Phenotyping of Methicillin-Resistant Staphylococcus aureus Using a Ratiometric Sensor Array.

Chemical tools capable of classifying multidrug-resistant bacteria (superbugs) can facilitate early-stage disease diagnosis and help guide precision therapy. Here, we report a sensor array that permits the facile phenotyping of methicillin-resistant Staphylococcus aureus (MRSA), a clinically common superbug. The array consists of a panel of eight separate ratiometric fluorescent probes that provide characteristic vibration-induced emission (VIE) profiles. These probes bear a pair of quaternary ammonium salts in different substitution positions around a known VIEgen core. The differences in the substituents result in varying interactions with the negatively charged cell walls of bacteria. This, in turn, dictates the molecular conformation of the probes and affects their blue-to-red fluorescence intensity ratios (ratiometric changes). Within the sensor array, the differences in the ratiometric changes for the probes result in "fingerprints" for MRSA of different genotypes. This allows them to be identified using principal component analysis (PCA) without the need for cell lysis and nucleic acid isolation. The results obtained with the present sensor array agree well with those obtained using polymerase chain reaction (PCR) analysis.

New Insights into the Accumulation, Transport, and Distribution Mechanisms of Hexafluoropropylene Oxide Homologues, Important Alternatives to Perfluorooctanoic Acid, in Lettuce (Lactuca sativa L.).

Hexafluoropropylene oxide (HFPO) homologues, which are important alternatives to perfluorooctanoic acid, have been frequently identified in crops. Although exposure to HFPO homologues via crops may pose non-negligible threats to humans, their impact on crops is still unknown. In this study, the accumulation, transport, and distribution mechanisms of three HFPO homologues in lettuce were investigated at the plant, tissue, and cell levels. More specifically, HFPO trimer acid and HFPO tetramer acid were primarily fixed in roots and hardly transported to shoots (TF, 0.06-0.63). Conversely, HFPO dimer acid (HFPO-DA) tended to accumulate in lettuce shoots 2-264 times more than the other two homologues, thus resulting in higher estimated daily intake values. Furthermore, the dissolved organic matter derived from root exudate enhanced HFPO-DA uptake by increasing its desorption fractions in the rhizosphere. The transmembrane uptake of HFPO homologues was controlled by means of a transporter-mediated active process involving anion channels, with the uptake of HFPO-DA being additionally facilitated by aquaporins. The higher accumulation of HFPO-DA in shoots was attributed to the larger proportions of HFPO-DA in the soluble fraction (55-74%) and its higher abundance in both vascular tissues and xylem sap. Our findings expand the understanding of the fate of HFPO homologues in soil-crop systems and reveal the underlying mechanisms of the potential exposure risk to HFPO-DA.

Highly sensitive liquid chromatographic method combined with online ion suppression to remove interfering anions and mass spectrometry for impurity profiling of paromomycin sulfate.

A previously developed high-performance liquid chromatography method combined with pulsed amperometric detection allowed to separate many impurities of paromomycin. However, due to the presence of ion pairing agents and sodium hydroxide in the mobile phase, direct coupling to mass spectrometry for the identification of the chemical structures of the impurities was not an option. Indeed, ion suppression was encountered by trifluoroacetic acid and pentafluoroproponic acid in the mobile phase. A cation self-regenerating suppressor, which was originally designed for increasing analyte conductivity of ammonia and amines analysis in ion chromatography, was coupled between the liquid chromatography and ion trap-time of flight-mass spectrometry and almost all trifluoroacetic acid and pentafluoroproponic acid in the mobile phase was removed. The limit of detection of paromomycin in this integrated system improved significantly to 20 ng/ml (0.4 ng). The chemical structures of 19 impurities were elucidated and seven impurities were reported for the first time.

Anti-bacterial monoclonal antibodies: next generation therapy against superbugs.

