Parageobacillus thermoglucosidasius NCIMB 11955 was engineered to produce 3-hydroxybutanone (acetoin) and 2,3-butanediol , organic compounds with commercial applications. Using heterologous acetolactate synthase (ALS) and acetolactate decarboxylase (ALD) enzymes, a functional 2,3-BDO biosynthetic pathway was constructed. The formation of by-products was minimized by the deletion of competing pathways surrounding the pyruvate node. Redox imbalance was addressed through autonomous overexpression of the butanediol dehydrogenase and by investigating appropriate aeration levels. Through this, we were able to produce 2,3-BDO as the predominant fermentation metabolite, with up to 6.6 g/L 2,3-BDO (0.33 g/g glucose) representing 66% of the theoretical maximum at 50°C. In addition, the identification and subsequent deletion of a previously unreported thermophilic acetoin degradation gene (acoB1) resulted in enhanced acetoin production under aerobic conditions, producing 7.6 g/L (0.38 g/g glucose) representing 78% of the theoretical maximum. Furthermore, through the generation of a ΔacoB1 mutant and by testing the effect of glucose concentration on 2,3-BDO production, we were able to produce 15.6 g/L of 2,3-BDO in media supplemented with 5% glucose, the highest titre of 2,3-BDO produced in Parageobacillus and Geobacillus species to date.The current climate crisis has emphasised the need to achieve global net-zero by 2050, with countries being urged to set considerable emission reduction targets by 2030. Exploitation of a fermentative process that uses a thermophilic chassis can represent a way to manufacture chemicals and fuels through more environmentally friendly routes with a net reduction in greenhouse gas emissions. In this study, the industrially relevant thermophile  Its gloCurrently, both chemicals are primarily derived from petrochemicals by chemical-synthesis. However, concerns over the finite nature of fossil fuels and the current climate crisis, coupled with increasing demands, have provided a greater incentive to produce 2,3-BDO and acetoin via sustainable and renewable approaches. Microbial-based fermentations using lignocellulose, a renewable feedstock, has begun to attracted considerable attention in recent years . This isKlebsiella penumoniae, Klebsiella oxytoca and Paenibacillus polymyxa, which were known to produce significant amounts . In both cases, with all major competing pathways disrupted, including ethanol production, up to 90% of the carbon flux entered the acetoin/2,3-BDO pathway, giving scope for further improvement.To achieve high titre 2,3-BDO and acetoin production, three interrelated approaches were taken by: 1) implementing an efficient acetoin/2,3-BDO pathway through the assembly and expression of genes encoding acetolactate synthase [EC 2.2.1.6] (ALS), acetolactate decarboxylase [EC 4.1.1.5] (ALD) and acetoin reductase/2,3-BDO-dehydrogenase [EC 1.1.1.4] or AR/BDH ; 2) inacAll chemicals and reagents were obtained from Sigma-Aldrich Ltd., . DNA polymerases, restriction enzymes, T4 ligase and DNA ladders were purchased from New England Biolabs or Promega . EDTA-Free Protease Inhibitor Cocktail Tablets were purchased from Roche E. coli Top 10 was used as a host for plasmid construction and E. coli B21 (DE3) was used for protein expression. P. thermoglucosidasius NCIMB 11955 was used for 2,3-BDO and acetoin production. All E. coli strains were grown in Luria-Bertani (LB) agar or LB broth containing appropriate antibiotics  at 30°C or 37°C with agitation of 200 rpm. P. thermoglucosidasius strains were routinely grown on TSA agar or in 2SPYNG broth  with appropriate carbon sources at 1%.To assess growth on acetoin and 2,3-BDO, WT n in CBM   at a 10% (v/v). Fermentation was carried out on a rotatory shaker at 250 rpm at 50°C for 48 h in sealed 50 mL Falcon tubes with various volumes as indicated before analysis.For acetoin and 2,3-BDO production, typically, appropriate strains from a 10 mL overnight (O/N) preculture were inoculated to prewarmed modified ASYE medium (2 mM MgSOE. coli Top10 cells produced in-house were used to propagate the constructed plasmids and plasmids were harvested using Monarch® Plasmid Miniprep Kit (New England BioLabs). All cloning procedures were conducted following the manufacturer’s instructions. Sanger sequencing  were routinely performed to confirm the authenticity of plasmid constructs. Preparation of P. thermoglucosidasius electro-competent cells and electroporation procedure using Genepulser electroporator  was undertaken in accordance with the method previously described  was fused with the ALD(n) gene concomitant with the introduction of a synthetic RBS (ribosome binding site). Primers used were GPAD_F_NotI, GPAD_R_Bot9, ALD(n)_Bot9_F and ALD(n)_R_XhoI. The resultant DNA fragment, flanked by NotI and XhoI restriction sites, was combined with the previous fragment carrying ALS to give plasmid pMTL-BD(n)  Plasmid pMTL-BD3b could not be propagated in E. coli due to cell toxicity, instead, inserts containing the promoter of the rplS gene fused to the native bdh gene  were ligated with pMTL-BD3 after restriction digest with XhoI and NheI and electroporated directly into NICMB 11955 following dialysis treatment. Colony PCR and Sanger sequencing were used to verify that each plasmid was correct.All of the plasmid constructs carrying the genes of the 2,3-BDO biosynthesis pathway are based on pMTL61110 , which cStreptococcus thermophilus Cas9-3. Synthetic knockout cassettes contained target specific sgRNA, identified using Benchling CRISPR Guide Design software (www.benchling.com), and homology arms (HA) synthesized by Genewiz Ltd. HA consists of 501 bp regions of DNA from up- (5′) and down- (3′) stream of the gene to be inactivated. Ligation of the fragments between the BamHI and AscI restriction sites of pMTL67555 yielded the knockout plasmids. Deletion cassettes were designed to generated an in-frame deletion of the targeted gene comprising just 3 codons, including the start and stop codons. Following the successful transformation of the knockout plasmids into NICMB 11955, transformants are passaged on TSA-kanamycin agar for 3 days before verification of gene knockout by colony PCR. Once verified, the mutants were passaged for at least 6 times in 2SPYNG without selection  for plasmid curing.All constructs for various targeted gene knockouts were based on pMTL67555  which is+ was monitored spectrophotometrically by the AR dependent rate of A340 decrease. Acetolactate decarboxylase activity was assayed as previously described  creatinine. After incubation at 40 min at room temperature, A522 was used for quantification of the final acetoin concentration using an acetoin standard curve. Negative controls included reaction with and without substrate in the absence of ALD. One Unit of ALD activity is defined as the concentration of acetoin by ALD (absorbance/a constant molar coefficient ε) per 1 mg of protein.Crude cell extracts were prepared using BugBuster 10X protein extraction reagent (Merck). The protein extraction buffer (1X) consisted of BugBuster 10X (2 mL), sodium phosphate buffer 50 mM (18 mL), EDTA-free protease inhibitor tablet (Roche), and lysozyme (20 mg/mL). Total protein concentrations of the soluble fraction of cell lysates were determined using BCA-kit  following the manufacturer’s instructions and a standard curve generated from BSA  was used to calculate the protein concentrations. Assays for acetoin reductase activity were carried out at 60°C, initiated by adding 10 μL crude cell lysate to a solution containing 50 mM acetoin, 0.5 mM NADH or NADPH and 1 mM TCEP . Oxidatiescribed . In shor600) using a Pharmacia Novaspec II. Metabolites were analysed using high-performance liquid chromatography (HPLC-UV and RI), performed using the Dionex UltiMate 3,000 System and 5 mM H2SO4 as the mobile phase. Valeric acid at 50 mM was used as an internal standard. The sample supernatants were filtered with 0.2 μm filters and mixed in 1:1 ratio with diluent. The Bio-Rad Aminex HPX-87H 300 mm × 7.8 mm × 9 μm column was used for sample elucidation, running at a flow rate of 0.5 mL/min, at 35°C for 55 min. All experiments were performed in triplicate and data analysed using PRISM  and Excel (Microsoft).Growth of all bacterial cultures was monitored by measuring optical density at 600 nm (ODP. thermoglucosidasius NICMB 11955 genome possesses genes encoding homologues of ALS (BCV53_05415) and BDH (BCV53_03685) but not ALD. Although it may still be able to produce 2,3-BDO via the oxygen-dependent, spontaneous oxidation of acetolactate to di-acetyl, no trace was evident in culture supernatants following growth of the WT in ASYE medium. The major metabolites  concentrations . Enzyme assays with acetoin as the substrate were conducted at 60°C using crude cell extracts of the E. coli clone generated. These showed  is a suitable candidate  (The native hibition . Essentiandidate . Interes6 (ALS2) , a speci6 (ALS2) . ALS2 isThe conversion of acetolactate to acetoin also needs to be efficiently catalyzed. An effective ALD is, therefore, essential. Furthermore, it is desirable to have a wide selection of pathway components to give greater flexibility for a more integrated approach to metabolic engineering, as sometimes the introduction of high enzyme activities throughout a pathway, especially in heterologous hosts, can lead to depletion of intermediates, causing diminished growth, cell toxicity, and reduced product yields .Exiguobacterium acetylicum (formerly Brevibacterium acetylicum). However, previous reports have indicated that its ALD (ALD1) possessed promising properties including stability at 60°C, high activity at 50°C and a broad pH range of 5–8 (B. coagulans) (Bacillus cereus) (P. thermopropionicum) (Chaetomium thermophilum) (Thielavia terrestris) (Genome sequences of the two thermophilic producers  were unae of 5–8 . Others agulans)  and ALD3 cereus) ; one froionicum)  and two ophilum)  and ALD6restris) .P. thermoglucosidasius expression vector pMTL61110_pldh between its XbaI and KpnI restriction sites. This positioned the ALD genes between the strong Pldh promoter of the G. stearothermophilus ldh (lactate dehydrogenase) gene . Significant activities were observed in extracts made from cells producing three of the ALDs. ALD2 had the highest activity (0.81 ± 0.00 U/mg), followed by ALD6 and ALD4, which exhibited 73% and 70%, respectively, of the activity of ALD1 (The synthetic genes (Genewiz Ltd.) encoding the ALD candidates were codon optimized and cloned into the se) gene  and the rnB gene . The 3’  of ALD1 .ldh and Pgapd promoters, respectively  were tested and only JM109 and NEB F− allowed propagation of the desired plasmids. No growth was evident in the case of DH5α, while although Top10 transformants grew, the plasmids were found to have acquired mutations effecting the integrity of the cloned genes. Insertions/deletions and nucleotide substitutions that respectively caused frameshifts and nonsense mutations in the ALD gene proved to be a frequent occurrence when plasmids were introduced into P. thermoglucosidasius. In total, just three cassettes  were obtained that were cloneable in E.coli and which maintained structural integrity (verified by Sanger sequencing) for at least three passages following transformation into P. thermoglucosidasius. Plasmids carrying these cassettes (encoded enzymes in brackets), were pMTL-BD2 (ALS2-ALD1), pMTL-BD3 (ALS1-ALD3) and pMTL-BD5 (ALS1-ALD5).During the assembly of the various combinations of ALS and ALD genes, a major obstacle observed was cell toxicity both when cloning, in P. thermoglucosidasius WT and the LS100 mutant carrying plasmids pMTL-BD2, pMTL-BD3 and pMTL-BD5 to identify which combination of ALS and ALD was the most productive  of competing pathways (lactate and ethanol formation) are essential for elevated production of 2,3-BDO (The Embden-Meyerhof-Parnas (EMP) pathway requires two NAD + for the production of ATP. In aerobes, NAD+ is recycled by the oxidation of NADH by molecular oxygen. Under oxygen-limiting conditions, a number of fermentative routes in P. thermoglucosidasius are utilized for redox recycling. While they help to maintain redox balance, carbons diverted into various chemicals severely impact the yield of the desired product. A number of studies have shown that deleting genes  as reported previously  and sharing no sequence similarities to other non-P. thermoglucosidasius strains. The gene was designated ldh2. In the case of glycerol, biosynthesis can occur either via glycerol-3-phosphate dehydrogenase (G3PDH)  are present in P. thermoglucosidasius NICMB 11955. G3PDH1, also annotated