We established that the strain reported in Sasaki et al., 2019, C. glutamicum (previously referred to as ATCC 13032 NHRI 1.1.2) outperformed another isolate, ATCC 13032 ∆cglIM ∆cgLIR ∆cgLIIR (referred to as “∆mrr”) (Figure 1A). C. glutamicum ∆mrr was first described in Baumgart et al., 2013 and is a methylation-deficient strain widely used due to its improved plasmid transformation and genomic integration rate (Schäfer et al., 1997; Baumgart et al., 2013). When C. glutamicum BRC-JBEI 1.1.2 is used in conjunction with an IP production pathway, it can produce 300 mg/L IP from pure glucose, but the product titers are near the lower detection limit by GC-FID in the C. glutamicum ATCC 13032 ∆mrr strain. While only C. glutamicum BRC-JBEI 1.1.2 produced IP, both the type strain and this specific isolate tolerate high concentrations of exogenous ILs (Figure 1B), suggesting that IL tolerance was a shared feature between these two isolates despite differences in IP production.

We also confirmed the ability of C. glutamicum BRC-JBEI 1.1.2 to handle renewable carbon streams from sorghum biomass using an improved carbon extraction protocol enhanced by the use of ensiled biomass (Magurudeniya et al., 2021). The ensiling process enables naturally occurring lactic-acid secreting bacteria to partially decompose the hemicellulose in sorghum while stored in a silo before downstream processing. After ensiling, the biomass was pretreated with [Ch][Lys] followed by enzymatic saccharification (Materials and Methods). This hydrolysate contained 48.7 g/L glucose, 17.9 g/L xylose, and trace concentrations of aromatic compounds. Our optimized C. glutamicum BRC-JBEI 1.1.2 with an optimized IP production system had no detected growth defects when grown with 58% (v/v) hydrolysate supplemented media and produced 1 g/L IP from pure glucose or ∼600 mg/L IP from sorghum hydrolysate (Figure 1C). These results showcase its versatility with handling actual plant biomass derived carbon streams. For the remainder of this study, we focus on characterizing the genetic differences present in C. glutamicum BRC-JBEI 1.1.2 relative to other closely related C. glutamicum strains that might explain the IP production values between these two strains.

While 16S rRNA sequencing suggested that C. glutamicum ∆mrr strain as in the C. glutamicum ATCC 13032 strain background, this same method indicated that C. glutamicum BRC-JBEI 1.1.2 is related to C. glutamicum CICC10112 or SCgG1/SCgG2. To overcome the limitation inherent to 16S rRNA-based identification, we turned to using whole genome sequencing. Only SCgG1 and SCgG2 have been characterized with whole-genome sequencing, and to our knowledge there was no additional information about C. glutamicum CICC10112 beyond the partial 16S ribosomal sequence. As 16S rRNA is inconclusive for isolate-level identification (Sabat et al., 2017; Hahne et al., 2018; Johnson et al., 2019), we reasoned that the whole-genome sequencing in this IP producing strain would ensure an accurate reference genome in downstream RNAseq analysis if the improved performance observed in this strain was due to variations in the strain background. One of the major limitations in short-read sequencing is the difficulty in assembling overlapping contigs to generate a high-quality de novo assembly of a single contiguous read. Therefore, we chose PacBio long-read sequencing (Koren and Phillippy, 2015) for optimal coverage over short read sequencing as a potential solution. However, routine methods for lysing and isolating C. glutamicum genomic DNA were insufficient for building high-quality genome assemblies since the physical lysis method we employed (Eng et al., 2018) shears DNA to fragments ranging from 2 to 8 kb in size. Detergent-based lysis methods failed to extract genomic DNA, even with prolonged incubation times. We developed a method to isolate larger DNA fragments approximately 20 kb in size for the PacBio Sequel (Pacific Biosciences) assembly pipeline using a zymolyase protease treatment for cell lysis (see Materials and Methods). This modified DNA extraction protocol enabled us to use PacBio long read sequencing to generate a high-quality de novo genome assembly.

