Enzymatic treatment to improve permeability and quality of cherry tomatoes for production of dried products.

BACKGROUND: Cherry tomatoes are nutritious and favored by consumers. Processing them into dried cherry tomatoes can prolong their storage life and improve their flavor. The pretreatment of tomato pericarp is crucial for the subsequent processing. However, the traditional physical and chemical treatments of tomato pericarp generally cause nutrient loss and environmental pollution. RESULTS: In this study, a novel enzymatic method for cherry tomatoes was performed using mixed enzymes containing cutinase, cellulase and pectinase. Results showed that the pericarp permeability of cherry tomatoes was effectively improved due to enzymatic treatment. Changes in the microscopic structure and composition of the cuticle were revealed. After treatment with different concentrations of enzymes, cherry tomatoes exhibited higher pericarp permeability and sensory quality to varying degrees. The lycopene content and total polyphenol content significantly increased 2.4- and 1.45-fold, respectively. In addition, the satisfactory effect of the six-time reuse of enzymes on cherry tomatoes could still reach the same level as the initial effect, which effectively reduced the cost of production. CONCLUSIONS: This study revealed for the first time that a mixed enzymatic treatment consisting of cutinase, pectinase and cellulase could effectively degrade the cuticle, enhance the pericarp permeability and improve the quality of cherry tomatoes, with the advantages of being mildly controllable and environmentally friendly, providing a new strategy for the processing of dried cherry tomatoes. © 2023 Society of Chemical Industry.

Conformational Switch of the 250s Loop Enables the Efficient Transglycosylation in GH Family 77.

Amylomaltases (AMs) play important roles in glycogen and maltose metabolism. However, the molecular mechanism is elusive. Here, we investigated the conformational dynamics of the 250s loop and catalytic mechanism of Thermus aquaticus TaAM using path-metadynamics and QM/MM MD simulations. The results demonstrate that the transition of the 250s loop from an open to closed conformation promotes polysaccharide sliding, leading to the ideal positioning of the acid/base. Furthermore, the conformational dynamics can also modulate the selectivity of hydrolysis and transglycosylation. The closed conformation of the 250s loop enables the tight packing of the active site for transglycosylation, reducing the energy penalty and efficiently preventing the penetration of water into the active site. Conversely, the partially closed conformation for hydrolysis results in a loosely packed active site, destabilizing the transition state. These computational findings guide mutation experiments and enable the identification of mutants with an improved disproportionation/hydrolysis ratio. The present mechanism is in line with experimental data, highlighting the critical role of conformational dynamics in regulating the catalytic reactivity of GHs.

Producing 2-O-α-D-glucopyranosyl-L-ascorbic acid by modified cyclodextrin glucosyltransferase and isoamylase.

In this study, site saturation mutagenesis was performed on the - 3 (R44, D86, S90, and D192) and - 6 subsite (Y163, G175, G176, and N189) of Bacillus stearothermophilus NO2 cyclodextrin glucosyltransferase to enhance its specificity for the donor substrate maltodextrin for 2-O-α-D-glucopyranosyl-L-ascorbic acid (AA-2G) preparation. The AA-2G yields produced by the mutants S90D, G176H, and S90D/G176H were 181, 171, and 185 g/L, respectively. Our previous study found that the mutant K228R/M230L also increased the AA-2G yield. Therefore, the mutants S90D, G176H, S90D/G176H, and K228R/M230L were further used to generate combinatorial mutants. Among these mutants, the highest AA-2G yield (217 g/L) was produced by S90D/K228R/M230L with 500 g/L maltodextrin as the glucosyl donor, which was 56 g/L higher than that produced by wild-type CGTase. In addition, AA-2G was prepared by adding isoamylase to hydrolyze α-1,6 glucosidic linkages in maltodextrin that could not be utilized by CGTase to improve the utilization rate of maltodextrin. The addition of isoamylase reduced the concentration of maltodextrin from 500 to 350 g/L, while the AA-2G yield remained high (208 g/L). The preparation of AA-2G by complexing isoamylase with mutant S90D/K228R/M230L reduced the maltodextrin concentration by 150 g/L, while the AA-2G yield increased by 47 g/L than preparation with wild-type CGTase alone, which laid a foundation for the large-scale preparation of AA-2G. KEY POINTS: • Mutants exhibited improved maltodextrin specificity. • Mutant S90D/K228R/M230L produced high yield of AA-2G with maltodextrin as substrate. • AA-2G was first synthesized by a combination of isoamylase and CGTase.

