Point mutation of V252 in Neomycin C epimerase enlarges substrate binding pocket and improve the accumulation of Neomycin B in Streptomyces fradiae

doi:10.21203/rs.3.rs-2083578/v1

Download PDF

Research Article

Point mutation of V252 in Neomycin C epimerase enlarges substrate binding pocket and improve the accumulation of Neomycin B in Streptomyces fradiae

https://doi.org/10.21203/rs.3.rs-2083578/v1

This work is licensed under a CC BY 4.0 License

You are reading this latest preprint version

Neomycin, aminoglycoside antibiotics with broad-spectrum antibacterial resistance, is widely used in pharmaceutical and agricultural fields. However, the separation and purification process of neomycin B as an active substance is complicated in Streptomyces fradiae. Although NeoN can catalyze neomycin C to generate neomycin B, the catalytic mechanism of NeoN is still unclear. In this study, whole genomics sequencing clarified the genomic information of high-yielding mutant SF-2. Subsequently, the mechanism of NeoN in catalyzing neomycin C synthesis of neomycin B was resolved based on the NeoN-SAM-neomycin C ternary complex. Finally, mutant NeoN^V252A improved the activity of NeoN and the recombinant strain SF-2-NeoN^V252A accumulated neomycin B 16766.6 U/mL and the ratio of neomycin C decreased from 16.1–6.28% relative to the starting strain SF-2. In summary, this work had analyzed the catalytic mechanism of NeoN, which had certain reference significance for rationally design NeoN to improve the production of neomycin B and weaken the proportion of neomycin C.

S. fradiae

Neomycin

NeoN

catalytic mechanism

Neomycin, the first 2-deoxystreptamine (2-DOS) aminoglycoside antibiotic, was isolated from the product of Streptomyces fradiae in 1949 (Zheng et al., 2020). Neomycin products were approved by the U.S. Food and Drug Administration (FDA) in 1964 and are widely used in animal husbandry to prevent disease and improve feed utilization. In recent years, neomycin topical products, such as neomycin cream, neomycin ophthalmic suspension and neomycin ointment, have been used to treat human diseases (Hanko et al., 2010). Current studies have shown that neomycin could inhibit the binding of HIV-related polypeptides to trans-activation response (TAR) RNA, thereby achieving anti-HIV effects (Chen et al., 2018). In addition, neomycin can activate the inhibitory factor p53 in tumor cells and induce tumor cell apoptosis (Zhang et al., 2019). Neomycin can also be used in combination with other drugs to improve the killing activity of the drug on lung cancer cells NCI-H460, thereby achieving anti-cancer effects (Cuccarese et al., 2013; Swiatkowska et al., 2020). Neomycin is a glycoside antibiotic formed by the conversion of carbohydrates through the HMP pathway, including A, B, and C 3 classes whose chemical structures and biological activities are not similar. The antibacterial activity of neomycin mainly produces is binding to the 16S rRNA site of the 30S ribosome, leading to protein mistranslation, inhibiting the assembly of 30S ribosomal subunits, generating hydroxyl radicals, and inhibiting the activity of ribonucleases, among which neomycin B had the highest activity (Homa, 2010; Waksman et al., 1949). However, since neomycin B and neomycin C are stereoisomers of each other, the process of separation and purification of neomycin B components is relatively complicated. Therefore, metabolic engineering and enzyme engineering can be used to promote the accumulation of neomycin B and weaken the production of neomycin C as a by-product more effectively.

The biosynthetic pathway of neomycin B is mainly distributed on the neo gene cluster. On the neo gene cluster of S. fradiae, a total of 12 genes from neoE to neoD are form an operon, including various structural genes in the process of neomycin biosynthesis; gene neoGH-aphA and other regulatory genes form another operon (Zheng et al., 2020) (Fig. 1C). At present, the modification of neomycin biosynthesis pathway by molecular biology and other technical means to improve the production of neomycin has also been widely studied by researchers. The overexpression of two regulatory genes afsA-g and neoR in the biosynthetic pathway of neomycin B, increased the production of neomycin to 722.9 ± 20.1 mg/L and 564.7 ± 32.5 mg/L, respectively (Meng et al., 2017). Zheng et al. constructed the plasmid PKCZ03 to enhance the expression of neomycin biosynthesis gene cluster and obtained the S. fradiae engineering strain SF/PKCZ03 (CGMCC4.576), which increased the yield of neomycin by 36%, and by adding key precursors N-acetylglucosamine and L-glutamine, the production of neomycin increased by 62% and 107%, respectively (Zheng et al., 2019). The microgene cluster (P_kasO*-neoN-metK-P_kasO*-neoGH-aphA) was overexpressed in S. fradiae CGMCC 4.576 to reduce the accumulation of neomycin C from 19.1–12.7% effectively, while increasing the content of neomycin B by about 13.1% (Zheng et al., 2020). At the same time, knockout of NeoN would make S. fradiae CGMCC 4.576 lose the ability to synthesize neomycin B. It was elucidated that NeoN was the key enzyme in the synthesis of neomycin B and that overexpression of NeoN could enhance the accumulation of neomycin B and attenuate the production of neomycin C.

NeoN encoded by neoN is a putative free radical SAM-dependent epimerase that catalyzes the conversion of neomycin C to neomycin B in the final step of neomycin B biosynthesis (Kudo et al., 2014; Kudo et al., 2016). It belongs to the free radical SAM protein family and contains the characteristic CxxxCxxC motif for stabilization of 4Fe-4S clusters (Besandre et al., 2021; Goldman et al., 2013; Grell et al., 2018). There is a unique iron atom that lacks cysteine coordination, coordinated by the amino and carboxylic acid moieties of the SAM, and this SAM-cluster interaction plays a central role in catalysis (Bauerle et al., 2015; Kuhner et al., 2016). Free radical SAM enzymes have a wide range of catalytic types, catalyzing steps in metabolism, DNA repair, vitamin and coenzyme biosynthesis, and the biosynthesis of many antibiotics, such as biotin synthase (BioB), fatty acyl synthase (LipA), pyruvate formatting enzyme (PFL), coporphyrinogen oxidase (HemN), lysine 2,3-amino dismutase (LAM), Anaerobic ribonucleotide reductase (ARR) and an enzyme that biosynthesizes molybdenum pterin (MoaA), etc. These reactions typically begin with the reductive cleavage of SAM by catalytic iron-sulfur clusters coordinated at the active site of the enzyme, followed by the resulting L-methionine, 5′-deoxyadenosine radical equivalents used to initiate radical-mediated conversion of substrate to product. In the biosynthesis of avilamycin, the free radical SAM enzyme is involved in the related epimerization reaction (Ayikpoe et al., 2019; Grove et al., 2013; Ruszczycky et al., 2018). The biosynthetic pathway of butirosin also has this family of enzymes (2-DOIA dehydrogenase BtrN), and have been characterized in vitro. In 2014, Kudo et al. determined the direction of reaction catalyzed by isomerase NeoN by radioisotope labeling method, which could catalyze neomycin C to generate neomycin B (Kudo et al., 2014). The reaction occurs at the C-5' position, and Cys249 in NeoN provides a hydrogen atom to complete the isomerization of neomycin C during the reaction. In 2020, Zheng et. al. verified that NeoN catalyzes the conversion of neomycin C to neomycin B by knocking out neoN, and the overexpression of NeoN could enhance the yield of neomycin B (Zheng et al., 2020).