Prior to the nineteenth century, infectious disease was one of the leading causes of death. Human life expectancy has roughly doubled over the past century as a result of the development of antibiotics and vaccines. However, the emergence of antibiotic-resistant superbugs brings new challenges. The side effects of broad-spectrum antibiotics, such as causing antimicrobial resistance and destroying the normal flora, often limit their applications. Furthermore, the development of new antibiotics has lagged far behind the emergence and spread of antibiotic resistance. On the other hand, the genome complexity of bacteria makes it difficult to create effective vaccines. Therefore, novel therapeutic agents in supplement to antibiotics and vaccines are urgently needed to improve the treatment of infections. In recent years, monoclonal antibodies (mAbs) have achieved remarkable clinical success in a variety of fields. In the treatment of infectious diseases, mAbs can play functions through multiple mechanisms, including toxins neutralization, virulence factors inhibition, complement-mediated killing activity, and opsonic phagocytosis. Toxins and bacterial surface components are good targets to generate antibodies against. The U.S. FDA has approved three monoclonal antibody drugs, and there are numerous candidates in the preclinical or clinical trial stages. This article reviews recent advances in the research and development of anti-bacterial monoclonal antibody drugs in order to provide a valuable reference for future studies in this area. KEY POINTS: • Novel drugs against antibiotic-resistant superbugs are urgently required • Monoclonal antibodies can treat bacterial infections through multiple mechanisms • There are many anti-bacterial monoclonal antibodies developed in recent years and some candidates have entered the preclinical or clinical stages of development.

A new aminoglycoside etimicin shows low nephrotoxicity and ototoxicity in zebrafish embryos.

Aminoglycoside antibiotics are widely used for many life-threatening infections. The use of aminoglycosides is often comprised by their deleterious side effects to the kidney and inner ear. A novel semisynthetic antibiotic, etimicin, has good antimicrobial activity against both gram-positive and gram-negative bacteria. But its toxicity profile analysis is still lacking. In the present study, we compared the in vivo toxic effects of three aminoglycosides, gentamicin, amikacin, and etimicin, in zebrafish embryos. We examined the embryotoxicity, nephrotoxicity, and the damage to the neuromast hair cells. Our results revealed that etimicin and amikacin exhibit more developmental toxicities to the young embryos than gentamicin. But at subtoxic doses, etimicin and amikacin show significantly reduced toxicities towards kidney and neuromast hair cells. We further demonstrated that fluorescently conjugated aminoglycosides (gentamicin-Texas red [GTTR], amikacin-Texas red [AMTR], and etimicin-Texas red [ETTR]) all enter the hair cells properly. Inside the hair cells, gentamicin, not etimicin and amikacin, displays robust reactive oxygen species generation and induces apoptosis. Our data support that the different intracellular cytotoxicity underlies the different ototoxicity of the three aminoglycosides and that etimicin is a new aminoglycoside with reduced risk of nephrotoxicity and ototoxicity.

Protective effects of anti-alginate monoclonal antibody against Pseudomonas aeruginosa infection of HeLa cells.

Pseudomonas aeruginosa is an opportunistic pathogen that is highly resistant to antibiotics, especially when it grows in biofilms. As an alternative to antibiotic intervention, antimicrobial antibody drugs have drawn attention in recent years due to their immunotherapeutic functions. In this study, we designed a monoclonal scFv-Fc-form antibody, MFb, targeting P. aeruginosa antigen alginate and investigated its function against this bacterium in vitro. MFb was generated by transient gene expression in HEK293 cells and purified by one-step protein A affinity chromatography. Experiments showed that MFb could recognize alginate specifically based on enzyme-linked immunosorbent assays. Its KD value of 8.31 nM was determined by surface plasmon resonance, demonstrating its high affinity for alginate. Further detailed studies revealed that the antibody exerted antibacterial effects by three mechanisms: 1) MFb inhibited P. aeruginosa biofilm formation with an IC50 of 0.58 µg/mL; 2) MFb reduced P. aeruginosa adhesion to HeLa cells, and successfully prevented its invasion on epithelial cells; 3) based on an in vitro macrophage phagocytosis assay, MFb could enhance the phagocytotic capacity of macrophages for P. aeruginosa in a concentration-dependent manner. Taken together, our work demonstrated that the antimicrobial monoclonal antibody MFb has a protective effect on HeLa cells, and it may be a promising novel strategy to treat P. aeruginosa infection.

