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Article

Genomic Comparison of Lactobacillus casei AP and Lactobacillus plantarum DR131 with Emphasis on the Butyric Acid Biosynthetic Pathways

by
Widodo Widodo
1,2,*,
Aditya Lutfe Ariani
1,
Donny Widianto
1,3 and
Dietmar Haltrich
4
1
Graduate School of Biotechnology, Universitas Gadjah Mada, Yogyakarta 55281, Indonesia
2
Faculty of Animal Science, Universitas Gadjah Mada, Yogyakarta 55281, Indonesia
3
Department of Microbiology, Faculty of Agriculture, Universitas Gadjah Mada, Yogyakarta 55281, Indonesia
4
Food Biotechnology Laboratory, University of Natural Resources and Life Sciences, 1190 Vienna, Austria
*
Author to whom correspondence should be addressed.
Microorganisms 2021, 9(2), 425; https://0-doi-org.brum.beds.ac.uk/10.3390/microorganisms9020425
Submission received: 8 January 2021 / Revised: 2 February 2021 / Accepted: 8 February 2021 / Published: 19 February 2021
(This article belongs to the Special Issue Functional Characterization of Lactic Acid Bacteria)
Versions Notes

Abstract

:
Butyric acid is known to possess anticarcinogenic and antioxidative properties. The local lactic acid bacteria (LAB) strains Lactobacillus casei AP isolated from the digestive tract of healthy Indonesian infants and L. plantarum DR131 from indigenous fermented buffalo milk (dadih) can produce butyric acid in vitro. However, the genes and metabolic pathways involved in this process remain unknown. We sequenced and assembled the 2.95-Mb L. casei AP and 4.44-Mb L. plantarum DR131 draft genome sequences. We observed that 98% of the 2870 protein-coding genes of L. casei AP and 97% of the 3069 protein-coding genes of L. plantarum DR131 were similar to those of an L. casei strain isolated from infant stools and an L. plantarum strain in sheep milk, respectively. Comparison of the genome sequences of L. casei AP and L. plantarum DR131 led to the identification of genes encoding butyrate kinase (buk) and phosphotransbutyrylase (ptb), enzymes involved in butyric acid synthesis in L. casei AP. In contrast, a medium-chain thio-esterase and type 2 fatty acid synthase facilitated butyric acid synthesis in L. plantarum DR131. Our results provide new insights into the physiological behavior of the two LAB strains to facilitate their use as probiotics.

1. Introduction

Lactic acid bacteria (LAB) have been widely used for the fermentation and production of a variety of food items for human and animal consumption. These bacteria enhance the flavor, texture, nutritional value, and safety of fermented food items. In addition, specific strains of LAB are known to have probiotic potential owing to their beneficial effects on the health of consumers [1]. Previous studies have reported that the beneficial health effects of LAB are associated with its ability to produce many bioactive compounds, including exopolysaccharides [2,3], riboflavin [4], gamma-aminobutyric acid (GABA) [5], and short-chain fatty acids (SCFAs) [6]. Recent advances in microbial genomics, such as genome sequencing and functional genomic analyses, have broadened our knowledge regarding the diversity and evolution of LAB strains. Further, they have aided in the analysis of important food traits, such as flavor formation, sugar metabolism, stress response, adaptation, and molecular-level interactions [7].
Butyric acid (butyrate) is an SCFA synthesized by the microflora present in the intestine [8]. Butyrogenesis (or butyrate production) has been widely researched in several gut-related studies [9,10] as well as in biotechnology [11]. In bacteria, butyric acid can be synthesized either directly from carbohydrates via butyrate kinase or indirectly from acetate, succinate, and lactate via butyryl-coenzyme A (CoA): acetate-CoA transferase, succinyl-CoA synthetase, and lactate dehydrogenase, respectively, together with butyrate kinase [12]. Due to its wide range of beneficial roles, including as an essential energy source for epithelial cells of the large intestine and for the prevention of inflammatory bowel disease and colorectal cancer [13], butyric acid has huge potential for use in probiotics. However, studies related to butyric acid biosynthetic pathways in L. casei species have been limited to their physiology. Therefore, we selected two local LAB strains, namely Lactobacillus casei AP, isolated from the digestive tract of a healthy infant aged <1 month, and L. plantarum DR131, obtained from indigenous fermented buffalo milk (dadih) in Indonesia. Both L. casei AP and L. plantarum DR 131 can potentially be developed into fermented dairy products as a health food. Widodo et al. [14] reported that the use of human-origin L. casei AP in fermented dairy products could reduce hyperglycemia and cholesterol in Sprague Dawley rats. Further, L. plantarum DR131 has also been used to produce fermented buffalo milk in Indonesia.
Kusmiyati et al. [15] demonstrated that L. casei AP produced butyric acid in media containing inulin, whereas Pessione et al. [6] reported that L. plantarum was capable of producing butyric acid. Based on these findings, we explored the genes, pathways, and mechanisms involved in butyric acid synthesis using genome sequencing and annotation to verify the experimental outcomes in both strains.

