Corrigendum: Positive effects of Cordyceps cateniannulata colonization in tobacco: growth promotion and resistance to abiotic stress.

[This corrects the article DOI: 10.3389/fmicb.2023.1131184.].

Ascorbate peroxidase catalyses synthesis of protocatechualdehyde from p-hydroxybenzaldehyde in Lycoris aurea.

Protocatechualdehyde is a plant natural phenolic aldehyde and an active ingredient with important bioactivities in traditional Chinese medicine. Protocatechualdehyde is also a key intermediate in the synthesis of Amaryllidaceae alkaloids for supplying the C6-C1 skeleton. However, the biosynthesis of protocatechualdehyde in plants remains obscure. In this study, we measured the protocatechualdehyde contents in the root, bulb, scape and flower of the Amaryllidaceae plant Lycoris aurea (L'Hér.) Herb., and performed the correlation analysis between the protocatechualdehyde contents and the transcriptional levels of the phenolic oxidization candidate protein encoding genes. We found that a novel ascorbate peroxidase encoded by the contig_24999 in the L. aurea transcriptome database had potential role in the biosynthesis of protocatechualdehyde. The LauAPX_24999 gene was then cloned from the cDNA of the scape of L. aurea. The transient expression of LauAPX_24999 protein in Arabidopsis protoplasts demonstrated that LauAPX_24999 protein was localized in the cytoplasm, thus belonging to Class II L-ascorbate peroxidase. Subsequently, LauAPX_24999 protein was heterogenously expressed in Escherichia coli, and identified that LauAPX_24999 biosynthesized protocatechualdehyde from p-hydroxybenzaldehyde using L-ascorbic acid as the electron donor. The protein structure modelling and molecular docking indicated that p-hydroxybenzaldehyde could access to the active pocket of LauAPX_24999 protein, and reside at the δ-edge of the heme group while L-ascorbic acid binds at the γ-heme edge. To our knowledge, LauAPX_24999 is the first enzyme discovered in plants able to biosynthesize protocatechualdehyde from p-hydroxybenzaldehyde, and offers a competent enzyme resource for the biosynthesis of Amaryllidaceae alkaloids via synthetic biology.

Positive effects of Cordyceps cateniannulata colonization in tobacco: Growth promotion and resistance to abiotic stress.

Background: Entomopathogenic fungi can live in insects to cause disease and death and are the largest group of entomopathogenic microorganisms. Therefore, these fungi are best known for their microbial control potential. Importantly, they also have other beneficial effects, including promoting plant growth and development by colonizing plant. Here, the study sought to identify specific strains of the entomopathogenic fungus, Cordyceps cateniannulata that would form endophytic associations with tobacco, thus benefiting plant growth and resistance to abiotic stresses, thereby highlighting the application of entomopathogenic fungi in tobacco. Methods: The C. cateniannulata-tobacco symbiont was constructed by root irrigation. The effects of C. cateniannulata on tobacco growth were evaluated by measuring the maximum leaf length, maximum leaf width, number of leaves, plant height, stem thickness, stem circumference, dry and fresh shoot weight 7, 14, 21, and 28 days after colonization. The peroxidase, catalase, superoxide dismutase, and malondialdehyde were measured to observe the impact of C. cateniannulata on tobacco defense enzyme activity. Finally, high-throughput sequencing was used to access microbial communities in the rhizosphere, with data subsequently linked to growth indicators. Results: After tobacco was inoculated with C. cateniannulata X8, which significantly promoted growth and related enzyme activity, malondialdehyde was decreased. The most significant impact was on peroxidase, with its activity being upregulated by 98.20, 154.42, 180.65, and 170.38% in the four time periods, respectively. The high throughput sequencing results indicated that C. cateniannulata had changed the rhizosphere microbial relative abundances, such as increasing Acidobacteria and Ascomycetes, and decreasing Actinomycetes and Basidiomycetes. The redundancy analysis showed that C. cateniannulata significantly boosted tobacco growth by reducing the abundance of specific dominant genera such as Stachybotrys, Cephalotrichum, Streptomyces, Isoptericola, and Microbacterium. Conclusion: Specific strains of C. cateniannulata can be introduced into host plants as endophytes, resulting in promotion of host plant growth and increased resistance to abiotic stress and microbial pathogens. The study provides a foundation for future studies of C. cateniannulata as an ecological agent.

[Expression optimization and molecular modification of heparin C5 epimerase].

