Expanding the horizons of levan: from microbial biosynthesis to applications and advanced detection methods.

Levan, a ß-(2,6)-linked fructose polymer, exhibits diverse properties that impart versatility, rendering it a highly sought-after biopolymer with various industrial applications. Levan can be produced by various microorganisms using sucrose, food industry byproducts and agricultural wastes. Microbial levan represents the most potent cost-effective process for commercial-scale levan production. This study reviews the optimization of levan production by understanding its biosynthesis, physicochemical properties and the fermentation process. In addition, genetic and protein engineering for its increased production and emerging methods for its detection are introduced and discussed. All of these comprehensive studies could serve as powerful tools to optimize levan production and broaden its applications across various industries.

Functional Endophytes Regulating Plant Secondary Metabolism: Current Status, Prospects and Applications.

Endophytes, which are widely found in host plants and have no harmful effects, are a vital biological resource. Plant endophytes promote plant growth and enhance plants' resistance to diseases, pests, and environmental stresses. In addition, they enhance the synthesis of important secondary metabolites in plants and improve the potential applicability of plants in agriculture, medicine, food, and horticulture. In this review, we summarize the recent progress in understanding the interaction between endophytes and plants and summarize the construction of synthetic microbial communities (SynComs) and metaomics analysis of the interaction between endophytes and plants. The application and development prospects of endophytes in agriculture, medicine, and other industries are also discussed to provide a reference for further study of the interaction between endophytes and plants and further development and utilization of endophytes.

Combined transcriptome and widely targeted metabolome analysis reveals the potential mechanism of HupA biosynthesis and antioxidant activity in Huperzia serrata.

Introduction: Huperzia serrata is a traditional Chinese herb that has gained much attention for its production of Huperzine A (HupA). HupA has shown promise on treating Alzheimer's disease (AD). However, the biosynthetic pathway and molecular mechanism of HupA in H. serrata are still not well understood. Methods: Integrated transcriptome and metabolome analysis was performed to reveal the molecular mechanisms related to HupA biosynthesis and antioxidant activity in Huperzia serrata. Results: HT (in vitro H. serrata thallus) exhibits higher antioxidant activity and lower cytotoxicity than WH (wild H. serrata). Through hierarchical clustering analysis and qRT-PCR verification, 7 important enzyme genes and 13 transcription factors (TFs) related to HupA biosynthesis were detected. Among them, the average |log2FC| value of CYP (Cytochrome P450) and CAO (Copper amine oxidase) was the largest. Metabolomic analysis identified 12 metabolites involved in the HupA biosynthesis and 29 metabolites related to antioxidant activity. KEGG co-enrichment analysis revealed that tropane, piperidine and pyridine alkaloid biosynthesis were involved in the HupA biosynthesis pathway. Furthermore, the phenylpropanoid, phenylalanine, and flavonoid biosynthesis pathway were found to regulate the antioxidant activity of H. serrata. The study also identified seven important genes related to the regulation of antioxidant activity, including PrAO (primary-amine oxidase). Based on the above joint analysis, the biosynthetic pathway of HupA and potential mechanisms of antioxidant in H. serrata was constructed. Discussion: Through differential transcriptome and metabolome analysis, DEGs and DAMs involved in HupA biosynthesis and antioxidant-related were identified, and the potential metabolic pathway related to HupA biosynthesis and antioxidant in Huperzia serrata were constructed. This study would provide valuable insights into the HupA biosynthesis mechanism and the H. serrata thallus medicinal value.

Advancements in heparosan production through metabolic engineering and improved fermentation.

Heparin is one of the most widely used natural drugs, and has been the preferred anticoagulant and antithrombotic agent in the clinical setting for nearly a century. Heparin also shows increasing therapeutic potential for treating inflammation, cancer, and microbial and viral diseases, including COVID-19. With advancements in synthetic biology, heparin production through microbial engineering of heparosan offers a cost-effective and scalable alternative to traditional extraction from animal tissues. Heparosan serves as the starting carbon backbone for the chemoenzymatic synthesis of bioengineered heparin, possessing a chain length that is critically important for the production of heparin-based therapeutics with specific molecular weight (MW) distributions. Recent advancements in metabolic engineering of microbial cell factories have resulted in high-yield heparosan production. This review systematically analyzes the key modules involved in microbial heparosan biosynthesis and the latest metabolic engineering strategies for enhancing production, regulating MW, and optimizing the fermentation scale-up of heparosan. It also discusses future studies, remaining challenges, and prospects in the field.

