A tRNA modification-based regulatory strategy for lincomycin biosynthesis in Streptomyces lincolnensis

doi:10.21203/rs.3.rs-8038474/v1

Lincomycin is a clinically important lincosamide antibiotic. Its biosynthetic efficiency is limited by the presence of the rare UUA codon in the key positive regulators LmbU and AdpA. In this study, we developed a universal metabolic optimization strategy based on tRNA modification engineering. Co-overexpression of the UUA-decoding tRNA gene bldA and its post-transcriptional modification enzyme gene miaA markedly increased lincomycin production. Further optimization was achieved by expressing miaA under the phase-dependent promoter PlmbU. Mechanistic analysis showed that this strategy enhances the decoding efficiency of the UUA codon, thereby improving translation of LmbU and AdpA. Because bldA and miaA are highly conserved among species of Streptomyces, this approach may be broadly applicable to other rare-codon-dependent secondary metabolites. Our work provides a new and rational strategy for microbial secondary metabolic engineering through precise control of tRNA modification.

Streptomyces lincolnensis

Lincomycin

bldA

tRNA modification

Metabolic engineering

Streptomyces serves as a major producer of various secondary metabolites, with approximately 75% of known natural products originating from this genus (Genilloud 2017, Liu et al. 2018, Alam et al. 2022). To efficiently harness the potential of these valuable resources, it is particularly important to develop a method that can be universally applied to Streptomyces for enhancing the production of natural products. This study focuses on lincomycin produced by Streptomyces lincolnensis to explore such universal strategies. Lincomycin is a broad-spectrum lincosamide antibiotic that exhibits significant clinical activity against both Gram-positive bacteria and anaerobes (Spizek and Rezanka 2004).

The biosynthesis of lincomycin is precisely controlled by a multi-layered regulatory network (Meng et al. 2017, Hou et al. 2018, Li et al. 2019, Xu et al. 2019, Xu et al. 2020, Tu et al. 2023, Wang et al. 2023, Wang et al. 2023, Zou et al. 2023, Wang et al. 2025). LmbU, a key positive regulator within the lincomycin biosynthesis cluster (lmb), is indispensable for lincomycin synthesis, as its deletion completely halts production (Hou et al. 2018). Mechanistically, LmbU directly activates the transcription of structural genes within the cluster and also regulates multiple genes outside it (Mao et al. 2024). This demonstrates its dual regulatory functions, encompassing both pathway-specific and global regulation. In addition, the global regulatory factor AdpA is also a key positive regulator of lincomycin synthesis. Deletion of adpA similarly results in a complete loss of lincomycin production and severely affects morphological differentiation, preventing the formation of spores (Kang et al. 2019). It is noteworthy that both lmbU and adpA contain a UUA rare codon in their coding regions. In Streptomyces, the effective decoding of this codon strictly depends on tRNA^Leu_UUA encoded by the bldA gene, which is the only tRNA responsible for recognizing the UUA codon (Hackl and Bechthold 2015). Therefore, increasing the abundance of tRNA^Leu_UUA is considered a potential strategy to alleviate the translational limitation of key regulators such as LmbU and AdpA, thereby enhancing lincomycin production.

The tRNA^Leu_UUA encoded by bldA undergoes post-transcriptional modifications catalyzed sequentially by the MiaA, MiaB, and MiaE enzymes, with the modification site located at the adenosine at position 37 (A37) (Koshla et al. 2019, Koshla et al. 2023). Previous studies have shown that this modification pathway plays an important role in the metabolic regulation of Streptomyces. For example, in Streptomyces ghanaensis ATCC14672, deletion of miaB leads to a significant decrease in moenomycin production (Sehin et al. 2019). In Streptomyces albus J1074, deletion of either miaA or miaB causes significant changes in morphological differentiation and secondary metabolism, with miaA deletion proven to affect the translation efficiency of specific genes (Koshla et al. 2019). Furthermore, overexpression of bldA and miaA in Streptomyces venezuelae significantly enhances jadomycin B production (Qiu et al. 2024). These findings collectively reveal that the tRNA modification system has a conserved and important regulatory role in the genus Streptomyces, suggesting its potential as a new target for metabolic engineering interventions.

