Efficient gene targeting in ligase IV-deficient Monascus ruber M7 by perturbing the non-homologous end joining pathway

Inactivating the non-homologous end joining (NHEJ) pathway is a well established method to increase gene replacement frequency (GRF) in filamentous fungi because NHEJ is predominant for the repair of DNA double strand breaks (DSBs), while gene targeting is based on homologous recombination (HR). DNA ligase IV, a component of the NHEJ system, is strictly required for the NHEJ in Saccharomyces cerevisiae and Neurospora crassa. To enhance the GRF in Monascus ruber M7, we deleted the Mrlig4 gene encoding a homolog of N. crassa DNA ligase IV. The obtained mutant (MrΔlig4) showed no apparent defects in vegetative growth, colony phenotype, microscopic morphology, spore yield, and production of Monascus pigments and citrinin compared with the wild-type strain (M. ruber M7). Gene targeting of ku70 and triA genes revealed that GRF in the MrΔlig4 strain increased four-fold compared with that in the wild-type strain, reached 68 % and 85 %, respectively. Thus, the MrΔlig4 strain is a promising host for efficient genetic manipulation. In addition, the MrΔlig4 strain is more sensitive than M. ruber M7 to a DNA-damaging agent, methyl methanesulfonate.     

Enhanced gene targeting frequency in ku70 and ku80 disruption mutants of Aspergillus sojae and Aspergillus oryzae

In the koji molds Aspergillus sojae and Aspergillus oryzae, exogenous DNA is integrated in the genome, in most cases irrespective of the sequence homology, suggesting that DNA integration occurs predominantly through a nonhomologous end joining pathway where two ku genes, namely, ku70 and ku80, play a key role. To determine the effect of ku gene disruption on the gene targeting frequency, we constructed ku70-, ku80-, and ku70–ku80-disrupted strains of A. sojae and A. oryzae. The gene targeting frequency of the tannase gene in ku70 and ku80 strains of both Aspergillus species was markedly enhanced as compared with that of the parental strains. The gene targeting frequency of the aflR and ku80 genes was also enhanced in an A. sojae ku70 background. Therefore, the koji mold strains with ku-disrupted genes will be excellent tools as hosts for efficient gene targeting.     

MpigE, a gene involved in pigment biosynthesis in Monascus ruber M7

Monascus pigments (MPs) have been used as food colorants for several centuries in Asian countries. However, MP biosynthesis pathway is still a controversy, and only few related genes have been reported. In this study, the function of MpigE, a gene involved in MP biosynthesis in Monascus ruber M7, was analyzed. The results revealed that the disruption, complementation, and overexpression of MpigE in M. ruber M7 had very little effects on the growth and phenotypes except MPs. The MpigE deletion strain (?MpigE) just yielded four kinds of yellow MPs and very little red pigments, while the wild-type strain M. ruber M7 produced a MP complex mixture including three (orange, red, and yellow) categories of MP compounds. Two of the four yellow MPs produced by ?MpigE were the same as those yielded by M. ruber M7. The MpigE complementation strain (?MpigE::MpigE) recovered the ability to generate orange and red MPs as M. ruber M7. The MP types produced by the MpigE overexpression strain (M7::PtrpC-MpigE) were consistent with those of M. ruber M7, while the color value was about 1.3-fold as that of M. ruber M7 (3,129 U/g red kojic). For the production of citrinin, the disruption of MpigE almost had no influence on the strain, whereas the overexpression of MpigE made citrinin decrease drastically in YES fermentation. This work will make a contribution to the study on the biosynthesis pathway of MPs in M. ruber.     

