Growth and the production of CA
As shown in Fig. 1a, cells took approximately 18 h to grow to the stationary phase. Although the maximum OD600 of the culture with lactate supplementation was slightly higher than that of the control (without lactate supplementation) at the stationary phase (1.25 vs 1.16), both cultures showed similar growth kinetics, indicating that lactate supplementation had little impact on cell growth. The CA production was started to be observed in the culture with lactate supplementation after 6 h of cultivation, and the CA titer continued to increase and reached 1717.2 mg L− 1 at 21 h (Fig. 1b), while CA production was not detected until 15 h of cultivation in the control, the CA titer of which only reached 618 mg L− 1 at 21 h. In both cultures, while more significantly in the culture with lactate supplementation, the main endproduct was CA together with small amount of butyrate, suggesting that the carbon flux of acetyl-CoA may be inclined to be channelled to CA synthesis, especially in the presence of lactate. This is consistent with our previous studies [15].
In sum, compared to the control without lactate supplementation, the lactate supplementation had little effect on the cell growth, but led to earlier initiation for CA production (6 vs 15 h), higher final CA titer (1717 vs 618 mg L− 1) and higher CA productivity (81.8 vs 29.4 mg L− 1 h− 1).
RNA-Seq statistics
Samples were taken for RNA-Seq analysis from both the growth (12 h) and stationary (18 h) phases for both the culture with lactate supplementation and the control. For each culture, independent biological triplicates (a, b, and c) were included (Table 1). Therefore, a total of twelve samples were taken for cDNA libraries construction and sequencing on the Illumina HiSeq platform (Illumina, San Diego, USA). The number of raw reads generated from the sequencing for each library was from 15.7 to 23.5 million (Table S1 in Additional files). A total of 224 Mb sequence reads from 12 cDNA libraries were mapped to the genome of strain CPB6. Only those reads that mapped unambiguously to the CPB6 genome were used for further analysis.
Table 1
Summary of RNA-Seq sequencing and data analysis results
Sample Name | L1 | L2 | C1 | C2 |
a | b | c | a | b | c | a | b | c | a | b | c |
Total reads | 22982792 | 18614536 | 18087962 | 16881648 | 16844058 | 15343814 | 18684112 | 19108148 | 19099082 | 19909258 | 23032120 | 18662552 |
No. of read mapped | 22684858 | 18390709 | 17865180 | 16713319 | 16606726 | 15098988 | 18437261 | 18893873 | 18905208 | 19631686 | 22572873 | 18285805 |
Ratio of reads mapped (%) | 98.7 | 98.8 | 98.77 | 99 | 98.59 | 98.4 | 98.68 | 98.88 | 98.98 | 98.61 | 98.01 | 97.98 |
No. of unique reads mapped | 22339068 | 17915954 | 17559600 | 16540060 | 16324768 | 14862898 | 18050316 | 18505697 | 18508661 | 19247256 | 22187272 | 17856892 |
No. of genes with detectable expression | 1969 | 1968 | 1968 | 1968 | 1969 | 1968 | 1968 | 1968 | 1968 | 1968 | 1969 | 1969 |
Range in expression levels (TPM) | 8.3–2.7 × 104 | 8.0-1.7 × 104 | 11.5–1.7 × 104 | 3.2–5.3 × 104 | 26.4–3.7 × 104 | 6.6–7.3 × 104 | 4.3-2.0 × 104 | 3.8–1.8 × 104 | 4.1–1.9 × 104 | 5.2–2.4 × 104 | 10.8–1.8 × 104 | 24.5–1.9 × 104 |
L1: cell culture with lactate supplementation from the growth phase; |
L2: cell culture with lactate supplementation from the stationary phase; |
C1: Control culture without lactate supplementation from the growth phase; |
C2: Control culture without lactate supplementation from the stationary phase. |
a, b and c represented the biological triplicate samples. |
Overall, out of reads derived from all the samples, 15.1 to 22.7 million reads were unambiguously mapped to the CPB6 genome, and over 98% reads were mapped (Table 1). A total of 1968/1969 out of 2045 protein-coding genes had detectable expression in both conditions (with or without lactate supplementation) (Table 1), indicating that the RNA-Seq analysis in this study achieved comprehensive coverage of the CPB6 transcriptome. The transcription levels (the number of transcripts per million, TPM) of most active protein-coding genes were in the range of 3.2 × 104–7.3 × 104.
As illustrated in Fig. 2, the gene expression could be classified into four levels: low (TPM < 30), moderate (TPM: 30–150), high (TPM: 150–1000), and very high (TPM > 1000). The number of genes at some specific expression levels was significantly different for the two cultures (with or without lactate supplementation). For the growth phase, there were slightly more genes in the moderate and high and very high expression level in the culture with lactate supplementation than in the control, but lowly expressed genes were significantly decreased. While for the stationary phase, the culture with lactate supplementation had more genes in the moderate expression level, but fewer genes in the high and very high expression level. These results suggested that more changes in gene expression are triggered by the addition of lactate at the stationary phases.
