Jiaming Qi2,3, Dongxu Zhang1,2,3
, Shaoli Wang1, Libin Huang1, Lili Xia1, Wangyang Dong1, Qiang Zheng1, Qing'e Liu1, Jianzhong Xiao1, Zhiwen Xu2,3 1. College of Ecology, Lishui University, Lishui 323000, Zhejiang Province, China;
2. Leading Bio-Agricultural Co., Ltd., Qinhuangdao 066000, Hebei Province, China;
3. Engineering & Technology Research Center of Agricultural Biotechnology of Hebei Province, Qinhuangdao 066000, Hebei Province, China
Received: 6 August 2019; Revised: 2 October 2019; Published online: 4 January 2020
Foundation item: Supported by the Zhejiang Provincial Natural Science Foundation of China (LY15C200012), by the Foundation for High-Level Talents of Lishui City (2016RC01) and by the Key Research and Development Project of Zhejiang Province (2018C02031)
Corresponding author: Dongxu Zhang, Tel:+86-578-2271308, E-mail:zhangdongxu@lsu.edu.cn; Zhiwen Xu, Tel:+86-335-8500208, E-mail:xu.zhiwen@leadst.cn.
Abstract: [Objective] The prebiotic effect of xylo-oligosaccharides (XOS) has obtained much attention in recent years due to their fermentation properties. In this study, a kimchi-derived strain, Weissella confusa XU1 grew better on XOS than on glucose and xylose. The mechanism of XOS utilization by W. confusa XU1 was further explored.[Methods] Differential transcriptomes of W. confusa XU1, induced by XOS, glucose and xylose, were analyzed to identify the genetic loci involved in the uptake and catabolism of XOS.[Results] Transcriptome analysis reveals that several major facilitator superfamily (MFS) transporters and glycoside hydrolases were involved in the uptake and hydrolysis of XOS. In addition, glycolysis pathway and pentose phosphate pathway in W. confusa XU1 were enhanced when XOS were present, indicating that XOS were utilized more efficiently compared with other carbon sources.[Conclusion] This study reveals a proposed XOS utilization mechanism in W. confusa XU1.
Keywords: major facilitator superfamily (MFS) transporterprobioticstranscriptomeWeissella confusaxylo-oligosaccharides
基于转录组测序数据对Weissella confusa XU1中低聚木糖代谢系统的分析
戚家明2,3, 张东旭1,2,3
, 王少丽1, 黄立斌1, 夏丽丽1, 董汪洋1, 郑强1, 刘青娥1, 肖建中1, 徐志文2,3 1. 丽水学院生态学院, 浙江 丽水 323000;
2. 领先生物农业股份有限公司, 河北 秦皇岛 066000;
3. 河北省农业生物技术工程技术研究中心, 河北 秦皇岛 066000
收稿日期：2019-08-06；修改日期：2019-10-02；网络出版日期：2020-01-04
基金项目：浙江省自然科学基金（LY15C200012）；丽水市高层次人才项目（2016RC01）；浙江省重点研发计划（2018C02031）
通信作者：张东旭, Tel:+86-578-2271308, E-mail:zhangdongxu@lsu.edu.cn; 徐志文, Tel:+86-335-8500208, E-mail:xu.zhiwen@leadst.cn.
摘要：[目的] 近年来由于在发酵方面的良好特性，低聚木糖的益生作用越来越得到公众的关注。研究发现相比于葡萄糖和木糖Weissella confusa XU1在以低聚木糖为唯一碳源时生长情况最好。本文将对Weissella confusa XU1中低聚木糖的代谢机制进行研究。[方法] 本研究分别以葡萄糖、木糖和低聚木糖作为唯一碳源对Weissella confusa XU1进行转录组测序并进行比较分析。[结果] 通过转录组分析发现以低聚木糖为唯一碳源的处理中部分编码MFS转运蛋白和糖基水解酶的基因转录水平显著上升，Weissella confusa XU1中的糖酵解过程和磷酸戊糖途径也得到显著增强。[结论] 本研究根据转录组数据分析得出Weissella confusa XU1中的低聚木糖代谢机制。本研究首次在革兰氏阳性菌中发现MFS转运蛋白参与到低聚木糖转运的过程，为提高微生物对木聚糖利用效率进行分子改造提供了改造方向，该机制为低聚木糖代谢的研究和Weissella的工业化应用提供了新的思路。
关键词：MFS转运蛋白益生菌转录组Weissella confusa低聚木糖
Probiotics are beneficial microorganisms which are used for the purpose of improving general health conditions of hosts including human beings and farm animals[1-2]. Lactic acid bacteria Weissella together with Bifidobacterium, and Lactobacillus are employed as the most important probiotics for their long history of use in fermented foods with much positive effects on the health[3-4]. Previous studies have reported that some Weissella strains could be used as probiotics[5] and are important starters for fermented foods including kimchi and soy sauce[6]. The Weissella species are Gram-positive, non-endospore forming cells with rod-shaped or coccoid morphology[7-8]. Weissella species have been found in a variety of environments, including traditional fermented foods such as kimchi, and the gastrointestinal tracts of human and animals[9], some of which have enzymatic activities that play important roles in food fermentation. For example, β-glucosidases of Weissella confusa 31 and Weissella cibaria 33 converts glycosides of foods into aglycones which show more bioactive than glycosides in human body[10]. Dextran and fructan synthesized by Weissella have the function of relieving gastrointestinal discomfort and increasing the absorption of trace elements[11].
