D-allulose 3-epimerase for low-calorie D-allulose synthesis: microbial production, characterization, and applications.

D-allulose, an epimer of D-fructose at C-3 position, is a low-calorie rare sugar with favorable physiochemical properties and special physiological functions, which displays promising perspectives in the food and pharmaceutical industries. Currently, D-allulose is extremely sparse in nature and is predominantly biosynthesized through the isomerization of D-fructose by D-allulose 3-epimerase (DAEase). In recent years, D-allulose 3-epimerase as the key biocatalyst for D-allulose production has received increasing interest. The current review begins by providing a summary of D-allulose regarding its characteristics and applications, as well as different synthesis pathways dominated by biotransformation. Then, the research advances of D-allulose 3-epimerase are systematically reviewed, focusing on heterologous expression and biochemical characterization, crystal structure and molecular modification, and application in D-allulose production. Concerning the constraint of low yield of DAEase for industrial application, this review addresses the various attempts made to promote the production of DAEase in different expression systems. Also, various strategies have been adopted to improve its thermotolerance and catalytic activity, which is mainly based on the structure-function relationship of DAEase. The application of DAEase in D-allulose biosynthesis from D-fructose or low-cost feedstocks through single- or multi-enzymatic cascade reaction has been discussed. Finally, the prospects for related research of D-allulose 3-epimerase are also proposed, facilitating the industrialization of DAEase and more efficient and economical bioproduction of D-allulose.

Physiological and transcriptome analyses reveal tissue-specific responses of Leucaena plants to drought stress.

Leucaena leucocephala (Leucaena) is a leguminous tree widely cultivated in tropical and subtropical regions due to its strong environmental suitability for abiotic stresses, especially drought. However, the molecular mechanisms and key pathways involved in Leucaena's drought response require further elucidation. Here, we comparatively analyzed the physiological and early transcriptional responses of Leucaena leaves and roots under drought stress simulated by polyethylene glycol (PEG) treatments. Drought stress induced physiological changes in Leucaena seedlings, including decreases in relative water content (RWC) and increases in relative electrolyte leakage (REL), malondialdehyde (MDA), proline contents as well as antioxidant enzyme activities. In response to drought stress, 6461 and 8295 differentially expressed genes (DEGs) were identified in the leaves and roots, respectively. In both tissues, the signaling transduction pathway of plant hormones was notably the most enriched. Specifically, abscisic acid (ABA) biosynthesis and signaling related genes (NCED, PP2C, SnRK2 and ABF) were strongly upregulated particularly in leaves. The circadian rhythm, DNA replication, alpha-linolenic acid metabolism, and secondary metabolites biosynthesis related pathways were repressed in leaves, while the glycolysis/gluconeogenesis and alpha-linolenic acid metabolism and amino acid biosynthesis processes were promoted in roots. Furthermore, heterologous overexpression of Leucaena drought-inducible genes (PYL5, PP2CA, bHLH130, HSP70 and AUX22D) individually in yeast increased the tolerance to drought and heat stresses. Overall, these results deepen our understanding of the tissue-specific mechanisms of Leucaena in response to drought and provide target genes for future drought-tolerance breeding engineering in crops.

Final piece to the Fusarium pigmentation puzzle - Unraveling of the phenalenone biosynthetic pathway responsible for perithecial pigmentation in the Fusarium solani species complex.

The Fusarium solani species complex (FSSC) is comprised of important pathogens of plants and humans. A distinctive feature of FSSC species is perithecial pigmentation. While the dark perithecial pigments of other Fusarium species are derived from fusarubins synthesized by polyketide synthase 3 (PKS3), the perithecial pigments of FSSC are derived from an unknown metabolite synthesized by PKS35. Here, we confirm in FSSC species Fusarium vanettenii that PKS35 (fsnI) is required for perithecial pigment synthesis by deletion analysis and that fsnI is closely related to phnA from Penicillium herquei, as well as duxI from Talaromyces stipentatus, which produce prephenalenone as an early intermediate in herqueinone and duclauxin synthesis respectively. The production of prephenalenone by expression of fsnI in Saccharomyces cerevisiae indicates that it is also an early intermediate in perithecial pigment synthesis. We next identified a conserved cluster of 10 genes flanking fsnI in F. vanettenii that when expressed in F. graminearum led to the production of a novel corymbiferan lactone F as a likely end product of the phenalenone biosynthetic pathway in FSSC.

