Optimisation of Recombinant Myrosinase Production in Pichia Pastoris

International Journal of Molecular Sciences Article Optimisation of Recombinant Myrosinase Production in Pichia pastoris Zuzana Rosenbergová 1, Kristína Kántorová 1, Martin Šimkoviˇc 2, Albert Breier 2 and Martin Rebroš 1,* 1 Institute of Biotechnology, Faculty of Chemical and Food Technology, Slovak University of Technology, Radlinského 9, 812 37 Bratislava, Slovakia; [email protected] (Z.R.); [email protected] (K.K.) 2 Institute of Biochemistry and Microbiology, Faculty of Chemical and Food Technology, Slovak University of Technology, Radlinského 9, 812 37 Bratislava, Slovakia; [email protected] (M.Š.); [email protected] (A.B.) * Correspondence: [email protected] Abstract: Myrosinase is a plant defence enzyme catalysing the hydrolysis of glucosinolates, a group of plant secondary metabolites, to a range of volatile compounds. One of the products, isothiocyanates, proved to have neuroprotective and chemo-preventive properties, making myrosinase a pharmaceutically interesting enzyme. In this work, extracellular expression of TGG1 myrosinase from Arabidopsis thaliana in the Pichia pastoris KM71H (MutS) strain was upscaled to a 3 L laboratory fermenter for the first time. Fermentation conditions (temperature and pH) were optimised, which resulted in a threefold increase in myrosinase productivity compared to unoptimised fermentation conditions. Dry cell weight increased 1.5-fold, reaching 100.5 g/L without additional glycerol feeding. Overall, a specific productivity of 4.1 U/Lmedium/h was achieved, which was 102.5-fold higher compared to flask cultivations. Citation: Rosenbergová, Z.; Keywords: myrosinase; Pichia pastoris; fermentation; upscale; Arabidopsis thaliana Kántorová, K.; Šimkoviˇc,M.; Breier, A.; Rebroš, M. Optimisation of Recombinant Myrosinase Production in Pichia pastoris. Int. J. Mol. Sci. 2021, 22, 3677. https://doi.org/10.3390/ 1. Introduction ijms22073677 Myrosinase (EC 3.2.1.147), a β-thioglucosidase, is a part of the plant defence system. Depending on the reaction conditions, it catalyses the hydrolysis of glucosinolates into Academic Editor: Sergio F. Sousa a range of products (Figure1)[ 1]. One family of products, isothiocyanates (ITCs), is reported to have antimutagenic, neuroprotective, and chemo-preventive effects. ITCs Received: 1 March 2021 have been previously linked with the reduction of cancer occurrence [2]. In addition Accepted: 26 March 2021 to their cancer-preventive role, there is evidence showing their anti-tumorigenic action Published: 1 April 2021 against different types of cancer, both in vitro and in vivo. Tumour inhibition by ITCs is mediated through the modulation of critical cancer-related pathways. Due to their low Publisher’s Note: MDPI stays neutral toxicity profile, ITCs are considered to be excellent chemotherapeutic candidates against with regard to jurisdictional claims in tumour initiation and progression [3]. Moreover, increasing attention has been given to published maps and institutional affil- diet-derived molecules that could prevent or suppress carcinogenesis [4], which makes the iations. glucosinolate-myrosinase system an interesting alternative to current cancer treatments. Since its discovery more than 150 years ago, myrosinase has been identified in more than 15 different plants, gut microbes, and fungi. In plants, myrosinases are generally encoded by a multigene family [5]. Arabidopsis thaliana possesses six different myrosinase Copyright: © 2021 by the authors. genes, while Brassica napus was reported to have at least 20 myrosinase isoenzymes, which Licensee MDPI, Basel, Switzerland. are divided into three subclasses based on enzyme activity, solubility, and molecular This article is an open access article weight [6,7]. Therefore, purification of these isoenzymes from their native host is very distributed under the terms and challenging. Recombinant production of plant myrosinases has proven to be a powerful conditions of the Creative Commons tool in the production of a single myrosinase isoenzyme to facilitate their purification and Attribution (CC BY) license (https:// characterisation. Moreover, for pharmaceutical applications, large quantities of myrosinase creativecommons.org/licenses/by/ 4.0/). have to be produced, which would be impossible to achieve by isolating it from plants. Int. J. Mol. Sci. 2021, 22, 3677. https://doi.org/10.3390/ijms22073677 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 2 of 15 Int. J. Mol. Sci. 2021, 22, 3677 2 of 14 Figure 1. Scheme of glucosinolate hydrolysis. Bioactive products formation after myrosinase- Figure 1. Scheme of glucosinolate hydrolysis. Bioactive products formation after myrosinase-cata- catalysed hydrolysis of glucosinolates, which is affected by pH, the