Fish Uses CTLA-4 Immune Checkpoint to Suppress mTORC1-Controlled T-Cell Glycolysis and Immunity.

As an immune checkpoint, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) suppresses the activation, proliferation, and effector function of T cells, thus preventing an overexuberant response and maintaining immune homeostasis. However, whether and how this immune checkpoint functions in early vertebrates remains unknown. In the current study, using a Nile tilapia (Oreochromis niloticus) model, we investigated the suppression of T cell response by CTLA-4 in bony fish. Tilapia CTLA-4 is constitutively expressed in lymphoid tissues, and its mRNA and protein expression in lymphocytes are upregulated following PHA stimulation or Edwardsiella piscicida infection. Blockade of CTLA-4 signaling enhanced T cell activation and proliferation but inhibited activation-induced T cell apoptosis, indicating that CTLA-4 negatively regulated T cell activation. In addition, blocking CTLA-4 signaling in vivo increased the differentiation potential and cytotoxicity of T cells, resulting in an enhanced T cell response during E. piscicida infection. Tilapia CTLA-4 competitively bound the B7.2/CD86 molecule with CD28, thus antagonizing the CD28-mediated costimulatory signal of T cell activation. Furthermore, inhibition of mammalian/mechanistic target of rapamycin complex 1 (mTORC1) signaling, c-Myc, or glycolysis markedly impaired the CTLA-4 blockade-enhanced T cell response, suggesting that CTLA-4 suppressed the T cell response of tilapia by inhibiting mTORC1/c-Myc axis-controlled glycolysis. Overall, the findings indicate a detailed mechanism by which CTLA-4 suppresses T cell immunity in tilapia; therefore, we propose that early vertebrates have evolved sophisticated mechanisms coupling immune checkpoints and metabolic reprogramming to avoid an overexuberant T cell response.

IL-10 Negatively Controls the Primary T Cell Response of Tilapia by Triggering the JAK1/STAT3/SOCS3 Axis That Suppresses NF-κB and MAPK/ERK Signaling.

The braking mechanisms to protect the host from tissue damage and inflammatory disease caused by an overexuberant immune response are common in many T cell subsets. However, the negative regulation of T cell responses and detailed mechanisms are not well understood in early vertebrates. In the current study, using a Nile tilapia (Oreochromis niloticus) model, we investigated the suppression of T cell immunity by IL-10. Tilapia encodes an evolutionarily conserved IL-10, whose expression in lymphocytes is markedly induced during the primary adaptive immune response against Aeromonas hydrophila infection. Activated T cells of tilapia produce IL-10, which in turn inhibits proinflammatory cytokine expression and suppresses PHA-induced T cell activation. Moreover, administration of IL-10 impairs the proliferation of tilapia T cells, reduces their potential to differentiate into Th subsets, and cripples the cytotoxic function, rendering the animals more vulnerable to pathogen attack. After binding to its receptor IL-10Ra, IL-10 activates the JAK1/STAT3 axis by phosphorylation and enhances the expression of the suppressor of cytokine signaling 3 (SOCS3), which in turn attenuates the activation of the NF-κB and MAPK/ERK signaling pathways, thus suppressing the T cell response of tilapia. Our findings elucidate a negative regulatory mechanism of T cell immunity in a fish species and support the notion that the braking mechanism of T cells executed through IL-10 existed prior to the divergence of the tetrapod lineage from teleosts. Therefore, this study, to our knowledge, provides a novel perspective on the evolution of the adaptive immune system.

TGF-ß1 suppresses the T-cell response in teleost fish by initiating Smad3- and Foxp3-mediated transcriptional networks.