Construction of an expression platform for fungal secondary metabolite biosynthesis in Penicillium crustosum.

Filamentous fungi are prolific producers of bioactive natural products and play a vital role in drug discovery. Yet, their potential cannot be fully exploited since many biosynthetic genes are silent or cryptic under laboratory culture conditions. Several strategies have been applied to activate these genes, with heterologous expression as one of the most promising approaches. However, successful expression and identification of new products are often hindered by host-dependent factors, such as low gene targeting efficiencies, a high metabolite background, or a lack of selection markers. To overcome these challenges, we have constructed a Penicillium crustosum expression host in a pyrG deficient strain by combining the split-marker strategy and CRISPR-Cas9 technology. Deletion of ligD and pcribo improved gene targeting efficiencies and enabled the use of an additional selection marker in P. crustosum. Furthermore, we reduced the secondary metabolite background by inactivation of two highly expressed gene clusters and abolished the formation of the reactive ortho-quinone methide. Finally, we replaced the P. crustosum pigment gene pcr4401 with the commonly used Aspergillus nidulans wA expression site for convenient use of constructs originally designed for A. nidulans in our P. crustosum host strain. As proof of concept, we successfully expressed a single polyketide synthase gene and an entire gene cluster at the P. crustosum wA locus. Resulting transformants were easily detected by their albino phenotype. With this study, we provide a highly efficient platform for heterologous expression of fungal genes. KEY POINTS: Construction of a highly efficient Penicillium crustosum heterologous expression host Reduction of secondary metabolite background by genetic dereplication strategy Integration of wA site to provide an alternative host besides Aspergillus nidulans.

Engineering Streptomyces albus B4 for Enhanced Production of (R)-Mellein: A High-Titer Heterologous Biosynthesis Approach.

The natural compound (R)-(-)-mellein exhibits antiseptic and fungicidal activities. We investigated its biosynthesis using the polyketide synthase encoded by SACE_5532 (pks8) from Saccharopolyspora erythraea heterologously expressed in Streptomyces albus B4, a chassis chosen for its fast growth, genetic manipulability, and ample large short-chain acyl-CoA precursor supply. High-level heterologous (R)-(-)-mellein yield was achieved by pks8 overexpression and duplication. The precursor supply pathways were strengthened by overexpression of SACE_0028 (encoding acetyl-CoA carboxylase) and four genes involved in ß-oxidation (fadD, fadE, fadB, and fadA). Cell growth inhibition by (R)-(-)-mellein production at high concentration was relieved by in situ adsorption using Amberlite XAD16 resin. The final strain, B4mel12, produced (R)-(-)-mellein at 6395.2 mg/L in shake-flask fermentation. Overall, this is the first report of heterologous (R)-(-)-mellein synthesis in microorganism with a high titer. (R)-(-)-mellein prototype in this study opens a possibility for the overproduction of valuable melleins in S. albus B4.

Human ß-defensins: The multi-functional natural peptide.

The increasing threat of antibiotic resistance among pathogenic microorganisms and the urgent demand for new antibiotics require immediate attention. Antimicrobial peptides exhibit effectiveness against microorganisms, fungi, viruses, and protozoa. The discovery of human ß-defensins represents a major milestone in biomedical research, opening new avenues for scientific investigation into the innate immune system and its resistance mechanisms against pathogenic microorganisms. Multiple defensins present a promising alternative in the context of antibiotic abuse. However, obstacles to the practical application of defensins as anti-infective therapies persist due to the unique properties of human ß-defensins themselves and serious pharmacological and technical challenges. To overcome these challenges, diverse delivery vehicles have been developed and progressively improved for the conjugation or encapsulation of human ß-defensins. This review briefly introduces the biology of human ß-defensins, focusing on their multistage structure and diverse functions. It also discusses several heterologous systems for producing human ß-defensins, various delivery systems created for these peptides, and patent applications related to their utilization, concluding with a summary of current challenges and potential solutions.

Improvement of the nitrogenase activity in Escherichia coli that expresses the nitrogen fixation-related genes from Azotobacter vinelandii.