Deletion of ku homologs increases gene targeting frequency in Streptomyces avermitilis

Streptomyces avermitilis is an industrially important soil bacterium known for production of avermectins, which are antiparasitic agents useful in animal health care, agriculture, and treatment of human infections. ku genes play a key role in the non-homologous end-joining pathway for repair of DNA double strand breaks. We identified homologs of eukaryotic ku70 and ku80 genes, termed ku1 and ku2, in S. avermitilis. Mutants with deletion of ku1, ku2, and both genes were constructed and their phenotypic changes were characterized. Deletion of ku genes had no apparent adverse effects on growth, spore formation, or avermectin production. The ku mutants, in comparison to wild-type strain, were slightly more sensitive to the DNA-damaging agent ethyl methanesulfonate, but not to UV exposure or to bleomycin. Gene targeting frequencies by homologous recombination were higher in the ku mutants than in wild-type strain. We conclude that ku-deleted strains will be useful hosts for efficient gene targeting and will facilitate functional analysis of genes in S. avermitilis and other industrially important bacterial strains.     

Efficient homologous recombination with short length flanking fragments in Ku70 deficient Yarrowia lipolytica strains

In Yarrowia lipolytica, targeted gene replacement occurs only with long length (1 kb) homologous flanking fragments, as this yeast preferentially uses the non-homologous end-joining mechanism (NHEJ) for DNA repair over homologous recombination (HR). To improve the frequency of HR, we identified and disrupted the KU70 and KU80 genes responsible for double strand break repair in the NHEJ pathway in Y. lipolytica. In ku70? HR of URA3 marker at the ADE2 locus occurred with 43 % frequency with as little as 50 bp long flanking regions. The number of Ura⁺ transformants was reduced to 1 % of the Po1d (ura3-302) wild type-like strain level, regardless of the flanking fragment length. On the contrary, even though HR was not improved in ku80?, Ura⁺ transformants was 60 % lower compared to the wild type.     

A highly efficient gene-targeting system for Aspergillus parasiticus

Aims: To establish a system that greatly increases the gene‐targeting frequency in Aspergillus parasiticus. Methods and Results: The ku70 gene, a gene of the nonhomologous end‐joining (NHEJ) pathway, was replaced by the nitrate reductase gene (niaD) in A. parasiticus RHN1 that accumulates O‐methylsterigmatocystin (OMST). The NHEJ‐deficient strain, RHΔku70, produced conidia, sclerotia and OMST normally. It had identical sensitivity as RHN1 to the DNA‐topoisomerase I complex inhibitor, camptothecin, and the DNA‐damaging agent, melphalan. For targeting an aflatoxin biosynthetic pathway gene, adhA, partial restriction enzyme recognition sequences in its flanking regions were manipulated to create homologous ends for integration. Using a linearized DNA fragment that contained Aspergillus oryzae pyrithiamine resistance gene (ptr) marker the adhA‐targeting frequency in RHΔku70 reached 96%. Conclusions: The homologous recombination pathway is primarily responsible for repair of DNA damages in A. parasiticus. The NHEJ‐deficient RHΔku70, easy creation of homologous ends for integration, and the ptr‐based selection form a highly efficient gene‐targeting system. It substantially reduces the time and workload necessary to obtain knockout strains for functional studies. Significance and Impact of the Study: The developed system not only streamlines targeted gene replacement and disruption but also can be used to target specific chromosomal locations like promoters or intergenic regions. It will expedite the progresses in the functional genomic studies of A. parasiticus and Aspergilllus flavus.     

Deletion of a KU80 homolog enhances homologous recombination in the thermotolerant yeast Kluyveromyces marxianus

Targeted gene replacement in the thermotolerant yeast Kluyveromyces marxianus KCTC 17555 has been hampered by its propensity to non-homologous end joining (NHEJ). To enhance homologous recombination (HR) by blocking NHEJ, we identified and disrupted the K. marxianus KU80 gene. The ku80 deletion mutant strain (Kmku80?) of K. marxianus KCTC 17555 did not show apparent growth defects under several conditions with the exception of exposure to tunicamycin. The targeted disruption of the three model genes, KmLEU2, KmPDC1, and KmPDC5, was increased by 13–70 % in Kmku80?, although the efficiency was greatly affected by the length of the homologous flanking fragments. In contrast, the double HR frequency was 0–13.7 % in the wild-type strain even with flanking fragments 1 kb long. Therefore, Kmku80? promises to be a useful recipient strain for targeted gene manipulation.     