Functional annotation and classification
In the transcriptome of strain CPB6, a total of 1122 expressed genes were allocated into three primary Gene Ontology (GO) categories (Fig. 3), including the category of biological process (601 genes), cellular component (524 genes), and molecular function (916 genes). In each category, the genes were further assigned into 28 functional groups, such as metabolic process (478 genes), cellular process (440 genes), cell part (307 genes), membrane part (297 genes), catalytic activity (654 genes), binding (561genes), and etc. The analysis of the genes based on the KEGG annotation identified a total of 1046 unigenes allocated into six primary KEGG categories including 35 subcategories (Figure. S1 in Additional files). The top 5 categories of genes were: carbohydrate metabolism, amino acid metabolism, membrane transport, translation, and metabolism of cofactors and vitamins, respectively. The top 10 enriched pathways included ABC transporters (58 genes), Amino sugar and nucleotide sugar metabolism (27 genes), Starch and sucrose metabolism (26 genes), Glycolysis / Gluconeogenesis (24 genes), Purine metabolism (40 genes), Pyrimidine metabolism (37 genes), Peptidoglycan biosynthesis, Aminoacyl-tRNA biosynthesis (28 genes), Ribosome (52 genes), and Quorum sensing (26 genes). The analysis based on the Clusters of Orthologous Groups (COGs) showed that 1785 unigenes were allocated to four primary COG categories containing 20 COG functional clusters. The top 5 annotated genes corresponding to the KEGG pathways were Replication recombination and repair (155 genes), Translation, ribosomal structure and biogenesis (142 genes), Amino acid transport and metabolism (126 genes), Carbohydrate transport and metabolism (110 genes), and Inorganic ion transport and metabolism (Figure S2 in Additional files).
Differential expression of global genes
The correct identification of differentially expressed genes (DEGs) between specific conditions is a key in the understanding phenotypic variation of organisms under environmental stress. As shown in Table 2, there were only 34 differentially expressed genes (DEGs, FC ≥ 2 or ≤ 0.5 with P-value < 0.05) identified during the growth phase between two culture conditions, of which 15 genes were upregulated, and 19 genes were downregulated more than two-fold. In addition, a total of 245 DEGs were identified at the stationary phase, of which 123 genes were significantly upregulated and 122 genes were downregulated (Table S2 in Additional files). It suggested that the addition of lactate led to differences in gene expression between the two cultures (with and without lactate supplementation) during different growth phases.
Table 2
The differentially expressed genes in culture with/without lactate supplementation during the growth phase
No. | Gene_ID | Gene name | Gene description | TPM | FC (L1/C1) | P-value |
C1 | L1 |
15 upregulated genes (FC ≥ 2.0); all statistically significant (P < 0.05) |
1 | B6259_RS06365 | atoB | acetyl-CoA C-acetyltransferase | 1224 | 5204 | 3.45 | 7.9E-39 |
2 | B6259_RS06360 | crt | enoyl-CoA hydratase | 795 | 3434 | 3.46 | 2.4E-33 |
3 | B6259_RS06355 | hbd | 3-hydroxybutyryl-CoA dehydrogenase | 1418 | 6306 | 3.49 | 2.3E-27 |
4 | B6259_RS07830 | pta | phosphate acetyltransferase | 271 | 666 | 2.09 | 4.3E-24 |
5 | B6259_RS00440 | - | methionine ABC transporter ATP-binding protein | 51 | 504 | 5.25 | 6.2E-18 |
6 | B6259_RS00450 | - | metal ABC transporter substrate-binding protein | 30 | 699 | 5.69 | 5.9E-15 |
7 | B6259_RS00445 | - | ABC transporter permease | 27 | 446 | 5.17 | 6.9E-14 |
8 | B6259_RS08190 | cysK | cysteine synthase A | 390 | 7426 | 4.07 | 6.4E-10 |
9 | B6259_RS08440 | - | unknown function | 1048 | 3598 | 2.33 | 5.2E-06 |
10 | B6259_RS06010 | - | hypothetical protein | 21 | 89 | 2.52 | 8.1E-06 |
11 | B6259_RS07140 | - | hypothetical protein | 154 | 470 | 2.17 | 3.0E-05 |
12 | B6259_RS01720 | cadA | cadmium-translocating P-type ATPase | 22 | 66 | 2.16 | 3.9E-05 |
13 | B6259_RS06870 | - | Hsp20/alpha crystallin family protein | 315 | 1102 | 2.21 | 1.6E-04 |
14 | B6259_RS00455 | pepT | peptidase T | 37 | 576 | 2.26 | 2.0E-04 |
15 | B6259_RS02585 | bdh | butanol dehydrogenase | 82 | 242 | 2.04 | 3.1E-04 |
19 downregulated genes (FC ≤ 0.5); all statistically significant (P < 0.05) |
