定向进化法<em>酿酒酵母</em>：突变体库的创建和筛选
Summary
We present a detailed protocol to construct and screen mutant libraries for directed evolution campaigns in Saccharomyces cerevisiae.
Abstract
设计用于生物技术应用的酶时，涉及建设，克隆和突变体库的表情，加上高频同源DNA重组在体内的过程在酿酒酵母定向进化提供了许多吸引人的优点。在这里，我们提出了一个协议，以创建一个基于真菌芳基醇氧化酶（AAO）的例子来提高其总活性酵母和屏幕突变体库。两种蛋白质的片段，用随机突变和体内 DNA重组进行集中定向进化。的〜50碱基对的侧翼每段悬允许AAO融合基因的正确重新装配在一个线性化的载体引起全自主复制质粒。与功能AAO变种丰富突变体文库的S.筛选酵母上清与基于芬顿反应灵敏的高通量测定法。的一般过程图书馆建设S.酵母这里描述可容易地应用于演进许多其他的真核基因，避免额外的PCR反应， 在体外 DNA重组和连接步骤。
Introduction
定向分子进化是设计酶1,2健壮的，快速和可靠的方法，通过迭代轮次的随机突变，重组和筛选，可以产生改进的酶的版本的作用于新底物，在新颖的反应，在非天然的环境中，或甚至以协助细胞来实现新的代谢目标3-5。间在定向进化中使用的宿主，啤酒酵母酿酒酵母提供了不在原核生物同行6,7-否则可用复杂的真核蛋白质的功能性表达的解决方案的一个曲目。
在细胞生物学研究中详尽使用时，该小的真核模型在翻译后修饰，易于操纵和转换效率，所有这一切都是由定向进化8工程师酶重要性状方面有许多优点。此外，高频率在S同源DNA重组酵母加上其高效的校对设备打开一个广阔的图书馆创建和组装的基因在体内的可能性阵列，促进由单一的酶来复杂的人工途径9-12不同系统的演变。我们的实验室已经度过了过去十年的设计工具和策略在酵母中不同的木质素酶的分子进化（天然木材腐烂过程中参与木质素的降解氧化还原酶）13-14。在这种沟通中，我们提出了详细的方案编制和S.屏幕突变体文库酵母为一个模型flavooxidase， -芳基醇氧化酶（AAO 15） – ，可以很容易地转换为许多其它的酶。该协议涉及的聚焦定向进化方法：通过酵母细胞装置16，辅助（MORPHING同源体内编组诱变有组织重组处理）基于以检测分泌到培养液中17 AAO活性Fenton反应哒非常敏感筛选测定。
Protocol
1突变体库建设选择的区域要进行与基于所述可用的晶体结构或同源性模型18的计算算法的帮助下变形。 这里，目标从杏鲍菇 AAO的两个区域为随机诱变和重组（蛋氨酸[α1] -Val109，Phe392-Gln566），而扩增基因（844碱基对）的通过高保真PCR，其余（ 图1）。 注意：几个片段可以通过以独立或组合方式16变形进行研究。 通?…
Representative Results
从AAO P.杏鲍菇是一种细胞外flavooxidase供给真菌过氧化物用H 2 O 2与开始攻击木质素。 AAO的两段被以变形，以提高其活性及其在S.表达受到关注，定向进化酵母 19。由S.窝藏外国酶无关酵母 ，当在酵母构建突变体文库涉及特定重叠区域的工程有利于片段和其克隆入线性化载体之间的拼接的最关键的问题。在当前的例子…
Discussion
在这篇文章中，我们总结了大部分的技巧和窍门在我们的实验室中通过S.定向进化工程师使用的酶酵母 （使用氧化铝作为一个例子），以便它们可以通过简单地按照此处描述的常用的方法适于使用与许多其它真核酶系统。
在库创建而言，变形是一个快速单罐法引进和重组中的小蛋白伸展随机突变，同时留下未改变16蛋白质的剩余区域。与几个突变负载库?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was supported by the European Commission project Indox-FP7-KBBE-2013-7-613549; a Cost-Action CM1303-Systems Biocatalysis; and the National Projects Dewry [BIO201343407-R] and Cambios [RTC-2014-1777-3].