Efficient gene targeting in a Candida guilliermondii non-homologous end-joining pathway-deficient strain

The yeast, Candida guilliermondii, has been widely studied due to its biotechnological interest as well as its biological control potential. It integrates foreign DNA predominantly via ectopic events, likely through the well-known non-homologous end-joining (NHEJ) pathway involving the Ku70p/Ku80p heterodimer, Lig4p, Nej1p and Lif1p. This phenomenon remains highly deleterious for targeted gene knock-out strategies that require the homologous recombination process. Here, we have constructed a ku70 mutant strain derived from the ATCC 6260 reference strain of C. guilliermondii. Following a series of disruption attempts of various genes (FCY1, ADE2 and TRP5), using several previously described dominant selectable markers (URA5, SAT-1 and HPH ^# ), we demonstrated that the efficiencies of homologous gene targeting in such a NHEJ-deficient strain was very high compared to the wild type strain. The C. guilliermondii ku70 deficient mutant thus represents a powerful recipient strain to knock-out genes efficiently in this yeast.     

High efficient gene targeting on the AGAMOUS gene in an ArabidopsisAtLIG4 mutant

Gene targeting induced by homologous integration of a foreign DNA segment into a chromosomal target sequence enables precise disruption or replacement of genes of interest and provides an effective means to analyze gene function, and also becomes an useful technique for breeding. But, integration of introduced DNA fragments is predominantly non-homologous in most species. However, we presented high-efficient homologous integration in disruptants of non-homologous end joining (NHEJ), that is, the Ku70-, Ku80- or Lig4-homologs deficient strain, in a model fungus Neurospora crassa. When the effect of NHEJ-defective plants for gene targeting was therefore examined in a model plant Arabidopsis (Arabidopsis thaliana), the efficiencies of gene targeting in the Atlig4/Atlig4 plant were 2/7 (28.6%) against calli obtained a selection-marker gene, 2/16 (12.5%) against selected calli, and about 2/540 (0.004%) against total cell particles at the starting point for transformation. The results of this paper show that the NHEJ-deficient system might cause a decrease in the efficiency of transformation but gives true targeted transformants with high efficiency in plant cell.     

Mitogen-activated protein kinase-defective Candida albicans is avirulent in a novel model of localized murine candidiasis

Candida albicans strains with a deletion of the mitogen-activated protein kinase CEK1 gene are defective in the yeast to hyphal transition on solid surfaces in vitro. The virulence of a cek1Δ/cek1Δ null mutant strain was compared with its wild-type parent strain (WT) in a novel model of localized candidiasis. The mammary glands of lactating mice (at day 5 postpartum) were infected for 2, 4 and 6 days with 50 μl suspension containing 1×10⁵, 1×10⁶ and 1×10⁷ blastospores before death. Infected and non-infected control glands were evaluated pathologically. All animals infected with cek1Δ/cek1Δ null mutant strains showed no lesions while 65% of animals infected with the WT strain had severe lesions characterized by widespread heterophilic infiltration, necrosis, and abscess formation. As an additional control, animals infected with the disrupted strain complemented with the WT CEK1, on a replicating plasmid, also showed severe pathological changes similar to the WT strain. These results clearly demonstrate that the CEK1 gene codes for a virulence determinant of C. albicans and that the mouse mastitis model is well suited for the discriminative study of the pathogenicity of different C. albicans strains.     

Ku80 gene is related to non-homologous end-joining and genome stability in Aspergillus niger

In this study, the ku70 and ku80 homologs from the Aspergillus niger genome were identified and their function was analyzed using targeted mutagenesis. The role of the ku80 gene in non-homologous end-joining (NHEJ) was investigated by calculating the frequency of homologous recombination. The transformation test verified that the frequency of homologous recombination significantly increased, from 1.78 to 65.6% in ku80 single deletion strains and to 100% in ku70/ku80 double deletion strains. These results suggest that the ku80 gene is important for non-homologous end-joining. Although the morphology of the ku deletion strains colonies was similar to that of the wildtype strain, mutants were more sensitive to the mutagen phleomycin. Furthermore, the purified ku80 deletion strain produced some sectored colonies on hygromycin B-containing plates. This result suggests that the ku80 gene deletion leads to genomic instability in A. niger.     