The transfer of nitrogen fixation (nif) genes from diazotrophs to non-diazotrophic hosts is of increasing interest for engineering biological nitrogen fixation. A recombinant Escherichia coli strain expressing Azotobacter vinelandii 18 nif genes (nifHDKBUSVQENXYWZMF, nifiscA, and nafU) were previously constructed and showed nitrogenase activity. In the present study, we constructed several E. coli strain derivatives in which all or some of the 18 nif genes were additionally integrated into the fliK locus of the chromosome in various combinations. E. coli derivatives with the chromosomal integration of nifiscA, nifU, and nifS, which are involved in the biosynthesis of the [4Fe-4S] cluster of dinitrogenase reductase, exhibited enhanced nitrogenase activity. We also revealed that overexpression of E. coli fldA and ydbK, which encode flavodoxin and flavodoxin-reducing enzyme, respectively, enhanced nitrogenase activity, likely by facilitating electron transfer to dinitrogenase reductase. The additional expression of nifM, putatively involved in maturation of dinitrogenase reductase, further enhanced nitrogenase activity and the amount of soluble NifH. By combining these factors, we successfully improved nitrogenase activity 10-fold.

Expression and characterization of monofunctional alcohol dehydrogenase enzymes in Clostridium thermocellum.

Clostridium thermocellum is a thermophilic anaerobic bacterium that could be used for cellulosic biofuel production due to its strong native ability to consume cellulose, however its ethanol production ability needs to be improved to enable commercial application. In our previous strain engineering work, we observed a spontaneous mutation in the native adhE gene that reduced ethanol production. Here we attempted to complement this mutation by heterologous expression of 18 different alcohol dehydrogenase (adh) genes. We were able to express all of them successfully in C. thermocellum. Surprisingly, however, none of them increased ethanol production, and several actually decreased it. Our findings contribute to understanding the correlation between C. thermocellum ethanol production and Adh enzyme cofactor preferences. The identification of a set of adh genes that can be successfully expressed in this organism provides a foundation for future investigations into how the properties of Adh enzymes affect ethanol production.

Advancing cyanobacterial biotechnology: new frontiers in natural product discovery and production.

Cyanobacteria, as oxygenic phototrophs, offer significant potential for sustainable biotechnology applications. Cyanobacterial natural products, with antimicrobial, anticancer, and plant growth-promoting properties, hold promise in pharmaceuticals, agriculture, and environmental remediation. By leveraging advanced technologies, cyanobacteria can significantly impact various industries, supporting the green biotechnology agenda. Recent advancements in integrated omics, orphan gene cluster activation, genetic manipulation, and chemo-enzymatic methods are expanding their biotechnological relevance. Omics technologies revolutionize cyanobacterial natural product research by facilitating biosynthetic gene cluster identification. Heterologous expression and pathway reconstitution enable complex natural product production, while high-titer strategies like metabolic engineering enhance yields. Interdisciplinary research and technological progress position cyanobacteria as valuable sources of bioactive compounds, driving sustainable biotechnological practices forward.

Microbial creation of ß-Nicotinamide mononucleotide and its regulation of lipid metabolism in the liver of high-fat diet mice.

ß-Nicotinamide mononucleotide (NMN) is a biologically active nucleotide that regulates the physiological metabolism of the body by rapidly increasing nicotinamide adenine dinucleotide (NAD+). To determine the safety and biological activity of NMN resources, we constructed a recombinant strain of P. pastoris that heterologously expresses nicotinamide-phosphate ribosyltransferase (NAMPT), and subsequently catalyzed and purified the expressed product to obtain NMN. Consequently, this study established a high-fat diet (HFD) obese model to investigate the lipid-lowering activity of NMN. The findings showed that NMN supplementation directly increased the NAD+ levels, and reduced HFD-induced liver injury and lipid deposition. NMN treatment significantly decreased total cholesterol (TC) and triglyceride (TG) in serum and liver, as well as alanine aminotransferase (ALT) and insulin levels in serum (p < .05 or p < .01). In conclusion, this study combined synthetic biology with nutritional evaluation to confirm that P. pastoris-generated NMN modulated lipid metabolism in HFD mice, offering a theoretical framework and evidence for the application of microbially created NMN.