Enhancing 2-O-α-D-glucopyranosyl-L-ascorbic acid synthesis by weakening the acceptor specificity of CGTase toward glucose and maltose.

2-O-α-D-glucopyranosyl-L-ascorbic acid (AA-2G) is a stable derivative of L-ascorbic acid (L-AA), which has been widely used in food and cosmetics industries. Sugar molecules, such as glucose and maltose produced by cyclodextrin glycosyltransferase (CGTase) during AA-2G synthesis may compete with L-AA as the acceptors, resulting in low AA-2G yield. Multiple sequence alignment combined with structural simulation analysis indicated that residues at positions 191 and 255 of CGTase may be responsible for the difference in substrate specificity. To investigate the effect of these two residues on the acceptor preference and the AA-2G yield, five single mutants Bs F191Y, Bs F255Y, Bc Y195F, Pm Y195F and Pm Y260F of three CGTases from Bacillus stearothermophilus NO2 (Bs), Bacillus circulans 251 (Bc) and Paenibacillus macerans (Pm) were designed for AA-2G synthesis. Under optimal conditions, the AA-2G yields of the mutants Bs F191Y and Bs F255Y AA-2G were 34.3% and 7.9% lower than that of Bs CGTase, respectively. The AA-2G yields of mutant Bc Y195F, Pm Y195F and Pm Y260F were 45.8%, 36.9% and 12.6% higher than those of wild-type CGTases, respectively. Kinetic studies revealed that the residues at positions 191 and 255 of the three CGTases were F, which decreased glucose and maltose specificity and increased L-AA specificity. This study not only proposes for the first time that the AA-2G yield can be improved by weakening the acceptor specificity of CGTase toward sugar byproducts, but also provides new insight on the modification of CGTase that catalyze the double-substrate transglycosylation reaction.

Enhanced extracellular α-amylase production in Brevibacillus choshinensis by optimizing extracellular degradation and folding environment.

A strategy for optimizing the extracellular degradation and folding environment of Brevibacillus choshinensis has been used to enhance the extracellular production of recombinant α-amylase. First, a gene (bcp) encoding an extracellular protease and another encoding an extracellular chaperone (prsC) were identified in the genome of B. choshinensis HPD31-SP3. Then, the effect of extracellular protein degradation on recombinant α-amylase production was investigated by establishing a CRISPR/Cas9n system to knock out bcp. The effect of extracellular folding capacity was investigated separately by coexpressing extracellular chaperones genes from different sources (prsA, prsC, prsL, prsQ) in B. choshinensis. The final recombinant strain (BCPPSQ), which coexpressed prsQ in a genetic background lacking bcp, produced an extracellular α-amylase activity of 6940.9 U/ml during shake-flask cultivation. This was 2.1-fold greater than that of the original strain BCWPS (3367.9 U/ml). Cultivation of BCPPSQ in a 3-l fermenter produced an extracellular α-amylase activity of 17925.6 U/ml at 72 h, which was 7.6-fold greater than that of BCWPS (2358.1 U/ml). This strategy demonstrates its great potential in enhancing extracellular α-amylase production in B. choshinensis. What's more, this study provides a strategic reference for improving the extracellular production of other recombinant proteins in B. choshinensis.

Effect of Leu277 on Disproportionation and Hydrolysis Activity in Bacillus stearothermophilus NO2 Cyclodextrin Glucosyltransferase.