Materials
| 1. Culture media | |||
| Ampicillin sodium salt | Sigma-Aldrich | A0166 | CAS Nº 69-52-3 M.W. 371.39 |
| Bacto Agar | Difco | 214010 | |
| Cloramphenicol | Sigma-Aldrich | C-0378 | CAS Nº 56-75-7 M.W. 323.13 |
| D-(+)-Galactose | Sigma-Aldrich | G0750 | CAS Nº 59-23-4 M.W. 180.16 |
| D-(+)-Glucose | Sigma-Aldrich | G5767 | CAS Nº 50-99-7 M.W. 180.16 |
| D-(+)-Raffinose pentahydrate | Sigma-Aldrich | 83400 | CAS Nº 17629-30-0 M.W. 594.51 |
| Peptone | Difco | 211677 | |
| Potassium phosphate monobasic | Sigma-Aldrich | P0662 | CAS Nº 7778-77-0 M.W. 136.09 |
| Uracil | Sigma Aldrich | U1128 | |
| Yeast Extract | Difco | 212750 | |
| Yeast Nitrogen Base without Amino Acids | Difco | 291940 | |
| Yeast Synthetic Drop-out Medium Supplements without uracil | Sigma-Aldrich | Y1501 | |
| Name | Company | Catalog Number | Comments |
| 2. PCR Reactions | |||
| dNTP Mix | Agilent genomics | 200415-51 | 25 mM each |
| iProof High-Fidelity DNA polymerase | Bio-rad | 172-5301 | |
| Manganese(II) chloride tetrahydrate | Sigma-Aldrich | M8054 | CAS Nº 13446-34-9 M.W. 197.91 |
| Taq DNA Polymerase | Sigma-Aldrich | D4545 | For error prone PCR |
| Name | Company | Catalog Number | Comments |
| 3. Plasmid linearization | |||
| BamHI restriction enzyme | New England Biolabs | R0136S | |
| Bovine Serum Albumin | New England Biolabs | B9001S | |
| XhoI restriction enzyme | New England Biolabs | R0146S | |
| Gel Red | Biotium | 41003 | For staining DNA |
| Name | Company | Catalog Number | Comments |
| 4. FOX assays | |||
| Ammonium iron(II) sulfate hexahydrate | Sigma-Aldrich | F3754 | CAS Nº 7783-85-9 M.W. 392.14 |
| Anysil Alcohol | Sigma Aldrich | W209902 | CAS Nº 105-13-5 M.W. 138.16 |
| D-Sorbitol | Sigma-Aldrich | S1876 | CAS Nº 50-70-4 M.W. 182.17 |
| Hydrogen peroxide 30% | Merck Millipore | 1072090250 | FOX standard curve |
| Xylenol Orange disodium salt | Sigma-Aldrich | 52097 | CAS Nº 1611-35-4 M.W. 716.62 |
| Agarose gel stuff | – | – | – |
| Agarose | Norgen | 28035 | CAS Nº 9012-36-6 |
| Gel Red | Biotium | 41003 | DNA analysis dye |
| GeneRuler 1Kb Ladder | Thermo Scientific | SM0311 | DNA M.W. standard |
| Loading Dye 6x | Thermo Scientific | R0611 | |
| Low-melting temperature agarose | Bio-rad | 161-3112 | CAS Nº 39346-81-1 |
| Name | Company | Catalog Number | Comments |
| 5. Kits and cells | |||
| S. cerevisiae strain BJ5465 | LGC Promochem, Spain | ATTC 208289 | Protease deficient strain with genotype: MATα ura3-52 trp1 leu2-delta1 his3-delta200 pep4::HIS3 prb1-delta1.6R can1 GAL |
| E. coli XL2-Blue competent cells | Agilent genomics | 200150 | For plasmid purification and amplification |
| NucleoSpin Gel and PCR Clean-up Kit | Macherey-Nagel | 740,609,250 | DNA gel extraction |
| NucleoSpin Plasmid Kit | Macherey-Nagel | 740,588,250 | Column miniprep Kit |
| Yeast Transformation Kit | Sigma-Aldrich | YEAST1-1KT | Included DNA carrier (Salmon testes) |
| Zymoprep yeast plasmid miniprep I | Zymo research | D2001 | Plasmid extraction from yeast |
| Name | Company | Catalog Number | Comments |
| 6. Plates | |||
| 96-well plates | Greioner Bio-One | 655101 | Clear, non-sterile, Polystyrene (for activity measurements) |
| 96-well plates | Greioner Bio-One | 655161 | Clear, sterile, Polystyrene (for microfermentations) |
| 96-well plate lid | Greioner Bio-One | 656171 | Clear, sterile, Polystyrene (for microfermentations) |
References
- Jäckel, C., Hilvert, D. Biocatalysts by evolution. Curr. Opin. Biotechnol. 21 (6), 753-759 (2010).
- Bornscheuer, U. T. Engineering the third wave of biocatalysis. Nature. 485 (7397), 185-194 (2012).