Heparin and heparan sulfate are a class of glycosaminoglycans for clinical anticoagulation. Heparosan N-sulfate-glucuronate 5-epimerase (C5, EC 5.1.3.17) is a critical modifying enzyme in the synthesis of heparin and heparan sulfate, and catalyzes the inversion of carboxyl group at position 5 on D-glucuronic acid (D-GlcA) of N-sulfoheparosan to form L-iduronic acid (L-IdoA). In this study, the heparin C5 epimerase gene Glce from zebrafish was expressed and molecularly modified in Escherichia coli. After comparing three expression vectors of pET-20b (+), pET-28a (+) and pCold Ⅲ, C5 activity reached the highest ((1 873.61±5.42) U/L) with the vector pCold Ⅲ. Then we fused the solution-promoting label SET2 at the N-terminal for increasing the soluble expression of C5. As a result, the soluble protein expression was increased by 50% compared with the control, and the enzyme activity reached (2 409±6.43) U/L. Based on this, site-directed mutations near the substrate binding pocket were performed through rational design, the optimal mutant (V153R) enzyme activity and specific enzyme activity were (5 804±5.63) U/L and (145.1±2.33) U/mg, respectively 2.41-fold and 2.28-fold of the original enzyme. Modification and expression optimization of heparin C5 epimerase has laid the foundation for heparin enzymatic catalytic biosynthesis.

[Production and application of 3-phosphoadenosine-5- phosphosulfate].

Sulfated compounds are widely present in cytoplasm, on cell surface, and in extracellular matrix. These compounds play important roles in cell development, differentiation, immune response, detoxication, and cell signal transduction. 3-Phosphoadenosine-5-phosphosulfate (PAPS) is the universal sulfate group donor for the biosynthesis of sulfated compounds. Up to now, the synthesis of PAPS is still too expensive for industrial applications. This review focuses on the recent progress of PAPS production and summaries the application of PAPS, particularly in the production of glucosinolate, heparin, condroitin sulfate, and oxamniquine production.

Secretory expression of the rat aryl sulfotransferases IV with improved catalytic efficiency by molecular engineering.

The rat aryl sulfotransferases IV (AST IV) has been used to catalyze 3'-phosphoadenosine-5'-phosphate (PAP) into the sulfuryl group donor 3'-phosphoadenosine-5'-phosphosulfate (PAPS) in biotechnological production of glycosaminoglycans. The performance of native AST IV is not satisfying due to the lower catalytic activity with substrate PAP. In the present study, we achieved secretory expression of the AST IV and improved its catalytic efficiency by molecular engineering. Fusion with signal peptides Cex, YebF and PelB allow for secretory expression of AST IV and the secreted AST IV yield reached 4.21 ± 0.23 U/mL, 8.67 ± 0.34 U/mL and 21.35 ± 0.87 U/mL, respectively. Modification of PelB further increased the secretory expression by more than fourfold, to 89.67 ± 1.34 U/mL. On this basis, molecular evolution of the predicted PAP-binding pocket gate loop was performed and a positive mutant L89S/E90L with higher activity was identified. Considering the importance of the sites Leu89 and Glu90, we performed site-saturation mutagenesis and found the mutant L89M/E90Q with much higher PAP affinity (K m= 0.46 ± 0.02 mM) and catalytic efficiency (k cat/K m = 1816.32 ± 12.72/s/M). The secretory expression of the AST IV variant L89M/E90Q with higher catalytic efficiency should facilitate the studies on biosynthesis of sulfated polysaccharides.

Engineering of multiple modular pathways for high-yield production of 5-aminolevulinic acid in Escherichia coli.

5-aminolevulinic acid (ALA), an important precursor of tetrapyrroles, has various applications in medicine and agriculture fields. Several methods have been adopted to enhance ALA synthesis in our previous studies. In this study, systematic metabolic engineering strategies were implemented to further improve ALA production in Escherichia coli. Firstly, hemA and hemL with different strength of RBS from the artificially constructed mutation libraries were randomly assembled to balance metabolic flux. Then the expression of ALA dehydratase was rationally regulated by replacing promoter with fliCp to weaken ALA catabolism. Besides, the activity of glutamate-1-semialdehyde aminotransferase was increased through strengthening the native biosynthesis pathway of cofactor pyridoxal 5'-phosphate. Moreover, plasmid stability was improved by 21.4% by deleting recA and endA in the recombinant. Finally, stepwise improvements in ALA production were increased to 5.25 g/L with a pH two-stage strategy in a 3-L fermenter. This study proved the importance of metabolic balance in the pathway.

[Optimized expression of heparin sulfotransferases and their application in sulfation of animal derived heparin].

Heparin is a very important anticoagulant drug. Currently, heparin is mainly extracted from porcine mucosa. However, animal-derived heparin shows low anticoagulant activity due to the low proportion of the anticoagulant active unit, the GlcNS6S-GlcA-GlcNS6S3S-Ido2S-GlcNS6S pentasaccharide. In this study we proposed an enzymatic strategy to sulfate the animal-sourced heparin to increase the proportion of anticoagulant pentasaccharide and the anticoagulant activity. First, three sulfotransferases HS2ST, HS6ST, and HS3ST were expressed tentatively in Escherichia coli and Pichia pastoris. After measuring the sulfotransferase activity, we confirmed P. pastoris GS115 is the better host for sulfotransferases production. Then, the maltose binding protein (MBP) and thioredoxin (TrxA) were fused separately to the N-terminal of sulfotransferases to increase enzyme solubility. As a result, the yields of HS2ST and HS6ST were increased to (839±14) U/L and (792±23) U/L, respectively. Subsequent sulfation of the animal-sourced heparin with the recombinant HS2ST, HS6ST and HS3ST increased the anticoagulant activity from (76±2) IU/mg to (189±17) IU/mg.