In this study, firstly, whole-genome sequencing was used to analyse the high-yielding neomycin-producing strain SF-2, which was obtained by early mutagenesis in the laboratory. Then, the genetic manipulation system of the SF-2 strain was optimized. Subsequently, it was found that NeoN was also a key enzyme for the synthesis of neomycin B by overexpressing NeoN and knocking out NeoN in SF-2. Next, based on the NeoN-SAM-neomycin C ternary complex docking model, the catalytic mechanism of NeoN catalyzing the synthesis of neomycin C to neomycin B was analyzed. Finally, the recombinant strain SF-2-NeoN^V252A increased the yield of neomycin B by 45.8% compared with SF-2, and the proportion of neomycin C was reduced to 6.28%. This study interpreted the whole genome information of neomycin high-producing mutant strain SF-2, and analyzed the catalytic mechanism of NeoN, which had certain reference significance for systematic metabolic engineering and rational modification of enzymes to improve the production of neomycin.

Whole genome sequencing of neomycin high-producing mutant strain SF-2

In the early stage of the laboratory, a neomycin high-yielding mutant strain Streptomyces fradiae SF-2 was obtained through ARTP for 6 consecutive rounds of mutagenesis screening, which could accumulate 10849 U/mL of neomycin (Yu et al., 2022). In order to further analyze the mechanism of high neomycin production by SF-2 and provide accurate theoretical guidance for subsequent systematic metabolic engineering, the whole genome sequencing and assembly of SF-2 was performed. The results showed that, as shown in Fig. 1A, the full-length genome of SF-2 was 6127725 bp, of which the GC content was 74.75%. A total of 6431 protein-coding genes, 62 tRNA genes, and 16s rRNA genes were identified in the genome. The relevant whole genome sequence information has been uploaded to the NCBI database (GenBank: CP072209.1). The KEGG metabolic pathway classification of SF-2 (Fig. 1B) was mainly divided into six categories: metabolic system, pathogenic system, genetic information processing system, cell differentiation system, environmental information processing and biological system. The annotated genes are 3309, 97, 239, 114, 214 and 86, respectively. Carbohydrate metabolism and amino acid metabolism account for the largest proportions in the metabolic system, indicating that the growth of SF-2 required a large amount of carbon and nitrogen sources, and the complex metabolic network also revealed the complexity of the strain's metabolic system, indicating that the SF-2 may accumulate a variety of metabolites. The genetic information processing system mainly includes transcription and translation, replication and repair, protein folding, classification and degradation, which is closely related to the complex morphological differentiation and powerful post-translational modification system of SF-2. SF-2 germinated from spores to form intrabasal hyphae and regenerates into aerial hyphae, and finally aerial hyphae form mature spores, which involved the assembly and distribution of a large number of DNA and proteins. At the same time, in the process of spore synthesis, the bacteria pass through complex translation. Post-modification systems synthesize complex and diverse metabolites such as virulence factors and antibiotics, which are beneficial to their ability to compete with other organisms in complex natural environments. Cell differentiation showed that SF-2 not only had the cell community characteristics of prokaryotes, such as biofilm formation and a strong quorum sensing system, but also had the characteristics of eukaryotic local adhesion, which facilitated their response to various changes in the natural environment. In addition, the environmental information processing system of SF-2 showed that the strain had strong signal transduction and membrane transport functions, especially the two-component system and ABC transporter. As the basic stimulus-response coupling mechanism, the two-component system regulated secondary metabolism and morphological differentiation by participating in core processes such as glycolysis, gluconeogenesis, stress signaling pathway, protein secretion and cell envelope metabolism. At present, the functions of many pairs of two-component signal transduction systems have been explained, such as AbsAl/A2, PhoP-R, MtrAB, AfsQ1/Q2, DraR-K, MacRS, RspA1/A2 (Bo et al., 2014; Nguyen et al., 2010; Yu et al., 2012; Zhang et al., 2021), etc., almost all aspects of secondary metabolism and growth and development of S. fradiae were covered. At the same time, ABC transporters could also change the primary metabolic process by transporting nutrients, thereby affecting the morphological differentiation and secondary metabolism (Higgins, 2001), so that the bacteria could quickly adapt to the complex environment. In addition, by analyzing the genes related to the neomycin synthesis gene cluster (Fig. 1C), it was found that there were 30 amino acid residue mutations, one-site deletion mutations and three amino acid insertion mutations (Table 2), mainly distributed in NeoM, NeoP , NeoX, NeoF, NeoD, NeoL and NeoA. Therefore, it was speculated that the mutation of these genes involving in the neomycin synthesis gene cluster may endow the mutant SF-2 with the ability to produce high neomycin, and also provide theoretical guidance for systematic metabolic engineering and rational design to further improve the biosynthesis of neomycin.

