analyzed using colony PCR with primers P1/P2.

Construction of the signal peptide library for Mta expression
The plasmid pBE-SP1mta, an E. coli-B.subtilis shuttle plasmid which contains the codon-optimized mta gene inserted into the Nde I/EcoR I sites and a chloramphenicol (Cm) resistance gene that replaces the original kanamycin resistance gene in the vector and functions in B. subtilis, was constructed based on the plasmid pBE-S and stored in our laboratory.The plasmid pBE-SP1mta was sequentially digested with restriction enzymes Quick-Cut™ Mlu I and Eco52 I, followed by purification.The linearized pBE-SP1mta without signal peptide sequence was ligated with the SP DNA mixture, which contains DNA fragments encoding 173 different signal peptides (obtained from the B. subtilis Secretory Protein Expression System, Takara) using In-Fusion® HD Cloning Kit, and then transformed into the E. coli HST08 competent cells.The resulting transformants were harvested and resuspended in LB medium, after which the recombinant plasmids were extracted.The recombinant plasmid mixture was transformed into B. subtilis WB800N(amyEΔ1) competent cells according to the method described in Takara's B. subtilis Secretory Protein Expression manual, and spread on LB plates supplemented with 5 µg/mL Cm as a selection marker.

Screening of the signal peptide library in deep-well plates
Following cultivation on LB plates supplemented with Cm, colonies harboring different signal peptides were selected and transferred into 96 deep-well plates containing 0.5 mL of 2×YT medium supplemented with 5 µg/ mL Cm.These cultures were incubated for 12 h at 37℃.Subsequently, 30-µL of the seed cultures were transferred into 48 deep-well plates containing 1.5 mL of 2×YT medium supplemented with 5 µg/mL Cm.The cultures

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were incubated for 3 h at 37℃, followed by 72 h of cultivation at 30℃.The screening of the signal peptide library was performed by measuring the Mta activity in the culture supernatant.Several strains exhibiting high Mta activity were selected for further verification in the shake flasks, and the plasmids were extracted and sequenced to determine the signal peptide sequences.

Optimization of the promoters for Mta expression
All recombinant plasmids containing different dual-promoters were constructed using the In-Fusion® HD Cloning Kit, which enables the ligation of two DNA fragments with 15-bp homologous arm sequences or the ligation of three DNA fragments with 20-bp homologous arm sequences.The DNA fragments of promoters P 43 , P HpaII , and promoter-signal peptide sequence P amyE -SPamyE were synthesized by Synbio Technologies (Suzhou, China).To construct plasmids pBE-du1SP7mta and pBE-du2SP7mta, three DNA fragments including a linearized vector, a constitutive promoter, and a promoter-signal peptide sequence were amplified and ligated.In brief, DNA fragment one, which contained the entire vector sequence except promoter and signal peptide, was amplified from plasmid pBE-SP1mta using primers P3/ P4.DNA fragment two, which contained either promoter P 43 or P HpaII , was amplified using primers P5/P6 or P9/ P10, respectively.DNA fragment three, which contained P amyE -SPamyE, was amplified using primers P7/P8.The three DNA fragments were purified and then ligated, yielding the plasmids pBE-du1SP7mta and pBE-du2SP-7mta.To construct plasmids pBE-du1SP3mta and pBE-du2SP3mta, two DNA fragments including a linearized vector containing the signal peptide yhcR, and a dualpromoter were amplified and ligated.In brief, DNA fragment one, which contained the entire vector sequence except promoter, was amplified from pBE-SP3mta using primers P11/P4.DNA fragment two of the dual-promoter was amplified using plasmids pBE-du1SP7mta or pBE-du2SP7mta as the templates with primers P12/P13 or P14/P13, respectively.The dual-promoter fragments were then ligated to the linearized vector fragment, yielding plasmids pBE-du1SP3mta and pBE-du2SP3mta.All plasmids containing different dual-promoters were verified by colony PCR and DNA sequencing (Beijing Genomics Institution, Beijing, China).For protein expression, the plasmids containing different dual-promoters were transformed into B. subtilis WB800N(amyEΔ1) competent cells using a method previously reported.A single colony was selected and inoculated into 5 mL of 2×YT medium supplemented with 5 µg/mL Cm, and cultured for 12 h at 37℃.Then 1 mL of the seed culture was transferred to a new 50 mL of 2×YT medium containing 5 µg/mL Cm, and incubated for 3 h at 37℃ followed by a 72 h of cultivation at 30℃.The culture supernatant was collected by centrifuging the culture medium at 10,000 rpm for 10 min at 4℃.