Based on this background, this study proposes a metabolic engineering strategy centered on the tRNA^Leu_UUA modification pathway, aiming to enhance lincomycin production by improving the biosynthesis and functional modification of tRNA. We constructed a series of combinatorial expression strains and systematically compared the effects of single, double, and multiple gene co-expression of bldA, miaA, miaB, and miaE under different combinations to clarify the contribution of each gene to lincomycin synthesis. After identifying the optimal gene combination, we further employed promoter engineering to optimize the expression of key genes, screening multiple promoters including constitutive and phase-specific promoters to achieve fine regulation. Finally, by introducing a Flag tag into the engineered strains, we validated the enhancing effect of this strategy on the expression levels of key regulators containing UUA codons at both transcriptional and translational levels. In summary, this study not only aims to improve the production of lincomycin but also seeks to use tRNA modification engineering as a starting point to explore a universal metabolic optimization strategy applicable to other Streptomyces, providing new insights for the rational design of secondary metabolic regulation.

Bacterial strains, plasmids and culture conditions

The strains and plasmids used in this study are listed in Table 1. Escherichia coli and S. lincolnensis were cultured according to the methods described in our previously published work (Mao et al. 2024).

Table 1 Strains and plasmids.

Strains or plasmids		Description	Source
Strains
E. coli
DH5α		Routine cloning host	Lab stocking
S17-1		Donor of conjugation transfer	Lab stocking
Streptomyces coelicolor
M145		Wild-type	Lab stocking
M145::pXYpE		M145 φC31 attB::pXYpE	This study
M145::pXYpK		M145 φC31 attB::pXYpK	This study
M145::pXYpthM		M145 φC31 attB::pXYpthM	This study
M145::pXYpY		M145 φC31 attB::pXYpY	This study
M145::pXYplmbU		M145 φC31 attB::pXYplmbU	This study
S. lincolnensis
xbl1		Wild-type (WT), lincomycin producer	Lab stocking
OpNbldA		xbl1 φC31 attB::pIBpNbldA	This study
ST1		xbl1 φC31 attB::pIBpNbldApEA	This study
ST2		xbl1 φC31 attB::pIBpNbldApEB	This study
ST3		xbl1 φC31 attB::pIBpNbldApEE	This study
ST4		xbl1 φC31 attB::pIBpNbldApEAB	This study
ST5		xbl1 φC31 attB::pIBpNbldApEAE	This study
ST6		xbl1 φC31 attB::pIBpNbldApEBE	This study
ST7		xbl1 φC31 attB::pIBpNbldApEABE	This study
xbl1::pXYpE		xbl1 φC31 attB::pXYpE	This study
xbl1::pXYpK		xbl1 φC31 attB::pXYpK	This study
xbl1::pXYpthM		xbl1 φC31 attB::pXYpthM	This study
xbl1::pXYpY		xbl1 φC31 attB::pXYpY	This study
xbl1::pXYplmbU		xbl1 φC31 attB::pXYplmbU	This study
STY		xbl1 φC31 attB::pIBpNbldApYA	This study
STK		xbl1 φC31 attB::pIBpNbldApKA	This study
STU		xbl1 φC31 attB::pIBpNbldApUA	This study
STM		xbl1 φC31 attB::pIBpNbldApMA	This study
STU::pBTflag-lmbU		STU φBT1 attB::pBTflag-lmbU	This study
STU::pBTflag-adpA		STU φBT1 attB::pBTflag-adpA	This study
Plasmids
pIB139	Integrative vector based on φC31 integrase, PermE*, Am^r		Lab stocking
pIBpNbldA	pIB139 harboring bldA driven by PbldA		This study
pIBpNbldApEA	pIB139 harboring bldA driven by PbldA and miaA driven by PermE*		This study
pIBpNbldApEB	pIB139 harboring bldA driven by PbldA and miaB driven by PermE*		This study
pIBpNbldApEE	pIB139 harboring bldA driven by PbldA and miaE driven by PermE*		This study
pIBpNbldApEAB	pIB139 harboring bldA driven by PbldA as well as miaA and miaB driven by PermE* respectively		This study
pIBpNbldApEAE	pIB139 harboring bldA driven by PbldA as well as miaA and miaE driven by PermE* respectively		This study
pIBpNbldApEBE	pIB139 harboring bldA driven by PbldA as well as miaB and miaE driven by PermE* respectively		This study
pIBpNbldApEABE	pIB139 harboring bldA driven by PbldA as well as miaA, miaB and miaE driven by PermE* respectively		This study
pXYpE	pIB139 harboring xylTE driven by PermE*		This study
pXYpK	pIB139 harboring xylTE driven by PkasO*		This study
pXYptM	pIB139 harboring xylTE driven by PthlM4		This study
pXYpY	pIB139 harboring xylTE driven by PstnY		This study
pXYplmbU	pIB139 harboring xylTE driven by PlmbU		This study
pIBpNbldApYA	pIB139 harboring bldA driven by PbldA and miaA driven by PermE*		This study
pIBpNbldApKA	pIB139 harboring bldA driven by PbldA and miaA driven by PkasO*		This study
pIBpNbldApMA	pIB139 harboring bldA driven by PbldA and miaA driven by PthlM4		This study
pIBpNbldApUA	pIB139 harboring bldA driven by PbldA and miaA driven by PlmbU		This study
pBTneo	Integrative vector based on φBT1 integrase, PermE*, Km^r		This study
pBTflag-lmbU	pBT harboring lmbU with a Flag tag at the 5' end under control of PlmbU		This study
pBTflag-adpA	pBT harboring adpA with a Flag tag at the 3' end under control of PadpA		This study

