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Feb 2009

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Development of an Efficient Transformation System for Halotolerant Yeast Debaryomyces hansenii CBS767
耐盐汉斯德巴氏酵母菌CBS767的一种高效转化体系的建立   

Anu P. MinhasAnu P. Minhas* Dipanwita   BiswasDipanwita Biswas* (*共同第一作者)
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Abstract

Debaryomyces hansenii is one of the most osmotolerant and halotolerant yeasts. Further, its association with traditional cheese and meat products imparting special flavors to these products project this yeast with enormous biotechnological potential in the agrofood sector. However, lack of an efficient transformation system in D. hansenii still direct the complementation based assay in S. cerevisiae mutants for functional analysis of D. hansenii genes. Here, we have described the development of an efficient transformation system for D. hansenii that is based on a histidine auxotrophic recipient strain, DBH9 (generated by UV induced random mutagenesis), and the DhHIS4 gene as the selectable marker (Minhas et al., 2009). Moreover, the same method has also been employed for gene disruption in D. hansenii by homologous recombination.

Keywords: Debaryomyces hansenii (汉斯德巴氏酵母菌), Transformation (转化), Gene disruption (基因破坏), Halotolerant yeast (耐盐酵母), Electroporation (电穿孔), Auxotroph (营养缺陷型)

Background

Debaryomyces hansenii CBS767 is one of the most osmotolerant and halotolerant yeasts and can tolerate salinity levels up to 4 M NaCl, whereas growth of Saccharomyces cerevisiae is inhibited when salinity reaches above 1.7 M NaCl. Debaryomyces hansenii is frequently associated with traditional cheese and meat products imparting special flavors to these products. Therefore, this yeast is not only an excellent model to study salt tolerance mechanisms in eukaryotic cells but it also has enormous biotechnological potential in the agrofood sector. However, lack of efficient transformation system and gene disruption tools required for gene manipulation in D. hansenii is a big lacuna behind the molecular analysis of its halotolerant feature. Ricaurte and Govind (1999) developed an ura3 mutant based transformation system for D. hansenii; however this system exhibited low-efficiency. Despite the development of transformation system for Candida famata (Voronovsky et al., 2002), an anamorph of D. hansenii, functional studies of the genes from D. hansenii are still based on complementation of S. cerevisiae mutants. Our transformation system is based on a histidine auxotrophic strain, DBH9 and the DhHIS4 gene as the selectable marker. The same method has also been demonstrated for gene disruption in D. hansenii by homologous recombination (Minhas et al., 2009).

Materials and Reagents

  1. Pipettes (Thermo Fisher Scientific, catalog numbers: 4652080, 4652050, 4652020, 4652010)
  2. Flask (Borosil, catalog numbers: 4980009, 4980012, 4980016, 4980021)
  3. Graduated cylinder (Borosil, catalog number: 3021029)
  4. Centrifuge tube (Oak Ridge,catalog number: 541040)
  5. Safe-Lock tubes 2.0 ml (Eppendorf, catalog number: 0030120094)
  6. Disposable Petri plates (Tarson, catalog number: 460090)
  7. Pipette tips (Tarson, catalog numbers: 521000, 521010, 521020)
  8. 0.2 µm syringe filter (Millipore, catalog number: SLGP033RS)
  9. 0.2 cm Electroporation cuvette (BioRad, catalog number: 1652086)
  10. DBH9: D. hansenii strain CBS767 (MTCC 3461) ∆dhhis4 (Original manuscript, Minhas et al., 2009) 
  11. Plasmid pDH4 (for cloning details refers to materials and methods section in Minhas et al., 2009)
  12. Sodium phosphate, dibasic (Merck, catalog number: 567550)
  13. Sodium phosphate, monobasic (Merck, catalog number: 567549)
  14. DL-Dithiothreitol (DL- DTT) (Sigma, catalog number: 43815)
  15. Glycerol (Sigma-Aldrich, catalog number:G9012)
  16. Sucrose (Sigma, catalog number: S8501)
  17. Glucose (Himedia, catalog number: MB037)
  18. Agar (Himedia, catalog number: GRM026)
  19. YPD broth (BD Difco, catalog number: DF0428-17-5)
  20. Yeast minimal media-SD base (Clontech, catalog number: 630411)
  21. Sterile water (Lab Pure-Series Model-Analytic UV)
  22. YPD broth medium (see Recipes)
  23. SD minimal agar medium with 0.3 M sucrose (see Recipes)
  24. YPD agar medium (see Recipes)
  25. 1 M sodium phosphate buffer, pH 7.5 (see Recipes)
  26. 1 M D L- DTT (see Recipes)
  27. 1 M Sucrose (see Recipes)

