Secretory expression of amylosucrase in Bacillus licheniformis through twin-arginine translocation pathway.

Amylosucrase (EC 2.4.1.4) is a versatile enzyme with significant potential in biotechnology and food production. To facilitate its efficient preparation, a novel expression strategy was implemented in Bacillus licheniformis for the secretory expression of Neisseria polysaccharea amylosucrase (NpAS). The host strain B. licheniformis CBBD302 underwent genetic modification through the deletion of sacB, a gene responsible for encoding levansucrase that synthesizes extracellular levan from sucrose, resulting in a levan-deficient strain, B. licheniformis CBBD302B. Neisseria polysaccharea amylosucrase was successfully expressed in B. licheniformis CBBD302B using the highly efficient Sec-type signal peptide SamyL, but its extracellular translocation was unsuccessful. Consequently, the expression of NpAS via the twin-arginine translocation (TAT) pathway was investigated using the signal peptide SglmU. The study revealed that NpAS could be effectively translocated extracellularly through the TAT pathway, with the signal peptide SglmU facilitating the process. Remarkably, 62.81% of the total expressed activity was detected in the medium. This study marks the first successful secretory expression of NpAS in Bacillus species host cells, establishing a foundation for its future efficient production. ONE-SENTENCE SUMMARY: Amylosucrase was secreted in Bacillus licheniformis via the twin-arginine translocation pathway.

Phenotypic Array for Identification and Screening of Antifungals against Aspergillus Isolates from Respiratory Infections in KwaZulu Natal, South Africa.

The rapid emergence of invasive fungal infections correlates with the increasing population of immunocompromised individuals, with many cases leading to death. The progressive increase in the incidence of Aspergillus isolates is even more severe due to the clinical challenges in treating invasive infections in immunocompromised patients with respiratory conditions. Rapid detection and diagnosis are needed to reduce mortality in individuals with invasive aspergillosis-related infections and thus efficient identification impacts clinical success. The phenotypic array method was compared to conventional morphology and molecular identification on thirty-six Aspergillus species isolated from patients with respiratory infections at the Inkosi Albert Luthuli Hospital in Kwa-Zulu Natal. In addition, an antimicrobial array was also carried out to screen for possible novel antimicrobial compounds for treatment. Although traditional morphological techniques are useful, genetic identification was the most reliable, assigning 26 to Aspergillus fumigatus species, 8 Aspergillus niger, and 2 Aspergillus flavus including cryptic species of A. niger, A. tubingensis and A. welwitschiae. The phenotypic array technique was only able to identify isolates up to the genus level due to a lack of adequate reference clinical species in the database. However, this technique proved crucial in assessing a wide range of possible antimicrobial options after these isolates exhibited some resistance to azoles. Antifungal profiles of the thirty-six isolates on the routine azole voriconazole showed a resistance of 6%, with 61% having moderate susceptibility. All isolates resistant to the salvage therapy drug, posaconazole pose a serious concern. Significantly, A. niger was the only species resistant (25%) to voriconazole and has recently been reported as the species isolated from patients with COVID-19-associated pulmonary aspergillosis (CAPA). Phenotypic microarray showed that 83% of the isolates were susceptible to the 24 new compounds and novel compounds were identified for potentially effective combination treatment of fungal infections. This study also reports the first TR34/98 mutation in Aspergillus clinical isolates which is located in the cyp51A gene.

Draft Genome Sequence of Pectobacterium brasiliense Isolated from Potato in South Africa.

This study reports a draft genome of a phytopathogenic bacterium, Pectobacterium brasiliense, isolated from potato in South Africa. The total reported length of the genome is 4,897,858 bp, contained in 172 contigs with 4,378 genes. The GC content of the genome is 51.6%.

Pullulanase with high temperature and low pH optima improved starch saccharification efficiency.