Transforming growth factor-ß1 (TGF-ß1) can suppress the activation, proliferation, and function of many T-cell subsets, protecting organisms from inflammatory and autoimmune disease caused by an overexuberant immune response. However, whether and how TGF-ß1 regulates T-cell immunity in early vertebrates remain unknown. Here, using a Nile tilapia (Oreochromis niloticus) model, we investigated suppression of the T-cell response by TGF-ß1 in teleost species. Tilapia encodes an evolutionarily conserved TGF-ß1, the expression of which in lymphocytes is significantly induced during the immune response following Edwardsiella piscicida infection. Once activated, tilapia T cells increase TGF-ß1 production, which in turn suppresses proinflammatory cytokine expression and inhibits T-cell activation. Notably, we found administration of TGF-ß1 cripples the proliferation of tilapia T cells, reduces the potential capacity of Th1/2 differentiation, and impairs the cytotoxic function, rendering the fish more vulnerable to bacterial infection. Mechanistically, TGF-ß1 initiates the TGF-ßR/Smad signaling pathway and triggers the phosphorylation and nuclear translocation of Smad2/3. Smad3 subsequently interacts with several transcriptional partners to repress transcription of cytokines IL-2 and IFN-γ but promote transcription of immune checkpoint regulator CTLA4 and transcription factor Foxp3. Furthermore, TGF-ß1/Smad signaling further utilizes Foxp3 to achieve the cascade regulation of these T-cell genes. Taken together, our findings reveal a detailed mechanism by which TGF-ß1 suppresses the T cell-based immunity in Nile tilapia and support the notion that TGF-ß1 had already been employed to inhibit the T-cell response early in vertebrate evolution, thus providing novel insights into the evolution of the adaptive immune system.

Inducible IL-2 production and IL-2+ cell expansion are landmark events for T-cell activation of teleost.

As a multipotent cytokine, interleukin (IL)-2 plays important roles in activation, differentiation and survival of the lymphocytes. Although biological characteristics and function of IL-2 have been clarified in several teleost species, evidence regarding IL-2 production at the cellular and protein levels is still scarce in fish due to the lack of reliable antibody. In this study, we developed a mouse anti-Nile tilapia IL-2 monoclonal antibody (mAb), which could specifically recognize IL-2 protein and identify IL-2-producing lymphocytes of tilapia. Using this mAb, we found that CD3+ T cells, but not CD3- lymphocytes, are the main cellular source of IL-2 in tilapia. Under resting condition, both CD3+CD4-1+ T cells and CD3+CD4-1- T cells of tilapia produce IL-2. Moreover, the IL-2 protein level and the frequency of IL-2+ T cells significantly increased once T cells were activated by phytohemagglutinin (PHA) or CD3 plus CD28 mAbs in vitro. In addition, Edwardsiella piscicida infection also induces the IL-2 production and the expansion of IL-2+ T cells in the spleen lymphocytes. These findings demonstrate that IL-2 takes part in the T-cell activation and anti-bacterial adaptive immune response of tilapia, and can serve as an important marker for T-cell activation of teleost fish. Our study has enriched the knowledge regarding T-cell response in fish species, and also provide novel perspective for understanding the evolution of adaptive immune system.

Excellent ratiometric two-photon fluorescent probes for hydrogen sulfide detection based on the fluorescence resonance energy transfer mechanism.

Fluorescence resonance energy transfer (FRET) is an important mechanism to design ratiometric fluorescent probes that are able to detect analytes quantitatively according to the ratio of two well-resolved emission signals. Two-photon (TP) fluorescent probes can realize the detection in living cells and tissues with deeper penetration depth, higher resolution, and lower photodamage in contrast to one-photon fluorescent probes. However, to date, fabricating TP-FRET ratiometric fluorescent probes possessing large two-photon absorption (TPA), high fluorescence quantum yield and perfect FRET efficiency is still challenging. Consequently, to develop excellent TP-FRET ratiometric probes and explore the relationship between their molecular structures and TP fluorescence properties, in this paper, we designed a series of H2S-detecting TP fluorescent probes employing the FRET mechanism based on an experimental probe BCD. Thereafter, we comprehensively evaluated the TP sensing performance of these probes by means of time-dependent density functional theory and quadratic response theory. Furthermore, we determined energy transfer efficiency and fluorescence quantum yield. Significantly, through regulating benzene-fused positions, we successfully improved fluorescence quantum yield and TPA cross-section simultaneously. Large spectral overlap between energy donor emission and acceptor absorption was achieved and near perfect energy transfer efficiency was acquired for all the studied probes. We revealed that these probes exhibit two well-resolved TPA bands, which are contributed by FRET donors and acceptors, respectively. Especially, both the wavelengths and the cross-sections of the two TPA bands agree well with those of energy donors and acceptors, which is the unique TPA spectral profile of FRET probes and has never been previously reported. Moreover, we proposed an excellent TP-FRET probe BCD3 and its product molecule BCD3-H2S, which exhibit large Stokes (141 nm and 88 nm) and emission shifts (5931 cm-1), as well as greatly increased TP action cross-sections (24-fold and 60-fold) in the near-infrared region with respect to BCD and BCD-H2S. Our detailed study can give an insight into the efficient design of novel TP-FRET fluorescent probes.

