Bacterial biofilm is a community of bacteria that are embedded and structured in a self-secreted extracellular matrix. An important clinical-related characteristic of bacterial biofilms is that they are much more resistant to antimicrobial agents than the planktonic cells (up to 1,000 times), which is one of the main causes of antibiotic resistance in clinics. Therefore, infections caused by biofilms are notoriously difficult to eradicate, such as lung infection caused by Pseudomonas aeruginosa in cystic fibrosis patients. Understanding the resistance mechanisms of biofilms will provide direct insights into how we overcome such resistance. In this review, we summarize the characteristics of biofilms and chronic infections associated with bacterial biofilms. We examine the current understanding and research progress on the major mechanisms of antibiotic resistance in biofilms, including quorum sensing. We also discuss the potential strategies that may overcome biofilm-related antibiotic resistance, focusing on targeting biofilm EPSs, blocking quorum sensing signaling, and using recombinant phages.

More and more available fungal genome sequence data reveal a large amount of secondary metabolite (SM) biosynthetic 'dark matter' to be discovered. Heterogeneous expression is one of the most effective approaches to exploit these novel natural products, but it is limited by having to clone entire biosynthetic gene clusters (BGCs) without errors. So far, few effective technologies have been developed to manipulate the specific large DNA fragments in filamentous fungi. Here, we developed a fungal BGC-capturing system based on CRISPR/Cas9 cleavage in vitro. In our system, Cas9 protein was purified and CRISPR guide sequences in combination with in vivo yeast assembly were rationally designed. Using targeted cleavages of plasmid DNAs with linear (8.5 kb) or circular (8.5 kb and 28 kb) states, we were able to cleave the plasmids precisely, demonstrating the high efficiency of this system. Furthermore, we successfully captured the entire Nrc gene cluster from the genomic DNA of Neosartorya fischeri. Our results provide an easy and efficient approach to manipulate fungal genomic DNA based on the in vitro application of Cas9 endonuclease. Our methodology will lay a foundation for capturing entire groups of BGCs in filamentous fungi and accelerate fungal SMs mining.

Hepatitis B virus (HBV) genome P-encoded protein HBV DNA polymerase (Pol) has long been known as a reverse transcriptase during HBV replication. In this study, we investigated the impact of HBV Pol on host cellular processes, mainly apoptosis, and the underlying mechanisms. We showed a marked reduction in apoptotic rates in the HBV Pol-expressed HepG2 cells compared to controls. Moreover, a series of assays, i.e., yeast two-hybrid, GST pull-down, co-immunoprecipitation, and confocal laser scanning microscopy, identified the host factor eEF1A2 to be associated with HBV Pol. Furthermore, knockdown of eEF1A2 gene by siRNA abrogated the HBV Pol-mediated anti-apoptotic effect with apoptosis induced by endoplasmatic reticulum (ER) stress-inducer thapsigargin (TG), thus suggesting that the host factor eEF1A2 is essential for HBV Pol's anti-apoptosis properties. Our findings have revealed a novel role for HBV Pol in its modulation of apoptosis through integrating with eEF1A2.

Inflammatory reactions activated by lipopolysaccharide (LPS) of gram-negative bacteria can lead to severe septic shock. With the recent emergence of multidrug-resistant gram-negative bacteria and a lack of efficient ways to treat resulting infections, there is a need to develop novel anti-endotoxin agents. Antimicrobial peptides have been noticed as potential therapeutic molecules for bacterial infection and as candidates for new antibiotic drugs. We previously designed the 9-meric antimicrobial peptide Pro9-3 and it showed high antimicrobial activity against gram-negative bacteria. Here, to further examine its potency as an anti-endotoxin agent, we examined the anti-endotoxin activities of Pro9-3 and elucidated its mechanism of action. We performed a dye-leakage experiment and BODIPY-TR cadaverine and limulus amebocyte lysate assays for Pro9-3 as well as its lysine-substituted analogue and their enantiomers. The results confirmed that Pro9-3 targets the bacterial membrane and the arginine residues play key roles in its antimicrobial activity. Pro9-3 showed excellent LPS-neutralizing activity and LPS-binding properties, which were superior to those of other peptides. Saturation transfer difference-nuclear magnetic resonance experiments to explore the interaction between LPS and Pro9-3 revealed that Trp3 and Tlr7 in Pro9-3 are critical for attracting Pro9-3 to the LPS in the gram-negative bacterial membrane. Moreover, the anti-septic effect of Pro9-3 in vivo was investigated using an LPS-induced endotoxemia mouse model, demonstrating its dual activities: antibacterial activity against gram-negative bacteria and immunosuppressive effect preventing LPS-induced endotoxemia. Collectively, these results confirmed the therapeutic potential of Pro9-3 against infection of gram-negative bacteria.