Parameters affecting the efficiency of conjugation between E. coli and SF-2

At present, the method of conjugation is the main genetic manipulation method of S. fradiae. However, the processing conditions of conjugation also have an important influence on the efficiency of conjugation. Therefore, in our work, to improve the conjugation efficiency between E. coli and SF-2, various parameters, including the type and concentration of metal ions, heat shock temperature and time, ratio of donor and acceptor cells and antibiotic solution addition time were optimized in detail. The medium of conjugation needs to meet the growth requirements of both the donor and the acceptor cells, so four solid mediums, MS, 2CMY, RG, and AS-1 were selected, and their effects on conjugation were compared. The results showed that the positive transformants on AS-1 medium had the highest frequency of conjugation (Fig. 2A). The type and concentration of metal ions also affect the conjugation efficiency. By analyzing the effects of MgCl₂, CaCl₂ and MgSO₄ on the conjugation efficiency, it was found that MgCl₂, CaCl₂ and MgSO₄ all had a certain role in promoting the conjugation efficiency. Among them, MgCl₂ had a greater impact on the frequency of conjugation SF-2. And when AS-1 medium was supplemented with 75 mM MgCl₂, the frequency of conjugation was highest (Fig. 2B). Appropriate heat shock treatment of spores could promote spore germination. In this study, five temperatures gradients of 40 ℃, 45 ℃, 50 ℃, 55 ℃, and 60 ℃ and four time gradients of 5 min, 10 min, 15 min, and 20 min were selected for improving the frequency of conjugation between E. coli and SF-2, respectively. Fig. 2C showed that the optimal heat shock temperature was 50 ℃ for 10 minutes. The ratio of donor and acceptor also had an important influence on the efficiency of conjugation, and the optimal donor-to-receive ratio of different S. fradiae during conjugation process was also different. Therefore, by optimizing the ratio of donor to recipient and analyzing the efficiency of conjugation frequency, it was found that the conjugation frequency was the highest when the ratio of donor to recipient was 10:1. When the number of donor cells was insufficient, the efficiency of conjugation would be seriously affected (Fig. 2D). The growth rate of different S. fradiae on conjugation medium was different, and the optimal time of antibiotic addition was also different. In this study, apramycin was added at 10 h, 12 h, 14 h, 16 h and 18 h after conjugation. As shown in Fig. 2E, the premature addition of apramycin inhibited the growth of SF-2 and was not conducive to conjugation. If the addition time was too late, the donor cells would overgrow and inhibit the growth of SF-2. The frequency of conjugation was the highest when the addition time of apramycin was 14 h after conjugation. At last, by designing orthogonal tables (Table. S2), orthogonal optimization determines optimal combinations found conjugation frequency was most affected by donor-receptor ratio, followed by MgCl₂ concentration in the medium, and the addition time of apramycin had the smallest effect. Under the conditions of the optimal combination A₂B₂C₂, the conjugation efficiency reached to 14.32×10^-6, which was 7.53-fold higher than the initial condition (Table. S3).

The effects of NeoN on neomycin biosynthesis

Neomycin includes three components A, B and C with different chemical structures and biological activities (Zheng et al., 2020). Among them, component A is extremely small, component B has the highest antibacterial activity, and component C is more toxic, about 300-fold than that of component B, and is considered to be the main impurity. Since neomycin B and neomycin C are stereoisomers of each other, it is difficult to separate them in industry. NeoN encoded by neoN is a SAM-dependent epimerase that catalyzes the conversion of neomycin C to neomycin B, thereby enriching the production of neomycin B and attenuating the accumulation of neomycin C and enhancing the expression of NeoN can increase the proportion of neomycin B in the final product (Kudo et al., 2014). Therefore, in order to verify whether NeoN also affected the proportion of neomycin B in SF-2, the NeoN overexpression strain SF-NeoN and the NeoN knockout strain SF-2ΔNeoN were constructed based on the optimized conjugation system, respectively. The accumulation of neomycin B was determined by fermentation. The results showed that, the recombinant strain SF-NeoN could accumulate neomycin B 14149 U/mL, which was 23.07% higher than the original strain SF-2. Strain SF-2ΔNeoN basically lost the synthesis of neomycin B, indicating that NeoN was essential for the synthesis of neomycin B in strain SF-2, which was consistent with the existing reports (Fig. 3).

Analysis of the catalytic mechanism of NeoN

In order to further analyze the catalytic mechanism of NeoN catalyzing the synthesis of neomycin C to neomycin B, the three-dimensional structural model of NeoN was established by using Alpha Fold prediction (Fig. 4A). The analysis results of ramachandran diagram was shown that 91.82% of the amino acid residues fall in the optimal area (green area), indicating that this protein model was reasonable and could be used for subsequent work and research (Fig. 4A). The CDD database of NCBI was used to predict its structure domain, and its E-value was less than the threshold value of 10^-6, indicating that the prediction result of structure domain was reliable. The results showed that amino acids (aa) from 20 to 189 of the sequence belonged to the free radical-dependent SAM superfamily, and 26 to 33aa were the binding sites for the formation of iron-sulfur clusters and SAM. The cysteine site of this conserved sequence CxxxCxxC coordinates with the 4Fe-4S cluster to jointly transfer an electron from the iron-sulfur cluster to SAM, which results in its reductive cleavage to methionine and 5' -deoxyadenosine, which extract a hydrogen atom from the appropriate position, the SAM is either consumed or recovered and reused in the process. Subsequently, the online software DoGSiteScorer was used to calculate a "catalytic pocket" with a volume of 912.64 Å³ formed by Ile28 ~ Leu181 in the space of NeoN (Fig. 4B and Table 3). Based on the analysis of the above information, this catalytic pocket may be used for binding with SAM and substrate molecules. Therefore, the small molecule ligand SAM and the receptor NeoN were docked through Discovery Studio to obtain the complex NeoN-SAM (Fig. 4C). The docked SAM molecules bind around the sites of Cys26, Cys30, and Cys33 located in the conserved sequence CxxxCxxC, which are used to form 4Fe-4S clusters, with a binding energy of -7.1 kcal/mol. The obtained NeoN structure was overlapped with the composite NeoN-SAM structure (Fig. 2D). The RMSD value of the NeoN structure before and after docking with SAM was about 0.216 Å, indicating that the overall structure of the NeoN and NeoN-SAM was similar. With the docking of SAM to NeoN, there were three distinct regions flanking the binding region, namely region 1 (L174 ~ F177), region 2 (E278 ~ S282) and region 3 (D226 ~ N230). In the complex NeoN-SAM structure, the β-sheet of region 1 was folded and expanded outward; the loop region of region 2 and region 3 were significantly increased, and these changes all increased the internal space of the catalytic pocket. Combined with the analysis of surface electrostatic potential energy, it could be seen that after SAM was docked, the original acidic environment in the catalytic pocket become a neutral environment (Fig. 4E). In summary, the change of NeoN after SAM binding increased the internal space of NeoN and changed the catalytic environment, which was more conducive to the recognition and binding of the substrate neomycin C, and provided conditions for the entry and binding of neomycin C molecules later. Subsequently, on the basis of obtaining the NeoN-SAM complex, Dsicovery Studio was further used to dock neomycin C to obtain the NeoN-SAM-neomycin C complex (Fig. 4F). The structure of NeoN-SAM-neomycin C ternary complex was analyzed by surface potential distribution map, and it was found that the presence of several amino acid residues (Glu34, Lys36, Ser251, Val252, etc.) in the binding pocket made the surface of the protein bulge, and the entrance was thus very narrow. Deep in the binding pocket was the SAM molecule binding cavity, the SAM molecule penetrated deep into the binding pocket, and the neomycin C molecule was tightly bound in the NeoN binding pocket near the entrance. By analyzing its binding force, it was found that neomycin C was adjacent to the SAM molecule and tightly bound to the catalytic pocket. There were 14 amino acid residues within 3Å of the neomycin C molecule in the NeoN substrate-binding pocket, and amino acid residues capable of hydrogen bonding interactions included Thr38, Gly24, Asp68, Asp23, Gln236, Arg243, Asp248, Tyr250 (Fig. 4F). These hydrogen bonds were the key to the binding of the neomycin C molecule to the NeoN-SAM complex, which catalyzed by stabilizing the conformation of neomycin C. Among them, different from other amino acid residues, Asp68 could form three hydrogen bonds and two salt bridges with the neomycin C molecule. Thr38 could form three hydrogen bonds with the neomycin C molecule and Val252 provided an unfavorable Molecular binding force. By analyzing the catalytic mechanism of NeoN, it had important guiding significance for rationally transforming NeoN to promote the accumulation of neomycin B.