Lycorine ameliorates bleomycin-induced pulmonary fibrosis via inhibiting NLRP3 inflammasome activation and pyroptosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic and irreversible lung disease with limited therapeutic strategies. Lycorine (LYC), an alkaloid isolated from Amaryllidaceae family plants, exhibits effective anti-inflammatory, antiviral, and anti-tumor activities. In this study, we attempted to determine the effect of LYC on bleomycin (BLM)-induced IPF and NLRP3 inflammasome activation. Our results demonstrated that the LYC treatment ameliorated BLM-induced pulmonary fibrosis and inflammation in mice. LYC inhibited active Caspase-1 expression and lactate dehydrogenase (LDH) release during BLM-induced acute lung injury (ALI) in mice. Furthermore, our in vitro assay showed that LYC inhibited LPS/Nigericin- or LPS/ATP-induced NACHT, LRP and PYD domains-containing protein 3 (NLRP3) inflammasome activation, and pyroptosis in bone marrow-derived macrophages (BMDMs). Mechanically, LYC could disturb the interaction of NLRP3 with apoptosis-associated speck-like protein containing a CARD (ASC) by targeting the pyrin domain (PYD) on Leu9, Leu50, and Thr53. Our findings indicate that LYC ameliorated BLM-induced pulmonary fibrosis by inhibiting NLRP3 inflammasome activation and pyroptosis through targeting the PYD domain of ASC. Thus, LYC might be a potential therapeutic agent for pulmonary inflammation and fibrosis.

Mutational biosynthesis to generate novel analogs of nosiheptide featuring a fluorinated indolic acid moiety.

Nosiheptide (NOS) is a member of bicyclic thiopeptides possessing a biologically important indolic acid (IA) moiety appended onto the family-characteristic core system. The IA formation relies primarily on NosL, a radical S-adenosylmethionine (SAM) protein that catalyzes a complex rearrangement of the carbon side chain of l-tryptophan, leading to the generation of 3-methyl-2-indolic acid (MIA). Here, we establish an efficient mutational biosynthesis strategy for the structural expansion of the side-ring system of NOS. The nosL-deficient mutant Streptomyces actuosus SL4005 complemented by chemically feeding 6-fluoro-MIA is capable of accumulating two new products. The target product 6'-fluoro-NOS contains an additional fluorine atom at C6 of the IA moiety, in contrast with an unexpected product 6'-fluoro-NOSint that features an open side ring and a bis-dehydroalanine (Dha) tail. The newly obtained 6'-fluoro-NOS displayed equivalent or slightly reduced activities against the tested drug-resistant pathogens compared with NOS, but dramatically decreased water solubility compared with NOS. Our results indicate that the modification of the IA moiety of NOS not only affects its biological activity but also affects its activity which will be key considerations for further modification.

Mobility of metal(loid)s in Pb/Zn tailings under different revegetation strategies.

Metal tailings are potential sources of strong environmental pollution. In situ remediation involves the installation of a plant cover to stabilize materials and pollutants. Whether metal(loid)s are effectively immobilized in remediated tailing ponds submitted to heavy rainfall remains uncertain. In this study, tailing materials were collected from bare tailings (control), grass-planted (G) and grass-shrub planted (GS) areas on a former Pb/Zn mine site. Batch column experiments were performed with three rainfall intensities of 0.36, 0.48, and 0.50 mL min-1 for 18 d in the lab. The pH, Eh, Cd, Pb, Zn and As concentration in leachate were recorded. Selected leached tailing materials were finally characterized. Results showed that leachates from control were strongly acidic (pH 3.11-4.65), and that Cd, Pb, Zn and As were quickly released at high rate (e.g., 945 mg L-1 Zn). During the experiment up to 4% Cd present in the material was released and almost 1% Zn. With material collected from the G area, leachates were even more acidic (2.16-2.84) with a rainfall intensity of 0.50 mL min-1 and exhibited a high redox potential (588-639 mV). However, concentrations of metals in leachates were much lower than that in the control, except for Zn (e.g., 433 mg L-1), and they tended to decrease with time. Cumulative leaching rate was still relatively high (e.g., 0.68% Cd; 0.75% Zn) during the first eight days (stage I). However, with the GS treatment, leachate pH gradually raised from acid to alkaline values (3.9-8.2) during stage I, then remained high until the end of the experiment (stage II). Also, amounts of elements released during the 18 d were low in general. The releasing ratios of Cd (R2 > 0.95), Pb (R2 > 0.95), As (R2 > 0.87), and Zn (R2 > 0.90) fitted well with a two-constant model. In conclusion, under subtropical climate with heavy rainfall, phytostabilization is effective but immobilization of metals is higher with a combination of grass and shrub than with only grass to reduce leaching of As and Zn.

