Genome-Wide Transcriptomic Analysis of N-Caproic Acid Production in Ruminococcaceae Bacterium CPB6 with Lactate Supplementation

doi:10.21203/rs.3.rs-23672/v1

Download PDF

Research

Genome-Wide Transcriptomic Analysis of N-Caproic Acid Production in Ruminococcaceae Bacterium CPB6 with Lactate Supplementation

https://doi.org/10.21203/rs.3.rs-23672/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Background: n-Caproic acid (CA) is gaining increased attention due to its high value as a chemical feedstock. Our recent studies have demonstrated that lactate can be an attractive energy substrate for the production of CA. However, little is known about the potential molecular mechanism for CA production triggered by the supplementation of exogenous lactate at the gene transcriptional level.

Results: 5% lactate was supplemented into the fermentation with Ruminococcaceae bacterium CPB6 for CA production. Results showed that lactate supplementation led to earlier CA production and higher final CA titer and productivity. Transcriptional analysis was carried out using RNA-Seq for the culture with lactate supplementation compared to the control (without lactate supplementation). It has been indicated that there were only 34 differentially expressed genes (DEGs) between the two groups at the exponential phase, of which 15 were upregulated, and 19 were downregulated by more than two-fold. A total of 245 DEGs were identified between the two groups at the stationary phase, of which 123 were upregulated and 122 were downregulated. These DEGs cover crucial functional categories. Specifically, 5 genes responsible for the reverse β-oxidation pathway, 11 genes encoding ATP-binding cassette (ABC) transporters, 6 genes encoding substrate-binding protein (SBP) and 4 genes encoding phosphotransferase system (PTS) transporters were strikingly upregulated in response to the addition of lactate. These genes would be candidates for future studies aiming at understanding the regulatory mechanism of lactate conversion into CA, as well as for the improvement of CA production in strain CPB6.

Conclusion: This study suggested that lactate supplementation can promote CA production by altering the expression patterns of genes involved in the essential metabolic pathways, such as central pyruvate metabolism, the reverse β-oxidation pathway, ABC and PTS transports. The findings presented herein reveal unique insights into the biomolecular effects of lactate on CA production at the gene transcriptional level.

Biotechnology and Bioengineering

n-Caproic acid

Lactate

Chain elongation

Transcriptome

RNA-Seq

The increasing demand for fuels and chemicals and the scarcity of fossil resources necessitate the development of sustainable and innovative strategies for the industrial production. Organic residual streams (e.g., food waste and brewery wastewater) have great potential to be employed as feedstock for the production of biofuels and biochemicals [1, 2].

n-Caproic acid (CA) is a medium chain fatty acid of 6-carbon, which has recently gained considerable attention due to its high value as a fuel and chemical feedstock [3]. Biosynthesis of CA can be achieved in some anaerobic bacteria (e.g., Clostridium kluyveri) via the chain elongation pathway with ethanol as reduced substrate (electron donor) [4, 5], in which the oxidation of ethanol provides energy and acetyl-CoA for the reverse β-oxidation pathway [6]. Many studies show that the addition of ethanol during the acidification of wastes can promote chain elongation, and lead to higher volumetric production rates and a high CA selectivity [7, 8], indicating that ethanol is an efficient reduced substrate for CA production. In addition, researchers explore other chemicals used as electron donors for CA production, including hydrogen [9], methanol [10], propanol [11], and D-galactitol [12], but the yield of CA from these substrates is much lower than that from ethanol.

Recently, lactate was showed to be a potential alternative to ethanol for the production of CA by reactor microbiome systems [13, 14]. Furthermore, our study demonstrates that CA is efficiently produced from lactate by the pure culture of a Ruminococcaceae bacterium CPB6 [15]. Moreover, strain CPB6 can produce CA (C6) from lactate (as electron donor) with C2-C4 carboxylic acids as electron acceptors, and heptoic acid (C7) with C3-C5 carboxylic acids [16]. More recently, we identified sets of genes correlated with the chain elongation pathway by sequencing and annotating the whole genome of the CPB6 strain [17]. However, the detailed molecular mechanism of lactate conversion into CA still remains unknown, and there is no genome-wide transcriptomic analysis of the CPB6 strain concerning its metabolism for CA production using lactate as the electron donor. This makes it impossible to clarify the effect of lactate on the metabolic pathway shift for CA production at the molecular level.