as a FAD dependent oxidoreductase, encodes a 550 AA protein sharing 67% and 30% sequence identity to B. subtilis and S. cerevisiae, respectively. G3PDH2 codes for a 348 AA enzyme that is highly conserved across species (97% in bacilli and 87% in S. cerevisiae). GDH1 encodes a 363 AA protein sharing 61% identity to the GDH of K. pneumoniae and 50% to that of B. subtilis while GDH2 is 358 AA with no significant sequence identity to proteins in any other genus.Prevention of ethanol formation could be most simply achieved  by knockeviously . Althoug (G3PDH)  or via g (G3PDH) . Two copynthesis . When coynthesis . Two G3PΔldhΔpflΔg3pdh1); LS102 (ΔldhΔpflΔgdh1); LS103 (ΔldhΔpflΔldh2), LS104 (ΔldhΔpflΔg3pdh1Δgdh1); LS105 (ΔldhΔpflΔg3pdh1Δgdh1Δldh2); LS200 (LS100ΔadhE); LS201 (LS101ΔadhE); LS202 (LS102ΔadhE); LS204(LS104ΔadhE) and LS205 (LS105ΔadhE). Despite numerous attempts, the two genes g3pdh2 and gdh2 could not be deleted. Temporary deletions were observed and confirmed by PCR, however, curing the deletion vector proved problematic and when the plasmid was lost reversion back to the parental strain was always observed. It is likely that those are essential genes, for example, the highly conserved g3pdh2 could be used for the biosynthesis of phospholipid membrane  for LS103 and LS105, respectively. No differences were observed for the single and double glycerol pathway mutants. Ethanol levels were also increased, with up to a 3-fold increase with LS105. For the AdhE mutants, ethanol production was largely eliminated, but 2,3-BDO levels were much reduced to levels of between 13 and 19 mM. Lactate levels increased slightly (1.5–2.0 mM) with single g3pdh1 deletions and were reduced in the single ldh2 mutant (0.8 mM). However, with both g3pdh1 and gdh1 deleted, the lactate level increased significantly (up to 9 mM) in strains with or without Δldh, indicating the presence of alternative routes. Other studies , three glyoxylase enzymes  and methylglyoxal reductase (BCV53_06315).The various mutant strains obtained were transformed with pMTL-BD3 and the fermentation profiles of the resultant transformants analyzed . Other t studies  suggest adhE strains, with the LS102 and LS104 producing only 1.1 and 0.6 mM, respectively, most likely a consequence of the redox imbalance caused by the loss of the capacity to regenerate NAD+ with the loss of the ethanol pathway. Thus, in the ΔadhE strains, both lactate and glycerol levels were significantly increased, despite almost double the amount of residual glucose being left in the media compare to the corresponding non-adhE mutants.Glycerol levels were reduced in all non-ΔadhE reduces growth and productivity. Whereas P. thermoglucosidasius strain DSM 2542, we observed both a decrease in titre and yield. However, it is still desirable to maximize carbon flux to the acetoin/2,3-BDO pathway necessitating alternative ways of co-factor regeneration.With the disruption of lactate production, the acetyl-CoA pathway plays a major role in maintaining redox balance under oxygen limitation. It follows that ethanol is a major by-product during 2,3-BDO fermentation and deletion of ΔadhE strains. In terms of 2,3-BDO production, the highest titre achieved was 34 mM, using strain LS205 habouring pMTL-BD3. With more aeration, production switched to acetoin, and in 2 mL cultures up to 52 mM was produced , which is reported to give strong, constitutive expression levels under varied growth conditions  under limited aeration, the levels of 2,3-BDO decreased. There was also a 2-fold decrease in ethanol produced and glucose consumed (Data not shown). We concluded that this is because increased bdh expression shifts the carbon flux towards the acetoin/2,3-BDO pathway, but without sufficient aeration, NAD+ deficiency brings about growth termination. Clearly, 2,3-BDO levels are determined by the intricate balance of oxygen supply and acetoin conversion. To optimize further, a wider range of fermentation volumes were analyzed, and at 6 mL, the highest amount of 2,3-BDO was produced at 63.4 mM  acetoin or 2,3-BDO as the sole carbon source in baffled-flasks at 60°C. NICMB 11955 utilized both acetoin and 2,3-BDO rapidly, reaching a maximum OD600 of >8.0 in 12 h. A protein BLAST (BLASTp) search of the P. thermoglucosidasius genome identified three potential candidates involved in catabolism. The first candidate AcuABC (locus BCV53_18410-18420) is annotated as acetoin utilization gene ABC and comprises a polycistronic operon with AcuA separated by 34 bp from AcuB and AcuB overlapped by 4 bp with AcuC. The further two candidates were annotated as (acetyl-transferring) pyruvate dehydrogenase complex (PDHC) E1 component subunits (BCV53_10285 and 10935).To test whether B. subtilis acetoin utilization proteins, AcuA (ACDH), AcuB, and AcuC (histone deacetylase), one of two separate operons (AcuABC and AcoABCL) responsible for acetoin consumption in this bacillus (P. thermoglucosidasius NICMB 11955 acuC gene is the a gene coding for the catabolite control protein A (CCPA), which in B. subtilis was reported to negatively regulate acetoin utilization by repression of AcoR, the transcriptional activator of AcuABC  oxidoreductase subunit alpha. A study by The two identified PDHCs belong to the 2-oxoacid hydrogenase complex (OADHCs.) family, a super-family containing many different dehydrogenase complexes, including the acetoin dehydrogenase system (ADHC) . The NCBacoB1), LS107 (ΔacoB2), LS108 (ΔacuA), LS109 (ΔacuB), LS110 (ΔacuC) and LS111 (ΔacuA-C). Deletion of acoB1 had a dramatic effect, with no growth evident on media containing either acetoin or 2,3-BDO as the sole carbon source. In contrast, all other mutants had a similar growth profile to the WT. Using glucose, slower growth was observed with all mutants, and the maximum OD600 of LS106 (ΔacoB1) and LS111 (ΔacuC) were slightly reduced compared to WT. For further validation, we expressed the AcoB1 gene in the ΔacoB1 strain under the control of the Pldh promoter using the expression plasmid pMTL61110_pldh. Complementation of the WT growth profile were observed for both acetoin and 2,3-BDO  (BCV53_03505) and acetate kinase (ack) (BCV53_18310) were targeted for deletion in strains LS205 and LS305. Only ack mutants, LS206 and LS306, were obtained. Both strains were transformed with pMTL-BD3 to allow investigation of the effect of the ack mutation on acetoin/2,3-BDO production.Thus far, for both acetoin and 2,3-BDO production, acetate was the only by-product measured that was still produced in significant amounts. In other studies, disruption of acetate production has been shown to enhanced 2,3-BDO yields . Accordi+. Acetaldehyde is further oxidised to acetyl-CoA through the activity of acetaldehyde dehydrogenase (AldDH)  (LS306 (pMTL-BD3) failed to grow under the conditions tested. This may be partly due the level of acetyl-CoA in this strain being insufficient. Acetyl-CoA can be reassimilated through the AcDH-mediated conversion of acetoin to acetaldehyde using cofactor CoA and NAD (AldDH) . Analysise (ACS) . Since mack, acetate was still present, though at a slightly lower level compared to LS205b, probably due to the presence of alternative routes to its formation, involving, for instance, acetyl-CoA ligase . Next, 2,3-BDO level was measured in LS206b. Again, the decoupling of bdh expression positively affected 2,3-BDO production, with up to 73 mm obtained, representing 0.33 g/g yield, the highest yield obtained in this study and 66% of the TMY. In addition, up to 25.9 mM acetoin was obtained, indicating that up to 89% of total carbon flux went into the acetoin/2,3-BDO pathway (In LS206 (pMTL-BD3), 2,3-BDO the titre was comparable to that of LS205 habouring pMTL-BD3b (LS205b) when using 5–7.5 mL fermentation volumes, and much higher under more oxygen-limited conditions . HoweverA ligase  and acetA ligase , both of pathway .Geobacillus  2,3-BDO is produced as the major product at 6.6 g/L, representing a yield of 0.33 g/g and 66% of TMY. Although this failed to exceed the 7.2 g/L titre in P. thermoglucosidasius DSM 2542 reported previously  and LS305 (with acoB1 deletion). However, when using 2 mL, acetoin production increased from 43.7 ± 2.7 mM to 86.0 ± 0.1 mM, respectively, for the same strains, representing a near 100% increase in titre.AcDH belongs to the family of 2-oxo acid dehydrogenase complexes which uses the reducing power of NAD and CO2 . During bdh, and varied glucose concentration were unsuccessful. Deletion of the native bdh gene resulted in lower acetoin production levels, while its production decreased proportionally to glucose concentration, suggesting that unknown mechanisms such as osmotic stress can regulate 2,3-BDO and acetoin biosynthesis. Remarkably, under aerobic conditions, the predominant metabolite in LS305 shifted from acetoin to 2,3-BDO with increasing glucose concentration, amounting to 15.6 g/L at 5% glucose, representing 64% TMY. The yield obtained here is comparable to what was achieved with LS206 (pMTL-BD3b), (0.32 g/g vs. 0.33 g/g), with the benefit of being able to metabolize more glucose. Moreover, carbon fluxes to the acetoin/2,3-BDO pathway is also comparable, if not better, compared to LS206 (pMTL-BD3b), , with 76% being 2,3-BDO, of which 97% is the R-R enantiomer (acoB1 deletion mutation in strains to support high 2,3-BDO titres under more aerated conditions.Further strategies to enhance acetoin production, including deletion of antiomer . These dP. thermoglucosidasius NCIMB 11955 we demonstrated high yields of acetoin, on par with the highest reported in the literature. This work also serves as a foundation for the optimization of fermentation at a larger scale, with much potential for further improvement.In conclusion, we have created platform strains for the production of acetoin and 2,3-BDO at thermophilic temperatures. By addressing issues of redox imbalance and product utilization, substantial 2,3-BDO was produced with minimal by-product formation. In parallel, for the first time in the industrially relevant strain


Malacosteusniger using long-read sequencing technologies. The 21,263 bp mitogenome features a complex structure with two copies of a 1198-bp inverted-repeat and a region of 2616-bp containing alternating copies of 16 and 26 bp repeat elements. Whole mitogenome phylogenies inferred from both nucleotide and amino-acid datasets place M.niger among Melanostomiinae. The need for additional complete mitogenome sequences from the subfamily Malacosteinae is discussed.We determined the complete mitogenome sequence of the bioluminescent fish  Sunlight is filtered by seawater and only blue/green light with wavelengths between 460 and 490 nm penetrates into the deep sea. Many deep-sea animals with well-adapted eyes can see this very weak light down to about 1500 m . Most ofMalacosteusnigerBRET) reaction  to its blue opsin proteins , a feature which comes in addition to the unique occipito-vertebral gap observed in several Stomiidae . Pieces of the caudal fin and muscle were sent to the “Plateforme d’Analyses Génomiques” of the “Institut de Biologie Intégrative et des Systèmes” of the Université Laval  for DNA library preparation and sequencing. For DNA extraction, 300 mg of muscle were crushed in liquid nitrogen and digested at 65 °C for 30 min in 1.0 ml lysis buffer containing 50 mM Tris-HCl pH 8.0, 200 mM NaCl, 20 mM EDTA, 2.0% SDS and 20 mg/ml proteinase K. An equal volume of CTAB buffer containing 50 mM Tris-HCl pH 8.0, 1.4 M NaCl, 20 mM EDTA, 2.0% CTAB, 1.0% PVP 40,000 was added to the lysate and incubation was pursued for an additional 30 min at 65 °C. This mixture was extracted with phenol: chloroform: isoamylalcohol (25:24:1), and following centrifugation, 5 µl of RNase A (100 mg/ml) was added to the aqueous phase and incubated at room temperature for 20 min. This mixture was then extracted twice with an equal volume of chloroform: isoamylalcohol (24:1) and DNA was precipitated with two volumes of EtOH, dried and dissolved in 100 µl of TE buffer .The The library preparation protocol for short reads sequencing was as follows. Genomic DNA (500 ng in 55 ul TE buffer) was mechanically fragmented for 40 s using a Covaris M220  with default settings. Fragmented DNA was transferred to PCR tubes and library synthesis was performed using a NEB Next Ultra II kit (New England Biolabs) according to the manufacturer’s instructions. To barcode the samples, TruSeq HT adapters  were used. The library was sequenced on the Illumina MiSeq platform (300-bp paired-end reads). Of the 15 335 342 raw paired-end reads obtained, 11 121 576 remained after elimination of low-quality reads.For long-read sequencing, DNA was quantified using a Qubit fluorometer (ThermoFisher) and quality checked on a Femto Pulse System with a genomic DNA 165-kb kit . A DNA aliquot of 8 µg was fragmented with a Covaris g-tube  and small fragments were removed using Short Read Eliminator XS . The library for Oxford Nanopore MinIon sequencing was prepared using 1.69 µg of DNA and the LSK-109 ligation sequencing kit , following repair and end-polishing of the sheared DNA using the NEBNext Companion Module for Oxford Nanopore Technologies ligation kit . Finally, 0.595 µg of library were loaded on a R9.4.1 MinION flow cell and sequencing was performed on a GridIon benchtop platform (Oxford Nanopore).https://github.com/rrwick/Filtlong), with the two contigs obtained from short reads as a reference. The filtered reads were assembled using Canu v.2.2  were first concatenated for each species/dataset and then aligned using MAFFT 7  contains 46% G+C and encodes 13 proteins, 22 tRNAs and 2 rRNAs , forming a clade with the MelanostomiinaePhotonectesmargarita  (AP018417) and Trigonolampamiriceps Regan & Trewavas, 1930 (AP012961). This group of taxa is sister to a clade containing the StomiinaeStomiasatriventer Garman, 1899 (MG321595) and the ChauliodontinaeChauliodussloani Bloch & Schneider, 1801 (AP002915) in the amino-acid phylogeny  in the nucleotide phylogeny   (CM035931)  and the ActinopterygiiDrepanepunctata  (KM273123), which reach 28 757 and 23 152 bp, respectively.With 21 263 bp, the mitogenome of N794187) , closelyM035931) . Among CPelagophyceae  that harbor a total of 14 valid species. These studies are expected to shed light not only on the phylogenetic positions of these bioluminescent fishes but also on the putative presence, origin and evolution of the inverted-repeat structure among the mitogenomes of Malacosteinae.Additional mitogenome sequences from the