The disproportionation activity of cyclodextrin glucosyltransferase (CGTase; EC 2.4.1.19) can be used to convert small molecules into glycosides, thereby enhancing their solubility and stability. However, CGTases also exhibit a competing hydrolysis activity. The +2 subsite of the substrate binding cleft plays an important role in both the disproportionation and hydrolysis activities, but almost all known mutations at this site decrease disproportionation activity. In this study, Leu277 of the CGTase from Bacillus stearothermophilus NO2, located near both the +2 subsite and the catalytic acid/base Glu253, was modified to assess the effect of side chain size at this position on disproportionation and hydrolysis activities. The best mutant, L277M, exhibited a reduced Km for the acceptor substrate maltose (0.48 mM versus 0.945 mM) and an increased kcat/Km (1,175 s-1 mM-1 versus 686.1 s-1 mM-1), compared with those of the wild-type enzyme. The disproportionation-to-hydrolysis ratio of L277M was 2.4-fold greater than that of the wild type. Existing structural data were combined with a multiple-sequence alignment and Gly282 mutations to examine the mechanism behind the effects of the Leu277mutations. The Gly282 mutations were included to aid a molecular dynamics (MD) analysis and the comparison of crystal structures. They reveal that changes to a hydrophobic cluster near Glu253 and the hydrophobicity of the +2 subsite combine to produce the observed effects. IMPORTANCE In this study, mutations that enhance the disproportionation to hydrolysis ratio of a CGTase have been discovered. For example, the disproportionation-to-hydrolysis ratio of the L277M mutant of Bacillus stearothermophilus NO2 CGTase was 2.4-fold greater than that of the wild type. The mechanism behind the effects of these mutations is explained. This paper opens up other avenues for future research into the disproportionation and hydrolysis activities of CGTases. Productive mutations are no longer limited to the acceptor subsite, since mutations that indirectly affect the acceptor subsite also enhance enzymatic activity.

Improved production of cyclodextrin glycosyltransferase from Bacillus stearothermophilus NO2 in Escherichia coli via directed evolution.

Cyclodextrin glycosyltransferases (CGTases) are widely used in starch deep processing, so reducing their cost by improving their production is of significant industrial interest. The CGTase from Bacillus stearothermophilus NO2 possesses excellent catalytic properties but suffers from low production in E. coli. In this study, directed evolution was used to create three point mutants (I631T, I641T and K647E) that were produced in E. coli with shake-flask yields 1.7-, 2.1-, and 2.2-fold higher than that of wild-type, respectively. The wild-type and K647E were then produced in a 3-L fermenter. The CGTase activity of the K647E (1904 U mL-1) was 2.0-fold higher than that of the wild-type. The K647E fermentation supernatant could be used directly to prepare 2-O-α-D-glucopyranosyl-L-ascorbic acid, reducing the costs associated with its production. Structural modeling of the three mutants suggested that hydrophilicity, hydrogen bonding, and negative charge may be responsible for their improved production. Since K647 is conserved in the CGTase family, the corresponding residues in the CGTases from Bacillus circulans 251, Paenibacillus macerans, and Anaerobranca gottschalkii were changed to glutamic acid. Productions of the resulting K647E mutants were 2.0-, 1.5-, and 1.0-fold higher than those of their respective wild-types. Electrostatic protein surface analysis suggested that mutations occurring at low negative surface charge may increase CGTase production.

Recombinant expression, characterization and application of maltotetraohydrolase from Pseudomonas saccharophila.