Bio-Based Strategies for Producing Glycosaminoglycans and Their Oligosaccharides.

Glycosaminoglycans (GAGs) and their oligosaccharides have attracted extensive attention because of their wide applications in cosmetics, health, and clinical treatment of, for example, thrombus, osteoarthritis, rheumatism, and cancer. Compared to conventional preparation approaches, bio-based strategies for producing GAGs and their oligosaccharides are more promising because they avoid cross-infection of human and animal diseases, use green production processes, and can precisely control the degree of sulfation and molecular weight. This review summarizes recently developed bio-based strategies for producing GAGs, highlighting in particular enzymatic, metabolic engineering, and synthetic biology strategies. Directions and prospects for near-future research are also outlined and discussed.

A microbial-enzymatic strategy for producing chondroitin sulfate glycosaminoglycans.

Chondroitin sulfate has been widely used in both medical and clinical applications. Commercial chondroitin sulfate has been mainly acquired from animal tissue extraction. Here we report a new two-step biological strategy for producing chondroitin sulfate A and chondroitin sulfate C. First, the chondroitin biosynthesis pathway in a recombinant Bacillus subtilis strain using sucrose as carbon source was systematically optimized and the titer of chondroitin was significantly enhanced to 7.15 g/L. Then, specific sulfation transformation systems were successfully constructed and optimized by combining the purified aryl sulfotransferase IV (ASST IV), chondroitin 4-sulfotransferase (C4ST) and chondroitin 6-sulfotransferase (C6ST). Chondroitin sulfate A and C were enzymatically transformed from chondroitin at conversion rates of 98% and 96%, respectively. The present biological strategy has great potential to be scaled up for biosynthesis of chondroitin sulfate A and C from cheap carbon sources.

[Optimization of heparosan synthetic pathway in Bacillus subtilis 168].

Heparosan is the start point for chemoenzymatic synthesis of heparin and it is of great significance to efficiently synthesize heparosan in microorganisms. The effects of overexpressing key enzyme genes of the UDP-glucuronic acid (UDP-GlcUA) pathway (pgcA, gtaB and tuaD) or the UDP-N-acetyl-glucosamine (UDP-GlcNAc) pathway (glmS, glmM and glmU) on the heparosan production and molecular mass were analyzed in the constructed heparosan-producing Bacillus subtilis ((1.71±0.08) g/L). On this basis, heparosan production was increased to (2.89±0.11) g/L with the molecular mass of (75.90±1.18) kDa through co-overexpressing the tuaD, gtaB, glmU, glmM and glmS genes in shake flask cultivation. In the 3 L fed-batch fermentation, heparosan production was improved to (7.25±0.36) g/L with the molecular mass of (46.66±2.71) kDa, providing the potential for heparosan industrial production.

Efficient transformation of Rhizopus delemar by electroporation of germinated spores.

High efficient transformation of mycelial fungi is essential to both metabolic engineering and physiological analysis of these industrially important microorganisms. However, transformation efficiencies for mycelial fungi are highly restricted by difficulties in colony formation and competent cell preparation. In this work, an innovative transformation procedure that could significantly improve the efficiency of colony formation and transformation process has been established for a typical mycelial fungus, Rhizopus delemar. Single colonies of R. delemar were obtained with the addition of sodium deoxycholate. Fresh germinated spores of R. delemar were successfully transformed by electroporation. In addition, by pretreatment of the germinated spores with 0.05M lithium acetate (LiAc) and 20mM dithiothreitol (DTT) before electroporation, the transformation efficiency was further improved by 9.5-fold. The final transformation efficiency at optimal conditions reached 1239 transformants/µg DNA. The method described here would facilitate more efficient metabolic engineering and investigation of physiological functions in R. delemar or other similar mycelial fungi.

Optimization of fumaric acid production by Rhizopus delemar based on the morphology formation.

The effects of temperature, agitation rate and medium composition, including concentrations of glucose, soybean peptone, and inorganic ions, on pellet formation and pellet diameter of Rhizopus delemar (Rhizopus oryzae) NRRL1526 during pre-culture were studied. Inorganic ions and soybean peptone had negative and positive effects on pellet formation, respectively. The initial glucose and soybean peptone concentrations directly affected pellet diameter. Within a certain range, pellet diameter decreased with increased initial substrate concentrations; however, above this range there was an opposite trend. Thus, optimal concentrations of substrate during pre-culture were beneficial for producing small pellets of R. delemar. Furthermore, dry cell mass and yield of fumaric acid tended to increase with decreased pellet diameter. Based on the pellet morphology optimization, the final fumaric acid concentration was improved by 46.13% when fermented in a flask and 31.82% in stirred bioreactor tank fermentation.