1 | B6259_RS08515 | - | peptide ABC transporter substrate-binding protein | 98 | 53 | 0.48 | 1.7E-23 |
2 | B6259_RS09280 | - | PTS glucose transporter subunit IIA | 1200 | 484 | 0.37 | 5.5E-23 |
3 | B6259_RS09735 | ilvH | acetolactate synthase small subunit | 564 | 302 | 0.48 | 9.4E-19 |
4 | B6259_RS06995 | - | hypothetical protein | 276 | 43 | 0.20 | 2.7E-18 |
5 | B6259_RS08565 | - | hypothetical protein | 143 | 79 | 0.50 | 2.3E-13 |
6 | B6259_RS07000 | - | sugar ABC transporter permease | 113 | 31 | 0.30 | 9.2E-13 |
7 | B6259_RS01525 | - | unknown function | 2683 | 1010 | 0.37 | 8.4E-12 |
8 | B6259_RS03200 | - | unknown function | 2683 | 1010 | 0.37 | 8.4E-12 |
9 | B6259_RS07010 | tag | glycosylase | 144 | 46 | 0.34 | 9.9E-11 |
10 | B6259_RS07005 | - | carbohydrate ABC transporter permease | 90 | 33 | 0.37 | 1.1E-09 |
11 | B6259_RS01865 | - | DUF2520 domain-containing protein | 260 | 85 | 0.36 | 6.7E-09 |
12 | B6259_RS01880 | panD | aspartate 1-decarboxylase | 444 | 156 | 0.37 | 9.5E-09 |
13 | B6259_RS01870 | panB | 3-methyl-2-oxobutanoate hydroxymethyltransferase | 314 | 105 | 0.37 | 1.1E-08 |
14 | B6259_RS01875 | panc | pantoate-beta-alanine ligase | 350 | 115 | 0.37 | 1.2E-08 |
15 | B6259_RS01760 | - | hypothetical protein | 820 | 369 | 0.44 | 8.9E-08 |
16 | B6259_RS02315 | - | basic amino acid ABC transporter substrate-binding protein | 147 | 79 | 0.50 | 1.8E-07 |
17 | B6259_RS00100 | fruK | 1-phosphofructokinase | 1256 | 276 | 0.35 | 1.7E-06 |
18 | B6259_RS00095 | - | PTS fructose transporter subunit IIC | 1273 | 372 | 0.37 | 1.8E-06 |
19 | B6259_RS00105 | - | DeoR/GlpR transcriptional regulator | 1304 | 278 | 0.36 | 3.6E-06 |
L1: lactate-supplemented cells at growth phase |
C1: no-lactate-supplemented cells (controls) at growth phase |
The COG distribution of the DEGs at both the growth phase and the stationary phase was illustrated in Fig. 4. It revealed the potential genes related to the pathways and bioprocesses for the utilization of lactate for CA production. At the growth phase, predominant number of DEGs belong to the ‘inorganic ion transport and metabolism, [P]’ group and the ‘carbohydrate transport and metabolism, [G]’ group. While at stationary phase, most upregulated genes belong to the ‘carbohydrate transport and metabolism, [G]’ group, which play important roles for the degradation and utilization of carbohydrate substrates [20, 21]. It was worth noting that a large number of DEGs (for both upregulated and downregulated ones) fall into the ‘function unknown, [S]’ group.), meaning that their functions are unknown. This may be because CPB6 belongs to a novel species or clade (Clostridium cluster IV) of the family Ruminococcaceae, sharing low 16S rRNA sequence similarity (< 92.6%) with the other organisms in GenBank and RDP [15, 17].
Cluster analysis of the DEGs between the culture with lactate supplementation and the control was showed in Fig. 5. The results showed that the gene expression of the triplicate (a, b and c) of each sample demonstrated very similar expression patterns. According to Fig. 5, L1 (with lactate supplementation at the growth phase) cluster was most closely with C1 (without lactate supplementation at the growth phase) cluster, indicating the presence of a small amount of DEGs caused by the addition of lactate at this phase. However, the distinct difference of gene expression was observed between the two cultures at the stationary phase (L2 vs C2), Further, venn diagram showed that 295 genes whose changes in expression patterns were substrate and/or growth dependent, of which 31 genes were substrate (lactate) dependent, 228 genes were growth dependent, and 36 genes were substrate-growth dependent (Fig. 6a). Specifically, 11 and 20 lactate-dependent genes were significantly upregulated and downregulated, as well as 98 and 130 growth-dependent genes were significantly upregulated and downregulated, respectively (Fig. 6b). It was suggested that the differences in gene expression are stronger for stationary phase vs growth phase than for plus/minus lactate. Similar results was observed for Clostridium thermocellum, in which growth rate had stronger effects on gene expression than substrate type (insoluble cellulose vs soluble cellobiose) [22]. These DEGs were described in detail in later section.
