Biomimetic natural biomaterials for tissue engineering and regenerative medicine:new biosynthesis methods,recent advances,and emerging applications

Biomimetic materials have emerged as attractive and competitive alternatives for tissue engineering(TE)and regenerative medicine.In contrast to conventional biomaterials or synthetic materials,biomimetic scaffolds based on natural biomaterial can offer cells a broad spectrum of biochemical and biophysical cues that mimic the in vivo extracellular matrix(ECM).Additionally,such materials have mechanical adaptability,micro-structure interconnectivity,and inherent bioactivity,making them ideal for the design of living implants for specific applications in TE and regenerative medicine.This paper provides an overview for recent progress of biomimetic natural biomaterials(BNBMs),including advances in their preparation,functionality,potential applications and future challenges.We highlight recent advances in the fabrication of BNBMs and outline general strategies for functionalizing and tailoring the BNBMs with various biological and physicochemical characteristics of native ECM.Moreover,we offer an overview of recent key advances in the functionalization and applications of versatile BNBMs for TE applications.Finally,we conclude by offering our perspective on open challenges and future developments in this rapidly-evolving field.

Rational Engineering of Secondary Metabolic Pathways in a Heterologous Host to Enable the Biosynthesis of Hibarimicin Derivatives with Enhanced Anti-Melanomic Activity

A 61-kb biosynthetic gene cluster(BGC),which is accountable for the biosynthesis of hibarimicin(HBM)B from Microbispora rosea subsp.hibaria TP-A0121,was heterologously expressed in Streptomyces coelicolor M1154,which generated a trace of the target products but accumulated a large amount of shunt products.Based on rational analysis of the relevant secondary metabolism,directed engineering of the biosynthetic pathways resulted in the high production of HBM B,as well as new HBM derivates with improved antitumor activity.These results not only establish a biosynthetic system to effectively synthesize HBMs-a class of the largest and most complex Type-Ⅱpolyketides,with a unique pseudo-dimeric structure-but also set the stage for further engineering and deep investigation of this complex biosynthetic pathway toward potent anticancer drugs.

Chain Elongation Using Native Soil Inocula:Exceptional n-Caproate Biosynthesis Performance and Microbial Mechanisms

This study demonstrates the feasibility and effectiveness of utilizing native soils as a resource for inocula to produce n-caproate through the chain elongation(CE)platform,offering new insights into anaerobic soil processes.The results reveal that all five of the tested soil types exhibit CE activity when supplied with high concentrations of ethanol and acetate,highlighting the suitability of soil as an ideal source for n-caproate production.Compared with anaerobic sludge and pit mud,the native soil CE system exhibited higher selectivity(60.53%),specificity(82.32%),carbon distribution(60.00%),electron transfer efficiency(165.00%),and conductivity(0.59 ms∙cm^(-1)).Kinetic analysis further confirmed the superiority of soil in terms of a shorter lag time and higher yield.A microbial community analysis indicated a positive correlation between the relative abundances of Pseudomonas,Azotobacter,and Clostridium and n-caproate production.Moreover,metagenomics analysis revealed a higher abundance of functional genes in key microbial species,providing direct insights into the pathways involved in n-caproate formation,including in situ CO_(2)utilization,ethanol oxidation,fatty acid biosynthesis(FAB),and reverse beta-oxidation(RBO).The numerous functions in FAB and RBO are primarily associated with Pseudomonas,Clostridium,Rhodococcus,Stenotrophomonas,and Geobacter,suggesting that these genera may play roles that are involved or associated with the CE process.Overall,this innovative inoculation strategy offers an efficient microbial source for n-caproate production,underscoring the importance of considering CE activity in anaerobic soil microbial ecology and holding potential for significant economic and environmental benefits through soil consortia exploration.