Introduction: Muscle glycolytic potential (GP) is a key factor affecting multiple meat quality traits. It is calculated based on the contents of residual glycogen and glucose (RG), glucose-6-phosphate (G6P), and lactate (LAT) contents in muscle. However, the genetic mechanism of glycolytic metabolism in skeletal muscle of pigs remains poorly understood. With a history of more than 400 years and some unique characteristics, the Erhualian pig is called the “giant panda” (very precious) in the world’s pig species by Chinese animal husbandry.Methods: Here, we performed a genome-wide association study (GWAS) using 1.4M single nucleotide polymorphisms (SNPs) chips for longissimus RG, G6P, LAT, and GP levels in 301 purebred Erhualian pigs.Results: We found that the average GP value of Erhualian was unusually low (68.09 μmol/g), but the variation was large (10.4–112.7 μmol/g). The SNP-based heritability estimates for the four traits ranged from 0.16–0.32. In total, our GWAS revealed 31 quantitative trait loci (QTLs), including eight for RG, nine for G6P, nine for LAT, five for GP. Of these loci, eight were genome-wide significant (p < 3.8 × 10−7), and six loci were common to two or three traits. Multiple promising candidate genes such as FTO, MINPP1, RIPOR2, SCL8A3, LIFR and SRGAP1 were identified. The genotype combinations of the five GP-associated SNPs also showed significant effect on other meat quality traits.Discussion: These results not only provide insights into the genetic architecture of GP related traits in Erhualian, but also are useful for pig breeding programs involving this breed. Meat quality significantly influences consumers’ preference and purchasing behavior, thus improving meat quality has been being a major goal of pig breeding . In receThe degree of post-mortem pH decline in porcine skeletal muscle is an important factor accounting for the occurrence of inferior meat, as it significantly affects meat color development, water-holding capacity and tenderness . The ultPRKAG3 is the first causal gene proved to affect GP level, and its R200Q mutation significantly increases muscle glycogen content by up to ∼70%, consequently causing acidic meat in Hampshire pigs , a smaller extent of pH decline (pHd) from post-mortem 45 min to 24 h, and a greater variation in the two pH index  and then stored at −80°C until assay.The Erhualian population was established as described previously . Brieflyi) and 24 h (pHu) after slaughter using a Delta 320 pH meter, and their mean values were calculated separately. The difference between pHi and pHu was taken as pH decline (pHd). Drip loss was assayed using an EZ-Drip Loss method. Three color parameters L*, a* and b* on the surface cuts of LT were objectively evaluated with a CM-2600d/2500d Minolta Chroma meter , glucose-6-phosphate (G6P) and lactate (LAT) in LT muscle were determined using the glycogen assay kits (E2GN-100) BioAssay Systems, the glucose-6-phosphate assay kits (EG6P-100) BioAssay Systems and the lactic acid assay kits (A019-2) from the Nanjing Jiancheng Bioengineering Institute, respectively. GP were calculated as the sum of: 2 × (RG + G6P) + Lactate  and exprma meter . The corGenomic DNA was extracted from pig ear tissue using standard phenol/chloroform extraction. In order to meet the genotyping requirements, all DNA samples were standardized to a final concentration of 50 ng/μL and quality controlled. Totally, 301 Erhualian pigs were genotyped using the 1.4M SNP Beadchips, which is a customized Affymetrix Axiom chip. The SNPs on the chip were found from whole genome sequence data of 150 Chinese indigenous pigs and 38 International commercial pigs, and were evenly distributed across the pig genome . PLINK vY is a vector of residual phenotypic values that were corrected for fixed effects (sex and slaughter batch) using lm function in R program. S is the incidence vector for a, and a is the additive genetic effect of the SNP under test; u is a vector of random polygenic effects that is assumed to follow a multivariate normal distribution MVN method for genetic association analyses and SNP-based heritability estimation, which were conducted using the “−lmm 1” and “−gk 1” commands of GEMMA software . GEMMA er2 < 0.3) by the command “indep-pairwise 50 5 0.3” in PLINK v1.9. Therefore, a SNP was considered to be genome-wide significance at p < 3.8 × 10−7 , and to be suggestive significance at p < 7.6 × 10−6 . The impact of population stratification was estimated by the quantile-quantile (Q-Q) plot and genotype data PCA analysis (reduce - Vfull)/Vreduce, where Vfull and Vreduce are residual variances of models for association analysis with and without the SNP term, respectively. Haplotype block or LD analysis was performed for the chromosomal regions with multiple significant SNPs clustered around the peak SNP. Haplotype blocks were identified using the HAPLOVIEW v4.2 software with default settings (Bonferroni correction method was used to set the genome-wide significant (0.05/N) and suggestive (1/N) association thresholds, where N is the number of independent association tests or SNPs . Consideanalysis . The phesettings .The descriptive statistical results of four GP-related traits  in the longissimus muscle of 301 Erhualian pigs were given in p < 0.001; d) and a lower ultimate pH (pHu) value  but strongly associated with pHd , which is located in the intron of SLC8A3 gene on Sus scrofa chromosome 7 (SSC7), exceeded the genome-wide significance threthold (r2 = 0.63) (SLC8A3–SYNJ2BP gene region (93.78–93.95 Mb) on SSC7  , and a c on SSC7 . The min on SSC7 . In addip < 3.8 × 10−7) with the peak SNPs rs320783325, rs330779127 and rs333704759 on SSC8, 9 and 13, respectively. Among them, the rs320783325, an intron variant in LIMCH1 gene, had the most significant association with G6P (p = 9.81 × 10−8). In addition, we found that on SSC14, the GWAS peak SNP rs337801210 for G6P was located 92.7 kb proximal to the GWAS peak SNP rs331183308 for RG (r2 = 0.21) were observed between rs337801210 and rs331183308, they and nine other significant SNPs (2 for G6P and seven for RG) linked to them were concentrated in the 99.52–99.64 Mb region, which only contains the MINPP1 gene. Moreover, the minor alleles of rs337801210 and rs331183308 were both associated with increased phenotypic values , and another on SSC12 with the peak SNP rs345106152 (p = 3.27 × 10−7). Moreover, these two peak SNPs were also significantly with GP. Notably, the pleiotropic locus rs345106152 explained the largest portion of phenotypic variance for LAT (10%) and GP (12.2%)  .p = 3.06 × 10−7) and rs318410870 (p = 3.77 × 10−7) represented the genome-wide significant QTLs on SSC5 and SSC7, respectively. For rs697205060, an intron variant of SRGAP1, its minor allele was associated with increased concentrations of GP and LAT, while the minor allele at rs318410870, an intron variant of the RIPOR2 gene, was associated with reduced concentrations of GP, RG and LAT . The GWAS peak SNPs rs697205060 ( and LAT . FurtherOR2 gene . In addi2) that increase GP values at the five peak SNPs in each individual’s genotype combination, we divided all individuals into three groups: group A with 0–2 alleles2, group B with three to seven alleles2, and group C with 8–10 alleles2. Analysis of variance showed that there were significant differences in GP, pHi, pHu, pHd, drip loss, redness (a*) and yellowness (b*) of meat between the three groups, but no significant differences in lightness (L*). Moreover, the GP, pHd, drip loss, a* and b* values tended to increase in the genotype combination groups with more allele2, while their corresponding pHi and pHu showed a significant downward trend  in breeding. To this end, we first identified the genome combination types of these five SNPs in the Erhualian population. 73 genotype combinations were found in this population . Accordird trend .p < 0.001) correlated with pHu, which is consistent with the notion that higher glycogen or GP at slaughter leads to greater pHu decline   . In addis. ∼5.5) , which m. Unlike most pig GWAS using 50 K or 60 K SNP chips, this study used 1.4 M SNP chips, which improved the statistical power and QTL mapping resolution. In fact, we not only detected 31 QTLs (8 loci per trait on average), but also found QTLs in some regions with small LD.By comparing with the QTLs for GP deposited in pig QTL database (pigQTLdb), we found that all QTLs detected in this study, except for two, had not been previously reported. On SSC7, the GWAS peak locus (rs332736034) we identified for LAT at 109.12 Mb is close to the QTL for the same trait detected in a Meishan × Pietrain F2 family . SimilarMINPP1 gene. The protein encoded by MINPP1 is multiple inositol polyphosphate phosphatase, which can remove 3-phosphate from inositol phosphate substrates and convert 2,3 bisphosphoglycerate  to 2-phosphoglycerate that is part of the glycolytic pathway  for RG and G6P found on SSC14 was adjacent to the  pathway . The reltabolism . Thus, Mtabolism , and SLCeostasis . TherefoRIPOR2 gene. RIPOR2 can inhibit the activity of small GTPase RhoA and regulate myoblast differentiation  in cellular differentiation and proliferation  for G6P was present in the LIMCH1 gene. This gene encodes calponin homology domains-containing protein 1 which enables myosin II head/neck binding activity. The absence of LIMCH1 can affects the formation of actin stress fibers as well as the stability of focal adhesions, which are basic structures to ensure benign contraction and expansion in skeletal muscle (The most significant GWAS signal (rs320783325 with l muscle . Fiuza-ll muscle . HoweverPRKAG3 and PHKG1, two known major genes in pig breeding (It is very important to identify genetic loci affecting GP phenotypes for genetic improvement of meat quality traits, as demonstrated by the application of mutations in breeding . In thisbreeding , indicatbreeding . TherefoSCL8A3, MINPP1, SRGAP1, RIPOR2, and LIFR near the pleiotropic QTL peaks are highlighted as novel candidate genes related to glucose metabolism in muscle, which are worthy of further study. We also demonstrate the potential application of GP-associated SNPs in improving a variety of meat quality traits. Therefore, this study not only advances our understanding of the genetic architecture of GP related traits but also provide genetic markers for improving the pork quality of Erhualian.We found that the GP content in the skeletal muscle of Erhualian pigs was low and had a wide range of phenotypic variation, which may play a role in the formation of specific meat quality traits of this breed. Further, our GWAS analysisin the Erhualian population identified 31 loci significantly associated with the GP related traits, including 29 novel loci. Among them, six loci exhibited pleiotropic effects on these GP related traits.