Due to the potential of antioxidants to scavenge free radicals in human body, it is important to be able to prepare antioxidant peptides that meet the industrial requirements for cosmetics and food. Here, we determined in vivo/in vitro activities of antioxidant peptide from P. fucata (PFAOP) prepared by bio-fermentation method. The antioxidant property test results showed the DPPH, hydroxyl, superoxide radical-scavenging, and cellular antioxidant activity. EC50 values of PFAOPs were 0.018 ± 0.005, 0.126 ± 0.008, 0.168 ± 0.005, and 0.105 ± 0.005 mg/ml, respectively, exhibiting higher antioxidant activities than glutathione (p < 0.05). Moreover, anti-proliferation and cytotoxicity activity results illustrated PFAOP has a potent anti-proliferative activity against HepG2, Caco-2, and MCF-7 carcinoma cells with no cytotoxicity. Moreover, the protocols we developed in this work demonstrated several excellent advantages in PFAOP preparation compared to enzymatic hydrolysis or chemical synthesis methods and provide a theoretical foundation for higher-value application of marine-derived functional peptides.

A newly cloned 4-α-glucanotransferase (αGT) from Deinococcus geothermalis and two typical bacterial αGTs from Thermus scotoductus and Escherichia coli (MalQ) were investigated. Among 4 types of catalysis, the cyclization activity of αGTs that produces cycloamylose (CA), a valuable carbohydrate making inclusion complexes, was intensively studied. The new αGT, DgαGT, showed close protein sequence to the αGT from T. scotoductus (TsαGT). MalQ was clearly separated from the other two αGTs in the phylogenetic and the conserved regions analyses. The reaction velocities of disproportionation, cyclization, coupling, and hydrolysis of three αGTs were determined. Intriguingly, MalQ exhibited more than 100-fold lower cyclization activity than the others. To lesser extent, the disproportionation activity of MalQ was relatively low. DgαGT and TsαGT showed similar kinetics results, but TsαGT had nearly 10-fold lower hydrolysis activity than DgαGT. Due to the very low cyclizing activity of MalQ, DgαGT and TsαGT were selected for further analyses. When amylose was treated with DgαGT or TsαGT, CA with a broad DP range was generated immediately. The DP distribution of CA had a bimodal shape (DP 7 and 27 as peaks) for the both enzymes, but larger DPs of CA quickly decreased in the DgαGT. Cyclomaltopentaose, a rare cyclic sugar, was produced at early reaction stage and accumulated as the reactions went on in the both enzymes, but the increase was more profound in the TsαGT. Taken together, we clearly demonstrated the catalytic differences between αGT groups from thermophilic and pathogenic bacteria that and showed that αGTs play different roles depending on their lifestyle.

Here, we investigated the prebiotic and antioxidant effects of Actinidia arguta sprout water extract (AASWE) on lipopolysaccharide (LPS)-induced cognitive deficit mice. AASWE increased viable cell count, titratable acidity, and acetic acid production in Lactobacillus reuteri strain and showed a cytoprotective effect on LPS-induced inflammation in HT-29 cells. We assessed the behavior of LPS-induced cognitive deficit mice using Y-maze, passive avoidance and Morris water maze tests and found that administration of AASWE significantly improved learning and memory function. The AASWE group showed antioxidant activity through downregulation of malondialdehyde levels and upregulation of superoxide dismutase levels in brain tissue. In addition, the AASWE group exhibited activation of the cholinergic system with decreased acetylcholinesterase activity in brain tissue. Furthermore, AASWE effectively downregulated inflammatory mediators such as phosphorylated-JNK, phosphorylated-NF-κB, TNF-α and interleukin-6. The major bioactive compounds of AASWE were identified as quercetin-3-O-arabinopyranosyl(1→2)-rhamnopyranosyl(1→6)-glucopyranose, quercetin-3-O-apiosyl(1 → 2)-galactoside, rutin, and 3-caffeoylquinic acid. Based on these results, we suggest that AASWE not only increases the growth of beneficial bacteria in the intestines, but also shows an ameliorating effect on LPS-induced cognitive impairment.