Equipment

  1. Incubator, New BrunswickTM Innova® 44 (Eppendorf, model: M1282-0002)
  2. UV-VIS spectrophotometer (Shimadzu, model: UV 1900)
  3. Microcentrifuge (Eppendorf, model: 5415R)
  4. Centrifuge (REMI, model: CPR-24Plus)
  5. Water bath (Julabo, model: PURA 4)
  6. Gene Pulser (BioRad, model: GenePulserXcell) 
  7. Vortex (DLAB, model: MX-S)
  8. Laminar air flow (Ocean life science corp, model: OLSC 113-4)
  9. L- spreader (Himedia, model: PW1085)
  10. Autoclave

Software

  1. TBLASTN search tool on NCBI website (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=tblastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome)

Procedure

Notes:

  1. Perform all steps involving microbial culture under sterilie environment in laminar air flow.
  2. Use autoclaved water in all the steps.

  1. Streak DBH9 strain on an YPD agar plate from frozen glycerol stock (-70 °C). 
  2. Incubate streaked plate at 25 °C in an incubator for 3-4 days or till colonies appear.
    Note: Always start with freshly streaked culture for enhanced transformation efficiency.
  3. Inoculate a single colony of DBH9 from the plate in 10 ml YPD broth in a flask and incubate for appx. 24 h at 25 °C at 200 rpm for growth (saturated culture).
  4. Check the absorbance of the culture (after 10 times dilution in water) using spectrophotometer at 600 nm. 
  5. Re-inoculate 120 µl of above grown saturated culture (if O.D. of culture is 3.0) into 30 ml of YPD broth in a flask (so that an initial A600nm of 0.045 is acheived).
    Note: The volume of re-inoculation may vary with O.D. of culture.
  6. Grow the re-inoculated culture at 25 °C at 200 rpm for ~16 h till the A600nm reached 2.6-2.8 (appx. 4.3 x 107 cells ml-1).
  7. Confirm the absorbance using spectrophotometer.
    Note: A drastic reduction in the number of transformants has been observed when the A600nm of the harvested cells was > 2.8.
  8. Harvest cells from the liquid culture by centrifugation in a centrifuge at 4,500 x g using centrifuge tube for 10 min at room temperature (RT). 
  9. Discard the flow-through.
  10. Re-suspend cells pellet in 6 ml of 50 mM sodium phosphate buffer, pH 7.5 (containing 25 mM DL-Dithiothreitol).
    Note: Vortex the cell suspension at low rpm for 5-10 s for re-suspension.
  11. Incubate cells suspension in a 30 °C water bath (pre-warm) for 15 min. 
  12. Again harvest cells from the suspension by centrifugation at 4,500 x g using centrifuge tube for 10 min at RT.
  13. Discard the flow-through.
  14. Wash the cells by re-suspending cell pellet in 15 ml sterile water and harvest the cell pellet by centrifugation at 4,500 x g for 10 min at RT. 
  15. Discard the flow-through.
  16. Repeat Steps 14-15.
  17. Re-suspend the cell pellet in 1.0 ml ice-cold 1 M sucrose and transfer the entire chilled cell suspension to pre-chilled 2.0 ml safe-lock tube.
  18. Harvest the cell pellet again by centrifugation at 4,500 x g for 10 min at 4 °C in a microcentrifuge. 
  19. Discard the flow-through.
  20. Finally to the cell pellet, add 175 µl ice-cold 1 M sucrose to make the final volume of chilled cell suspension to 200 µl in a safe-lock tube.
  21. Uniformly re-suspend the cells by pipetting. 
  22. To this chilled cell suspension, add 0.25-1.5 µg DNA (pDH4 plasmid) and transfer the entire mixture to 0.2 cm electroporation cuvette. 
  23. Electroporate the mixture using Gene Pulser at a setting of 2.3 kV, 50 mFd and 100 Ω.
    Note: Perform Steps 17-22 strictly on ice.
  24. To the electroporation cuvette, immediately add 800 µl of 1 M sucrose solution (stored at room temperature).
  25. Spread 200 µl of the cells transformed with pDH4 on SD minimal medium (containing 0.3 M sucrose) plates using L-spreader. 
  26. Incubate these plates at 25 °C for 3-4 days for appearance of transformants positive for histidine prototrophy (as replicating vector pDH4 has DhHIS4 gene as the selection marker).
    Note: We haven’t used DhHIS4 as the selection marker in other yeast lines.
  27. An extra sample is always used as a control where no DNA will be added.
  28. Around 3,500 colonies would appear on the plate transformed with 0.25 µg of pDH4 plasmid DNA. No colony will appear in control transformation without DNA.