Pullulanase, a starch debranching enzyme, is required for the preparation of high glucose/maltose syrup from starch. In order to expand its narrow reaction conditions and improve its application value, Bacillus naganoensis pullulanase (PulA) was mutated by site-directed mutagenesis and the biochemical characteristics of the mutants were studied. The mutant PulA-N3 with mutations at asparagine 467, 492 and 709 residues was obtained. It displayed the activity maximum at 60 °C and pH 4.5 and exceeded 90% activities between 45 and 60 °C and from pH 4.0 to pH 5.5, which was improved greatly compared with wild-type PulA. Its thermostability and acidic pH stability were also remarkably improved. Its catalytic rate (kcat/Vmax) was 2.76 times that of PulA. In the preparation of high glucose syrup, the DX (glucose content, %) values of glucose mediated by PulA-N3 and glucoamylase reached 96.08%, which were 0.82% higher than that of PulA. In conclusion, a new pullulanase mutant PulA-N3 was successfully developed, which has high debranching activity in a wide range of temperature and pH, thereby paving the way for highly efficient starch saccharification.

High-efficiency chromosomal integrative amplification strategy for overexpressing α-amylase in Bacillus licheniformis.

Bacillus licheniformis is a well-known platform strain for production of industrial enzymes. However, the development of genetically stable recombinant B. licheniformis for high-yield enzyme production is still laborious. Here, a pair of plasmids, pUB-MazF and pUB'-EX1, were firstly constructed. pUB-MazF is a thermosensitive, self-replicable plasmid. It was able to efficiently cure from the host cell through induced expression of an endoribonuclease MazF, which is lethal to the host cell. pUB'-EX1 is a nonreplicative and integrative plasmid. Its replication was dependent on the thermosensitive replicase produced by pUB-MazF. Transformation of pUB'-EX1 into the B. licheniformis BL-UBM harboring pUB-MazF resulted in both plasmids coexisting in the host cell. At an elevated temperature, and in the presence of isopropyl-1-thio-ß-d-galactopyranoside and kanamycin, curing of the pUB-MazF and multiple-copy integration of pUB'-EX1 occurred, simultaneously. Through this procedure, genetically stable recombinants integrated multiple copies of amyS, from Geobacillus stearothermophilus ATCC 31195 were facilely obtained. The genetic stability of the recombinants was verified by repeated subculturing and shaking flask fermentations. The production of α-amylase by recombinant BLiS-002, harboring five copies of amyS, in a 50-l bioreactor reached 50 753 U/ml after 72 hr fermentation. This strategy therefore has potential for production of other enzymes in B. licheniformis and for genetic modification of other Bacillus species.

A novel aminopeptidase with potential debittering properties in casein and soybean protein hydrolysates.

A new aminopeptidase (An-APa) was identified and biochemically characterized from Aspergillus niger CICIM F0215. It had maximal activity at 40 °C and pH 7.0 and exhibited a broad substrate specificity both on hydrophilic and hydrophobic amino acid residues at N-terminals. With An-APa hydrolysis for 1 h, the casein-pepsin and soybean protein isolates (SPI)-pepsin hydrolysates released both hydrophilic and hydrophobic amino acids and the hydrophobic amino acids having Q values (degree of hydrophobicity) greater than 1500 cal/mol were remarkably released. Leu, Ile, Phe, Tyr, Trp, Pro, Val and Lys in the casein hydrolysate after treatment with An-APa increased 18.61, 0.84, 11.35, 13.18, 3.34, 6.30, 7.46, and 8.19 mg/100 mL, respectively, and 19.72, 1.47, 18.37, 11.72, 4.61, 4.10, 8.13, and 5.85 mg/100 mL, respectively, in the SPI hydrolysate. Both accounted for 65.0% and 64.4% of total released free amino acids from casein and SPI hydrolysates, respectively. This indicated that An-APa could be potentially applicable in debittering protein hydrolysates.

Biochemical characterization of two new Aspergillus niger aspartic proteases.