Fermentation of S. lincolnensis

The spore suspension of S. lincolnensis was evenly spread on MS solid medium (20 g/L soybean meal, 20 g/L mannitol, and 20 g/L agar) and cultured at 28 °C for 96 h. Subsequently, a 1 cm × 1 cm agar block bearing mycelia was excised and inoculated into SFPⅠ liquid medium (1 g/L soya flour, 20 g/L soluble starch, 10 g/L glucose, 30 g/L corn steep liquor, 1.5 g/L (NH₄)₂SO₄, and 4 g/L CaCO₃, pH 7.2). The culture was incubated at 28 °C with shaking at 200 rpm for 96 h to obtain the seed culture. This seed culture was then transferred at a 4% (v/v) inoculation rate into SFPⅡ medium (105 g/L glucose, 3.5 g/L corn steep liquor, 20 g/L soybean meal, 8 g/L CaCO₃, 6 g/L (NH₄)₂SO₄, 5 g/L NaCl, 0.025 g/L K₂HPO₄, and 8 g/L NaNO₃, pH 7.8) and further cultivated at 28 °C with shaking at 200 rpm for 144 h.

RNA Extraction, Reverse Transcription, and Quantitative Real-time PCR (qRT-PCR)

S. lincolnensis cultured in SFPII medium for 96 h were collected for transcriptomic analysis. Total RNA was extracted using the Trizol method (Thermo Fisher Scientific). The specific procedure included rapid freezing in liquid nitrogen, grinding to lyse cells, followed by phase separation with chloroform and purification via isopropanol precipitation. Residual genomic DNA in the extracted RNA was removed using DNase I (TaKaRa). First-strand cDNA was synthesized using Reverse Transcriptase M-MLV (RNase H⁻) (TaKaRa).

RT-qPCR amplification was performed with Hieff UNICON® Universal Blue qPCR SYBR Green Master Mix (Yeasen) on ABI-7500 Fast system (Thermo Fisher Scientific). The housekeeping gene hrdB was used as an internal control. Primer specificity was verified by melt curve analysis, and the relative expression levels of target genes were calculated using the 2^-ΔΔCT method. Three biological replicates were included for each sample to ensure data reliability.

Lincomycin analysis

The lincomycin content was analyzed using a Shimadzu LC-20A high-performance liquid chromatography system. An Agilent ZORBAX Eclipse XDB-C18 column (4.6 mm×150 mm, 5 μm) served as the stationary phase, with a mobile phase consisting of 50 mM ammonium acetate buffer-methanol (40:60, v/v). The analytical conditions included: column temperature of 30°C, flow rate of 0.6 mL/min, detection wavelength of 214 nm, and injection volume of 10 μL.

