Development of CRISPR Interference (Crispri) Platform for Metabolic Engineering of Leuconostoc Citreum and Its Application for Engineering Riboﬂavin Biosynthesis

International Journal of Molecular Sciences

Article Development of CRISPR Interference (CRISPRi) Platform for Metabolic Engineering of Leuconostoc citreum and Its Application for Engineering Riboﬂavin Biosynthesis

Jaewoo Son 1, Seung Hoon Jang 1, Ji Won Cha 1 and Ki Jun Jeong 1,2,*

1 Department of Chemical and Biomolecular Engineering, BK21 Plus program, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon 34141, Korea; [email protected] (J.S.); [email protected] (S.H.J.); [email protected] (J.W.C.) 2 Institute for The BioCentury, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon 34141, Korea * Correspondence: [email protected]; Tel.: +82-42-350-3934

Received: 1 July 2020; Accepted: 3 August 2020; Published: 5 August 2020

Abstract: Leuconostoc citreum, a hetero-fermentative type of lactic acid bacteria, is a crucial probiotic candidate because of its ability to promote human health. However, inefficient gene manipulation tools limit its utilization in bioindustries. We report, for the first time, the development of a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) interference (CRISPRi) system for engineering L. citreum. For reliable expression, the expression system of synthetic single guide RNA (sgRNA) and the deactivated Cas9 of Streptococcus pyogenes (SpdCas9) were constructed in a bicistronic design (BCD) platform using a high-copy-number plasmid. The expression of SpdCas9 and sgRNA was optimized by examining the combination of two synthetic promoters and Shine–Dalgarno sequences; the strong expression of sgRNA and the weak expression of SpdCas9 exhibited the most significant downregulation (20-fold decrease) of the target gene (sfGFP), without cell growth retardation caused by SpdCas9 overexpression. The feasibility of the optimized CRISPRi system was demonstrated by modulating the biosynthesis of riboflavin. Using the CRISPRi system, the expression of ribF and folE genes was downregulated (3.3-fold and 5.6-fold decreases, respectively), thereby improving riboflavin production. In addition, the co-expression of the rib operon was introduced and the production of riboflavin was further increased up to 1.7 mg/L, which was 1.53 times higher than that of the wild-type strain.

Keywords: lactic acid bacteria; Leuconostoc citreum; CRISPRi; synthetic biology; riboﬂavin

1. Introduction Leuconostoc spp. is a hetero-fermentative type of lactic acid bacteria that plays a significant role in the fermentation of products such as kimchi, milk, vegetables and meat [1]. Recently, Leuconostoc spp. has been in the spotlight as one of the crucial candidates for use as a probiotic, as it has the ability to produce vitamins and bioactive antimicrobial peptides, and has also been shown to promote human health by regulating immune responses [2–4]. Furthermore, it has distinct advantages, such as its categorization as a “Generally Recognized As Safe” (GRAS) strain [5], its enhanced protein secretion ability [6,7], and its capacity to produce exopolysaccharides that are recognized to have therapeutic properties [8–10]; thus, Leuconostoc spp. is desirable as a next-generation probiotic that can be used to deliver pharmaceutical proteins in humans or animals and can be applied in various biomedical fields. Although Leuconostoc spp. has potential advantages in a variety of bioindustry fields, extensive research has not been conducted on the engineering of Leuconostoc spp. due to the lack of genetic

Int. J. Mol. Sci. 2020, 21, 5614; doi:10.3390/ijms21165614 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 5614 2 of 13 manipulation tools. Particularly, the engineering of bacterial hosts, including Leuconostoc spp., can enhance their performance by giving them desired properties, which can be achieved by the extensive manipulation of the host genome, such as the elimination or downregulation of unnecessary genes and/or the overexpression of essential genes. However, such manipulation of genes requires very precise tools for genome editing [11,12]. Recently, various genome-editing tools that can be used in lactic acid bacteria (LAB) have been introduced, including homologous recombination and the recombinase-mediated insertion or deletion of target genes [13–15]. The development of the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)–Cas9 system as a counter-selection tool in combination with existing recombineering tools has improved the genome editing efficiency in LAB [16]. Indirect genome modification tools using catalytically deactivated Cas9 (dCas9) can make it possible to effectively regulate the expression of target genes without permanently removing the genes from chromosomes. This system, called CRISPR interference (CRISPRi), can regulate the expression of a target gene by blocking RNA polymerase through the interaction of dCas9 with synthetic guide RNA, which has a complementary sequence to the target gene (Figure1A) [17,18]. However, most of these tools are available only for Lactococcus spp. and Lactobacillus spp.; genome-editing tools that can be optimized and used for Leuconostoc spp. are very limited. Here, we developed a CRISPRi system for regulating the knock-down of gene expression in L. citreum and used this tool for the engineering of L. citreum to increase riboflavin biosynthesis. Recently, we developed several synthetic parts (high-copy-number plasmids, and promoters and Shine–Dalgarno sequences (SD) with different strengths) for the fine tuning of gene expression in L. citreum [19,20]. Using these synthetic parts, we first developed CRISPRi for the reliable expression of CRISPRi components such as synthetic single guide RNA (sgRNA) and the catalytically deactivated Cas9 of Streptococcus pyogenes (SpdCas9) [21,22]. To reduce the toxicity caused by the overexpression of SpdCas9, the expression of SpdCas9 is optimized by combining synthetic expression parts (promoters and SDs) to produce a bicistronic design (BCD) expression platform. Finally, to demonstrate the feasibility of optimized CRISPRi on the modulation of metabolite biosynthesis, we engineered the riboflavin biosynthesis pathway in L. citreum. The titer of riboflavin is remarkably increased by employing a combination of two strategies: (i) regulating the knock-down of the branched folate synthesis pathway and a loss of the capability of riboflavin to convert into a flavin mononucleotide (FMN) using CRISPRi and (ii) the overexpression of the rib operon (ribA, ribG, ribH, and ribB).

2. Results and Discussion

2.1. Construction of CRISPRi in the BCD Platform To implement the CRISPRi platform in L. citreum, we employed SpdCas9 derived from a type II CRISPR system [23–25]. It is generally known that a high concentration of sgRNA in cells is required for the effective operation of CRISPRi [26]. Therefore, to provide a high amount of sgRNA, we first constructed sgRNA and SpdCas9 expression cassettes in a high-copy-number plasmid (pCB4270) that is present at about 60 copies per cell in L. citreum [19]. For the reliable expression of SpdCas9, the expression cassette was constructed in a bicistronic design (BCD) platform, which we had previously developed to induce the reliable expression of recombinant proteins in L. citreum [20]. In the BCD, the first cistron consisting of the first SD sequence (SD1) and a short peptide (17 amino acids) was cloned in a downstream region of the high strength constitutive promoter (P710V4) (Figure1B). An engineered second SD sequence (eSD2) that had a high translational initiation property [20] was cloned in the 3’-end of the first cistron. It served as a ribosome binding site (RBS) for the translation of SpdCas9 (second cistron) by translational coupling (Figure1B). To evaluate the operation of the CRISPRi platform in L. citreum, an expression cassette for sgRNA targeting the superfolder green fluorescent protein (sfGFP) gene was constructed upstream of the SpdCas9 expression cassette in the same plasmid (Figure1B). Previous studies have shown that targeting the non-template strand of a target gene in a type II CRISPR system shows higher transcriptional repression efficiency than targeting the template strand [21,22]. Int. J. Mol. Sci. 2020, 21, 5614 3 of 13 Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 3 of 13 We constructed GFP-sgRNA comprising a 20-base sequence complementary to the non-template target comprising a 20-base sequence complementary to the non-template target region next to the region next to the protospacer adjacent motif (PAM) sequence, located at the most upstream position protospacer adjacent motif (PAM) sequence, located at the most upstream position in the sfGFP in the sfGFP coding sequence (Table S3 and Figure S1A). coding sequence (Table S3 and Figure S1A).

