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A natural variant of arylsulfatase from Kluyveromyces lactis shows no formylglycine modification and has no enzyme activity

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Kluyveromyces lactis is a common fungal microorganism used for the production of enzyme preparations such as β-galactosidases (native) or chymosin (recombinant). It is generally important that enzyme preparations have no unwanted side activities. In the case of β-galactosidase preparations produced from K. lactis, an unwanted side activity could be the presence of arylsulfatase (EC Due to the action of arylsulfatase, an unpleasant “cowshed-like” off-flavor would occur in the final product. The best choice to avoid this is to use a yeast strain without this activity. Interestingly, we found that certain natural K. lactis strains express arylsulfatases, which only differ in one amino acid at position 139. The result of this difference is that K. lactis DSM 70799 (expressing R139 variant) shows no arylsulfatase activity, unlike K. lactis GG799 (expressing S139 variant). After recombinant production of both variants in Escherichia coli, the R139 variant remains inactive, whereas the S139 variant showed full activity. Mass spectrometric analyses showed that the important posttranslational modification of C56 to formylglycine was not found in the R139 variant. By contrast, the C56 residue of the S139 variant was modified. We further investigated the packing and secondary structure of the arylsulfatase variants using optical spectroscopy, including fluorescence and circular dichroism. We found out that the inactive R139 variant exhibits a different structure regarding folding and packing compared to the active S139 variant. The importance of the amino acid residue 139 was documented further by the construction of 18 more variants, whereof only ten showed activity but always reduced compared to the native S139 variant.

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  • Almeida CM, Gomes D, Faro C, Simões I (2015) Engineering a cardosin B-derived rennet for sheep and goat cheese manufacture. Appl Microbiol Biotechnol 99:269–281

    Article  CAS  PubMed  Google Scholar 

  • Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410

    Article  CAS  PubMed  Google Scholar 

  • Barbeyron T, Potin P, Richard C, Collin O, Kloareg B (1995) Arylsulphatase from Alteromonas carrageenovora. Microbiology 141(Pt 1):2897–2904

  • Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 72:248–254

    Article  CAS  PubMed  Google Scholar 

  • Cregut M, Piutti S, Slezack-Deschaumes S, Benizri E (2013) Compartmentalization and regulation of arylsulfatase activities in Streptomyces sp., Microbacterium sp. and Rhodococcus sp. soil isolates in response to inorganic sulfate limitation. Microbiol Res 168:12–21

    Article  CAS  PubMed  Google Scholar 

  • Daniels DL, Plunkett G, Burland V, Blattner FR (1992) Analysis of the Escherichia coli genome: DNA sequence of the region from 84.5 to 86.5 minutes. Science 257:771–778

    Article  CAS  PubMed  Google Scholar 

  • de Hostos EL, Schilling J, Grossman AR (1989) Structure and expression of the gene encoding the periplasmic arylsulfatase of Chlamydomonas reinhardtii. Mol Gen Genet MGG 218:229–239

    Article  PubMed  Google Scholar 

  • Dierks T, Miech C, Hummerjohann J, Schmidt B, Kertesz MA, Von Figura K (1998) Posttranslational formation of formylglycine in prokaryotic sulfatases by modification of either cysteine or serine. J Biol Chem 273:25560–25564

    Article  CAS  PubMed  Google Scholar 

  • Edelheit O, Hanukoglu A, Hanukoglu I (2009) Simple and efficient site-directed mutagenesis using two single-primer reactions in parallel to generate mutants for protein structure-function studies. BMC Biotechnol 9:61

  • Fairbanks G, Steck TL, Wallach DFH (1971) Coomassie blue R250 used in isopropanol-acetic acid. Biochemistry 10:2602–2618

    Article  Google Scholar 

  • Gish W, States DJ (1993) Identification of protein coding regions by database similarity search. Nat Genet 3:266–272

    Article  CAS  PubMed  Google Scholar 

  • Gygi SP, Rochon Y, Franza BR, Aebersold R (1999) Correlation between protein and mRNA abundance in yeast. Mol Cell Biol 19:1720–1730

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Harada T, Spencer B (1962) The effect of sulphate assimilation on the induction of arylsulphatase synthesis in fungi. Biochem J 82:148–156

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Inchaurrondo VA, Yantorno OM, Voget CE (1994) Yeast growth and β-galactosidase production during aerobic batch cultures in lactose-limited synthetic medium. Process Biochem 29:47–54

  • Jo H-J, Noh J-S, Kong K-H (2013) Efficient secretory expression of the sweet-tasting protein brazzein in the yeast Kluyveromyces lactis. Protein Expr Purif 90:84–89

    Article  CAS  PubMed  Google Scholar 

  • Keller A, Nesvizhskii AI, Kolker E, Aebersold R (2002) Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search. Anal Chem 74:5383–5392

    Article  CAS  PubMed  Google Scholar 

  • Kertesz MA (2000) Riding the sulfur cycle—metabolism of sulfonates and sulfate esters in gram-negative bacteria. FEMS Microbiol Rev 24:135–175

    CAS  PubMed  Google Scholar 

  • Kim J-H, Byun D-S, Godber JS, Choi J-S, Choi W-C, Kim H-R (2004) Purification and characterization of arylsulfatase from Sphingomonas sp. AS6330. Appl Microbiol Biotechnol 63:553–559

    Article  CAS  PubMed  Google Scholar 

  • Kim D-E, Kim K-H, Bae Y-J, Lee J-H, Jang Y-H, Nam S-W (2005) Purification and characterization of the recombinant arylsulfatase cloned from Pseudoalteromonas carrageenovora. Protein Expr Purif 39:107–115