Identification of key enzymes in lignocellulose biosynthesis from dynamic observations in maize stalks

Maize stalk lignin and cellulose contents are linked to lodging resistance,disease resistance,feed quality and ethanol conversion efficiency.After the six-leaf stage of maize(V6),these constituents are biosynthesized and accumulated under the control of related enzymes and genes.However,the key enzymes,critical MYB transcription factors,and their dynamic alterations pattern under natural field circumstances are still unknown.Hence,we selected five cultivars with significant differences in lignocellulose content and lodging resistance as testing materials,performed field experiments for two years,and investigated the dynamics of lignin and cellulose content,related enzyme concentrations,and gene expression levels in the 3^(rd) and 5^(th) internodes above the ground after V6.The results showed that lignin and cellulose content increased after V6,stabilizing during the silking stage.This study identified COMT(caffeic acid 3-Omethyltransferase),TAL(tyrosine ammonia-lyase)and PAL(phenylalanine ammonia-lyase)as the key enzymes of lignin biosynthesis,while ZmCOMT,ZmCesA10 and ZmCesA8 were identified as essential genes.ZmMYB8,ZmMYB31 and ZmMYB39 were involved in regulating the expression of genes related to lignin synthesis,with ZmMYB31 potentially acting as a key negative regulator,while ZmMYB39 and ZmMYB8 acting as positive regulators.The study also found that around 14 d after V6 was a critical stage for regulating lignocellulose synthesis in the 3^(rd) to 5^(th) basal internode.This provides a theoretical foundation for developing regulatory techniques and breeding new cultivars to enhance lodging and disease resistance as well as the utility of maize stalks.

Transcriptome analysis reveals steroid hormones biosynthesis pathway involved in abdominal fat deposition in broilers

Excessive abdominal fat deposition reduces the feed efficiency and increase the cost of production in broilers.Therefore,it is an important task for poultry breeders to breed broilers with low abdominal fat.Abdominal fat deposition is a highly complex biological process,and its molecular basis remains elusive.In this study,we performed transcriptome analysis to compare gene expression profiles at different stages of abdominal fat deposition to identify the key genes and pathways involved in abdominal fat accumulation.We found that abdominal fat weight(AFW)increased gradually from day 35(D35)to 91(D91),and then decreased at day 119(D119).Accordingly,after detecting differentially expressed genes(DEGs)by comparing gene expression profiles at D35 vs.D63 and D35 vs.D91,and identifying gene modules associated with fat deposition by weighted gene co-expression network analysis(WGCNA),we performed intersection analysis of the detected DEGs and WGCNA gene modules and identified 394 and 435 intersecting genes,respectively.The results of the Gene Ontology(GO)functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analyses showed that the steroid hormone biosynthesis and insulin signaling pathways were co-enriched in all intersecting genes,steroid hormones have been shown that regulated insulin signaling pathway,indicating the importance of the steroid hormone biosynthesis pathway in the development of broiler abdominal fat.We then identified 6 hub genes(ACTB,SOX9,RHOBTB2,PDLIM3,NEDD9,and DOCK4)related to abdominal fat deposition.Further analysis also revealed that there were direct interactions between 6 hub genes.SOX9 has been shown to bind to proteins required for steroid hormone receptor binding,and RHOBTB2 indirectly regulates the steroid hormones biosynthesis through cyclin factor,and ultimately affect fat deposition.Our results suggest that the genes RHOBTB2 and SOX9 play an important role in fat deposition in broilers,by regulating steroid hormone synthesis.These findings provide new targets and directions for further studies on the mechanisms of fat deposition in chicken.

Wetting alternating with partial drying during grain filling increases lysine biosynthesis in inferior rice grain

Lysine content is a criterion of the nutritional quality of rice.Understanding the process of lysine biosynthesis in early-flowering superior grain(SG)and late-flowering inferior grain(IG)of rice would advance breeding and cultivation to improve nutritional quality.However,little information is available on differences in lysine anabolism between SG and IG and the underlying mechanism,and whether and how irrigation regimes affect lysine anabolism in these grains.A japonica rice cultivar was grown in the field and two irrigation regimes,continuous flooding(CF)and wetting alternating with partial drying(WAPD),were imposed from heading to the mature stage.Lysine content and activities of key enzymes of lysine biosynthesis,and levels of brassinosteroids(BRs)were lower in the IG than in the SG at the early grainfilling stage but higher at middle and late grain-filling stages.WAPD increased activities of these key enzymes,BR levels,and contents of lysine and total amino acids in IG,but not SG relative to CF.Application of 2,4-epibrassinolide to rice panicles in CF during early grain filling reproduced the effects of WAPD,but neither treatment altered the activities of enzymes responsible for lysine catabolism in either SG or IG.WAPD and elevated BR levels during grain filling increased lysine biosynthesis in IG.Improvement in lysine biosynthesis in rice should focus on IG.