2. Materials and Methods

2.1. Bacterial Strain Identification

L. casei AP was isolated from the fecal sample of a healthy infant [16] and L. plantarum DR131 was isolated from indigenous fermented buffalo milk (dadih). These strains were identified by their 16S rRNA using 27 F and 1492 R primers. A DNA fragment of approximately 500–1000 kb was amplified using the Bio-Rad Thermal Cycler (Bio-Rad, Hercules, CA, USA). Polymerase chain reaction (PCR) was performed under the following conditions: initial denaturation at 96 °C for 4 min followed by 30 cycles of denaturation at 94 °C for 1 min, annealing at 52 °C for 1 min 50 s, and a final extension at 68 °C for 8 min. The PCR products were analyzed using 1.0% (w/v) agarose gel electrophoresis (Bio-Rad) in 1× Tris/borate/ethylenediaminetetraacetic acid buffer at 100 V for 30 min and visualized on a gel documentation system (BioDocAnalyze; Biometra GmbH, Gottingen, Germany). The purified PCR products were sequenced using 16S rRNA primers. Whole-genome sequences were used for similarity searches against the NCBI GenBank database using the Basic Local Alignment Search Tool (BLAST) program available at the website (http://blast.ncbi.nlm.nih.gov/BLAST.cgi (accessed on 6 January 2021)).

2.2. Genome Sequencing and Assembly

The DNA from L. casei AP and L. plantarum DR131 was extracted using the PrestoTM Mini gDNA Bacteria Kit (Geneaid) according to the manufacturer’s instructions. The final DNA concentration was determined using the Qubit 2.0 Fluorometer (Life Technology, Carlsbad, CA, USA). Whole-genome sequencing was performed on the Illumina platform using the massively parallel sequencing technology. Paired-end (PE) adapters ligated to A-tailed fragments and PCR amplified with a 500-bp insert and a mate-pair (MP) library with a 5-kb insert were used for the construction of the genome library at Novogene Bioinformatics Technology Co., Ltd. (Beijing, China). The PE and MP libraries were sequenced on the Illumina HiSeq platform using the PE150 strategy. We used our own compiling pipeline to filter the Illumina PCR adapter reads and low-quality reads from the PE and MP libraries. All good quality PEs were assembled into several scaffolds using the SOAPdenovo genome assembler (http://soap.genomics.org.cn/soapdenovo.html (accessed on 6 January 2021)) [17,18]. The reads were filtered during the gap-closing step.

2.2.1. Genome Component Prediction

Genome component prediction included the prediction of coding genes, repetitive sequences, and non-coding RNA. For bacteria, we used the GeneMarkS (http://topaz.gatech.edu/GeneMark/ (accessed on 6 January 2021)) program to retrieve the related coding genes [19]. The interspersed repetitive sequences were predicted using RepeatMasker (http://www.repeatmasker.org/ (accessed on 6 January 2021)) [20]. Tandem repeats were analyzed by the tandem repeats finder (http://tandem.bu.edu/trf/trf.html (accessed on 6 January 2021)) [21]. Transfer RNA (tRNA) genes were predicted using tRNAscan-SE [22]. Ribosomal RNA (rRNA) genes were analyzed using rRNAmmer [18]. Small nuclear RNAs (snRNA) were predicted by BLAST against the Rfam database [23,24].

