Computer-Aided Reconstruction and Application of <i>Bacillus halodurans</i> S7 Xylanase with Heat and Alkali Resistance

β-1,4-Endoxylanase is the most critical hydrolase for xylan degradation during lignocellulosic biomass utilization. However, its poor stability and activity in hot and alkaline environments hinder its widespread application. In this study, BhS7Xyl from Bacillus halodurans S7 was improved using a computer-aided design through isothermal compressibility (βT) perturbation engineering and by combining three thermostability prediction algorithms (ICPE-TPA). The best variant with remarkable improvement in specific activity, heat resistance (70 °C), and alkaline resistance (both pH 9.0 and 70 °C), R69F/E137M/E145L, exhibited a 4.9-fold increase by wild-type in specific activity (1368.6 U/mg), a 39.4-fold increase in temperature half-life (458.1 min), and a 57.6-fold increase in pH half-life (383.1 min). Furthermore, R69F/E137M/E145L was applied to the hydrolysis of agricultural waste (corncob and hardwood pulp) to efficiently obtain a higher yield of high-value xylooligosaccharides. Overall, the ICPE-TPA strategy has the potential to improve the functional performance of enzymes under extreme conditions for the high-value utilization of lignocellulosic biomass

Directed Evolution of the UDP-Glycosyltransferase UGT_BL1 for Highly Regioselective and Efficient Biosynthesis of Natural Phenolic Glycosides

The O-glycosylation of polyphenols for the synthesis of glycosides has garnered substantial attention in food research applications. However, the practical utility of UDP-glycosyltransferases (UGTs) is significantly hindered by their low catalytic efficiency and suboptimal regioselectivity. The concurrent optimization of the regioselectivity and activity during the glycosylation of polyphenols presents a formidable challenge. Here, we addressed the long-standing activity–regioselectivity tradeoff in glycosyltransferase UGTBL1 through systematic enzyme engineering. The optimal combination of mutants, N61S/I62M/D63W/A208R/P218W/R282W (SMWRW1W2), yielded a 6.1-fold improvement in relative activity and a 17.3-fold increase in the ratio of gastrodin to para-hydroxybenzyl alcohol-4′-O-β-glucoside (with 89.5% regioselectivity for gastrodin) compared to those of the wild-type enzyme and ultimately allowed gram-scale production of gastrodin (1,066.2 mg/L) using whole-cell biocatalysis. In addition, variant SMWRW1W2 exhibited a preference for producing phenolic glycosides from several substrates. This study lays the foundation for the engineering of additional UGTs and the practical applications of UGTs in regioselective retrofitting

MoYvh1 translocates into the nucleus in response to the oxidative stress.

(A) Fluorescence observation of conidia untreated (upper panels) and treated with 5 mM H2O2 for 2 h (lower panels). 4’,6-Diamidino-2-phenylindole (DAPI) was added to the cultures 5 min prior to the observation of the nuclei. The merged images of GFP and DAPI staining showed that MoYvh1-GFP is localized in the nucleus when treated with H2O2. Bar = 5 μm. (B) Fluorescence observation of mycelia contain MoYvh1-GFP and H1-RFP were untreated (left panels) and treated with 5 mM H2O2 for 2 h (right panels). “green line” represents MoYvh1-GFP, “red line” represents H1-RFP. Insets highlight areas analyzed by line-scan. Bar = 5 μm.</p

Host derived ROS induces MoYvh1 nuclear accumulation during infection.

<p>(A) Whole-plant assays with cultivars LTH and K23 inoculated with the Δ<i>Moyvh1/MoYVH1</i> strain. The strain formed rare, small lesions on K23 which were different from the lesions on LTH. Plants were photographed at 7 d after inoculation. (B) DAB staining of the excised leaf sheath of cultivars LTH and K23 infected by the Δ<i>Moyvh1/MoYVH1</i> strain 30 h after inoculation. Bar = 5 μm. (C) Rice leaves were incubated with Δ<i>Moyvh1/MoYVH1-GFP-H1-RFP</i> strain for 30 h. Equal weight of rice leaves (LTH and K23) were divided into three parts for extraction of total, nuclear and cytoplasm proteins. Equal amounts of total, nuclear and cytoplasm proteins were separated by SDS-PAGE, and the presence of MoYvh1 was detected by Western blotting using the anti-GFP antibody. The intensity of Western blotting bands was quantified with the ODYSSEY infrared imaging system (application software Version 2.1). The intensity of MoYvh1 was compared between the cv. LTH and cv. K23 among total proteins, nuclear proteins, and cytoplasmic proteins. H1 (a nucleus marker) and actin (a cytoplasm marker) were detected by Western blotting analysis using the anti-RFP or anti-Actin antibodies. Bars denote standard errors from three independent experiments. Asterisk indicates significant differences (Duncan’s new multiple range test p < 0.01) (D) Localization of MoYvh1 during infection. Infection hyphae contain MoYvh1 and H1-RFP were observed by confocal fluorescence microscopy in the sheath of cultivars of LTH and K23 at 30 hpi. “green line” represents MoYvh1-GFP, “red line” represents H1-RFP. Insets highlight areas analyzed by line-scan. Bars = 5μm.</p