BACKGROUND: Maltotetraohydrolase, widely used in food and medical fields, possesses the ability to hydrolyze starch to produce maltooligosaccharides with maltotetraose as the main product. It also has the potential usage in delaying bread aging. RESULTS: Pseudomonas saccharophila maltotetraohydrolase was expressed in Bacillus subtilis WS11. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed obvious bands at 57 kDa (maltotetraohydrolase I) and 47 kDa (maltotetraohydrolase II). Both showed similar enzymatic properties, although the catalytic efficiency of maltotetraohydrolase I was 4.93 fold higher than that of maltotetraohydrolase II using soluble starch as substrate. In addition, the maltotetraohydrolase production was further scaled up in a 3-L fermentor, and the highest activity reached 1907 U mL-1 . Then, the recombinant maltotetraohydrolase was used to produce maltotetraose. The maltotetraose yields catalyzed by maltotetraohydrolase I and II reached 73.2% and 69.7%, respectively. Finally, when recombinant maltotetraohydrolase was used in bread-making, texture profile analysis of the bread indicated recombinant maltotetraohydrolase I exhibited a significant anti-aging effect. CONCLUSION: This is the first describing high-efficient expression of P. saccharophila maltotetraohydrolase in the food safety strain B. subtilis, and the yield represented the highest level ever reported. Excellent results were also obtained with respect to the preparation of maltotetraose and delaying bread aging using the recombinant maltotetraohydrolase. The present study will help lay the foundation for the industrial production and application of maltotetraohydrolase. © 2020 Society of Chemical Industry.

Current studies on the enzymatic preparation 2-O-α-d-glucopyranosyl-l-ascorbic acid with cyclodextrin glycosyltransferase.

2-O-α-d-glucopyranosyl-l-ascorbic acid (AA-2G) is one of the most important l-ascorbic acid derivatives because of its resistance to reduction and oxidation and its easy degradation by α-glucosidase to release l-ascorbic acid and glucose. Thus, AA-2G has commercial uses in food, medicines and cosmetics. This article presents a review of recent studies on the enzymatic production of AA-2G using cyclodextrin glycosyltransferase. Reaction mechanisms with different donor substrates are discussed. Protein engineering, physical and biological studies of cyclodextrin glycosyltransferase are introduced from the viewpoint of effective AA-2G production. Future prospects for the production of AA-2G using cyclodextrin glycosyltransferase are reviewed.

Enhanced extracellular expression of Bacillus stearothermophilus α-amylase in Bacillus subtilis through signal peptide optimization, chaperone overexpression and α-amylase mutant selection.

BACKGROUND: Our laboratory has constructed a Bacillus stearothermophilus α-amylase (AmyS) derivative with excellent enzymatic properties. Bacillus subtilis is generally regarded as safe and has excellent protein secretory capability, but heterologous extracellular production level of B. stearothermophilus α-amylase in B. subtilis is very low. RESULTS: In this study, the extracellular production level of B. stearothermophilus α-amylase in B. subtilis was enhanced by signal peptide optimization, chaperone overexpression and α-amylase mutant selection. The α-amylase optimal signal peptide (SPYojL) was obtained by screening 173 B. subtilis signal peptides. Although the extracellular α-amylase activity that was produced by the resulting recombinant strain was 3.5-fold greater than that of the control, significant quantities of inclusion bodies were detected. Overexpressing intracellular molecular chaperones significantly reduced inclusion body formation and further increased α-amylase activity. Error-prone PCR produced an amylase mutant K82E/S405R (AmySA) with enzymatic activity superior to that of AmyS. Expression of the amySA gene with the SPYojL while overexpressing molecular chaperones resulted in a 7.1-fold improvement in α-amylase activity. When the final expression strain (WHS11YSA) was cultivated in a 3-L fermenter for 92 h, the α-amylase activity of the culture supernatant was 9201.1 U mL-1, which is the highest level that has been reported to date. CONCLUSIONS: This is the first report that describes an improvement of B. stearothermophilus α-amylase extracellular production levels in B. subtilis using these strategies, and this represents the highest extracellular production level ever reported for α-amylase from B. stearothermophilus in B. subtilis. This high-level production provides a basis for enhanced industrial production of α-amylase. These extracellular production level improvement approaches are also expected to be valuable in the expression of other enzymes in B. subtilis.