Polymerase chain reaction (PCR) kits have been used ascommon diagnosingtools during the outbreak of the severe acute respiratory syndromecoronavirus 2 (SARS-CoV-2) pandemic, with daily worldwide usage inthe millions. It is well known that at the beginning of the pandemic,there was a shortage of PCR kits. So far, the ecosystem of a PCR kitis linear use; that is, kits are produced, used once, and disposedof as biolab waste. Here, we show that to mitigate the risk of futureshortages, it is possible to envision recyclable PCR kits based ona more sustainable use of nucleic acid resources. A PCR kit is mainlycomposed of primers, nucleotides, and enzymes. In the case of a positivetest, the free nucleotides are polymerized onto the primers to formlonger DNA strands. Our approach depolymerizes such strands, keepingthe primers and regenerating the nucleotides, i.e., returning thenucleic acid materials to the original state. The polymerized longDNA strands are hydrolyzed into nucleotide monophosphates that arethen phosphorylated into triphosphates using a method that is developedfrom a recent publication. We used oligonucleotides with a 3′-terminalphosphorothioate (PS) backbone modification as nonhydrolyzable PCRprimers, which are able to undergo the recycling process unchanged.The nuclease resistance of oligonucleotides with a ribose sugar modificationwas also evaluated, which showed worse recycling efficiency than PS-modifiedoligonucleotides. We successfully recycled both PCR primers and nucleotidemonomers (∼75% yield). We demonstrate that the method allowsfor the direct reuse of PCR kits. We also show that the recycled primerscan be isolated and then added to endpoint or quantitative PCR. Thisrecycling approach provides a new path for circularly reusing nucleicacid materials in PCR kits. Theprimers and nucleotides of a PCR kit could be recycledusing nonhydrolyzable primers with a 3′-terminal phosphorothioate(PS) modification. Although many countries announced the endemic state of the SARS-CoV-2pandemic in 2022, PCR and rapid tests are still provided due to thethreat of reinfection and the emergence of new variants or other humaninfectious diseases .6 PCR is a polymerase-catalyzed,templated, temperature-controlled, precise DNA polymerization andamplification process. It seems necessary to consider the usage ofnucleic acid materials in PCR kits in a more sustainable way by transitioningPCR testing kits from a linear economy to a circular economy.Since the worldwide outbreak of the severeacute respiratory syndromecoronavirus 2 (SARS-CoV-2) pandemic, massive polymerase chain reaction(PCR) testing has been required in many countries for diagnosing andscreening.7 For circular recycling of polymer materials , depolymerization is required to break them down into smallerunits (often the starting oligomers or monomers), which, in turn,can be reused to produce new polymers or new molecules.9 In PCR kits, the initial nucleic acid materials are primers (oligonucleotideswith a length of about 20 nucleotides) and nucleotide triphosphatemonomers (dNTPs). The function of primers is sequential recognitionand complementary binding to template DNA . Under the catalysis of DNA polymerases,DNA chain elongation is processed are the copies of the targeted DNA strand, which are polynucleotidesconjugated by phosphodiester bonds. To circularly recycle the nucleicacid materials in PCR kits, a controlled depolymerization method isrequired, which should proceed in the 3′-to-5′ direction(the opposite direction of DNA polymerization). This reaction wouldbreak the phosphodiester bonds and release monomeric nucleotide monophosphates(dNMPs). Also, the depolymerization process should terminate at the3′ terminus of PCR primers, leaving them intact to be reused.A “circular economy” would change the economic logicby turning linear used old goods into new resources with the aim toform closed loops in the industrial ecosystem, minimize waste, andreduce the need to make the originals from scratch.rocessed 1, step 111 and DNA.12 Specifically, NaCRe can depolymerize DNA completelyand recycle the nucleotide monomers into any new desired DNA sequence.12 NaCRe cannot be directly used for circular recyclingof all nucleic acid materials from PCR kits. As described, it wouldhydrolyze (depolymerize) the entire chain of the DNA polymer, includingthe PCR primers. To circularly recycle the PCR primers, the depolymerizationof the DNA polymer by the NaCRe system should terminate before the3′ terminus of the primer, leaving the primer intact and readyto be reused  able to recycle natural sequence-definedpolymers, such as proteinse reused 1, step 214 To improve the nuclease resistanceof primers and keep them intact for recycling, a 3′-terminalmodification with the ability to resist hydrolyzation is required.In this paper, we replaced the natural phosphodiester with phosphorothioate(PS15) at the 3′ terminus of primersto obtain a nonhydrolyzable backbone. PS modification is more commonlyused for developing a nuclease-resistant backbone17 for oligonucleotide drugs. The phosphodiesterbonds of the nonbridging oxygen atoms were substituted with sulfur as an arbitrary example of a biologically meaningful DNA sequence.Primers without the PS modification were applied for PCR as controlsto compare the polymerization efficiency. The PCR amplification productswere purified using a standard PCR extraction kit and quantified byNanodrop.20 We found only a slight decreasein the PCR yield with PS-modified primers , showing that the PS modification did not have a significantimpact on the affinity of the primers to their DNA targets or thechain extension mediated by polymerase.To establish our method for recyclingPCR kits, we first needed to determine whether the modified hydrolysis-resistantoligonucleotides could be used as primers in a PCR experiment. Forwardand reverse primers were designed with a 4-PS tail  were incubated together with nucleases toevaluate their nuclease tolerance. The reaction mixture was analyzedusing agarose gel electrophoresis. We found that the PS-modified primerscan tolerate more than 24 h of nuclease hydrolysis , while the primers without PS modification werenearly completely hydrolyzed after 2 h. The PS-modified oligonucleotidesshowed excellent hydrolysis resistance, making them suitable for recycling.To test the hydrolysisresistance of backbone-modified primers, we used a one-pot DNA hydrolysismethod that was developed recently.S2a). In such plots, when comparing a fresh PCRkit with a used one , we find that about two-thirds of the dNTPs are consumeddue to polymerization , as expected.We also find new peaks of nucleotide diphosphates . We believe that these dNDPsare derived from dNTPs due to their partial hydrolysis induced byheating under PCR conditions. Next, we applied the second NaCRe stepto the PCR waste (see BanI and BstYI) with multiple cleavage sites of the amplified lucsequences were added to the PCR product so that the amplified DNAsequences can be fragmented into shorter pieces. Subsequently, theDNA fragments were hydrolyzed by a mixture of exonucleases to releasedNMPs, as in the established method.12 Afterthe enzymatic depolymerization process, the amount of dNMPs in therecycled PCR kit was highly increased . Further, the released dNMPs were phosphorylatedto generate dNTPs , withacetyl-phosphate (AceP)/ATP as the dual phosphate donor. After phosphorylation,the dNMP peaks almost disappeared, and the intensity of dNTP peaksincreased . Overall,through this hydrolysis–phosphorylation process, monomericdNTPs in the PCR substrate were regenerated from PCR products/waste.We thenused such PS-modified primers in a PCR kit that led to successfulDNA elongation 1, step 1aste see 1, step 2te dNTPs 1, step 3Figure S2b). After all of the steps of dNTP recycling, the reactionmixture was diluted 1.47 times due to the components added in theNaCRe steps . The average concentrationfor each dNTP was decreased to ∼100 μM . The average recycling efficiency of each nucleotidemonomer was calculated to be 74.5 ± 2% , which is similar to thephosphorylation yield of dNMPs reported by us.12 In the regenerated PCR kit, the dNDP residue was 5.94 ±0.04%, the ATP/ADP residue was 12.23 ± 0.02%, and dNTP contentwas 81.81 ± 0.02% .Concentrations of dNTPs from the fresh and regeneratedPCR kitswere determined S2b. AfteS4a–c), showing that the 4-PS tail couldeffectively protect primers from the enzymatic hydrolysis step. Unexpectedly,we found that the enzymatic hydrolysis was “slowed down”instead of totally “stopped” by the 4-PS tail. For thepreserved primers with 0–3 terminal nucleotides lost, the enzymatichydrolysis was relatively slow, yet the remaining length of primerscould be recycled. These observations lead us to believe that onceall of the 4PS-modified nucleotides are hydrolyzed, the primers quicklydegrade. A possible reason for the incomplete protection against hydrolysiscould be the chiral configuration of phosphorothioate. Indeed, S-PS can tolerate enzymatic hydrolysis better than R-PS.21 As the 4PS-modified primerswere achieved by solid-phase synthesis, it is difficult to determinethe stereoselectivity of the PS modification at the current stage.Stereopure phosphorothioate-modified primers are reported,22 which could be more resistant to enzymatic hydrolysis.Overall, the recovery yields of primers were found to be 60.85 ± 0.01% (mass ratio) and 64.38± 0.01% , which is rather promising. We should state that, due tothe limited number of primers (∼0.7 μg), there was asignificant loss of material in the oligo extraction process; hence,the yields we mention are lower estimates.We then evaluated whetherthe PS-modified primers can be preserved through this enzymatic hydrolysisand recycling process. After all three steps of NaCRe, we isolatedthe primers using a commercial oligo extraction kit. The molecularmass of the extracted primers was measured by matrix-assisted laserdesorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry.A mixture of recycled primers with a fully preserved sequence andshortened length  was observed. In support of this conclusion,we should mention that when the recycled primers were purified usingan Oligo extraction kit and applied for another round of PCR amplification,the additional band was absent .In an ideal situation, we would liketo establish a method to regenerate and circularly reuse PCR kitsby simply adding polymerization and depolymerization enzymes whenneeded 2a.There23 Consequently, in this circular recycling process,the quality of recycled primers (purity and sequence length) woulddirectly affect the performance of qPCR. Earlier, we have shown thatthe 4PS modification can protect primers from enzymatic hydrolysis,but the recycled primers have different lengths (with 0–3 nucleotideloss); this, in theory, should lead to the use of a more complex annealingtemperature profile for optimized recycled PCR use.We show that the nucleicacid materials in the PCR kit can be recycled using PS-modified primers.However, for the detection of specific DNA, an additional step ofgel electrophoresis is required to show the band of the amplicon.This endpoint PCR is time-consuming and lacks efficiency. For moleculardiagnostics, quantitative PCR (qPCR) is widely used as