Analysis of the catalytic mechanism of NeoN

Based on the NeoN-SAM-neomycin C ternary complex model obtained above, Asp68 could form both hydrogen bonds and salt bridges with substrate molecules, and Thr38 participated in the formation of three hydrogen bonds, which contributed to the binding of NeoN to the substrate. These two amino acids contributed a large proportion of the force to the binding of NeoN to the substrate. Therefore, these two amino acids were mutated to the uncharged, hydrophobic amino acid alanine, which had less influence on the protein structure, to study its role in the catalytic reaction. On the other hand, the previous analysis showed that the catalytic pocket of NeoN was both small and narrow, while neomycin C was a larger molecule composed of four sugar rings, and the protruding amino acid side chain at the entrance of the pocket made it difficult for neomycin C to enter. In this study, by enlarging catalytic pockets, Glu34, Lys36, Ser251 and Val252 at the entrance of binding pockets were selected to mutate into alanine with methyl side chain and low steric hindrance to increase the area and volume of substrate binding pockets and make more substrate molecules enter catalytic pockets for catalytic reactions. Therefore, heteroexpression of NeoN and its mutants in E. coli BL21 was performed (Fig. 5A), and the enzyme activity of NeoN and its mutants was determined. The results showed that the enzyme activities of the mutant NeoN^T38A and NeoN^D68A were decreased compared with the wild-type NeoN. Among them, the relative enzymatic activity of NeoN^T38A and NeoN^D68A was 72% and 39% that of wild-type NeoN, respectively. For the mutant enzymes that changed the catalytic pocket, except for NeoN^E34A, which had no enzyme activity detected, the enzyme activities of NeoN^K36A and NeoN^S251A were decreased to varying degrees, and the relative enzyme activities were 87.5% and 66.9%, respectively. The relative enzyme activity of NeoN^V252A was 115.2% of wild-type NeoN (Fig. 5B). By analyzing the catalytic pocket of the mutant NeoN^V252A with DoGSiteScorer, it was found that the volume of the catalytic pocket of NeoN^V252A was increased by 51.01 Å³ relative to that of NeoN (Table 3). Therefore, it was speculated that NeoN^V252A could expand the catalytic pocket, which facilitated the entry and binding of the substrate molecule neomycin C. Subsequently, the recombinant strain SF-2-NeoN^V252A was constructed by overexpressing the mutant NeoN^V252A in the SF-2 strain. Finally, the engineered strain SF-2-NeoN^V252A was able to accumulate neomycin B 16766.6 U/mL, which was 18.5% higher than those of SF-2-NeoN (Fig. 5C).

SF-2 was obtained through ARTP for 6 consecutive rounds of mutagenesis screening, which could accumulate 10849 U/mL of neomycin (Yu et al., 2022). In this work, whole-genome sequencing was used to analyse the high-yielding neomycin-producing strain SF-2 and the genetic manipulation system of the SF-2 strain was also optimized. Subsequently, the catalytic mechanism of NeoN catalyzing the synthesis of neomycin C to neomycin B was analyzed and the recombinant strain SF-2-NeoN^V252A increased the yield of neomycin B by 45.8% compared with SF-2, and the proportion of neomycin C was reduced to 6.28%.

With the rapid development of whole-genome sequencing technology, the cost of genome sequencing has dropped significantly. At present, a large number of microbial genome data have been widely published and reported, which provides a lot of material for the functional study of microbial genes (Choo et al., 2016; Sandoval et al., 2015). These studies will also make great contributions to changing public health (Kwong et al., 2015), epidemiology (Llarena et al., 2017), health economics (Frank et al., 2013), and more. At present, the use of genetic engineering and other methods to modify related genes and metabolic pathways in cells to improve the yield and synthesis efficiency of specific metabolites has become a very important way of breeding Streptomyces industrial strains (Xu et al., 2022). Sequencing, annotation and analysis of the S. fradiae genome could understand its genetic variation laws, important metabolic pathways and regulatory mechanisms at the molecular level, and provide a theoretical basis for the construction of high-yielding strains. In addition, the discovery of biosynthetic gene clusters of important metabolites from the Streptomyces genome is of great significance for many aspects such as antibiotic development and synthetic biology research. In this study, the whole genome of SF-2 was sequenced and assembled. And finally a complete genome sequence with a length of 6127725 bp was obtained, which was provided a theoretical basis for the construction of neomycin engineering strains. What’s more, the point mutations of NeoM, NeoP, NeoX, NeoF, NeoD, NeoL and NeoA in the neomycin synthesis pathway gene cluster and the neomycin synthesis regulation gene NeoR were found. Therefore, it was speculated that mutations in these key enzymes may play an important role in neomycin synthesis. It has been reported that the transcription factor NeoR regulated the biosynthesis of neomycin by regulating NeoG and NeoH. So, in the follow-up research, the catalytic mechanism of key enzymes in the neomycin synthesis gene cluster could be further studied, and the level of neomycin synthesis by SF-2 could be further improved. Meanwhile, transcription factors play an important role in the metabolism of cells through regulating the metabolic network (Li et al., 2021). Therefore, NeoR could be overexpressed in the SF-2 and comparative transcriptomic analysis of the key genes regulated by NeoR and the interaction between with NeoR and its binding promoters by EMSA experiments could be analyzed, which will be helpful to regulate the biosynthesis of neomycin.

