Reducing Yield of Fusel Alcohols by Saccharomyces cerevisiae of Compound Mutagenesis Through UV-MPMS Method

Excessive fusel alcohol contents will cause the beer to produce off-flavors and cause dizziness and headaches.Reducing the contents of fusel alcohols in beer is very important to people's health.The excessive fusel alcohol contents in beer is a common problem in the industry.How to control the contents of fusel alcohols in a reasonable range is of great significance for improving beer quality.After one round of ultraviolet(UV)and one round of multifunctional plasma mutagenesis system(MPMS)mutagenesis,the yeast strains with lower fusel oil yield and more stablility could be screened.According to the relationship between the fusel alcohol Harris metabolic pathway of brewer's yeast and lactic acid metabolism,excellent strains were obtained by triple screening with lactic acid medium,calcium carbonate medium and 2,3,5-triphenyl tetrazolium chloride upper medium.The content of fusel alcohol in the finished beer fermentation test of screened strain Z43 was 52.1±0.142 mg•L^(-1),which was 43%lower than that of the starting strain,and other fermentation properties remained unchanged.After eight passages,it was verified that the strain was stable and heritable.These results showed that strain Z43 presented promising characteristics for use in the production of beer with a potentially low contents of fusel alcohols.

Screening for the Strain Highly Producing Antagonistic Substance from Bacillus subtilis B47 by UV Mutagenesis

[Objective] The aim of this study was to breed new strains which have higher inhibitory effects on the pathogens of watermelon fusarium wilt.[Method] The endophytic Bacillus subtilis B47 strain was obtained from tomato stems by UV mutagenesis for two consecutive times,then genetic stability as well as physiological and biochemical properties of mutant strains were studied.[Result] The antibacterial activity of all the three mutant strains F303,F304 and F305 was higher than that of B74 strain.After subculture of 10 successive generations,the antibacterial activity of all the three mutant strains for the pathogens of watermelon fusarium wilt decreased,but the antibacterial activity of F305 strain decreased the least,indicating its best genetic stability among the tested strains.The antibacterial circle diameter of F305 strain was 5 mm larger than that of wild strain B47 under the same condition.The mutant strain F305 was in logarithmic growth phase within 36 h and in stationary phase within 36-96 h,while its optimum growth temperature was 35 ℃.F305 strain could grow in sodium salt with the concentration of 1%-10%,but it grew best at the concentration of 1%.Physiological and biochemical responses of F305 strain were in accordance with those of wild strain B47.[Conclusion] This study lays the foundation for the factorial production of antagonistic substance by B47 strain and new methods of preventing from the pathogens watermelon fusarium wilt.

Screening for a Novel Trichoderma vride Strain Highly Producing Cellulase via Ultraviolet Mutagenesis

[Objective] The aim of this study was to find out a new Trichoderma vride K strain highly producing cellulase.[Method] Ultraviolet（UV） was used to induce mutagenesis on T.vride K and to select out a new Trichoderma vride strain highly producing cellulase from the first round and further selection.[Result] A new T.vride strain K6 with high yield of cellulase was obtained with the enzyme production amount of 1.39 times over that of starting strain K.This strain showed highest cellulase yield under the culture condition of 28 ℃ for 96 h.[Conclusion] The strain K6 selected out from induced mutation is endowed with better capacity of producing cellulase,which provides a new method for the utilization of straw.

Construction of Mutant Population and Analysis of Dwarf Mutants in "6421"(Capsicum annuum L.)through EMS Mutagenesis

[Objective] The aim was to improve the genetic property of peppers, the mutant population of Capsicum annuum L cultivar ＂6421＂ was constructed. [Method] The seeds of ＂6421＂ were treated with 0.2% to 1.2% ethyl methane sulfonate to identified LD50, and then 10 000 LD^o of treated seeds were sowed to construct mutant population. The agronomic characters and genetic regularity of dwarf mutants in M4 generation were analyzed. [Result] Our results showed that GR and SSR were 45.2% and 40.2% respectively at 1.0% EMS, close to LD50, with GI （17.6） and seed Ⅵ （19.7） being half of that of control; 562 M4 mutants were identified in 2015, and the mutation could be characterized according 11 major categories and 32 subcategories; Simultaneously, we found that plant height, plant width, diameter of mainstem, length of main-stem, the number of main-stem nodes and branch of lines E29, E58, E142 and E312 were all significantly different from that of the control. The mutation of lines E29, E58 and E312 was all controlled by a single recessive gene. [Conclusionl The study first created a pepper mutant population, which provides not only the germplasm resources for further breeding but also direct and effective materials for genomic study of the pepper.