DNA amplificationcan be monitored in real time by measuring the fluorescence signalof the DNA intercalation dye. Primers are critical components in qPCR,as their functions of recognizing and binding with target DNA determinethe precision and sensitivity of the molecular diagnostic assay.Figure S6a). The 2PS-modified primers also showed excellentnuclease hydrolysis tolerance . Further, the qPCR product was incubated with a mixture of nucleasesfor different periods  to hydrolyze the amplicon andrecycle the primers. The recycled primers were then purified usinga commercial oligo extraction kit. Next, the Agilent Oligo Pro IIsystem was applied to analyze the recycling efficiency of the 2PS-modifiedqPCR primers. As a capillary electrophoresis analytical tool, theOligo Pro II system uses a denaturing gel that allows single-strandedoligonucleotide samples to migrate through a capillary and separateby size, allowing the direct detection of oligos with single-nucleotideresolution.To maintainthe length of the recycled primers without changing their annealingtemperature significantly, primers with a 2-PS tail at the 3′-terminalmodification were used for qPCR (amplicon length 133 nucleotides)and recycled by the abovementioned method. The qPCR performance wasnot affected by primers with a 2PS modification  values were obtained for the qPCRassay prepared with fresh primers and recycled primers , indicating the presence of unwanted residues. Althoughthis NTC result should not affect the detection limit of the qPCRkit, because it is higher than the CT value of 0.001 ngfound for the DNA template with the recycled primers  position generatesexcellent nuclease resistance.26 We testedthe nuclease resistance of four types of ribose sugar-modified primers, while the CT valueof 2′-OMe primers was increased. The 2′-MOE modificationled to no detectable amplification. These results show that 2′-Lock,2′-F, and 2PS are the most suitable modifications for qPCRprimers as they showed a PCR efficiency similar to that of unmodifiedprimers. After nuclease treatment, primers with a 2′-Lock modificationcould tolerate Exo I treatment for 1.5 h with a slightly increasedCT value and Exo III treatment for 4 h without any obviouschange in the CT value. They cannot tolerate 4 h of ExoI treatment as the CT value was beyond the detection limit,which corresponds to the MALDI-TOF result . Primers with a 2′-F modification could not toleratethe hydrolysis by both Exo I and Exo III for 1.5 h. In conclusion,primers with the 2PS modification showed the best nuclease resistance.After 1.5 and 4 h of Exo I and Exo III treatment, respectively, therewere no obvious changes in the CT values for qPCR, indicatingthat the concentration of primers was not drastically decreased bynuclease resistance. Although it is difficult to determine the recyclingyield, qPCR results showed relatively good nuclease tolerance of the2PS-modified primers. Both the intact primers and primers with oneterminal nucleotide lost were detected by MALDI-TOF , showing that the 2PS modification can effectivelyprotect the primers from nucleases hydrolysis. After nuclease treatment,the concentration of 2PS-modified primers should remain the same,given that the qPCR sensitivity remains unchanged.In additionto the PS modification (phosphodiester backbone), ribose sugar modificationcan also improve the nuclease tolerance of oligonucleotides by increasingsteric hindrance. primers5a in ter27 The final ΔRn value is correlated to theDNA synthesis yield of the PCR reaction, which is determined by theconcentration of reactive agents in the PCR reaction . As shown in ΔRn indicates the magnitude of the signal generated under the given set of PCR conditions.22 Yet, large-scale recycling by this method willrequire the optimization of all steps and will not be easy. Overall,the daily accumulated PCR waste during the current pandemic couldbecome a valuable resource for the circular recycling of PCR reagents.In this work, we discussed the possibilityto circularly recyclethe nucleic acid materials in PCR kits. Using PS modifications, theprimers can be protected from the nucleotide recycling process, preserved,and extracted for circular usage. Together with the recycled monomericnucleotides with good yield, the PCR substrate was regenerated andreused for the detection of targeted DNA. The PS-modified primerscan also be recycled separately by the same method and reused as newmaterials for targeted DNA detection by qPCR with relatively goodsensitivity. The quality (length and purity) of recycled primers canbe evaluated by Oligo pro II capillary electrophoresis with single-nucleotideresolution. The method we established here shows the possibility ofusing PCR reagents in a smarter way. With the PS modification, itis possible to design nonhydrolyzable, recyclable primers to easethe problem of PCR kit shortage. It is possible to improve the recyclingefficiency of primers using stereopure PS modifications in the future.28 As DNA technology has become a very well-establishedtechnique with DNA materials used for sequencing, bioengineering,molecular diagnoses, DNA origami, DNA hydrogel, and DNA for data storage,35 our established DNA recycling methodology could also be adaptedfor the circular recycling of modified nucleotides and oligonucleotideproducts from the abovementioned waste to render more sustainableapplications.So far, the established method was tested by endpoint PCR and qPCR, but it might also be applicable to recycleoligo- and mononucleotides from other DNA polymerization systems suchas the loop-mediated isothermal amplification (LAMP) system.Ethanol, isoproponal, and2-mercaptoethanol (for oligo-extraction) were purchased from Sigma-Aldrich.2′-Deoxyguanosine 5′-monophosphate sodium salt hydrate(dGMP), thymidine 5′-monophosphate disodium salt hydrate (dTMP),2′-deoxycytidine 5′-monophosphate sodium salt (dCMP),2′-deoxyadenosine 5′-monophosphate (dAMP), acetylephosphatelithium potassium salt (AceP), sodium chloride , sodium hydroxide solution (5.0 M), acetic acid, tetrabutylammoniumdihydrogenphosphate solution (1.0 M in water), deoxynucleotide mixreagent , and adenosine 5′-triphospate disodium were purchased from Sigma-Aldrich. Nuclease-free water(10 × 50 mL) was purchased from Qiagen.HEPES buffer (1 M) was purchased fromThermo Fisher Scientific.For gel electrophoresis,400 μL SYBR Safe DNA Gel Stain, DNA loading dye, SDS solution(6×), TAE buffer (Tris–acetate–EDTA) (50×),TrackIt 100 bp DNA Ladder, and Ultra Low Range DNA Ladder were purchasedfrom Thermo Fisher Scientific. Agarose was purchased from Bio-RadLaboratories. NuPAGE 4–12%, Bis–Tris, 1.0 mm, and MiniProtein Gel (12-well) were purchased from Invitrogen.DreamTaq Green PCR Master Mix(for the first round of PCR), DreamTaq polymerase (for the secondround of PCR), and PowerTrack SYBR Green Master Mix (for qPCR) werepurchased from Thermo Fisher Scientific. Amicon Ultra 0.5 mL CentrifugalFilters (3 kD cutoff) were purchased from Merck. The Oligo Clean-Upand Concentration Kit was purchased from Norgen Biotek.DdeI), BshNI (BanI), PsuI (BstYI), and Kpn2I (BspEI) were purchased from Thermo Fisher Scientific. Hydrolysisenzymes exonuclease III (200 U/μL) and exonuclease I (20 U/μL)were purchased from Thermo Fisher Scientific. T4 NMP (nucleotide monophosphate)kinase was purchased from Jena Bioscience. E. coli S30 Extract System (with luciferase plasmid DNA template) was purchasedfrom Promega Corporation.Restriction enzymes HpyF3I (E. coli S30 extraction kit. Plasmid DNA template encoding the SARS-CoV-2envelope sequence (2019-nCoV_E Positive) was purchased from IntegratedDNA Technologies company. Primers were designed from published sequences19 without 5′ NdeI and3′ BamHI restriction site sequences. The amplificationlength is 1653 base pairs (16–1668) of the luc sequence. Primers(oligonucleotides) with or without the 5′-terminal Fam modification,and with or without the 3′-phosphorothiolate (PS) modification,were purchased from Biomers Gmbh, Germany. The 3′-terminalPS tail was labeled by “*” in the flowing sequences(forward primer (Fam): 5′-atg gaa gac gcc aaa aac at*a *a*a*g-3′;reverse primer (Fam): 5′-tta caa ttt gga ctt tcc gc*c *c*t*t-3′).Plasmid luciferaseDNA  was from the 12). Primers werepurchased from Biomers Gmbh. The 3′-terminal PS tail was labeledby “*” in the flowing sequences (forward primer (Fam):5′-cgc atg cca gag atc cta *t*t-3′; reverse primer (Fam):5′-aga cga ctc gaa atc cac ata *t*c-3′). Primers witha sugar modification for the amplification of 2019-nCoV_E Positiveplasmid DNA by qPCR  were purchased from Biomers, Gmbh. The modifications,2′-Lock (2′-locked nucleic acid), 2′-OMe (2′-O-methyl), 2′-MOE (2′-O-methoxyethyl),and 2′-F (2′-deoxy-2′-fluoro), are at the penultimateposition . The backbone2PS modification is labeled with *, between the last three nucleobases.The DNA templatefor qPCR is the same as that for PCR (luc template). Primers weredesigned by IDT PrimerQuest Tool, with the forward primer startingfrom 673 of the luciferase sequence, the reverse primer starting from805 of the luciferase sequence, and the length of amplicon being 133base pairs  was purchased from Eppendorf. The horizontal gelelectrophoresis system was purchased from Bio-Rad. Gel images wereobtained with a GelDoc Go system, Bio-Rad. PCR was performed usinga Proflex 3 × 32-well PCR thermal cycler system (Thermo FisherScientific). qPCR was performed using a QuantStudio 7 qPCR instrument(Applied Biosystems). HPLC was performed using an Infinite 1260 HPLCwith C 18 column, Agilent.Initial denaturing for one cycle, amplification  for 35 cycles, final extension (72 °Cfor 10 min) for one cycle, and cooling to 4 °C. The PCR amplificationproducts were used for the recycling of dNTPs, regeneration of thePCR kit, and recycling of primers. Unless specified, all PCRs wereprocessed under the same thermocycling conditions.The agarose gel was run in 1×TAE buffer at 120 V for 40 min. Afterward, the gel was stained by1× Sybr safe solution for 40 min under slow shaking. Thereafter,the image of the gel was obtained by a GelDoc Go under Sybr safe channelwith 1 s exposure time. Unless specified, all agarose gel experimentswere processed under the same conditions.Initial denaturing for one cycle, amplification  for 35 cycles, final extension (72 °C for10 min) for one cycle, and cooling to 4 °C. The PCR amplificationproducts were used for the recycling of dNTPs, regeneration of thePCR kit, and recycling of primers. Unless specified, all PCRs wereprocessed under the same thermocycling conditions.All MALDI-TOF spectra were collected usinga Bruker AutoFlex Speed instrument .The AgilentOligo Pro II system with DN-415 OLIGEL ssDNA Gel was used in the correspondingmethods.Figure S1b), the FAMdye-labeled forward and reverse primers with PS protection (Fam-FP-PSand Fam-RP-PS) can tolerate enzymatic hydrolysis for 24 h (lanes 1–3(Fam-FP-PS) and lanes 7–9 (Fam-RP-PS)). In contrast, the forwardand reverse primers without a PS tail (Fam-FP and Fam-RP) were hydrolyzedwith very less residues after 2 h (lane 6 (Fam-FP) and lane 9 (Fam-RP)).This shows that the primers with a 4PS tail modification can resistnuclease-catalyzed hydrolysis.Thenuclease-resistance capacity of PS-modified primers was tested bymixing Fam-FP-PS, Fam-RP-PS, Fam-FP, and