    Article  CAS  PubMed  Google Scholar 

  • Lachance M-A (2007) Current status of Kluyveromyces systematics. FEMS Yeast Res 7:642–645

    Article  CAS  PubMed  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  CAS  PubMed  Google Scholar 

  • Lee D-G, Shin JG, Jeon MJ, Lee S-H (2013) Heterologous expression and characterization of a recombinant thermophilic arylsulfatase from Thermotoga maritima. Biotechnol Bioprocess Eng 18:897–902

    Article  CAS  Google Scholar 

  • Lim J-M, Jang Y-H, Kim H-R, Kim YT, Choi TJ, Kim JK, Nam S-W (2004) Overexpression of arylsulfatase in E. coli and its application to desulfatation of agar. J Microbiol Biotechnol 14:777–782

    CAS  Google Scholar 

  • Miech C, Dierks T, Selmer T, Von Figura K, Schmidt B (1998) Arylsulfatase from Klebsiella pneumoniae carries a formylglycine generated from a serine. J Biol Chem 273:4835–4837

    Article  CAS  PubMed  Google Scholar 

  • Murooka Y, Yim MH, Harada T (1980) Formation and purification of Serratia marcescens arylsulfatase. Appl Environ Microbiol 39:812–817

    CAS  PubMed  PubMed Central  Google Scholar 

  • Naumov GI, Naumova ES, Barrio E, Querol A (2006) Genetic and molecular study of inability of the yeast Kluyveromyces lactis var drosophilarum to ferment lactose. Mikrobiologiia 75:299–304

    CAS  PubMed  Google Scholar 

  • Nesvizhskii AI, Keller A, Kolker E, Aebersold R (2003) A statistical model for identifying proteins by tandem mass spectrometry. Anal Chem 75:4646–4658

    Article  CAS  PubMed  Google Scholar 

  • Nonklang S, Abdel-Banat BMA, Cha-aim K, Moonjai N, Hoshida H, Limtong S, Yamada M, Akada R (2008) High-temperature ethanol fermentation and transformation with linear DNA in the thermotolerant yeast Kluyveromyces marxianus DMKU3-1042. Appl Environ Microbiol 74:7514–7521

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Olsen JV, de Godoy LMF, Li G, Macek B, Mortensen P, Pesch R, Makarov A, Lange O, Horning S, Mann M (2005) Parts per million mass accuracy on an Orbitrap mass spectrometer via lock mass injection into a C-trap. Mol Cell Proteomics 4:2010–2021

    Article  CAS  PubMed  Google Scholar 

  • Schestag F, Yaghootfam A, Habetha M, Poeppel P, Dietz F, Klein RA, Zlotogora J, Gieselmann V (2002) The functional consequences of mis-sense mutations affecting an intra-molecular salt bridge in arylsulphatase A. Biochem J 367:499–504

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Shevchenko A, Wilm M, Vorm O, Mann M (1996) Mass spectrometric sequencing of proteins from silver-stained polyacrylamide gels. Anal Chem 68:850–858

    Article  CAS  PubMed  Google Scholar 

  • Smith PK, Krohn RI, Hermanson GT (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150:76–85

    Article  CAS  PubMed  Google Scholar 

  • Spohner SC, Schaum V, Quitmann H, Czermak P (2016) Kluyveromyces lactis: an emerging tool in biotechnology. J Biotechnol 222:104–116

    Article  CAS  PubMed  Google Scholar 

  • Stressler T, Leisibach D, Lutz-Wahl S, Kuhn A, Fischer L (2016a) Homologous expression and biochemical characterization of the arylsulfatase from Kluyveromyces lactis and its relevance in milk processing. Appl Microbiol Biotechnol 100:5401–5414

    Article  CAS  PubMed  Google Scholar 

  • Stressler T, Pfahler N, Merz M, Hubschneider L, Lutz-Wahl S, Claaßen W, Fischer L (2016b) A fusion protein consisting of the exopeptidases PepN and PepX—production, characterization, and application. Appl Microbiol Biotechnol 100:7499–7515

    Article  CAS  PubMed  Google Scholar 

  • Stressler T, Seitl I, Kuhn A, Fischer L (2016c) Detection, production, and application of microbial arylsulfatases. Appl Microbiol Biotechnol 100:9053–9067

    Article  CAS  PubMed  Google Scholar 

  • Stressler T, Tanzer C, Ewert J, Claaßen W, Fischer L (2017) Simple purification method for a recombinantly expressed native His-tag-free aminopeptidase A from Lactobacillus delbrueckii. Protein Expr Purif 131:7–15

    Article  CAS  PubMed  Google Scholar 

  • Taylor WR (1986) The classification of amino acid conservation. J Theor Biol 119:205–218

    Article  CAS  PubMed  Google Scholar 

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The authors would like to thank Wolfgang Claaßen (University of Hohenheim) for his support during the bioreactor cultivations. We also thank Susanne Herr and Sabine Lutz-Wahl (University of Hohenheim) for their support during the construction of the arylsulfatase variants. In addition, many thanks to Veronika Volk and Desirée Leisibach (University of Hohenheim) for performing preliminary experiments.

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Correspondence to Timo Stressler.

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Stressler, T., Reichenberger, K., Glück, C. et al. A natural variant of arylsulfatase from Kluyveromyces lactis shows no formylglycine modification and has no enzyme activity. Appl Microbiol Biotechnol 102, 2709–2721 (2018).

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