High-efficiency expression of Sulfolobus acidocaldarius maltooligosyl trehalose trehalohydrolase in Escherichia coli through host strain and induction strategy optimization.

Maltooligosyl trehalose trehalohydrolase (MTHase, EC 3.2.1.141) catalyzes the release of trehalose, a novel food ingredient, by splitting the α-1,4-glucosidic linkage adjacent to the α-1,1-glucosidic linkage of maltooligosyl trehalose. However, the high-yield preparation of recombinant MTHase has not yet been reported. In this study, a codon-optimized synthetic gene encoding Sulfolobus acidocaldarius MTHase was expressed in Escherichia coli. In initial expression experiments conducted using pET-24a (+) and E. coli BL21 (DE3), the MTHase activity was 10.4 U/mL and a large amount of the expression product formed inclusion bodies. The familiar strategies, including addition of additives, co-expression with molecular chaperones, and expression with a fusion partner, failed to enhance soluble MTHase expression. Considering the intermolecular disulfide bond of MTHase, expression was investigated using a system comprising plasmid pET-32a (+) and host E. coli Origami (DE3), which is conducive to cytoplasmic disulfide bond formation. The MTHase activity increased to 55.0 U/mL, a 5.3-fold increase. Optimization of the induction conditions in a 3-L fermentor showed that when the lactose was fed at 0.2 g/L/h beginning at an OD600 of 40 and the induction temperature was maintained at 30 °C, the MTHase activity reached a maximum of 204.6 U/mL. This is the first report describing a systematic effort to obtain high-efficiency MTHase production. The high yield obtained using this process provides the basis for the industrial-scale production of trehalose. This report is also expected to be valuable in the production of other enzymes containing disulfide bonds.

Highly efficient production of Clostridium cellulolyticum H10 D-psicose 3-epimerase in Bacillus subtilis and use of these cells to produce D-psicose.

BACKGROUND: D-Psicose 3-epimerase (DPEase) catalyzes the isomerization of D-fructose to the rare sugar D-psicose, which may help prevent obesity, reduce blood sugar and blood fat, and inhibit intra-abdominal fat accumulation. RESULTS: In this study, the DPEase of Clostridium cellulolyticum H10 was expressed in the food-grade host Bacillus subtilis. Optimization of the culture medium during shake-flask experiments yielded a DPEase activity of 314 U/mL. The optimal medium included 20 g/L peptone, 15 g/L corn steep powder, 5 g/L glycerol, and 1 mM Ca2+. Controlling the carbon source concentration was important because elevated concentrations can result in catabolite metabolic suppression (CCR). To avoid CCR and increase DPEase expression, we developed a fed-batch strategy in a 3.6-L fermenter. We altered the ratio of carbon source to nitrogen source (C/N) in the feeding medium and employed a constant feeding rate (6 g/L/h). This strategy improved the DPEase activity to 2246 U/mL (7.8 g/L), which is almost 15 times higher than that observed in the original shake-flask cultures. Finally, we used the DPEase-expressing B. subtilis cells to produce D-psicose from D-fructose, and a 28% conversion yield was achieved with these cells, demonstrating their potential use in D-psicose production. CONCLUSIONS: This is the first report to enhance recombinant DPEase production in B. subtilis using efficient and convenient fermentation strategy, and the DPEase yield is three times higher than the highest yield reported to date. The recombinant B. subtilis cells were further used in the efficient synthesis of D-psicose. This study provides a basis for the industrial production of D-psicose.

Enhanced extracellular pullulanase production in Bacillus subtilis using protease-deficient strains and optimal feeding.