A Simple and Easy Method for Site-specific Mutagenesis Using Long-distance Inverse PCR in the Presence of Pfu-DNA Polymerase

Site_specific mutagenesis has been widely used in molecular biology and biochemistry. The authors have developed a simple and easy method for site_specific mutagenesis of any genes on plasmids using long distance inverse PCR in the presence of Pfu_DNA polymerase. The efficiency of this method is higher than 90% and the entire procedure can be performed just in one tube. No subcloning is needed. This method is especially useful for obtaining mutant genes on large plasmids such as Ti plasmids used for plant transformation.

Site-directed Mutagenesis of Streptomyces avermitilis aveD Gene

[Objective] The aim of this study was to produce Streptomyces avermitilis strain with site-directed mutagenesis in aveD gene,and so as to provide theoretical basis for genetic breeding of S.avermitilis.[Method] PCR-driven overlap extension was conducted for the site-directed mutagenesis in aveD gene;the mutated aveD gene then was used to construct vector pDC3（pKC1139∷aveD） via molecular manipulations like in vitro enzyme digestion and ligation;the vector pDC3（pKC1139∷aveD） was then introduced to aveD deletion mutant 489 of avermectin-producing strain S.avermitilis 76-9.[Result] Mutant strain 536 of site-directed mutagenesis of S.avermitilis 76-9 was obtained by homologous recombination.The sequencing results show that the sixty-ninth base C in aveD-coding region of mutant 536 was substituted by T,and the corresponding amino acid Thr was mutated to be Ile.[Conclusion] This study laid basis for the development of strains specifically producing avermectin B.

Site-directed Mutagenesis Based on Overlap Extension PCR

[Objective] To establish an efficient, convenient and economical method for site-directed mutagenesis. [Method] The target mutation was introduced into primers designed by DNAMAN5.0 software. Through overlap extension PCR for twice obtained the mutation gene which of the full length of the recombinant Human Tissue type plasminogen activator （Reteplase）. The mutation gene cloned it into pEASY- blunt simple cloning vector for sequencing. [Result] The sequencing results showed that three site mutations were fully consistent with the expected results （10~ site had been added a base-pair of A, C had been changed into G at 137~ site, G had been changed into A at 686~ site）.Three site mutations were introduced by using overlap extension PCR on one-step. The overall rate of obtaining the mutant sites was 100%. Site-directed mutagenesis will clone the recombinant Human Tissue type plas- minogen activator and laid the basis for the functional study. [Conclusion] Site-directed mutagenesis was successfully implemented based on the overlap extension PCR which is an efficient, convenient and economical DNA-directed mutagenesis method.

Effects of Mutagenesis by UV Irradiation and ^(60)Co-γ Irradiation on Fermentation of Xylose to Ethanol by Pichia stipitis

ln this study, effects of UV irradiation and 60Co-γ irradiation on fermenta-tion of xylose to ethanol by Pichia stipitis were analyzed to investigate the optimal mutagenic conditions. According to the growth curve and fermentation curve of P. stipi-tis, the optimal incubation duration and fermentation duration of P. stipitis mutant strain were 18 and 48 h, respectively. The cel concentration of original yeast liquid was 107 cel s/ml. After mutagenesis by UV irradiation and 60Co-γ irradiation, yeast liquid was incubated in 20 g/L xylose media for 48 h. According to the results, after UV irradiation for 45-75 s, transformation efficiency reached 0.3794 g/g, which was 74.39% of the theoretical value; after irradiation with 800-1 000 Gy 60Co-γ, transforma-tion efficiency reached 0.3165 g/g, which was 62.06% of the theoretical value. Therefore, both UV irradiation and 60Co-γ irradiation could improve the efficiency of xylose fermentation to ethanol by P. stipitis under appropriate conditions.

A Non-Marker Mutagenesis Strategy to Generate Poly-hrp Gene Mutants in the Rice Pathogen Xanthomonas oryzae pv. oryzicola

Xanthomonas oryzae pv.oryzicola （Xoc）,the critical pathogen causing bacterial leaf streak in rice,possesses a hrp cluster that is responsible for triggering hypersensitive response （HR） in non-host tobacco and pathogenicity in host rice,and is considered to be one of the model pathogens in the rice model plant.Here,we developed a high-throughput mutagenesis system using a two-step integration mediated by a novel suicide vector pKMS1.It was used to generate single or poly-gene mutants of hpa1,hpa2,hrcV,hrpE,hpaB,and hrpF gene for functional analysis.In total,five single,four double,and two triple hrp gene mutants were constructed.The double and triple hrp gene deletion mutants triggered novel phenotypes in planta.Our data suggest that pKMS1 is a useful tool for non-marker mutagenesis of multiple genes in Xoc.