Fam-RP  with exonuclease III  and exonucleaseI ; 10 μL of 10× exonuclease IIIbuffer, 79.5 μL of nuclease-free water ,and the nuclease hydrolysis mixtures were incubated at 37 °Cand 350 rpm for 2, 6, and 24 h, respectively. Further, the hydrolysisreaction mixtures (5 μL each) were loaded onto 2% agarose gel.As shown in the gel , forward primer ,reverse primer , Luc DNA template , and nuclease-free water (48.5 μL)were mixed in a total of 100 μL. The PCR products  were extracted using a PCRextraction kit, and the concentration of PCR products was quantifiedby Nanodrop.The PCR product was first heatedto 100 °C and incubated for 15 min to deactivate polymerase.Further, 50 μL of the PCR product was mixed with 3.25 μLof Dedl (32.5 Units), 4 μL of Exo III, and 4 μL of ExoI and incubated at 37 °C and 350 rpm overnight. At the cleavagesite of Dedl , 5′-terminaloverhangs with a length of four nucleotides were generated, whichare suitable for the binding of Exo III to the cleaved PCR productand initiation of hydrolysis. Afterward, the hydrolysis mixture washeated to 80° and incubated for 15 min to inactivate the restrictionand hydrolysis enzymes. As a result of the increase in volume by addingthe cleavage and hydrolysis enzymes, there was a 1.225× dilutionof the reaction mixture.E. coli S30 extract (20× dilution), 1 μL of T4 dNMP kinase , 1 μL of ATP (3 mM), and 0.77 μL ofacetylphosphate lithium potassium , in a total of 24μL. The phosphorylation reaction mixture with a final volumeof 24 μL, with estimated amounts of dNMPs , ATP (0.125 mM), and AceP , was incubatedin a thermomixer at 400 rpm at 37 °C for 4 h. Afterward, allhydrolysis and phosphorylation enzymes and nonhydrolyzed DNA wereremoved by ultrafiltration . As a result of the increase in volume by adding thephosphorylation reagents, there was a 1.2× dilution of the reactionmixture.The PCR product hydrolysis mixture(20 μL) was mixed with 1.2 μL of t-butyl ammoniumphosphate, 10 mM KH2PO4, and 0.25% methanoladjusted to pH 6.9. Buffer B: 5 mM t-butyl ammoniumphosphate, 50 mM KH2PO4, and 30% methanol (pH7.0). From 0 to 15 min, the gradients of buffer A/B changed from 40/60to 20/80%, were run under the same gradient conditions until 20 min,and then changed back to the starting condition of 40/60%, with aflow rate of 0.5 mL/min.The concentrationof recycled dNTPs was determined by HPLC with a C-18 reverse-phasecolumn. Mobile-phase Buffer A: 5 mM Figure S2a. The calibration curve was achievedby serial dilution of dNTPs and ATP . The final concentration of dNTPswas multiplied with the dilution factor during the PCR kit regenerationprocess (1.225× post hydrolysis and 1.2× for phosphorylation).After PCR amplification, about 70% of the dNTPs was consumed . In addition, new peaks were generated, which wereattributed to hydrolysis products of dNTPs (dNMPs and dNDPs) probablyinduced by the heating under PCR conditions. After enzymatic hydrolysis,the amount of dNMPs was highly increased . There is a slight decrease in the dNTP residue,which might be caused by enzymatic hydrolysis. After phosphorylation,the dNMP peaks almost disappeared, and the dNTP peaks were regenerated,showing excellent phosphorylation efficiency . The recycling efficiencyof dNTPs is 74.49 ± 2.09%, and the final concentration of alldNTPs is 101.35 ± 2.84 μM .Further, the filtered reactionmixture was diluted (50×) and injected into the HPLC column (50 μL)for the quantification of dNTPs. The mixture of dNMPs (2.5 μMeach) and dNTPs from the original PCR kit  was injected into the HPLC column (50 μL) as a positivecontrol. For retention times of the above five samples, see The ratio of DNA templateand primers used for PCR amplification was the same as that mentionedabove. Although a relatively good monomer recycling efficiency isachieved, we cannot confirm that all PCR residues have been removedand no contaminants will be carried over to the next round of PCR.To improve the hydrolytic efficiency, we further split the hydrolysisof PCR product into two steps with the restriction enzymes added onlyin the second step.The PCR product was first heatedto 100 °C and incubated for 15 min to deactivate polymerase.Further, 50 μL of the PCR product was mixed with 1.5 μLof Exo III and 1.5 μL of Exo I and incubated at 37 °C and350 rpm overnight. Afterward, the hydrolysis mixture was heated to80 °C and incubated for 15 min to inactivate the hydrolysis enzymes.Next, 1 μL of DedI (10 Units), 1.5 μL of Exo III, and1.5 μL of Exo I were added to the hydrolysis mixture and incubatedat 37 °C and 350 rpm for 8 h. Afterward, the cleavage and hydrolysismixture was heated to 80 °C and incubated for 15 min to inactivatethe restriction and hydrolysis enzymes. As a result of the increasein volume by the two steps of hydrolysis and cleavage, there was a1.12× (50–56 μL) dilution of the reaction mixture.The phosphorylation step was processedby a method similar to that mentioned above. As the phosphorylationmixture would increase the total volume of the reaction mixture anddecrease the concentration of all reagents in the PCR substrate, wewould like to minimize the amount of added phosphorylation mixture.Therefore, we first prepared a phosphorylation reagent mixture (10μL) with T4 60× dilution, S30 20× dilution, ATP 2.46mM, and AceP 31.5 mM. The PCR hydrolysis mixture (30 μL) wasmixed with 2 μL of the prepared phosphorylation reagents, andthe phosphorylation mixture was incubated in a thermomixer at 400rpm at 37 °C for 4 h. Afterward, the reaction mixture was directlysubjected to PCR amplification. As a result of the increase in volumeby adding the phosphorylation reagents, there was a 1.066× dilution(30–32 μL) of the reaction mixture.From the recyclingefficiency of monomers as well as the dilution factor for each stepof PCR kit regeneration, the final concentration of dNTPs in the regeneratedPCR kit is calculated as follows:The regeneratedPCR substrate (20 μL), 0.5 μL of luc DNA template (20ng/μL), and 0.5 μL of DreamTaq polymerase (diluted to1 U/μL) were mixed in a total volume of 21 μL.Preparationof the No-DNA Template ControlThe regenerated PCR substrate(20 μL), 0.5 μL of nuclease-freewater, and 0.5 μL of DreamTaq polymerase (diluted to 1 U/μL)were mixed in a total volume of 21 μL. As a result of the increasein volume by adding the DNA template and the polymerase, there wasa 1.05× dilution (21–20 μL) of the reaction mixture.The whole process for PCR amplification, regeneration, and secondround of PCR was processed by primers without PS modification as anegative control.After the PCR thermocycles, thefirst and second rounds of the PCR product as well as the PCR hydrolysismixture were loaded onto 2% agarose gel. All samples were mixed with2 μL of 6× DNA loading dye. Lane 1, TrackIt 100 bp ladder,2 μL; lane 2, first round of PCR product, 8.4 + 1.6 μLwater; lane 3, PCR hydrolysis mixture, 9.375 + 0.625 μL water;lane 4, second round of PCR product with DNA template added, 10 μL;lane 5, second round of PCR product without DNA template added, 10μL. Lanes 6–8, first and second rounds of PCR products(primers without PS modification); lane 6, first round of PCR product,8.4 + 1.6 μL water; lane 7, PCR hydrolysis mixture, 9.375 +0.625 μL water; lane 8, second round of PCR product with DNAtemplate added, 10 μL.The ratio of DNA templateand primers used for PCR amplification (duplicate) was the same asthat mentioned above. The concentration of primers was determinedby Nanodrop , andthe amount of added primers was 716.5 ng .BanI) . At the cleavage site ofBshNI (BanI) ,5′-terminal overhangs with a length of four nucleotides weregenerated. Another restriction enzyme PsuI (BstYI) with multiple cleavage sites  was also added to cleavethe PCR product. Also, the cleaved fragment with the forward primerwas shortened to 33 nt (16–48), and the cleaved fragment witha reverse primer was shortened to 43 nt (1625–1668) for betterhydrolysis and recycling efficiency. The cleavage mixture was incubatedat 37 °C and 350 rpm for 5 h. Afterward, the hydrolysis mixturewas heated to 80 °C and incubated for 20 min to inactivate thecleavage enzymes. Next, 15 μL of Exo III and 15 μL ofExo I were added to the hydrolysis mixture and incubated at 37 °Cand 350 rpm for 8 h. Afterward, the hydrolysis mixture was heatedto 80 °C and incubated for 15 min to inactivate the hydrolysisenzymes. Further, the PCR cleavage–hydrolysis mixture was concentratedby ultrafiltration  to 50μL. The mixture of primers was extracted using a “OligoClean-Up and Concentration Kit.” The concentration of extractedprimers was quantified by Nanodrop (ssDNA method) with a final amountof 436 ± 7.2 ng .The PCR product (100 μL)was first heated to 100 °C and incubated for 15 min to deactivatepolymerase. Further, the PCR product was mixed with BshNI .The forward primer (2.5 μM in water), reverse primer (2.5 μMin water), and recycled primers (10.90 ± 0.18 ng/μL inwater) were mixed with an equal volume of the matrix solution. Foreach sample, a 1 μL aliquot of such a solution mixture was depositedand dried onto a stainless ground steel target plate. Measurementswere performed in positive ionization mode and operated in the linearmode in the 1–14 K m/z massrange. The laser intensity was kept at around 80% for all measurements.Typically, around 1000 shots were accumulated for each spectrum. Massspectra were processed with Flex Analysis (Bruker) software.The molecular masses of fresh primers andrecycled primers were characterized by MALDI-TOF. The matrix was preparedas follows: a 90:10 mixture of (1) 50 mg/mL 3-hydroxy picolinic acidin 1:1 water/acetonitrile and (2) 100 mg/mL diammonium hydrogen citratein water.Because of the shortenedlength of primers, the molecular weight of recycled primers was decreasedfrom 7.5 and 7.3 to 6.9 kD (on average). Therefore, the recyclingyield calculated by the molar ratio is 64.38 ± 0.01%. The concentrationof recycled primers thus calculated from the molar ratio was 0.82μM.