We now report a new genome assembly of a single contiguous scaffold of 3,352,276 bases with 53.83% GC content (Figure 2). Genome-wide average nucleotide identity (ANI) confirmed this isolate was 99.9987% identical to C. glutamicum SCgG1 and SCgG2 as well as another sequenced C. glutamicum isolate, Z188. The average nucleotide identity alignment for the 28 sequenced C. glutamicum isolates has been deposited at the database of the Joint Genome Institute (https://genome.jgi.doe.gov/portal/), Project ID 1203597 and is also included in Supplementary Table S1. C. glutamicum BRC-JBEI 1.1.2 differs from SCgG1 only by a few single nucleotide polymorphisms (∼10) and two additional genes that are absent from SCgG1, a putative transposase and a hypothetical protein coding sequence that is 414 bp in length. When C. glutamicum BRC-JBEI 1.1.2 was compared with more commonly used reference strains, C. glutamicum R and 13032 (Bielefeld), we identified genomic islands encoding genes unique to BRC-JBEI 1.1.2. Genome topology analysis also identified a 140 kb inversion in the genome of BRC-JBEI 1.1.2 isolate (Figure 2A). Out of 3,097 genes, homology mapping indicated that 85% (2,641 genes) were at least 80% identical to known genes in C. glutamicum ATCC 13032. With a less restrictive % identity threshold of 50%, the identical ratio could account for 89% (2,777 genes). Nonetheless, 320 genes did not meet the minimum % identity threshold and could not be annotated with this reference genome (Supplementary Figure S1).

Some of these unknown genes that were unique to BRC-JBEI 1.1.2 might be related to the catabolism of IL. Intriguingly, a putative choline dehydrogenase, Ga0373873_2846, showed only 40% identity to other known choline dehydrogenases primarily found in Gram-negative microbes such as Burkholderia phytofirmans PsJN and Cupriavidus basilensis FW507-4G11. Meta-COG analysis of these four C. glutamicum genomes revealed that C. glutamicum BRC-JBEI 1.1.2 contains over 100 additional genes related to the transport or metabolism of inorganic ions, carbohydrates, and amino acids, suggesting a broader metabolic capacity to utilize a more significant number of substrates than the type strain (Figure 2). In summary, this genome sequencing analysis was valuable for characterizing differences between C. glutamicum BRC-JBEI 1.1.2 and the more routinely studied type strain ATCC 13032. Due to its similarity with SCgG1 and SCgG2, C. glutamicum BRC-JBEI 1.1.2 is likely an industrial glutamate overproducing isolate but has more annotations in the inorganic ion and amino acid transport and metabolism COG categories than its nearest neighbors, SCgG1, SCgG2, and Z188 that need further characterization.

Next, we sought to build a systems-level understanding of C. glutamicum gene expression changes in bioreactors upon exogenous IL treatment. This data could be useful for subsequent Design-Build-Test-Learn (DBTL) cycles in providing the diagnostic information for future strain optimization strategies (Opgenorth et al., 2019). We prepared samples from sequential time points during a scaleup campaign to analyze shifts in gene expression as a proxy for changes in metabolic and regulatory behavior in both [Ch][Lys] treated and untreated runs. First, we determined if the failure to produce IP was due to loss of the production pathway, possibly due to loss of the plasmid-borne IP pathway genes. The IP production pathway is composed of 5 genes in 2 adjacent operons under the trc and lacUV5 promoters, namely mk, pmd and atoB, hmgS, hmgR respectively. Using the transcripts per million (TPM) metrics, we examined absolute gene expression levels as well as changes over the course of the production campaign. The IP pathway started off high for both hmgR and hmgS in the shake flask (200,000 TPM), but expression of these two genes decreased between 10-16x over the duration of the 65-h fed batch. Expression amounts of atoB in the shake flask were comparatively lower (1,500 TPM) but decreased 4x at the shake flask to bioreactor transition. atoB TPM counts remained low for the duration of the subsequent time points. Since the pathway genes were still expressed during this run, we then focused on analyzing gene expression changes in the native C. glutamicum genome.

To interpret the differential gene expression results with genes identified in the new assembly for C. glutamicum BRC-JBEI 1.1.2, we mapped gene names and identifiers from C. glutamicum ATCC 13032 back onto the open reading frames (ORFs) in C. glutamicum BRC-JBEI 1.1.2 as genes in the type strain genome have been broadly characterized. We used a medium confidence cutoff of 70% identity to capture most homologs when analyzing this dataset. First, we characterised gene expression upon inoculating cells from the seed culture in a shake flask to the bioreactor. This differential gene expression (DEG) was calculated as the ratio of an early time point in the bioreactor (6.5 h post inoculation in the stirred tank) divided by values from the seed culture immediately before transfer. This time point was chosen to give cells approximately three doublings to ensure the cells were rapidly growing under these new conditions. The result showed differential expression of 258 genes after 6.5 h (Figure 3, and Supplementary Dataset S1).