Effect of ultraviolet mutagenesis on heterotrophic strain mutation and bioleaching of low grade copper ore

The effect of ultraviolet mutagenesis on a heterotrophic strain(Providencia JAT-1) mutation was studied and bioleaching of low grade copper ore with mutant bacteria was investigated. The results show that the activity of bacteria was improved after ultraviolet mutagenesis; the best irradiation time was 120 s. Compared to the original bacteria, the cells density of mutant bacteria at stationary phase increased by 26% and ammonia produced by mutant bacteria increased by 12%. Higher activity of bacteria leads to a higher copper extraction rate. The bioleaching performance of Providencia JAT-1 was improved after UV mutagenesis. The copper extraction rate with mutant bacteria increased by 10.6% compared to the original bacteria. The ore surface was corroded and the fine particles were absent after bioleaching. Free copper oxide and copper silicates could be leached out easily by using JAT-1; a small part of the copper sulfide can also be leached out. Bioleaching using JAT-1 is more effective than ammonia leaching and copper extraction rate with mutant bacteria was 21.1% higher than that by ammonia leaching under the same condition.

Determination of Endophytic Bacteria Resistance against Rifampin and UV-mutagenesis

The study aimed to isolate and screen endophytic bacteria from the plant in order to understand their colonization dynamics in plants. [Method] 5 kinds of endophytic bacteda including H1, DP1, CJL1, DJL12 and YC1 were selected as the original strains, and conducted UV mutagenesis studies on H1 and DJL12 with weak resistance, while the resistance of them against rifampicin was confirmed. [Result] Through preliminary screening, five kinds of endophytic bacteria could grew well in the plate without rifampin, among them, H1 and DLJ12 were unable to survive under 10 μg/ml concentration of rifampicin, and the other three strains could survive under 50 μg/ml concentration of rifampicin. After UV mutagenesis, DLJ12 strain with dfampicin resistance concentration of 80 μg/ml was obtained. [ Conclusion] The study could provide theoretical and practical basis for development and utilization of endophytic bacteria.

Increased L-arginine Production by Site-directed Mutagenesis of N-acetyl-L-glutamate Kinase and pro B Gene Deletion in Corynebacterium crenatum

Objective In Corynebacterium crenatum,the adjacent D311 and D312 of N-acetyl-L-glutamate kinase(NAGK),as a key rate-limiting enzyme of L-arginine biosynthesis under substrate regulatory control by arginine,were initially replaced with two arginine residues to investigate the L-arginine feedback inhibition for NAGK.Methods NAGK enzyme expression was evaluated using a plasmid-based method.Homologous recombination was employed to eliminate the pro B.Results The IC50 and enzyme activity of NAGK M4,in which the D311 R and D312 R amino acid substitutions were combined with the previously reported E19 R and H26 E substitutions,were 3.7-fold and 14.6% higher,respectively,than those of the wild-type NAGK.NAGK M4 was successfully introduced into the C.crenatum MT genome without any genetic markers;the L-arginine yield of C.crenatum MT-M4 was 26.2% higher than that of C.crenatum MT.To further improve upon the L-arginine yield,we constructed the mutant C.crenatum MT-M4 ?pro B.The optimum concentration of L-proline was also investigated in order to determine its contribution to L-arginine yield.After L-proline was added to the medium at 10 mmol/L,the L-arginine yield reached 16.5 g/L after 108 h of shake-flask fermentation,approximately 70.1% higher than the yield attained using C.crenatum MT.Conclusion Feedback inhibition of L-arginine on NAGK in C.crenatum is clearly alleviated by the M4 mutation of NAGK,and deletion of the pro B in C.crenatum from MT to M4 results in a significant increase in arginine production.