Unraveling the evolutionary patterns and phylogenomics of coronaviruses: A consensus network approach.

The COVID-19 pandemic emphasizes the significance of studying coronaviruses (CoVs). This study investigates the evolutionary patterns of 350 CoVs using four structural proteins (S, E, M, and N) and introduces a consensus methodology to construct a comprehensive phylogenomic network. Our clustering of CoVs into 4 genera is consistent with the current CoV classification. Additionally, we calculate network centrality measures to identify CoV strains with significant average weighted degree and betweenness centrality values, with a specific focus on RaTG13 in the beta genus and NGA/A116E7/2006 in the gamma genus. We compare the phylogenetics of CoVs using our distance-based approach and the character-based model with IQ-TREE. Both methods yield largely consistent outcomes, indicating the reliability of our consensus approach. However, it is worth mentioning that our consensus method achieves an approximate 5000-fold increase in speed compared to IQ-TREE when analyzing the data set of 350 CoVs. This improved efficiency enhances the feasibility of conducting large-scale phylogenomic studies on CoVs.

Rational design of HJH antimicrobial peptides to improve antimicrobial activity.

Antimicrobial peptides (AMPs) have attracted considerable interest in the past decade due to their advantages for tackling antibiotic resistance. They exhibit potential antimicrobial activity through unique cell membrane destruction mechanism based on their net charge, hydrophobic properties, and α-helix. In this work, a series of HJH peptides was rationally designed and synthesized. The antimicrobial activity and cytotoxicity assays indicated that HJH-5 and HJH-6 containing hydrophobic residues and helices displayed prominent antimicrobial activity and mild cytotoxicity, respectively. These peptides may be developed for combatting microbial infections.

Interleukin-12 induces IFN-γ secretion and STAT signaling implying its potential regulation of Th1 cell response in Nile tilapia.

As a pleiotropic cytokine consisting of IL-12p35 and IL-12p40, Interleukin-12 (IL-12) features in inflammation regulation and anti-bacterial immunity. While IL-12 homologs have been identified in non-mammalian species, the precise mechanisms by which IL-12 contributes to early adaptive immune responses in vertebrates remain incompletely understood. Herein, an evolutionary conserved Oreochromis niloticus IL-12 (defined as OnIL-12) was identified by synteny characterization, structural comparisons and phylogenetic pattern of IL-12p35b and IL-12p40a. IL-12p35b and IL-12p40a exhibited widespread expression in lymphoid-related tissues of tilapia, while their mRNA expression in head-kidney demonstrated a significant increase after Edwardsiella piscicida infection. Compared with other lymphocytes, recombinant OnIL-12 (rOnIL-12) displayed stronger affinity binding to T cells. Although stimulation of lymphocytes with the p35b or p40a subunit resulted in a significant induction of IFN-γ expression, rOnIL-12 showed stronger potential to promote IFN-γ expression than these subunits. rOnIL-12 not only elevated the mRNA expression level Th1 cell-associated transcription factor T-bet in lymphocytes, but also increased the proportion of CD4-1+IFN-γ+ lymphocytes. Moreover, the mRNA and phosphorylation levels of STAT1, STAT3, STAT4 and STAT5 were enhanced by rOnIL-12. These findings will offer previous evidence for further exploration into the regulatory mechanisms of Th1 cellular immunity in early vertebrates.

Exploring site-specific N application rate to reduce N footprint and increase crop production for green manure-rice rotation system in southern China.

Milk vetch (Astragalus sinicus L.) is leguminous green manure (GM) which produces organic nitrogen (N) for subsequent crops and is widely planted and utilized to simultaneously reduce the use of synthetic N fertilizer and its environmental costs in rice systems. Determination of an optimal N application rate specific to the GM-rice system is challenging because of the large temporal and spatial variations in soil, climate, and field management conditions. To solve this problem, we developed a framework to explore the site-specific N application rate for the low-N footprint rice production system in southern China based on multi-site field experiments, farmer field survey, and process-based model (WHCNS_Rice, soil water heat carbon nitrogen simulator for rice). The results showed that a process-based model can explain >83.3% (p < 0.01) of the variation in rice yield, aboveground biomass, crop N uptake, and soil mineral N. Based on the scenario analysis of the tested WHCNS_Rice model, the simple regression equation was developed to implement site-specific N application rates that considered variations in GM biomass, soil, and climatic conditions. Simulation evaluation on nine provinces in southern China showed that the site-specific N application rate reduced regional synthetic N fertilizer input by 29.6 ± 17.8% and 65.3 ± 23.0% for single and early rice, respectively; decreased their total N footprints (NFs) by 23.4% and 49.3%, respectively; and without reduction in rice yield, compared with traditional farming N practices. The reduction in total NF was attributed to the reduced emissions from ammonia volatilization by 35.2%, N leaching by 28.4%, and N runoff by 32.7%. In this study, we suggested a low NF rice production system that can be obtained by combining GM with site-specific N application rate in southern China.

