Extracellular Overexpression of a Neutral Pullulanase in Bacillus subtilis through Multiple Copy Genome Integration and Atypical Secretion Pathway Enhancement.

Neutral pullulanases, having a good application prospect in trehalose production, showed a limited expression level. In order to address this issue, two approaches were utilized to enhance the yield of a new neutral pullulanase variant (PulA3E) in B. subtilis. One involved using multiple copies of genome integration to increase its expression level and fermentation stability. The other focused on enhancing the PulA-type atypical secretion pathway to further improve the secretory expression of PulA3E. Several strains with different numbers of genome integrations, ranging from one to four copies, were constructed. The four-copy genome integration strain PD showed the highest extracellular pullulanase activity. Additionally, the integration sites ytxE, ytrF, and trpP were selected based on their ability to enhance the PulA-type atypical secretion pathway. Furthermore, overexpressing the predicated regulatory genes comEA and yvbW of the PulA-type atypical secretion pathway in PD further improved its extracellular expression. Three-liter fermenter scale-up production of PD and PD-ARY yielded extracellular pullulanase activity of 1767.1 U/mL at 54 h and 2465.1 U/mL at 78 h, respectively. Finally, supplementing PulA3E with 40 U/g maltodextrin in the multi-enzyme catalyzed system resulted in the highest trehalose production of 166 g/L and the substrate conversion rate of 83%, indicating its potential for industrial application.

N-Terminal Sequences of Signal Peptides Assuming Critical Roles in Expression of Heterologous Proteins in Bacillus subtilis.

The N-terminal sequences of proteins and their corresponding encoding sequences may play crucial roles in the heterologous expression. In this study, the secretory expression of alkaline pectin lyase APL in B. subtilis was investigated to explore the effects of the N-terminal 5-7 amino acid sequences of different signal peptides on the protein expression and secretion. It was identified for the first time that the first five amino acid sequences of the N-terminal of the signal peptide (SP-LipA) from Bacillus subtilis lipase A play an important role in promoting the expression of APL. Furthermore, it was revealed that SP-LipA resulted in higher secretory expression compared to other signal peptides in this study primarily due to its encoding of N-terminal amino acids with relatively higher transcription levels and its efficient secretion capacity. Based on this foundation, the recombinant strain constructed in this work achieved a new record for the highest extracellular yields of APL in B. subtilis, reaching 12,295 U/mL, which was 1.9-times higher than that expressed in the recombinant Escherichia coli strain previously reported. The novel theories uncovered in this study are expected to play significant roles in enhancing the expression of foreign proteins both inside and outside of cells.

Enhancing Poly-γ-glutamic Acid Production in Bacillus tequilensis BL01 through a Multienzyme Assembly Strategy and Expression Features of Glutamate Synthesis from Corynebacterium glutamicum.

The enhancement of intracellular glutamate synthesis in glutamate-independent poly-γ-glutamic acid (γ-PGA)-producing strains is an essential strategy for improving γ-PGA production. Bacillus tequilensis BL01ΔpgdSΔggtΔsucAΔgudB:P43-ppc-pyk-gdhA for the efficient synthesis of γ-PGA was constructed through expression of glutamate synthesis features of Corynebacterium glutamicum, which increased the titer of γ-PGA by 2.18-fold (3.24 ± 0.22 g/L) compared to that of B. tequilensis BL01ΔpgdSΔggtΔsucAΔgudB (1.02 ± 0.11 g/L). To further improve the titer of γ-PGA and decrease the production of byproducts, three enzymes (Ppc, Pyk, and AceE) were assembled to a complex using SpyTag/Catcher pairs. The results showed that the γ-PGA titer of the assembled strain was 31.31% higher than that of the unassembled strain. To further reduce the production cost, 25.73 ± 0.69 g/L γ-PGA with a productivity of 0.48 g/L/h was obtained from cheap molasses. This work provides new metabolic engineering strategies to improve the production of γ-PGA in B. tequilensis BL01. Furthermore, the engineered strain has great potential for the industrial production of γ-PGA from molasses.

A novel starch-active lytic polysaccharide monooxygenase discovered with bioinformatics screening and its application in textile desizing.