Carotenoids, which have biologically beneficial effects and occur naturally in microorganisms and plants, are pigments widely applied in the food, cosmetics and pharmaceutical industries. The compound 4,4'-diaponeurosporene is a C30 carotenoid produced by some Lactobacillus species, and Lactobacillus plantarum is the main species producing it. In this study, the antioxidant activity of 4,4'-diaponeurosporene extracted from L. plantarum subsp. plantarum KCCP11226 was examined. Maximum carotenoid content (0.74 ± 0.2 at A470) was obtained at a relatively low temperature (20℃). The DPPH radical scavenging ability of 4,4'-diaponeurosporene (1 mM) was approximately 1.7-fold higher than that of butylated hydroxytoluene (BHT), a well-known antioxidant food additive. In addition, the ABTS radical scavenging ability was shown to be 2.3- to 7.5-fold higher than that of BHT at the range of concentration from 0.25 mM to 1 mM. The FRAP analysis confirmed that 4,4'-diaponeurosporene (0.25 mM) was able to reduce Fe3+ by 8.0-fold higher than that of BHT. Meanwhile, 4,4'-diaponeurosporene has been confirmed to be highly resistant to various external stresses (acid/bile, high temperature, and lysozyme conditions). In conclusion, L. plantarum subsp. plantarum KCCP11226, which produces 4,4'-diaponeurosporene as a functional antioxidant, may be a potentially useful strain for the development of functional probiotic industries.

Identifying the extracellular metabolites of microorganisms in fresh vegetables is industrially useful for assessing the quality of processed foods. Pectobacterium carotovorum subsp. carotovorum (PCC) is a plant pathogenic bacterium that causes soft rot disease in cabbages. This microbial species in plant tissues can emit specific volatile molecules with odors that are characteristic of the host cell tissues and PCC species. In this study, we used headspace solid-phase microextraction followed by gas chromatography coupled with mass spectrometry (HS-SPME-GC-MS) to identify volatile compounds (VCs) in PCC-inoculated cabbage at different storage temperatures. HS-SPME-GC-MS allowed for recognition of extracellular metabolites in PCC-infected cabbages by identifying specific volatile metabolic markers. We identified 4-ethyl-5-methylthiazole and 3-butenyl isothiocyanate as markers of fresh cabbages, whereas 2,3-butanediol and ethyl acetate were identified as markers of soft rot in PCC-infected cabbages. These analytical results demonstrate a suitable approach for establishing non-destructive plant pathogen-diagnosis techniques as alternatives to standard methods, within the framework of developing rapid and efficient analytical techniques for monitoring plant-borne bacterial pathogens. Moreover, our techniques could have promising applications in managing the freshness and quality control of cabbages.

γ-Glutamylcysteine synthetase (Gcs1) and glutathione reductase (Glr1) activity maintains minimal levels of cellular methylglyoxal in Candida albicans. In glutathione-depleted Δgcs1, we previously saw that NAD(H)-linked methylglyoxal oxidoreductase (Mgd1) and alcohol dehydrogenase (Adh1) are the most active methylglyoxal scavengers. With methylglyoxal accumulation, disruptants lacking MGD1 or ADH1 exhibit a poor redox state. However, there is little convincing evidence for a reciprocal relationship between methylglyoxal scavenger genes-disrupted mutants and changes in glutathione-(in)dependent redox regulation. Herein, we attempt to demonstrate a functional role for methylglyoxal scavengers, modeled on a triple disruptant (Δmgd1/Δadh1/Δgcs1), to link between antioxidative enzyme activities and their metabolites in glutathione-depleted conditions. Despite seeing elevated methylglyoxal in all of the disruptants, the result saw a decrease in pyruvate content in Δmgd1/Δadh1/Δgcs1 which was not observed in double gene-disrupted strains such as Δmgd1/Δgcs1 and Δadh1/Δgcs1. Interestingly, Δmgd1/Δadh1/Δgcs1 exhibited a significantly decrease in H2O2 and superoxide which was also unobserved in Δmgd1/Δgcs1 and Δadh1/Δgcs1. The activities of the antioxidative enzymes erythroascorbate peroxidase and cytochrome c peroxidase were noticeably higher in Δmgd1/Δadh1/Δgcs1 than in the other disruptants. Meanwhile, Glr1 activity severely diminished in Δmgd1/Δadh1/Δgcs1. Monitoring complementary gene transcripts between double gene-disrupted Δmgd1/Δgcs1 and Δadh1/Δgcs1 supported the concept of an unbalanced redox state independent of the Glr1 activity for Δmgd1/Δadh1/Δgcs1. Our data demonstrate the reciprocal use of Eapx1 and Ccp1 in the absence of both methylglyoxal scavengers; that being pivotal for viability in non-filamentous budding yeast.