After inoculation into the bioreactors, we benchmarked the bioreactor run with online and offline measurements including growth, glucose consumption, and organic acid secretion, with and without [Ch][Lys]. We noted several differences between cells grown in the control reactor and the [Ch][Lys] treated reactor. While cells were pulse-fed the same feed solution to restore glucose levels back to 60 g/L, the [Ch][Lys] treated engineered strain produced much less acetate and succinate than the control (Figures 4A, 5A). Overall OD₆₀₀ measurements indicated similar initial growth patterns before the first feeding, but after feeding, OD₆₀₀ measurements did not appreciably increase further and instead we detected overflow metabolite accumulation above 10 g/L of succinate and acetate (Figure 4A). The control reactor decreased in OD₆₀₀ from a high of 49 to a 21 OD₆₀₀. The [Ch][Lys] reactor also decreased in OD₆₀₀, but from a similar high of 50 to 36 OD₆₀₀ (Supplementary Figure S2). We correlated gene expression changes during this campaign for both reactors using RNAseq analysis to understand how glucose was redirected from growth to the generation of these overflow metabolites (Supplementary Dataset S2).

Next we analyzed differential gene expression when cells were grown in the presence of 50 mM [Ch][Lys], simulating hydrolysate prepared under a water-conservation regimen (Neupane et al., 2017). ILs have been reported to increase osmotic pressure, interact with lipid structures and consequently disrupt microbial membranes (Thuy Pham et al., 2010; Khudyakov et al., 2012; Yu et al., 2016). C. glutamicum exhibited differential expression of 727 genes (Supplementary Dataset S3), during the [Ch][Lys] treated fed-batch bioreactor cultivation in comparison to the untreated culture at the time-matched samples (Figure 5A). While both bioreactors consumed the initial glucose in the reactor at similar rates, their response to the first feeding at 24 h differed. The [Ch][Lys] reactor showed maximum accumulation of 4 g/L succinate and 6 g/L acetate over the duration of this time course, a 4-fold decrease for both organic acids in the absence of [Ch][Lys] (compare Figures 4A, 5A). In the presence of [Ch][Lys], genes encoding for succinate utilization such as sdhA, sdhB and sdhC were all upregulated at 24 and 41 h in contrast to the control reactor. Similarly, genes encoding for pyruvate decarboxylation to acetyl CoA (instead of acetate) via aceE and aceF were also highly upregulated at later time points.

During the early cultivation time points (6.5–16.5 h), only 1.5% of the total pool of differentially expressed genes changed in response specifically to [Ch][Lys], but the datasets diverged after the first feeding at 24 h as biomass formation reached its maximum (Figure 5B). Only two genes were upregulated at the 6.5 h time point: a MFS transporter (Cgl2611) and its transcriptional regulator (Cgl2612) (Supplementary Dataset S3). The BRC-JBEI 1.1.2 homolog is 97.37% identical to Cgl2611 which exports cadaverine, a L-lysine derived product (Kind et al., 2011; Adkins et al., 2012; Jones et al., 2015; Tsuge et al., 2016). Cgl2611 expression was not detected at the control 6.5 h time point, but both genes are highly upregulated with or without [Ch][Lys] treatment in the remaining time points. Cgl1203, which encodes a phospho-N-acetylmuramoyl- pentapeptide-transferase associated with cell wall biosynthesis, was only upregulated at 16.5 h.

Early transcriptome changes in C. glutamicum during bioreactor cultivation post [Chl][Lys] exposure included overexpression of MFS transporters along with repression of mechanosensitive channels that were consistent with IL tolerance mechanisms reported in other microbes (Khudyakov et al., 2012; Martins et al., 2013; Yu et al., 2016). Many genes were downregulated in response to exogenous [Ch][Lys] in the bioreactor and represented 25% of DEGs. Cgl0879/mscL, a large-conductance mechanosensitive channel, that is related to osmotic regulation (Krämer, 2009), was uniquely downregulated at 16.5 h.