Optimization of the culture medium of the recombinant B. subtilis strain
The B. subtilis strain was cultured in 5 mL of 2×YT medium supplemented with 5 µg/mL Cm, and incubated for 12 h at 37℃.Then, 1 mL of the seed culture was transferred to a new 50 mL of fermentation medium, which contained various carbon source and nitrogen source.Four different carbon sources, including 5 g/L of glucose, lactose, sucrose, and glycerol, were selected to determine the optimal carbon source for Mta expression.
The concentrations of the carbon source tested for optimization were 0 g/L, 5 g/L, 10 g/L and 15 g/L, respectively.Five different nitrogen sources, including 25 g/L of soya peptone, fish peptone, beef extract, yeast extract, and corn steep liquor, were selected to determine the optimal nitrogen source for Mta expression.The concentrations of the nitrogen source tested for optimization were 15 g/L, 25 g/L, 35 g/L and 45 g/L, respectively.

Enzymatic activity determination
The Mta activity was determined by measuring the amount of reducing sugar released from the hydrolysis of soluble starch according to previously reported literatures [14,27].In briefly, a 500 µL of 10 g/L of soluble starch was mixed with 450 µL of 10 mM NaH 2 PO 4 -Na 2 HPO 4 buffer solution (pH 7.5) and a 50 µL of appropriately diluted crude enzyme solution.The reaction mixture was incubated for 10 min at 55℃, after which the amount of released reducing sugar was determined using the Dinitrosalicylic acid (DNS) method [28].One unit (U) of Mta activity was defined as the amount of the enzyme that produce 1 µmol of reducing sugar (using glucose as the standard) per min under the specified conditions.

Time-course analysis of maltotetraose production using recombinant mta
For the time-course study, the reaction was performed by incubating a crude enzyme solution (50 U/g of substrate) with maltodextrin (DE = 7 ~ 9) solution at a final concentration of 80 g/L, 120 g/L, and 200 g/L for 24 h at 50℃, while shaking at 200 rpm.At specific time intervals, aliquots of the reaction mixture were taken and boiled for 15 min to terminate the reaction.After that, the samples were centrifuged at 10,000 rpm for 15 min, and the supernatant obtained was filtered using a 0.22 μm membrane filter.The amount of maltotetraose in the filtrate was determined using an Hypersil APS-2 column coupled to an Agilent 1260 system equiped with a refractive index detector (Agilent, Waldbronn, Germany).A solution consisted of acetonitrile and water (70:30 v/v) was used as the mobile phase.The flow rate was set at 0.8 mL/ min.The yield of maltotetraose was calculated based on the absorption peak area of the samples and the maltotetraose standard product.

Statistical analysis
The statistical analysis was performed using Graph-Pad Prism 9 (GraphPad Software, San Diego, USA).For comparisons of multiple groups, One-way ANOVA with Tukey's post hoc test was used.Data are presented as mean ± standard deviation (SD).P < 0.05 is considered to indicate statistically significant differences.