FigureFigure 1. 1.( A(A)) Schematic Schematic of of the the CRISPRi CRISPRi system. system (B. (B)) Schematic Schematic diagram diagram of the of the plasmid plasmid pGFP–sgR–D1–Em pGFP–sgR–D1– constructedEm constructed for the for CRISPRithe CRISPRi system system for L.for citreum. L. citreum.Dashed Dashed and and solid solid boxes boxes in in the the DNA DNA sequences sequences indicateindicate the the stop stop codon codon of of the the ﬁrst first cistron cistron and and start start codon codon of of second second cistron cistron (SpdCas9), (SpdCas9), respectively. respectively.

TheThe abilityability ofof thethe SpdCas9-mediatedSpdCas9-mediated CRISPRiCRISPRi systemsystem (pGFP–sgR–D1–Em)(pGFP–sgR–D1–Em) toto regulateregulate thethe expressionexpression ofof thethe sfGFPsfGFP genegene waswas evaluatedevaluated usingusing L.L. citreumcitreum JW001,JW001, inin whichwhich thethe sfGFPsfGFP genegene expressionexpression cassette cassette was was integrated integrated into into the ldhDthe locusldhD oflocusL. citreum of L. CB2567.citreum L.CB2567. citreum L.JW001 citreum harboring JW001 pGFP–sgR–D1–Emharboring pGFP–sgR–D1–Em was cultured, was and thecultured, change and of fluorescence the change intensity of fluorescence (FI) was analyzedintensity by(FI) a flowwas cytometer.analyzed by As a shown flow cytometer. in Figure2 A,As cellsshown harboring in Figure pGFP–sgR–D1–Em 2A, cells harboring showed pGFP–sgR–D1–Em a significantly showed reduced a FIsignificantly compared toreduced that of FIL. compared citreum JW001 to that without of L. citreum the plasmid JW001 without used as the a positiveplasmid control,used as a and positive also showedcontrol, a and similar also FIshowed as that a of similar wild-type FI as (WT) that L.of citreumwild-type(no (WT) expression L. citreum of sfGFP) (no expression as a negative of sfGFP) control. as Ina contrast,negative thecontrol. cells harboringIn contrast, pGFP–sgR–Em the cells harbor (expressioning pGFP–sgR–Em of sgRNA alone)(expression or pD1–Em of sgRNA (expression alone) ofor SpdCas9pD1–Em alone) (expression exhibited of SpdCas9 similar fluorescence alone) exhibited intensities similar (FIs) fluorescence as those of theintensities positive (FIs) control as those (L. citreum of the JW001)positive (Figure control2A). (L. These citreum results JW001) indicated (Figure that 2A). the These reduced results sfGFP indicated expression that level the wasreduced due tosfGFP the eexpressionffective operation level was of CRISPRi,due to the and effective was not operat a phenomenonion of CRISPRi, that occurs and was by thenot expressiona phenomenon of either that sgRNAoccurs by or SpdCas9.the expression of either sgRNA or SpdCas9. InIn thethe CRISPRiCRISPRi system,system, anan RNA–proteinRNA–protein complexcomplex ofof sgRNAsgRNA andand SpdCas9SpdCas9 preventsprevents RNARNA polymerasepolymerase fromfrom binding binding toto the the target target DNA, DNA, resulting resulting in in the the repression repression of of target target gene gene transcription transcription (Figure(Figure1 A)1A) [ 22[22].]. InIn ourour study,study, this this repression, repression, which which occurred occurred due due to to the the cooperation cooperation of of sgRNA sgRNA and and SpdCas9,SpdCas9, was was confirmed confirmed by analyzingby analyzing the transcriptionthe transcription level oflevel the sfGFPof the gene sfGFP under gene the under co-expression the co- ofexpression sgRNA and of/ orsgRNA SpdCas9. and/or After SpdCas9. the cultivation After the of cells,cultivation total mRNA of cells, was total purified mRNA from was each purified strain, from and theeach transcription strain, and levelthe transcription of sfGFP was level quantified of sfGFP by wa quantitatives quantified reverse by quantitative transcription reverse PCR transcription (qRT-PCR). ComparativePCR (qRT-PCR). analysis Comparative of the mRNA analysis clearly of indicatedthe mRNA that clearly the mRNA indicated transcription that the mRNA level for transcription the sfGFP genelevel was for reducedthe sfGFP by gene more was than reduced 90% in the by cells more expressing than 90% both in the sgRNA cells and expressing SpdCas9 both compared sgRNA to theand positiveSpdCas9 control compared (L. citreum to theJW001) positive (Figure control2B). (L. We citreum concluded JW001) that (Figure the constructed 2B). We concluded CRISPRi systemthat the introducedconstructed in CRISPRiL. citreum systemeffectively introduced regulated in the L. expressioncitreum effectively of the chromosomal regulated the gene expression in L. citreum of. the In thischromosomal experiment, gene however, in L. citreum one unexpected. In this experiment, result—an however, abnormal one elevated unexpected level ofresult—an mRNA transcript abnormal forelevated the target level gene of mRNA (sfGFP)—was transcript identified for the intargetL. citreum gene (sfGFP)—wasJW001, harboring identified pD1–Em, in L. expressing citreum JW001, only SpdCas9harboring without pD1–Em, sgRNA, expressing while L.only citreum SpdCas9JW001 without harboring sgRNA, pGFP–sgR–Em while L. citreum expressing JW001 only harboring sgRNA pGFP–sgR–Em expressing only sgRNA without SpdCas9 exhibited a similar level to the positive control (Figure 2B). In addition, when we checked the cell growth during flask cultivation, the

Int. J. Mol. Sci. 2020, 21, 5614 4 of 13

Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 4 of 13 without SpdCas9 exhibited a similar level to the positive control (Figure2B). In addition, when we Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 4 of 13 retardation ofchecked cell growth the cell under growth the duringoverexpression ﬂask cultivation, of SpdCas9 the retardation (L. citreum of JW001 cell growth harboring under either the overexpression pGFP–sgR–D1–Emofretardation SpdCas9 or pD1–Em) ( L.of citreumcell was growth JW001also observedunder harboring the (Figure overexpression either 2C). pGFP–sgR–D1–Em of SpdCas9 (L. or citreum pD1–Em) JW001 was harboring also observed either (FigurepGFP–sgR–D1–Em2C). or pD1–Em) was also observed (Figure 2C).