Xylo-oligosaccharides (XOS) are prebiotics and can not be hydrolyzed by enzymes or the low pH in human body. So XOS are not absorbed by human during transit through the small intestine. Based on special structure characteristics, XOS are easy to pass through the foregut and reach the colon, where they serve as fermentable substrates for the resident beneficial bacterium in the hindgut[12-13]. Therefore, XOS can increase the indigenous Bifidobacterium spp. biomass in gastrointestinal tract[14-15] and promote the growth of probiotics in human gut microbiota to make the bacteria stay within a healthy balance[16]. Moreover, studies have shown that XOS ingestion contributes to anti-oxidant, anti-bacterial, immune-modulatory and anti-diabetic activities[17-18]. XOS can be digested by prebiotics but the underlying molecular mechanism remains elusive. Recently, several researches reported that XOS could be utilized by Bifidobacterium and Pediococcus and the utilization pathways of XOS in these microbes were found out by transcriptome analysis[19-20]. XOS are the results of partial hydrolysis of xylan, which are the major components of most plant cell walls. The main classes of xylan are the glucuronoxylans, arabinoxylans and glucuronoarabinoxylans, which are main cell wall components of birchwood, wheat flour and corn bran, respectively[21]. The XOS used in the current study have a degree of polymerization ranging from 2-7 and were hydrolyzed from corn glucuronoarabinoxylan. Because of the complexity of xylan structure, XOS used in the current study contains various side chains such as arabinose, galactose and xylose. Therefore, the degradation and utilization of XOS requires the synergistic activity of several glycoside hydrolases.
In the current study, Weissella confusa XU1 (W. confusa XU1) isolated from kimchi was found to be able to grow on the medium that XOS are the sole carbon and energy source. Similar to Bifidobacterium and Pediococcus, Weissella is able to utilize XOS. Transcriptome analysis was employed to identify W. confusa XU1 genes which are involved in XOS utilization. The molecular mechanism of XOS utilization in W. confusa XU1 may reveal novel pathways.
1 Materials and Methods 1.1 Bacterial strains and bacterial growth curve assay W. confusa XU1 previously isolated from kimchi bought from supermarket and was identified by 16S rRNA sequence analysis. Pre-grown W. confusa XU1 (1%, V/V) was inoculated to modified MRS medium (Oxoid peptone 10 g/L, XOS/glucose/ xylose 20 g/L, K2HPO4 2 g/L, CH3COONa·3H2O 5 g/L, triammonium citrate 2 g/L, MgSO4·7H2O 0.2 g/L, MnSO4·4H2O 0.05 g/L, pH 6.2) containing XOS (LongLive Biotechnology), glucose (Sigma Aldrich) or xylose (Sigma Aldrich) as the only carbon source, respectively. Aliquots of cultures were sampled at regular intervals. All the cultures were incubated at 30 ℃ under anaerobic conditions. For anaerobic growth, media was incubated at 30 ℃ in a coy anaerobic chamber (10% CO2, 10% H2 and 80% N2) for 16 hours before inoculation, and cultures were grown at 30 ℃ in flasks without shaking. The microbial growth was monitored by dry cell weight at every hour.
1.2 RNA isolation and library preparation for strand-specific transcriptome sequencing W. confusa XU1 cells for RNA isolation were harvested independently from triplicate cultures of each carbon source MRS medium at estimated early mid-exponential growth phase by centrifugation. Total RNA was extracted using Trizol and used for library preparation and Illumina sequencing. Sequencing and analysis of transcriptome of the samples were performed in Beijing Novogene Bioinformation Technology Co. Ltd (Beijing, China).
For each sample, mRNAs were purified using oligo (dT)-attached magnetic beads. The mRNA was fragmented and primed with random hexamers, and then submitted for the synthesis of the first-strand and second-strand cDNA. The cDNA fragments were processed for end repair and finally ligated to paired-end adaptors. The cDNA fragments with 150-200 bp size were selected and enriched by PCR amplification. The cDNA libraries were constructed and adopted the paired-end sequencing on an Illumina Hiseq-PE150.
1.3 Data analysis Clean data (clean reads) were obtained by removing reads containing adapter, reads containing ploy-N and low quality reads from raw data. Bowtie2-2.2.3 was used to build index of reference genome (Weissella confusa DSM 20196) and align clean reads to reference genome.