Understanding bacteriocin heterologous expression: A review.

Bacteriocins are a diverse group of ribosomally synthesised antimicrobial peptides/proteins that play an important role in self-defence. They are widely used as bio-preservatives and effective substitutes for disease eradication. They can be used in conjunction with or as an alternative to antibiotics to minimize the risk of resistance development. There are remarkably few reports indicating resistance to bacteriocins. Although there are many research reports that emphasise heterologous expression of bacteriocin, there are no convincing reports on the significant role that intrinsic and extrinsic factors play in overexpression. A coordinated and cooperative expression system works in concert with multiple genetic elements encoding native proteins, immunoproteins, exporters, transporters and enzymes involved in the post-translational modification of bacteriocins. The simplest way could be to utilise the existing E. coli expression system, which is conventional, widely used for heterologous expression and has been further extended for bacteriocin expression. In this article, we will review the intrinsic and extrinsic factors, advantages, disadvantages and major problems associated with bacteriocin overexpression in E. coli. Finally, we recommend the most effective strategies as well as numerous bacteriocin expression systems from E. coli, Lactococcus, Kluveromyces lactis, Saccharomyces cerevisiae and Pichia pastoris for their suitability for successful overexpression.

Effects of Heterologous Expression of Genes Related L-Malic acid Metabolism in Saccharomyces uvarum on Flavor Substances Production in Wine.

During malolactic fermentation (MLF) of vinification, the harsh L-malic acid undergoes transformation into the milder L-lactic acid, and via decarboxylation reactions it is catalyzed by malolactic enzymes in LAB. The use of bacterial malolactic starter cultures, which usually present challenges in the industry as the suboptimal conditions after alcoholic fermentation (AF), including nutrient limitations, low temperatures, acidic pH levels, elevated alcohol, and sulfur dioxide concentrations after AF, lead to "stuck" or "sluggish" MLF and spoilage of wines. Saccharomyces uvarum has interesting oenological properties and provides a stronger aromatic intensity than Saccharomyces cerevisiae in AF. In the study, the biological pathways of deacidification were constructed in S. uvarum, which made the S. uvarum carry out the AF and MLF simultaneously, as different genes encoding malolactic enzyme (mleS or mleA), malic enzyme (MAE2), and malate permease (melP or MAE1) from Schizosaccharomyces pombe, Lactococcus lactis, Oenococcus oeni, and Lactobacillus plantarum were heterologously expressed. For further inquiry, the effect of L-malic acid metabolism on the flavor balance in wine, the related flavor substances, higher alcohols, and esters production, were detected. Of all the recombinants, the strains WYm1SN with coexpression of malate permease gene MAE1 from S. pombe and malolactic enzyme gene mleS from L. lactis and WYm1m2 with coexpression of gene MAE1 and malate permease gene MAE2 from S. pombe could reduce the L-malic acid contents to about 1 g/L, and in which the mutant WYm1SN exhibited the best effect on the flavor quality improvement.

Heme o Isolation and Analysis Using Heme o Synthase Heterologously Expressed in Escherichia coli.

Heme o is an Fe-porphyrin involved in the majority of aerobic respiration pathways found in all three domains of life. In eukaryotes and most aerobic prokaryotes, heme o functions solely as the precursor for the synthesis of heme a, a necessary cofactor for most heme-copper terminal oxidases. In some prokaryotes, such as Escherichia coli (E. coli), heme o can serve as a cofactor for heme-copper oxidases instead of heme a. Given its role as a key substrate or cofactor, purified heme o promises to be a valuable resource for the study of heme-copper oxidase assembly and activity. However, commercially available heme o is sold in limited quantities at a relatively high cost (compared to the prototypical heme b), making the use of heme o purchased from suppliers unfeasible for such studies. In this chapter, we present step-by-step methods both for heme o isolation from E. coli overexpressing heme o synthase and for HPLC analysis of cellular hemes (i.e., heme o and heme b).

Optimizing Systems for Robust Heterologous Production of Biosurfactants Rhamnolipid and Lyso-Ornithine Lipid in Pseudomonas putida KT2440.