Expression of glycolysis genes
An overview of the metabolic pathway in strain CPB6 and the expression levels of genes involved in key metabolic processes with their fold change (FC) were shown in Fig. 6 and Table 3. Generally, in clostridia, glucose is converted into pyruvate via glycolysis, and the produced pyruvate is further converted into acetyl-CoA for the production of acetate and butyrate at acidogenic phase [23–25]. In the present study, the expression of genes for glycolysis was detected in the CPB6 transcriptome, which reinforced its genome annotation [17]. Most glycolytic genes were expressed at a relatively high level (TPM > 150) between the culture with lactate supplementation and the control, but there was no significant difference between them at the growth phase. Three glycolytic genes exhibited different expression patterns at the stationary phase. Gene encoding phosphofructokinase (PFK, B6259_RS06095) was significantly downregulated, while genes encoding glucose-1-phosphate adenylyltransferase (GlgC, B6259_RS09035) and 1, 4-alpha-glucan branching enzyme (GlgB, B6259_RS09040) were upregulated by 4.58 and 3.42-fold in the culture with lactate supplementation than in the control, respectively. GlgB and GlgC are typically associated with glycogen synthesis, why expression of these genes be affected by lactate supplementation remains unclear. Overall, the addition of lactate has little impact on the expression of glycolytic genes.
Table 3
The differentially expressed genes within the important metabolic pathways in culture with/without lactate supplementation
Functional description | Gene_ID | TPM of genes from culture with lactate supplementationa | TPM of genes from the Controla | RNA relative fold change (Treatment/Control) |
12 h | 18 h | 12 h | 18 h | 12 h | 18 h |
Glycolysis | | | | | | | |
PTS-Glc-EIIA, PTS glucose transporter subunit IIA | B6259_RS09280 | 484 | 260 | 1200 | 517 | 0.37b | 0.62 |
GlgC, glucose-1-phosphate adenylyltransferase | B6259_RS09035 | 153 | 1323 | 236 | 241 | 0.57 | 4.58c |
GlgB, 1,4-alpha-glucan branching enzyme | B6259_RS09040 | 194 | 745 | 236 | 201 | 0.72 | 3.42 c |
sugar phosphate isomerase/epimerase | B6259_RS06500 | 181 | 175 | 150 | 233 | 1.05 | 0.88 |
PGM, phosphoglucomutase | B6259_RS09200 | 95 | 189 | 127 | 113 | 0.66 | 1.80 |
GPI, glucose-6-phosphate isomerase | B6259_RS04825 | 2015 | 1789 | 1833 | 1818 | 0.96 | 1.12 |
PFK, phosphofructokinase | B6259_RS06095 | 426 | 97 | 580 | 516 | 0.66 | 0.23 b |
ALDO, fructose-bisphosphate aldolase | B6259_RS00415 | 749 | 402 | 800 | 891 | 0.83 | 0.57 |
TPI, triose-phosphate isomerase | B6259_RS09105 | 224 | 229 | 315 | 493 | 0.65 | 0.58 |
GapA, glyceraldehyde phosphate dehydrogenase | B6259_RS09050 | 5322 | 4284 | 4790 | 7732 | 0.98 | 0.70 |
PGK, phosphoglycerate kinase | B6259_RS09100 | 523 | 524 | 705 | 1029 | 0.67 | 0.65 |
gpmI, 2,3-bisphosphoglycerate-independent phosphoglycerate mutase | B6259_RS09110 | 203 | 200 | 284 | 469 | 0.66 | 0.55 |
ENO, phosphopyruvate hydratase | B6259_RS04810 | 41 | 65 | 30 | 52 | 1.14 | 1.46 |
PK, pyruvate kinase | B6259_RS02335 | 254 | 102 | 293 | 228 | 0.77 | 1.46 |
Central pyruvate metabolism | | | | | | | |
PpdK, pyruvate phosphate dikinase | B6259_RS00120 | 1301 | 823 | 1163 | 1535 | 0.99 | 0.69 |
Pfor, pyruvate: ferredoxin (flavodoxin) oxidoreductase | B6259_RS09135 | 4329 | 4382 | 2044 | 1225 | 1.83 | 3.26 c |
PCK, phosphoenolpyruvate carboxykinase | B6259_RS09255 | 368 | 159 | 554 | 1031 | 0.62 | 0.23 b |
PflD, formate C-acetyltransferase | B6259_RS09900 | 98 | 188 | 107 | 471 | 0.83 | 0.54 |
ADH, alcohol dehydrogenase | B6259_RS03100 | 200 | 116 | 163 | 159 | 1.07 | 0.84 |
Incomplete TCA cycle | | | | | | | |