2.2.2. Genome Function Annotation

Protein-coding genes were predicted using GeneMarkS (http://topaz.gatech.edu/GeneMark/ (accessed on 6 January 2021)) [25]. We used the following seven databases to predict gene functions: Gene Ontology [26], the Kyoto Encyclopedia of Genes and Genomes (KEGG) [27,28], Clusters of Orthologous Groups [29], the Non-Redundant Protein Database (NR) [18], Swiss-Prot [30], TrEMBL Uniport [31], Brenda enzymes (www.brenda-enzymes.org (accessed on 6 January 2021)), and MetaCyc (https://metacyc.org/ (accessed on 6 January 2021)). A whole-genome BLAST search (E-value < 1 × 10−5 [32], minimal alignment length > 40%) was performed against these seven databases.

3. Results

3.1. Genomic Characteristics of L. casei AP and L. plantarum DR131

The genomes of L. casei AP and L. plantarum DR131 were sequenced using the whole-genome shotgun strategy to produce clean data after filtering low-quality reads and reads with adapter contamination. First, the genome of L. casei AP was assembled using the SOAPdenovo (version 2.04) assembler to generate 79 contigs (>500 bp) with N50 of 83,564 base pairs (bp) [18,33] followed by assembly into 71 scaffolds (>500 bp) with N50 of 83,564 bp. The lengths of the scaffolds ranged from 557 bp to 277,281 bp. For L. plantarum DR131, 87 contigs (>500 bp) with N50 of 120,340 bp assembled into 67 scaffolds (>500 bp) with N50 of 128,698 bp were generated. The lengths of the scaffolds ranged from 503 bp to 262,440 bp. We could not assemble the scaffolds into chromosomes via K-mer analysis for L. casei AP and L. plantarum DR131. However, for L. casei AP, we obtained a draft genome sequence of 2.95 Mb as compared to the expected genome size of 3.05 Mb; this indicates that the scaffolds covered 96.72% of the whole genome with a K-mer depth of 85.87. In the case of L. plantarum DR131, we obtained a draft genome sequence of 4.44 Mb compared to the expected genome size of 4.47 Mb, indicating that the scaffolds covered 99.33% of the whole genome with a K-mer depth of 60.38. The G + C content of L. casei AP and L. plantarum DR131 was 46.34% and 44.42%, respectively.

3.2. Identification of the Butyric Acid Biosynthetic Pathways

We analyzed the genes involved in the butanoate metabolism (map00650) pathway in the genomes of L. casei AP and L. plantarum DR131. The genes encoding for butyrate kinase (buk) (locus = Scaffold14:33935:35056:+) and phosphotransbutyrylase (ptb) (locus = Scaffold14:33042:33938:+), enzymes responsible for butyric acid synthesis, were found in L. casei AP, but not in L. plantarum DR131. The results of KEGG annotation of the genes presumably involved in butanoate metabolism in L. casei AP are presented in Table 1.
After excluding these terminal genes and their respective butanoate metabolism pathways, we searched all the genes responsible for the enzymatic reactions involved in butyric acid production (www.brenda-enzymes.org (accessed on 6 January 2021); http://www.genome.jp/kegg/annotation/enzyme.html (accessed on 6 January 2021); https://metacyc.org/ (accessed on 6 January 2021)). Recently, Botta et al. [34] reported that butyrogenesis in L. plantarum occurs via the complementary activities of a medium-chain thio-esterase and type 2 fatty acid synthase (FASII). This pathway is also involved in the biosynthesis of hexanoic acid, octanoic acid, decanoic acid, dodecanoic acid, and tetradecanoic acid. The butyrogenic capability of L. plantarum varies based on the strain and is highly dependent on the substrate type, with glutamine/glutamate playing an important role. However, this has not been assigned to any specific metabolic pathway. A previous study by Botta et al. [34] linked the loss of the butyrogenic capability of L. plantarum to deleterious functional mutations in the enzyme glutamate decarboxylase and a glutamine ABC transporter. We found similar results for L. plantarum DR131, confirming that butyrogenesis can only occur via the complementary activities of a medium-chain thioesterase and FASII in L. plantarum. In addition, the evaluation of butanoate metabolism in L. plantarum DR131 revealed the involvement of two glutamate decarboxylase (gad) genes. GAD is the key enzyme involved in GABA biosynthesis [35] and is linked to stress resistance and pH homeostasis in the cytosol via protein translocation [36].
The results of the KEGG annotation of the genes involved in the proposed butanoate metabolism in L. plantarum DR131 are shown in Table 2.
By integrating the KEGG annotation of butanoate metabolism in L. plantarum DR131 and previously published GABA biosynthesis in L. plantarum, we proposed the potential involvement of the GABA pathway in L. plantarum DR131 in butanoate synthesis via an unknown mechanism. The results of KEGG annotation of the genes involved in butyric acid production can be attributed to the complementary activities of a medium-chain thio-esterase and FASII in L. plantarum DR131 (Table 3).