In this work, the catalytic mechanism of NeoN was resolved and the finally recombinant strain SF-2-NeoN^V252A accumulated neomycin B 16766.6 U/mL, which was 45.8% higher than that of SF-2 and the ratio of neomycin C decreased from 16.1–6.28% relative to the starting strain. It was reported that the higher enzymatic activity of NeoN could promote the biosynthesis of neomycin B and weaken the proportion of neomycin C in S. fradiae (Zheng et al., 2020). However, the enzyme activity of the mutant NeoN^V252A was only increased by 115.2% relative to NeoN. Therefore, the enzymatic activity of NeoN could be further enhanced by site-directed saturation mutation of V252, thereby enhancing the biosynthesis of neomycin B. At the same time, it was also found that the volume and surface area of the catalytic pocket of the mutant NeoN^S251A were significantly increased, but the enzyme activity was significantly decreased. It may be due to the obvious enhancement of the surface area of the catalytic pocket, which reduced its binding specificity, thereby reducing its enzymatic activity. Therefore, in the future work, it is possible to enhance the surface area and volume of its binding pocket appropriately by mutating S251 with greater steric hindrance, which should be more conducive to maintaining the catalytic specificity of NeoN and promoting the enzymatic activity of NeoN, thereby enhancing the biosynthesis of neomycin.

In conclusion, this study clarified the whole gene information of neomycin high-producing mutant strain SF-2, and the catalytic mechanism of NeoN was also resolved. The finally recombinant strain SF-2-NeoN^V252A accumulated neomycin B 16766.6 U/mL, which was 45.8% higher than that of SF-2 and the ratio of neomycin C decreased from 16.1–6.28% relative to the starting strain. This work had analyzed the catalytic mechanism of NeoN, which had certain reference significance for rationally design NeoN to improve the production of neomycin B and weaken the proportion of neomycin C.

Strains, plasmids and growth conditions

The strains and plasmids used in this work are listed in Table1. The primers used in this study are listed in supplementary materials (Table. S1). Streptomyces fradiae SF-2, Escherichia coli DH5α, E. coli BL21 and E. coli ET12567 were deposited in the laboratory. E. coli DH5α and E. coli BL21 were used as a host cell for cloning and expression of target protein, respectively. E. coli ET12567 was used for conjugative transfer with actinomycetes. Streptomyces fradiae SF-2 was grown with AS-1 solid medium or YEME lipid medium at 35 ℃. Escherichia coli DH5α, E. coli BL21 and E. coli ET12567 were grown in LB medium at 37℃. Streptomyces fradiae SF-2 was first cultivated in seed medium at 35℃, 260 rpm to log phase (40~50 h), and then inoculated (1% v/v) into fermentation medium at 35℃, 260 rpm, 75% RH for 7 days. MS medium could be referred to the literature (Huang et al., 2002). AS-1 medium (g/L): Yeast powder 1, L-alanine 0.2, L-arginine 0.2, L-aspartate 0.5, NaCl 2.5, Na₂SO₄ 10, soluble starch 5, agar power 20, pH 7.3 to 7.8. Seed medium (g/L): (NH₄)₂SO₄ 1, Yeast powder 20, groundnut meal 10, soluble starch 10, glucose 30, corn steep liquor 10, trypsin 5, Na₂HPO₄ 1, CaCO₃ 10, bean oil 2, pH 7.3 to 7.8.

Fermentation medium (g/L): soluble starch 70, groundnut meal 28, Yeast powder 6, (NH₄)₂SO₄ 6, glucose 20, corn steep liquor 2.5, trypsin 9, medium-temperature bean cake powder 5, NaCl 4.5, High-temperature amylase 0.3, Na₂HPO₄ 0.4, CaCO₃ 4, bean oil 3, pH 6.8 to 7.3.

Whole genome sequencing

SF-2 preserved in -80℃ was cultured on MS medium and grown in 30℃ incubator for 5-7d. Then single colonies were inoculated in seed medium in 35℃, 260 rpm to logarithm phase. Then the cultured cells were collected at 4℃, 8000 rpm for 10 min and transferred to quick freezing with liquid nitrogen and send to Shanghai Megji Biomedical Technology Co in dry ice for whole genome sequencing. For annotation, S. fradiae DSM 40063 (GenBank: AJ629247.1) was used as the reference. Glimmer (Delcher et al., 2007), GeneMarkS (Besemer et al., 2005) and Prodigal software were used to predict the coding sequence (CDS) in the genome. tRNAscan-SE (Chan et al., 2019) software was used to predict the tRNA contained in the genome to obtain the nucleotide sequence information, anticodon information and secondary structure information of the tRNA in the genome of each sample. The rRNA included in the genome was predicted using the Barrnap software to obtain the species, location, and sequence information of all of the rRNA in the genome of each sample.

Construction of Plasmids and the recombinant strains

The target fragments NeoN was amplified with the genome of SF-2 as a template with the primer pair NeoN F/NeoN R. T4 DNA ligase (TakaRa) was used to connect the obtained target DNA fragments and the linearized vector pSET152 (PPR) that both had been digested and purified with NotI and EcoRV to construct recombinant plasmids PPR-NeoN. SF-2 chromosome was used as a template, and primer pairs 28a-NeoN F/28a-NeoN R was used to amplify the target fragments. Then T4 DNA ligase (Takara) was used to connect the obtained target DNA fragments and the purified linearized vector pET28a that had been digested with EcoRI and XhoI to construct recombinant plasmids pET28a-NeoN. recombinant plasmids pET28a-NeoN was used as template, and primer pairs V252A-F/V252A-R was used to amplify and obtained the the linearized fragments. Then, DpnI was used to digest the template and ClonExpress II One Step Cloning Kit (Vazyme, Nanjing, China) was used to connect the obtained target DNA fragments to construct the recombinant plasmids pET28a-NeoN^V252A. The other five mutant NeoN were constructed in the same way as the pET28a-NeoN^V252A, except that the corresponding primer pairs are required. The target fragments NeoN^V252A was amplified with the plasmid of pET28a-NeoN^V252A as a template with the primer pair NeoN F/NeoN R. T4 DNA ligase (TakaRa) was used to connect the obtained target DNA fragments and the linearized vector pSET152 (PPR) that both had been digested and purified with NotI and EcoRV to construct recombinant plasmids PPR-NeoN^V252A. The target fragments ΔNeoN was amplified with the genome of SF-2 as a template with the primer pair ΔNeoN F/ΔNeoN R. T4 DNA ligase (TakaRa) was used to connect the obtained target DNA fragments and the linearized vector pKC1139 that both had been digested and purified with XbaI and BamHI to construct recombinant plasmids PKC1139-ΔNeoN. All the recombinant plasmids constructed must be transformed into E. coli DH5α and identified by colony PCR and sequencing at the beginning.

