molecules Article Constructing an Efficient Bacillus subtilis Spore Display by Using Cohesin−Dockerin Interactions He Wang 1,*,†, Xiaomin Jiang 2,†, Yongchang Qian 3 and Lianghong Yin 3 1 School of Grain Science and Technology, Jiangsu University of Science and Technology, Zhenjiang 212100, China 2 School of Agricultural and Food Sciences, Zhejiang Agriculture and Forestry University, Hangzhou 311300, China; jiang_xiaomin@hs-biopharm.com 3 School of Forestry and Biotechnology, Zhejiang Agriculture and Forestry University, Hangzhou 311300, China; qian3906@zafu.edu.cn (Y.Q.); ylh4@163.com (L.Y.) * Correspondence: wh2989@just.edu.cn; Tel.: +86-511-84423559 † These authors contributed equally to this work. Abstract: Bacillus subtilis spore display has become a field of increasing interest in the past two decades. To improve the efficiency of B. subtilis spore display, its directed modification was performed based on the cellulosome architecture by introducing onto them divergent cohesin (Coh) modules that can specifically bind to the target enzyme bearing the matching dockerins (Doc). In this study, five different pairs of cohesins and dockerins, selected from four cellulolytic microbes, were examined for their capabilities in displaying a tetrameric enzyme β-galactosidase from Bacillus stearothermophilus IAM11001 on the surface of B. subtilis WB600 spores. Immunofluorescence microscopy, western blotting, dot blotting, and enzyme assay was applied to confirm its surface expression. All the resultant five Coh–Doc based spore display can hydrolyze o-nitrophenyl-β-D-galactopyranoside. Citation: Wang, H.; Jiang, X.; Qian, Further, the optimized Coh–Doc based spore display exhibited the highest display efficiency. Overall, Y.; Yin, L. Constructing an Efficient the results of current study may open new perspectives on the use of Coh–Doc interaction, which Bacillus subtilis Spore Display by will find application in improving the efficiency of B. subtilis spore display. Using Cohesin−Dockerin Interactions. Molecules 2021, 26, 1186. Keywords: spore display; Bacillus subtilis; cohesin dockerin interaction; β-galactosidase; display effi- https://doi.org/10.3390/ ciency molecules26041186 Academic Editor: Roberto Fernandez-Lafuente 1. Introduction It has been 20 years since Isticato and colleagues described the use of CotB to anchor Received: 13 January 2021 the C-terminal fragment of tetanus toxin on the Bacillus subtilis spores for developing a Accepted: 21 February 2021 Published: 23 February 2021 vaccine against tetanus [1]. B. subtilis spore surface display, referring to the expression of desired enzyme or antigen (target protein) on the surface of spore via fusion with a Publisher’s Note: MDPI stays neutral spore coat protein (anchoring motif), has been proven to be a powerful tool in fields of with regard to jurisdictional claims in vaccine development, enzyme immobilization, as well as the delivery of cancer drug and published maps and institutional affil- human-related proteins [2,3]. The B. subtilis spore surface display offers natural advantages iations. that include high safety, outstanding stability, and unique ability to display a broad variety of biomolecules, like antigens, tetrameric enzymes and proteins [4]. Currently, spore surface display can be accomplished through non-genetic and genetic approaches [5]. The non-genetic method highly depends on passive adsorption between the spore surface and the target protein, which involves electrostatic forces, hydrogen Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. bonding, and hydrophobic interactions. However, those nonspecific affinities lead to This article is an open access article uncontrollable adsorption, and the storage stability of the adsorbed protein is sometimes distributed under the terms and unsatisfactory [6]. The genetic method is completed by directly fusing anchoring motifs conditions of the Creative Commons such as CotB, CotC, and CotG with target proteins. In this manner, a strong covalent Attribution (CC BY) license (https:// bond between the anchoring motif and target protein can be created. However, the loss of creativecommons.org/licenses/by/ catalytic activity is challenging, since most of the spore displayed proteins are buried in 4.0/). the coat layer, which makes it inaccessible for the binding of substrates [7]. Molecules 2021, 26, 1186. https://doi.org/10.3390/molecules26041186 https://www.mdpi.com/journal/molecules Molecules 2021, 26, x FOR PEER REVIEW 2 of 17 Molecules 2021, 26, 1186 between the anchoring motif and target protein can be created. However, the loss of2 ofcata- 16 lytic activity is challenging, since most of the spore displayed proteins are buried in the coat layer, which makes it inaccessible for the binding of substrates [7]. Despite the great strides achieved in the past two decades, B. subtilis spore display Despite the great strides achieved in the past two decades, B. subtilis spore display stillstill suffers suffers from from low low expression expression level level of of the the protein protein of of interest, interest, thus thus rendering rendering it unsuitable it unsuita- forble industrialfor industrial applications. applications. Tavassoli Tavassoli et al.et al [8. ][8] observed observed that that the the CotC CotC deficient deficient spores spores displayingdisplaying B.B. subtilis 168 ββ-galactosidase-galactosidase retained retained 1.6-fold 1.6-fold higher higher residual residual activity activity than than the thewild-type wild-type spores spores after after three three reuses, reuses, although although both both exhibited exhibited the the same same initial initial enzyme enzyme ac- activitytivity and and expression expression level. level. The The work work of ofNg Nguyenuyen and and Schumann Schumann [9] [ also9] also indicated indicated that that the isopropyl-β-D-thiogalactoside (IPTG)-inducible promoter PSgrac could greatly increase the the isopropyl-β-D-thiogalactoside (IPTG)-inducible promoter PSgrac could greatly increase theexpression expression of Bacillus of Bacillus amyloliquefaciens amyloliquefaciens α-amylaseα-amylase Q (AmyQ), Q (AmyQ), but it but gave it gavethe lowest the lowest activ- ity (1.5 U) as compared to those obtained with Pgrac (3 U) and PcotB (2.5 U). It is noted that activity (1.5 U) as compared to those obtained with Pgrac (3 U) and PcotB (2.5 U). It is noted thatthere there was wasa big a difference big difference in the in number the number of CotB of CotB∆–AmyQD–AmyQ molecule molecule displayed displayed per spore per grac cotB sporebetween between the P the andPgrac Pand promoter,PcotB promoter, in which in which the former the former was wascalculated calculated to be to almost be almost two twotimes times higher higher than thanthe latter. the latter. Collectively, Collectively, these theseobservations observations suggest suggest that a small that a portion small portionof fusion of fusionproteins proteins could exist could in exist an enzymatically in an enzymatically inactive inactive form formsurrounding surrounding the spore the sporesurface, surface, a restriction a restriction likely likely be due be dueto the to result the result of low of lowcatalytic catalytic efficiency efficiency arising arising from from the thesteric steric hindrance hindrance between between anchoring anchoring motif motif and and the the displayed displayed enzyme. enzyme. CellulosomesCellulosomes are extracellular extracellular large large multienzyme multienzyme complexes complexes that that are arecommonly commonly pro- producedduced by bymany many different different anaerobic anaerobic cellulolytic cellulolytic bacteria bacteria [10]. [10 ].Each Each of ofthem them contains contains one one or ormore more cohesin cohesin modules modules that that interact interact with with complementary complementary modules modules termed termed dockerins. dockerins. The Thecohesin-dockerin cohesin-dockerin (Coh–Doc) (Coh–Doc) interaction interaction is considered is considered to be to beone one of ofnature’s nature’s strongest strongest bi- bimolecularmolecular interactions. interactions. Furthermore, Furthermore, thethe bindingbinding affinity affinity between between cohesin cohesin and and dockerin dockerin is notis not influenced influenced by the byfused the fused enzyme enzyme and the and linker the [linker11]. The [11]. architecture The architecture and unique and subunit unique arrangementsubunit arrangement of the cellulosome of the cellulosome can have thecan genetichave the potential genetic topotential create a to robust createB. a subtilis robust sporeB. subtilis display spore platform display by platform the Coh–Doc by the interaction. Coh–Doc interaction. Inspired from Inspired these observations,from these obser- the firstvations, evidence the first is the evidence expression is the of expressionEscherichia coliof Escherichiaβ-galactosidase coli β-galactosidase on the spore surface on the spore with asurface controllable with mannera controllable through manner Coh–Doc through interaction Coh–Doc [7]. Introducinginteraction [7]. Coh–Doc Introducing interaction Coh– mightDoc interaction relieve the might spatial relieve barrier the between spatial barrier the anchoring between motif the anchoring and the displayed motif and protein, the dis- thusplayed contributing protein, thus to its contributing elevated catalytic to its elevated efficiency. catalytic efficiency. TheThe objectiveobjective ofof thisthis researchresearch waswas toto improveimprove thetheperformance performance ofofB.