BACKGROUND: Lytic polysaccharide monooxygenases (LPMOs) catalyzing the oxidative cleavage of different types of polysaccharides have potential to be used in various industries. However, AA13 family LPMOs which specifically catalyze starch substrates have relatively less members than AA9 and AA10 families to limit their application range. Amylase has been used in enzymatic desizing treatment of cotton fabric for semicentury which urgently need for new assistant enzymes to improve reaction efficiency and reduce cost so as to promote their application in the textile industry. RESULTS: A total of 380 unannotated new genes which probably encode AA13 family LPMOs were discovered by the Hidden Markov model scanning in this study. Ten of them have been successfully heterologous overexpressed. AlLPMO13 with the highest activity has been purified and determined its optimum pH and temperature as pH 5.0 and 50 °C. It also showed various oxidative activities on different substrates (modified corn starch > amylose > amylopectin > corn starch). The results of enzymatic textile desizing application showed that the best combination of amylase (5 g/L), AlLPMO13 (5 mg/L), and H2O2 (3 g/L) made the desizing level and the capillary effects increased by 3 grades and more than 20%, respectively, compared with the results treated by only amylase. CONCLUSION: The Hidden Markov model constructed basing on 34 AA13 family LPMOs was proved to be a valid bioinformatics tool for discovering novel starch-active LPMOs. The novel enzyme AlLPMO13 has strong development potential in the enzymatic textile industry both concerning on economy and on application effect.

Trehalose Production Using Three Extracellular Enzymes Produced via One-Step Fermentation of an Engineered Bacillus subtilis Strain.

At present, the double-enzyme catalyzed method using maltooligosyltrehalose synthase (MTSase) and maltooligosyltrehalose trehalohydrolase (MTHase) is the mainstream technology for industrial trehalose production. However, MTSase and MTHase are prepared mainly using the heterologous expression in the engineered Escherichia coli strains so far. In this study, we first proved that the addition of 3 U/g neutral pullulanase PulA could enhance the trehalose conversion rate by 2.46 times in the double-enzyme catalyzed system. Then, a CBM68 domain was used to successfully assist the secretory expression of MTSase and MTHase from Arthrobacter ramosus S34 in Bacillus subtilis SCK6. At the basis, an engineered strain B. subtilis PSH02 (amyE::pulA/pHT43-C68-ARS/pMC68-ARH), which co-expressed MTSase, MTHase, and PulA, was constructed. After the 24 h fermentation of B. subtilis PSH02, the optimum ratio of the extracellular multi-enzymes was obtained to make the highest trehalose conversion rate of 80% from 100 g/L maltodextrin. The high passage stability and multi-enzyme preservation stability made B. subtilis PSH02 an excellent industrial production strain. Moreover, trehalose production using these extracellular enzymes produced via the one-step fermentation of B. subtilis PSH02 would greatly simplify the procedure for multi-enzyme preparation and be expected to reduce production costs.

Self-Assembly-Based Nanoprobes for the Simultaneous Detection and Downregulation of HSP90α mRNA to Enhance Photothermal Therapy.

Although photothermal therapy (PTT) has been widely applied for tumor treatment, tumor cells thermotolerance still limits PTT efficiency. Since the overexpressed HSP90α in tumor cells further enhances thermotolerance and protects them from PTT damage, a new nanoprobe that can specifically detect and downregulate HSP90α mRNA was developed to enhance the PTT effect. Based on the HSP90α mRNA sequence, the nanoprobe Au-DNA1/DNA2 can specifically bind to HSP90α mRNA for recovering its fluorescence and further inhibit the synthesis of HSP90α to reduce tumor heat tolerance. Moreover, another nanoprobe, Au-DNA3, can self-assemble with the Au-DNA1 nanoprobe after the detection to form Au aggregations to enhance PTT afterward for better efficiency. Simultaneously, such a design improves tissue penetration and tumor retention, thereby reducing the damage to the surrounding normal tissues. Both in vitro and in vivo experiments showed that the nanoprobes have excellent tumor diagnosis and cancer treatment capabilities, which is of great significance for clinical translational applications.

Porous Organic Polymers Derived from Ferrocene and Tetrahedral Silicon-Centered Monomers for Carbon Dioxide Sorption.