Lactobacillus casei, one of the most widely used probiotics, has been reported to alleviate multiple diseases. However, the effects of this species on intestinal diseases are strain-specific. Here, we aimed to screen L. casei strains with inflammatory bowel disease (IBD)-alleviating effects based on in vitro physiological characteristics. Therefore, the physiological characteristics of 29 L. casei strains were determined, including gastrointestinal transit tolerance, oligosaccharide fermentation, HT-29 cell adhesion, generation time, exopolysaccharide production, acetic acid production, and conjugated linoleic acid synthesis. The effects of five candidate strains on mice with induced colitis were also evaluated. The results showed that among all tested L. casei strains, only Lactobacillus casei M2S01 effectively relieved colitis. This strain recovered body weight, restored disease activity index score, and promoted anti-inflammatory cytokine expression. Gut microbiota sequencing showed that L. casei M2S01 restored a healthy gut microbiome composition. The western blotting showed that the alleviating effects of L. casei M2S01 on IBD were related to the inhibition of the NF-κB pathway. A good gastrointestinal tolerance ability may be one of the prerequisites for the IBD-alleviating effects of L. casei. Our results verified the efficacy of L. casei in alleviating IBD and lay the foundation for the rapid screening of L. casei strain with IBD-alleviating effects.

Petroleum-contaminated soil is considered among the most important potential anthropogenic atmospheric methane sources. Additionally, various rhizoremediation factors can affect methane emissions by altering soil ecosystem carbon cycles. Nonetheless, greenhouse gas emissions from soil have not been given due importance as a potentially relevant parameter in rhizoremediation techniques. Therefore, in this study we sought to investigate the effects of different plant and soil amendments on both remediation efficiencies and methane emission characteristics in diesel-contaminated soil. An indoor pot experiment consisting of three plant treatments (control, maize, tall fescue) and two soil amendments (chemical nutrient, compost) was performed for 95 days. Total petroleum hydrocarbon (TPH) removal efficiency, dehydrogenase activity, and alkB (i.e., an alkane compound-degrading enzyme) gene abundance were the highest in the tall fescue and maize soil system amended with compost. Compost addition enhanced both the overall remediation efficiencies, as well as pmoA (i.e., a methane-oxidizing enzyme) gene abundance in soils. Moreover, the potential methane emission of diesel-contaminated soil was relatively low when maize was introduced to the soil system. After microbial community analysis, various TPH-degrading microorganisms (Nocardioides, Marinobacter, Immitisolibacter, Acinetobacter, Kocuria, Mycobacterium, Pseudomonas, Alcanivorax) and methane-oxidizing microorganisms (Methylocapsa, Methylosarcina) were observed in the rhizosphere soil. The effects of major rhizoremediation factors on soil remediation efficiency and greenhouse gas emissions discussed herein are expected to contribute to the development of sustainable biological remediation technologies in response to global climate change.

Phenol-soluble modulins (PSMs) are responsible for regulating biofilm formation, persister cell formation, pmtR expression, host cell lysis, and anti-bacterial effects. To determine the effect of psm deletion on methicillin-resistant Staphylococcus aureus, we investigated psm deletion mutants including Δpsmα, Δpsmβ, and Δpsmαβ. These mutants exhibited increased β-lactam antibiotic resistance to ampicillin and oxacillin that was shown to be caused by increased N-acetylmannosamine kinase (nanK) mRNA expression, which regulates persister cell formation, leading to changes in the pattern of phospholipid fatty acids resulting in increased anteiso-C15:0, and increased membrane hydrophobicity with the deletion of PSMs. When synthetic PSMs were applied to Δpsmα and Δpsmβ mutants, treatment of Δpsmα with PSMα1-4 and Δpsmβ with PSMβ1-2 restored the sensitivity to oxacillin and slightly reduced the biofilm formation. Addition of a single fragment showed that α1, α2, α3, and β2 had an inhibiting effect on biofilms in Δpsmα; however, β1 showed an enhancing effect on biofilms in Δpsmβ. This study demonstrates a possible reason for the increased antibiotic resistance in psm mutants and the effect of PSMs on biofilm formation.