Recent advances on bioactive compounds, biosynthesis mechanism, and physiological functions of Nelumbo nucifera.

Nelumbo nucifera Gaertn, commonly known as lotus, is a genus comprising perennial and rhizomatous aquatic plants, found throughout Asia and Australia. This review aimed to cover the biosynthesis of flavonoids, alkaloids, and lipids in plants and their types in different parts of lotus. This review also examined the physiological functions of bioactive compounds in lotus and the extracts from different organs of the lotus plant. The structures and identities of flavonoids, alkaloids, and lipids in different parts of lotus as well as their biosynthesis were illustrated and updated. In the traditional medicine systems and previous scientific studies, bioactive compounds and the extracts of lotus have been applied for treating inflammation, cancer, liver disease, Alzheimer's disease, etc. We suggest future studies to be focused on standardization of the extract of lotus, and their pharmacological mechanisms as drugs or functional foods. This review is important for the lotus-based food processing and application.

Multi-omics combined with MALDI mass spectroscopy imaging reveals the mechanisms of biosynthesis of characteristic compounds in Tetrastigma hemsleyanum Diels et Gilg.

Tetrastigma hemsleyanum Diels et Gilg is recognized as a source of extracts with various desirable bioactivities. However, current knowledge regarding the mechanisms of biosynthesis of flavonoids, phenolic compounds, and other bioactive chemicals is limited. We conducted comprehensive tissue distribution studies and biosynthetic analyses of the 26 main bioactive compounds of this plant. The majority of flavonoids exhibited higher concentrations in the cortex (CT) compared to the vascular cylinder (VC). The expression levels of genes and proteins in CT and VC were quantified using mRNA sequencing and isobaric tags for relative and absolute quantification (iTRAQ). A total of 31,700 genes were identified, among which 4921 exhibited differential expression between CT and VC. A total of 13,996 proteins were identified in the proteomes of CT and VC, with 927 showing differential expression. Co-expression network analyses of DEGs and DEPs from multiple sites demonstrated substantial pathway variations linked to flavonoid biosynthesis. Through differential enrichment analysis, a total of 32 genes involved in the flavone biosynthesis pathway were identified, with iTRAQ specifically detecting C3'H, F3H and FLS. Pearson correlation analysis revealed a strong association between the expression levels of C3'H, F3H, and FLS and the concentrations of flavonoids. The validation of multiple genes encoding pivotal enzymes was conducted using real-time fluorescence quantitative PCR (RT-qPCR). The findings provide a foundation for future investigations into the molecular mechanisms and functional characterization of T. hemsleyanum candidate genes associated with characteristic compounds.

Precise Regulation of Juvenile Hormone III R-Stereoisomer Synthesis by Apis mellifera through Specifically Binding Methyl-(2E,6E)-farnesoate and Strictly Controlling Its Titer.

Juvenile hormone III (JH III) is a crucial hormone synthesized exclusively as R-stereoisomer in most insects. Herein, we established a mature Tris-HCl culture system for essential biochemical reactions and applied stable instrumental detection methods to analyze JH III, methyl farnesoate (MF) and juvenile hormone acid (JHA) using UPLC-MS/MS. Our results revealed that the R-JH III terminal synthesis pathway in Apis mellifera follows the "esterify then epoxidize" sequence, with precise methyl-(2E,6E)-farnesoate titer regulation and its spatial cis-trans isomerism, achieving selective R-JH III synthesis. Furthermore, we observed that the preferred generation of S/R-JH III chiral enantiomers varied depending on the spatial cis-trans isomerism of different MFs. Our results suggest that S-JH III could theoretically exist in insects, offering a novel perspective for understanding the synthesis mechanism of diverse complex juvenile hormones in different insect species.

Biosynthetic Mechanisms of Secondary Metabolites Promoted by the Interaction Between Endophytes and Plant Hosts.

Endophytes is a kind of microorganism resource with great potential medicinal value. The interactions between endophytes and host not only promote the growth and development of each other but also drive the biosynthesis of many new medicinal active substances. In this review, we summarized recent reports related to the interactions between endophytes and hosts, mainly regarding the research progress of endophytes affecting the growth and development of host plants, physiological stress and the synthesis of new compounds. Then, we also discussed the positive effects of multiomics analysis on the interactions between endophytes and their hosts, as well as the application and development prospects of metabolites synthesized by symbiotic interactions. This review may provide a reference for the further development and utilization of endophytes and the study of their interactions with their hosts.