Surfactants are amphiphilic molecules that are capable of mixing water and oil. Biosurfactants are eco-friendly, low-toxicity, and stable to a variety of environmental factors. Optimizing conditions for microorganisms to produce biosurfactants can lead to improved production suitable for scaling up. In this study, we compared heterologous expression levels of the luminescence system luxCDABE operon controlled by regulatable promoters araC-PBAD and its strong version araC-PBAD-SD in Escherichia coli K12, Pseudomonas aeruginosa PAO1, and P. putida KT2440. Real-time monitoring of luminescence levels in the three strains indicated that luxCDABE controlled by araC-PBAD-SD promoter with 0.2% arabinose supplementation in P. putida produced the highest level of luminescence. By using the araC-PBAD-SD promoter-controlled rhlAB expression in P. putida, we were able to produce mono-rhamnolipid at a level of 1.5 g L-1 when 0.02% arabinose was supplemented. With the same system to express olsB, lyso-ornithine lipid was produced at a level of 10 mg L-1 when 0.2% arabinose was supplemented. To our knowledge, this is the first report about optimizing conditions for lyso-ornithine lipid production at a level up to 10 mg L-1. Taken together, our results demonstrate that regulatable araC-PBAD-SD promoter in P. putida KT2440 is a useful system for heterologous production of biosurfactants.

Stepwise increase of fidaxomicin in an engineered heterologous host Streptomyces albus through multi-level metabolic engineering.

The anti-Clostridium difficile infection (CDI) drug fidaxomicin is a natural polyketide metabolite mainly produced by Micromonosporaceae, such as Actinoplanes deccanensis, Dactylosporangium aurantiacum, and Micromonospora echinospora. In the present study, we employed a stepwise strategy by combining heterologous expression, chassis construction, promoter engineering, activator and transporters overexpression, and optimization of fermentation media for high-level production of fidaxomicin. The maximum yield of 384 mg/L fidaxomicin was achieved with engineered Streptomyces albus D7-VHb in 5 L-tank bioreactor, and it was approximately 15-fold higher than the native strain Actinoplanes deccanensis YP-1 with higher strain stability and growth rate. This study developed an enhanced chassis strain, and for the first time, achieved the heterologous synthesis of fidaxomicin through a combinatorial metabolic engineering strategy.

Gene function characterization in Aspergillus niger using a dual resistance marker transformation system mediated by Agrobacterium tumefaciens.

Aspergillus niger is a well-known workhorse for the industrial production of enzymes and organic acids. This fungus can also cause postharvest diseases in fruits. Although Agrobacterium tumefaciens-mediated transformation (ATMT) based on antibiotic resistance markers has been effectively exploited for inspecting functions of target genes in wild-type fungi, it still needs to be further improved in A. niger. In the present study, we re-examined the ATMT in the wild-type A. niger strains using the hygromycin resistance marker and introduced the nourseothricin resistance gene as a new selection marker for this fungus. Unexpectedly, our results revealed that the ATMT method using the resistance markers in A. niger led to numerous small colonies as false-positive transformants on transformation plates. Using the top agar overlay technique to restrict false positive colonies, a transformation efficiency of 87 ± 18 true transformants could be achieved for 106 conidia. With two different selection markers, we could perform both the deletion and complementation of a target gene in a single wild-type A. niger strain. Our results also indicated that two key regulatory genes (laeA and veA) of the velvet complex are required for A. niger to infect apple fruits. Notably, we demonstrated for the first time that a laeA homologous gene from the citrus postharvest pathogen Penicillium digitatum was able to restore the acidification ability and pathogenicity of the A. niger ΔlaeA mutant. The dual resistance marker ATMT system from our work represents an improved genetic tool for gene function characterization in A. niger.

WONOEP appraisal: Modeling early onset epilepsies.