4. Discussion

LABs are widely used in the fermentation and production of a wide range of food products due to their ability to impart flavor and texture to the fermented products. In addition, specific LAB strains are known to have the ability to act as probiotics owing to their health-promoting effects in animals and humans [1]. The sequencing and annotation of LAB genomes are crucial not only for their functional application, but also for comparative genomics research. In this study, we selected two LAB strains, namely L. casei AP, a local strain isolated from the digestive tract of a healthy infant aged <1 month, and L. plantarum DR131, a LAB isolated from the indigenous fermented buffalo milk (dadih) of Indonesia, for genome sequencing based on Illumina technology and comparative analyses.
For L. casei AP, we assembled the genome sequences into 71 scaffolds (>500 bp) of a 2.95-Mb sequence, which represented approximately 96.72% of the entire genome, and annotated 2870 protein-coding genes at the genome level. For L. plantarum DR131, we assembled the sequence into 67 scaffolds (>500 bp) of a 4.44-Mb sequence, representing approximately 99.33% of the whole genome, and annotated 3069 protein-coding genes at the genome level. Junior et al. [37] reported the draft genome sequence of the L. paracasei strain DTA83 isolated from the stool sample of healthy infants in Rio de Janeiro (Brazil). The 2.8-Mb genome of this strain possessed 2825 protein-coding sequences distributed in 330 SEED subsystems. When we compared the results of our sequencing analysis, we found 98% similarity with the genome sequence of the L. paracasei strain DTA83 considering that they shared the same origin—the digestive tract of healthy infants. Patil et al. [38] studied the L. plantarum strain JDARSH, a potential probiotic with a wide range of functions isolated from sheep milk. The draft genome in our study was 3.20 Mb in size with 2980 protein-coding sequences. Comparison of the genome sequence of the L. plantarum DR131 used in our study with that of the L. plantarum strain JDARSH revealed a similarity of 97%. The predicted gene models of our sequenced genome and sequencing results were highly similar with JGI genome annotation, indicating that the quality of our sequence and annotation was reliable.
In the genome of L. casei AP, we only detected the genes buk (Scaffold14:33935:35056:+) and ptb (Scaffold14:33042:33938:+), which are involved in the butyric acid biosynthesis pathway for butanoate metabolism. These genes were not detected in L. plantarum DR131. In L. casei AP, the metabolic routes for butyrate production directly from glucose generate 2 mol H2/mol of butyrate in accordance with the equation C6H12O6 → butyric acid + 2H2 + 2CO2 [34]. Butyrogenesis proceeds through butyryl-coenzyme A (butyryl-CoA) generation from acetoacetyl-CoA via the intermediates β-hydroxybutyryl-CoA and crotonyl-CoA. Butyryl-CoA is then converted to butyrate via two pathways, one of which involves the generation of butyrate-phosphate via ptb, which is further converted to butyrate via buk [12].
Recently, medium-chain acyl–acyl carrier protein thio-esterase was proposed as the only terminal enzyme capable of producing butyric acid in L. plantarum. In addition to this enzyme, the enzymes present in L. equisimilis GGS 124 have been demonstrated to be capable of truncating the fatty acid biosynthesis pathway of FASII in engineered E. coli to release butyric acid and other medium-chain fatty acids [39]. Notably, the complementary activities of a medium-chain thio-esterase and FASII are also involved in the synthesis of hexanoic acid, octanoic acid, decanoic acid, dodecanoic acid, and tetradecanoic acid. Nevertheless, there is no explanation regarding the mechanism underlying butyric acid production in L. plantarum.
The enzymes and proteins involved in the fatty acid synthesis of FASII in L. plantarum DR131 (Table 2) can roughly be assigned to two categories: (i) those with highly conserved protein sequences and (ii) those with less conserved sequences, such as enoyl reductase, which comprises three discrete enzymes. Notably, except for E. coli, much of our discussions on proteins and genes were based on the DNA sequence data rather than on genetic or biochemical analyses [40]. The highly conserved proteins are often encoded within gene clusters.
Based on this study, we propose that L. casei AP employs the butanoate pathway as the main pathway in butyric acid synthesis. Meanwhile, butyric acid production in L. plantarum DR131 involves the GABA pathway in butanoate synthesis via an unknown mechanism, which was attributed to the complementary activities of medium-chain thio-esterase and FASII.
The above-mentioned analyses verify the outcomes of the previous experimental study regarding the capability of both LAB strains (L. casei AP and L. plantarum DR131) to synthesize butyric acid. The present study showed metabolic flexibility between species of the same lactobacilli genus in synthesizing the same organic acid. The data of genome sequencing revealed the metabolic pathways of butyric acid synthesis in two different species of lactobacilli, leading to a better understanding of how to optimize these pathways for butyric acid production.