Secretion expression of SOD1 and its overlapping function with GSH in brewing yeast strain for better flavor and anti-aging ability

Superoxide dismutase (SOD) is a significant antioxidant, but unlike glutathione (GSH), SOD cannot be secreted into beer by yeast cells during fermentation, this directly leads to the limited application of SOD in beer anti-aging. In this investigation, we constructed the SOD1 secretion cassette in which strong promoter PGK1p and the sequence of secreting signal factor from Saccharomyces cerevisiae were both harbored to the upstream of coding sequence of SOD1 gene, as a result, the obtained strains carrying this cassette successfully realized the secretion of SOD1. In order to overcome the limitation of previous genetic modification on yeast strains, one new comprehensive strategy was adopted targeting the suitable homologous sites by gene deletion and SOD1 + GSH1 co-overexpression, and the new strain ST31 (Δadh2::SOD1 + Δilv2::GSH1) was constructed. The results of the pilot-scale fermentation showed that the diacetyl content of ST31 was lower by 42 % than that of the host, and the acetaldehyde content decreased by 29 %, the GSH content in the fermenting liquor of ST31 increased by 29 % compared with the host. Both SOD activity test and the positive and negative staining assay after native PAGE indicated that the secreted active SOD in the fermenting liquor of ST31 was mainly a dimer with the size of 32,500 Da. The anti-aging indexes such as the thiobarbituric acid and the resistance staling value further proved that the flavor stability of the beer brewed with strain ST31 was not only better than that of the original strain, but also better than that of the previous engineering strains. The multi-modification and comprehensive improvement of the beer yeast strain would greatly enhance beer quality than ever, and the self-cloning strain would be attractive to the public due to its bio-safety.     

Multiplex gene editing of the <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> 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.     

Haloplanus litoreus sp. nov. and Haloplanus ruber sp. nov., from a marine solar saltern and an aquaculture farm,respectively

Two halophilic archaea, strains GX21^T and R35^T, were isolated from a marine solar saltern and an aquaculture farm in China, respectively. Cells of the two strains were observed to be pleomorphic, flat, to contain gas vesicles, stain Gram-negative and produce red-pigmented colonies. Strain GX21^T was found to be able to grow at 25–50 °C (optimum 37 °C), at 2.6–4.8 M NaCl (optimum 3.4 M NaCl), at 0.05–1.0 M MgCl₂ (optimum 0.1 M MgCl₂) and at pH 6.0–8.5 (optimum pH 6.5) while strain R35^T was found to be able to grow at 25–45 °C (optimum 37 °C), at 2.1–4.8 M NaCl (optimum 3.1 M NaCl), at 0–0.7 M MgCl₂ (optimum 0.03 M MgCl₂) and at pH 5.5–9.5 (optimum pH 6.5–7.0). The cells of both isolates were observed to lyse in distilled water. The minimum NaCl concentrations that prevented cell lysis were determined to be 15 % (w/v) for strain GX21^T and 12 % (w/v) for strain R35^T. The major polar lipids of the two strains were identified as phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate, one major glycolipid and a minor lipid chromatographically identical to sulfated mannosyl glucosyl diether and mannosyl glucosyl diether, respectively. 16S rRNA gene sequence analysis revealed that strains GX21^T and R35^T show 97.1 % sequence similarity to each other and are closely related to Haloplanus aerogenes TBN37^T (96.8 and 95.8 %), Haloplanus vescus RO5-8^T (96.7 and 96.1 %), Haloplanus salinus YGH66^T (96.4 and 95.8 %) and Haloplanus natans JCM 14081^T (96.3 and 95.4 %). The rpoB′ gene similarity between strains GX21^T and R35^T is 90.5 % and show 88.5–90.8 % similarity to the Haloplanus species with validly published names. The DNA G+C content of strain GX21^T and R35^T were determined to be 65.8 and 66.0 mol%, respectively. The DNA–DNA hybridization values between strain GX21^T and strain R35^T, and the two strains with the Haloplanus species with validly published names, showed less than 50 % DNA–DNA relatedness. It was concluded that strain GX21^T (=CGMCC 1.10456^T = JCM 17092^T) and strain R35^T (=CGMCC 1.10594 ^T = JCM 17271^T) represent two new species of Haloplanus, for which the names Haloplanus litoreus sp. nov. and Haloplanus ruber sp. nov. are proposed.     