Herein, we present two novel ferrocene-containing porous organic polymers, FPOP-1 and FPOP-2, by the Heck reactions of 1,1'-divinylferrocene with two tetrahedral silicon-centered units, i.e., tetrakis(4-bromophenyl)silane and tetrakis(4'-bromo-[1,1'-biphenyl]-4-yl)silane. The resulting materials possess high thermal stability and moderate porosity with the Brunauer-Emmer-Teller (BET) surface areas of 499 m2 g-1 (FPOP-1) and 354 m2 g-1 (FPOP-2) and total pore volumes of 0.43 cm3 g-1 (FPOP-1) and 0.49 cm3 g-1 (FPOP-2). The porosity is comparable to previously reported ferrocene-containing porous polymers. These materials possess comparable CO2 capacities of 1.16 mmol g-1 (5.10 wt%) at 273 K and 1.0 bar, and 0.54 mmol g-1 (2.38 wt%) at 298 K and 1.0 bar (FPOP-1). The found capacities are comparable to, or higher than many porous polymers having similar or higher surface areas. They have high isosteric heats of up to 32.9 kJ mol-1, proving that the affinity between the polymer network and CO2 is high, which can be explained by the presence of ferrocene units in the porous networks. These results indicate that these materials can be promisingly utilized as candidates for the storage or capture of CO2. More ferrocene-containing porous polymers can be designed and synthesized by combining ferrocene units with various aromatic monomers under this strategy and their applications could be explored.

Regulate the hydrophobic motif to enhance the non-classical secretory expression of Pullulanase PulA in Bacillus subtilis.

Bacillus subtilis has been widely used as a prokaryotic host for the secretory expression of heterologous proteins. In this study, a pullulanase (PulA) from Anoxybacillus sp. LM18-11 was firstly identified to be expressed in Bacillus subtilis 1A751 through non-classical secretion pathway. Results showed that both the N- and C-terminal regions of PulA were essential for its soluble expression. To explore its specific structural basis of secretion in B. subtilis, we revealed a hydrophobic motif A501-H507 which is vital for the secretion of the whole protein of PulA. Through a series of site-specific mutagenesis, the triple-sites mutants R503E/I506E/H507E and R503E/I506Y/H507E showed the highest extracellular activity (160.07 U/mL) and total activity (243.37 U/mL) which was 1.71 times and 1.55 times higher than those of PulA. The highest secretion rate of mutant I506E/H507E was more than 50% which was 34.72% higher comparing with that of PulA. The glutamic acid substitution on these three key surface sites which decreased the surface hydrophobicity of that region was confirmed to be beneficial to improve the secretory expression of PulA. This novel discovery for the secretory expression of PulA in B. subtilis would make a new perspective on regulating a kind of non-classical secretion in B. subtilis.

Multiplex genetic engineering improves endogenous expression of mesophilic α-amylase gene in a wild strain Bacillus amyloliquefaciens 205.

A wild strain Bacillus amyloliquefaciens 205 was screened for its high activity of α-amylase. A mesophilic α-amylase encoding gene amyE-205 was revealed and analyzed by genome sequencing. In order to facilitate plasmid transformation to strain 205, an interspecific plasmid transformation method was improved with 5-13 times higher in transformants than that of electronic transformation. A series of CRISPR genome editing tools have been successfully constructed for gene knockout, transcript repression and activation in 205 genome. At this basis, sporulation related genes spo0A and spoIIAC were knockout and suppressed with CRISPR/Cas9 and CRISPR/dCas9 respectively. The double knockout strain 205spo- was eliminated sporulation with 22.8% increasing of α-amylase activity. The optimal binding site G8 for dCas9-ω has been confirmed in the transcript activation. When amyE-205 was over-expressed with high copy plasmid pUC980-2, its whole upstream sequences containing G8 were also cloned. Whereafter, dCas9-ω was used to activate amyE-205 expression both at genome and plasmid. The final engineered strain 205PG8spo- achieved 784.3% promotion on α-amylase activity than the starting strain 205. The novel genetic tool box containing an efficient interspecific transformation method and functional CRISPR systems, superadded the multiplex regulation strategies used in strain modification would be also applicative in many Bacillus species.

Enhanced Heterologous Production of Glycosyltransferase UGT76G1 by Co-Expression of Endogenous prpD and malK in Escherichia coli and Its Transglycosylation Application in Production of Rebaudioside.