Conjugation and optimization between E. coli and SF-2

The preparation of donor of conjugation: Donor cells E. coli ET12567 preserved in -80℃ was cultured on LB medium in 37℃. Then single colonies were inoculated in LB medium in 37℃, 180 rpm to OD₆₀₀= 0.4~0.6. Then, cells were collected by centrifugation at 8000 rpm for 10 min at 4℃, re-suspended and washed twice with sterile saline to maintain the concentration of cell at about 10⁸cfu/mL.

The preparation of receptor of conjugation: SF-2 preserved in -80℃ was cultured on MS medium and grown in 30℃ incubator for 5-7d. The mature spores were wished with an appropriate amount of YEME liquid medium, and then the spores were dispersed with sufficient oscillation with sterile glass beads. Subsequently, the hyphae was removed by filtration using 8 layers of gauze to obtain a spore suspension, controlled a concentration of 10⁸ cfu/mL.

The method of conjugation: The spore suspension of SF-2 were placed at 50℃ for 10 min and preincubated in 37℃, 200 rpm for 3 h. Then, the donor cells were mixed with cultured spore suspension of SF-2 (donor cells: receptor cells=10:1). Subsequently, the mixture was collected by centrifugation at 4000 rpm for 5 min at 4℃ and spread on the AS-1 solid plate with 75mM MgCl₂ in 30℃ for 14 h. Next, 50 μg/mL apramycin and 500 μg/mL nalidixic acid were used to cover the AS-1 plate and cultured in 30℃ for another 4~5 days. The frequency of conjugation was calculate as the number of zygotes/the number of initial spores.

Bioinformatics tools

Alpha Fold database (https://alphafold.com/) was used to obtain the 3D structural model of NeoN. The VERIFY_3D Server (http://services.mbi.ucla.edu/Verify_3D/) was used to check the quality of the generated model. CDD database (https://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml) and Uniport (https://www.uniprot.org/) were used to analyse of the classes, domains and characteristic amino acid positions of the protein superfamily to which NeoN belongs. DoGSiteScorer was used to calculate protein catalytic pocket. Discovery Studio (DS) was used to analyse the interactions. Gromas 2019.5 was used for molecular dynamics simulations. The simulation temperature was 300 K and Gromacs' all-atom force field and SPC water Model were selected.

Analytical methods

The spectrophotometer (UV-1800) was used to determine the Optical Density (OD₆₀₀). The method to detect Neomycin B and Neomycin B were preformed as described (Zheng et al., 2020). Method for determination of NeoN enzyme activity was performed as described (Kudo et al., 2014). All of the assays were performed in triplicate.

Ethics approval and consent to participate

Not applicable.

Consent for publication

All authors have seen and approved the manuscript before submission to Bioresources and Bioprocessing.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests

The authors declare no conflicts of interest.

Funding

This work was supported by the National Natural Science Foundation of China (31471615, 31871781, 31772081).

Authors’ contributions

XL preformed all the experimental works, analyzed the data and coordinated the manuscript draft and revision. FY and SJ designed and revised the manuscript critically. FW, WS and RH helped with data analysis. YC, ZX and MZ were involved in proofreading the manuscript. All authors read and approved the final manuscript.

Acknowledgements

Not applicable.