Figure 2. (A) FigureThe histogram 2. (A) The histogramof fluorescence-activated of fluorescence-activated cell-sorting cell-sorting (FACS) (FACS) analysis analysis for sfGFP for sfGFP expression. expression. TheThe foldFigure fold changes changes2. (A) of offluorescenceThe fluorescence histogram intensit intensity of fluorescence-activatedy in in the the FACS FACS analysis analysis werecell-sorting were plotted plotted (FACS)in the right analysis graph. for (B )sfGFP The graph. (B) The quantificationquantificationexpression. The of sfGFP sfGFPfold changes transcripts transcripts of fluorescence by by qRT-PCR. qRT-PCR. intensit NS NS represen representsy in thets nonsignificantFACS nonsignificant analysis (werep-value (p-value plotted > 0.05),in the ****right > 0.05), **** p-valuep-valuegraph. < 0.0001< (B)0.0001 The based quantification based on Student on Student of t-test. sfGFPt-test. N, transcripts L. N,citreumL. citreum byCB2567; qRT-PCR.CB2567; P, L. NS citreum P, represenL. citreum JW001;ts nonsignificantJW001; 1, L. 1, L. citreum (p-value citreum JW001 JW001harboring> 0.05), harboring **** pGFP–sgR–D1–Em; p-value pGFP–sgR–D1–Em; < 0.0001 based 2, L. 2,oncitreumL. Student citreum JW001 JW001t-test. harboring N, harboring L. citreum pGFP–sgR–Em; pGFP–sgR–Em; CB2567; P, L.3, 3,citreum L.L. citreum JW001;JW001 1, L. citreum JW001 harboringcitreum JW001pD1–Em. harboring (C) (C) Time Time pGFP–sgR–D1–Em; profile profile of cell growth. 2, L.The The citreum growth growth JW001 curve curve harboring was was presented presented pGFP–sgR–Em; as an average 3, L. as an average valuecitreum from JW001 the biologicalbiologic harboringal replicates’ pD1–Em. cell(C) Time culture.culture. profile Symbols: of cell ■ growth.; ;L.L. citreum citreum The JW001,JW001,growth ◆curve;; L. citreum was presentedJW001 harboringas an average pGFP–sgR–D1–Em, value from the Nbiological; L. citreum replicates’JW001 harboring cell culture. pGFP–sgR–Em, Symbols: ■; L. citreum; L. citreum JW001,JW001 ; L. citreum JW001 harboring pGFP–sgR–D1–Em, ▲; L. citreum JW001 harboring pGFP–sgR–Em, •●; L. ◆ citreum JW001 harboringcitreum JW001pD1–Em. harboring pGFP–sgR–D1–Em, ▲; L. citreum JW001 harboring pGFP–sgR–Em, ●; L. citreum JW001 harboring pD1–Em. 2.2. Optimization of CRISPRi System Using Synthetic Parts 2.2. Optimization of CRISPRi System Using Synthetic Parts 2.2.Previous Optimization studies of CRISPRi have confirmed System Using that Synthetic the overexpression Parts of SpdCas9 in many bacteria causes Previous studies have confirmed that the overexpression of SpdCas9 in many bacteria causes cytotoxicity to host cells, which results in a significant reduction of cell growth [27]. In the case of cytotoxicity to host cells,Previous which studies results have in a confirmed significant that reduction the overex of cellpression growth of [27]. SpdCas9 In the casein many of E. bacteria causes E. coli, abnormal morphological changes of cells were induced, and transcriptome analysis showed coli, abnormal morphologicalcytotoxicity to changeshost cells, of whichcells were results induced, in a significant and transcriptome reduction analysis of cell growth showed [27]. that In the case of E. that the overexpression of SpdCas9 affected the transcriptional levels of various genes in the host [28]. the overexpressioncoli, abnormalof SpdCas9 morphological affected the transcriptional changes of cells levels were of induced, various genesand transcriptome in the host [28]. analysis As showed that As shown in Figure2, we also had a similar growth retardation problem and abnormal gene expression shown in Figurethe 2, overexpressionwe also had a similar of SpdCas9 growth affect retardationed the transcriptional problem and levelsabnormal of various gene expression genes in the host [28]. As under the overexpression of SpdCas9. The exact mechanism of the effects of SpdCas9 overexpression under the overexpressionshown in Figure of SpdCas9. 2, we alsoThe hadexact a mesimilarchanism growth of the retardation effects of problemSpdCas9 andoverexpression abnormal gene expression on cells is not yet clearly identified; however, it is known that cytotoxicity due to the overexpression on cells is not yetunder clearly the identified;overexpression however, of SpdCas9. it is known The thatexact cytotoxicity mechanism due of the to theeffects overexpression of SpdCas9 overexpression of SpdCas9 places limitations on the effective introduction of the CRISPRi system into hosts [29]. of SpdCas9 placeson cells limitations is not yet on clearly the effective identified; intr however,oduction itof is the known CRISPRi that cytotoxicitysystem into duehosts to [29].the overexpression Therefore, we decided to optimize the CRISPRi system by fine tuning the expression level of SpdCas9 Therefore, we decidedof SpdCas9 to optimize places thelimitations CRISPRi on syst theem effective by fine tuning introduction the expression of the CRISPRi level of SpdCas9 system into hosts [29]. with synthetic parts to induce the effective operation of the CRISPRi system while reducing the with synthetic Therefore,parts to induce we decided the effective to optimize oper theation CRISPRi of the syst CRISPRiem by finesystem tuning while the reducing expression the level of SpdCas9 cytotoxicity of SpdCas9 in L. citreum. cytotoxicity of SpdCas9with synthetic in L. citreum parts .to induce the effective operation of the CRISPRi system while reducing the To controlcytotoxicity SpdCas9 expressionof SpdCas9 in L.a citreumgradient. manner, we constructed four sets of BCD combinations (D1, D2,To D3,control and D4)SpdCas9 that enable expression the expression in a gradient of SpdCas9 manner, (without we theconstructed co-expression four sets of BCD combinations (D1, D2, D3, and D4) that enable the expression of SpdCas9 (without the co-expression of sgRNA) at various intensities using two different strength promoters (strong P710V4 and weak P710)

Int. J. Mol. Sci. 2020, 21, 5614 5 of 13

To control SpdCas9 expression in a gradient manner, we constructed four sets of BCD combinations (D1, D2, D3, and D4) that enable the expression of SpdCas9 (without the co-expression of sgRNA) at Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 5 of 13 various intensities using two diﬀerent strength promoters (strong P710V4 and weak P710) and SD parts (strong eSD2 and weak SD2): D1 was constructed with P710V4 and eSD2; D2 with P710V4 and SD2; D3 and SD parts (strong eSD2 and weak SD2): D1 was constructed with P710V4 and eSD2; D2 with P710V4 with P710 and eSD2; and D4 with P710 and SD2 (Figure3A). After the cultivation of the cells in each Int. J. Mol. Sci. 2019, 20, x FOR PEERand SD2;REVIEW D3 with P710 and eSD2; and D4 with P710 and SD2 (Figure 3A).4 of After13 the cultivation of the combination, the SpdCas9 expression level in each cell group was determined by SDS-PAGE, and cells in each combination, the SpdCas9 expression level in each cell group was determined by SDS- we found that the expression of SpdCas9 could be ﬁne tuned by a combination of synthetic parts in retardation of cell growthPAGE, under and the overexpressionwe found that ofthe SpdCas9 expression (L. citreumof SpdCas9 JW001 could harboring be fine either tuned by a combination of BCD platforms. D1 expressed the highest expression of SpdCas9, and a moderate expression was pGFP–sgR–D1–Em or pD1–Em)synthetic was parts also in observed BCD platforms. (Figure 2C).D1 expressed the highest expression of SpdCas9, and a moderate observed in D2 and D3; above all, the combination of weak P710 and weak SD2 (D4) exhibited the expression was observed in D2 and D3; above all, the combination of weak P710 and weak SD2 (D4) weakest expression of SpdCas9 (Figure3B). exhibited the weakest expression of SpdCas9 (Figure 3B).