Cellular senescence and organismal ageing in the absence of p21CIP1/WAF1 in ku80−/− mice

Ku80 is important in the repair of DNA double-strand breaks by its essential function in non-homologous end-joining. The absence of Ku80 causes the accumulation of DNA damage and leads to premature ageing in mice. We showed that mouse embryonic fibroblasts (MEFs) from ku80^−/− mice senesced rapidly with elevated levels of p53 and p21. Deletion of p21 delayed the early senescence phenotype in ku80^−/− MEFs, despite an otherwise intact response of p53. In contrast to ku80^−/−p53^−/− mice, which die rapidly primarily from lymphomas, there was no significant increase in tumorigenesis in ku80^−/−p21^−/− mice. However, ku80^−/−p21^−/− mice showed no improvement with respect to rough fur coat or osteopaenia, and even showed a shortened lifespan compared with ku80^−/− mice. These results show that the increased lifespan of ku80^−/− MEFs owing to the loss of p21 is not associated with an improvement of the premature ageing phenotypes of ku80^−/− mice observed at the organismal level.     

Further enhanced production of heterologous proteins by double-gene disruption (ΔAosedD ΔAovps10) in a hyper-producing mutant of Aspergillus oryzae

The filamentous fungus Aspergillus oryzae is used as one of the most favored hosts for heterologous protein production due to its ability to secrete large amounts of proteins into the culture medium. We previously generated a hyper-producing mutant strain of A. oryzae, AUT1, which produced 3.2- and 2.6-fold higher levels of bovine chymosin (CHY) and human lysozyme (HLY), respectively, compared with the wild-type strain. However, further enhancement of heterologous protein production by multiple gene disruption is difficult because of the low gene-targeting efficiency in strain AUT1. Here, we disrupted the ligD gene, which is involved in nonhomologous recombination, and the pyrG gene to create uridine/uracil auxotrophy in strain AUT1, to generate a hyper-producing mutant applicable to pyrG marker recycling with highly efficient gene targeting. We generated single and double disruptants of the tripeptidyl peptidase gene AosedD and vacuolar sorting receptor gene Aovps10 in the hyper-producing mutant background, and found that all disruptants showed significant increases in heterologous protein production. Particularly, double disruption of the Aovps10 and AosedD genes increased the production levels of CHY and HLY by 1.6- and 2.1-fold, respectively, compared with the parental strain. Thus, we successfully generated a fungal host for further enhancing the heterologous protein production ability by combining mutational and molecular breeding techniques.     

Roles of the three Mycobacterium smegmatis katG genes for peroxide detoxification and isoniazid susceptibility

Three different katG sequences (katGI, katGII and katGIII) were identified in the Mycobacterium smegmatis genome. The contributions of the three katG genes to survival of the bacterium were examined by constructing disruptants of these three genes. The katGIII sequence did not produce a functional catalase‐peroxidase. Analyses of peroxidase activity and mRNA expression revealed that in wild type M. smegmatis, expression dominance between KatGI and KatGII was switched in the exponential and stationary growth phases. Susceptibility of the M. smegmatis gene disruptants to hydrogen peroxide (H₂O₂) was tested in two growth phases. In the exponential phase, the katGI‐null strain was more susceptible to H₂O₂ than the katGII‐null strain, indicating that KatGI plays a more important role in survival than KatGII in this growth phase. In contrast, in the stationary phase, growth of the katGII‐null strain was inhibited at lower concentrations of H₂O₂. These results suggest that M. smegmatis has two types of catalase‐peroxidases, expressions of which are controlled under different gene regulatory systems. Isoniazid (INH) susceptibilities of the katG‐null strains were also examined and it was found that katGI is a major determinant of M. smegmatis susceptibility to INH.