CS, citrate synthase, citrate lyase | B6259_RS03360 | 936 | 187 | 543 | 642 | 1.42 | 0.39 b |
ACO, aconitate hydratase | B6259_RS05795 | 227 | 162 | 153 | 201 | 1.27 | 0.94 |
IDH, isocitrate dehydrogenase | B6259_RS05805 | 237 | 232 | 197 | 291 | 1.04 | 0.93 |
FUM, fumarate hydratase | B6259_RS07270 | 310 | 186 | 260 | 437 | 1.04 | 0.49 b |
PCK, phosphoenolpyruvate carboxykinase | B6259_RS09255 | 368 | 159 | 554 | 1031 | 0.62 | 0.23 b |
Hydrogen production | | | | | | | |
HydE, [FeFe] hydrogenase H-cluster | B6259_RS02550 | 113 | 73 | 174 | 44 | 1.44 | 2.24 c |
HydF, [FeFe] hydrogenase H-cluster | B6259_RS09690 | 67 | 40 | 50 | 24 | 1.43 | 1.17 |
Lactate fermentation pathway | | | | | | | |
D-ldh, D-lactate dehydrogenase | B6259_RS06770 | 76 | 88 | 58 | 108 | 1.14 | 0.95 |
L-ldh, L-lactate dehydrogenase | B6259_RS09845 | 79 | 111 | 119 | 295 | 0.59 | 0.44 b |
Acetate fermentation pathway | | | | | | | |
PTA, phosphate acetyltransferase | B6259_RS07830 | 666 | 697 | 271 | 321 | 2.09 c | 2.23 c |
ACK, acetate kinase | B6259_RS03430 | 290 | 297 | 288 | 233 | 0.88 | 1.41 |
The reverse β-oxidation pathway | | | | | | | |
AtoB, acetyl-CoA C-acetyltransferase | B6259_RS06365 | 5204 | 9909 | 1224 | 1077 | 3.45 c | 6.31 c |
HBD, 3-hydroxybutyryl-CoA dehydrogenase | B6259_RS06355 | 6306 | 13975 | 1418 | 1022 | 3.49 c | 8.59 c |
CRT, enoyl-CoA hydratase | B6259_RS06360 | 3434 | 7348 | 795 | 647 | 3.46 c | 7.34 c |
BCD1, butyryl-CoA dehydrogenase | B6259_RS01790 | 3278 | 3104 | 3787 | 3014 | 0.76 | 1.19 |
BCD2, butyryl-CoA dehydrogenase | B6259_RS02600 | 42 | 313 | 41 | 66 | 0.90 | 4.49c |
EtfA, electron transfer flavoprotein subunit alpha | B6259_RS01785 | 2657 | 2968 | 3175 | 2572 | 0.73 | 1.31 |
EtfB, electron transfer flavoprotein subunit beta | B6259_RS01780 | 3996 | 4830 | 4357 | 4169 | 0.71 | 1.31 |
CAT, butyryl-CoA: acetate CoA-transferase | B6259_RS06345 | 521 | 1497 | 283 | 330 | 1.55 | 4.01c |
Fructose fermentation pathway | | | | | | | |
PPF, 1-phosphofructokinase | B6259_RS00100 | 276 | 2174 | 1256 | 239 | 0.35 | 7.33 |
Starch and sucrose metabolism | | | | | | | |
PYG, glycogen phosphorylase | B6259_RS00300 | 90 | 163 | 121 | 103 | 0.66 | 1.71 |
MalQ, 4-alpha-glucanotransferase | B6259_RS07805 | 53 | 270 | 55 | 61 | 0.85 | 4.34 |
PGM, Phosphoglucomutase | B6259_RS09200 | 95 | 189 | 127 | 113 | 0.66 | 1.80 |
Energy conservation | | | | | | | |
energy-coupling factor transporter ATPase | B6259_RS02790 | 141 | 104 | 117 | 159 | 1.04 | 0.76 |
electron transport complex protein RnfA | B6259_RS06245 | 230 | 162 | 357 | 362 | 0.58 | 0.52 |
Sporulation | | | | | | | |
stage 0 sporulation protein | B6259_RS00205 | 379 | 279 | 233 | 252 | 0.97 | 0.82 |
stage II sporulation protein D | B6259_RS09065 | 98 | 59 | 96 | 53 | 0.97 | 1.29 |
stage III sporulation protein AD | B6259_RS03910 | 126 | 54 | 87 | 26 | 1.67 | 1.27 |
stage IV sporulation protein A | B6259_RS04975 | 65 | 30 | 58 | 16 | 1.42 | 1.58 |
stage V sporulation protein AC | B6259_RS09190 | 89 | 46 | 77 | 40 | 0.99 | 1.42 |
stage V sporulation protein AD | B6259_RS09195 | 69 | 41 | 66 | 34 | 1.05 | 1.57 |
stage V sporulation protein AE | B6259_RS00500 | 292 | 226 | 200 | 167 | 1.15 | 1.02 |
sporulation transcription factor Spo0A | B6259_RS05505 | 127 | 115 | 83 | 106 | 0.94 | 0.94 |
sporulation transcriptional regulator SpoIIID | B6259_RS01550 | 213 | 188 | 140 | 207 | 0.79 | 1.01 |
sporulation protein YtfJ | B6259_RS04885 | 291 | 183 | 145 | 159 | 1.00 | 0.65 |
Transporter genes | | | | | | | |
ABC transporter permease | B6259_RS00445 | 446 | 274 | 27 | 235 | 5.17 c | 1.27 |
metal ABC transporter | B6259_RS00450 | 699 | 628 | 30 | 457 | 5.69 c | 1.52 |
ABC transporter permease | B6259_RS02670 | 296 | 130 | 441 | 387 | 0.60 | 0.40 b |