A Supramolecular-Based Dual-Wavelength Phototherapeutic Agent with Broad-Spectrum Antimicrobial Activity Against Drug-Resistant Bacteria.

With the ever-increasing threat posed by the multi-drug resistance of bacteria, the development of non-antibiotic agents for the broad-spectrum eradication of clinically prevalent superbugs remains a global challenge. Here, we demonstrate the simple supramolecular self-assembly of structurally defined graphene nanoribbons (GNRs) with a cationic porphyrin (Pp4N) to afford unique one-dimensional wire-like GNR superstructures coated with Pp4N nanoparticles. This Pp4N/GNR nanocomposite displays excellent dual-modal properties with significant reactive-oxygen-species (ROS) production (in photodynamic therapy) and temperature elevation (in photothermal therapy) upon light irradiation at 660 and 808 nm, respectively. This combined approach proved synergistic, providing an impressive antimicrobial effect that led to the complete annihilation of a wide spectrum of Gram-positive, Gram-negative, and drug-resistant bacteria both in vitro and in vivo. The study also unveils the promise of GNRs as a new platform to develop dual-modal antimicrobial agents that are able to overcome antibiotic resistance.

Dose Optimization of Colistin Combinations against Carbapenem-Resistant Acinetobacter baumannii from Patients with Hospital-Acquired Pneumonia in China by Using an In Vitro Pharmacokinetic/Pharmacodynamic Model.

Colistin-based combination therapy has become an important strategy to combat the carbapenem-resistant Acinetobacter baumannii (CRAB). However, the optimal dosage regimen selection for the combination with maximum efficacy is challenging. Checkerboard assay was employed to evaluate the synergy of colistin in combination with meropenem, rifampin, fosfomycin, and minocycline against nine carbapenem-resistant A. baumannii isolates (MIC of meropenem [MICMEM], ≥32 mg/liter) isolated from Chinese hospital-acquired pneumonia (HAP) patients. A static time-kill assay, in vitro dynamic pharmacokinetic/pharmacodynamic (PK/PD) model, and semimechanistic PK/PD modeling were conducted to predict and validate the synergistic effect of the most efficacious combination. Both checkerboard and static time-kill assays demonstrated the superior synergistic effect of the colistin-meropenem combination against all CRAB isolates. In the in vitro PK/PD model, the dosage regimen of 2 g meropenem daily via 3-h infusion combined with steady-state 1 mg/liter colistin effectively suppressed the bacterial growth at 24 h with a 2-log10 decrease, compared with the initial inocula against two CRAB isolates. The semimechanistic PK/PD model predicted that more than 2 mg/liter colistin combined with meropenem (2 g, 3-h infusion) was required to achieve the killing below the limit of detection (

MicroRNA-145 regulates immune cytokines via targeting FSCN1 in Staphylococcus aureus-induced mastitis in dairy cows.

Dairy cow mastitis is a detrimental factor in milk quality and food safety. Mastitis generally refers to inflammation caused by infection by pathogenic microorganisms. Our studies in recent years have revealed the role of miRNA regulation in Staphylococcus aureus-induced mastitis. In the present study, we overexpressed and suppressed miR-145 to investigate the function of miR-145 in Mac-T cells. Flow cytometry, ELISA and EdU staining were used to detect changes in the secretion of several Mac-T cytokines and in cell proliferation. We found that overexpression of miR-145 in Mac-T cells significantly reduced the secretion of IL-12 and TNF-α, but increased the secretion of IFN-γ; the proliferation of bovine mammary epithelial cells was also inhibited. Using quantitative real-time PCR (qRT-PCR), Western blotting and luciferase multiplex verification techniques, we found that miR-145 targeted and regulated FSCN1. Knock-down of FSCN1 significantly increased the secretion of IL-12, while the secretion of TNF-α was significantly downregulated in Mac-T cells. Upon S. aureus infection of mammary gland tissue, the body initiated inflammatory responses; Bta-miR-145 expression was downregulated, which reduced the inhibitory effect on the FSCN1 gene; and upregulation of FSCN1 expression promoted mammary epithelial cell proliferation to allow the recovery of damaged tissue. The results of the present study will aid in understanding the immune mechanism opposing S. aureus infection in dairy cows and will provide a laboratory research basis for the prevention and treatment of mastitis.