OsFK1 encodes C-14 sterol reductase,which is involved in sterol biosynthesis and affects premature aging of leaves in rice

The enzyme C-14 sterol reductase is involved in biosynthesis of brassinosteroids(BR)and sterols,as well as plant development.OsFK1,a member of the sterol biosynthesis pathway located in the endoplasmic reticulum(ER),encodes C-14 sterol reductase.However,there is little research on the function of C-14 sterol reductase in rice.Compared with the wild type,an osfk1 mutant showed dwarf phenotype and premature aging in the second leaf during the trefoil stage,and abnormal development of leaf veins during the tillering stage.The osfk1 mutant showed signs of aberrant PCD,as evidenced by TUNEL staining.This suggested that high ROS buildup caused DNA damage and ROS-mediated cell death in the mutant.The osfk1 mutant also showed decreased chlorophyll content and aberrant chloroplast structure.Sequencing of the osfk1 mutant allele revealed a non-synonymous G to A mutation in the final intron,leading to early termination.Here,we identified the OsFK1 allele,cloned it by Mutmap sequencing,and verified it by complementation.HPLC-MS/MS assays demonstrated that the osfk1 mutation caused lower phytosterol levels.These findings showed that the OsFK1 allele encoding C-14 sterol reductase is involved in phytosterol biosynthesis and mediates normal development of rice plants.

The biosynthesis of alarm pheromone in the wheat aphid Rhopalosiphum padi is regulated by hormones via fatty acid metabolism

Aphids are major insect pests in agriculture and forestry worldwide.Following attacks by natural enemies,many aphids release an alarm pheromone to protect their population.In most aphids,the main component of the aphid alarm pheromone(AAP)is the sesquiterpene hydrocarbon(E)-β-farnesene(EβF).However,the mechanisms behind its biosynthesis and regulation remain poorly understood.In this study,we used the bird cherry–oat aphid Rhopalosiphum padi,which is an important wheat aphid,to investigate the regulatory mechanisms of EβF biosynthesis.Our results showed that EβF biosynthesis occurs during the mature embryo period and the molting period of the 1st-and 2nd-instar nymphs.Triglycerides provide the prerequisite material for EβF production and release.Based on transcriptome sequencing,RNAi analysis,hormone treatments,and quantitative measurements,we found that the biosynthesis of EβF utilizes acetyl coenzyme A produced from fatty acid degradation,which can be suppressed by juvenile hormone but it is promoted by 20-hydroxyecdysone through the modulation of fatty acid metabolism.This is the first systemic study on the modulation of EβF production in aphids.The results of our study provide insights into the molecular regulatory mechanisms of AAP biosynthesis,as well as valuable information for designing potential aphid control strategies.

Identification and Molecular Characterization of the Alkaloid Biosynthesis Gene Family in Dendrobium catenatum

As one of the main active components of Dendrobium catenatum, alkaloids have high medicinal value. The physicochemicalproperties, conserved domains and motifs, phylogenetic analysis, and cis-acting elements of the genefamily members in the alkaloid biosynthesis pathway of D. catenatum were analyzed by bioinformatics, and theexpression of the genes in different years and tissues was analyzed by qRT-PCR. There are 16 gene families,including 25 genes, in the D. catenatum alkaloid biosynthesis pathway. The analysis of conserved domains andmotifs showed that the types, quantities, and orders of domains and motifs were similar among members ofthe same family, but there were significant differences among families. Phylogenetic analysis indicated that thegene family members showed some evolutionary conservation. Cis-acting element analysis revealed that therewere a large number of light-responsive elements and MYB (v-myb avian myeloblastosis viral oncogene homolog)-related elements in these genes. qRT-PCR showed that expressions of gene family members involved in alkaloidsynthesis were different in different years and tissues of D. catenatum. This study provides a theoretical basisfor further exploration of the regulatory mechanisms of these genes in the alkaloid biosynthesis of D. catenatum.