A comprehensive analysis of upregulated DEGs at more than one time point represented around 59% of the total upregulated genes in the presence of IL (Figure 5B). Nearly 15% of those genes showed consistent overexpression from 24 h through 65 h (Figure 5C). This differential transcript profile reflects the metabolic perturbation over the course of the fed-batch cultivation after the initial glucose exhaustion followed by glucose pulse feeding and is depicted in Figure 6. Prominent DEGs include those encoding for energy metabolism, amino acids biosynthesis, response to oxidative and other environmental stress conditions (Supplementary Dataset S4).

Our transcriptome analysis identified differential profiles for energy metabolism, amino acid biosynthesis and redox related genes as discussed in the previous section (Figure 6). Several genes encoding for metabolic reactions related to acetoin and TMP accumulation were specifically upregulated in the presence of 50 mM of [Ch][Lys] at the 24 h or 41 h time points (Supplementary Dataset S3, Supplementary Figure S4) when compared to the control samples at the same time points. Of the two subunits of the acetolactate synthase (ALS) ilvB and ilvN, the smaller regulatory subunit, Cgl1272/ilvN was upregulated in the presence of IL fed-batch cultivation when compared to the absence of IL at 24 h. Acetolactate synthase in C. glutamicum takes part in diverting pyruvate flux towards branched chain amino acids biosynthesis and acetoin biosynthesis and could be a precursor to TMP (Eng et al., 2020) (Figure 5). Although branched chain amino acid biosynthesis has been extensively researched for engineering branched chain alcohol (e.g. isobutanol) producing C. glutamicum strains (Hasegawa et al., 2020) the branched chain amino acid degradation towards isopentenol biosynthesis (through HMG-CoA) and TCA through acetyl CoA still remains to be fully investigated. The other proposed enzyme in TMP accumulation is the NADH consuming acetoin reductase (AR, Cgl2674) and was also significantly upregulated (log₂ > 4) at 41 h in presence of 50 mM of [Ch][Lys] compared to fed-batch cultivation in the absence of IL at similar time points. Genes encoding mechanisms that divert pyruvate flux towards acetyl CoA (Cgl2248/aceE and Cgl2207/aceF) were also upregulated along with genes for pyruvate kinase (Cgl2089/pyk) and citrate synthase (Cgl0829/gltA).

In a previous report (Sasaki et al., 2019), we referred to the IP producing C. glutamicum strain as ATCC 13032 NHRI 1.1.2, as indicated in our archival notes. As we cannot confirm the provenance of C. glutamicum BRC-JBEI 1.1.2 and how it may have been derived from its closest relatives C. glutamicum SCgG1 or SCgG2, we opted to give this strain a unique identifier to avoid further confusion.

Unless indicated elsewhere, all reagents used were molecular biology grade or higher. Primers were synthesized by IDT DNA Technologies (Coralville, IA). CGXII media was prepared as previously described (Sasaki et al., 2019; Keilhauer et al., 1993). All strains and plasmids used in this study are described in Supplementary Table S2. C. glutamicum strains were struck to single colonies from glycerol stock on LB plates containing the appropriate antibiotic and prepared for production runs as previously described (Eng et al., 2020). The fed-batch cultivation with 50 mM of [Ch][Lys] supplementation was previously described in (Eng et al., 2020). The control bioreactor without [Ch][Lys] was conducted at the same time and the glucose feeding regime was identical to that of the ionic liquid (IL) supplemented reactor. For RNAseq extraction, 5 ml culture samples were harvested in 1 ml aliquots, collected by centrifugation at 14,000 × g for 3 min, and stored at −80°C until subsequent RNA extraction. The supernatant from one of the appropriate time point aliquots was processed for organic acid analysis as described previously (Eng et al., 2020). Lab-scale IP production in deep well plates or 5 ml culture tubes were conducted as previously described (Eng et al., 2020). Isopentenol titers reported for the deep well plate format were corrected for evaporation at the 48 h time point as conducted previously (Sasaki et al., 2019). Exogenous [Ch][Lys] toxicity against C. glutamicum ATCC13032 and BRC-JBEI 1.1.2 was analyzed in a 48-well microtiter dish format. Cells were first adapted two times in CGXII minimal media with 4% (w/v) D-glucose. When cells were back diluted into fresh media in the microtiter dish, the starting Optical Density (OD) was set to 0.1 with a fill volume of 200 μl. The plate was incubated with shaking at 30°C and exogenous [Ch][Lys] added at the start of the time course. OD was monitored at 600 nm on a Synergy 4 plate reader (BioTek Instruments, Winooski VT) with the continuous shaking setting.