- Renata, H., Wang, Z. W., Arnold, F. H. Expanding the enzyme universe: accessing non-natural reactions by mechanism-guided directed evolution. Angew. Chem. Int. Ed. 54 (11), 3351-3367 (2015).
- Cobb, R. E., Chao, R., Zhao, H. Directed evolution: past, present and future. AIChE J. 59 (5), 1432-1440 (2013).
- Abatemarco, J., Hill, A., Alper, H. S. Expanding the metabolic engineering toolbox with directed evolution. Biotechnol. J. 8 (12), 1397-1410 (2013).
- Pourmir, A., Johannes, T. W. Directed evolution: selection of the host organism. Comput Struct Biotechnol J. 2 (3), e201209012 (2012).
- Krivoruchko, A., Siewers, V., Nielsen, J. Opportunities for yeast metabolic engineering: lessons from synthetic biology. Biotechnol J. 6 (3), 262-276 (2011).
- Gonzalez-Perez, D., Garcia-Ruiz, E., Alcalde, M. Saccharomyces cerevisiae in directed evolution: an efficient tool to improve enzymes. Bioeng Bugs. 3, 172-177 (2012).
- Alcalde, M. Mutagenesis protocols in Saccharomyces cerevisiae by In Vivo Overlap Extension. Methods Mol. Biol. 634, 3-14 (2010).
- Bulter, T., Alcalde, M. Preparing libraries in Saccharomyces cerevisiae. Methods. Mol. Biol. 231, 17-22 (2003).
- Ostrov, N., Wingler, L. M., Cornish, W. Gene assembly and combinatorial libraries in S. cerevisiae via reiterative recombination. Methods. Mol. Biol. 978, 187-203 (2013).
- Shao, Z., Zhao, H., Zhao, H. DNA assembler, an in vivo genetic method for rapid construction of biochemical pathways. Nucleic Acids Res. 37 (2), e16 (2009).
- Alcalde, M. Engineering the ligninolytic enzyme consortium. Trends Biotechnol. 33 (3), 155-162 (2015).
- Garcia-Ruiz, E. Directed evolution of ligninolytic oxidoreductases: from functional expression to stabilization and beyond. Cascade Biocatalysis: integrating stereoselective and environmentally friendly reactions. , 1-22 (2014).
- Hernandez-Ortega, A., Ferreira, P., Martinez, A. T. Fungal aryl-alcohol oxidase: a peroxide-producing flavoenzyme involved in lignin degradation. Appl. Microbiol. Biotechnol. 93 (4), 1395-1410 (2012).
- Gonzalez-Perez, D., Molina-Espeja, P., Garcia-Ruiz, E., Alcalde, M. Mutagenic organized recombination process by homologous in vivo grouping (MORPHING) for directed enzyme evolution. PLoS One. 9, e90919 (2014).
- Rhee, S. G., Chang, T., Jeong, W., Kang, D. Methods for Detection and Measurement of Hydrogen Peroxide Inside and Outside of Cells. Mol. Cells. 29 (6), 539-549 (2010).
- Sebestova, E., Bendl, J., Brezovsky, J., Damborsky, J. Computational tools for designing smart libraries. Methods. Mol. Biol. 1179, 291-314 (2014).
- Viña-Gonzalez, J., Gonzalez-Perez, D., Ferreira, P., Martinez, A. T., Alcalde, M. Focused directed evolution of aryl-alcohol oxidase in yeast using chimeric signal peptides. Appl. Environ. Microbiol. , (2015).
- Gay, C., Collins, J., Gebicki, J. M. Hydroperoxide Assay with the Ferric-Xylenol orange Complex. Anal. Biochem. 273 (2), 149-155 (1999).
- Reetz, M. T. Biocatalysis in organic chemistry and biotechnology: Past, present, and future. J. Am. Chem. Soc. 135 (34), 12480-12496 (2013).
- Mate, D. M., Gonzalez-Perez, D., Mateljak, I., Gomez de Santos, P., Vicente, A. I., Alcalde, M. The pocket manual of directed evolution: Tips and tricks. Biotechnology of Microbial Enzymes: Production, Biocatalysis and Industrial Applications. , .
- Chao, R., Yuan, Y., Zhao, H. Recent advances in DNA assembly technologies. FEMS Yeast Res. 15, 1-9 (2015).
Cite This Article
Viña-Gonzalez, J., Gonzalez-Perez, D., Alcalde, M. Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening. J. Vis. Exp. (110), e53761, doi:10.3791/53761 (2016).