Data analysis

For details, refers to Minhas et al. (2009).

Recipes

  1. YPD agar medium
    1. Add 5.0 g of YPD broth powder in 250 ml flask 
    2. Add 2.0 g of agar powder
    3. Dissolve the medium completely in 100 ml sterile water and sterilize by autoclaving at 15 lbs pressure (121 °C) for 15 min 
  2. YPD broth medium
    1. Suspend 5.0 g of YPD broth medium in 100 ml distilled water in 250 ml flask 
    2. Dissolve the medium completely and sterilize by autoclaving at 15 lbs pressure (121 °C) for 15 min
  3. SD minimal agar media with 0.3 M sucrose (100 ml)
    1. Add 0.67 g Yeast minimal media-SD base to 80 ml of distilled water in a 250 ml flask
    2. Add 2.0 g glucose, 2.0 g agar and 1.02 g sucrose to it
    3. Mix the solution uniformly
    4. Make up the final volume to 100 ml with distilled water
  4. 1 M Sodium phosphate buffer, pH 7.5
    1. Add 20.209 g of sodium phosphate dibasic to 800 ml of distilled water in a graduated cylinder (1000 ml capacity) to prepare 0.0754 M sodium phosphate dibasic solution
    2. Add 3.394 g of sodium phosphate monobasic (0.0246 M) to the above solution
    3. Mix the solution uniformly and adjust the final pH to 7.5 using HCl or NaOH (if required)
    4. Make up the final volume to 1 L with distilled water
  5. 1 M DL-DTT
    Note: Always use freshly prepared DL-DTT solution.
    1. Add 0.154 g of DL-DTT to 1.0 ml of sterile dH2O in a safe lock tube
    2. Dissolve completely by pipetting and sterilize the stock through a 0.2 µm syringe filter
  6. 1.0 M Sucrose
    1. Add 34.23 g of sucrose to 80 ml of sterile dH2O in 250 ml flask. Dissolve completely
    2. Make up the final volume to 100 ml and sterilize by autoclaving as directed above

Acknowledgments

The research was supported by Council of Scientific and Industrial Research, New Delhi, India. Special gratitude to Dr. Alok Mondal for guiding the original work and writing the published manuscript.
  We would like to acknowledge our original publication “Minhas, A., Biswas, D. and Mondal, A. K. (2009). Development of host and vector for high-efficiency transformation and gene disruption in Debaryomyces hansenii. FEMS Yeast Res 9 (1): 95-102.

Competing interests

Authors have no competing interest.

References

  1. Minhas, A., Biswas, D. and Mondal, A. K. (2009). Development of host and vector for high-efficiency transformation and gene disruption in Debaryomyces hansenii. FEMS Yeast Res 9 (1): 95-102.
  2. Ricaurte, M. L. and Govind, N. S. (1999). Construction of plasmid vectors and transformation of the marine yeast Debaryomyces hansenii. Mar Biotechnol (NY) 1(1): 15-19.
  3. Voronovsky, A. A., Abbas, C. A., Fayura, L. R., Kshanovska, B. V., Dmytruk, K. V., Sybirna, K. A. and Sibirny, A. A. (2002). Development of a transformation system for the flavinogenic yeast Candida famata. FEMS Yeast Res 2(3): 381-388.