CGXII minimal media was supplemented with ensiled sorghum biomass hydrolysate to test the ability of C. glutamicum BRC-JBEI 1.1.2 to utilize carbon sources from renewable feedstock pretreated with IL. Briefly, the forage sorghum (NK300 type, grown in Fresno, CA) was planted in Spring 2020 and harvested in Fall 2020. A forage harvester was used to both harvest and chop the sorghum biomass, which was then loaded in a silage pit, inoculated, and covered to maintain anaerobic conditions. The pit was opened in November 2020 and a sample of the ensiled material was collected, packed with dry ice while in transit, and stored at 4°C. A 210 L scale Andritz Hastelloy C276 pressure reactor (AG, Graz, Austria) with a helical impeller was utilized to process ensiled sorghum for the pretreatment and saccharification processes. Ensiled sorghum biomass was pretreated at 20% w/w solid loading with 10% w/w [Ch][Lys] at 140°C for 3 h with a mixing speed of 30 rpm. Solid loading was calculated based on the dry matter content determined using a Binder VDL115 vacuum oven. After 3 h at the target temperature, the reactor was cooled to room temperature before proceeding with the next steps. The Andritz reactor is sealed during this process, preventing contamination until further processing. Following pretreatment, the pretreated materials were adjusted to pH 5.1 using 50% v/v sulfuric acid and an enzyme cocktail of Novozyme, Inc. Cellic Ctec3 and Cellic Htec3 commercial enzymes in a ratio of 9:1 was added. Concentration of the commercial stocks were determined using Bradford assays and bovine serum albumin as a reference. Enzyme load was conducted at a ratio of 10 mg enzyme per 1 g of dry weight biomass. Following pH adjustment and enzyme addition, RODI water was added to obtain a final solid loading of 18.70%. Saccharification by enzymatic hydrolysis was operated at 50°C, 30 rpm for 70 h (Barcelos et al., 2021). The hydrolysate was then sequentially filtered using a filter press through 5, 1, and 0.25 μm filters. Final filter sterilization was completed with a 0.2 μm filter and stored at −80°C until further use. This hydrolysate was thawed and added in place of water in CGXII media (amounting to 2.8 % (w/v) glucose), pH was adjusted to 7.4 and filter sterilized one additional time before use. We make the assumption the hydrolysate contained no biologically available nitrogen. To maintain a C/N ratio of glucose/ammonium sulfate + urea of 2.8, pure glucose powder was supplemented to the hydrolysate CGXII cultivation medium composition (Sasaki et al., 2019).

Genomic DNA from C. glutamicum BRC-JBEI 1.1.2 was isolated with the following protocol. In brief, strains from glycerol stocks were struck to single colonies on LB plates grown at 30°C overnight. A single colony was then inoculated into a 250 ml shake flask with 25 ml LB media and grown overnight to saturation. Cells were collected by centrifugation at 4,000 × g for 5 min. The cell pellet was then resuspended in 2 ml lysis buffer (2 mM EDTA, 250 mM NaCl, 2% (w/v) SDS, 2% (v/v) Triton-X 100, 2% (v/v) Tween-80, 5 mM DTT, 30 units Zymolase 100T, 1 mg/ml RNaseA). Zymolyase was supplied by US Biological (Salem, MA). The cells were initially incubated at 50°C for 30 min to promote protease activity and then incubated for an additional 3 h at 37°C with occasional mixing, at which point the lysate became noticeably viscous. DNA was extracted following standard protocols for isolation of DNA using phenol chloroform: isoamyl alcohol and subsequent isopropanol precipitation (Sambrook and Russell, 2001).

RNA was extracted from C. glutamicum samples using a Direct-Zol RNA Kit (Zymo Research, Irvine, CA) following the manufacturer’s protocol. C. glutamicum cells were lysed after initially resuspending the cell pellet in 500 µl TRI reagent and mixed with glass beads. This mixture was then subject to cell disruption using a bead-beater (Biospec Inc., Bartlesville, OK) with a 3-min homogenization time at maximum intensity. After bead beating, samples were collected following the manufacturer’s protocol without any additional modifications. RNA quality was assessed using a BioAnalyzer (Agilent Technologies, Santa Clara, CA) before RNA library preparation and downstream analysis.