Screening the efficient signal peptide for Mta secretion
The signal peptide is crucial for the expression and secretion of specific proteins in B. subtilis.Recent studies demonstrated that identifying a signal peptide suitable for heterologous proteins expression can be achieved using a strategy of signal peptide library construction followed by high-throughput screening [9,17,18].To enhance the expression and secretion of heterologous Mta in B. subtilis, a signal peptide library comprised of 173 different signal peptides was developed.The WB800N strain, a derivative of B. subtilis 168, is deficient in eight extracellular proteases, which promotes the stability of heterologous proteins during secretion and is frequently used in researches [29][30][31].However, WB800N secretes endogenous amylase, which is encoded by the amyE gene and can catalyze starch hydrolysis, thereby introducing background activity.To avoid this background effect and improve screening sensitivity, the amyE gene was inactivated following the method described in a previous study [26].As shown in Fig. 1, the results showed that there was no clear zone surrounding colony with an amyE knockout on the LB-plate supplemented with starch.The obtained amyE knockout mutant, designated as WB800N(amyEΔ1), was used as the expression host in subsequent experiments for signal peptide screening.Using In-Fusion cloning technology, a collection of DNA fragments encoding 173 different signal peptides was substituted for the original SPaprE and in-frame fused to the mta gene, which was expressed under the control of constitutive P aprE promoter in the vector.The resulting plasmids were then transformed into WB800N(amyEΔ1), and the colonies obtained were grown in 96-deep well plates with the WB1M strain as a control for screening.The extracellular Mta activity of 148 strains was found to be higher than that of the control strain WB1M.Five strains, designated as WB2M-6 M, exhibiting higher Mta activity than other strains, were selected for further validation in shake flasks to further confirm the effect of the signal peptides on the secretory expression of Mta.As shown in Fig. 2, after 72 h cultivation in shake flasks, the extracellular Mta activities of strains WB2M-6 M were 52.1, 75.4,63.8, 63.9, and 69.4 U/mL, respectively, all significantly greater than that of strain WB1M.DNA sequencing of the recombinant plasmids from these five strains revealed that the signal peptides present in WB2M-6 M were SPywfM, SPyhcR, SPabnA, SPmreC, and SPyqgA.The strain WB3M, which contained SPyhcR, exhibited the highest Mta activity, which was 3.3-fold higher than the control strain WB1M carrying SPaprE.While there was no significant statistical difference between WB3M and WB6M, WB3M consistently exhibited the highest Mta activity in both the shake flask cultivation and high-throughput screening.Therefore, due to its consistently good performance, the SPyhcR in WB3M was selected for the subsequent experiments.
Signal peptide optimization has been proven to be an effective method to enhance the secretion of recombinant proteins in B. subtilis.However, predicting the most suitable signal peptide for a specific target protein remains challenging.While the properties of signal peptides, such as the number of positive charges in the N-domain and the hydrophobicity of the H-domain, have been discussed in the literatures to assist in signal peptide selection [32,33], it has been suggested that developing a signal peptide library followed by screening is a more practical approach [17,34].This method has previously been shown to identify the suitable signal peptides for a range of proteins [17][18][19].In this study, through deepwell screening and shake flask verification, it was found that five signal peptides exhibited the highest extracellular Mta enzyme activity.Among these five signal peptides leading to higher Mta secretion levels, not every one has more positively charged N-domain and more hydrophobic amino acids in the H-domain compared to SPaprE (Table 2).Therefore, while analyzing signal peptide features can be informative, large-scale library screening is the preferred method to identify the optimal signal peptide for a specific heterologous protein.

Optimization of promoters for Mta expression
Enhancing the expression of heterologous proteins in B. subtilis can be achieved by optimizing the promoter.The strong constitutive promoter P amyE has been shown to effectively produce several heterologous proteins in B. subtilis [21,35].Inserting an additional promoter upstream of P amyE , generating a dual-promoter, can further improve the expression level of recombinant proteins in B. subtilis [21].In this study, we inserted strong constitutive promoters P 43 and P HpaII upstream of the promoter P amyE , respectively, to generate different dualpromoters.Moreover, previous study has suggested that using a promoter and signal peptide from the same gene may result in high-level expression of the target protein [24].Therefore, the signal peptide SPamyE was also employed in combination with the P amyE promoter.
The recombinant plasmids inserted with different dual-promoters combined with signal peptides were constructed (Fig. 3), and then transformed into B. subtilis WB800N(amyEΔ1) for Mta expression.As shown in The charge of the N-terminal region is calculated by defining amino acid D and E as -1, R and K as + 1, and any other amino acid as 0 The hydrophobic amino acids in the H-domain of the signal peptide include G, A, V, L, I, M, F, W, and P, while the other amino acids are hydrophilic Fig. 4, the strains WB7M, WB9M and WB10M exhibited significantly higher extracellular Mta activities (88.5 U/ mL, 89.7 U/mL and 100.3 U/mL, respectively) than strain WB3M after 72 h of cultivation in shake flasks.These strains contained different dual-promoters in place of the original P aprE promoter in the expression vector.Notably, WB10M, which carried the plasmid pBE-du2SP3mta inserted with the dual-promoter P HpaII P amyE and signal peptide SPyhcR, exhibited the highest Mta activity, which was 1.3-fold higher than WB3M which contained the original promoter P aprE and the screened signal peptide SPyhcR.
The initiation of gene transcription is dependent on the recognition of the promoter by the σ-factor of RNA polymerase.Dual-promoters possess multiple σ-factor binding sites, which could lead to enhanced transcriptional activity in comparison to single promoters.In addition, the expression of heterologous proteins under the control of dual-promoters may involve cooperation between the individual promoters.Previous study has found that a dual-promoter arrangement with the σB-recognized promoter upstream of the σA-recognized promoter can result in effective promoter synergism [36,37].The P amyE promoter is a σA-recognized promoter, while P 43 and P HpaII promoter have been reported to be recognized by both σA and σB [36].In our study, the strains WB9M and WB10M, which contained combinations of P 43 P amyE -SPyhcR and P HpaII P amyE -SPyhcR respectively, exhibited enhanced Mta production compared to WB3M, which contained combination P aprE -SPyhcR.This result could be attributed to an increased number of RNA polymerase binding sites and a good synergistic impact of the two individual single promoter in the dual-promoters.Further investigation is needed to clarify the molecular mechanism underlying the synergistic effect of the dualpromoters.Additionally, previous studies have shown that using tandem promoters that composed of more than two individual promoters can further increase the expression level of heterologous proteins [36,38].Therefore, it is worthwhile to explore the effect of inserting