Sortase A-mediated cyclization of novel polycyclic RGD peptides for ανß3 integrin targeting.

Cyclic arginine-glycine-aspartic (RGD) peptides that specifically bind to integrin ανß3 have been developed for drug delivery, tracers, and imaging for tumor diagnosis and treatment. Herein, a series of polycyclic RGD peptides containing dual, tri, and tetra rings were designed and synthesized through sortase A-mediated ligation. An in vitro test on cell adhesion inhibition indicated that the RGD peptide containing tricylic structure exhibited outstanding potency and selectivity for ανß3 integrin.

Activated T cells are the cellular source of IL-22 that enhances proliferation and survival of lymphocytes in Nile tilapia.

As a pleiotropic cytokine mainly secreted by CD4+ T cells, interleukin (IL)-22 plays an important role in immune regulation and infection elimination. Despite IL-22 homologues have been identified in non-mammal, whether and how IL-22 participates in the adaptive immune response of early vertebrates have not been fully addressed. In this study, we identified an evolutionarily conserved IL-22 from Nile tilapia Oreochromis niloticus (defined as OnIL-22), proved by its properties regarding sequence, gene structure, functional domain, tertiary structure and phylogeny. IL-22 was broadly expressed in lymphoid-related tissues of tilapia, and with relatively higher levels in skin, gill, intestine and liver. The expression of OnIL-22 in spleen lymphocytes was markedly induced at the adaptive immune stage after Streptococcus agalactiae infection. Moreover, once lymphocytes were activated by PMA plus ionomycin or T-cell specific mitogen PHA in vitro, OnIL-22 expression was obviously up-regulated at both mRNA and protein levels. These results thus suggest that activated T cells produce IL-22 to take part in the adaptive immune response of tilapia. Furthermore, treatment of lymphocytes with recombinant OnIL-22 increased the expression of genes related to proliferation and survival, and further promoted the proliferation and reduced the apoptosis of lymphocytes during bacterial infection or T-cell activation. These cellular effects of IL-22 seem to be associated with JAK1/STAT3 axis downstream of IL-22, because IL-22 application not only elevated the mRNA expression of JAK1 and STAT3, but also enhanced their phosphorylation in lymphocytes. Altogether, we suggest that activated T cells produce IL-22 to promote lymphocyte proliferation and survival probability via JAK1/STAT3 signaling pathway, thus participating in adaptive immune response of Nile tilapia. Our study therefore provides helpful perspective for understanding the function and mechanism of adaptive immune system in teleost.

Evolutionary history and functional characterization of Lj-TICAM-a and Lj-TICAM-b formed via lineage-specific tandem duplication in lamprey (Lampetra japonica).

Toll/interleukin-1 receptor domain-containing adaptor molecule (TICAM) genes respond to infections. We identified TICAM-a and TICAM-b in Lampetra japonica and investigated their evolutionary history and potential function via comparative genomics and molecular evolution analyses. They are arranged in tandem and evolved from a multi-exon to a single-exon structure. Lj-TICAM-a and Lj-TICAM-b might be the ancestral gene of the vertebrate TICAM genes. Lj-TICAM-b arose via a lamprey-specific tandem duplication event. Both genes are expressed in many tissues during an immune response, and exhibit different responses to peptidoglycan, indicating their functional divergence. Simultaneous overexpression of both proteins activated nuclear factor κB expression and co-immunoprecipitation assays indicated that they might form a complex for signal transduction. However, unlike in mammals, the TICAM-dependent signaling pathway in lamprey might rely on TRAF3 rather than on TRAF6. These results suggest that both Lj-TICAM-a and Lj-TICAM-b play a role in host defenses.

MSClustering: A Cytoscape Tool for Multi-Level Clustering of Biological Networks.