5. Conclusions

L. casei AP and L. plantarum DR131 are capable of producing butyric acid. The metabolic pathways of butyrogenesis in these two strains are different. Both strains have potential for the development of metabolic engineering strategies.

Author Contributions

Conceptualization, W.W., A.L.A., D.W., and D.H.; Data curation, W.W., A.L.A., D.W., and D.H.; Formal analysis, W.W., A.L.A., and D.W.; Funding acquisition, W.W. and D.W.; Methodology, W.W., A.L.A., D.W., and D.H.; Writing—original draft, W.W., A.L.A., and D.W.; Writing—review and editing, W.W. and D.H. All authors have read and agreed to the published version of the manuscript.

Funding

The study was supported by the Indonesian Ministry for Education and Culture. Part of the research reported in this publication was jointly supported by the ASEAN-European Academic University Network (ASEA-UNINET), the Austrian Federal Ministry of Education, Science and Research and the Austrian Agency for International Mobility and Cooperation in Education, Science and Research (OeAD-GmbH).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

All data generated or analyzed during this study are included. The raw data are available from the corresponding author upon reasonable request.

Acknowledgments

We would like to thank Kirana Kristina Mulyono for microbiological technical support and Ari Surya Sukarno for helping in molecular assessments.

Conflicts of Interest

The authors have no conflict of interest to declare.