RNA-Seq is a powerful method to elucidate the metabolism and identify specific genes/enzymes for the particular interesting metabolic pathways [18, 19]. In the present study, RNA-Seq for the transcriptomic analysis of strain CPB6 was carried out to investigate the effect of lactate

supplementation on cell growth, CA production and other essential metabolisms. The identified key genes herein provide candidates for the metabolic engineering in the future in order to develop more robust strains based on CPB6 for enhanced production of CA and relevant biochemicals.

Growth and fermentation kinetics

As shown in Figure 1a, cells took approximately 12 h to grow to the exponential phase and 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 21h (Figure1b), 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 21h. 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, 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 exponential (12h) and stationary (18h) 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
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 exponential phase;

L2: cell culture with lactate supplementation from the stationary phase;

C1: Control culture without lactate supplementation from the exponential 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 Figure 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 exponential 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 (Figure 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

Differential gene expression analysis is commonly used to reveal the molecular mechanisms of microbial adaptation to 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 exponential 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 exponential 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 exponential phase
C1: no-lactate-supplemented cells (controls) at exponential phase

The COG distribution of the DEGs at both the exponential phase and the stationary phase was illustrated in Figure 4. It revealed the potential genes related to the pathways and bioprocesses for the utilization of lactate for CA production. At the exponential 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 Figure 5. The results indicated that the gene expression of the triplicate (a, b and c) of each sample demonstrated very similar expression patterns (Figure 5). The two cultures (with or without lactate supplementation) were cluster together at the exponential phase, 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, implying that supplemented lactate induces greater metabolic shift at this phase. 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 Figure 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 [22-24]. 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 exponential phase. Three glycolytic genes exhibited different expression patterns at the stationary phase. 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. Gene encoding phosphofructokinase (PFK, B6259_RS06095) was significantly downregulated. Generally, the addition of lactate has little impact on the expression of glycolytic genes. Notably, the expression of the gene encoding glucokinase (GK) was not detected in the transcriptome under either condition. GK is a rate-limiting enzyme in glycolysis mediating glucose-induced insulin release by regulating intracellular ATP production. The reason for the absence of GK expression warrants further investigation.

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)
12h			18h				12h					18h				12h				18h
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
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, 25]. 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 exponential and stationary phase, respectively (Figure 6). 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), 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 exponential 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) 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 it showed no change in expression in the two cultures. B6259_RS02600 was expressed at relative low level at the exponential 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. 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 [25]. 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 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 caproy-CoA and CA formation [6]. However, Clostridium sp. BPY5 and C. tyrobutyricum, which contains these genes, only produce

butyric acid instead of CA [25, 26], 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 [27]. 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 [28]. 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 exponential 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 exponential 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 from certain doom when living conditions become intolerable [29]. 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 [30]. 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 [31]. But recent studies showed that the sporulation events were uncoupled from the induction of solventogenesis in C. beijerinckii [22]. 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.

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 [32]. 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 [33]. In the present study, most genes for ABC transporter and substrate-binding protein (SBP) were no significant change in the two cultures in the exponential phase, 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. This indicated 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 [34].

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 [35]. 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 [36]. 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 exponential 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. This is probably because PTS transporter-mediated sugar transport in membrane vesicles decreased with the exhaustion of glucose in the control. While in the culture with lactate supplementation, the utilization of glucose was slightly slowed down, and thus PTS transporter was upregulated to regain sugar transport as soon as lactate is depleted.