ABC transporter permease | B6259_RS02665 | 180 | 96 | 258 | 231 | 0.62 | 0.48 b |
carbohydrate ABC transporter permease | B6259_RS07005 | 33 | 124 | 90 | 41 | 0.37 b | 3.51 c |
carbohydrate ABC transporter permease | B6259_RS07905 | 71 | 744 | 71 | 40 | 0.90 | 12.71 c |
carbohydrate ABC transporter permease | B6259_RS07810 | 39 | 229 | 40 | 45 | 0.85 | 5.48 c |
carbohydrate ABC transporter permease | B6259_RS02030 | 26 | 71 | 16 | 39 | 1.35 | 2.14 c |
sugar ABC transporter permease | B6259_RS07910 | 82 | 1175 | 88 | 50 | 0.86 | 14.74 c |
sugar ABC transporter permease | B6259_RS03335 | 39 | 401 | 26 | 61 | 1.30 | 5.61 c |
sugar ABC transporter permease | B6259_RS07815 | 36 | 197 | 37 | 49 | 0.85 | 4.34 c |
sugar ABC transporter permease | B6259_RS07000 | 31 | 135 | 113 | 38 | 0.30 b | 3.48 c |
iron ABC transporter permease | B6259_RS00320 | 53 | 1278 | 77 | 89 | 0.62 | 10.05 c |
ABC transporter ATP-binding protein | B6259_RS00440 | 504 | 277 | 51 | 239 | 5.25 c | 1.39 |
ABC transporter ATP-binding protein | B6259_RS00325 | 60 | 2032 | 94 | 100 | 0.58 | 11.14 c |
ABC transporter ATP-binding protein | B6259_RS08900 | 153 | 682 | 233 | 214 | 0.58 | 3.13 |
ABC transporter ATP-binding protein | B6259_RS07940 | 190 | 40 | 259 | 94 | 0.66 | 0.42 |
carbohydrate ABC transporter substrate-binding protein | B6259_RS07915 | 216 | 3434 | 203 | 103 | 0.93 | 14.51 |
maltose ABC transporter substrate-binding protein | B6259_RS03345 | 30 | 501 | 22 | 37 | 1.15 | 7.65 |
ABC transporter substrate-binding protein | B6259_RS07820 | 372 | 1913 | 451 | 344 | 0.73 | 4.63 |
sugar ABC transporter substrate-binding protein | B6259_RS02005 | 30 | 93 | 29 | 48 | 0.92 | 2.29 |
peptide ABC transporter substrate-binding protein | B6259_RS08515 | 53 | 78 | 98 | 369 | 0.48 | 0.28 |
peptide ABC transporter substrate-binding protein | B6259_RS02685 | 1385 | 819 | 1442 | 2222 | 0.85 | 0.50 |
ABC transporter ATP-binding protein | B6259_RS02660 | 238 | 119 | 369 | 320 | 0.58 | 0.45 |
ABC transporter ATP-binding protein | B6259_RS07940 | 190 | 58 | 259 | 166 | 0.66 | 0.42 |
PTS fructose transporter subunit IIC | B6259_RS00095 | 372 | 2117 | 1273 | 485 | 0.37 | 3.87 |
PTS glucose transporter subunit IIA | B6259_RS09280 | 484 | 260 | 1200 | 517 | 0.37 | 0.62 |
PTS β-glucoside transporter subunit IIABC | B6259_RS01415 | 81 | 760 | 134 | 141 | 0.54 | 4.70 |
PTS mannitol transporter subunit IICBA | B6259_RS00370 | 29 | 89 | 19 | 44 | 1.26 | 2.34 |
ferrous iron transport protein B | B6259_RS03880 | 471 | 389 | 531 | 150 | 0.81 | 2.72 |
a, Data presented as mean of independent triplicates |
b, Significantly upregulated (FC ≥ 2.0, p < 0.05) |
c, Significantly downregulated (FC ≤ 0.5, p < 0.05) |
Expression of butyrate- and CA-producing genes
The bioproduction of CA is a well-known chain elongation process from acetate (C2) to caproate (C6) via the reverse β-oxidation pathway, in which an acetyl-CoA (from ethanol) unit is combined with another acetyl-CoA (from acetate), and consequently C2 is elongated to C4, and further C4 is elongated to C6 [4]. Thus, acetyl-CoA is a key intermediate of flux distribution for the chain elongation. The conversion of pyruvate into acetyl-CoA is mainly catalyzed by the pyruvate: ferredoxin (flavodoxin) oxidoreductase (Pfor) that is a flavodoxin- and NADPH-dependent enzyme [6, 26]. Here, the Pfor gene (B6259_RS09135) maintained at very high expression level (TPM > 4000) in the culture with lactate supplementation (Table 3), which were upregulated by 1.83- and 3.26-fold than that in the control in the growth and stationary phase, respectively (Fig. 7). High-level expression of the Pfor gene would result in increased acetyl-CoA, which provides the high amount of acetyl-CoA for chain elongation from acetate to butyrate and CA.