Polyhydroxyalkanoate (PHA) synthase is a key enzyme for PHA production in microorganisms. The class IV PHA synthase is composed of two subunits: PhaC and PhaR. The PhaR subunit, which encodes the phaR gene, is only present in class IV PHA synthases. Therefore, the phaR gene is used as a biomarker for bacteria that contain a class IV PHA synthase, such as some Bacillus spp. The phaR gene was developed to screen phaR-containing Bacillus spp. The phaR screening method involved two steps: phaR gene amplification by PCR and phaR amplicon detection using a DNA lateral flow assay. The screening method has a high specificity for phaR-containing Bacillus spp. The lowest amount of genomic DNA of B. thuringiensis ATCC 10792 that the phaR screening method could detect was 10 pg. This novel screening method improves the specificity and sensitivity of phaR gene screening and reduces the time and cost of the screening process, which could enhance the opportunity to discover good candidate PHA producers. Nevertheless, the screening method can certainly be used as a tool to screen phaR-containing Bacillus spp. from environmental samples.

Persister cell formation and biofilms of pathogens are extensively involved in the development of chronic infectious diseases. Eradicating persister cells is challenging, owing to their tolerance to conventional antibiotics, which cannot kill cells in a metabolically dormant state. A high frequency of persisters in biofilms makes inactivating biofilm cells more difficult, because the biofilm matrix inhibits antibiotic penetration. Fatty acids may be promising candidates as antipersister or antibiofilm agents, because some fatty acids exhibit antimicrobial effects. We previously reported that fatty acid ethyl esters effectively inhibit Escherichia coli persister formation by regulating an antitoxin. In this study, we screened a fatty acid library consisting of 65 different fatty acid molecules for altered persister formation. We found that undecanoic acid, lauric acid, and N-tridecanoic acid inhibited E. coli BW25113 persister cell formation by 25-, 58-, and 44-fold, respectively. Similarly, these fatty acids repressed persisters of enterohemorrhagic E. coli EDL933. These fatty acids were all medium-chain saturated forms. Furthermore, the fatty acids repressed Enterohemorrhagic E. coli (EHEC) biofilm formation (for example, by 8-fold for lauric acid) without having antimicrobial activity. This study demonstrates that medium-chain saturated fatty acids can serve as antipersister and antibiofilm agents that may be applied to treat bacterial infections.

Most cervical cancers are associated with high-risk human papillomavirus (HPV) infection. Currently, cervical cancer treatment entails surgical removal of the lesion, but treatment of infection and preventing tissue damage are issues that still remain to be addressed. Herbal medicine and biological studies have focused on developing antiviral drugs from natural sources. In this study, we analyzed the potential antiviral effects of Pinus densiflora Sieb. et Zucc. leaf extracts against HPV. The pine needle extracts from each organic solvent were analyzed for antiviral activity. The methylene chloride fraction (PN-MC) showed the highest activity against HPV pseudovirus (PV). The PN-MC extract was more effective before, rather than after treatment, and therefore represents a prophylactic intervention. Mice were pre-treated with PN-MC via genital application or oral administration, followed by a genital or subcutaneous challenge with HPV PV, respectively. The HPV challenge results showed that mice treated via genital application exhibited complete protection against HPV. In conclusion, PN-MC represents a potential topical virucide for HPV infection.