For sample preparation, the fermentation broth was centrifuged and the supernatant was mixed with mobile phase at a 1:3 (v/v) ratio. The mixture was vortex-mixed thoroughly and then filtered through a 0.22 μm membrane filter, with the resulting filtrate used for instrumental analysis.

USP-grade lincomycin standard (Sangon Biotech) was progressively diluted with mobile phase to prepare standard solutions at concentrations of 10, 20, 40, 80, 160, and 320 mg/L. A quantitative calibration curve was established by performing linear regression of peak area (Y) against standard solution concentration (X, mg/L). The method demonstrated excellent linearity within the specified concentration range, with R²≥0.999.

Catechol dioxygenase activity analysis

Promoter fragments of PermE*, PkasO*, PthlM4, and PstnY were obtained by PCR amplification based on sequences reported in the literature (Wang et al. 2013, Wang et al. 2019, Guo et al. 2023). The PlmbU promoter fragment was amplified using primer pair xylmbU-F/R with xbl1 genomic DNA as the template. The xylTE reporter gene was amplified using primer pair xylF/R with plasmid p0469TE as the template. The linearized pIB139 vector, digested with PvuII, was assembled with the above promoter fragments and the xylTE gene via multiF seamless assembly mix (ABclonal), resulting in the construction of reporter plasmids pXYpE, pXYpK, pXYpth4, pXTpY, and pXYplmbU.

The constructed reporter plasmids were introduced into S. lincolnensis xbl1 and S. coelicolor M145 via conjugal transfer, yielding the corresponding recombinant reporter strains. Reporter strains were inoculated from MS solid medium into YEME liquid medium and cultured in a shaker for 96 h. After measuring the OD₆₀₀ of the cultures, cell biomass equivalent to 2 OD was harvested, resuspended in 1 mL of YEME medium, and transferred into fresh YEME medium for an additional 24 h of cultivation. Three biological replicates were set up for each strain.

Cells were harvested and washed twice with washing buffer (20 mmol/L K₃PO₄, pH 7.2), followed by resuspension in resuspension buffer (100 mmol/L K₃PO₄, 20 mmol/L EDTA, 10% (v/v) acetone, pH 7.5) to adjust the cell density to 4 OD. Cell disruption was performed using an ultrasonic cell disruptor (SCIENTZ) with the following parameters: 28% power, 4 s pulse-on, 4 s pulse-off, for a total duration of 4 min. The lysate was centrifuged at 12,000 rpm for 30 min at 4°C, and the supernatant was collected as the whole-cell protein extract.

A volume of 20 μL of the whole-cell protein extract (appropriately diluted according to the detection range of the microplate reader) was thoroughly mixed with 180 μL of assay buffer (100 mmol/L K₃PO₄, 1 mmol/L catechol (added immediately before use), pH 7.5). The absorbance at 375 nm was immediately measured using a microplate reader (BioTek) at 0, 1, 2, 3, 4, 5, and 6 min. A reaction kinetic curve was plotted with reaction time as the X-axis and OD₃₇₅ as the Y-axis, where the slope represents the enzyme reaction rate (U/min). The total protein concentration of the whole-cell protein extract was determined using the Bradford method. The specific enzyme activity (U/min/mg) was calculated by dividing the enzyme reaction rate by the total protein concentration.

Western Blot Analysis

S. lincolnensis cultured in SFPⅡ medium for 96 h were collected, washed twice with PBS buffer (2 mmol/L KH₂PO₄, 10 mmol/L Na₂HPO₄, 137 mmol/L NaCl, 2.7 mmol/L KCl, pH 7.4), and stored at -85 ℃. The frozen cells were ground into a fine powder in a liquid nitrogen-pre-cooled mortar, resuspended in PBS buffer, and centrifuged at 12,000 rpm for 30 min at 4℃. The supernatant was collected as the total protein extract.

Protein concentration was determined using the Bradford method, and samples were diluted to 1 mg/mL with PBS buffer. A 20 μL aliquot of each protein sample was mixed with an equal volume of 2×SDS-PAGE loading buffer, boiled at 100℃ for 5 min, and centrifuged at 12,000 rpm for 10 min before SDS-PAGE analysis. After electrophoresis, the separation gel was excised for membrane transfer.