To study the effect of proteases on pullulanase production, six protease-encoding genes (nprB, bpr, mpr, epr, vpr, and wprA) in the genome of Bacillus subtilis strain WS5, which already lacks the protease-encoding genes nprE and aprE, were sequentially disrupted using a CRISPR/Cas9 system. This created strains WS6-WS11, respectively. The strains WS3 (none) and WS4 (ΔnprE) were constructed earlier. After addition of expression plasmid pHYPULd4 into the strains WS3-WS11, the pullulanase production levels of the resulting strains (WS3PUL-WS11PUL, respectively) were investigated in shake-flask cultivations and recombinant strain WS5PUL produced the highest pullulanase activity (148.2 U/mL). Then, the scale-up pullulanase production levels of four recombinant strains WS5PUL, WS9PUL, WS10PUL, and WS11PUL were investigated in the 3-L fermenter cultivations. Strain WS9PUL produced the highest pullulanase activity (2449.6 U/mL) when fed an inorganic nitrogen source. However, the specific activity of the pullulanase obtained in a 3-L fermenter generally decreased as the number of protease deletions increased. Meanwhile, using pullulanase, α-cyclodextrin glucosyltransferase and ß-cyclodextrin glucosyltransferase as reporter proteins, the protein production differences among strains WS3, WS9, and the widely used WB600 were investigated. Finally, the carbon to organic nitrogen source ratio of the feeding solution used in the 3-L fermenter was optimized. Recombinant strain WS9PUL fed with carbon and organic nitrogen sources in a ratio of 4:1 achieved a pullulanase activity of 5951.8 U/mL, the highest activity reported to date.

High-level extracellular protein production in Bacillus subtilis using an optimized dual-promoter expression system.

BACKGROUND: We recently constructed a Bacillus subtilis strain (CCTCC M 2016536) from which we had deleted the srfC, spoIIAC, nprE, aprE and amyE genes. This strain is capable of robust recombinant protein production and amenable to high-cell-density fermentation. Because the promoter is among the factors that influence the production of target proteins, optimization of the initial promoter, PamyQ from Bacillus amyloliquefaciens, should improve protein expression using this strain. This study was undertaken to develop a new, high-level expression system in B. subtilis CCTCC M 2016536. RESULTS: Using the enzyme ß-cyclodextrin glycosyltransferase (ß-CGTase) as a reporter protein and B. subtilis CCTCC M 2016536 as the host, nine plasmids equipped with single promoters were screened using shake-flask cultivation. The plasmid containing the PamyQ' promoter produced the greatest extracellular ß-CGTase activity; 24.1 U/mL. Subsequently, six plasmids equipped with dual promoters were constructed and evaluated using this same method. The plasmid containing the dual promoter PHpaII-PamyQ' produced the highest extracellular ß-CGTase activity (30.5 U/mL) and was relatively glucose repressed. The dual promoter PHpaII-PamyQ' also mediated substantial extracellular pullulanase (90.7 U/mL) and α-CGTase expression (9.5 U/mL) during shake-flask cultivation, demonstrating the general applicability of this system. Finally, the production of ß-CGTase using the dual-promoter PHpaII-PamyQ' system was investigated in a 3-L fermenter. Extracellular expression of ß-CGTase reached 571.2 U/mL (2.5 mg/mL), demonstrating the potential of this system for use in industrial applications. CONCLUSIONS: The dual-promoter PHpaII-PamyQ' system was found to support superior expression of extracellular proteins in B. subtilis CCTCC M 2016536. This system appears generally applicable and is amenable to scale-up.

Enhanced extracellular production of recombinant proteins in Escherichia coli by co-expression with Bacillus cereus phospholipase C.