Ayikpoe, R., Ngendahimana, T., Langton, M., Bonitatibus, S., Walker, L.M., Eaton, S.S., Eaton, G.R., Pandelia, M.E., Elliott, S.J., & Latham, J.A. (2019). Spectroscopic and Electrochemical Characterization of the Mycofactocin Biosynthetic Protein, MftC, Provides Insight into Its Redox Flipping Mechanism. Biochemistry, 58(7): 940-950.
Bauerle, M.R., Schwalm, E.L., & Booker, S.J. (2015). Mechanistic diversity of radical S-adenosylmethionine (SAM)-dependent methylation. J Biol Chem, 290(7): 3995-4002.
Besandre, R.A., Chen, Z., Davis, I., Zhang, J., Ruszczycky, M.W., Liu, A., & Liu, H.W. (2021). HygY Is a Twitch Radical SAM Epimerase with Latent Dehydrogenase Activity Revealed upon Mutation of a Single Cysteine Residue. J Am Chem Soc, 143(37): 15152-15158.
Besemer, J., & Borodovsky, M. (2005). GeneMark: web software for gene finding in prokaryotes, eukaryotes and viruses. Nucleic Acids Res, 33(Web Server issue): W451-4.
Bo, W.U., & Zhang, W.J. (2014). PhoP-PhoR two-component signal transduction systems in pathogenesis of Mycobacterium tuberculosis. Chinese Journal of Zoonoses.
Chan, P.P., & Lowe, T.M. (2019). tRNAscan-SE: Searching for tRNA Genes in Genomic Sequences. Methods Mol Biol, 1962: 1-14.
Chen, L., Feng, Z., Yue, H., Bazdar, D., Mbonye, U., Zender, C., Harding, C.V., Bruggeman, L., Karn, J., & Sieg, S.F. and others. (2018). Exosomes derived from HIV-1-infected cells promote growth and progression of cancer via HIV TAR RNA. Nat Commun, 9(1): 4585.
Choo, J.H., Hong, C.P., Lim, J.Y., Seo, J.A., Kim, Y.S., Lee, D.W., Park, S.G., Lee, G.W., Carroll, E., & Lee, Y.W. and others. (2016). Whole-genome de novo sequencing, combined with RNA-Seq analysis, reveals unique genome and physiological features of the amylolytic yeast Saccharomycopsis fibuligera and its interspecies hybrid. Biotechnol Biofuels, 9: 246.
Cuccarese, M.F., Singh, A., Amiji, M., & O'Doherty, G.A. (2013). A novel use of gentamicin in the ROS-mediated sensitization of NCI-H460 lung cancer cells to various anticancer agents. ACS Chem Biol, 8(12): 2771-7.
Delcher, A.L., Bratke, K.A., Powers, E.C., & Salzberg, S.L. (2007). Identifying bacterial genes and endosymbiont DNA with Glimmer. Bioinformatics, 23(6): 673-9.
Frank, M., Prenzler, A., Eils, R., & Graf, V.D.S.J. (2013). Genome sequencing: a systematic review of health economic evidence. Health Econ Rev, 3(1): 29.
Goldman, P.J., Grove, T.L., Booker, S.J., & Drennan, C.L. (2013). X-ray analysis of butirosin biosynthetic enzyme BtrN redefines structural motifs for AdoMet radical chemistry. Proc Natl Acad Sci U S A, 110(40): 15949-54.
Grell, T., Kincannon, W.M., Bruender, N.A., Blaesi, E.J., Krebs, C., Bandarian, V., & Drennan, C.L. (2018). Structural and spectroscopic analyses of the sporulation killing factor biosynthetic enzyme SkfB, a bacterial AdoMet radical sactisynthase. J Biol Chem, 293(45): 17349-17361.
Grove, T.L., Ahlum, J.H., Qin, R.M., Lanz, N.D., Radle, M.I., Krebs, C., & Booker, S.J. (2013). Further Characterization of Cys-Type and Ser-Type Anaerobic Sulfatase Maturating Enzymes Suggests a Commonality in the Mechanism of Catalysis. Biochemistry (Easton), 52(17): 2874-2887.
Hanko, V.P., & Rohrer, J.S. (2010). Suitability of a liquid chromatography assay of neomycin sulfate to replace the microbiological assay for neomycin in USP Monographs. J Pharm Biomed Anal, 51(1): 96-102.
Higgins, C.F. (2001). ABC transporters: physiology, structure and mechanism – an overview. Research in microbiology, 152(3): 205-210.
Homa, S.T. (2010). Neomycin, an inhibitor of phosphoinositide hydrolysis, inhibits the resumption of bovine oocyte spontaneous meiotic maturation. Journal of Experimental Zoology, 258(1): -.
Huang, F., Li, Y., Yu, J., & Spencer, J.B. (2002). Biosynthesis of aminoglycoside antibiotics: cloning, expression and characterisation of an aminotransferase involved in the pathway to 2-deoxystreptamine. Chemical communications (Cambridge, England),(23): 2860-2861.
Kudo, F., Hoshi, S., Kawashima, T., Kamachi, T., & Eguchi, T. (2014). Characterization of a RadicalS-Adenosyl-l-methionine Epimerase, NeoN, in the Last Step of Neomycin B Biosynthesis. Journal of the American Chemical Society, 136(39): 13909-13915.
Kudo, F., & Eguchi, T. (2016). Aminoglycoside Antibiotics: New Insights into the Biosynthetic Machinery of Old Drugs. Chem Rec, 16(1): 4-18.
Kuhner, M., Schweyen, P., Hoffmann, M., Ramos, J.V., Reijerse, E.J., Lubitz, W., Broring, M., & Layer, G. (2016). The auxiliary [4Fe-4S] cluster of the Radical SAM heme synthase from Methanosarcina barkeri is involved in electron transfer. Chem Sci, 7(7): 4633-4643.
Kwong, J.C., Mccallum, N., Sintchenko, V., & Howden, B.P. (2015). Whole genome sequencing in clinical and public health microbiology. Pathology, 47(3): 199-210.
Li, X., Bao, T., Osire, T., Qiao, Z., Liu, J., Zhang, X., Xu, M., Yang, T., & Rao, Z. (2021). MarR-type transcription factor RosR regulates glutamate metabolism network and promotes accumulation of L-glutamate in Corynebacterium glutamicum G01. Bioresource Technology, 342: 125945.
Llarena, A.K., Taboada, E., & Rossi, M. (2017). Whole-Genome Sequencing in Epidemiology of Campylobacter jejuni Infections. J Clin Microbiol, 55(5): 1269-1275.
Meng, X., Wang, W., Xie, Z., Li, P., Li, Y., Guo, Z., Lu, Y., Yang, J., Guan, K., & Lu, Z. and others. (2017). Neomycin biosynthesis is regulated positively by AfsA-g and NeoR in Streptomyces fradiae CGMCC 4.7387. Sci China Life Sci, 60(9): 980-991.
Nguyen, H.T., Wolff, K.A., Cartabuke, R.H., Ogwang, S., & Nguyen, L. (2010). A lipoprotein modulates activity of the MtrAB two-component system to provide intrinsic multidrug resistance, cytokinetic control and cell wall homeostasis in Mycobacterium. Molecular Microbiology, 76(2): 348-364.
Ruszczycky, M.W., Zhong, A., & Liu, H.W. (2018). Following the electrons: peculiarities in the catalytic cycles of radical SAM enzymes. Nat Prod Rep, 35(7): 615-621.
Sandoval, N.R., Venkataramanan, K.P., Groth, T.S., & Papoutsakis, E.T. (2015). Whole-genome sequence of an evolved Clostridium pasteurianum strain reveals Spo0A deficiency responsible for increased butanol production and superior growth. Biotechnol Biofuels, 8: 227.
Swiatkowska, A., Dutkiewicz, M., Machtel, P., Janecki, D.M., Kabacinska, M., Żydowicz-Machtel, P., & Ciesiołka, J. (2020). Regulation of the p53 expression profile by hnRNP K under stress conditions. RNA biology, 17(10): 1402-1415.
Waksman, Selman, A., Lechevalier, & Hubert, A. (1949). Neomycin, A New Antibiotic Against Streptomycin-Resistant Bacteria, including Tuberculosis Organisms. The Bulletin of the American Society of Hospital Pharmacists.
Xu, Z., Ji, L., Tang, W., Guo, L., Gao, C., Chen, X., Liu, J., Hu, G., & Liu, L. (2022). Metabolic engineering of Streptomyces to enhance the synthesis of valuable natural products. Engineering Microbiology, 2(2).
Yu, F., Zhang, M., Sun, J., Wang, F., Li, X., Liu, Y., Wang, Z., Zhao, X., Li, J., & Chen, J. and others. (2022). Improved Neomycin Sulfate Potency in Streptomyces fradiae Using Atmospheric and Room Temperature Plasma (ARTP) Mutagenesis and Fermentation Medium Optimization. Microorganisms (Basel), 10(1): 94.
Yu, Z., Zhu, H., Dang, F., Zhang, W., Qin, Z., Sheng, Y., Tan, H., Lu, Y., & Jiang, W. (2012). Differential regulation of antibiotic biosynthesis by DraR-K, a novel two-component system in Streptomyces coelicolor. Molecular Microbiology, 85(3).
Zhang, K., Mohsin, A., Dai, Y., Ali, M.F., Chen, Z., Zhuang, Y., Chu, J., & Guo, M. (2021). Role of a Two-Component Signal Transduction System RspA1/A2 in Regulating the Biosynthesis of Salinomycin in Streptomyces albus. Appl Biochem Biotechnol, 193(5): 1296-1310.
Zhang, T., Li, Y., Zhu, R., Song, P., Wei, Y., Liang, T., & Xu, G. (2019). Transcription Factor p53 Suppresses Tumor Growth by Prompting Pyroptosis in Non-Small-Cell Lung Cancer. Oxid Med Cell Longev, 2019: 8746895.
Zheng, J., Li, Y., Guan, H., Li, J., Li, D., Zhang, J., & Tan, H. (2020). Component Optimization of Neomycin Biosynthesis via the Reconstitution of a Combinatorial Mini-Gene-Cluster in Streptomyces fradiae. ACS synthetic biology, 9(9): 2493-2501.
Zheng, J., Li, Y., Guan, H., Zhang, J., & Tan, H. (2019). Enhancement of neomycin production by engineering the entire biosynthetic gene cluster and feeding key precursors in Streptomyces fradiae CGMCC 4.576. Appl Microbiol Biotechnol, 103(5): 2263-2275.