Optimization of the fermentation conditions for mta production
A cost-effective culture medium is crucial for the scaleup enzyme production for industrial application [39,40].To increase the expression level of recombinant Mta while reducing production costs, the culture medium for Mta production from the strain WB10M was optimized.Various carbon sources and nitrogen sources were selected for evaluation.Carbon sources are crucial for both substances production and cell growth.Four different carbon sources, including glucose, lactose, sucrose and glycerol, were investigated in shake flask cultivation to explore their impact on Mta synthesis.The results revealed that when glucose was employed as the carbon source, the strain WB10M exhibited the highest extracellular Mta activity, reaching 197.3 U/mL (as shown in Fig. 5A).Then, the effect of glucose on Mta expression was investigated in detail by varying its concentration within the range of 0-15 g/L.The results showed that the optimal glucose concentration for Mta expression was 5 g/L (Fig. 5B).Subsequently, five different nitrogen sources, including soya peptone, fish peptone, beef extract, yeast extract and corn steep liquor, were employed to investigated their effect on Mta expression in shake flask cultivation.As illustrated in Fig. 5C, among the nitrogen sources tested, soya peptone yielded the highest Mta expression with an extracellular activity of 200.1 U/mL, followed by yeast extract and corn steep liquor.Then, a detailed investigation was conducted by varying the concentration of soya peptone within the range of 15-45 g/L.The results indicated that the highest Mta activity was detected when 35 g/L of soya peptone was used.Therefore, after optimizing the fermentation medium, the recombinant strain WB10M produced the highest yield of recombinant Mta, with an extracellular activity of 288.9 U/mL in shake flask cultivation with 5 g/L of glucose and 35 g/L of soya peptone, which was 2.9-fold enhancment compared to the yield from the non-optimized culture medium.
The production of Mta has been explored using both wild-type and engineered strains.However, wild-type strains have typically produced insufficient amount of Mta.For example, Pseudomonas sp.IMD353 produced 29 U/mL of extracellular Mta activity after 69 h of shake flask cultivation [5].Pseudomonas stutzeri AS22 produced 6.8 U/mL of Mta [6].The limited yield and unclear safety status of wild-type strains have hampered their application in the industrial production of Mta.Efforts have been made to develop recombinant expression systems for Mta production using heterologous hosts.When Mta was produced in E. coli, a significant proportion of recombinant Mta misfolded and was found in inclusion bodies [7].Research on the expression and secretion of recombinant Mta in food-grade microorganism is promising for its industrial application.A Bacillus licheniformis strain and a xylose-inducible promoter were used to express Mta, resulting in an extracellular activity of Mta reached 168.2 U/mL in shake flask cultivation [41].A B. subtilis WB600 strain was employed to express the Mta, achieving an extracellular Mta activity of 230 U/mL in the shake flasks using TB medium [14].A B. subtilis WS11 strain was used to express the Mta, reaching an extracellular Mta activity of 236 U/mL using an optimized culture medium in shake flasks [15].In our study, through the optimization of the regulatory elements and the composition and concentration of the culture medium, an extracellular Mta activity of 288.9 U/mL was reached in shake flask cultivation.This not only demonstrates efficient expression and secretion of Mta in B. subtilis with cost savings and potential economic benefits but also validates Mta production using a food-grade strain.Further scale-up fermentation of strain WB10M in a fermenter would be carried out in the future to evaluate its industrial application potential.