HTSeq v0.6.1 was used to count the reads numbers mapped to each gene. And then FPKM of each gene was calculated based on the length of the gene and reads count mapped to this gene.
Differential expression analysis of three conditions (three biological replicates per condition) was performed using the DESeq R package (1.18.0). The resulting P-values were adjusted using the Benjamini and Hochberg's approach for controlling the false discovery rate. Genes with an adjusted P-value < 0.05 found by DESeq were assigned as differentially expressed.
Gene Ontology (GO) enrichment analysis of differentially expressed genes (DEGs) was implemented by the GOseq R package, in which gene length bias was corrected. GO terms with corrected P-value less than 0.05 were considered significantly enriched by differential expressed genes.
KOBAS software was used to test the statistical enrichment of differential expression genes in KEGG pathways.
1.4 Data archiving The raw transcriptome data generated during the current study are available in the National Center for Biotechnology Information Sequence Read Archive (SRA) under SRA accession number SRR9021087-SRR9021095.
2 Results 2.1 Growth on XOS, xylose or glucose The changes of W. confusa XU1 biomass with cultivation time of three different carbon sources were shown in Figure 1. Fermentation was completed within 12 hours. Unexpectedly, W. confusa XU1 grown on MRS media with XOS as the sole carbon source had the highest biomass in comparison with xylose or glucose as the sole carbon source. W. confusa XU1 was observed to grow best on XOS, followed by growth on the glucose. The biomass of W. confusa XU1 on XOS at 6 hour was about 1.51 and 2.63-fold greater than that on glucose and xylose, respectively.
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| Figure 1 Changes of W. confusa XU1 biomass with cultivation time of three different carbon sources. Mean values (±s.e.m.) of three replicates are shown. Experiments were repeated twice with similar results. XOS: xylo-oligosaccharides. |
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2.2 RNA sequencing and assembly of the transcriptomes To obtain a global view of W. confusa XU1 transcriptomes during different carbon sources utilization, RNA-seq was performed on W. confusa XU1 cultures containing XOS, xylose and glucose as the sole carbon source, and the gene expression profiles of each condition were compared. Nine cDNA libraries prepared from the pooled total RNA extracted from Weissella confusa XU1 grown under three different carbon conditions were paired-end sequenced on the Illumina Hiseq-PE150 platform. After filtering out repetitive, low-complexity, and low-quality reads, the clean reads were assembled according to the reference genome (Weissella confusa DSM 20196). The sequencing results for the nine libraries (designated Xylose_1, Xylose_2, Xylose_3, Glu_1, Glu_2, Glu_3, XOS_1, XOS_2 and XOS_3) are summarized in Table 1. For each library, all clean reads were mapped back to the Weissella confusa reference genome. The percentage of clean reads from each library that could be uniquely mapped ranged from 91.14% to 93.45%, thus providing good coverage of the transcript profiles.
Table 1. Statistics of nine libraries reads mapping
| Sample name | Xylose_1 | Xylose_2 | Xylose_3 | Glu_1 | Glu_2 | Glu_3 | XOS_1 | XOS_2 | XOS_3 |
| Total reads | 9800160 | 8798142 | 8558890 | 9789186 | 10406530 | 10107678 | 9970784 | 10524586 | 9914424 |
| Total mapped | 9407360 (95.99%) | 8447705 (96.02%) | 8241762 (96.29%) | 9508215 (97.13%) | 10107203 (97.12%) | 9791271 (96.87%) | 9526282 (95.54%) | 10062974 (95.61%) | 9473711 (95.55%) |
| Uniquely mapped | 9041236 (92.26%) | 8119380 (92.29%) | 7910181 (92.42%) | 9131155 (93.28%) | 9724725 (93.45%) | 9383840 (92.84%) | 9087176 (91.14%) | 9606691 (91.28%) | 9039402 (91.17%) |
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2.3 DEGs induced by different carbon sources Differentially expressed genes (DEGs) were analyzed via pairwise comparisons of XOS vs glucose, XOS vs xylose and xylose vs glucose to investigate which genes involved in XOS utilization when W. confusa XU1 grew in presence of XOS, xylose, or glucose as the sole carbon source. Cluster analysis of DEGs among xylose, glucose and XOS was shown in Figure 2.
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| Figure 2 Cluster analysis of DEGs in different treatments. Values of log10 (FPKM+1) were conducted normalized transformation before clustered. Red indicated high expressed genes; Blue indicated low expressed genes. |
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The expression model of DEGs in xylose showed the lowest and highest similarities with XOS and glucose treatment, respectively. In comparison of XOS vs glucose, most DEGs were related to metabolic pathways, biosynthesis of secondary metabolites and biosynthesis of antibiotics. In comparison of XOS vs xylose, most DEGs were related to metabolic pathways, biosynthesis of secondary metabolites and transporters. In comparison of xylose vs glucose, most DEGs were also related to metabolic pathways, biosynthesis of secondary metabolites and transporters.