A Water-Soluble, Green-Light Triggered, and Photo-Calibrated Nitric Oxide Donor for Biological Applications.

Nitric oxide (NO) is a versatile endogenous molecule, involved in various physiological processes and implicated in the progression of many pathological conditions. Therefore, NO donors are valuable tools in NO related basic and applied applications. The traditional spontaneous NO donors are limited in scenarios where flux, localization, and dose of NO could be monitored. This has promoted the development of novel NO donors, whose NO release is not only under control, but also self-calibrated. Herein, we reported a phototriggered and photocalibrated NO donor (NOD565) with an N-nitroso group on a rhodamine dye. NOD565 is nonfluorescent and could release NO efficiently upon irradiation by green light. A bright rhodamine dye is generated as a side-product and its fluorescence can be used to monitor the NO release. The potentials of NOD565 in practical applications are showcased in in vitro studies, e.g., platelet aggregation inhibition and fungi growth suppression.

Role of two 5-aminolevulinic acid biosynthetic pathways in heme and secondary metabolite biosynthesis in Amycolatopsis orientalis.

Analysis of the Amycolatopsis orientalis genome revealed that two genes, hemA1 and hemA2, belonging to divergent pathways, were involved in the biosynthesis of 5-aminolevulinic acid. The roles of hemA1 and hemA2 were elucidated via genetic manipulation and metabolite analysis. The disruption of hemA1, encoding the glutamyl-tRNAGlu reductase of the C5 pathway, was essential for cell growth and is used for heme synthesis. Overexpression of hemA1 resulted in elevated vancomycin and ECO-0501 production in Amycolatopsis orientalis, and it was also effective in increasing the production of daptomycin and natamycin in other Streptomycetes. The disruption of hemA2 indicated that it encodes the 5-aminolevulinic acid synthase of the Shemin pathway, serving as a key enzyme for the synthesis of the precursor aminohydroxycyclopentenone unit of ECO-0501. However, hemA2 disruption could not be complemented by the addition of 5-aminolevulinic acid or by the expression of hemA2 outside of the ECO-0501 gene cluster. The synthesis of ECO-0501 was only restored by the insertion of hemA2 at its original locus. The hemA2 gene could partly complement the hemA1 deficiency. Overexpression of hemA1, a key gene from the heme biosynthetic pathway, is proposed here as a new approach to improve the production of secondary metabolites in bacteria, whereas hemA2 plays different roles depending on its pattern of expression.

Iterative integration of multiple-copy pathway genes in Yarrowia lipolytica for heterologous ß-carotene production.

ß-Carotene is a terpenoid molecule with high hydrophobicity that is often used as an additive in foods and feed. Previous work has demonstrated the heterologous biosynthesis of ß-carotene from an intrinsic high flux of acetyl-CoA in 12 steps through 11 genes in Yarrowia lipolytica. Here, an efficient biosynthetic pathway capable of producing 100-fold more ß-carotene than the baseline construct was generated using strong promoters and multiple gene copies for each of the 12 steps. Using fed-batch fermentation with an optimized medium, the engineered pathway could produce 4g/L ß-carotene, which was stored in lipid droplets within engineered Y. lipolytica cells. Expansion of these cells for squalene production also demonstrated that Y. lipolytica could be an industrially relevant platform for hydrophobic terpenoid production.

Anaerobic biodegradation of spiramycin I and characterization of its new metabolites.

Activated sludge was used to treat the wastewater containing spiramycin I. Three new metabolites were isolated and identified, which produced by oxidation of C6-aldehyde, hydrolysis of C5-mycaminose-mycarose and macrolactone ring-open reaction of spiramycin I in anaerobic digestion. And their antimicrobial activities were inactivated. Our results indicated that anaerobic biodegradation metabolites of spiramycin I could not induce bacterial resistance in environment.

Production of ß-carotene by expressing a heterologous multifunctional carotene synthase in Yarrowia lipolytica.