MoSsb1 and MoSsz1 are required for the translocation of MoYvh1 to the nucleus under oxidative stress.

<p>(A) Yeast two-hybrid analysis of interactions between MoYvh1 and MoSsa1, MoSsb1, and MoSsz1. MoYvh1 cDNA was inserted into the vector pGADT7, whilst MoSsa1, MoSsb1, and MoSsz1 cDNA was inserted into pGBKT7. “P” represents positive controls and “N” represents negative controls. Yeast cells grown on synthetic dextrose (SD) medium lacking leucine (Leu), tryptophan (Trp) and Histidine (His) were investigated against positive and negative controls as indicated. Plates were incubated at 30°C for 3 days before being photographed. (B) Co-IP assay. Western blot analysis of total proteins (T) extracted from conidia of various transformants, suspension proteins (S) and elution proteins (E) eluted from anti-GFP beads. The presence of MoYvh1, MoSsa1, MoSsb1 and MoSsz1 was detected with the anti-GFP and anti-FLAG antibodies, respectively. (C) Bimolecular fluorescence complementation (BiFC) assays for interactions between MoYvh1 and Hsp70 homologs. Conidia of transformants expressing MoYvh1-^NYFP and Hsp70s-^CYFP constructs (left panels) were treated (right panels) with 5 mM H₂O₂ for 2 h before interference contrast (DIC) and epifluorescence microscopy. YFP, yellow fluorescent protein. Bar = 5 μm. (D) Localization of MoYvh1-GFP was visualized in conidia of the Δ<i>Mossb1</i>, Δ<i>Mossz1</i> and Δ<i>Moyvh1</i> mutants. “+” represents the samples treated with 5 mM H₂O₂ for 2 h. Bar = 5 μm.</p

MoMrt4 is important for vegetative growth, conidiation, and full virulence.

<p>(A) Comparison of the wild type, the Δ<i>Momrt4</i> and the complement strains in vegetative growth on various medium. (B) The pathogenicity assay on rice leaves. Conidial suspensions of strains were sprayed onto two-week old rice seedlings (CO-39). Diseased leaves were photographed after 7 days of inoculation. (C) Quantification of lesion type. Lesions were photographed and measured at 7 days post-inoculation (dpi), counted within an area of 4 cm² and experiments were repeated three times with similar results. Asterisk indicates significant differences at p = 0.01. (D) Conidia development of the wild type, Δ<i>Momrt4</i> and the complement strains on SDC medium for 7 days were examined by light microscopy. (E) Statistical analysis of conidia production. Conidia produced by the wild-type, the mutant and complemented strains on SDC medium for 10 days were collected, counted and analyzed. ±SD is calculated from three repeated experiments and asterisks indicate statistically significant differences (Duncan’s new multiple range test, p < 0.01). (F) Ribosome proteins of indicated strains were extracted. Equal amounts of supernatant (S) and pellet (P) were separated by SDS-PAGE, and MoRpp0 and MoMrt4 were detected by Western blotting analysis using the anti-GFP and anti-FLAG antibodies, respectively.</p

MoYvh1 controls ribosome maturation via the release of MoMrt4 to overcome host innate immunity.

<p>A proposed schematic representation of how MoYvh1 desponds to host-derived ROS by nuclear translocation to regulate ribosome maturation through the release of MoMrt4. MoYvh1 and MoMrt4 regulate extracellular protein synthesis that have a role in the evasion of rice innate immunity.</p

MoMrt4^G69D and MoMrt4^G69E bypass the requirement of MoYvh1.