Two new aspartic proteases, PepAb and PepAc (encoded by pepAb and pepAc), were heterologously expressed and biochemically characterized from Aspergillus niger F0215. They possessed a typical structure of pepsin-type aspartic protease with the conserved active residues D (84, 115), Y (131, 168) and D (281, 326), while their identity in amino acid sequences was only 19.0%. PepAb had maximum activity at pH 2.5 and 50 °C and PepAc at 3.0 and 50 °C. The specific activities of PepAb and PepAc toward casein were 1368.1 and 2081.4 U/mg, respectively. Their activities were significantly promoted by Cu2+ and Mn2+ and completely inhibited by pepstatin. PepAb exhibited higher catalytic efficiency (k cat/K m) toward soy protein isolates than casein, while PepAc showed higher catalytic efficiency toward casein. The hydrolysis capacities of PepAb and PepAc on soy protein isolates were slightly lower than that of previously identified A. niger aspartic protease, PepA (aspergillopepsin I), while the resultant peptide profiles were remarkably different for all three proteases.

Twin-arginine signal peptide of Bacillus licheniformis GlmU efficiently mediated secretory expression of protein glutaminase

Background: Protein glutaminase specifically deamidates glutamine residue in protein and therefore significantly improves protein solubility and colloidal stability of protein solution. In order to improve its preparation efficiency, we exploited the possibility for its secretory expression mediated by twin-arginine translocation (Tat) pathway in Bacillus licheniformis. Results: The B. licheniformis genome-wide twin-arginine signal peptides were analyzed. Of which, eleven candidates were cloned for construction of expression vectors to mediate the expression of Chryseobacterium proteolyticum protein glutaminase (PGA). The signal peptide of GlmU was confirmed that it significantly mediated PGA secretion into media with the maximum activity of 0.16 U/ml in Bacillus subtilis WB600. A mutant GlmU-R, being replaced the third residue aspartic acid of GlmU twin-arginine signal peptide with arginine by site-directed mutagenesis, mediated the improved secretion of PGA with about 40% increased (0.23 U/ml). In B. licheniformis CBBD302, GlmU-R mediated PGA expression in active form with the maximum yield of 6.8 U/ml in a 25-l bioreactor. Conclusions: PGA can be produced and secreted efficiently in active form via Tat pathway of B. licheniformis, an alternative expression system for the industrial-scale production of PGA.

Xylanase Superproducer: Genome Sequence of a Compost-Loving Thermophilic Fungus, Thermomyces lanuginosus Strain SSBP.

We report here the draft genome sequence of Thermomyces lanuginosus strain SSBP, which was isolated from soil in South Africa. This fungus produces the largest amount of xylanase ever reported in the literature.

Expression of an alkalo-tolerant fungal xylanase enhanced by directed evolution in Pichia pastoris and Escherichia coli.

The alkaline stability of the xylanase from Thermomyces lanuginosus was further improved by directed evolution using error-prone PCR mutagenesis. Positive clones were selected by their ability to produce zones of clearing on pH 9 and 12 xylan agar plates. Variant NC38 was able to withstand harsh alkaline conditions retaining 84% activity after exposure at pH 10 for 90 min at 60 degrees C, while the parent enzyme had 22% activity after 60 min. The alkaline stable variant NC38 was cloned into pBGP1 under the control GAP promoter and pET22b(+) for expression in Pichia pastoris and Escherichia coli BL21, respectively. Best extracellular expression of the recombinant xylanase was observed in P. pastoris (261.7+/-0.61 U ml(-1)) whereas intracellular activity was observed in E. coli (47.9+/-0.28 U ml(-1)) was low. Total activity obtained in P. pastoris was 545-fold higher than E. coli. The mutated alkaline stable xylanase from P. pastoris was secreted into the culture medium with little or no contamination by host proteins, which favours the application of this enzyme in the pulp and paper industry.