The global gene expression profiles of three comparisons were shown in Figure 3. Lots of DEGs found in each comparison suggests that different carbon sources have significant effects on global gene expression. Compared to xylose and glucose treatment, several highly expressed genes (listed by W. confusa XU1 locus tag numbers) with highlight molecular functions in XOS treatment would be related to XOS utilization mechanisms.
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| Figure 3 Volcano plots of pairwise comparisons of the carbon-induced differential global transcriptome in W. confusa XU1. A: XOS vs xylose; B: Xylose vs glucose; C: XOS vs glucose. |
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2.4 MFS transporters involved in XOS and xylose uptake Analysis of differentially up-regulated loci enabled the identification of several genes conferring the uptake of the XOS and xylose used in this study. As shown in Table 2, major facilitator superfamily (MFS) transporters were significantly up-regulated among three comparisons. Compared to xylose treatment, MFS transporters IV69_RS08185 and IV69_RS01700 were up-regulated 20.39-fold and 6.92-fold with q-value≤0.01 under XOS treatment, respectively.
Table 2. Statistically significant up-regulated genes involved in XOS uptake
| Gene Noa | Gene annotation | Log2FoldChange (XOS vs Xylose)b | -Log10 (q-value) | Log2FoldChange (Xylose vs Glu) | -Log10 (q-value) | Log2FoldChange (XOS vs Glu) | -Log10 (q-value) |
| 08185 | MFS transporter | 4.35 | 70.40 | 4.38 | 113.93 | 8.71 | 175.92 |
| 01700 | MFS transporter (Na+/xyloside symporter related) | 2.79 | 118.17 | 4.23 | 96.26 | 7.02 | q-value=0 |
| 01450 | Sugar porter family MFS transporter (D-xylose-proton symporter) | -0.30 | 1.16 | 2.64 | 34.66 | 2.33 | 53.98 |
| 01480 | Sugar porter family MFS transporter (D-xylose-proton symporter) | 0.11 | 0.30 | 8.13 | 118.06 | 8.23 | q-value=0 |
| a: Gene number referenced as IV69_RS XXXXX with XXXXX being a five-digit number; b: Significance of fold change data in XOS vs xylose is judged by having a q-value on no more than 0.05 (except 01450 and 01480). | |||||||
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Interestingly, another two MFS transporters (IV69_RS01480 and IV69_RS01450) were both significantly up-regulated more than 270-fold and 5-fold in xylose vs glucose and XOS vs glucose comparisons, respectively. However, there was no significant difference of these two MFS transporters (IV69_RS01480 and IV69_RS01450) in XOS vs xylose comparison (Table 2). These results indicated that these two MFS transporters (IV69_RS01480 and IV69_RS01450) were only related to xylose uptake.
2.5 DEGs involved in XOS catabolism Several genes related to XOS degradation were found in the comparison of XOS and xylose (Table 3). Under XOS treatment, the extracellular GH43 enzymes (IV69_RS09575 and IV69_RS02985) encoding β-xylosidase, and extracellular GH2 enzymes (IV69_RS05575 and IV69_RS05580) encoding β-galactosidase contributed to the degradation of the XOS generated in the periplasm.
Table 3. Statistically significant up-regulated genes involved in XOS hydrolyzation
| Gene Noa | Gene annotation | Sub-cellular localizationb | Log2FoldChange (XOS vs Xylose)c | -Log10 (q-value) | Log2FoldChange (Xylose vs Glu) | -Log10 (q-value) | Log2FoldChange (XOS vs Glu) | -Log10 (q-value) |
| 09575 | β-xylosidase | Extracellular space | 1.06 | 21.25 | 7.55 | q-value=0 | 8.59 | q-value=0 |
| 02985 | β-xylosidase | Extracellular space | 0.46 | 1.81 | 1.84 | 13.55 | 2.29 | 30.49 |
| 05580 | β-galactosidase | Extracellular space | 1.13 | 23.38 | 4.37 | 165.44 | 5.50 | q-value=0 |
| 05575 | β-galactosidase | Extracellular space | 0.73 | 9.49 | 4.36 | 122.72 | 5.08 | q-value=0 |
| a: Gene number referenced as IV69_RS XXXXX with XXXXX being a five-digit number; b: Sub-cellular localizations were predicted by "LocTree3" method[22]; c: Significance of fold change data in XOS vs xylose is judged by having a q-value on no more than 0.05. | ||||||||
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Several genes related to xylose catabolism were also found significantly up-regulated under XOS treatment in the comparison of XOS and xylose, such as xylose isomerase (IV69_RS08095 and IV69_RS09560) and xylulose kinase (IV69_RS09555). What's more, nine genes involved in pentose phosphate pathway (map00030) and glycolysis pathway (map00010) were also up-regulated in XOS treatment (Table 4).