Organophosphorus nerve agents (OPNAs), including both G- and V-type nerve agents such as sarin, soman, tabun and VX, are extremely neurotoxic organophosphorus compounds. Catalytic bioscavengers capable of hydrolyzing OPNAs are under development because of the low protective effects and adverse side effects of chemical antidotes to OPNA poisoning. However, these bioscavengers have certain limitations for practical application, including low catalytic activity and narrow specificity. In this study, we generated a fusion-hybrid form of engineered recombinant human paraoxonase 1 (rePON1) and bacterial organophosphorus hydrolase (OPH), referred to as GV-hybrids, using a flexible linker to develop more promising catalytic bioscavengers against a broad range of OPNAs. These GV-hybrids were able to synergistically hydrolyze both G-type OPNA analogs (paraoxon: 1.7 ~ 193.7-fold, p-nitrophenyl diphenyl phosphate (PNPDPP): 2.3 ~ 33.0-fold and diisopropyl fluorophosphates (DFP): 1.4 ~ 22.8-fold) and V-type OPNA analogs (demeton-S-methyl (DSM): 1.9 ~ 34.6-fold and malathion: 1.1 ~ 4.2-fold above) better than their individual enzyme forms. Among the GV-hybrid clones, the GV7 clone showed remarkable improvements in the catalytic activity toward both G-type OPNA analogs (kcat/Km (106 M-1 min-1): 59.8 ± 0.06 (paraoxon), 5.2 ± 0.02 (PNPDPP) and 47.0 ± 6.0 (DFP)) and V-type OPNA analogs (kcat/Km (M-1 min-1): 504.3 ± 48.5 (DSM) and 1324.0 ± 47.5 (malathion)). In conclusion, we developed GV-hybrid forms of rePON1 and bacterial OPH mutants as effective and suitable catalytic bioscavengers to hydrolyze a broad range of OPNA analogs.

L-5-methyltetrahydrofolate (5-MTHF) is one of the biological active forms of folate, which is widely used as a nutraceutical. However, low yield and serious pollution associated with the chemical synthesis of 5-MTHF hampers its sustainable supply. In this study, 5-MTHF production was improved by engineering the 5-MTHF biosynthesis pathway and NADPH supply in Lactococcus lactis for developing a green and sustainable biosynthesis approach. Specifically, overexpressing the key rate-limiting enzyme methylenetetrahydrofolate reductase led to intracellular 5-MTHF accumulation, reaching 18 ㎍/l. Next, 5-MTHF synthesis was further enhanced by combinatorial overexpression of 5-MTHF synthesis pathway enzymes with methylenetetrahydrofolate reductase, resulting in 1.7-fold enhancement. The folate supply pathway was strengthened by expressing folE encoding GTP cyclohydrolase I, which increased 5-MTHF production 2.4-fold to 72 ㎍/l. Furthermore, glucose-6-phosphate dehydrogenase was overexpressed to improve the redox cofactor NADPH supply for 5-MTHF biosynthesis, which led to a 60% increase in intracellular NADPH and a 35% increase in 5-MTHF production (97 ㎍/l). To reduce formation of the by-product 5-formyltetrahydrofolate, overexpression of 5-formyltetrahydrofolate cyclo-ligase converted 5-formyltetrahydrofolate to 5,10-methyltetrahydrofolate, which enhanced the 5-MTHF titer to 132 ㎍/l. Finally, combinatorial addition of folate precursors to the fermentation medium boosted 5-MTHF production, reaching 300 ㎍/l. To the best of our knowledge, this titer is the highest achieved by L. lactis. This study lays the foundation for further engineering of L. lactis for efficient 5-MTHF biosynthesis.

Enzyme replacement therapy for lysosomal storage diseases usually requires recombinant enzymes containing mannose-6-phosphate (M6P) glycans for cellular uptake and lysosomal targeting. For the first time, a strategy is established here for the in vitro mannosyl-phosphorylation of high-mannose type N-glycans that utilizes a recombinant Mnn14 protein derived from Saccharomyces cerevisiae. Among a series of N-terminal- or C-terminal-deleted recombinant Mnn14 proteins expressed in Pichia pastoris, rMnn1477-935 with deletion of N-terminal 76 amino acids spanning the transmembrane domain (46 amino acids) and part of the stem region (30 amino acids), showed the highest level of mannosyl-phosphorylation activity. The optimum reaction conditions for rMnn1477-935 were determined through enzyme assays with a high-mannose type N-glycan (Man8GlcNAc2) as a substrate. In addition, rMnn1477-935 was shown to mannosyl-phosphorylate high-mannose type N-glycans (Man7-9GlcNAc2) on recombinant human lysosomal alpha-glucosidase (rhGAA) with remarkably high efficiency. Moreover, the majority of the resulting mannosyl-phosphorylated glycans were bis-form which can be converted to bis-phosphorylated M6P glycans having a superior lysosomal targeting capability. An in vitro N-glycan mannosyl-phosphorylation reaction using rMnn1477-935 will provide a flexible and straightforward method to increase the M6P glycan content for the generation of "Biobetter" therapeutic enzymes.