epithiospecifier protein (ESP), lysed hydrolysis of glucosinolates, which is affected by pH, the epithiospecifier protein (ESP), and and the structure of the amino acid-derived side chain (R). the structure of the amino acid-derived side chain (R). The first recombinant production of myrosinase was published by Chen and Halkier [7], Since its discovery more than 150 years ago, myrosinase has been identified in more who reported heterologous expression of active MYR1 myrosinase from Brassica napus in thanSaccharomyces 15 different cerevisiae plants, .gut It wasmicrobes, the first and naturally fungi. In complex-bound plants, myrosinases myrosinase are generally to be pro-en- codedduced by with a multigene no cross-contamination family [5]. Arabidopsis by lectins thaliana [7]. This possesses work was six followed different by myrosinase Härtel and genes,Brandt while [8], who Brassica expressed napus was MYR1 reported from B. to napus have inat leastPichia 20 pastoris myrosinase. The previously isoenzymes, observed which areproblems divided with into myrosinase three subclasses expression based in S.on cerevisiae enzymewere activity, solved, solubility, including and the molecular toxicity of weightintracellular [6,7]. Therefore, expression purifi to S. cerevisiaecation ofcells these and isoenzymes low expression from levels.their native host is very challenging.P. pastoris Recombinanthas been used production as an expression of plant myrosinases host for recombinant has proven protein to be a production powerful toolfor morein the thanproduction 20 years of [ a9 ].si Thengle genemyrosinase of interest isoenzyme is usually to fa placedcilitate under their thepurification control of and the characterisation.methanol-inducible Moreover, AOX1 promoter,for pharmaceutical although applications, many other promoterslarge quantities are available. of myrosi- As nasean eukaryotic have to be organism, produced,P. which pastoris wouldcan perform be impossible posttranslational to achieve modifications by isolating withoutit from plants.hyper-glycosylation (common for S. cerevisiae) and can secrete expressed proteins into cultivationThe first medium. recombinant The ability production to grow of P.myrosinase pastoris to was high-cell published densities by Chen on minimal and Halkier media [7],often who leads reported to high heterologous yields of the expressi desiredon protein of active [10 MYR1]. myrosinase from Brassica napus in SaccharomycesAs mentioned cerevisiae previously,. It was myrosinasesthe first naturally have comp beenlex-bound expressed myrosinase in P. pastoris to(Table be pro-1), ducedonly in with Mut no+ strains cross-contamination and mostly intracellularly. by lectins [7]. For This industrial work was applications, followed by extracellular Härtel and Brandtprotein [8], expression who expressed is desirable MYR1 because from B. itnapus greatly in Pichia simplifies pastoris downstream. The previously processing observed [11]. problemsOnly two with cases myrosinase of extracellular expression myrosinase in S. productioncerevisiae were were solved, published: including TGG1 the and toxicity TGG2 ofmyrosinases intracellular from expressionA. thaliana to S.[ 12cerevisiae] and MYR1 cells and myrosinase low expression from B. levels. napus [8]. In both cases, theP. production pastoris has of been myrosinase used as itself an expression was not studied, host for optimised,recombinant or protein upscaled, production and was formostly more used thanfor 20 enzymeyears [9]. property The gene studies. of interest is usually placed under the control of the methanol-inducible AOX1 promoter, although many other promoters are available. As an eukaryoticTable 1. Recombinant organism, expression P. pastoris of can various perform myrosinase posttranslationa genes in Pichial modifications pastoris. without hyper-glycosylation (common for S. cerevisiae) and can secrete expressed proteins into culti- Gene Donor Gene Expression Vector Expression Host Reference vation medium. The ability to grow P. pastoris to high-cell densities on minimal media often leads to high yields of the desired proteinpHIL-S1 [10]. P. pastoris GS115 Arabidopsis thaliana TGG1, TGG2 + [12] As mentioned previously, myrosinases(extracellular) have been expressed (Mutin P. pastoris) (Table 1), TGG1, TGG2 pPIC3.5K P. pastoris GS115 onlyArabidopsis in Mut+ thalianastrains and mostly intracellularly. For industrial applications, extracellular[13] TGG4, TGG5 (intracellular) (Mut+) protein expression is desirable because it greatlypPIC3.5K simplifies downstreamP. pastoris GS115 processing [11]. Armoracia rusticana ArMY2 [14] Only two cases of extracellular myrosinase (intracellular)production were published:(Mut+ )TGG1 and TGG2 myrosinases from

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