MSClustering is an efficient software package for visualizing and analyzing complex networks in Cytoscape. Based on the distance matrix of a network that it takes as input, MSClustering automatically displays the minimum span clustering (MSC) of the network at various characteristic levels. To produce a view of the overall network structure, the app then organizes the multi-level results into an MSC tree. Here, we demonstrate the package's phylogenetic applications in studying the evolutionary relationships of complex systems, including 63 beta coronaviruses and 197 GPCRs. The validity of MSClustering for large systems has been verified by its clustering of 3481 enzymes. Through an experimental comparison, we show that MSClustering outperforms five different state-of-the-art methods in the efficiency and reliability of their clustering.

Density functional theory calculations and thermodynamic analysis of bridgmanite surface structure.

Bridgmanite, a high temperature and pressure form of MgSiO3, is believed to be Earth's most abundant mineral and responsible for the observed seismic anisotropy in the mantle. Little is known about surfaces of bridgmanite but knowledge of the most stable surface terminations is important for understanding various geochemical processes as well as likely slip planes. A density functional theory based thermodynamic approach is used here to establish the range of stability of bridgmanite as well as possible termination structures of the (001), (010), (100) and (011) surfaces as a function of the chemical potential of oxygen and magnesium. The vibrational contribution to the Gibbs free energy is found to be essential for obtaining a stability region of bridgmanite in the phase diagram. The most stable surface termination of bridgmanite varies between three different atomic structures depending on the chemical potential of oxygen and magnesium. The results presented provide a basis for further theoretical studies of the chemical processes on bridgmanite surfaces in the Earth's mantle and slip plane analysis.

Effects of gas reservoir configuration and pore radius on shale gas nanoflow: A molecular dynamics study.

We calculated methane transport through cylindrical graphite nanopores in cyclical steady-state flows using non-equilibrium molecular dynamics simulations. First, two typical gas reservoir configurations were evaluated: open (OS) and closed (CS) systems in which pores connect to the gas reservoir without/with a graphite wall parallel to the gas flow. We found that the OS configuration, which is commonly used to study nanoflows, exhibited obvious size effects. Smaller gas reservoir cross-sectional areas were associated with faster gas flows. Because Knudsen diffusion and slip flow in pores are interrupted in a gas reservoir that does not have walls as constraints, OSs cannot be relied upon in cyclical nanoflow simulations. Although CSs eliminated size effects, they introduced surface roughness effects that stem from the junction surface between the gas reservoir and the pore. To obtain a convergent nanoflow, the length of a side of the gas reservoir cross-section should be at least 2 nm larger than the pore diameter. Second, we obtained methane flux data for various pore radii (0.5-2.5 nm) in CSs and found that they could be described accurately using the Javadpour formula. This is the first direct molecular simulation evidence to validate this formula. Finally, the radial density and flow-velocity distributions of methane in CS pores were analyzed in detail. We tested pores with a radius between 0.5 nm and 2.5 nm and determined that the maximum ratio (∼34%) of slip flow to overall flow occurred in the pore with a radius of 1.25 nm. This study will aid in the design of gas reservoir configurations for nanoflow simulations and is helpful in understanding shale gas nanoflows.

Zebrafish Mutants Carrying Leptin a (lepa) Gene Deficiency Display Obesity, Anxiety, Less Aggression and Fear, and Circadian Rhythm and Color Preference Dysregulation.

Leptin, a hormone secreted by peripheral adipose tissues, regulates the appetite in animals. Recently, evidence has shown that leptin also plays roles in behavioral response in addition to controlling appetite. In this study, we examined the potential function of leptin on non-appetite behaviors in zebrafish model. By using genome editing tool of Transcription activator-like effector nuclease (TALEN), we successfully knocked out leptin a (lepa) gene by deleting 4 bp within coding region to create a premature-translation stop. Morphological and appetite analysis showed the lepa KO fish display a phenotype with obese, good appetite and elevation of Agouti-related peptide (AgRP) and Ghrelin hormones, consistent with the canonical function of leptin in controlling food intake. By multiple behavior endpoint analyses, including novel tank, mirror biting, predator avoidance, social interaction, shoaling, circadian rhythm, and color preference assay, we found the lepa KO fish display an anxiogenic phenotype showing hyperactivity with rapid swimming, less freezing time, less fear to predator, loose shoaling area forming, and circadian rhythm and color preference dysregulations. Using biochemical assays, melatonin, norepinephrine, acetylcholine and serotonin levels in the brain were found to be significantly reduced in lepa KO fish, while the levels of dopamine, glycine and cortisol in the brain were significantly elevated. In addition, the brain ROS level was elevated, and the anti-oxidative enzyme catalase level was reduced. Taken together, by performing loss-of-function multiple behavior endpoint testing and biochemical analysis, we provide strong evidence for a critical role of lepa gene in modulating anxiety, aggression, fear, and circadian rhythm behaviors in zebrafish for the first time.