Metabolomic Analysis of Biosynthesis Mechanism of ε-Polylysine Produced by Streptomyces diastatochromogenes.

ε-Polylysine (ε-PL), a natural preservative with broad-spectrum antimicrobial activity, has been widely used as a green food additive, and it is now mainly produced by Streptomyces in industry. In the previous study, strain 6#-7 of high-yield ε-PL was obtained from the original strain TUST by mutagenesis. However, the biosynthesis mechanism of ε-PL in 6#-7 is still unclear. In this study, the metabolomic analyses of the biosynthesis mechanism of ε-PL in both strains are investigated. Results show that the difference in metabolisms between TUST and 6#-7 is significant. Based on the results of both metabolomic and enzymatic activities, a metabolic regulation mechanism of the high-yield strain is revealed. The transport and absorption capacity for glucose of 6#-7 is improved. The enzymatic activity benefits ε-PL synthesis, such as pyruvate kinase and aspartokinase, is strengthened. On the contrary, the activity of homoserine dehydrogenase in the branched-chain pathways is decreased. Meanwhile, the increase of trehalose, glutamic acid, etc. makes 6#-7 more resistant to ε-PL. Thus, the ability of the mutagenized strain 6#-7 to synthesize ε-PL is enhanced, and it can produce more ε-PLs compared with the original strain. For the first time, the metabolomic analysis of the biosynthesis mechanism of ε-PL in the high-yield strain 6#-7 is investigated, and a possible mechanism is then revealed. These findings provide a theoretical basis for further improving the production of ε-PL.

Recent Strategies for the Biosynthesis of Ergothioneine.

Ergothioneine (EGT) is a unique naturally occurring amino acid that is usually biosynthesized by bacteria and fungi. As a food-derived antioxidant and cytoprotectant, it has several physiological benefits and has a wide range of applications in food, medicine, and cosmetics. Traditional production of EGT is mainly through biological extraction or chemical synthesis; however, these methods are inefficient, making large-scale production to meet the growing market demand difficult. Nowadays, the rapid development of synthetic biology has greatly accelerated the research on the EGT production by microbial fermentation. In this paper, the biological characteristics, applications, biosynthesis, separation, and detection methods of EGT were fully reviewed. Furthermore, the approaches and challenges for engineering microbial cells to efficiently synthesize EGT were also discussed. This work provides new ideas and future research potentials in EGT production.

Versatile strategies for bioproduction of hyaluronic acid driven by synthetic biology.

Owing to its outstanding water-retention ability, viscoelasticity, biocompatibility and non-immunogenicity, Hyaluronic acid (HA), a natural linear polymer alternating linked by d-glucuronic acid and N-acetylglucosamine, has been widely employed in cosmetic, medical and clinical applications. With the development of synthetic biology and bioprocessing optimization, HA production via microbial fermentation is an economical and sustainable alternative over traditional animal extraction methods. Indeed, recently Streptococci and other recombinant systems for HA synthesis has received increasing interests due to its technical advantages. This review summarizes the production of HA by microorganisms and demonstrates its synthesis mechanism, focusing on the current status in various production systems, as well as common synthetic biology strategies include driving more carbon flux into HA biosynthesis and regulating the molecular weight (MW), and finally discusses the major challenges and prospects.

Molecular insights into lipoxygenases for biocatalytic synthesis of diverse lipid mediators.

Oxylipins derived mainly from C20- and C22-polyunsaturated fatty acids (PUFAs), termed lipid mediators (LMs), are essential signalling messengers involved in human physiological responses associated with homeostasis and healing process for infection and inflammation. Some LMs involved in the resolution of inflammation and infection are termed specialized pro-resolving mediators (SPMs), which are generated by human M2 macrophages or polymorphonuclear leukocytes and have the potential to protect and treat hosts from bacterial and viral infections by phagocytosis activation. Lipoxygenases (LOXs) biosynthesize regio- and stereoselective LMs. Thus, understanding the regio- and stereoselectivities of LOXs for PUFAs at a molecular level is important for the biocatalytic synthesis of diverse LMs. Here, we elucidate the catalytic mechanisms and discuss regio- and stereoselectivities and their changes of LOXs determined by insertion direction and position of the substrate and oxygen at a molecular level for the biosynthesis of diverse human LMs. Recently, the biocatalytic synthesis of PUFAs to human LMs or analogues has been conducted using microbial LOXs. Such microbial LOXs involved in the biosynthesis of LMs are expected to exert significantly higher activity and stability than human LOXs. Diverse regio- and stereoselective LOXs can be obtained from microorganisms, which represent a wealth of genomic sources. We reconstruct the biosynthetic pathways of LOX-catalyzed LMs in humans and other organisms. Furthermore, we suggest the effective methods of biocatalytic synthesis of diverse human LMs from PUFAs or glucose by using microbial LOXs, increasing the stability and activity of LOXs, combining the reactions of LOXs, and constructing metabolic pathways.