简介

hansenii酵母是最耐渗透和耐盐的酵母之一。此外,它与传统奶酪和肉类产品的结合赋予了这些产品特殊的风味,使这种酵母在农业食品领域具有巨大的生物技术潜力。然而,由于缺乏一个高效的转化体系,目前仍缺乏一种基于互补的方法对酿酒酵母突变株进行功能分析。在此,我们描述了基于组氨酸营养不良受体菌株dbh9(由紫外线诱导的随机突变产生)和dhhis4基因作为可选择标记(minhas等,2009)的高效转化系统的开发。此外,同样的方法也被用于通过同源重组对d.hansenii的基因断裂。
【背景】hanseniicbs767是最耐渗透和耐盐的酵母菌之一,能耐受高达4 m nacl的盐度,而当盐度超过1.7 mnacl时,酿酒酵母的生长受到抑制。汉森放线菌常与传统奶酪和肉制品联系在一起,赋予这些产品特殊的风味。因此,该酵母不仅是研究真核细胞耐盐机制的良好模型,而且在农业食品领域具有巨大的生物技术潜力。然而,缺乏有效的转化系统和基因操作所需的基因打乱工具,是汉森地鼠耐盐特性分子分析的一大空白。Ricourte和Govind(1999)为d.Hansenii开发了一个基于突变的Ura3转化系统,但该系统效率较低。尽管famata假丝酵母(voronovskyet al.,2002)的转化系统已经发展成熟,但对hansenii基因的功能研究仍然是基于对酿酒酵母突变体的补充。我们的转化系统是以组氨酸营养不良株dbh9和dhhis4基因为选择标记。同样的方法也被证明可以通过同源重组(minhaset al.,2009)来破坏d.hansenii中的基因。

关键字:汉斯德巴氏酵母菌, 转化, 基因破坏, 耐盐酵母, 电穿孔, 营养缺陷型

材料和试剂

  1. 移液管(Thermo Fisher Scientific,目录号:4652080、4652050、4652020、4652010)
  2. 烧瓶(Borosil,目录号:4980009、4980012、4980016、4980021)
  3. 量筒(Borosil,目录号:3021029)
  4. 离心管(橡树岭,目录号:541040)
  5. 安全锁管2.0毫升(Eppendorf,目录号:0030120094)
  6. 一次性培养皿(Tarson,目录号:460090)
  7. 吸管头(Tarson,目录号:521000、521010、521020)
  8. 0.2μm注射器过滤器(微孔,目录号:SLGP033RS)
  9. 0.2 cm电穿孔反应杯(Biorad,目录号:1652086)
  10. DBH9:d.hansenii菌株CBS767(MTCC 3461)。/dhhis4(原稿,Minhas等人,2009年)
  11. 质粒pdh4(克隆细节参见Minhas等人2009年的材料和方法部分)
  12. 磷酸钠,二元酸(默克,目录号:567550)
  13. 一元磷酸钠(默克,产品目录号:567549)
  14. 二硫苏糖醇(DL-DTT)(西格玛,目录号:43815)
  15. 甘油(Sigma-Aldrich,目录号:G9012)
  16. 蔗糖(Sigma,目录号:S8501)
  17. 葡萄糖(Himedia,目录号:MB037)
  18. 琼脂(Himedia,目录号GRM026)
  19. YPD肉汤(bd difco,目录号:df0428-17-5)
  20. 酵母最小培养基-SD基(克隆泰克,目录号:630411)
  21. 无菌水(实验室纯系列模型-分析紫外线)
  22. YPD肉汤培养基(见配方)
  23. 含0.3m蔗糖的最小SD琼脂培养基(见配方)
  24. YPD琼脂培养基(见配方)
  25. 1 M磷酸钠缓冲液,pH值7.5(见配方)
  26. 1 m D L-DTT(见配方)
  27. 1米蔗糖(见配方)