Research Progress on Functions and Biosynthesis of D-Psicose

D-Psicose,a naturally occurring rare sugar,exhibits a sweetness approximately 70%that of sucrose.It possesses high solubility,antioxidant activity,anti-inflammatory properties,and the ability to regulate cholesterol levels and enhance insulin sensitivity.However,D-psicose is relatively scarce in nature,making large-scale extraction and utilization impractical.Consequently,the development of cost-effective synthetic strategies for D-psicose is pivotal for its industrial application.In recent years,the Izumoring strategy has emerged as an efficient alternative to chemical synthesis for producing D-psicose.Nonetheless,limitations in the biotransformation of D-psicose,primarily governed by the conversion rate of D-psicose 3-epimerase(DPEase)and enzyme yield,continue to pose challenges in achieving economically viable production.Enzyme engineering and the establishment of high-level expression systems remain crucial avenues for reducing the overall biosynthesis costs.

Biosynthesis of Silver Nanoparticles with Clerodendron phlomoides Leave Extract:Particle Morphology,Antimicrobial Potential and Application

Silver nanoparticles are versatile nanomaterials that have found numerous applications in various fields.The use of plant extract for the synthesis of silver is a green and sustainable approach.Clerodendron phlomoides leaves extract has been found to contain various phytochemicals,such as phenols,flavonoids,tannins,and alkaloids,which possess reducing and stabilizing properties that can aid the production of silver particles.In this paper,morphological and topographical analyses were performed on silver nanoparticles.The biosynthesized silver nanoparticles showed antimicrobial potential against wound pathogens.SEM and TEM micrographs revealed that the particles were sphere and nanosized,which makes them suitable for various biomedical applications.

EXPRESSION OF c－myc GENE AND BIOSYNTHESIS OF BIOLOGICAL MACROMOLECULES IN ANTISENSE TRANSFECTANT HL_（60）~R－9

The recombinant plasmid PGC was constructed for transcription unit of c-myc gene with diorientation in vitro, to make RNA probes for detection of c-myc mRNA and antisence RNA expression of tranfectant HL-9,which was obtained from HL60 cells transfected with inducible c-myc antisense RNA expression plasmid. The results from HL-9 cells induced by Cd2+ indicated that expression of c-myc antisense RNA increased with Cd2+ concentration and exposure time, while c-myc mRNA expression progressively reduced. Using immunohistochemical technique no c-myc P62 protein expression was detected. The incorporation of 3H-TdR, 3H-UR and 3H-Leu revealed significant suppression of DNA, RNA and protein biosynthesis. It is suggested that the reversion changes previously reported in malignant Phenotypes of HL-9 cells and the inhibition of macromolecular biosynthesis mentioned above were associated with the blockade of c-myc gene expression by its antisense RNA.

The Chlorophyll Biosynthesis in Lotus Embryo Is Light-dependent

Angiosperms need light to synthesize chlorophyll, but lotus (Nelumbo nucifera Gaertn.) embryo was suspected to have the ability to form chlorophyll in the dark because lotus embryo can turn into green under the coverage of four layers of integuments (cotyledon, seed coat, pericarp, lotus pod) which were thought impossible for light to pass through. The authors excluded this possibility based on two experimental results: First, enclosing the young lotus pod with aluminium foil, the growth of louts embryo continued, but the chlorophyll formation was seriously inhibited. A lot of protochlorophyllide, chlorophyll precursor, were accumulated, most of which were combined with LPOR (light dependent protochlorophyllide oxidoreductase). Second, DPOR (dark or light-independent protochlorophyllide oxidoreductase) was the enzyme necessary for chlorophyll synthesis in the dark. The genes encoding DPOR were conservative in many species, but no homologues could be found in lotus genome. Taken together, authers' results clearly demonstrated that lotus embryo synthesizes chlorophyll only through the light-dependent pathway.