The PVDF membrane was activated in methanol for 10 s and equilibrated in transfer buffer (24 mmol/L Tris, 192 mmol/L glycine, 20% methanol). The transfer sandwich was assembled in the following order from cathode to anode: filter paper – separation gel – PVDF membrane – filter paper. Transfer was performed in an ice-water bath at a constant current of 380 mA for 30 min.

After transfer, the PVDF membrane was blocked in blocking solution (50 mmol/L Tris-Cl, 150 mmol/L NaCl, 0.2% Tween-20, 5% skim milk powder, pH 7.4) at room temperature for 2 h. The membrane was then incubated sequentially with primary antibody (Mouse monoclonal Flag antibody, Proteintech; 1:10,000 dilution in blocking solution) for 1 h at room temperature and secondary antibody (HRP-conjugated Goat Anti-Mouse IgG, Proteintech; 1:10,000 dilution in blocking solution) for 1 h at room temperature. After each incubation, the membrane was washed three times with TBST buffer (50 mmol/L Tris-Cl, 150 mmol/L NaCl, 0.2% Tween-20, pH 7.4) for 10 min each. Finally, the membrane was developed using ECL chemiluminescence reagent (Beyotime), and images were captured with a chemiluminescence imaging system (Tanon).

Co-overexpression of bldA and miaA Enhances Lincomycin Production

The coding sequences of the positive regulators LmbU and AdpA in lincomycin biosynthesis contain the rare UUA codon, whose translation depends on the tRNA^Leu_UUA encoded by bldA. Previous studies have shown that overexpression of lmbU and adpA can significantly increase lincomycin production (Hou et al. 2018, Kang et al. 2019). Based on this, we sought to enhance lincomycin biosynthesis by improving the translational efficiency of these positive regulators.

Our results showed that overexpression of bldA under its native promoter (strain OpNbldA) did not significantly improve lincomycin production (Fig. 2a). We speculated that although the intracellular abundance of tRNA^Leu_UUA increased, its functional maturation still depended on a complete post-transcriptional modification process. In S. lincolnensis, tRNA^Leu_UUA undergoes sequential modifications by the enzymes MiaA, MiaB, and MiaE, whose genes are located at different genomic loci and are independently regulated by their own promoters (Fig. 1a, 1b). Therefore, overexpression of bldA alone may create an imbalance between tRNA^Leu_UUA abundance and its subsequent modification capacity, limiting the formation of functionally mature tRNA and thus weakening the overall effect of overexpression.

To examine the role of these modification enzymes, we constructed co-overexpression strains ST1, ST2, and ST3, in which bldA was co-expressed with miaA, miaB, or miaE, respectively. PermE* was used to individually drive the expression of modification enzyme genes. Lincomycin titers in ST1 and ST2 increased markedly, whereas production in ST3 decreased, suggesting that miaE may exert an inhibitory effect on lincomycin biosynthesis (Fig. 2a). Phenotypic analysis revealed that ST3 almost completely lost the ability to form aerial mycelia on MS agar, although its growth rate was comparable to that of the wild type (Fig. 2b, S1). This indicates that the decreased production may be related to impaired morphological differentiation.

To further optimize lincomycin production, we constructed double co-overexpression strains ST4, ST5, and ST6, expressing bldA in combination with pairs of miaA, miaB, and miaE. Lincomycin production in ST5 was not higher than in ST1, while both ST5 and ST6 showed significant decreases in production, further confirming the negative influence of miaE (Fig. 2a). Finally, the quadruple co-overexpression strain ST7, harboring bldA, miaA, miaB, and miaE, did not show any additional improvement in production (Fig. 2a).

In summary, the co-overexpression of bldA and miaA exerted the most pronounced positive effect on lincomycin biosynthesis. These findings provide clear guidance for subsequent metabolic engineering strategies aimed at enhancing antibiotic production. It is noteworthy that due to the high conservation of MiaA across various Streptomyces species, this strategy holds promise as a universal metabolic engineering approach (Fig. S2). By introducing or overexpressing miaA to activate silent gene clusters, it may open new avenues for the discovery of novel natural products.