Table 4. DEGs involved in XOS catabolism
| Gene Noa | Gene annotation | EC No. | Log2FoldChange (XOS vs Xylose)b | -Log10 (q-value) | Log2FoldChange (XOS vs Glu) | -Log10 (q-value) |
| 08095 | Xylose isomerase | 5.3.1.5 | 2.12 | 73.86 | 7.91 | q-value=0 |
| 09560 | Xylose isomerase | 5.3.1.5 | 2.31 | 95.53 | 10.54 | q-value=0 |
| 09555 | Xylulokinase | 2.7.1.17 | 2.34 | 95.81 | 9.35 | q-value=0 |
| 01395 | Xylulose-5-phosphate phosphoketolase | 4.1.2.9 | 0.31 | 2.01 | 0.49 | 7.04 |
| 08795 | Type I glyceraldehyde-3-phosphate dehydrogenase | 1.2.1.12 | 0.77 | 11.37 | 1.03 | 29.54 |
| 08800 | Phosphoglycerate kinase | 2.7.2.3 | 0.53 | 5.50 | 0.81 | 18.51 |
| 00890 | 2, 3-bisphosphoglycerate-dependent phosphoglycerate mutase | 5.4.2.11 | 1.04 | 20.74 | 0.74 | 14.99 |
| 05190 | Pphosphopyruvate hydratase | 4.2.1.11 | 0.49 | 4.90 | 0.41 | 5.16 |
| 03240 | Pyruvate kinase | 2.7.1.40 | 0.34 | 2.42 | -0.73 | 15.04 |
| 05010 | Glucose-6-phosphate isomerase | 5.3.1.9 | 0.67 | 8.39 | -0.03 | 0.08 |
| 00475 | Glucose-6-phosphate 1-dehydrogenase | 1.1.1.363 | 0.38 | 2.76 | -0.90 | 10.56 |
| 02530 | 6-phosphogluconolactonase | 3.1.1.31 | 0.98 | 18.06 | 2.69 | 170.22 |
| 00035 | 6-phosphogluconate dehydrogenase (decarboxylating) | 1.1.1.343 | 1.53 | 40.64 | -2.36 | 139.10 |
| 04535 | L-lactate dehydrogenase | 1.1.1.27 | -0.19 | 0.75 | -2.80 | 90.70 |
| a: Gene number referenced as IV69_RS XXXXX with XXXXX being a five-digit number; b: Significance of fold change data in XOS vs xylose is judged by having a q-value on no more than 0.05 (except 04535). | ||||||
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Compared to glucose treatment, genes (IV69_RS08795, IV69_RS08800, IV69_RS00890 and IV69_RS05190) related to phosphoenolpyruvate production (map00010) were up-regulated under XOS treatment. Moreover, a gene encoded L-lactate dehydrogenase (IV69_RS04535) was significantly down-regulated under XOS treatment.
3 Discussion 3.1 Debranch and hydrolysis of XOS backbone XOS are oligomers that consist of 2-10 xylose residues connected through β-(1-4)-linkages as the result of partial hydrolysis of xylan. So, the conserved β-1, 4-xylose backbone of XOS may be decorated with various kinds of short side chains, including L-arabinose and L-galactose.
Before transported into cells, the xylan-derived XOS may be degraded by a suite of primarily exo- acting enzymes, such as β-xylosidase (IV69_RS09575 and IV69_RS02985) and β-galactosidase (IV69_RS05580 and IV69_RS05575). Some β-galactosidase enzymes had been reported that could cleave α-L-arabinosides, β-D-fucosides and β-D-glucosides[23]. And the side chains of XOS may be cleaved by β-galactosidase (IV69_RS05580 and IV69_RS05575) and the conserved β-1, 4-xylose backbone of XOS was hydrolyzed by β-xylosidase (IV69_RS09575 and IV69_RS02985), after which XOS were converted to xylose and XOS with a low degree of polymerization outside cells.
Different from Pediococcus[20] and Bifidobacterim[24], XOS were hydrolyzed into xylose and XOS with a low degree of polymerization outside cells with exo-acting enzymes.
3.2 More energy-efficient XOS transportation by MFS transporters Several reports have shown that XOS are transported into cell membrane only by an ABC transporter sugar system in bacteria, such as P. acidilactici BCC-1[20] and B. lactis B1-04[24]. In this study, MFS transporters were observed to participate in XOS transportation. Proton-motive force (PMF) was utilized by many MFS transporters to drive the transport process[25-26]. Two ATP molecules (equivalent to about 6 protons) were consumed by ABC transporters per transport cycle. While, for MFS transporters, one proton was consumed to transport one molecule of an electroneutral substrate. Thus, MFS transporters appear to be more energy-efficient in terms of the stoichiometric ratio of substrate to protons than ABC transporters[27]. What's more, products hydrolyzed outside cells may be easier to be transported into cells than XOS.
Taken together, these findings suggested that W. confusa XU1 may possess more efficient catabolic pathway for XOS utilization than other lactic acid bacteria for its more energy-efficient transportation of XOS.