The Biosynthesis Mechanism Involving 2,3-Pentanedione and Aminoacetone Describes the Production of 2-Ethyl-3,5-dimethylpyrazine and 2-Ethyl-3,6-dimethylpyrazine by Bacillus subtilis.

2-Ethyl-3,5(3,6)-dimethylpyrazines (EDMPs) have a pleasant aroma of roasted cocoa or nuts with an extreme low odor threshold that have potential in industrial applications as food fragrances. The food fermentation process can accumulate EDMPs, and this might be the chance to study the biosynthesis mechanism of EDMPs under mild conditions for "natural" EDMPs' production. In this study, an EDMP-producing strain was isolated from baijiu fermentation. This strain was identified as Bacillus subtilis, a generally regarded as safe organism. After reasonable assumption and substrate addition and isotope-labeled experiments, we found that EDMPs are produced from l-threonine and d-glucose at environmental temperature and pressure. In addition, aminoacetone, the metabolite of l-threonine, and 2,3-pentanedione, the metabolite of l-threonine and d-glucose, are intermediates for the production of EDMPs. This study proposed and confirmed the biosynthesis pathway of EDMPs. It will be helpful for the industrial production of EDMPs and provides reference for the biosynthetic mechanism analysis of other valuable pyrazines.

Mycosynthesis of bactericidal silver and polymorphic gold nanoparticles: physicochemical variation effects and mechanism.

AIM: Extracellular synthesis of silver and gold nanoparticles using aqueous cell-free filtrate (CFF) of endophytic Chaetomium globosum and characterization of its bioactive proteins. METHODS: Temperature and pH gradients were used to assess their effects on dimensions of NPs. NPs were tested in vivo for antibacterial activity. MALDI-TOF-MS/MS was used for characterization of CFF proteins. RESULTS: Fungal CFF fabricated nanoparticles of various shape under varied physicochemical conditions. Silver nanoparticles showed significantly (p ≤ 0.5) enhanced antibacterial activity against Staphylococcus aureus and Klebsiella pneumoniae compared with AgNO3. Two prominent CFF proteins showed homology with benzoate 4-monooxygenase cytochrome P450 and ubiquinol-cytochrome c reductase. CONCLUSION: The study achieved controlled mycosynthesis of NPs and explains the hitherto poorly known mechanism of reduction, stabilization and antibacterial activity of nanoparticles.

Toward understanding the key enzymes involved in ß-poly (L-malic acid) biosynthesis by Aureobasidium pullulans ipe-1.

ß-poly (L-malic acid) (PMLA) is a biopolyester which has attracted industrial interest for its potential application in medicine and other industries. A high dissolved oxygen concentration (DO) was beneficial for PMLA production, while the mechanisms of DO in PMLA biosynthesis by Aureobasidium pullulans are still poorly understood. In this work, the amount of PMLA was first compared when A. pullulans ipe-1 were cultured under a high DO level (70% saturation) and a low DO level (10% saturation). Meanwhile, the key enzymes involved in different pathways of the precursor L-malic acid biosynthesis were studied. The results revealed that the activities of glucose-6-phosphate dehydrogenase (G6PDH) and phosphoenolpyruvate carboxylase (PEPC) were positively correlated with cell growth and PMLA production, while the activities of phosphofructokinases (PFK), pyruvic carboxylase (PC) and citrate synthetase (CS) did no show such correlations. It indicated that the Pentose Phosphate Pathway (PPP) may play a vital role in cell growth and PMLA biosynthesis. Moreover, the precursor L-malic acid for PMLA biosynthesis was mainly biosynthesized through phosphoenolpyruvic acid (PEP) via oxaloacetate catalyzed by PEPC. It was also found that low concentration of sodium fluoride (NaF) might impel carbon flux flow to the oxaloacetate through PEP, but inhibit the flux to the oxaloacetate via pyruvic acid.