团队

  1. 孵化器,新不伦瑞克TMInnova44(Eppendorf,型号:M1282-0002)
  2. 紫外可见分光光度计(岛津,型号:紫外1900)
  3. 微型离心机(Eppendorf,型号:5415R)
  4. 离心机(REMI,型号:CPR-24PLUS)
  5. 水浴(Julabo,型号:Pura 4)
  6. 基因脉冲发生器(Biorad,型号:Genepulserxcell)
  7. 涡流(DLAB,型号:MX-S)
  8. 层流(海洋生命科学公司,型号:OLSC 113-4)
  9. L-吊具(Himedia,型号:PW1085)
  10. 高压灭菌器

软件

  1. tblastn ncbi网站上的搜索工具(https://blast.ncbi.nlm.nih.gov/blast.cgi?程序=tblastn&page_type=blastsearch&link_loc=blasthome)

程序

注意:

  1. 在层流的立体环境下进行微生物培养的所有步骤。
  2. 在所有步骤中使用高压水。
< BR>
  1. 在YPD琼脂平板上从冷冻甘油原料(-70°C)中提取DBH9菌株。
  2. 将划线板在25°C的培养箱中培养3-4天或直到出现菌落。< BR> 注意:始终从新的条纹培养基开始,以提高转化效率。
  3. 将培养皿中的一个DBH9菌落接种在10毫升YPD肉汤中,并在烧瓶中培养用于APPX。在25℃下以200转/分的速度生长24小时(饱和培养)。
  4. 用分光光度计在600 nm处检查培养物的吸光度(在水中稀释10倍后)。
  5. 将120微升以上生长的饱和培养物(如果培养物的外径为3.0)重新接种到30毫升YPD肉汤中(以便获得0.045的初始a600纳米)。
    注:再接种量可能随培养物的外径而变化。
  6. 将重新接种的培养物在25°C下以200转/分的速度培养约16小时,直到A600nm达到2.6-2.8(约4.3 x 107细胞ml-1)。
  7. 用分光光度计确认吸光度。< BR> 注:当收获细胞的600纳米为2.8时,已观察到转化子的数量急剧减少。
  8. 在4500x g的离心机中,用离心管在室温(rt)下离心10分钟,从液体培养物中提取细胞。
  9. 丢弃直通管。
  10. 在6毫升50毫米磷酸钠缓冲液中重新悬浮细胞颗粒,pH值7.5(含25毫米二硫苏糖醇)。< BR> 注意:在低转速下使电池悬浮液涡流5-10秒,以便重新悬浮。
  11. 在30°C水浴(预热)中培养细胞悬浮液15分钟。
  12. 用离心管在室温下离心4500x g10分钟,再次从悬浮液中提取细胞。
  13. 丢弃流过的液体。
  14. 用15毫升无菌水重新悬浮细胞颗粒洗涤细胞,并在室温下4500x g离心10分钟以获得细胞颗粒。
  15. 丢弃流过的液体。
  16. 重复步骤14-15。
  17. 将细胞颗粒重新悬浮在1.0毫升冰冷1米蔗糖中,并将整个冷冻细胞悬浮液转移到预冷2.0毫升安全锁定管中。
  18. 在4500x g下,在4°C下,在微型离心机中离心10分钟,再次收获细胞颗粒。
  19. 丢弃流过的液体。
  20. 最后,向细胞颗粒中添加175微升冷1米蔗糖,使最终体积的冷冻细胞悬浮液在安全锁定管中达到200微升。
  21. 用移液管均匀地重新悬浮细胞。
  22. 将0.25-1.5微克DNA(PDH4质粒)加入到冷冻细胞悬液中,并将整个混合物转移到0.2厘米的电穿孔反应杯中。
  23. 在2.3kv、50mfd和100Ω的设置下,使用基因脉冲发生器对混合物进行电穿孔。< BR> 注意:严格在冰上执行步骤17-22。
  24. 立即向电穿孔反应杯中加入800微升1 M蔗糖溶液(室温下保存)。
  25. 将200微升用PDH4转化的细胞用L-Spreader涂在SD最小培养基(含0.3 M蔗糖)板上。
  26. 将这些培养板在25℃下培养3-4天,以观察组氨酸原生质体阳性转化子的出现(因为复制载体pdh4具有dhhis4基因作为选择标记)。< BR> 注意:我们没有在其他酵母系中使用dhhis4作为选择标记。
  27. 一个额外的样本总是被用作没有DNA被添加的对照。
  28. 用0.25μg pdh4质粒dna转化的平板上约有3500个菌落。没有dna的对照转化不会出现菌落。