3.3 XOS catabolism in W. confusa XU1 After transported into cells, xylose may be further translated to glyceraldehyde-3P with the action of xylose isomerase (IV69_RS08095 and IV69_RS09560), xylulose kinase (IV69_RS09555) and xylulose-5-phosphate phosphoketolase (IV69_RS01395).
Glyceraldehyde-3P was converted to pyruvate which then may participate in TCA cycle (map00020) and pyruvate metabolism (map00620) by up-regulated genes of glycolysis pathway (map00010) in XOS treatment (Table 4), including type I glyceraldehyde-3-phosphate dehydrogenase (IV69_RS08795), phosphoglycerate kinase (IV69_RS08800), 2, 3-bisphosphoglycerate-dependent phosphoglycerate mutase (IV69_RS00890), phosphopyruvate hydratase (IV69_RS05190) and pyruvate kinase (IV69_RS03240). Glycolysis pathway was enhanced under XOS treatment in W. confusa XU1 and there may be more pyruvate to participate in TCA cycle.
Pentose phosphate pathway can be divided into oxidative branch and non-oxidative branch. In this study, oxidative pentose phosphate pathway was also enhanced. Alpha-D-glucose-6-phosphate in glycolysis (map00010) was converted to D-ribulose-5- phosphate by related genes which were up-regulated in XOS vs xylose (Table 4), including glucose-6-phosphate isomerase (IV69_RS05010), glucose-6-phosphate 1-dehydrogenase (IV69_RS00475), 6-phosphogluconolactonase (IV69_RS02530), and 6-phosphogluconate dehydrogenase (IV69_RS00035).
The oxidative branch consists of three irreversible reactions, which result in NADPH and pentose phosphate production. NADPH is used as a reducing agent in many biosynthetic pathways and is also important for protection against oxidative damage[28]. Previous studies reported that xylose fermentation rate was controlled by pentose phosphate pathway[29]. A prime control point in xylose fermentation would be the oxidative pentose phosphate pathway, since this pathway is the principal NADPH-generating pathway in the cell[30-31]. And prior study has reported that xylose consumption rate drastically decreases with a non-functional oxidative pentose phosphate pathway[32].
Glycolysis pathway and oxidative pentose phosphate pathway were both enhanced by XOS stimulation in W. confusa XU1, which may be one of the reasons for the maximum biomass accumulation under XOS treatment.
3.4 Different biomass production on XOS, xylose and glucose In the current research, W. confusa XU1 with the growth capacity on XOS was reported (Figure 1). However, the findings of the current study are different from previous research[20]. XOS were more preferred by W. confusa XU1 than glucose and xylose.
According to DEGs analysis of XOS vs xylose, a lower expression of L-lactate dehydrogenase may also reduce the consumption of pyruvate to produce L-lactate and more pyruvate would participate in TCA cycle (Table 4), which may give an advantage to cell growth of W. confusa XU1.
A lower expression of L-lactate dehydrogenase, more energy-efficient transportation of XOS and enhancement of glycolysis pathway and oxidative pentose phosphate pathway, all these results illustrated that the maximum biomass production of W. confusa XU1 was observed when XOS were used as the solo carbon source. This character may be a results of growth competition happened in beneficial bacterium located hindgut of animal or human being, where XOS are more available than glucose or xylose.
Based on the results presented here, a possible pathway for XOS uptake and catabolism in W. confusa XU1 was proposed (Figure 4). XOS were first hydrolyzed by β-xylosidase and β-galactosidase outside cells to produce xylose and XOS with a low degree of polymerization. Then hydrolysis products were transported into cells by MFS transporters to participate in glycolysis pathway (map00010), pentose phosphate pathway (map00030) and TCA cycle (map00020), which could enhance sugar absorption and utilization.
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| Figure 4 Proposed pathways of XOS uptake and catabolism. XOS: Xylo-oligosaccharides; MFS: major facilitator superfamily; PEP: phosphoenolpyruvate; xpk: xylulose-5-phosphate phosphoketolase; TCA cycle: tricarboxylic acid cycle. |
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Weissella confusa is an important specific starter for food fermentation, which has potential in improving nutritional properties of foods. This study provides insights into the mechanism of utilization of XOS at molecular level in Weissella confusa, which may benefit to improve its fermentation process both in vivo and in vitro. Moreover, with energy-efficient transportation of XOS, W. confusa XU1 shows bright application prospects in food fermentation industry. XOS transportation pathway revealed here may be used as a referable tool to improve utilizing efficiency of xylan substrates for genetically engineered microorganism.
Conflict of interest The authors declare that they have no conflict of interest.
Acknowledgements We thank Na Yang (Leading Bio-agricultural Co., Ltd.) for technical assistance. We acknowledge Dr. Zhouxing Ding (Ngee Ann Polytechnic, Singapore) for the linguistic editing and proofreading during the preparation of this manuscript.