Steviol glycosides (SGs) with zero calories and high-intensity sweetness are the best substitutes of sugar for the human diet. Uridine diphosphate dependent glycosyltransferase (UGT) UGT76G1, as a key enzyme for the biosynthesis of SGs with a low heterologous expression level, hinders its application. In this study, a suitable fusion partner, Smt3, was found to enhance the soluble expression of UGT76G1 by 60%. Additionally, a novel strategy to improve the expression of Smt3-UGT76G1 was performed, which co-expressed endogenous genes prpD and malK in Escherichia coli. Notably, this is the first report of constructing an efficient E. coli expression system by regulating prpD and malK expression, which remarkably improved the expression of Smt3-UGT76G1 by 200% as a consequence. Using the high-expression strain E. coli BL21 (DE3) M/P-3-S32U produced 1.97 g/L of Smt3-UGT76G1 with a yield rate of 61.6 mg/L/h by fed-batch fermentation in a 10 L fermenter. The final yield of rebadioside A (Reb A) and rebadioside M (Reb M) reached 4.8 g/L and 1.8 g/L, respectively, when catalyzed by Smt3-UGT76G1 in the practical UDP-glucose regeneration transformation system in vitro. This study not only carried out low-cost biotransformation of SGs but also provided a novel strategy for improving expression of heterologous proteins in E. coli.

The effects of humidity and ammonia on the chemical composition of secondary aerosols from toluene/NOx photo-oxidation.

The secondary aerosol formation mechanism in the presence of ammonia (NH3), is poorly understood, especially under high relative humidity (RH) conditions. In this study, a total of seven experiments were conducted from toluene/NOx photo-oxidation in the presence/absence of NH3 under dry (~7% RH) and wet (>60% RH) conditions in a ~3 m3 smog chamber. A series of instruments including gas analysers, scanning mobility particle sizer (SMPS), aerosol mass spectrometry (HR-ToF-AMS) etc. were applied to measure the NOx and O3 concentrations, the mass concentration and chemical composition of secondary aerosol. It was found that NH3 could enhance the mass loading of secondary aerosol, especially under wet condition. However, the presence of NH3 or increasing RH did not have a significant influence on SOA yield. The organic aerosol mass spectrum from AMS showed that the most abundant fragment was at m/z = 44, which was mainly from the fragmentation of carboxylic acids. Compared to the absence of NH3, the fraction of fragment at m/z = 44 and O:C was higher in the presence of NH3, regardless of dry or wet conditions. The highest O:C value of 0.71-0.75 was observed in the presence of NH3 under wet condition, suggesting there could be a synergetic effect between the high RH and the presence of NH3, which jointly contributed to the photochemical aging process of SOA. The N:C increased in the presence of NH3 under both dry and wet conditions, which might be attributed to the carboxylates and organic nitrates formed from the reaction between NH3 and carboxylic acids. The results implied that SOA modelling should consider the role of NH3 and water vapour, which might fill the gap of O:C between laboratory studies and field measurements.

High copy number and highly stable Escherichia coli-Bacillus subtilis shuttle plasmids based on pWB980.

BACKGROUND: pWB980 derived from pUB110 is a promising expression vector in Bacillus for its high copy number and high stability. However, the low transformation rate of recombinant plasmids to the wild cells limited the application of it. On the basis of pWB980, constructing an E. coli-B. subtilis shuttle plasmid could facilitate the transformation rate to Bacillus cells. Because the insertion site for E. coli replication origin sequence (ori) is not unique in pWB980, in order to investigate the best insertion site, eight shuttle plasmids (pUC980-1 ~ pUC980-8) containing all possible insertion sites and directions were constructed. RESULTS: The results showed that all the selected insertion sites could be used to construct shuttle plasmid but some sites required a specific direction. And different insertion sites led to different properties of the shuttle plasmids. The best shuttle plasmids pUC980-1 and pUC980-2, which showed copies more than 450 per cell and segregational stabilities up to 98%, were selected for heterologous expressions of an alkaline pectate lyase gene pelN, an alkaline protease spro1 and a pullulanase gene pulA11, respectively. The highest extracellular activities of PelN, Spro1 and PulA11 were up to 5200 U/mL, 21,537 U/mL and 504 U/mL correspondingly after 54 h, 60 h and 48 h fermentation in a 10 L fermentor. Notably, PelN and Spro1 showed remarkably higher yields in Bacillus than previous reports. CONCLUSION: The optimum ori insertion site was the upstream region of BA3-1 in pWB980 which resulted in shuttle plasmids with higher copy numbers and higher stabilities. The novel shuttle plasmids pUC980-1 and pUC980-2 will be promising expression vectors in B. subtilis. Moreover, the ori insertion mechanism revealed in this work could provide theoretical guidance for further studies of pWB980 and constructions of other shuttle plasmids.