Temporal regulation of miaA expression enhances lincomycin production

Building on the synergistic improvement in lincomycin production achieved by co-overexpressing miaA and bldA, we next sought to enhance miaA transcription through promoter optimization. Guided by transcriptomic data and previous reports, we selected several candidate promoters, including the constitutive promoters PermE^⁎, PkasO^⁎, PthlM4, and PstnY, as well as the phase-dependent promoter PlmbU (Wang et al. 2013, Wang et al. 2019, Guo et al. 2023).

To compare their transcriptional strengths in Streptomyces, each promoter was fused to the xylTE reporter gene and integrated into both S. lincolnensis and S. coelicolor. Reporter activity, quantified via catechol 2,3-dioxygenase assays, revealed that PkasO^⁎ exhibited the highest transcriptional strength, exceeding that of PermE*. PthlM4 showed reduced activity, whereas PstnY was slightly stronger in S. lincolnensis. In contrast, PlmbU displayed minimal activity under YEME liquid culture, consistent with its phase-dependent expression profile (Fig. 3).

Each promoter was subsequently used to drive miaA overexpression, generating strains STY, STK, STU, and STM4. Despite its weak apparent strength, PlmbU-driven miaA expression yielded the highest lincomycin production (Fig. 4). This unexpected result indicates that maximal, constitutive miaA expression is not advantageous. Excessive expression may enhance translation of UUA-codon-containing repressors, counteracting antibiotic biosynthesis. In contrast, the temporally regulated PlmbU promoter aligns miaA expression with the critical phase of lincomycin biosynthesis, providing stage-specific enhancement and superior production efficiency.

Enhanced Translation of Positive Regulators via Co-expression of bldA and miaA

Based on the optimized strain STU, the translational effects on key UUA-containing regulators were examined. We introduced flag-tagged lmbU and adpA separately into both STU and xbl1 through BT1 site-specific homologous recombination and then compared their expression at both the transcriptional and translational levels.

qRT-PCR analysis showed no significant change in mRNA levels of adpA or lmbU between STU and xbl1 (Fig. 5a). In contrast, Western blot analysis revealed a substantial increase in the protein abundance of both regulators in STU (Fig. 5b). These results demonstrate that co-expression of bldA and miaA effectively improves the decoding efficiency of the rare UUA codon, enhancing translation of key positive regulators and ultimately promoting lincomycin biosynthesis.

This study developed a novel metabolic engineering strategy based on tRNA modification engineering. By coordinately overexpressing bldA and its key modifying enzyme gene miaA, we effectively enhanced the biosynthesis capacity of lincomycin. Our findings reveal that the success of this strategy depends not only on gene expression levels but, more critically, on the temporal regulation of expression—using the PlmbU promoter, which aligns with the lincomycin biosynthesis phase, to drive miaA expression resulted in superior production performance compared to traditional strong constitutive promoters. At the mechanistic level, this study is the first to demonstrate in S. lincolnensis that the strategy specifically enhances the decoding efficiency of the UUA rare codon, significantly increasing the protein expression of key positive regulators LmbU and AdpA at the translational level, thereby systematically alleviating the translational limitation in lincomycin biosynthesis. This research not only provides an effective approach for improving lincomycin yield but also offers theoretical insights and methodological references for the precise regulation of other rare-codon-dependent secondary metabolite biosynthesis.

CRediT authorship contribution statement

All authors contributed to the conception and design of the study. Material preparation, data collection, and analysis were performed by Yue Mao, Ruida Wang, and Haizhen Wu. Yue Mao drafted the initial version of the manuscript. Jiang Ye, Ruida Wang, Haizhen Wu, and Huizhan Zhang contributed to the review and editing of the manuscript. Ruida Wang, Haizhen Wu, and Huizhan Zhang supervised the project and provided critical revision of the work. All authors read and approved the final version of the manuscript.

Funding

This work was funded by the National Natural Science Foundation of China (NSFC) (42406126) and the National Key Research and Development Program of China (2021YFC2100600) and the President’s Fund of Tarim University (TDZKBS202536)

Conflict of interest

The authors have no relevant financial or non-financial interests to disclose.

Data availability

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

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No competing interests reported.

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A tRNA modification-based regulatory strategy for lincomycin biosynthesis in Streptomyces lincolnensis

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Abstract

Figures

1. Introduction

2. Materials and methods

3. Results and discussion

Conclusion

Declarations

References

Additional Declarations

Supplementary Files

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