Table 1

Strains and plasmids in this study.

	Characteristics	Source
Strains
E. coli DH5α E. coli BL21	General cloning host General expressing host	The lab The lab
E. coli ET12567	Demethylated strain containing pUZ8002 plasmid for conjugative transfer with actinomycetes	The lab
SF-2	Streptomyces fradiae, neomycin-producing strains induced by ARTP	(Yu et al., 2022)
BL21/pET28a	E. coli BL21 derivative harboring pET28a	The lab
BL21/pET28a-NeoN	E. coli BL21 derivative harboring pET28a-NeoN	The lab
BL21/pET28a-NeoN^E34A	E. coli BL21 derivative harboring pET28a-NeoN^E34A	This study
BL21/pET28a-NeoN^T38A	E. coli BL21 derivative harboring pET28a-NeoN^T38A	This study
BL21/pET28a-NeoN^D68A	E. coli BL21 derivative harboring pET28a-NeoN^D68A	This study
BL21/pET28a-NeoN^K36A	E. coli BL21 derivative harboring pET28a-NeoN^K36A	This study
BL21/pET28a-NeoN^S251A	E. coli BL21 derivative harboring pET28a-NeoN^S251A	This study
BL21/pET28a-NeoN^V252A	E. coli BL21 derivative harboring pET28a-NeoN^V252A	This study
ET12567/PPR-NeoN	E. coli ET12567 derivative harboring PPR-NeoN	This study
ET12567/PPR-NeoN^V252A	E. coli ET12567 derivative harboring PPR-NeoN^V252A	This study
ET12567/PKC1139-NeoN	E. coli ET12567 derivative harboring PKC1139-NeoN	This study
SF-2-NeoN	SF-2 derivative the expression of NeoN	This study
SF-2-NeoN^V252A	SF-2 derivative the expression of NeoN^V252A	This study
SF-2ΔNeoN	SF-2 derivative the deletion of neoN	This study
Plasmids
pET28a	Kan^r; Expression vector for the expression of target protein	The lab
PPR (pSET152-PermE*)	E. coli-S. fradiae shuttle vector for the expression of target protein	The lab
pKC1139	E. coli-S. fradiae shuttle vector for the deletion of target protein	The lab
pET28a-NeoN	Derived from pET28a, for induced expression of NeoN	This study
pET28a-NeoN^E34A	Derived from pET28a, for induced expression of NeoN^E34A
pET28a-NeoN^T38A	Derived from pET28a, for induced expression of NeoN^T38A	This study
pET28a-NeoN^D68A	Derived from pET28a, for induced expression of NeoN^D68A	This study
pET28a-NeoN^K36A	Derived from pET28a, for induced expression of NeoN^K36A	This study
pET28a-NeoN^S251A	Derived from pET28a, for induced expression of NeoN^S251A	This study
pET28a-NeoN^V252A	Derived from pET28a, for induced expression of NeoN^V252A	This study
PPR-NeoN	Derived from PPR, for the expression of NeoN	This study
PPR-NeoN^V252A	Derived from PPR, for the expression of NeoN^V252A	This study
PKC1139-ΔNeoN	Derived from PKC1139, for the deletion of neoN	This study

Table 2

Whole-genome sequencing analysis of sf-2 related to the biosynthesis pathway of neomycin.

Genes	Mutation base	Mutation amnio acid residue
NeoE	T91C, C112T, G895A	None
NeoS	G373A, C652T	None
NeoC	T412C, T958G, G1018T	None
NeoM	G247C, C585T, A1115G	V49A, A226T
NeoQ	C43G, C1366G	None
NeoN	G667A	None
NeoP	G241T, C261T, C448T, G518C	A62G, A148T
NeoX	G46C, C176T	C26Y
NeoF	T14C, C69T, A265C, G795T, C931T	A345T, E363G
NeoD	G108C, A205C,G332C, C470T, T501A, C832T	T113S, P170R, G124D, P245A
NeoL	G142C, T347C, T362C, G395C, C403G, G428A, C510G, T834G, C930T, A1485G, T1599C, A1602G, G1608C, T1659A, C1918G	E48Q, V116A, V121A, Q135E, R143H, P640A
NeoB	G61C, C475G, G1092C, G1219A	None
NeoA	T161C, G283C, A376T, G461A, G859C, T991C, C1111T, G1341C, G2099C, T2151C, G3396A, 2088 Del, 2113 Del, 2125 Del, 3649_3650 Indel CCCGGTGTT	P163S, T578A, R589V, G590A, S591V, P592A, A593R, W594L, T595E, R596A, P597A, R848G, A1141V, D1241G, 70_71 Indel GTP, 588 Del
NeoR	T876C, T995A, C1089G, G1272C, T1529C, C1617T, A1671G, T1989C, C2319G, C2331T, T2373C, G2374C, G2644A	V332D, T510I, G792R, E882K, 866_867 Indel LAAA
NeoG	G597C, C647T, G696A	A216V
NeoH	C81G	None
NeoI	None	None

Table 3

Information on the catalytic pocket of each mutant.

Size and Shape descriptors	Volume[Å³]	Surface[Å²]	Depth[Å]
NeoN	912.64	1370.45	22.03
NeoN^E34A	694.91	1053.27	19.94
NeoN^K36A	818.18	1498.79	21.50
NeoN^S251A	1349.63	1850.20	30.82
NeoN^V252A	963.65	1220.57	19.50

Download PDF

Reviewers invited by journal
30 Oct, 2022
Reviewers agreed at journal
11 Oct, 2022
Editor assigned by journal
10 Oct, 2022
First submitted to journal
19 Sep, 2022

You are reading this latest preprint version

Point mutation of V252 in Neomycin C epimerase enlarges substrate binding pocket and improve the accumulation of Neomycin B in Streptomyces fradiae

Status:

Version 1

Abstract

Figures

Introduction

Results

Discussion

Conclusions

Methods

Declarations

References

Tables

Supplementary Files

Status:

Version 1