Figure 2. (A) The histogramFigure of 3. fluorescence-activatedOptimization of the CRISPRi cell-sorting system. (FACS) (A) Schematicanalysis for illustration sfGFP of the BCD versions of expression. The fold changesSpdCas9.Figure of fluorescence 3. (B Optimization) Analysis intensit of SpdCas9 ofy inthe the CRISPRi in FACS the BCD analysis system. system were (A) by SDS-PAGE.Schematicplotted in theillustration Lane right M; molecular of the weightBCD versions markers of graph. (B) The quantification(kDa).SpdCas9. of sfGFP Lanes (B) transcripts D1–D4 Analysis represent by of qRT-PCR. SpdCas9L. citreum NS in represenJW001the BCD harboringts system nonsignificant pD1–Em,by SDS-PAGE. (p-value pD2–Em, Lane pD3–Em, M; molecular or pD4–Em, weight > 0.05), **** p-value < 0.0001respectively.markers based on (kDa). TheStudent arrowheadLanes t-test. D1–D4 N, (red L. represent triangle)citreum CB2567; indicatesL. citreum P, the JW001L. band citreum harboring of SpdCas9JW001; pD1–Em,1, (~158 L. kDa). pD2–Em, (C) Quantification pD3–Em, or citreum JW001 harboring ofpGFP–sgR–D1–Em;pD4–Em, sfGFP mRNA respectively. by qRT-PCR. 2, L. The citreum P;arrowheadL. citreumJW001 (red JW001.harboring triang D1–D4,le) pGFP–sgR–Em; indicates same as the (B). band (3,D )L. Time of SpdCas9 profiles of(~158 cell kDa) growth.. (C) citreum JW001 harboring pD1–Em.Symbols:Quantification (C); L.Time citreum of profile sfGFPCB2567 ofmRNA cell harboring growth. by qRT-PCR. The pCB4270, growth P; L. curve ;citreumL. citreum was JW001. presentedJW001 D1–D4, harboring same pCB4270–Em, as (B). (D) TimeN; as an average value fromL. thepro citreum biologicfiles CB2567of alcell replicates’ harboringgrowth. cellSymbols: pD1, culture.H; L.□ ; citreumSymbols: L. citreumCB2567 ■; CB2567L. citreum harboring harboring JW001, pD2, ◆pCB4270,;; L.L. citreum ■CB2567; L. citreum harboring JW001 pD3,harboring; L. citreum pCB4270–Em,CB2567 harboring ▲; L. citreum pD4. CB2567 The growth harboring curve pD1, was presented▼; L. citreum as anCB2567 average harboring value from pD2, citreum JW001 harboring pGFP–sgR–D1–Em,• ▲; L. citreum JW001 harboring pGFP–sgR–Em, ●; L. citreum JW001 harboring pD1–Em.the◆ biological; L. citreum replicates’ CB2567 harboring cell culture. pD3, ●; L. citreum CB2567 harboring pD4. The growth curve was presented as an average value from the biological replicates’ cell culture. 2.2. Optimization of CRISPRi WeSystem compared Using Synthetic the transcription Parts level of the sfGFP gene under each combination by qRT-PCR. The comparativeWe compared analysis the transcription of the mRNA level clearly of the indicated sfGFP gene that under the combination each combination of weak by expression qRT-PCR. Previous studies havepartsThe confirmed wascomparative highly that eff analysisective the overex in of the thepression down mRNA regulation of clearlySpdCas9 ofindi sfGFPincated many transcription that bacteria the combination causes (Figure 3C). of Compared weak expression with cytotoxicity to host cells,the partswhich D1 was combination, results highly in aeffective significant exhibiting in the reduction the down highest regulation of SpdCas9cell growth of expression,sfGFP [27]. transcription In the all case the otherof (Figure E. combinations 3C). Compared (D2, D3,with coli, abnormal morphologicalandtheD4) D1 changes combination, showed of cells remarkably wereexhibiting induced, reduced the andhighest levels transcriptome SpdCas of sfGFPs,9 expression, analysis which were showed all similarthe thatotherto combinations those of the positive (D2, D3, the overexpression of SpdCas9controland D4) (affectL. citreumshoweded theJW001) remarkably transcriptional (Figure reduced3C). levels Among levels of various the of foursfGF genes combinations,Ps, whichin the hostwere the [28]. similar D4 As combination to those of with the a positive weak shown in Figure 2, we alsopromotercontrol had a ( similar andL. citreum a weak growth JW001) SD retardation showed (Figure a 3C). higherproblem Among reduction and theabnormal four (65%) co gene inmbinations, the expression transcription the D4 levelcombination than D1, with but a under the overexpressionweak of SpdCas9. promoter The and exact a weak mechanism SD showed of the a effects higher of re SpdCas9duction (65%)overexpression in the transcription level than D1, on cells is not yet clearly butidentified; displayed however, a similar it is level known to thethat positive cytotoxicity contro duel (Figure to the overexpression 3C). With these four combinations, we of SpdCas9 places limitationsalso confirmed on the effective that the intr celloduction growth of gradually the CRISPRi recovered system as into the hostsexpression [29]. of SpdCas9 decreased. Therefore, we decided to Similaroptimize to the the CRISPRi qRT-PCR syst results,em by the fine D4 tuning combination the expression showed level the highest of SpdCas9 cell growth rate and recovery with synthetic parts to induceof cell growth,the effective with upoper toation 86% forof thethe CRISPRinegative (systemL. citreum while CB2567 reducing harboring the pCB4270) and positive cytotoxicity of SpdCas9 in L. citreum. To control SpdCas9 expression in a gradient manner, we constructed four sets of BCD combinations (D1, D2, D3, and D4) that enable the expression of SpdCas9 (without the co-expression

Int. J. Mol. Sci. 2020, 21, 5614 6 of 13 displayed a similar level to the positive control (Figure3C). With these four combinations, we also confirmedInt. J. Mol. Sci. that 2019 the, 20 cell, x FOR growth PEER graduallyREVIEW recovered as the expression of SpdCas9 decreased. Similar6 of 13 to the qRT-PCR results, the D4 combination showed the highest cell growth rate and recovery of cell growth,controls with (L. upcitreum to 86% JW001 for the harboring negative (pCB4270–Em)L. citreum CB2567 when harboring the stationary pCB4270) phase and was positive reached controls (18 h) (L.(Figure citreum 3D).JW001 harboring pCB4270–Em) when the stationary phase was reached (18 h) (Figure3D). WeWe examined examined whether whether the the gene gene knock-down knock-down effi efficiciencyency of of the the target target gene gene could could be be properly properly maintainedmaintained in in cells cells by by cooperating cooperating with with sgRNA sgRNA even even when when the the expression expression of of SpdCas9 SpdCas9 was was reduced. reduced. MaintainingMaintaining a propera proper concentration concentration between between sgRNA sgRNA and and SpdCas9 SpdCas9 is is important important for for the the eff effectiveective repressionrepression of of target target genes genes in in the the CRISPRi CRISPRi system system [30 [30].]. Therefore, Therefore, when when GFP-sgRNA GFP-sgRNA was was transcribed transcribed usingusing the the strong strong promoter promoter (P 710V4(P710V4),), the the downregulation downregulation of of the the sfGFP sfGFP gene gene in in each each SpdCas9 SpdCas9 expression expression setset (D1–D4) (D1–D4) occurred, occurred, which which was verifiedwas verified by qRT-PCR. by qRT-PCR. The comparative The comparative analysis ofanalysis the mRNA of the revealed mRNA thatrevealed the transcription that the transcription level of sfGFP level was of sfGFP remarkably was remarkably reduced by reduced the co-expression by the co-expression of GFP-sgRNA of GFP- andsgRNA SpdCas9, and SpdCas9, and its level and its was level gradually was gradually regulated regulated by the by SpdCas9 the SpdCas9 expression expression level level (Figure (Figure4). Cells4). harboringCells harboring pGFP–sgR–D1–Em pGFP–sgR–D1–Em (strong expression(strong expression of SpdCas9) of showed SpdCas9) the highestshowed transcription the highest level,transcription but the cells level, harboring but the cells pGFP–sgR–D4–Em harboring pGFP–s (weakgR–D4–Em expression (weak of expression SpdCas9) showed of SpdCas9) the lowest showed transcriptionthe lowest transcription level, which was level, below which 5% was of the below positive 5% of control the positive (L. citreum controlJW001) (L. citreum (Figure 4JW001)). We found (Figure that4). theWe combination found that the of combination sgRNA expression of sgRNA under expression strong P710V4 underwith strong D4 (weakP710V4 with expression D4 (weak of SpdCas9)expression wasof theSpdCas9) most optimized was the most system opti ofmized CRISPRi system for ofL. citreumCRISPRi. for L. citreum.