<p>(A) Comparison of the Δ<i>Moyvh1</i> and point mutation strains in colony morphology. Guy11, the Δ<i>Moyvh1</i> mutant, <i>MoMRT4</i> and mutated alleles complementation strains were cultured at 28°C in darkness for 7 days before being photographed. (B) Spraying assay. Conidial suspensions of each strain were sprayed on rice seedlings. Diseased leaves were photographed 7 days after inoculation. (C) Quantification of lesion type. Quantification of lesion type (0, no lesion; 1, pinhead-sized brown specks; 2, 1.5-mm brown spots; 3, 2–3-mm grey spots with brown margins; 4, many elliptical grey spots longer than 3 mm; 5, coalesced lesions) were measured at 7 days post-inoculation (dpi), counted within an area of 4 cm² and experiments were repeated three times with similar results. Asterisk indicates significant differences at p = 0.01. (D) DAB staining of the excised leaf sheath of rice infected by Guy11, the Δ<i>Moyvh1</i> mutant, the point mutation strains and complementation strain 36 h after inoculation. Bar = 5 μm.</p

MoYvh1 nuclear localization promotes extracellular proteins to evade host innate immunity.

<p>(A) Extracellular fluids from Guy11 appressoria suppress the defects in scavenging host-derived ROS of the Δ<i>Moyvh1</i> mutant, whereas boiled EF did not exhibit a similar defense response. “EF” represents the extracellular fluid. “BEF” represents the boiled extracellular fluid. Data represents observations from three independent experiments. Bar = 5 μm. (B) The infected cell stained by DAB. Three independent biological experiments were performed, with three replicates each time and yielded similar results in each independent biological experiment. Error bars represent standard deviation, and asterisks represent significant difference between the different strains (p < 0.01). (C) Laccase activity measured by ABTS oxidizing test without H₂O₂ and peroxidase activity measured by ABTS oxidizing test with H₂O₂. Bars denote standard errors from three independent experiments. Asterisk indicates significant differences (Duncan’s new multiple range test p < 0.01) (D) The conidial suspensions of each treatment were sprayed on the rice leaves. “EF” represents the extracellular fluid. “BEF” represents the boiled extracellular fluid. (E) Quantification of lesion type. Lesions were photographed and measured at 7 days post-inoculation (dpi), counted within an area of 4 cm² and experiments were repeated three times with similar results. Asterisk indicates significant differences at p = 0.01. (F) 1D gel analysis of Guy11 and the Δ<i>Moyvh1</i> mutant extracellular fluid proteins: Equal numbers of Guy11 and Δ<i>Moyvh1</i> conidia were harvested and equalized by using the actin antibody. The same number of conidia was used to extract the extracellular fluid. The extracellular fluid was concentrated to 100 μl. A 50 μl aliquot of each sample was then fractionated by 1D SDS-PAGE and proteins were stained by Coomassie brilliant blue. (G) GO functional analysis of extracellular fluid proteins.</p

Dissociation of MoMrt4 from the pre-ribosome in the nucleolus facilitated by MoYvh1 is required for the ribosome maturity.

<p>(A) The localization of Mrt4-GFP, MoMrt4^G69D-GFP and MoMrt4^G69E-GFP was observed in conidia of the Δ<i>Momrt4</i> and Δ<i>Moyvh1</i> mutants. DAPI was used to stain nuclei. The merged image of GFP and DAPI staining showed that Δ<i>Moyvh1</i>/<i>MoMRT4–GFP</i>, Δ<i>Moyvh1</i>/<i>MoMRT4</i>^<i>G69D</i><i>-GFP</i> and Δ<i>Moyvh1</i>/<i>MoMRT4</i>^<i>G69E</i><i>-GFP</i> strains were localized in the nucleus. Bar = 5 μm. (B) Ribosomal proteins were prepared from Δ<i>Moyvh1</i>/<i>MoMRT4–GFP</i> and Δ<i>Moyvh1/MoMRT4</i>^<i>G69E</i><i>-GFP</i> strains grown at as indicated salt concentrations. Free and ribosome-bound proteins were separated by sedimentation through sucrose cushions. Equal amounts of supernatant (S) and pellet (P) were separated by SDS-PAGE, and the presence of MoMrt4 and Rpl3 (a ribosome marker) was detected by Western blotting analysis using anti-GFP or anti-RPL3 antibodies. (C) Ribosome proteins were extracted from the strains as indicated. Free and ribosome-bound proteins were separated by sedimentation through sucrose cushions. Both the anti-GFP and anti-FLAG antibodies were added to detect the presence of MoMrt4 and MoYvh1 in supernatants (S) and pellets (P) following SDS-PAGE. RPL3 was used as a marker for ribosome. (D) Ribosome proteins of the indicated strains were extracted. Equal amounts of supernatant (S) and pellet (P) were separated by SDS-PAGE, and MoRpp0 and MoYvh1 were detected by Western blotting using anti-GFP and anti-FLAG antibodies.</p