References
| [1] | Fuller R. Probiotics in man and animals. Journal of Applied Bacteriology, 1989, 66(5): 365-378. DOI:10.1111/j.1365-2672.1989.tb05105.x |
| [2] | Ouwehand AC, Kirjavainen PV, Shortt C, Salminen S. Probiotics:mechanisms and established effects. International Dairy Journal, 1999, 9(1): 43-52. DOI:10.1016/S0958-6946(99)00043-6 |
| [3] | Chang YH, Kim JK, Kim HJ, Kim WY, Kim YB, Park YH. Selection of a potential probiotic Lactobacillus strain and subsequent in vivo studies. Antonie van Leeuwenhoek, 2001, 80(2): 193-199. DOI:10.1023/A:1012213728917 |
| [4] | Lee YK, Salminen S. The coming of age of probiotics. Trends in Food Science & Technology, 1995, 6(7): 241-245. |
| [5] | Lee KW, Park JY, Jeong HR, Heo HJ, Han NS, Kim JH. Probiotic properties of Weissella strains isolated from human faeces. Anaerobe, 2012, 18(1): 96-102. DOI:10.1016/j.anaerobe.2011.12.015 |
| [6] | Tanaka Y, Watanabe J, Mogi Y. Monitoring of the microbial communities involved in the soy sauce manufacturing process by PCR-denaturing gradient gel electrophoresis. Food Microbiology, 2012, 31(1): 100-106. |
| [7] | Collins MD, Samelis J, Metaxopoulos J, Wallbanks S. Taxonomic studies on some Leuconostoc-like organisms from fermented sausages:description of a new genus Weissella for the Leuconostoc paramesenteroides group of species. Journal of Applied Bacteriology, 1993, 75(6): 595-603. DOI:10.1111/j.1365-2672.1993.tb01600.x |
| [8] | Bj?rkroth J, Dicks LMT, Endo A. The genus Weissella//Holzapfel WH, Wood BJB. Lactic Acid Bacteria: Biodiversity and Taxonomy. Chichester: Wiley Blackwell, 2014: 418-428. |
| [9] | De Bruyne K, Camu N, De Vuyst L, Vandamme P. Weissella fabaria sp. nov., from a Ghanaian cocoa fermentation. International Journal of Systematic and Evolutionary Microbiology, 2010, 60(9): 1999-2005. DOI:10.1099/ijs.0.019323-0 |
| [10] | Izumi T, Piskula MK, Osawa S, Obata A, Tobe K, Saito M, Kataoka S, Kubota Y, Kikuchi M. Soy isoflavone aglycones are absorbed faster and in higher amounts than their glucosides in humans. The Journal of Nutrition, 2000, 130(7): 1695-1699. DOI:10.1093/jn/130.7.1695 |
| [11] | Bounaix MS, Robert H, Gabriel V, Morel S, Remaud-Siméon M, Gabriel B, Fontagné-Faucher C. Characterization of dextran-producing Weissella strains isolated from sourdoughs and evidence of constitutive dextransucrase expression. FEMS Microbiology Letters, 2010, 311(1): 18-26. DOI:10.1111/j.1574-6968.2010.02067.x |
| [12] | Grootaert C, van den Abbeele P, Marzorati M, Broekaert WF, Courtin CM, Delcour JA, Verstraete W, van de Wiele T. Comparison of prebiotic effects of arabinoxylan oligosaccharides and inulin in a simulator of the human intestinal microbial ecosystem. FEMS Microbiology Ecology, 2009, 69(2): 231-242. |
| [13] | Vázquez MJ, Alonso JL, Dom??nguez H, Parajó JC. Xylooligosaccharides:manufacture and applications. Trends in Food Science & Technology, 2000, 11(11): 387-393. |
| [14] | Campbell JM, Fahey Jr GC, Wolf BW. Selected indigestible oligosaccharides affect large bowel mass, cecal and fecal short-chain fatty acids, pH and microflora in rats. The Journal of Nutrition, 1997, 127(1): 130-136. DOI:10.1093/jn/127.1.130 |
| [15] | Hsu CK, Liao JW, Chung YC, Hsieh CP, Chan YC. Xylooligosaccharides and fructooligosaccharides affect the intestinal microbiota and precancerous colonic lesion development in rats. The Journal of Nutrition, 2004, 134(6): 1523-1528. DOI:10.1093/jn/134.6.1523 |
| [16] | Gibson GR, Probert HM, Van Loo J, Rastall RA, Roberfroid MB. Dietary modulation of the human colonic microbiota:updating the concept of prebiotics. Nutrition Research Reviews, 2004, 17(2): 259-275. |