Figure 4. Quantification of sfGFP mRNA by qRT-PCR. P, L. citreum JW001; D1, L. citreum JW001 harboringFigure 4. pGFP–sgR–D1–Em; Quantification of D2,sfGFPL. citreum mRNAJW001 by qRT-PCR. harboring P, pGFP–sgR–D2–Em; L. citreum JW001; D3,D1,L. L. citreum citreumJW001 JW001 harboringharboring pGFP–sgR–D3–Em; pGFP–sgR–D1–Em; D4, D2,L. L. citreum citreumJW001 JW001 harboring harboring pGFP–sgR–D4–Em. pGFP–sgR–D2–Em; All D3, error L. citreum bars representJW001 harboring the standard pGFP–sgR–D3–Em; deviation which was D4, calculatedL. citreum fromJW001 three harboring repeated pGFP–s experiments.gR–D4–Em. All error bars represent the standard deviation which was calculated from three repeated experiments. 2.3. Increase in Riboflavin Production via Optimized CRISPRi System for L. citreum 2.3. Increase in Riboflavin Production via Optimized CRISPRi System for L. citreum To demonstrate the feasibility of the optimized CRISPRi system in L. citreum, we engineered the riboflavinTo demonstrate biosynthesis the feasibility pathway of toward the optimized the enhanced CRISPRi production system in L. of citreum riboflavin., we engineered Riboflavin, the alsoriboflavin known biosynthesis as vitamin pathway B2, is an toward essential the enhanc componented production of cellular of metabolismriboflavin. Riboflavin, required byalso humansknown [as31 ].vitamin Recently, B2, is thean essential use of LAB component as a microbial of cellular cell metabolism factory for required producing by humans riboflavin [31]. hasRecently, been proposedthe use of toLAB obtain as a microbial fermented cell bio-enriched factory for producing food [32 –riboflavin34] as the has addition been proposed of the to riboflavin-producingobtain fermented bio-enriched LAB to fermented food [32–34] foods is as both the feasibleaddition and of economicallythe riboflavin-producing viable in the LAB dairy to fermented foods is both feasible and economically viable in the dairy industry [35]. In the riboflavin biosynthesis pathway, guanosine-5’-triphosphate (GTP) is converted to 2,5-diamino-6-ribosylamino- 4-(3H)-pyrimidinone-5’-phosphate by GTP cyclohydrolase Ⅱ and is subsequently transferred to the flavin mononucleotide (FMN) synthesis pathway [36] (Figure 5A). However, GTP, the main precursor, can be also converted to H2-neopterin triphosphate by GTP cyclohydrolase Ⅰ, and then

Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 7 of 13

transferred to the folate synthesis pathway (Figure 5A). Therefore, to enhance the biosynthesis of Int. J. Mol.riboflavin, Sci. 2020, 21it ,is 5614 necessary to reduce the expression of the folE gene encoding GTP cyclohydrolase Ⅰ7 of, 13 which can minimize the flux of GTP toward the folate synthesis pathway. In addition, a ribF gene, which encodes the riboflavin kinase responsible for the bioconversion of riboflavin to FMN, was industrychosen [35]. as In the the second riboflavin target biosynthesis for downregulation, pathway, in order guanosine-5’-triphosphate to reduce the loss of riboflavin (GTP) isdue converted to its to 2,5-diamino-6-ribosylamino-4-(3H)-pyrimidinone-5’-phosphateconversion into FMN (Figure 5A). We designed sgRNAs targeting by the GTP folE cyclohydrolaseand ribF genes, which II and is subsequentlycomprised transferred 20-base sequences, to the flavin complementary mononucleotide to the (FMN)non-template synthesis target pathway regions [next36] (Figureto PAM5A). sequences located at the most upstream position in each coding sequence (Table S3 and Figure S1B However, GTP, the main precursor, can be also converted to H2-neopterin triphosphate by GTP and S1C). cyclohydrolase I, and then transferred to the folate synthesis pathway (Figure5A). Therefore, to For the knock-down of both folE and ribF genes expressions by the CRISPRi, pFRdual–D4 was enhance the biosynthesis of riboflavin, it is necessary to reduce the expression of the folE gene encoding constructed, in which each gene-specific sgRNA was co-expressed together with the SpdCas9 (D4 GTP cyclohydrolaseplatform). As the I, controls, which cantwo minimizeplasmids, pFolE– the fluxsgR–D4 of GTP and towardpRibF–sgR–D4, the folate were synthesis also constructed pathway. In addition,for the atranscriptionribF gene, whichof either encodes folE- or ribF the-specific riboflavin sgRNA, kinase respectively. responsible After for the the cultivation bioconversion of each of riboflavinstrain, to FMN,the repression was chosen level asof each the second gene was target deter formined downregulation, by qRT-PCR. As in shown order toin reduceFigure 5B, the cells loss of riboflavinharboring due to either its conversion pFolE–sgR–D4 into or FMN pRibF–sgR–D4 (Figure5A). showed We designed a reduced sgRNAs transcription targeting level only the infolE theand ribF genes,target which genes comprised(5.6-fold decrease 20-base in folE sequences, and 3.3-fold complementary decrease in rib toF), thewhich non-template were orthogonal target to regionseach next totarget PAM without sequences any locatedinterference at the (Figure most 5B). upstream Moreover, position the cells in eachharboring coding the sequenceFRdual–D4 (Table exhibited S3 and Figurea S1B transcriptional and S1C). repression activity with a 3.9-fold decrease in folE and 6.3-fold decrease in ribF compared with those in WT (Figure 5B).

Figure 5. (A) Schematic diagram of the biosynthesis pathway of riboflavin in L. citreum. Blue and purple T-shaped bars represent the repression by CRISPRi with folE and ribF-specific sgRNA, respectively. Figure 5. (A) Schematic diagram of the biosynthesis pathway of riboflavin in L. citreum. Blue and (B) Transcription analysis of the folE and ribF genes by qRT-PCR. Blue and purple bars represent purple T-shaped bars represent the repression by CRISPRi with folE and ribF-specific sgRNA, the transcriptionrespectively. of (B)folE Transcriptionand ribF genes,analysis respectively. of the folE and All ribF the genes error by qRT-PCR. bars represent Blue and the purple value bars of the standardrepresent deviation, the transcription which was of calculated folE and ribF from genes, three respectively. repeated All experiments. the error bars represent (C) Identification the value of riboflavin accumulation via the designed CRISPRi system and the verification of the synergetic effect of riboflavin accumulation via combination with the CRISPRi system and the rib operon expression cassette. Wild-type (WT), L. citreum CB2567; D4, L. citreum CB2567 harboring plasmid pD4. The error bars represent the standard deviation of the samples. NS represents nonsignificant (p-value > 0.05), * p-value < 0.05, ** p-value < 0.01 based on the Student t-test. Int. J. Mol. Sci. 2020, 21, 5614 8 of 13

For the knock-down of both folE and ribF genes expressions by the CRISPRi, pFRdual–D4 was constructed, in which each gene-specific sgRNA was co-expressed together with the SpdCas9 (D4 platform). As the controls, two plasmids, pFolE–sgR–D4 and pRibF–sgR–D4, were also constructed for the transcription of either folE- or ribF-specific sgRNA, respectively. After the cultivation of each strain, the repression level of each gene was determined by qRT-PCR. As shown in Figure5B, cells harboring either pFolE–sgR–D4 or pRibF–sgR–D4 showed a reduced transcription level only in the target genes (5.6-fold decrease in folE and 3.3-fold decrease in ribF), which were orthogonal to each target without any interference (Figure5B). Moreover, the cells harboring the FRdual–D4 exhibited a transcriptional repression activity with a 3.9-fold decrease in folE and 6.3-fold decrease in ribF compared with those in WT (Figure5B). We examined whether the repression of folE and ribF expression via the CRISPRi system increased the production of riboflavin in L. citreum. After the cultivation of the cells in a shake flask, the yield of riboflavin was analyzed by HPLC. As shown in Figure5C, by repressing the expression of the target gene via the CRISPRi system, it was clearly observed that the changes in the amount of riboflavin production occurred in a gradient manner. The downregulation of either folE (pFolE–sgR–D4) or ribF (pRibF–sgR–D4) via the CRISPRi system increased riboflavin production by 1.13-fold (1.21 0.03 mg/L) ± or 1.17-fold (1.28 0.01 mg/L), respectively, compared with WT (1.08 0.02 mg/L) or with the strain ± ± that lacked the sgRNA (D4) (1.06 0.03 mg/L) (Figure5C). Although the repression of folE was higher ± than ribF via the CRISPRi system (Figure5B), relatively higher riboflavin accumulation was observed when ribF was repressed rather than folE (Figure5C). These results indicate that direct the inhibition of conversion of riboflavin to FMN is a more effective strategy to confirm the high productivity of riboflavin. Furthermore, the cultivation of L. citreum CB2567 harboring pFRdual–D4 for the downregulation of both the genes exhibited the enhanced production of riboflavin (1.30 0.01 mg/L), ± which was increased by 1.20-fold compared to WT (Figure5C). Therefore, these results verified that the developed system effectively performs multiplex gene expression regulation in L. citreum.