Advances in Trehalose Biosynthesis Pathways and Application of Molecular Biology Technique

This paper aims to provide better guidance for construction of trehalose-producing recombinant strains to further improve the yield of trehalose. The research progress on trehalose biosynthesis pathways and the application of molecular biology technique in trehalose study in recent 30 years, especially the last 10 years are reviewed. Results show that there are 5 pathways of trehalose synthesis. Although enzymes and genes of trehalose synthesis have been isolated and genetic engineering strains have increased gradually, the improvement of trehalose yield is still inadequate because most recombinant strains are limited to study the physicochemical properties of single enzyme. With the development of modern biological technology, especially the rapid development of DNA recombinant technology, metagenomics and synthetic biology, high expression of heterologous trehalose in recombinant strains would become a hot research topic in the future.

The Combinatorial Biosynthesis of＂Unnatural＂ Products with Polyketides

Polyketides have been widely used clinically due to their significant biological activities, but the needed structural and functional diversity cannot be achieved by common chemical synthetic methods. The tool of combinatorial biosynthesis provides the possibility to produce "unnatural" natural drugs, which has achieved initial success. This paper provides an overview for the strategies of combinatorial biosynthesis in producing the structural and functional diversity of polyketides, including the redesign of metabolic flow, polyketide synthase(PKS) engineering, and PKS post-translational modification. Although encouraging progress has been made in the last decade, challenges still exist regarding the rational combinatorial biosynthesis of polyketides. In this review, the perspectives of polyketide combinatorial biosynthesis are also discussed.

Functional Characterization of SmbHLH13 in Anthocyanin Biosynthesis and Flowering in Eggplant

Anthocyanin is abundant in a few vegetables,including eggplant.It protects plants from abiotic stress and benefits human health,making the research of anthocyanin biosynthesis increasingly important.Flowering time is an important reference for judging reproduction and adaptability,which can guide plant production.In this study,SmbHLH13 from eggplant was identified.Yeast one-hybrid and dual-luciferase assays showed that SmbHLH13 binded and activated the expression of structural genes SmCHS and SmF3H in anthocyanin biosynthesis and it also was bound to the promoter of the key gene SmFT in flowering.Furthermore,genetic transformation of Arabidopsis revealed that overexpression of SmbHLH13 enhanced anthocyanin accumulation and delayed flowering.These results demonstrated that SmbHLH13 might promote anthocyanin accumulation through positive regulation of SmCHS and SmF3H.Moreover,SmbHLH13 might have a role in delaying eggplant flowering.

Effects of uniconazole with or without micronutrient on the lignin biosynthesis,lodging resistance,and winter wheat production in semiarid regions

Lodging stress results in grain yield and quality reduction in wheat. Uniconazole, a potential plant growth regulator significantly enhances lignin biosynthesis and thus provides mechanical strength to plants in order to cope with lodging stress. A field study was conducted during the 2015–2016 and 2016–2017 growing seasons, to investigate the effects of uniconazole sole application or with micronutrient on the lignin biosynthesis, lodging resistance, and production of winter wheat. In the first experiment, uniconazole at concentrations of 0(CK), 15(US1), 30(US2), and 45(US3) mg L^-1 was applied as sole seed soaking, while in the second experiment with manganese(Mn) at concentration of 0.06 g L^-1 Mn, 0.06 g L^-1 Mn+ 15 mg L^-1 uniconazole(UMS1), 0.06 g L^-1 Mn+30 mg L^-1 uniconazole(UMS2), and 0.06 g L^-1 Mn+45 mg L^-1 uniconazole(UMS3), respectively. Uniconazole sole application or with micronutrient significantly increased the lignin content by improving the lignin-related enzyme activities of phenylalanine ammonia-lyase, cinnamyl alcohol dehydrogenase, tyrosine ammonialyase, and peroxidase, ameliorating basal internode characteristics, and breaking strength. The spike length, spike diameter, spikes/plant, weight/spike, yield/spike, and grain yield increased and then decreased with uniconazole application at a higher concentration, where their maximum values were recorded with UMS2 and US2 treatments. The lignin accumulation was positively correlated with lignin-related enzyme activities and breaking strength while, negatively correlated with lodging rate. Uniconazole significantly improved the lignin biosynthesis, lodging resistance, and grain yield of winter wheat and the treatments which showed the greatest effects were uniconazole seed soaking with micronutrient at a concentration of 30 mg L^-1 and 0.06 g L^-1 , and uniconazole sole seed soaking at a concentration of 30 mg L^-1 .