This study showed that lactate supplementation induced earlier CA production, higher CA titer and productivity, but had little impact on cell growth, suggesting that the supplemented lactate is more involved in secondary metabolism (e.g., the production of CA) than the carbon assimilation. The gene transcriptional profiles based on RNA-Seq demonstrated that supplemented lactate promoted CA production by altering the expression patterns of genes involved in crucial metabolic pathways. Specifically, the Pfor gene involved in central pyruvate metabolism, 5 genes (AtoB, HBD, CRT, BCD and CAT) involved in the reverse β-oxidation pathway, 11 genes encoding ABC transporter, 6 SBP genes, and 4 PTS transporter genes showed sharp upregulation in response to the addition of lactate. The findings presented herein provide unique insights into the metabolic effects of lactate on CA production at the gene regulation level.

Microorganisms, media and fermentation experiment

Ruminococcaceae bacterium CPB6 (GDMCC No.60133) was routinely cultured at 37 oC anaerobically in a modified tryptone-glucose-yeast extract (mTGY) medium containing the following compounds (g L-1, pH 6.0): tryptone, 5.0; glucose, 2.0; yeast extract, 5.0; sodium acetate, 3.5; K2HPO4·3H2O, 0.41; KH2PO4, 0.23; NH4Cl, 0.25; MgSO4·7H2O, 0.20; NaHCO3, 2.5; L-cysteine, 0.25; Na2S·9H2O, 0.25; resazurin, 0.0005; and 1ml of trace element solution SL-10 and 1ml of vitamin solution [14]. The suspension of activated strain CPB6 was inoculated with a 5% ratio into the same medium as described above and incubated for 12-15 h until the OD600 of the culture reached 0.8-1.0. Then the culture would be used as the seed inoculum (5% ratio, v/v) for batch experiments. To investigate the effect of lactate on cell growth and CA production, 5 g L-1 sodium lactate was supplemented into the modified TGY medium (mTGYL). Batch experiments were performed in 250 mL serum bottles containing 100 mL of modified mTGY or mTGYL media. The headspace of the bottle was filled with highly pure N2. Each fermentation was performed in triplicate. The fermentation was carried out at 37 oC for 24 h in an E500 anaerobic workstation (Gene Science, USA) under N2: CO2: H2 (volume ratio of 80:10:10) atmosphere.

Samples were taken at specific times and processed for cell concentration determination and HPLC analysis. Samples for RNA isolation were taken at the exponential and stationary phases of the cell growth.

Analytical methods

Culture growth was monitored by measuring the optical density at 600 nm (OD600) using a TU-1810 UV/Vis Spectrophotometer (Puxi Instrument Co. Ltd., Beijing). Lactic acid, acetic acid, butyric acid, and caproic acid were quantified using an HPLC system (Agilent 1260 Infinity, USA) equipped with a differential refraction detector (RID) and a Agilent Hi-Plex H column (300 × 6.5 mm) following the procedure as previously described [16].

RNA isolation, library construction, and sequencing

In preparation for RNA isolation, 10 ml cell culture was harvested at each time point, and centrifuged at 8,000 × g for 10 min at 4 °C. Cells were then frozen in liquid nitrogen prior to storage at -80°C. The RNA was extracted and purified using a RNA extraction kit (DP430, Tiangen Biotech, Beijing, China) following the manufacturer's protocol. RNA quality and quantity were characterized using a NanoDrop2000 (NanoDrop Technologies, Wilmington, DE), agarose gel electrophoresis (RNA integrity detection) and Agilent 2100 (RIN value measurement). Only the RNA samples with high-quality (≥5μg; ≥200ng/μL; OD260/280=1.8~2.2) were used for the cDNA library construction and sequencing. Before library construction, rRNAs were removed with the Ribo-Zero rRNA Removal Kit (Epicentre, San Diego, CA) following the manufacturer’s protocol. The enriched mRNA was fragmented into short fragments (approximately 200 bp) randomly using metal ions. Then the first strand cDNA was synthesized using the random hexamer-primer with the mRNA fragment as the template. After synthesizing the second strand cDNA using DNA polymerase I and RNase H, double-stranded cDNA was further end repaired, A-tailed, and indexed adapters ligated. The final cDNA library was constructed using the mRNA-Seq library construction kit (Illumina Inc., San Diego, CA), and then sequenced by Illumina platform (Illumina Inc., San Diego, CA).