2.4. Increase in Riboflavin Production by Co-Expression of the Rib Operon in L. citreum As mentioned above, the accumulation of riboflavin could be induced in cells by regulating the flux of the riboflavin synthesis pathway via the CRISPRi system. In the riboflavin synthesis pathway, the enzymatic activities required to activate the biosynthesis of riboflavin from GTP are encoded by four genes (ribA, ribG, ribH, and ribB), which are located in an operon assembly (rib operon) (Figure5A), and it was previously reported that the overexpression of rib operon could increase the production of riboflavin [37]. We further examined whether a combination of downregulation via the CRISPRi system and the overexpression of the rib operon gene could exert a synergetic effect on the production of riboflavin. The expression cassette of the rib operon gene was inserted into the pFRdual–D4 plasmid, yielding pFRdual–Rib–D4, and after flask cultivation, the titer of riboflavin produced was determined. First, we examined the production of riboflavin in L. citreum CB2567 harboring pH-rib (overexpression of the rib operon without the CRISPRi system), and a 1.2-fold increase in riboflavin (1.30 0.01 mg/L) ± was observed compared to the WT (Figure5C). L. citreum CB2567 harboring pFRdual–Rib–D4, in which both the repression by CRISPRi system and the overexpression of the rib operon were combined, and exhibited a dramatic increase in riboflavin production as high as 1.7 0.06 mg/L, which was a 1.53-fold ± increase compared to the WT (Figure5C).

3. Conclusions In the last decade, substantial advancements in the CRISPR/Cas system have provided a very powerful tool to edit the genome and regulate the expression of endogenous genes in various microorganisms. In this study, we successfully extended the potential of the dCas9-based regulation tool to L. citreum, a promising probiotic. It was clearly demonstrated that the transcription of target genes was significantly reduced, and metabolic pathways for riboflavin production were successfully modulated using optimized CRISPRi. The expression levels of major components (sgRNA and dCas9) Int. J. Mol. Sci. 2020, 21, 5614 9 of 13 of CRISPRi could be regulated by a combination of synthetic parts (promoters and SDs) of different strengths. Through the low-level expression of dCas9 and the high-level expression of sgRNA, toxicity caused by the overexpression of dCas9 could be overcome along with the downregulation of the target gene. The regulation of the gene expression using synthetic parts also allows the fine tuning of the CRISPRi system, which makes it possible to optimize metabolic pathways towards more enhanced production of the desired biomolecules. As the power of CRISPR/Cas9 is based on its easy implementation and programmability, we believe that the CRISPRi systems employed in this study will be a useful tool for the engineering of L. citreum and other lactic acid bacteria, which can be employed as beneficial cell factories in various bioindustry fields.

4. Materials and Methods

4.1. Bacterial Strains and Culture Conditions All the bacterial strains used in this study are listed in Table S1. E. coli XL1-blue was used as a host for the gene cloning and maintenance of plasmids. L. citreum CB2567 [38] was used as a major host for the development of the CRISPRi system and riboﬂavin production. E. coli was cultivated in a Luria–Bertani (LB) medium (tryptone 10 g/L, yeast extract 5 g/L, and sodium chloride 10 g/L, purchased as premixed media from BD, Franklin Lakes, NJ, USA) at 37 °C with shaking (200 rpm). L. citreum CB2567 was cultivated in Lactobacilli MRS (De Man, Rogosa and Sharpe) medium (proteose peptone no. 3 10 g/L, beef extract 10 g/L, yeast extract 5 g/L, dextrose 20 g/L, polysorbate 80 1 g/L, ammonium citrate 2 g/L, sodium acetate 5 g/L, magnesium sulfate 0.1 g/L, manganese sulfate 0.05 g/L, and dipotassium phosphate 2 g/L, purchased as premixed media from BD) at 30 °C with shaking (200 rpm). Ampicillin (Amp, 100 mg/L) was added for the selection and cultivation of E. coli. Chloramphenicol (Cm, 10 mg/L) and erythromycin (Em, 10 mg/L) were used for the selection and cultivation of L. citreum.

4.2. Plasmid Construction and Chromosome Integration All the plasmids used in this study are listed in Table S1. Polymerase chain reaction (PCR) was carried out using a C1000TM Thermal Cycler (Bio-Rad, Richmond, CA, USA) and primeSTAR HS Polymerase (Takara, Shiga, Japan). All the oligonucleotides used for PCR are listed in Table S2. The erythromycin resistance gene (Emr) was amplified from pSOS95 [39] by PCR with two primers (F-Erm and R-Erm). The PCR product was digested with two restriction enzymes (StuI and XbaI), and cloned into pCB4270 and pCB4270V4, yielding pCB4270–Em and pCB4270V4–Em, respectively. For the expression of the SpdCas9 gene under the strong control ofeSD2, the eSD2–dCas9 gene was amplified from pdCas9 by PCR using three primers (F1–eSD2dCas9, F2–eSD2dCas9, and R–Not–dCas9). The PCR product (eSD2–dCas9) was digested with two restriction enzymes (XhoI and NotI) and cloned into pCB4270V4, pCB4270, pCB4270V4–Em, and pCB4270–Em, yielding plasmids pD1, pD3, pD1–Em, and pD3–Em, respectively. For the expression of the SpdCas9 gene under the control of a weak SD2, the SD2–dCas9 gene was amplified from pdCas9 by PCR with three primers (F1–SD2dCas9, F2–SD2dCas9, and R–Not–dCas9). The PCR product (SD2–dCas9) was digested with two restriction enzymes (XhoI and NotI) and cloned into pCB4270V4, pCB4270, pCB4270V4–Em, and pCB4270–Em, yielding the plasmids pD2, pD4, pD2–Em, and pD4–Em, respectively. GFP–sgRNA was amplified from pgRNA-bacteria by PCR using four primers (F1–v4sfgsgR, F2–v4sfgsgR, F3–v4sfgsgR, and R–v4sfgsgR). The PCR product was digested with the NarI restriction enzyme and cloned into pCB4270–Em, pD1–Em, pD2–Em, pD3–Em, and pD4–Em, yielding plasmids pGFP–sgR–Em, pGFP–sgR–D1–Em, pGFP–sgR–D2–Em, pGFP–sgR–D3–Em, and pGFP–sgR–D4–Em, respectively. The folE–sgRNA was amplified from the plasmid pGFP–sgR–Em by PCR using four primers (F1–v4folEsgR, F2–v4folEsgR, F3–v4folEsgR, and R–v4sfgsgR). The PCR product was digested with the XbaI restriction enzyme, and cloned into the plasmid pD4, yielding pFolE–sgR–D4. A ribF–sgRNA was amplified from the plasmid pGFP–sgR–Em by PCR with three primers (F1–v4ribFsgR, F2–v4ribFsgR, and R–v4sfgsgR). The PCR product was digested with the NarI restriction enzyme, and cloned into the plasmid pD4, yielding Int. J. Mol. Sci. 2020, 21, 5614 10 of 13 pRibF–sgR–D4. Plasmid pFRdual-D4 was constructed by inserting the NarI-digested PCR product of ribF–sgRNA into the plasmid pFolE–sgR–D4. A rib operon was amplified from the genomic DNA of L. citreum CB2567 by PCR using four primers (F1–v4–ribD, F2–v4–ribD, F3–v4–ribD, and R–ribH). The PCR product was digested with the PstI restriction enzyme, and cloned into the plasmids pCB4270 and pFRdual–D4, yielding pH–rib and pFRdual–Rib–D4, respectively. A sfGFP gene was integrated into the chromosomal DNA of L. citreum CB2567 via the homologous recombination using a suicide vector pCB4270–InsfGFP. To construct pCB4270–InsfGFP, a 0.8 kb upstream region of ldhD gene was amplified from the chromosomal DNA of L. citreum CB2567 by PCR using F–LDHU and R–LDHU primers. The PCR product was digested with BamHI and then ligated into pCB4270–sfGFP yielding pHldhU–sfGFP. The ldh gene was amplified from the chromosomal DNA of L. citreum CB2567 by PCR using F–LDHD and R–LDHD primers. After digestion with PstI, the PCR product was cloned into pHldhU–sfGFP, yielding pH–InsfGFP. After electroporation, the integrated clones were screened on agar plates containing chloramphenicol. The generation of integrated clones was confirmed by PCR using IF–stCAT and OR–ldh primers. The correct clone was named L. citreum JW001.