Key enzymes involved in the butyrate formation include acetyl-CoA C-acetyltransferase (AtoB), 3-hydroxybutyryl-CoA dehydrogenase (HBD), enoyl-CoA hydratase (CRT), NAD-dependent butyryl-CoA dehydrogenase (BCD), Electron transfer flavoprotein (Etf) and butyryl-CoA: acetate CoA transferase (CAT) [6, 17]. Here, genes encoding AtoB (B6259_RS06365), CRT (B6259_RS06360) and HBD (B6259_RS06355) were identified from the transcriptomes of strain CPB6, whose expression levels maintained at very high levels (TPM > 3000) in the culture with lactate supplementation throughout the growth and stationary phases, and were upregulated by 3.5–8.6 folds compared to the control. It suggested that the high-level expression of the three genes can be induced by supplemented lactate. In addition, two BCD genes (B6259_RS01790 and _RS02600) and EtfAB (B6259_RS01785 and _RS01780) responsible for the conversion of crotonyl-CoA to butyryl-CoA showed different expression profiles. B6259_RS01790 was expressed at a very high level (TPM > 3000) throughout the fermentation phases, but showing no change in expression in the two cultures. B6259_RS02600 was expressed at relatively low level at the growth phase, but its expression was induced to high levels in the culture with lactate supplementation at the stationary phase, which was > 4.4-fold higher than that in the control. More research is needed to determine which BCD gene plays the key role in acidogenesis in the CPB6. EtfAB showed no significant change in the two cultures during the growth and stationary phases. A CAT gene (B6259_RS06345) was detected in the CPB6 transcriptome, and its expression was markedly upregulated by 4-fold in the culture with lactate supplementation than that in the control in the stationary phase, and kept at very high expression levels. CAT is key enzyme responsible for catalysing the last step of the butyrate formation [26]. High-level expression of CAT gene should theoretically result in a high concentration of butyric acid in the culture with lactate supplementation. Nevertheless, significant accumulation of CA instead of butyric acid was observed in the CPB6 culture with lactate supplementation, suggesting that the CAT is likely more intent to transforming caproy-CoA to caproate instead of converting butyryl-CoA to butyrate. Up to now, little is known about the key functional genes responsible for CA synthesis from butyryl-CoA. Genes involved in butyrate synthesis via the reverse β-oxidation (e.g., AtoB, CRT, HBD, BCD and CAT) are assumed to have the function in the caproyl-CoA and CA formation [6]. However, Clostridium sp. BPY5 and C. tyrobutyricum, which contains these genes, only produce butyric acid instead of CA [26, 27], while C. kluyveri and Ruminococcaceae bacterium CPB6, which contain these genes, can further elongate butyric acid to CA [6, 16]. It suggested that there may be differences in structure and function between these genes from different organisms. Therefore, the further study needs to be performed to explore the functions of these genes in strain CPB6.
Lactate is a major endproduct of glycolysis in the absence of oxygen [28]. Its formation or conversion requires lactate dehydrogenase (LDH) with the regeneration of NADH to NAD+. There are two LDH genes in the CPB6 genome [17], encoding L-lactate dehydrogenase (L-LDH) and D-lactate dehydrogenase (D-LDH), respectively. In this study, the expression of LDH genes (L-LDH, B6259_RS09845; D-LDH, B6259_RS06770) were also detected in the transcripts of strain CPB6. However, the two genes showed relatively low expression levels in both the culture with lactate supplementation and the control, except that slightly higher expression level was observed in the control at the stationary phase (Table 3). LDH catalyzes the reaction converts pyruvate to lactate or the reverse reaction that converts lactate to pyruvate [29]. This conversion is essential in hypoxic and anaerobic conditions when ATP production by oxidative phosphorylation is disrupted. The recent study showed that lactate can be transformed into CA in either mixed microbiome [2, 13, 14], or in the pure culture where the conversion of lactate to acetyl-CoA coupling with the reverse β-oxidation is speculated to result into chain elongation [15]. Interestingly, the lactate supplementation in this study did not lead to increased expression levels of LDHs in either growth or stationary phases, indicating that the expression of LDHs might be uncoupled from the utilization of lactate. It warrants further investigation concerning the function of LDH in the conversion of lactate to CA in the CPB6 strain.