数据分析

有关详细信息,请参阅Minhas等人(2009年)。

食谱

  1. ypd琼脂培养基
    1. 在250毫升烧瓶中加入5.0克YPD肉汤粉
    2. 加入2.0g琼脂粉
    3. 将培养基完全溶解在100毫升无菌水中,并在15磅压力(121°C)下高压灭菌15分钟;
  2. ypd肉汤培养基
    1. 将5.0 g YPD肉汤培养基悬浮在250 ml烧瓶中的100 ml蒸馏水中
    2. 完全溶解介质,并在15磅压力(121°C)下高压灭菌15分钟
  3. sd最小琼脂培养基,含0.3m蔗糖(100 ml)
    1. 将0.67 g酵母最小培养基SD碱添加到250 ml烧瓶中的80 ml蒸馏水中
    2. 加入2.0g葡萄糖、2.0g琼脂和1.02g蔗糖
    3. 把溶液混合均匀
    4. 用蒸馏水将最终体积补充到100毫升
  4. 1 M磷酸钠缓冲液,pH 7.5
    1. 将20.209 g磷酸二氢钠加入刻度量筒(1000 ml容量)中的800 ml蒸馏水中,制备0.0754 m磷酸二氢钠溶液
    2. 向上述溶液中添加3.394g磷酸二氢钠(0.0246m)
    3. 将溶液均匀混合,并使用HCl或NaOH(如需要)将最终pH值调整至7.5。
    4. 用蒸馏水将最终体积补充到1L
  5. 1米DL-DTT
    注意:务必使用新制备的DL-DTT溶液。
    1. 将0.154g dl-dtt加入1.0ml无菌dh2o的安全锁定管中
    2. 通过吸管完全溶解,并通过0.2μm注射器过滤器对原料进行消毒。
  6. 1.0米蔗糖
    1. 将34.23 g蔗糖添加到250 ml烧瓶中的80 ml无菌dh2o中。完全溶解
    2. 将最终体积补充至100毫升,并按照上述说明进行高压灭菌。

致谢

这项研究得到了印度新德里科学和工业研究理事会的支持。特别感谢阿洛克·蒙达尔博士指导原著并撰写已出版的手稿。
&nbsp;我们要感谢我们的原始出版物“Minhas,A.,Biswas,D.和Mondal,A.K.(2009)。汉森德巴酵母高效转化和基因断裂宿主及载体的构建。fems酵母res9(1):95-102。

相互竞争的利益

作者没有相互竞争的兴趣。

工具书类

  1. Minhas,A.,Biswas,D.和Mondal,A.K.(2009年)。在汉森德巴酵母fems酵母res9(1):95-102中高效转化和基因破坏的宿主和载体的开发。
  2. Ricourte,M.L.和Govind,N.S.(1999年)。质粒载体的构建和海洋酵母的转化hansenii德巴酵母mar biotechnol(ny)1(1):15-19。
  3. Voronovsky,A.A.,Abbas,C.A.,Fayura,L.R.,Kshanovska,B.V.,Dmytruk,K.V.,Sybirna,K.A.和Siberny,A.A.(2002)。黄原酵母famata转化系统的开发。fems酵母res2(3):381-388。
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引用:MINHAS, A. P. and Biswas, D. (2019). Development of an Efficient Transformation System for Halotolerant Yeast Debaryomyces hansenii CBS767. Bio-protocol 9(17): e3352. DOI: 10.21769/BioProtoc.3352.
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