RNA-Seq data analysis

High quality reads in each sample were aligned to the CPB6 genome using Bowtie2, and those that did not align uniquely to the genome were discarded using the default quality parameters. Each base was assigned a value based on the number of mapped sequence coverage. Gene expression levels were defined using the number of transcripts per million (TPM), which is proportional to the quantity of cDNA fragments derived from the gene transcripts. The quantitative gene expression values between samples were identified by calculating the number of unambiguous tags for each gene and then normalizing this to TPM, which was calculated following the method reported by Parto et al., [37]. The gene expression results were visualized using heatmap plots.

Statistical analysis

Significant differences of the gene expression between the culture from fermentation with lactate supplementation vs. the control were determined using ANOVA in R software (version 3.5.2). TPM values were first transformed to log10-scale. The log10-transformed TPM values were then properly centered for better representation of the data using the heatmap plots. Fold changes (FCs) as the ratio of

the TPM values were calculated following the method reported by Love et al. [38], and were used to compare the differentially expressed genes (DEGs) between the culture from fermentation with lactate supplementation and the control.

RNA-Seq data accession number

The RNA-Seq sequencing data have been deposited in the NCBI Sequence Read Archive (SRA) under the accession number PRJNA564589

CA: n-Caproic acid; DEG: differentially expressed gene; TPM: the number of transcripts per million; HPLC: high performance liquid chromatography; ABC: ATP-binding cassette; PTS: the phosphotransferase system; GO: Gene Ontology (GO); COGs: Clusters of Orthologous Groups

Authors' contributions

YT planned and analysed the results, and wrote the manuscript. SL performed the experiments. CW participated in the planning and revision of manuscript, CW and QY participated in the experiments. All authors read and approved the final manuscript.

Author details

1 CAS Key Laboratory of Environmental and Applied Microbiology & Environmental Microbiology Key Laboratory of Sichuan Province, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, People’s Republic of China. 2 Department of Biosystems

Engineering, Auburn University, Auburn, AL 36849, USA.

Acknowledgements

We thank professor Yi Wang and Jun Feng for their assistance and advice for the study.

Competing interests

The authors declare that they have no competing interests.

Availability of supporting data

The RNA-Seq sequencing data have been deposited in the NCBI Sequence Read Archive (SRA) under the accession number PRJNA564589

Consent for publication

All authors have read and approved this manuscript.

Ethics approval and consent to participate

Not applicable.

Funding

This work was financially supported by the Natural Science Foundation of China (31770090), the Open-foundation project of CAS Key Laboratory of Environmental and Applied Microbiology (KLCAS-2017-01).