Cloning, Functional Characterization and Site-Directed Mutagenesis of 4-Coumarate: Coenzyme A Ligase (4CL) Involved in Coumarin Biosynthesis in Peucedanum praeruptorum Dunn.

Coumarins are the main bioactive compounds in Peucedanum praeruptorum Dunn, a common Chinese herbal medicine. Nevertheless, the genes involved in the biosynthesis of core structure of coumarin in P. praeruptorum have not been identified yet. 4-Coumarate: CoA ligase (4CL) catalyzes the formation of hydroxycinnamates CoA esters, and plays an essential role at the divergence point from general phenylpropanoid metabolism to major branch pathway of coumarin. Here, three novel putative 4CL genes (Pp4CL1, Pp4CL7, and Pp4CL10) were isolated from P. praeruptorum. Biochemical characterization of the recombinant proteins revealed that Pp4CL1 utilized p-coumaric and ferulic acids as its two main substrates for coumarin biosynthesis in P. praeruptorum. Furthermore, Pp4CL1 also exhibited activity toward caffeic, cinnamic, isoferulic, and o-coumaric acids and represented a bona fide 4CL. Pp4CL7 and Pp4CL10 had no catalytic activity toward hydroxycinnamic acid compounds. But they had close phylogenetic relationship to true 4CLs and were defined as 4CL-like genes. Among all putative 4CLs, Pp4CL1 was the most highly expressed gene in roots, and its expression level was significantly up-regulated in mature roots compared with seedlings. Subcellular localization studies showed that Pp4CL1 and Pp4CL10 proteins were localized in the cytosol. In addition, site-directed mutagenesis of Pp4CL1 demonstrated that amino acids of Tyr-239, Ala-243, Met-306, Ala-309, Gly-334, Lys-441, Gln-446, and Lys-526 were essential for substrate binding or catalytic activities. The characterization and site-directed mutagenesis studies of Pp4CL1 lays a solid foundation for elucidating the biosynthetic mechanisms of coumarins in P. praeruptorum and provides further insights in understanding the structure-function relationships of this important family of proteins.

Hydroxy-pentanones production by Bacillus sp. H15-1 and its complete genome sequence.

Acyloins are useful organic compounds with reactive adjacent hydroxyl group and carbonyl group. Current research is usually constrained to acetoin (i.e. 3-hydroxy-2-butanone) and the biological production of other acyloins was scarcely reported. In this study, two hydroxy-pentanone metabolites (3-hydroxy-2-pentanone and 2-hydroxy-3-pentanone) of Bacillus sp. H15-1 were identified by gas chromatography-mass spectrometry and authentic standards. Then the complete genome of this strain was sequenced and de novo assembled to a single circular chromosome of 4,162,101bp with a guanine-cytosine content of 46.3%, but no special genes were found for the biosynthesis of the hydroxy-pentanones. Since hydroxy-pentanones are the homologues of acetoin, the two genes alsD and alsS (encoding α-acetolactate decarboxylase and α-acetolactate synthase, respectively) responsible for acetoin formation in this strain were respectively expressed in Escherichia coli. The purified enzymes were found to be capable of transforming pyruvate and 2-oxobutanoate to the two hydroxy-pentanones. This study extends the knowledge on the biosynthesis of acyloins and provides helpful information for further utilizing Bacillus sp. H15-1 as a source of valuable acyloins.

"Coding" and "Decoding": hypothesis for the regulatory mechanism involved in heparan sulfate biosynthesis.

Heparan sulfate (HS) is widely distributed in mammalian tissues in the form of HS proteoglycans, which play essential roles in various physiological and pathological processes. In contrast to the template-guided processes involved in the synthesis of DNA and proteins, HS biosynthesis is not believed to involve a template. However, it appears that the final structure of HS chains was strictly regulated. Herein, we report research based hypothesis that two major steps, namely "coding" and "decoding" steps, are involved in the biosynthesis of HS, which strictly regulate its chemical structure and biological activity. The "coding" process in this context is based on the distribution of sulfate moieties on the amino groups of the glucosamine residues in the HS chains. The sulfation of these amine groups is catalyzed by N-deacetylase/N-sulfotransferase, which has four isozymes. The composition and distribution of sulfate groups and iduronic acid residues on the glycan chains of HS are determined by several other modification enzymes, which can recognize these coding sequences (i.e., the "decoding" process). The degree and pattern of the sulfation and epimerization in the HS chains determines the extent of their interactions with several different protein factors, which further influences their biological activity.