| [17] | Gobinath D, Madhu AN, Prashant G, Srinivasan K, Prapulla SG. Beneficial effect of xylo-oligosaccharides and fructo-oligosaccharides in streptozotocin-induced diabetic rats. British Journal of Nutrition, 2010, 104(1): 40-47. |
| [18] | Lasrado LD, Gudipati M. Purification and characterization of β-D-xylosidase from Lactobacillus brevis grown on xylo-oligosaccharides. Carbohydrate Polymers, 2013, 92(2): 1978-1983. DOI:10.1016/j.carbpol.2012.11.081 |
| [19] | Amaretti A, Bernardi T, Leonardi A, Raimondi S, Zanoni S, Rossi M. Fermentation of xylo-oligosaccharides by Bifidobacterium adolescentis DSMZ 18350:kinetics, metabolism, and β-xylosidase activities. Applied Microbiology and Biotechnology, 2013, 97(7): 3109-3117. DOI:10.1007/s00253-012-4509-y |
| [20] | Lei Z, Wu YQ, Nie W, Yin DF, Yin XN, Guo YM, Aggrey SE, Yuan JM. Transcriptomic analysis of xylan oligosaccharide utilization systems in Pediococcus acidilactici strain BCC-1. Journal of Agricultural and Food Chemistry, 2018, 66(18): 4725-4733. DOI:10.1021/acs.jafc.8b00210 |
| [21] | Rogowski A, Briggs JA, Mortimer JC, Tryfona T, Terrapon N, Lowe EC, Baslé A, Morland C, Day AM, Zheng HJ, Rogers TE, Thompson P, Hawkins AR, Yadav MP, Henrissat B, Martens EC, Dupree P, Gilbert HJ, Bolam DN. Glycan complexity dictates microbial resource allocation in the large intestine. Nature Communications, 2015, 6(1): 7481. DOI:10.1038/ncomms8481 |
| [22] | Goldberg T, Hecht M, Hamp T, Karl T, Yachdav G, Ahmed N, Altermann U, Angerer P, Ansorge S, Balasz K, Bernhofer M, Betz A, Cizmadija L, Do KT, Gerke J, Greil R, Joerdens V, Hastreiter M, Hembach K, Herzog M, Kalemanov M, Kluge M, Meier A, Nasir H, Neumaier U, Prade V, Reeb J, Sorokoumov A, Troshani I, Vorberg S, Waldraff S, Zierer J, Nielsen H, Rost B. LocTree3 prediction of localization. Nucleic Acids Research, 2014, 42(Web Server issue): W350-W355. |
| [23] | BRENDA. Information on EC 3.2.1.23-beta-galactosidase.[2017-06-13]. http://www.brenda-enzymes.org/enzyme.php?ecno=3.2.1.23. |
| [24] | Andersen JM, Barrangou R, Hachem MA, Lahtinen SJ, Goh YJ, Svensson B, Klaenhammer TR. Transcriptional analysis of oligosaccharide utilization by Bifidobacterium lactis Bl-04. BMC Genomics, 2013, 14(1): 312. DOI:10.1186/1471-2164-14-312 |
| [25] | Paulsen IT, Brown MH, Skurray RA. Proton-dependent multidrug efflux systems. Microbiological Reviews, 1996, 60(4): 575-608. DOI:10.1128/MMBR.60.4.575-608.1996 |
| [26] | Poolman B, Konings WN. Secondary solute transport in bacteria. Biochimica et Biophysica Acta (BBA)-Bioenergetics, 1993, 1183(1): 5-39. |
| [27] | Zhang XC, Zhao Y, Heng J, Jiang DH. Energy coupling mechanisms of MFS transporters. Protein Science, 2015, 24(10): 1560-1579. DOI:10.1002/pro.2759 |
| [28] | Wamelink MMC, Struys EA, Jakobs C. The biochemistry, metabolism and inherited defects of the pentose phosphate pathway:a review. Journal of Inherited Metabolic Disease, 2008, 31(6): 703-717. DOI:10.1007/s10545-008-1015-6 |
| [29] | Johansson B, Hahn-H?gerdal B. The non-oxidative pentose phosphate pathway controls the fermentation rate of xylulose but not of xylose in Saccharomyces cerevisiae TMB3001. FEMS Yeast Research, 2002, 2(3): 277-282. |
| [30] | Bruinenberg PM, Van Dijken JP, Scheffers WA. An enzymic analysis of NADPH production and consumption in Candida utilis. Microbiology, 1983, 129(4): 965-971. |
| [31] | Nogae I, Johnston M. Isolation and characterization of the ZWF1 gene of Saccharomyces cerevisiae, encoding glucose-6-phosphate dehydrogenase. Gene, 1990, 96(2): 161-169. DOI:10.1016/0378-1119(90)90248-P |
| [32] | Jeppsson M, Johansson B, Hahn-H?gerdal B, Gorwa-Grauslund MF. Reduced oxidative pentose phosphate pathway flux in recombinant xylose-utilizing Saccharomyces cerevisiae strains improves the ethanol yield from xylose. Applied and Environmental Microbiology, 2005, 68(4): 1604-1609. |