BACKGROUND: Our laboratory has reported a strategy for improving the extracellular production of recombinant proteins through co-expression with Thermobifida fusca cutinase, which increases membrane permeability via its phospholipid hydrolysis activity. However, the foam generated by the lysophospholipid product makes the fermentation process difficult to control in a fermentor. Phospholipase C (PLC) catalyzes the hydrolysis of phospholipids to produce sn1,2-diacylglycerides and organic phosphate, which do not induce foam formation. Therefore, co-expression with Bacillus cereus PLC was investigated as a method to improve the extracellular production of recombinant proteins. RESULTS: When B. cereus PLC was expressed in Escherichia coli without its signal peptide, 95.3% of the total PLC activity was detected in the culture supernatant. PLC expression enhanced membrane permeability without obvious cell lysis. Then, six test enzymes, three secretory and three cytosolic, were co-expressed with B. cereus PLC. The enhancement of extracellular production correlated strongly with the molecular mass of the test enzyme. Extracellular production of Streptomyces sp. FA1 xylanase (43 kDa), which had the lowest molecular mass among the secretory enzymes, was 4.0-fold that of its individual expression control. Extracellular production of glutamate decarboxylase (51 kDa), which had the lowest molecular mass among the cytosolic enzymes, reached 26.7 U/mL; 88.3% of the total activity produced. This strategy was effectively scaled up using a 3-L fermentor. No obvious foam was generated during this fermentation process. CONCLUSIONS: This is the first study to detail the enhanced extracellular production of recombinant proteins through co-expression with PLC. This new strategy, which is especially appropriate for lower molecular mass proteins, allows large-scale protein production in an easily controlled fermentation process.

[Anchorage of cyclodextrin glycosyltransferase on outer membrane of Saccharomyces cerevisiae to produce 2-O-α-D-glucopyranosyl-L-ascorbic acid].

OBJECTIVE: Displaying cyclodextrin glycosyltransferase ( CGTase) from Bacillus circulans 251 on the cell surface of Saccharomyces cerevisiae to improve 2-O-α-D-glucopyranosyl-L-ascorbic acid (AA-2G) production. METHODS: CGTase encoding gene cgt was inserted into the 3' terminal of Aga2p of vector pYD1 and the obtained recombinant plasmid pYD1-cgt was then transformed into S. cerevisiae EBY100 to produce surface displayed CGTase and culture conditions (culture medium, inductive temperature and concentration of inducer galactose) were optimized. Moreover, resulted CGTase displayed on the yeast cell surface was used for the AA-2G biosynthesis under the optimized condition. RESULTS: CGTase activity on the cell surface of recombinant yeast, S. cerevisiae EBY100-pYD1-cgt, reached 0.5 U/ml in 48 h fermentation using Yeast Peptone Galactose culture medium with 20% galactose as sole carbon source and inducer at 25 degrees C. The displayed CGTase exhibited better thermostability and pH stability than that of free CGTase. The concentration of AA-2G produced by the surface displayed CGTase was 37% higher than that produced by free CGTase at its optimal transformation conditions of 30 degrees C and pH4. 5. CONCLUSION: The cell surface display system based on α-agglutinin is an effective system for displaying CGTase. During AA-2G production by surface displayed CGTase, the by-product glucose might be consumed by yeast cell and thus facilitated AA-2G production. The whole cell EBY100-pYD1-cgt will have better prospects for applications.

Genome-Wide CRISPRi Screening of Key Genes for Recombinant Protein Expression in Bacillus Subtilis.

Bacillus subtilis is an industrially important microorganism that is often used as a microbial cell factory for the production of recombinant proteins due to its food safety, rapid growth, and powerful secretory capacity. However, the lack of data on functional genes related to recombinant protein production has hindered the further development of B. subtilis cell factories. Here, a strategy combining genome-wide CRISPRi screening and targeted CRISPRa activation to enhance recombinant protein expression is proposed. First, a CRISPRi library covering a total of 4225 coding genes (99.7%) in the B. subtilis genome and built the corresponding high-throughput screening methods is constructed. Twelve key genes for recombinant protein expression are identified, including targets without relevant functional annotations. Meanwhile, the transcription of recombinant protein genes by CRISPRa is up-regulated. These screened or selected genes can be easily applied to metabolic engineering by constructing sgRNA arrays. The relationship between differential pathways and recombinant protein expression in engineered strains by transcriptome analysis is also revealed. High-density fermentation and generalisability validation results prove the reliability of the strategy. This method can be extended to other industrial hosts to support functional gene annotation and the design of novel cell factories.