Roghair M, Liu Y, Adiatma JC, Weusthuis RA, Bruins ME, Buisman CJN, Strik D: Effect of n-Caproate Concentration on Chain Elongation and Competing Processes. ACS Sustain Chem Eng 2018, 6(6):7499-7506.
Nzeteu CO, Trego AC, Abram F, O'Flaherty V: Reproducible, high-yielding, biological caproate production from food waste using a single-phase anaerobic reactor system. Biotechnol Biofuels 2018, 11:108.
Agler MT, Wrenn BA, Zinder SH, Angenent LT: Waste to bioproduct conversion with undefined mixed cultures: the carboxylate platform. Trends Biotechnol 2011, 29(2):70-78.
Spirito CM, Richter H, Rabaey K, Stams AJ, Angenent LT: Chain elongation in anaerobic reactor microbiomes to recover resources from waste. Curr Opin Biotechnol 2014, 27:115-122.
Weimer PJ, Stevenson DM: Isolation, characterization, and quantification of Clostridium kluyveri from the bovine rumen. Appl Microbiol Biot 2012, 94(2):461-466.
Seedorf H, Fricke WF, Veith B, Bruggemann H, Liesegang H, Strittimatter A, Miethke M, Buckel W, Hinderberger J, Li FL et al: The genome of Clostridium kluyveri, a strict anaerobe with unique metabolic features. P Natl Acad Sci USA 2008, 105(6):2128-2133.
Grootscholten TIM, Kinsky dal Borgo F, Hamelers HVM, Buisman CJN: Promoting chain elongation in mixed culture acidification reactors by addition of ethanol. Biomass and Bioenergy 2013, 48:10-16.
Roghair M, Liu Y, Strik D, Weusthuis RA, Bruins ME, Buisman CJN: Development of an Effective Chain Elongation Process From Acidified Food Waste and Ethanol Into n-Caproate. Front Bioeng Biotechnol 2018, 6:50.
Steinbusch KJJ, Hamelers HVM, Plugge CM, Buisman CJN: Biological formation of caproate and caprylate from acetate: fuel and chemical production from low grade biomass. Energy Environ Sci 2011, 4(1):216-224.
Chen WS, Ye Y, Steinbusch KJJ, Strik DPBTB, Buisman CJN: Methanol as an alternative electron donor in chain elongation for butyrate and caproate formation. Biomass Bioenerg 2016, 93:201-208.
Kenealy WR, Waselefsky DM: Studies on the Substrate Range of Clostridium Kluyveri the Use of Propanol and Succinate. Arch Microbiol 1985, 141(3):187-194.
Jeon BS, Kim BC, Um Y, Sang BI: Production of hexanoic acid from D-galactitol by a newly isolated Clostridium sp. BS-1. Applied microbiology and biotechnology 2010, 88(5):1161-1167.
Kucek LA, Nguyen M, Angenent LT: Conversion of l-lactate into n-caproate by a continuously fed reactor microbiome. Water research 2016, 93:163-171.
Zhu X, Tao Y, Liang C, Li X, Wei N, Zhang W, Zhou Y, Yang Y, Bo T: The synthesis of n-caproate from lactate: a new efficient process for medium-chain carboxylates production. Sci Rep 2015, 5:14360.
Zhu X, Zhou Y, Wang Y, Wu T, Li X, Li D, Tao Y: Production of high-concentration n-caproic acid from lactate through fermentation using a newly isolated Ruminococcaceae bacterium CPB6. Biotechnol Biofuels 2017, 10:102.
Wang H, Li X, Wang Y, Tao Y, Lu S, Zhu X, Li D: Improvement of n-caproic acid production with Ruminococcaceae bacterium CPB6: selection of electron acceptors and carbon sources and optimization of the culture medium. Microb Cell Fact 2018, 17(1):99.
Tao Y, Zhu XY, Wang H, Wang Y, Li XZ, Jin H, Rui JP: Complete genome sequence of Ruminococcaceae bacterium CPB6: A newly isolated culture for efficient n-caproic acid production from lactate. J Biotechnol 2017, 259:91-94.
Wang Y, Li XZ, Mao YJ, Blaschek HP: Single-nucleotide resolution analysis of the transcriptome structure of Clostridium beijerinckii NCIMB 8052 using RNA-Seq. Bmc Genomics 2011, 12.
Sedlar K, Koscova P, Vasylkivska M, Branska B, Kolek J, Kupkova K, Patakova P, Provaznik I: Transcription profiling of butanol producer Clostridium beijerinckii NRRL B-598 using RNA-Seq. Bmc Genomics 2018, 19.