For 16S ribosomal sequencing, C. glutamicum ATCC 13032 ∆mrr and C. glutamicum JBEI-BRC 1.1.2 were struck from glycerol stocks to single colonies on LB plates and incubated overnight at 30°C. A single colony was isolated and boiled in 50 µl dH₂0 for 10 min. 1 µl of the boiled colony was used for PCR with primer pair (JGI_27F: 5′-AGAGTTTGATCCTGGCTCAG-3′ and JGI_1391R: 5′-GACGGGCRGTGWGTRCA-3′) with NEB Q5 Polymerase (New England Biolabs, Ipswitch, MA). The PCR amplicon was confirmed by agarose gel electrophoresis and the sequence was determined using conventional Sanger Sequencing (Genewiz LLC, Chelmsford, MA).

DNA sequencing was generated at the DOE Joint Genome Institute (JGI) using the Pacific Biosciences (PacBio) sequencing technology. A Pacbio SMRTbell(tm) library was constructed and sequenced on the PacBio Sequel and PacBio RS II platforms, which generated 397,096 filtered subreads (1,418,602,725 subread bases) totaling 3,352,276 bp. The mean coverage for this genome was 432.21x. All general aspects of library construction and sequencing performed at the JGI can be found at http://www.jgi.doe.gov.

Stranded RNAseq library(s) were created and quantified by qPCR. Sequencing was performed using an Illumina instrument (refer to Supplementary Table S3 for specifics per library). Raw fastq file reads were filtered and trimmed using the JGI QC pipeline resulting in the filtered fastq file (*.filter-RNA.gz files). Using BBDuk (https://sourceforge.net/projects/bbmap/), raw reads were evaluated for artifact sequence by kmer matching (kmer = 25), allowing for 1 mismatch and detected artifacts which were trimmed from the 3′ end of the reads. RNA spike-in reads, PhiX reads and reads containing any Ns were removed. Quality trimming was performed using the phred trimming method set at Q6. Following trimming, reads that did not meet the length threshold of at least 50 bases were removed.

Filtered reads from each library were aligned to the reference genome using HISAT2 version 2.2.0 (Kim et al., 2015). Strand-specific coverage bigWig files were generated using deepTools v3.1 (Ramírez et al., 2014). Next, featureCounts (Liao et al., 2014) was used to generate the raw gene counts (counts.txt) file using gff3 annotations. Only primary hits assigned to the reverse strand were included in the raw gene counts (-s 2 -p --primary options). Raw gene counts were used to evaluate the level of correlation between biological replicates using Pearson’s correlation and determine which replicates would be used in the DEG analysis (Supplementary Figure S5). In the heatmap view, the libraries were ordered as groups of replicates. The cells containing the correlations between replicates have a purple (or white) border around them. For fragments per kilobase of transcript per million fragments mapped (FPKM) and TPM, normalized gene counts refer to SRA reads (Data availability section). A sample legend and description of RNAseq libraries used in this paper is described in Supplementary Table S3.

Global transcriptome response under various experiment conditions were measured using Geneious Prime 2021 (https://www.geneious.com). The normalized expression was calculated and the differentially expressed genes (DEGs) were filtered for absolute log₂ ratio >2 (i.e. a 4-fold up or down regulation), absolute confidence >3 (p < 0.001) and >90% sequence identity. The DEGs at various conditions were functionally annotated using Blast2GO suite (Götz et al., 2008) to assign GO annotations (Galperin et al., 2015). Each DEG was subjected to pathway analysis using the KEGG (Kyoto Encyclopedia of Genes and Genomes) database (http://www.kegg.jp/kegg/pathway.html) to explore the biological implications. Biocyc (https://biocyc.org/) was used to calculate pathway enrichment for the last 65 h/41 h time point and for additional gene orthologs identification. Pathways were considered significant if p < 0.05. Hierarchically clustered heat maps were generated with average linkage method and euclidean distance metric in Jupyter notebook using Python library Seaborn 0.11.1 (Waskom et al., 2020).