(1) Positivecontrol of PCR with fresh primers: DreamTaq master mix , a mixture of forward and reverse primers , Luc DNA template , and nuclease-freewater (1 μL) were mixed in a total volume of 20 μL. (2)PCR with recycled primers: DreamTaq master mix ,recycled primer mixture , Luc DNAtemplate , and nuclease-free water (1μL) were mixed in a total volume of 20 μL. The thermocyclerconditions are the same as the above settings. After the PCR amplification,PCR products from fresh and recycled primers (10 μL each) wereloaded onto 2% agarose gel.Figure S1), the FAMdye-labeled forward and reverse primers with PS protection (Fam-FP-PSand Fam-RP-PS) can tolerate enzymatic hydrolysis (lanes 2 and 4).There is no obvious difference in the bands in comparison with thecontrol samples of nuclease-free primers (lanes 1 and 3). In contrast,the forward and reverse primers without a PS tail (Fam-FP and Fam-RP)were hydrolyzed with very few residues (lanes 6 and 8) in comparisonwith the control samples of nuclease-free primers (lanes 5 and 8).This shows that the PS tail modification of primers could resist nuclease-catalyzedhydrolysis.Thenuclease-resistance capacity of PS-modified primers was tested bymixing 0.5 μL (100 μM) of Fam-FP-PS, Fam-RP-PS, Fam-FP,or Fam-RP with exonuclease III  and exonucleaseI , 10 μL of 10× exonuclease IIIbuffer, and 79.5 μL of nuclease-free water and incubating overnight at 37 °C and 350 rpm. Further, the hydrolysisreaction mixtures (5 μL each) were loaded onto 2% agarose gel.As shown in the gel , next, we triedto recover primers from PCR product/waste .PowerTrackSYBR Green Master Mix , a mixture of forwardand reverse primers  with a final concentrationof 400 nM, Luc DNA template , and nuclease-freewater (47.4 μL) were mixed in a total volume of 100 μL. The concentrationof primers was quantified by Nanodrop , and the amount of added primers was 577.79 ng/100μL of PCR mixture (1.6 μL).Initial denaturing for one cycle, amplification  for 35 cycles, and final extension (72 °C for 10 min) forone cycle and cooling to 4 °C.BspEI) . At the cleavage site ofKpn2I (BspEI) ,5′-terminal overhangs with a length of four nucleotides weregenerated. The amplified DNA was cleaved into two shorter fragments.The DNA cleavage mixture was incubated at 55 °C and 350 rpm overnight.Afterward, the reaction mixture was heated to 80 °C and incubatedfor 20 min to inactivate the cleavage enzymes. Next, 5 μL ofExo III and 5 μL of Exo I were added to the hydrolysis mixtureand incubated at 37 °C and 350 rpm for 1.5 h. Afterward, thehydrolysis mixture was heated to 80 °C and incubated for 15 minto inactivate the hydrolysis enzymes. Further, the PCR cleavage–hydrolysismixture  was concentrated by ultrafiltration to 50 μL. The mixtureof primers recycled from the quadruplicates was extracted using an“Oligo Clean-Up and Concentration Kit.” The concentrationof extracted primers was quantified by Nanodrop (ssDNA method) witha final amount of 873.2 ± 10.4 ng . The molecular masses of fresh primersand recycled primers were characterized by MALDI-TOF by the same methodas above.The PCR product was first heatedto 100 °C and incubated for 15 min to deactivate polymerase.Further, 100 μL of the PCR product was mixed with Kpn2I . Therefore,the recycling yield calculated by the molar ratio is 873.2 ±10.4 ng/(577.79 × 4)/(150/160)/0.976 = 40.93 ± 0.48%. Asa result of the loss during oligo extraction (up to 90% recovery ratewith at least 10% loss), the preserved primers should be more than50%. The as-obtained recycled primers were further diluted to a stocksolution (1.33 μM).All samples were dilutedto 0.25 μM by adding 5.0 μL of 1.0 μM stock to 15.0μL of nuclease-free water with mineral oil overlay. All sampleswere analyzed on the Agilent Oligo Pro II system with DN-415 OLIGELssDNA Gel using the corresponding methods.Mixture of fresh primers: 7 kV for5 s; sample separation: 12 kV for 90 min. Sample injection for themixture of recycled primers: 10 kV for 15 s; sample separation: 12kV for 90 min.The Agilent Oligo Pro II data analysissoftware with the following integration parameters 1 was use(1) Fresh primersfor qPCR: PowerTrack SYBR Green Master Mix ,a fresh mixture of primers , Luc DNA template,1 μL of serial dilutions of 1, 0.1, 0.01, and 0.001 ng/μL,1 μL of nuclease-free water as the no-template control (NTC)sample, and 3.4 μL of nuclease-free water were mixed in a totalvolume of 10 μL. In the final qPCR mixture, the concentrationof primers is 400 nM. (2) Recycled primers for qPCR: PowerTrack SYBRGreen Master Mix , recycled primers, , Luc DNA template, 1 μL of serial dilutions of 1,0.1, 0.01, and 0.001 ng/μL, and 1 μL of nuclease-freewater as the NTC were mixed in a total volume of 10 μL. In thefinal qPCR mixture, the concentration of primers is 400 nM.The qPCR amplificationwas performed by a QuantStudio 7 qPCR system: initial denaturing  for one cycle and amplification  for 40 cycles.Six groups of primer mixtures are prepared. The hydrolysis mixture  is prepared.The mixture is incubated at 37 °C and 350 rpm for 1.5 or 4 h.Afterward, the temperature is increased to 80 °C and incubatedfor 15 min to inactivate the hydrolysis enzymes. Fast centrifugationis carried out to cool down the reaction mixture. The reaction mixtureis stored for qPCR and Maldi experiments.The qPCR mixture was prepared in triplicateas follows: PowerTrack SYBR Green Master Mix and a mixture of six groups of fresh NaCRe primers  were combined. In the final qPCR mixture, with the concentrationof primers of 400 nM, DNA template, 1 μL of serial 10×dilutions of 200,000, 20,000, and 2000 copies or 1 μL of nuclease-freewater as the no-template control (NTC) sample, and 2.4 μL ofnuclease-free water are added in a total of 10 μL.TheqPCR mixture using primers with nuclease treatment was prepared intriplicate as follows: PowerTrack SYBR Green Master Mix , a mixture of six groups of NaCRe primers after 1.5 or4 h of nuclease treatment were mixed .In the final qPCR mixture, , DNAtemplate, 1 μL of serial 10× dilutions of 200,000, 20,000,and 2000 copies or 1 μL of nuclease-free water as the no-templatecontrol (NTC) sample, and 2.4 μL of nuclease-free water wereadded in a total of 10 μL.The qPCRamplification was performed by a QuantStudio 7 qPCR system with initialdenaturing  for one cycle and amplification  for 40 cycles.


HSPB1) is a crucial biomarker for pathological processes in various cancers. However, the clinical value and function of HSPB1 in breast cancer has not been extensively explored. Therefore, we adopted a systematic and comprehensive approach to investigate the correlation between HSPB1 expression and clinicopathological features of breast cancer, as well as determine its prognostic value. We also examined the effects of HSPB1 on cell proliferation, invasion, apoptosis, and metastasis.Heat shock protein beta-1 (HSPB1 in patients with breast cancer using The Cancer Genome Atlas and immunohistochemistry. Chi-squared test and Wilcoxon signed-rank test were used to examine the relationship between HSPB1 expression and clinicopathological characteristics.We investigated the expression of HSPB1 expression was significantly correlated with the stage N, pathologic stages, as well as estrogen and progesterone receptors. Furthermore, high HSPB1 expression resulted in a poor prognosis for overall survival, relapse-free survival, and distant metastasis-free survival. Multivariable analysis showed that patients with poor survival outcomes had higher tumor, node, metastasis, and pathologic stages. Pathway analysis of HSPB1 and the altered neighboring genes suggested that HSPB1 is involved in the epithelial-to-mesenchymal transition. Functional analysis revealed showed that transient knockdown of HSPB1 inhibited the cell migration/invasion ability and promoted apoptosis.We observed that HSPB1 may be involved in breast cancer metastasis. Collectively, our study demonstrated that HSPB1 has prognostic value for clinical outcomes and may serve as a therapeutic biomarker for breast cancer.The online version contains supplementary material available at 10.1186/s12885-023-10983-3. Breast cancer is one of the most common malignant tumors in women. The incidence rate is rising each year, which has a significant negative impact on women's health and quality of life –3. DespiHSPB1 regulates many pathological processes in cancer, including drug resistance, apoptosis, and metastasis [HSPB1 is considered an important molecular target for tumor growth inhibition and apoptosis induction [HSPB1 has a worse prognosis [HSPB1 is related to poor overall survival in hepatocellular carcinoma and promotes tumorigenesis [HSPB1 knockout led to a decrease in insulin levels and expression of growth factor-like binding protein 2, which may promote the proliferation and metastasis of hepatocellular carcinoma [HSPB1 expression is related to the epithelial-mesenchymal transition (EMT). Through the upregulation of Snail1 and PRRX1, HSPB1 overexpression promotes EMT and drives the migration and invasion of salivary adenoid cystic carcinoma cells [HSPB1 interferes with bone metastasis in breast cancer [PTEN levels in human breast cancer cells [HSPB1 and its underlying mechanisms in the development of breast cancer have yet to be elucidated.Heat shock protein 27, also referred to as heat shock protein beta-1 (HSPB1), belongs to the small HSP family. Its purpose is to stop or prevent cellular proteins from denaturing or unfolding in response to stress or elevated temperatures . HSPB1 rtastasis –8. HSPB1nduction  and is vnduction –12. For rognosis . Upreguligenesis . Furtherarcinoma . Moreovema cells . Additiot cancer  and moduer cells . The potHSPB1 and tumorigenesis has been demonstrated, limited evidence has illustrated the clinical significance and function of HSPB1 in breast cancer. In this study, we used a systematic and comprehensive approach to assess the relationship between HSPB1 expression and clinicopathological characteristics in patients with breast cancer. We also determined the prognostic value of HSPB1. Additionally, we examined the effects of HSPB1 on cell proliferation, invasion, apoptosis, and metastasis. We believe that this study has identified HSPB1 as a therapeutic target for breast cancer.While the relationship between n = 15) and normal adjacent tissue (n = 3) were collected from patients undergoing surgery for breast cancer at the First Affiliated Hospital of Shenzhen University from April 2019 to February 2021. These samples were taken from tissues removed surgically. In addition, we collected 20 samples of in situ breast cancer and lung metastases. The tissues were quickly stored at − 80 °C until use, according to the Tumor Bank protocol [A total of 18 cancerous tissues , disease-specific survival (DSS), and progress free interval (PFI). Patients with breast cancer were examined to determine whether HSPB1 expression was correlated with clinicopathological variables to better understand the prognostic value of HSPB1 based on status , patient age (≤ 60 or > 60 years of age), tumor (T) stage , node (N) stage , metastasis (M) stage (M0 or M1), pathological stage , estrogen receptors , progesterone receptors , and human epidermal growth factor receptor 2 . We examined the association between HSPB1 expression and clinical characteristics using Chi-square and Wilcoxon signed-rank tests.We obtained data with the relevant clinical characteristics from The Cancer Genome Atlas (TCGA). TCGA is an open-access resource, that includes 33 types of cancer from approximately 20,000 patients [HSPB1 siRNAs were synthesized by Gene Pharma : si-1: 5′-GCCAUUAUUAGAGACCUCATT-3′ and si-2: 5′-UCACCAUCCCAGUCACCUUTT-3′. Cells were transfected using Lipofectamine 2000 . The Pure Link RNA Mini Kit  was used to extract total RNA. Quantitative real-time PCR (RT-PCR) was performed under the following conditions: 95 °C for 5 min, and 40 cycles of 95 °C for 30 s, 60 °C for 45 s and 72 °C for 30 s. Primers were purchased from Gene Pharma  using the following sequences: HSPB1, 5′-CTCTGAAGGGTCCGAAGTGAT-3′ and 5′-ATTCCTGTGGTGGTCCAAAAC-3′; Actin: 5′-CACCATTGGCAATGAGCGGTTC-3′ and 5′-AGGTCTTTGCGGATGTCCACGT-3′.We obtained six cell lines from the Chinese Academy of Sciences, including human normal mammary epithelial cells: MCF10A, and all breast cancer cell line types: luminal A: T47D, luminal B: BT474, HER-2 positive: SK-BR-3, Triple-negative A: MDA-MB-453, Triple-negative B: MDA-MB-231. A mix of 10% fetal bovine serum (FBS) and RPMI Medium 1640  was used to culture each cell line. Two sequences targeting HSPB1 (1: 1000 dilution), glyceraldehyde-3-phosphate