High-level extracellular production of an alkaline pectate lyase in E. coli BL21 (DE3) and its application in bioscouring of cotton fabric.

A high heterologous expression of an alkaline pectate lyase (APL) pelNK93I in E. coli was obtained through optimizing the lactose feeding and fed-batch fermentation. The highest soluble APL activity produced by E. coli BL21 (pET22b-pelNK93I) was 10,181 U/mL which is the highest level so far. On this basis, to improve the extracellular yield of APL, optimized glycine feeding was used to achieve elevated extracellular production of pelNK93I. The highest extracellular APL activity produced by E. coli BL21 (pET22b-pelNK93I) was 6357 U/mL which was also relatively higher than that in previous reports. The final productivity of APL was 282.8 U/mL/h in the fermentation of E. coli BL21 (pET22b-pelNK93I) in a 10 L fermenter. Thus the current study has provided a cost-effective method for the over-expression and preparation of alkaline pectate lyase pelNK93I for its industrial applications. Moreover, pelNK93I (4 U/mL) used for bioscouring increased cottonseed husk removal and radial capillary effect of cotton fabric by 37.63% and 47.06%, respectively, making it a promising enzyme in green textile technology.

Ethanol effects on the overexpression of heterologous catalase in Escherichia coli BL21 (DE3).

A novel method involving ethanol-induced increase in the heterologous recombinant protein expression in E. coli cells was commonly used in recent studies. However, the detailed mechanism of this method is still to be revealed. This work used comparative transcriptomic analysis and numerous experiments to uncover the mechanism of ethanol effects on the expression of heterologous catalase in the recombinant strain E. coli BL21 (pET26b-katA). The key regulatory genes malK and prpD were found to have the most significant effects on the expression of heterologous catalase. Thus, the maltose ABC transporter and carbon metabolism from propanoate metabolism to citrate cycle were found to be the main regulatory pathways activated by ethanol to enhance the synthesis of heterologous proteins. Based on these mechanisms, a universally applicable E. coli expression host strain for improving the expression of heterologous proteins might be constructed.

Construction of a plasmid interspecific transfer system in Bacillus species with the counter-selectable marker mazF.

Bacillus sp. strains as attractive hosts for the production of heterologous secretory proteins usually play important roles in bio-industry. However, low transformation efficiency of exogenous plasmids limited the application of Bacillus species. Here, a novel plasmid interspecific transfer system, with high transformation efficiency, high positive rate, and convenient manipulation, has been successfully constructed. A high electrotransformation efficiency strain Bacillus subtilis F-168 containing the counter-selectable marker mazF was used as the plasmid donor strain in this transfer method. A shuttled plasmid pBE980 and its recombinant plasmids pBE980::pulA and pBE980::HSPA were successfully transferred into the recipient Bacillus strains (Bacillus amyloliquefaciens 66, Bacillus licheniformis 124 and Bacillus megaterium 258) by this method. After co-culturing the donor cells (OD600nm = 1.3-1.7) and the recipient cells (OD600nm = 0.5-0.9) for 24 h in 22 °C, more than 1.0 × 105 positive transformants were obtained and a interspecific transformation efficiency of 1.0 × 10-3. It would provide a new approach for genetic manipulation in Bacillus strains and accelerate the research progress of the wild Bacillus strains in bio-industry.

High level extracellular production of a recombinant alkaline catalase in E. coli BL21 under ethanol stress and its application in hydrogen peroxide removal after cotton fabrics bleaching.

The effects of induction parameters, osmolytes and ethanol stress on the productivity of the recombinant alkaline catalase (KatA) in Escherichia coli BL21 (pET26b-KatA) were investigated. The yield of soluble KatA was significantly enhanced by 2% ethanol stress. And a certain amount of Triton X-100 supplementation could markedly improved extracellular ratio of KatA. A total soluble catalase activity of 78,762U/mL with the extracellular ratio of 92.5% was achieved by fed-batch fermentation in a 10L fermentor, which was the highest yield so far. The purified KatA showed high stability at 50°C and pH 6-10. Application of KatA for elimination of H2O2 after cotton fabrics bleaching led to less consumption of water, steam and electric power by 25%, 12% and 16.7% respectively without productivity and quality losing of cotton fabrics. Thus, the recombinant KatA is a promising candidate for industrial production and applications.