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Table
Table 1. KEGG annotation of the genes involved in butanoate metabolism in Lactobacillus casei AP.
Table 1. KEGG annotation of the genes involved in butanoate metabolism in Lactobacillus casei AP.
KOAbbreviationGene NamesEnzymePutative Gene
K01641E2.3.3.10hydroxymethylglutaryl-CoA synthase 2.3.3.10LCAP_GM000043
K00626E2.3.1.9, atoBacetyl-CoA C-acetyltransferase 2.3.1.9LCAP_GM000041
K00656E2.3.1.54, pflDformate C-acetyltransferase 2.3.1.54LCAP_GM001343
K00929bukbutyrate kinase2.7.2.7LCAP_GM001757
K04072adhEacetaldehyde dehydrogenase/alcohol dehydrogenase 1.2.1.10 1.1.1.1LCAP_GM000697
K00135gabDsuccinate-semialdehyde dehydrogenase/glutarate-semialdehyde dehydrogenase 1.2.1.16 1.2.1.79 1.2.1.20LCAP_GM002304
K00634ptbphosphate butyryltransferase 2.3.1.19LCAP_GM001756
K01575alsD, budA, aldCacetolactate decarboxylase4.1.1.5LCAP_GM001865
K01652E2.2.1.6L, ilvB, ilvG, ilvIacetolactate synthase I/II/III large subunit 2.2.1.6LCAP_GM001866
K00244frdAfumarate reductase flavoprotein subunit 1.3.5.4LCAP_GM002528
Table
Table 2. KEGG annotation of the genes involved in butanoate metabolism in Lactobacillus plantarum DR131.
Table 2. KEGG annotation of the genes involved in butanoate metabolism in Lactobacillus plantarum DR131.
KOAbbreviationGene NamesEnzymePutative Gene
K01641E2.3.3.10hydroxymethylglutaryl-CoA synthase 2.3.3.10LPDR_GM000252
K00656E2.3.1.54, pflDformate C-acetyltransferase 2.3.1.54LPDR_GM001714
K00135gabDsuccinate-semialdehyde dehydrogenase/glutarate-semialdehyde dehydrogenase 1.2.1.16 1.2.1.79 1.2.1.20LPDR_GM000712
K01575alsD, budA, aldCacetolactate decarboxylase4.1.1.5LPDR_GM000217
K01652E2.2.1.6L, ilvB, ilvG, ilvIacetolactate synthase I/II/III large subunit 2.2.1.6LPDR_GM000843
K00244frdAfumarate reductase flavoprotein subunit 1.3.5.4LPDR_GM001916
K04072adhEacetaldehyde dehydrogenase/alcohol dehydrogenase1.2.1.10 1.1.1.1LPDR_GM000307
K01580E4.1.1.15, gadB, gadA, GAD glutamate decarboxylase 4.1.1.15LPDR_GM001627
K03778ldhAD-lactate dehydrogenase1.1.1.28LPDR_GM001493
K00016LDH, ldhL-lactate dehydrogenase1.1.1.27LPDR_GM002271
K00625E2.3.1.8, ptaphosphate acetyltransferase2.3.1.8LPDR_GM001551
K00925ackAacetate kinase2.7.2.1LPDR_GM001960
Table
Table 3. KEGG annotation of the genes involved in the complementary activities of a medium-chain thio-esterase and type 2 fatty acid synthase (FASII) in Lactobacillus plantarum DR131.
Table 3. KEGG annotation of the genes involved in the complementary activities of a medium-chain thio-esterase and type 2 fatty acid synthase (FASII) in Lactobacillus plantarum DR131.
KOAbbreviationGene NamesEnzymesPutative Gene
K01071MCHmedium-chain acyl-[acyl-carrier-protein] hydrolase 3.1.2.21LPDR_GM001386
K00208 fabIenoyl-[acyl-carrier protein] reductase I 1.3.1.9 1.3.1.10LPDR_GM002661
K02372fabZ3-hydroxyacyl-[acyl-carrier-protein] dehydratase 4.2.1.59LPDR_GM002650
K09458fabF3-oxoacyl-[acyl-carrier-protein] synthase II 2.3.1.179LPDR_GM002655
K00059fabG3-oxoacyl-[acyl-carrier protein] reductase 1.1.1.100LPDR_GM000931
K00645fabD[acyl-carrier-protein] S-malonyltransferase 2.3.1.39LPDR_GM002653
K00648fabH3-oxoacyl-[acyl-carrier-protein] synthase III 2.3.1.180LPDR_GM001441
K01962 accAacetyl-CoA carboxylase carboxyl transferase subunit alpha 6.4.1.2LPDR_GM002658
K02160accB, bccPacetyl-CoA carboxylase biotin carboxyl carrier protein--LPDR_GM001440
K01961accCacetyl-CoA carboxylase, biotin carboxylase subunit 6.4.1.2 6.3.4.14LPDR_GM002658
K01963accDacetyl-CoA carboxylase carboxyl transferase subunit beta 6.4.1.2LPDR_GM002658
K00997acpSholo-[acyl-carrier protein] synthase2.7.8.7LPDR_GM002257
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Widodo, W.; Ariani, A.L.; Widianto, D.; Haltrich, D. Genomic Comparison of Lactobacillus casei AP and Lactobacillus plantarum DR131 with Emphasis on the Butyric Acid Biosynthetic Pathways. Microorganisms 2021, 9, 425. https://0-doi-org.brum.beds.ac.uk/10.3390/microorganisms9020425

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Widodo W, Ariani AL, Widianto D, Haltrich D. Genomic Comparison of Lactobacillus casei AP and Lactobacillus plantarum DR131 with Emphasis on the Butyric Acid Biosynthetic Pathways. Microorganisms. 2021; 9(2):425. https://0-doi-org.brum.beds.ac.uk/10.3390/microorganisms9020425

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Widodo, Widodo, Aditya Lutfe Ariani, Donny Widianto, and Dietmar Haltrich. 2021. "Genomic Comparison of Lactobacillus casei AP and Lactobacillus plantarum DR131 with Emphasis on the Butyric Acid Biosynthetic Pathways" Microorganisms 9, no. 2: 425. https://0-doi-org.brum.beds.ac.uk/10.3390/microorganisms9020425

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