Isolation of Trichoderma reesei pyrG Negative Mutant by UV Mutagenesis and Its Application in Transformation

Two uridine auxotrophic mutants of Trichoderma reesei were isolated by resistance to 5-fluoroorotic acid after UV mutagenesis. One mutant, called M23, was complemented with the Aspergillus niger pyrG gene carried by plasmid pAB4-1. A mutated pyrG gene of M23 was cloned and DNA sequencing analysis indicated that a cytosine was inserted into the 934―939 oligo dC position of the pyrG coding region, resulted in a frameshift mutation. Transformation efficiency was approximately 200―300 transformants per microgram of DNA with plasmid pAB4-1. Stable transformants were obtained by monosporic culture and showed to be prototroph after successive propagation. Vitreoscilla hemoglobin expression plasmid pUCVHb was cotransformed with plasmid pAB4-1 and attained a transformation efficiency of 71.8% or of 26.1% with pAN7-1. Southern blot analysis of the transformants demonstrated that plasmid pUCVHb was integrated into the chromosomal DNA. The experimental results demonstrated that the pyrG-based system was more efficient and timesaving than the conventional hygromycin B resistance-based transformation system.

Radiation-induced in vitro mutagenesis system for salt tolerance and other agronomic characters in sugarcane(Saccharum officinarum L.)

Gamma ray-induced in vitro mutagenesis and selection for salt(NaC l) tolerance were investigated in sugarcane(Saccharum officinarum L.). Embryogenic callus cultures were irradiated(10 to 80 Gy) and subjected to in vitro selection by exposure of irradiated callus to NaC l(0, 50, 100,150, 200, and 250 mmol L-1). Increasing NaC l concentrations resulted in growth reduction and increased membrane damage. Salt-selected callus lines were characterized by the accumulation of proline, glycine betaine, and Na+and K+concentration. Higher accumulation of proline and glycine betaine was observed in NaC l stressed callus irradiated at 20 Gy. Na+concentration increased and K+concentration decreased with increasing salt level. Irradiated callus showed50–60% regeneration under NaC l stress, and in vitro-regenerated plants were acclimatized in the greenhouse, with 80–85% survival. A total of 138 irradiated and salt-selected selections were grown to maturity and their agronomic performance was evaluated under normal and saline conditions. Of these, 18 mutant clones were characterized for different agro-morphological characters and some of the mutant clones exhibited improved sugar yield with increased Brix%,number of millable canes, and yield. The result suggest that radiation-induced mutagenesis offers an effective way to enhance genetic variation in sugarcane.

Ethyl Methane Sulphonate (EMS) Induced Mutagenesis in Malaysian Rice (cv. MR219) for Lethal Dose Determination

Chemical and physical mutagenesis has been used to increase genetic variability in crop plants. More than 430 new varieties have been derived as mutants of rice (Oryza sativa L.) via the application of different mutagenic agents. Chemical mutagens such as ethyl methane sulphonate (EMS), diepoxybutane-derived (DEB), sodium azide and irradiation (Gamma rays, X-rays and fast neutrons) have been widely used to induce a large number of functional variations in rice and others crops. Among chemical mutagens, the alkylating agent, ethyl methane sulfonate (EMS) is the most commonly used in plants as it causes a high frequency of nucleotide substitutions, as detected in different genomes. In this study, seeds of potential genotype of the popular variety, (Oryza sativa L. spp. Indica cv. MR219) were treated with EMS at concentrations of 0.25%, 0.50%, 0.75%, 1%, 1.25%, 1.5% and 2%. Sensitivity to EMS was determined by various measurements on the M1 generation. As concentration of applied EMS increased, will decrease in germination, seedling height, root length and emergence under field conditions was observed in M1 generation as compared to the non-treatment control. Plant height and root length also decreased with increases in EMS mutagenesis in an approximately linear fashion. The LD25 and LD50 values were observed based on growth reduction of seedlings after EMS treatment with 0.25% and 0.50% on the rice variety (Oryza sativa L. spp. Indica cv. MR219).

Screening of Prolific Micromonospora carbonacea in Antibiotics Production by Sodium Nitrite Mutagenesis

[Objective] The purpose of the study is to breed Micromonospora car- bonacea highly producing antibiotics and then to improve the antibiotic production. [Method] Sodium Nitrite mutagenesis, combined with rifampicin resistance screening, was used in mutation breeding of M. carbonacea highly producing antibiotics from the strain of M. carbonacea JXNU-I. [Result] The overproducing strain JXNU-1-16- Y65 was screened with the production of antibiotics 266.05% more than that of the original strain. [Conclusion] The effectiveness of sodium nitrite mutation in breeding microorganisms highly producing antibiotic was proved, and the study may lay the foundation on further development and application of the antibiotic from M. car- bonacea JXNU-1.