Epilepsy has a peak incidence during the neonatal to early childhood period. These early onset epilepsies may be severe conditions frequently associated with comorbidities such as developmental deficits and intellectual disability and, in a significant percentage of patients, may be medication-resistant. The use of adult rodent models in the exploration of mechanisms and treatments for early life epilepsies is challenging, as it ignores significant age-specific developmental differences. More recently, models developed in immature animals, such as rodent pups, or in three-dimensional organoids may more closely model aspects of the immature brain and could result in more translatable findings. Although models are not perfect, they may offer a more controlled screening platform in studies of mechanisms and treatments, which cannot be done in pediatric patient cohorts. On the other hand, more simplified models with higher throughput capacities are required to deal with the large number of epilepsy candidate genes and the need for new treatment options. Therefore, a combination of different modeling approaches will be beneficial in addressing the unmet needs of pediatric epilepsy patients. In this review, we summarize the discussions on this topic that occurred during the XVI Workshop on Neurobiology of Epilepsy, organized in 2022 by the Neurobiology Commission of the International League Against Epilepsy. We provide an overview of selected models of early onset epilepsies, discussing their advantages and disadvantages. Heterologous expression models provide initial functional insights, and zebrafish, rodent models, and brain organoids present increasingly complex platforms for modeling and validating epilepsy-related phenomena. Together, these models offer valuable insights into early onset epilepsies and accelerate hypothesis generation and therapy discovery.

TED: Enhancing Translation Efficiency in Bacterial Expression Systems.

Genetic engineering enables the forced expression of desired products in bacteria, which can then be used for a variety of applications, including functional analysis and pharmaceuticals. Here, we describe a method for tuning translation in bacteria, including Escherichia coli and Rhodobacter capsulatus, based on a phenomenon known as TED (translation enhancement by a Dictyostelium gene sequence). This method promotes translation of mRNA encoded by downstream genes by inserting a short nucleotide sequence into the 5' untranslated region between the promoter and the Shine-Dalgarno (SD) sequence. Various expression levels can be observed depending on the inserted sequence and its length, even with an identical promoter.

Heterologous expression and antimicrobial potential of class II bacteriocins.

Gut bacteria are known to produce bacteriocins to inhibit the growth of other bacteria. Consequently, bacteriocins have attracted increased attention as potential microbiome-editing tools. In this study we examine the inhibitory spectrum of 75 class II bacteriocins against 48 representative gut microbiota species. The bacteriocins were heterologously expressed in Escherichia coli and evaluated in vitro, ex vivo and in vivo. In vitro assays revealed 22 bacteriocins to inhibit at least one species and showed selective inhibition patterns against species implicated in certain disorders and diseases. Three bacteriocins were selected for ex vivo assessment on mouse feces. Based on 16S rRNA sequencing of the cultivated feces we showed that the two bacteriocins: Actifencin (#13) and Bacteroidetocin A (#22) selectively inhibited the growth of Lactobacillus and Bacteroides, respectively. The probiotic: E. coli Nissle 1917 was engineered to express these two bacteriocins in mice. However, the selective inhibitory patterns found in the in vitro and ex vivo experiments could not be observed in vivo. Our study describes a methodology for heterologous high throughput bacteriocin expression and screening and elucidates the inhibitory patterns of class II bacteriocins on the gut microbiota.

Characterization of a novel carboxylesterase from Streptomyces lividans TK24 and site-directed mutagenesis for its thermostability.

As an industrial enzyme that catalyzes the formation and cleavage of ester bonds, carboxylesterase has attracted attention in fine chemistry, pharmaceutical, biological energy and bioremediation fields. However, the weak thermostability limits their further developments in industrial applications. In this work, a novel carboxylesterase (EstF) from Streptomyces lividans TK24, belonging to family XVII, was acquired by successfully heterologous expressed and biochemically identified. The EstF exhibited optimal activity at 55 °C, pH 9.0 and excellent catalytic performances (Km = 0.263 mM, kcat/Km = 562.3 s-1 mM-1 for p-nitrophenyl acetate (pNPA2) hydrolysis). Besides, the EstF presented exceptionally high thermostability with a half-life of 387.23 h at 55 °C and 2.86 h at 100 °C. Furthermore, the EstF was modified to obtain EstFP144G using the site-directed mutation technique to investigate the effect of single glycine on thermostability. Remarkably, the mutant EstFP144G displayed a 5.10-fold increase of half-life at 100 °C versus wild-type without affecting catalytic performance. Structural analysis implied that the glycine introduction could release a steric strain and induce cooperative effects between distal residues to increase the thermostability. Therefore, the thermostable EstF and EstFP144G with prominently catalytic characteristics have potential industrial applications and the introduction of a single glycine strategy opens up alternative avenues for the thermostability engineering of other enzymes.