Production of maltotetraose using recombinant Mta
The time-course profile of maltotetraose production using a crude enzyme solution containing recombinant Mta from strain WB10M was evaluated at varying  (a, b, c, d and e) indicate statistically significant group differences (P < 0.05) maltodextrin concentrations.The biotransformation reaction was carried out for 24 h at 50℃, and the reaction product was analyzed by HPLC.The maximum maltotetraose yields of 66.8%, 66.5%, and 70.3% were detected at maltodextrin concentrations of 80 g/L, 120 g/L and 200 g/L, respectively (Fig. 6).The results indicated that a high maltotetraose yield can be achieved using a crude enzyme solution containing Mta in the presence of a high maltodextrin concentration.The rate of maltotetraose yield was high during the initial 6 h of the biotransformation reaction but then decreased as the reaction progressed.This phenomenon may be explained by a decrease in Mta stability in the reaction mixture with high viscosity.For economical industrial maltotetraose production, high substrate concentrations are necessary.In our study, the highest maltotetraose yield obtained with crude enzyme solution containing Mta produced by strain WB10M was 70.3%, which was comparable to the yields from other strains.For example, the Mta from Pseudomonas saccharophila STB07 and its variants produced in B. subtilis WB600 had the highest conversion rate of 73.1% [14].Similarly, Mta from Pseudomonas saccharophila produced in Bacillus licheniformis reached the maximum maltotetraose yield of 72.4% [41].Therefore, our findings indicated that producing maltotetraose using a crude enzyme solution of strain WB10M in the presence of high substrate concentration can simplify the maltotetraose production and reduce costs, which would be favorable to its commercial application.
In this study, a high-level extracellular production of the codon-optimized mta gene from Pseudomonas saccharophila in Bacillus subtilis was achieved by optimizing the regulatory elements and culture medium.After screening various promoter and signal peptide combinations, the dual-promoter P HpaII P amyE with signal peptide SPyhcR was proven to be the most effective combination for Mta production, which resulted in an extracellular Mta activity of 100.3 U/mL.After further optimizing the culture medium, the strain WB10M harboring the optimized regulatory elements produced a highest Mta Fig. 6 Time-course analysis of maltotetraose production using crude enzyme solution containing recombinant Mta from strain WB10M.Data represent the mean ± SD from three independent experiments activity of U/mL in the culture medium.Moreover, the crude enzyme solution of the engineered B. subtilis strain WB10M demonstrated a high yield of maltotetraose under high substrate concentrations, resulting in a highest yield of 70.3% with 200 g/L of maltodextrin as the substrate.These findings demonstrate the efficient expression and secretion of Mta in B. subtilis, providing an expression system for scale-up production and application of Mta.

Fig. 1
Fig. 1 Amylase activity detection.(A) Both B. subtilis WB800N and WB800N(amyEΔ1) were grown on LB plate containing 10 g/L starch.Then, (B) the plate was stained with iodine

Fig. 2
Fig. 2 Secretion of Mta by recombinant B. subtilis strains harboring various signal peptides in shake flask cultivation.Extracellular Mta activity was determined using soluble starch as the substrate for 10 min reaction at 55℃.Data are the mean ± SD from three independent experiments and were analyzed using One-way ANOVA with Tukey's post hoc test.Different letters (a, b, c and d) indicate statistically significant group differences (P < 0.05)

Fig. 3
Fig. 3 Schematic diagram of the recombinant plasmids.The promoters, signal peptides, and mta gene are represented by white, black, and gray arrows, respectively

Fig. 4
Fig. 4 Production of Mta by recombinant B. subtilis strains containing various dual-promoters and signal peptides in the shake flask cultivation.The Mta activity was measured by using soluble starch as the substrate for 10 min reaction at 55℃.Data are the mean ± SD from three independent experiments and were analyzed using One-way ANOVA with Tukey's post hoc test.Different letters (a, b and c) indicate statistically significant group differences (P < 0.05)

Fig. 5
Fig. 5 Optimization of carbon sources and nitrogen sources in the culture medium for Mta production by strain WB10M in the shake flasks.(A) Effects of different types of carbon sources on Mta production.1-4: glucose, lactose, sucrose and glycerol.(B) Effect of varying glucose concentrations on Mta production.(C) Effects of different types of nitrogen sources on Mta production.1-5: soya peptone, fish peptone, beef extract, yeast extract and corn steep liquor.(D) Effect of varying soya peptone concentrations on Mta production.Data are the mean ± SD from three independent experiments and were analyzed using One-way ANOVA with Tukey's post hoc test.Different letters(a, b, c, d and e) indicate statistically significant group differences (P < 0.05)

Table 1
Strains, plasmids and primers used in this study

Table 2
Characteristics of the selected best-performing signal peptides for Mta secretion