A Simple Method to Decode the Complete 18-5.8-28S rRNA Repeated Units of Green Algae by Genome Skimming.

Green algae, Chlorella ellipsoidea, Haematococcus pluvialis and Aegagropila linnaei (Phylum Chlorophyta) were simultaneously decoded by a genomic skimming approach within 18-5.8-28S rRNA region. Whole genomic DNAs were isolated from green algae and directly subjected to low coverage genome skimming sequencing. After de novo assembly and mapping, the size of complete 18-5.8-28S rRNA repeated units for three green algae were ranged from 5785 to 6028 bp, which showed high nucleotide diversity (π is around 0.5-0.6) within ITS1 and ITS2 (Internal Transcribed Spacer) regions. Previously, the evolutional diversity of algae has been difficult to decode due to the inability design universal primers that amplify specific marker genes across diverse algal species. In this study, our method provided a rapid and universal approach to decode the 18-5.8-28S rRNA repeat unit in three green algal species. In addition, the completely sequenced 18-5.8-28S rRNA repeated units provided a solid nuclear marker for phylogenetic and evolutionary analysis for green algae for the first time.

Visualizing and Clustering Protein Similarity Networks: Sequences, Structures, and Functions.

Research in the recent decade has demonstrated the usefulness of protein network knowledge in furthering the study of molecular evolution of proteins, understanding the robustness of cells to perturbation, and annotating new protein functions. In this study, we aimed to provide a general clustering approach to visualize the sequence-structure-function relationship of protein networks, and investigate possible causes for inconsistency in the protein classifications based on sequences, structures, and functions. Such visualization of protein networks could facilitate our understanding of the overall relationship among proteins and help researchers comprehend various protein databases. As a demonstration, we clustered 1437 enzymes by their sequences and structures using the minimum span clustering (MSC) method. The general structure of this protein network was delineated at two clustering resolutions, and the second level MSC clustering was found to be highly similar to existing enzyme classifications. The clustering of these enzymes based on sequence, structure, and function information is consistent with each other. For proteases, the Jaccard's similarity coefficient is 0.86 between sequence and function classifications, 0.82 between sequence and structure classifications, and 0.78 between structure and function classifications. From our clustering results, we discussed possible examples of divergent evolution and convergent evolution of enzymes. Our clustering approach provides a panoramic view of the sequence-structure-function network of proteins, helps visualize the relation between related proteins intuitively, and is useful in predicting the structure and function of newly determined protein sequences.

In vivo investigation to the macrolide-glycosylating enzyme pair DesVII/DesVIII in Saccharopolyspora erythraea.

Glycosyltransferase DesVII and its auxiliary partner DesVIII from Streptomyces venezulae, homologs of EryCIII and EryCII in Saccharopolyspora erythraea, have previously been demonstrated to be flexible on their substrates in vitro. Herein, we investigated their in vivo function by interspecies complementation in the mutant strains of Sac. erythraea A226. As desVII and desVIII were concomitantly expressed in the ΔeryCIII mutant, the erythromycin A (Er-A) production was restored. Interestingly, co-expression of desVII and desVIII in the ΔeryBV mutant exhibited an increased Er-A yield by 15 % in comparison to A226. Hence, DesVII/DesVIII not only replaced EryCIII to upload D-desosamine to C5 position of 3-O-mycarosyl erythronolide B (MEB) but also in vivo attached L-mycarose, not D-desosamine to C3 position of erythronolide B (EB) with a higher activity than EryBV. Furthermore, expression of desVII in ΔeryCIII and ΔeryBV-CIII partially restored the Er-A production; however, no Er-A was detected while desVII was expressed in ΔeryBV. It was implicated that DesVII coupled with EryCII to form the DesVII/EryCII complex for attaching above two deoxysugars in the absence of EryCIII in Sac. erythraea. In addition, when desVII and desVIII were co-expressed in ΔeryBV-CII, Er-A was recovered with a lower yield than ΔeryBV-CIII. Our study presents an opportunity with Sac. erythraea as a cell factory for macrolide glycodiversification.