dehydrogenase , vimentin (1: 1000 dilution), N-cadherin (1: 1000 dilution) and E-cadherin (1: 1000 dilution) were used. All the antibodies used in this study were purchased from CST (USA). The labeled proteins were visualized via chemiluminescent imaging.Samples were tested for total protein concentration using the bicinchoninic acid method. A 12% SDS-PAGE gel was used to separate 20 µg of each protein sample. We then transferred the separated proteins onto polyvinylidene fluoride membranes for primary antibody detection. The membranes were then incubated for 1 h at room temperature with alkaline phosphatase-conjugated secondary antibodies , followed by three washes with Tris-buffered saline with Tween 20 for 15 min. Primary antibodies against HSPB1 (1: 1000 dilution) for 12 h, the sections were counterstained with hematoxylin to label the nuclei. Two pathologists blindly evaluated and scored all stained sections to determine the degree of immunostaining.Immunohistochemical assays were performed on human breast cancer and adjacent tissues. All fresh tissues were cryopreserved before being processed into histology blocks for sectioning. Briefly, after deparaffinization and rehydration, antigen retrieval was performed by heating 5μm -thick sections at 95 °C for 15 min in 10 mM citrate buffer (pH 6.0). After incubation with the primary antibody against HSPB1 transcription levels and OS, relapse-free survival (RFS), and distant metastasis-free survival (DMFS) in patients with breast cancer using the Kaplan–Meier plotter database (http://www.kmplot.com/) [p < 0.05. The sample sizes of the high- and low-HSPB1 groups were 939 and 940 for OS, 2,465 and 2,464 for RFS, and 1,383 and 1,382 for DMFS, respectively.We analyzed the correlation between ot.com/) . For allhttp://dna00.bio.kyutech.ac.jp/PrognoScan/index.html) [HSPB1 expression and prognosis in patients with breast cancer, including OS, RFS, and DMFS (these data from the GSE1456-GPL96 and GSE2990 cohorts). Samples were divided into groups of high or low HSPB1 expression levels, with the lowest 50% and the top 50% considered low and high expression, respectively.The PrognoScan database (ex.html)  was usedHSPB1 were constructed using GeneMANIA (http://www.genemania.org) and STRING online (https://string-db.org/) [HSPB1 and the altered neighboring genes, a breast cancer cohort from the TCGA database was analyzed using bc-GenExMinerv 4.8 (http://bcgenex.ico.unicancer.fr/BC-GEM/GEM-Accueil.php?js=1) [http://bioinfo.life.hust.edu.cn/web/GSCALite/) [The gene–gene interaction network and protein–protein interaction network of db.org/) , 24. To hp?js=1)  and RNA-CALite/) , pathway2, each well was incubated for 2 h with 10 μL of CCK-8 reagent. Measurement of absorbance at 450 nm and assessment of proliferation ability were conducted using a spectrophotometer . The colony-forming potential of all cancer cells was assessed by seeding them onto six-well plates at a density of 300 cells/well. After 10–14 days of culture, cells were removed from the medium, fixed with 4% paraformaldehyde, stained with 0.1% crystal violet, and washed thrice with phosphate buffered saline (PBS) before imaging.We tested the viability of cells using a Cell Counting Kit-8  in accordance with the manufacturer's protocol. In 96-well plates, culture media (100μL) and 3,000 cells were plated. After culturing for 0 to 72 h at 37 °C in a humidified incubator with 5% COHSPB1) were seeded and cultured in an FBS containing medium at 37 °C until 100% confluence. A straight scratch was made through each culture using a 200 μL pipette tip. After wounding, cells were washed three times with PBS and then replenished with fresh serum-free media. The wound area was photographed immediately (t = 0 h) and after 72 h (t = 72 h) using an inverted microscope.Transfected SK-BR-3 and MDA-MB-231 cells .HSPB1 expression between normal (n = 113) and tumor tissues . We examined the relationship between HSPB1 expression and clinicopathological characteristics using the Chi-squared test and Wilcoxon signed-rank test. We used Kaplan–Meier to evaluate the prognostic value of HSPB1 expression. All tests were defined as significant at p < 0.05.All statistical analyses were conducted using R version 3.6.3. The Wilcoxon rank sum test was used to analyze the difference in HSPB1 expression was increased in the breast cancer cell lines SK-BR-3, T47D, MDA-MB-231, MDA-MB-453, and BT474, especially in SK-BR-3 and MDA-MB-231 cells, compared with that in normal breast MCF10A cells , pathologic stage (p = 0.032), ER status (p < 0.001), and PR status (p < 0.001), but there was no significant difference between HSPB1 expression and age, T stage, M stage, and HER2 status (All p > 0.05). We further examined the relationship between HSPB1 expression and clinical characteristics, including the patient status  . Additionally, based on ER, PR, and HER2 expression, we observed that HSPB1 expression was significantly higher in receptor-positive samples than in receptor-negative samples .To better understand the relevance and underlying mechanisms of  p = 0.03, ER stat p = 0.03, ER stat p = 0.03, ER stat p = 0.03, ER stat p = 0.03, ER stat p = 0.03, ER stat p = 0.03, ER stat p = 0.03, ER stat p = 0.03, ER statHSPB1 expression levels are intimately related to breast cancer progression, Kaplan–Meier survival curves were used to compare the expression levels of HSPB1 with prognosis . A prognostic database, PrognoScan, was employed to test the clinical outcome effect of HSPB1. As shown in Fig. HSPB1 were significantly worse than low-HSPB1 on OS, RFS, and DMFS.As sis Fig. A. The exHSPB1 expression and N stage , pathologic stage , ER status , and PR status , but there was no significant difference between HSPB1 expression and age, T stage, M stage, HER2 status, and radiation_therapy (All p > 0.05).Logistic regression analysis Table  showed ap = 0.004). In multivariable Cox regression analysis, age and M stage were independent predictors of OS. Significant difference in HSPB1 expression observed in univariate Cox regression (p = 0.037), but there was no significant difference in multivariate analysis of factors potentially predictive of OS.We conducted univariate and multivariate Cox regression analyses of OS, DSS, PFI, RFS, and DMFS to investigate the relationship between prognostic factors and clinical outcomes , but there was no significant difference in multivariate analysis of factors potentially predictive of DSS, PFI, RFS, and DMFS.More advanced T, N, M, and pathologic stages were associated with worse DSS, and ER or PR positive statuses were associated with better DSS. The advanced N and M stages were independent predictors of DSS Table , PFI Ta, RFS Ta, and DMFHSPB1 expression differences observed in the univariate Cox regression and multivariate analyses were not significant.HSPB1 expression have a poor prognosis. HSPB1 is not an independent marker for OS, DSS, PFI, RFS, or DMFS.These data suggest that patients with breast cancer with high HSPB1 and the altered neighboring genes , death domain-associated protein (DAXX), MAPKAPK5, heat shock protein family A member 1A (HSPA1A), MAPKAPK3, and heat shock protein family A member 8 (HSPA8)) were found to interact with HSPB1 in the STRING database, with correlation scores of 0.998, 0.996, 0.992, 0.964, 0.981, and 0.972, respectively , and moderate associated with MAPKAPK2 . Our study used the GSCALite database to analyze pathways of altered neighboring genes  and non-triple-negative breast cancer cells . Concordantly, HSPB1 expression was significantly increased in triple-negative breast cancer cells (p < 0.01)  reported that HSPB1 was overexpressed in gastric adenocarcinoma tissue and that serum levels of HSPB1 were increased in patients with gastric adenocarcinoma, which may indicate gastric malignancy and thus its detection may be helpful for screening gastric adenocarcinoma [HSPB1 may be used to predict poor prognosis and transfer tendency in prostate cancer and demonstrated that HSPB1 was promoted in an insulin-like growth factor 1-dependent manner [HSPB1 expression in prostate cancer cells is significantly increased after androgen deprivation and chemotherapy and acts as a molecular chaperone for cell protection, making cells resistant to drugs [HSPB1 could be used as a target for radiotherapy sensitization in prostate cancer. Furthermore, HSPB1 antibody levels are elevated in patients with breast cancer [HSPB1 in breast cancer has not yet been conducted, and it remains unclear how HSPB1 affects breast cancer occurrence and development [HSPB1 was considerably upregulated in breast cancer tissues, which is consistent with a previous report [HSPB1 expression was associated with the clinical features of patients with breast cancer. Higher HSPB1 expression was closely correlated with pathologic stage, ER, and PR. The increased expression of HSPB1 observed in late-stage malignancies suggests that HSPB1 may contribute to cancer development. Therefore, we propose HSPB1 as a marker of poor survival in patients with breast cancer.Many studies have investigated arcinoma . Anothert manner . They alto drugs . These rt cancer . Despiteelopment . Accordis report . NotablyHSPB1 in breast cancer, we used the Kaplan–Meier survival curve to analyze the effect of HSPB1 expression level on survival. Patients with higher HSPB1 level had remarkably worse OS, RFS, and DMFS. Consistent results were obtained using the online tool PrognoScan. However, HSPB1 expression differences were not significant in univariate logistic regression and multivariate analyses for OS, DSS, PFI, RFS, and DMFS. We confirmed that HSPB1 is not an independent marker for OS, DSS, PFI, RFS, or DMFS. According to these findings, HSPB1 may be a prognostic biomarker for breast cancer and may facilitate the development of targeted precision oncology.To evaluate the prognostic potential of HSPB1 directly inhibits the activation of caspases to inhibit cell apoptosis [HSPB1 inhibited cell proliferation and migration/invasion activity and promoted apoptosis of breast cancer cells. EMT is the most crucial pathway for tumor cell invasion and metastasis [HSPB1 significantly reduced the expression of vimentin, and N-cadherin, but upregulated E-cadherin expression. As an oncogene, HSPB1 promotes the activity of breast cancer cells by regulating the EMT process, thus establishing HSPB1 as a promising biomarker for the diagnosis and treatment of breast cancer; however, the potential mechanism by which HSPB1 affects cell proliferation and EMT requires further investigation.Apoptosis plays a vital role in cancer . HSPB1 dpoptosis , preventpoptosis . In thistastasis –41. Duritastasis . Similartastasis , we confHSPB1 expression predicted poor clinical outcomes, meaning that it holds potential as a novel prognostic biomarker for breast cancer. HSPB1 knockdown inhibited the proliferation, migration, invasion, and apoptosis of breast cancer cells. This study advances our current understanding of the role of HSPB1 as a prognostic marker for breast cancer treatment.Overall, high Additional file 1.Additional file 2. Additional file 3. Additional file 4. Additional file 5. Additional file 6. Additional file 7. Additional file 8. Additional file 9. Additional file 10. Additional file 11.