The gene encoding phosphate acetyltransferase (PTA, B6259_RS07830), one important enzyme involved in acetate formation, was remarkably upregulated in the culture with lactate supplementation than in the control. However, the expression of acetate kinase (ACK, B6259_RS03430) showed no change in response to the addition of lactate. By including the production of H2 and CO2 into the loop, it could provide a whole picture for carbon balance for the substrate utilization and cell biomass production. Unfortunately, the production of H2 and CO2 was not monitored in this study. In the future studies, this should be taken into consideration for improvement.
Expression of putative ABC transporter and sporulation genes
Strain CPB6, affiliated with Clostridium cluster IV of the family Ruminococcaceae in the order of Clostridiales, is a spore-forming, obligate anaerobic bacterium that can produce CA from lactate (Zhu et al. 2017). As shown in Table 3, sporulation genes showed similar expression patterns in both groups, e.g., spo0, spoIIID, spoV, spoYtfJ, were induced to high expression under both conditions (with or without lactate supplementation) at the growth and stationary phases, while spoIID, spoIIIAD, spoIVA, spoVAC, spoVAD spoVAE were expressed at low or moderate levels. Some bacteria, such as bacilli and clostridia, develop into highly resistant spores to protect their genome and cell from certain doom when living conditions become intolerable [30]. It ensures bacterial survival under adverse environmental conditions. Sporulation in Clostridium spp. is ordinarily not triggered by starvation but by cessation of growth in the presence of excess carbon source or exposure to oxygen [31]. The two most critical factors involved in the shift to solventogenesis, a decrease in external pH and accumulation of acidic fermentation products, are generally assumed to be associated with the initiation of sporulation in Clostridium spp., to some extent [32]. But recent studies showed that the sporulation events were uncoupled from the induction of solventogenesis in C. beijerinckii [23]. In this study, the sporulation genes showed no significant difference between the culture with lactate supplementation and the control, indicating that the sporulation events are not associated with the production of CA in the CPB6 until the stationary phase. This may be because low concentrations of CA (1717 mg/L) are not sufficient to initiate sporulation for the CPB6. It will be investigated in the future whether high concentrations of CA trigger sporulation.
In the transcriptome of strain CPB6, most genes encoding ATP-binding cassette (ABC) transporters and substrate-bind proteins (SBP) maintained at low expression levels in the control, but were induced to relatively high expression levels (particularly upregulated by 2–14 folds at the stationary phase) in the culture with lactate supplementation. ABC transporters are ubiquitous membrane proteins that couple the transport of diverse substrates across cellular membranes to the hydrolysis of ATP [33]. ABC transporters are generally divided into importers and exporters on the basis of the polarity of solute movement. ABC importers are found mostly in bacteria and are crucial in mediating the uptake of solutes including sugar, metal ions and vitamins [34]. In the present study, most genes for ABC transporter and substrate-binding protein (SBP) were no significant change in the two cultures, except two ABC transporter genes (B6259_RS00445, B6259_RS00450), and one SBP gene (B6259_RS00440) which were upregulated by more than 2-fold in the culture with lactate supplementation than in the control. It was suggested that the three genes might be related to the intake and use of lactic acid. In addition, nine ABC transporter genes and six SBP genes were markedly upregulated at the stationary phase. Specially, B6259_RS07905, _RS07910, _RS00320, _RS00325 and B6259_RS07915 were increased over 10-fold in the culture with lactate supplementation than in the control, demonstrating that these genes are associated with the extrusion of CA from the cell and the maintenance of osmotic homeostasis in cytoplasm [35].
In addition, two phosphotransferase system (PTS) transporter genes (B6259_RS01415 and B6259_RS00370) and one ferrous iron transporter gene (B6259_RS03880) were upregulated by 2- to 4-fold in the culture with lactate supplementation than in the control. PTS is a multiple-component carbohydrate uptake system that drives specific saccharides across the bacterial inner membrane while simultaneously catalyzing sugar phosphorylation [36]. Five distinct subfamilies of proteins related to PTS have been identified within the glucose superfamily: the lactose family, the glucose family, the β-glucoside family, the mannitol family, and the fructose family [37]. In this study, four PTS transporter genes were detected in the transcriptome of strain CPB6, including PTS fructose transporter subunit IIC (B6259_RS00095), PTS glucose transporter subunit IIA (B6259_RS09280), PTS β-glucoside transporter subunit IIABC (B6259_RS01415), and PTS mannitol transporter subunit IICBA (B6259_RS00370). Genes encoding PTS fructose and glucose transporters were highly expressed under both conditions, but the two genes were significantly downregulated at the growth phase in the culture with lactate supplementation than in the control, indicating that PTS transporter-mediated sugar transport in membrane vesicles in CPB6 is inhibited by the lactate supplementation. However, at the stationary phase, genes encoding PTS fructose, β-glucoside and mannitol transporters were all strikingly upregulated.
Overall, the addition of lactate caused distinct changes in gene expression of the CPB6 strain, especially at stationary phase. The detailed mechanism remains to be further studied.