OsHemA gene, encoding glutamyl-tRNA reductase(GluTR) is essential for chlorophyll biosynthesis in rice(Oryza sativa)

Chlorophyll(Chl) biosynthesis is essential for photosynthesis and plant growth.Glutamyl-tRNA reductase(GluTR) catalyzes glutamyl-tRNA into glutamate-1-semialdehyde(GSA) and initiates the chlorophyll biosynthesis.Even though the main role of GluTR has been established,the effects caused by natural variations in its corresponding gene remain largely unknown.Here,we characterized a spontaneous mutant in paddy field with Chl biosynthesis deficiency,designated as cbd1.With intact thylakoid lamellar structure,the cbd1 plant showed light green leaves and reduced Chl and carotenoids(Cars) content significantly compared to the wild type.By map-based gene cloning,the mutation was restricted within a 57-kb region on chromosome 10,in which an mPingA miniature inverted-repeat transposable element(MITE) inserted in the promoter region of OsHemA gene.Both leaf color and the pigment contents in cbd1 were recovered in a complementation test,confirming OsHemA was responsible for the mutant phenotype.OsHemA was uniquely predicted to encode GluTR and its expression level was dramatically repressed in cbd1.Transient transformation in protoplasts demonstrated that GluTR localized in chloroplasts and a signal peptide exists in its N-terminus.A majority of Chl biosynthesis genes,except for POR and CHLG,were down-regulated synchronously by the repression of OsHemA,suggesting that an attenuation occurred in the Chl biosynthesis pathway.Interestingly,we found major agronomic traits involved in rice yield were statistically unaffected,except for the number of full grains per panicle was increased in cbd1.Collectively,OsHemA plays an essential role in Chl biosynthesis in rice and its weak allele can adjust leaf color and Chls content without compromise to rice yield.

How rice organs are colored: The genetic basis of anthocyanin biosynthesis in rice

Anthocyanins are a major subclass of flavonoids that have diverse biological functions and benefit human health.In rice(Oryza sativa),the various colors shown by organs are due mainly to the accumulation of anthocyanins and are traits associated with domestication.Elucidating the genetic basis of anthocyanin biosynthesis in rice would support the engineering of anthocyanins as well as shedding light on the evolutionary history of O.sativa.We summarize recent progress in rice anthocyanin biosynthesis research,including gene cloning,biosynthetic pathway discovery,and study of the domestication process.We discuss the application of anthocyanin biosynthesis genes in rice breeding.Our object is to broaden knowledge of the genetic basis of anthocyanin biosynthesis in rice and support the breeding of novel rice cultivars.

AtTHIC, a gene involved in thiamine biosynthesis in Arabidopsis thaliana

Thiamine （vitamin B1） is an essential compound for organisms. It contains a pyrimidine ring structure and a thiazole ring structure. These two moieties of thiamine are synthesized independently and then coupled together. Here we report the molecular characterization of AtTHIC, which is involved in thiamine biosynthesis in Arabidopsis. AtTHIC is similar to Escherichia coli ThiC, which is involved in pyrimidine biosynthesis in prokaryotes. Heterologous expression of AtTHIC could functionally complement the thiC knock-out mutant of E. coll. Downregulation of AtTHIC expression by T-DNA insertion at its promoter region resulted in a drastic reduction of thiamine content in plants and the knock-down mutant thicl showed albino （white leaves） and lethal phenotypes under the normal culture conditions. The thicl mutant could be rescued by supplementation of thiamine and its defect functions could be complemented by expression ofAtTHIC cDNA. Transient expression analysis revealed that the AtTHIC protein targets plastids and chloroplasts. AtTHIC was strongly expressed in leaves, flowers and siliques and the transcription of AtTHIC was downregulated by extrinsic thiamine. In conclusion, AtTHIC is a gene involved in pyrimidine synthesis in the thiamine biosynthesis pathway of Arabidopsis, and our results provide some new clues for elucidating the pathway of thiamine biosynthesis in plants.