4.3. Fluorescence-Activated Cell-Sorting (FACS) Analysis After the overnight cultivation of the recombinant L. citreum, the cells were transferred to 5 mL of fresh MRS medium in a 50 mL tube at 1:50 dilution and grown at 30 °C with shaking (200 rpm). After cultivation for 12 h, the cells were harvested by centrifugation at 13,000 rpm for 5 min at 4 °C and washed twice with phosphate-buffered saline (PBS, containing 135 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, pH 7.2). After suspension in the same buffer, the cells were identified using a high-speed flow cytometer (MoFlo XDP, Beckman Coutler, Miami, FL, USA). For the FACS analysis, the cells were analyzed on the basis of high fluorescence intensity detected through a 530/40 band-pass filter for the GFP emission spectrum.

4.4. Protein Preparation and Analysis After culturing at 30 °C for 12 h in a shake ﬂask, the cells were harvested by centrifugation (13,000 rpm, 5 min, 4 °C) and washed with PBS. Later, the cells were disrupted by sonication (7 min with a 5 s pulse and 3 s cooling time, 20% amplitude). After harvesting the total fractions, insoluble pellets were eliminated by centrifugation (13,000 rpm, 5 min, 4 °C) and the soluble fractions were collected from the supernatants. The protein samples were analyzed by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

4.5. Quantitative Reverse Transcription PCR (qRT-PCR) mRNA transcription levels were quantiﬁed by qRT-PCR. L. citreum harboring each plasmid was cultivated in MRS medium for 12 h, and then the cells were harvested by centrifugation (13,000 rpm, 5 min, 4 °C) and washed with PBS. Total RNA was extracted from the cells using the Qiagen RNeasy Mini Kit (Qiagen, Valencia, CA, USA), and the puriﬁed RNA was stored at –80 °C for further use. The primers sets for qRT-PCR were designed using Primer3web (http://primer3.ut.ee); the resulting primer sets are listed in Table S2. One-step qRT-PCR was performed using the One Step SYBR PrimeScript RT-PCR Kit (Takara, Japan) following the manufacturer’s instructions. qRT-PCR was carried out on a CFX-96 real-time PCR system (Bio-Rad, CA, USA) and the Cq values were determined by CFX Manager Software (Bio-Rad, CA, USA)).

4.6. High-Performance Liquid Chromatography (HPLC) Analysis To measure the amount of riboflavin in the medium, the cells were pelleted by centrifugation at 13,000 rpm for 5 min at 4 ◦C, and the supernatant thus obtained was filtered using a 0.22 µm syringe filter (Futecs, Daejeon, Korea). The filtrated supernatant was diluted 1:10 in distilled water and analyzed by HPLC. The HPLC system (Shimadzu, Kyoto, Japan) consisted of a pump (LC-20AD), autosampler Int. J. Mol. Sci. 2020, 21, 5614 11 of 13

(SIL-30AC), column oven (CTO-20A), and a refractive index detector (RID-10A). A Zorbax 300SB-C18 column (150 4.6 mm; Agilent Technologies, PA, CA, USA), and was eluted with methanol:water (75:25 × v/v), used as the mobile phase at a ﬂow rate of 0.5 mL/min, operating at 65 ◦C. The chromatographic software Lab Solution (Shimadzu, Kyoto, Japan) was used for the sample acquisition and data analysis.

Supplementary Materials: Supplementary Materials can be found at http://www.mdpi.com/1422-0067/21/16/ 5614/s1. Author Contributions: Conceptualization, K.J.J.; methodology, J.S. and S.H.J.; formal analysis, J.S., and S.H.J.; investigation, J.S. and J.W.C.; data curation, J.S. and J.W.C.; writing—original draft preparation, J.S.; writing—review and editing, K.J.J; supervision, K.J.J.; funding acquisition, K.J.J. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by the Intelligent Synthetic Biology Center of the Global Frontier Project (Grant no. NRF-2014M3A6A8066443) funded by the Ministry of Science and ICT (MSIT) & the National Research Foundation of Korea (Grant no. NRF-2020R1A2C2012537) funded by the Ministry of Science and ICT (MSIT). Acknowledgments: The authors wish to thank Nam Soo Han for providing Leuconostoc citreum CB2567. Conﬂicts of Interest: The authors declare no conﬂict of interest.

Abbreviations

LAB lactic acid bacteria CRISPRi CRISPR interference sgRNA single guide RNA SpdCas9 catalytically deactivated Cas9 of Streptococcus pyogenes BCD bicistronic design RBS ribosome binding sites sfGFP superfolder green fluorescent protein PAM protospacer adjacent motif qRT-PCR quantitative reverse transcription PCR HPLC high performance liquid chromatography PBS phosphate-buffered saline TBS-T Tris-buffered saline with Tween-20 solution

References

1. Hemme, D.; Foucaud-Scheunemann, C. Leuconostoc, characteristics, use in dairy technology and prospects in functional foods. Int. Dairy J. 2004, 14, 467–494. [CrossRef] 2. Sybesma, W.; Starrenburg, M.; Tijsseling, L.; Hoefnagel, M.H.; Hugenholtz, J. Effects of cultivation conditions on folate production by lactic acid bacteria. Appl. Environ. Microbiol. 2003, 69, 4542–4548. [CrossRef] [PubMed] 3. Lee, W.-K.; Ahn, S.-B.; Lee, S.-M.; Shon, M.-Y.; Kim, S.-Y.; Shin, M.-S. Immune-enhancing effects of Leuconostoc strains isolated from Kimchi. J. Biomed. Res. 2012, 13, 353–356. 4. Li, L.; Shin, S.Y.; Lee, K.; Han, N. Production of natural antimicrobial compound d-phenyllactic acid using Leuconostoc mesenteroides ATCC 8293 whole cells involving highly active d-lactate dehydrogenase. Lett. Appl. Microbiol. 2014, 59, 404–411. [CrossRef][PubMed] 5. de Vos, W.M. Systems solutions by lactic acid bacteria: From paradigms to practice. Microb. Cell. Fact. 2011, 10, S2. [CrossRef] 6. Eom, H.-J.; Moon, J.-S.; Seo, E.-Y.; Han, N.S. Heterologous expression and secretion of Lactobacillus amylovorus α-amylase in Leuconostoc citreum. Biotechnol. Lett. 2009, 31, 1783. [CrossRef][PubMed] 7. García-Fruitós, E. Lactic acid bacteria: A promising alternative for recombinant protein production. Microb. Cell. Fact. 2012, 11, 157. [CrossRef] 8. Caggianiello, G.; Kleerebezem, M.; Spano, G. Exopolysaccharides produced by lactic acid bacteria: From health-promoting benefits to stress tolerance mechanisms. Appl. Microbiol. Biotechnol. 2016, 100, 3877–3886. [CrossRef] Int. J. Mol. Sci. 2020, 21, 5614 12 of 13