Quantitative evaluation of DNA damage caused by atmospheric and room-temperature plasma (ARTP) and other mutagenesis methods using a rapid umu-microplate test protocol for microbial mutation breeding

Mutagenesis is an important technique for microbial mutation breeding.As the source of mutations,DNA damage extent is a key indicator for the effectiveness of mutagenesis.Therefore,a rapid and easy DNA damage quantification method is required for the comparison of mutagenesis effects and development of mutagenesis tools.Here,we used the umu-microplate test system to quantitatively compare the DNA damage strength caused by atmospheric and room-temperature plasma(ARTP)and other traditional mutagenesis methods including:ultraviolet radiation(UV),diethyl sulfate(DES)and 4-nitroquinoline-1-oxide(4-NQO).The test strain of Salmonella typhimurium TA1535/pSK1002 was used to monitor the time-course profile of b-galactosidase activity induced by DNA damage caused by different mutagenesis methods using a microplate reader.The umu-microplate test results showed that ARTP caused higher extent of DNA damage than UV and chemical mutagens,which agrees well with the result obtained by SOS-FACS-based quantification method as reported previously.This umu-microplate test is accessible for broad researchers who are lack of the expensive FACS instruments and allows the quick quantitative evaluation of DNA damage among living cells for different mutagenesis methods in the study of the microbial mutation breeding.

Transposon mutagenesis of Psychrobacter cryohalolentis PAMC 21807 by tri-parental conjugation

Random mutagenesis is commonly used to study gene function. The screening of mutants exhibiting specific pheno- types assists in the identification of phenotype-related genes. In the current study, we isolated Antarctic bacteria, and developed a transposon Tn5 mutagenesis system. A total of 26 strains were isolated from seawater and freshwater near Antarctic King Sejong Research Station, King George Island. Six Psychrobacter strains were identified as psychrophilic, with optimal growth tempera- tures of 10~C or 15~C Psychrobacter cryohalolentis PAMC 21807 with a high growth rate at 4~C was selected for transposon mutagenesis. Tri-parental conjugation with a plasmid containing Tn5 produced 13 putative recombinants containing the selectable marker. Genomic Southern hybridization confirmed Tn5 existed as episomes for seven recombinants, and for a single recombinant, Tn5 was integrated into the genome of Psychrobacter cryohalolentis PAMC 21807. The result indicates that the mutagenesis method, although successful, has a relatively low rate. The psychrophilic bacteria isolated in this study may be a useful resource for studying cold adaptation mechanisms, and the mutagenesis method can be applied to genetic analysis.

Development of peanut varieties with high oil content by in vitro mutagenesis and screening

Peanut (Arachis hypogaea L.) is an important oil crop globally and high oil content is one of the major targets in peanut breeding programs. Previous studies indicated that the osmotic pressure (OP) of the leaves of peanut plants subjected to drought stress was negatively correlated with kernel oil content. Based on this knowledge, we established a practical and reliable method for creating new peanut varieties with high oil content using in vitro mutagenesis and directional OP-based selection. Using embryonic leaflets of peanut variety Huayu 20 as explants, pingyangmycin (PYM) as the mutagen, and hydroxyproline (HYP) as the OP regulator, we developed 15 HYP-tolerant regenerated plants. For each regenerated plant, we selected offspring with oil content>55% (relative to 49.5% for Huayu 20). We developed and released three new peanut varieties with high yield and high oil content from the offspring of the HYP-tolerant regenerated plants. The three new varieties were named as Yuhua 4, Yuhua 9 and Yuhua 14 and their oil contents were 57.7, 61.1 and 59.3%, respectively. The results indicate that in vitro mutagenesis with PYM followed by directed screening with HYP is a useful approach for breeding peanut varieties with high oil contents.

Targeted mutagenesis of amino acid transporter genes for rice quality improvement using the CRISPR/Cas9 system

High grain protein content(GPC) reduces rice eating and cooking quality(ECQ). We generated OsAAP6 and OsAAP10 knockout mutants in three high-yielding japonica varieties and one japonica line using the CRISPR/Cas9 system. Mutation efficiency varied with genetic background in the T_0 generation, and GPC in the T_1 generation decreased significantly,owing mainly to a reduction in glutelin content. Amylose content was down-regulated significantly in some Osaap6 and all Osaap10 mutants. The increased taste value of these mutants was supported by Rapid Visco Analysis(RVA) profiles, which showed higher peak viscosity and breakdown viscosity and lower setback viscosity than the wild type. There were no significant deficiencies in agronomic traits of the mutants. Targeted mutagenesis of OsAAP6 and OsAAP10, especially OsAAP10, using the CRISPR/Cas9 system can rapidly reduce GPC and improve ECQ of rice, providing a new strategy for the breeding cultivars with desired ECQ.