Wei H, Fu Y, Magnusson L, Baker JO, Maness PC, Xu Q, Yang SH, Bowersox A, Bogorad I, Wang W et al: Comparison of transcriptional profiles of Clostridium thermocellum grown on cellobiose and pretreated yellow poplar using RNA-Seq. Front Microbiol 2014, 5.
Wang Y, Li XZ, Mao YJ, Blaschek HP: Genome-wide dynamic transcriptional profiling in clostridium beijerinckii NCIMB 8052 using single-nucleotide resolution RNA-Seq. Bmc Genomics 2012, 13.
Wang Y, Li XZ, Blaschek HP: Effects of supplementary butyrate on butanol production and the metabolic switch in Clostridium beijerinckii NCIMB 8052: genome-wide transcriptional analysis with RNA-Seq. Biotechnol Biofuels 2013, 6.
Patakova P, Linhova M, Rychtera M, Paulova L, Melzoch K: Novel and neglected issues of acetone-butanol-ethanol (ABE) fermentation by clostridia: Clostridium metabolic diversity, tools for process mapping and continuous fermentation systems. Biotechnology advances 2013, 31(1):58-67.
Jiao SY, Zhang Y, Wan CX, Lv J, Du RJ, Zhang RJ, Han B: Transcriptional analysis of degenerate strain Clostridium beijerinckii DG-8052 reveals a pleiotropic response to CaCO(3)associated recovery of solvent production. Sci Rep-Uk 2016, 6.
Lee J, Jang YS, Han MJ, Kim JY, Lee SY: Deciphering Clostridium tyrobutyricum Metabolism Based on the Whole-Genome Sequence and Proteome Analyses. Mbio 2016, 7(3).
Tao Y, Hu X, Zhu X, Jin H, Xu Z, Tang Q, Li X: Production of Butyrate from Lactate by a Newly Isolated Clostridium sp. BPY5. Appl Biochem Biotechnol 2016.
Rogatzki MJ, Ferguson BS, Goodwin ML, Gladden LB: Lactate is always the end product of glycolysis. Front Neurosci-Switz 2015, 9.
Skory CD: Isolation and expression of lactate dehydrogenase genes from Rhizopus oryzae. Applied and environmental microbiology 2000, 66(6):2343-2348.
Piggot PJ, Moran CPJ, Youngman P (eds.): Regulation of Bacterial Differentiation: Washington, D.C.; 1994.
Sauer U, Santangelo JD, Treuner A, Buchholz M, Durre P: Sigma factor and sporulation genes in Clostridium. Fems Microbiol Rev 1995, 17(3):331-340.
Woods DR, Jones DT: Physiological responses of Bacteroides and Clostridium strains to environmental stress factors. Adv Microb Physiol 1986, 28:1-64.
Hollenstein K, Dawson RJ, Locher KP: Structure and mechanism of ABC transporter proteins. Curr Opin Struct Biol 2007, 17(4):412-418.
Cui J, Davidson AL: ABC solute importers in bacteria. Essays Biochem 2011, 50(1):85-99.
Jones PM, George AM: The ABC transporter structure and mechanism: perspectives on recent research. Cell Mol Life Sci 2004, 61(6):682-699.
Jason G. McCoy, Elena J. Levin, Zhou M: Structural insight into the PTS sugar transporter EIIC. Biochim Biophys Acta 2015, 1850(3):577-585.
Nguyen TX, Yen MR, Barabote RD, Saier MH, Jr.: Topological predictions for integral membrane permeases of the phosphoenolpyruvate:sugar phosphotransferase system. J Mol Microbiol Biotechnol 2006, 11(6):345-360.
Patro R, Mount SM, Kingsford C: Sailfish enables alignment-free isoform quantification from RNA-seq reads using lightweight algorithms. Nat Biotechnol 2014, 32(5):462-U174.
38. Love MI, Huber W, Anders S: Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 2014, 15(12).

Download PDF

Version 1

posted

You are reading this latest preprint version

Genome-Wide Transcriptomic Analysis of N-Caproic Acid Production in Ruminococcaceae Bacterium CPB6 with Lactate Supplementation

Status:

Version 1

Abstract

Figures

Background

Results And Discussion

Growth and fermentation kinetics

RNA-Seq statistics

Functional annotation and classification

Differential expression of global genes

Expression of glycolysis genes

Expression of putative ABC transporter and sporulation genes

Conclusions

Materials And Methods

Microorganisms, media and fermentation experiment

Analytical methods

RNA isolation, library construction, and sequencing

RNA-Seq data analysis

Statistical analysis

Abbreviations

Declaration

References

Supplementary Files

Status:

Version 1