9. Domingos-Lopes, M.; Lamosa, P.; Stanton, C.; Ross, R.; Silva, C. Isolation and characterization of an exopolysaccharide-producing Leuconostoc citreum strain from artisanal cheese. Lett. Appl. Microbiol. 2018, 67, 570–578. [CrossRef] 10. Yang, Y.; Feng, F.; Zhou, Q.; Zhao, F.; Du, R.; Zhou, Z.; Han, Y. Isolation, purification, and characterization of exopolysaccharide produced by Leuconostoc citreum N21 from dried milk cake. Trans. Tianjin Univ. 2019, 25, 161–168. [CrossRef] 11. Oh, Y.H.; Choi, J.W.; Kim, E.Y.; Song, B.K.; Jeong, K.J.; Park, K.; Kim, I.-K.; Woo, H.M.; Lee, S.H.; Park, S.J. Construction of synthetic promoter-based expression cassettes for the production of cadaverine in recombinant Corynebacterium glutamicum. Appl. Biochem. Biotechnol. 2015, 176, 2065–2075. [CrossRef][PubMed] 12. Ko, Y.J.; You, S.K.; Kim, M.; Lee, E.; Shin, S.K.; Park, H.M.; Oh, Y.; Han, S.O. Enhanced Production of 5-aminolevulinic Acid via Flux Redistribution of TCA Cycle toward l-Glutamate in Corynebacterium glutamicum. Biotechnol. Bioprocess Eng. 2019, 24, 915–923. [CrossRef] 13. Van Pijkeren, J.-P.; Britton, R.A. High efficiency recombineering in lactic acid bacteria. Nucleic Acids Res. 2012, 40, e76. [CrossRef][PubMed] 14. Pines, G.; Freed, E.F.; Winkler, J.D.; Gill, R.T. Bacterial recombineering: Genome engineering via phage-based homologous recombination. ACS Synth. Biol. 2015, 4, 1176–1185. [CrossRef] 15. Zhu, D.; Zhao, K.; Xu, H.; Zhang, X.; Bai, Y.; Saris, P.E.; Qiao, M. Construction of thyA deficient Lactococcus lactis using the Cre-loxP recombination system. Ann. Microbiol. 2015, 65, 1659–1665. [CrossRef] 16. Oh, J.-H.; van Pijkeren, J.-P. CRISPR–Cas9-assisted recombineering in Lactobacillus reuteri. Nucleic Acids Res. 2014, 42, e131. [CrossRef] 17. Berlec, A.; Škrlec, K.; Kocjan, J.; Olenic, M.; Štrukelj, B. Single plasmid systems for inducible dual protein expression and for CRISPR-Cas9/CRISPRi gene regulation in lactic acid bacterium Lactococcus lactis. Sci. Rep. 2018, 8, 1009. [CrossRef] 18. Zheng, Y.; Su, T.; Qi, Q. Microbial CRISPRi and CRISPRa Systems for Metabolic Engineering. Biotechnol. Bioprocess Eng. 2019, 24, 579–591. [CrossRef] 19. Son, Y.J.; Ryu, A.J.; Li, L.; Han, N.S.; Jeong, K.J. Development of a high-copy plasmid for enhanced production of recombinant proteins in Leuconostoc citreum. Microb. Cell. Fact. 2016, 15, 12. [CrossRef] 20. Jang, S.H.; Cha, J.W.; Han, N.S.; Jeong, K.J. Development of bicistronic expression system for the enhanced and reliable production of recombinant proteins in Leuconostoc citreum. Sci. Rep. 2018, 8, 8852. [CrossRef] 21. Larson, M.H.; Gilbert, L.A.; Wang, X.; Lim, W.A.; Weissman, J.S.; Qi, L.S. CRISPR interference (CRISPRi) for sequence-specific control of gene expression. Nat. Protoc. 2013, 8, 2180. [CrossRef][PubMed] 22. Qi, L.S.; Larson, M.H.; Gilbert, L.A.; Doudna, J.A.; Weissman, J.S.; Arkin, A.P.; Lim, W.A. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 2013, 152, 1173–1183. [CrossRef][PubMed] 23. Bikard, D.; Jiang, W.; Samai, P.; Hochschild, A.; Zhang, F.; Marraffini, L.A. Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system. Nucleic Acids Res. 2013, 41, 7429–7437. [CrossRef][PubMed] 24. Choudhary, E.; Thakur, P.; Pareek, M.; Agarwal, N. Gene silencing by CRISPR interference in mycobacteria. Nat. Commun. 2015, 6, 1–11. [CrossRef] 25. Kim, S.K.; Han, G.H.; Seong, W.; Kim, H.; Kim, S.-W.; Lee, D.-H.; Lee, S.-G. CRISPR interference-guided balancing of a biosynthetic mevalonate pathway increases terpenoid production. Metab. Eng. 2016, 38, 228–240. [CrossRef] 26. Su, K.-C.; Tsang, M.-J.; Emans, N.; Cheeseman, I.M. CRISPR/Cas9-based gene targeting using synthetic guide RNAs enables robust cell biological analyses. Mol. Biol. Cell 2018, 29, 2370–2377. [CrossRef] 27. Nielsen, A.A.; Voigt, C.A. Multi-input CRISPR/Cas genetic circuits that interface host regulatory networks. Mol. Syst. Biol. 2014, 10, 763. [CrossRef] 28. Cho, S.; Choe, D.; Lee, E.; Kim, S.C.; Palsson, B.; Cho, B.-K. High-level dCas9 expression induces abnormal cell morphology in Escherichia coli. ACS Synth. Biol. 2018, 7, 1085–1094. [CrossRef] 29. Zhang, S.; Voigt, C.A. Engineered dCas9 with reduced toxicity in bacteria: Implications for genetic circuit design. Nucleic Acids Res. 2018, 46, 11115–11125. [CrossRef] 30. Wu, X.; Kriz, A.J.; Sharp, P.A. Target specificity of the CRISPR-Cas9 system. Quant Biol. 2014, 2, 59–70. [CrossRef] Int. J. Mol. Sci. 2020, 21, 5614 13 of 13

31. LeBlanc, J.; Laiño, J.E.; del Valle, M.J.; Vannini, V.V.; van Sinderen, D.; Taranto, M.P.; de Valdez, G.F.; de Giori, G.S.; Sesma, F. B-Group vitamin production by lactic acid bacteria–current knowledge and potential applications. J. Appl. Microbiol. 2011, 111, 1297–1309. [CrossRef][PubMed] 32. Capozzi, V.; Menga, V.; Digesu, A.M.; Vita, P.D.; van Sinderen, D.; Cattivelli, L.; Fares, C.; Spano, G. Biotechnological production of vitamin B2-enriched bread and pasta. J. Agric. Food. Chem. 2011, 59, 8013–8020. [CrossRef][PubMed] 33. Samaniego-Vaesken, M.d.; Alonso-Aperte, E.; Varela-Moreiras, G. Vitamin food fortification today. Food Nutr. Res. 2012, 56, 5459. [CrossRef][PubMed] 34. Laiño, J.E.; del Valle, M.J.; de Giori, G.S.; LeBlanc, J.G.J. Development of a high folate concentration yogurt naturally bio-enriched using selected lactic acid bacteria. LWT Food. Sci. Technol. 2013, 54, 1–5. [CrossRef] 35. LeBlanc, J.; Burgess, C.; Sesma, F.; de Giori, G.S.; van Sinderen, D. Ingestion of milk fermented by genetically modified Lactococcus lactis improves the riboflavin status of deficient rats. J. Dairy Sci. 2005, 88, 3435–3442. [CrossRef] 36. Thakur, K.; Tomar, S.K.; De, S. Lactic acid bacteria as a cell factory for riboflavin production. Microb. Biotechnol. 2016, 9, 441–451. [CrossRef] 37. Perkins, J.; Sloma, A.; Hermann, T.; Theriault, K.; Zachgo, E.; Erdenberger, T.; Hannett, N.; Chatterjee, N.; Williams, V., II; Rufo, G.J. Genetic engineering of Bacillus subtilis for the commercial production of riboflavin. J. Ind. Microbiol. Biotechnol. 1999, 22, 8–18. [CrossRef] 38. Otgonbayar, G.-E.; Eom, H.-J.; Kim, B.S.; Ko, J.-H.; Han, N.S. Mannitol production by Leuconostoc citreum KACC 91348P isolated from kimchi. J. Microbiol. Biotechnol. 2011, 21, 968–971. [CrossRef] 39. Raynaud, C.; Sarçabal, P.; Meynial-Salles, I.; Croux, C.; Soucaille, P. Molecular characterization of the 1,3-propanediol (1,3-PD) operon of Clostridium butyricum. Proc. Natl. Acad. Sci. USA 2003, 100, 5010–5015. [CrossRef]

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