Extracellular location of Thermobifida fusca cutinase expressed in Escherichia coli BL21(DE3) without mediation of a signal peptide.

Cutinase is a multifunctional esterase with potential industrial applications. In the present study, a truncated version of the extracellular Thermobifida fusca cutinase without a signal peptide (referred to as cutinase(NS)) was heterologously expressed in Escherichia coli BL21(DE3). The results showed that the majority of the cutinase activity was located in the culture medium. In a 3-liter fermentor, the cutinase activity in the culture medium reached 1,063.5 U/ml (2,380.8 mg/liter), and the productivity was 40.9 U/ml/h. Biochemical characterization of the purified cutinase(NS) showed that it has enzymatic properties similar to those of the wild-type enzyme. In addition, E. coli cells producing inactive cutinase(NS)S130A were constructed, and it was found that the majority of the inactive enzyme was located in the cytoplasm. Furthermore, T. fusca cutinase was confirmed to have hydrolytic activity toward phospholipids, an important component of the cell membrane. Compared to the cells expressing the inactive cutinase(NS)S130A, the cells expressing cutinase(NS) showed increased membrane permeability and irregular morphology. Based on these results, a hypothesis of "cell leakage induced by the limited phospholipid hydrolysis of cutinase(NS)" was proposed to explain the underlying mechanism for the extracellular release of cutinase(NS).

A novel strategy for enhancing extracellular secretion of recombinant proteins in Escherichia coli.

Secretion of cytoplasmic expressed proteins into culture medium has significant commercial advantages in large-scale production of proteins. Our previous study demonstrated that the membrane permeability of Escherichia coli could be significantly improved when Thermobifida fusca cutinase, without a signal peptide, was expressed in cytoplasm. This study investigated the extracellular production of other recombinant proteins, including both secretory and cytosolic proteins, with co-expression of cutinase. When the secretory enzymes, xylanase and α-amylase, were co-expressed with cutinase, the culture period was shortened by half, and the productivity was 7.9 and 2.0-fold to that of their individual control without co-expression, respectively. When the normally cytosolic proteins, xylose isomerase and trehalose synthase, were co-expressed with cutinase, more than half of the target proteins were "secreted" into the culture medium. Moreover, by using ß-galactosidase to detect membrane leakage, the improved secretion of the above model proteins was confirmed not to be due to cell lysis. The study provides a novel strategy for enhancing extracellular secretion of recombinant proteins in E. coli.

Heterologous expression and biochemical characterization of an endo-ß-1,4-glucanase from Thermobifida fusca.

The endoglucanase Cel5A from Thermobifida fusca was cloned and expressed in Escherichia coli BL21(DE3). The carboxymethyl cellulase (CMCase) activity in shake flasks and 3-L fermentation scale reached 46.8 and 656.6 IU/mL, respectively. The CMCase activity in 3-L fermentation scale represented the highest yield of T. fusca Cel5A reported so far. Recombinant Cel5A was purified and characterized in detail. The optimum temperature of recombinant enzyme was 80 °C, and the half-life of the enzyme was 132 H at 50 °C and 65 H at 60 °C. The activity of recombinant Cel5A was retained more than 90% over the range of pH 5.0-10.0 with maximal activity at pH 5.5. Using carboxymethyl cellulose as the substrate, the Km and Vmax values were 5.1 mg/mL and 48.7 IU/mg, respectively. The enzyme showed superstability in surfactants and was retained above 90% activity after treatment with sodium dodecyl sulfate, linear alkyl benzene sulfonate, fatty alcohol polyoxyethylene (9) ether, and polyoxyethylene (10) nonyl phenyl ether at 25 °C for 1 H, indicating that the enzyme could be a valuable component in detergents. The potential mechanism of this stability was investigated by analysis of the electrostatic potential of the surface of the enzyme.