OBJECTIVES: To obtain functional expression of a heterologous multifunctional carotene synthase containing phytoene synthase, phytoene dehydrogenase, and lycopene ß-cyclase activities encoded by carS from Schizochytrium sp. in order to allow Yarrowia lipolytica to produce ß-carotene. RESULTS: To increase the integration efficiency of a 3.8 kb carS under the control of P GPD promoter with a 2 kb selection marker, ura3, along with a geranylgeranyl diphosphate synthase (GGS1) expression cassette (~10 kb in total), was inserted into the Y. lipolytica chromosome, and the DNA assembler method was combined with double chromosomal deletions of ku70 and ku80. This method resulted in a 13.4-fold increase in integration efficiency compared with the original method, reaching 63% (10/16). The resulting recombinant Y. lipolytica produced 0.41 mg ß-carotene per g dry cell weight, while the wild type did not produce any indicating the functionality of the multifunctional carotene synthase in Y. lipolytica. CONCLUSION: Expression of GGS1 and a multifunctional carotene synthase from Schizochytrium sp. in Y. lipolytica led to ß-carotene production. DNA assembler efficiency was greatly increased by the deletion of ku70 and ku80, which resulted in decreased in vivo nonhomologous end-joining (NHEJ) in Y. lipolytica.

CRISPR/Cas9-based efficient genome editing in Staphylococcus aureus.

Staphylococcus aureus is an important pathogenic bacterium prevalent in nosocomial infections and associated with high morbidity and mortality rates, which arise from the significant pathogenicity and multi-drug resistance. However, the typical genetic manipulation tools used to explore the relevant molecular mechanisms of S. aureus have multiple limitations: leaving a scar in the genome, comparatively low gene-editing efficiency, and prolonged experimental period. Here, we present a single-plasmid based on the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system which allows rapid and efficient chromosomal manipulation in S. aureus. The plasmid carries the cas9 gene under the control of the constitutive promoter Pxyl/tet, a single guide RNA-encoding sequence transcribed via a strong promoter Pspac, and donor DNA used to repair the double strand breaks. The function of the CRISPR/Cas9 vector was demonstrated by deleting the tgt gene and the rocA gene, and by inserting the erm R cassette in S. aureus. This research establishes a CRISPR/Cas9 genome editing tool in S. aureus, which enables marker-free, scarless and rapid genetic manipulation, thus accelerating the study of gene function in S. aureus.

Investigation of halogenation during the biosynthesis of ramoplanin in Actinoplanes sp. ATCC33076.

Ramoplanin and enduracidin are lipopeptide antibiotics effective against Gram-positive pathogens, which share close similarity in structure and biosynthetic pathway. Both compounds have chlorine atoms attached to 4-hydroxyphenylglycine (Hpg) but with different chlorinating sites and levels. Here, to probe the factor affecting the site and level of halogenation, gene inactivation and heterologous expression were carried out in Actinoplanes sp. ATCC33076 by homologous recombination. Metabolite analysis confirmed that ram20 encodes the only halogenase in ramoplanin biosynthetic pathway, and enduracidin halogenase End30 could heterologously complement the ram20-deficient mutant. Additionally, the mannosyltransferase-deficient mutant produces a dichlorinated ramoplanin aglycone with the halogenation site at Hpg(13). This study has refined our understanding of how halogenation occurs in ramoplanin biosynthetic pathway, and lays the foundation for further exploitation of ramoplanin and enduracidin halogenase in combinatorial biosynthesis.

Multiplex gene editing of the Yarrowia lipolytica genome using the CRISPR-Cas9 system.

Yarrowia lipolytica is categorized as a generally recognized as safe (GRAS) organism and is a heavily documented, unconventional yeast that has been widely incorporated into multiple industrial fields to produce valuable biochemicals. This study describes the construction of a CRISPR-Cas9 system for genome editing in Y. lipolytica using a single plasmid (pCAS1yl or pCAS2yl) to transport Cas9 and relevant guide RNA expression cassettes, with or without donor DNA, to target genes. Two Cas9 target genes, TRP1 and PEX10, were repaired by non-homologous end-joining (NHEJ) or homologous recombination, with maximal efficiencies in Y. lipolytica of 85.6 % for the wild-type strain and 94.1 % for the ku70/ku80 double-deficient strain, within 4 days. Simultaneous double and triple multigene editing was achieved with pCAS1yl by NHEJ, with efficiencies of 36.7 or 19.3 %, respectively, and the pCASyl system was successfully expanded to different Y. lipolytica breeding strains. This timesaving method will enable and improve synthetic biology, metabolic engineering and functional genomic studies of Y. lipolytica.