Complete genome sequence of functional probiotic candidate Lactobacillus amylovorus CACC736

Soyeon Park1, Jung-Ae Kim1,2, Hyun-Jun Jang1, Dae-Hyuk Kim1,3, Yangseon Kim1,*
Author Information & Copyright
1Department of Research and Development, Center for Industrializaton of Agricultural and Livestock Microorganisms, Jeongeup 56212, Korea
2Department of Bioactive Material Science, Jeonbuk National University, Jeonju 54896, Korea
3Department of Molecular Biology, Department of Bioactive Material Science, Institute for Molecular Biology and Genetics, Jeonbuk National University, Jeonju 54896, Korea
*Corresponding author: Yangseon Kim, Department of Research and Development, Center for Industrialization of Agricultural and Livestock Microorganisms, Jeongeup 56212, Korea. Tel: +82-63-536-6712, E-mail:

© Copyright 2023 Korean Society of Animal Science and Technology. This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Received: Sep 28, 2022; Revised: Oct 10, 2022; Accepted: Oct 13, 2022

Published Online: Mar 31, 2023


Lactobacillus amylovorus CACC736 was originated from swine feces in Korea. The complete genome sequences of the strain contained one circular chromosome (2,057,809 base pair [bp]) with 38.2% guanine-cytosine (GC) content and two circular plasmids, namely, pCACC736-1 and pCACC736-2. The predicted protein-coding genes, which are encoding the clustered regularly interspaced short palindromic repeats (CRISPR)-associated proteins, biosynthesis of bacteriocin (helveticin J), and the related proteins of the bile, acid tolerance. Notably, the genes related to vitamin B-group biosynthesis (riboflavin and cobalamin) were also found in L. amylovorus CACC736. Collectively, the complete genome sequence of the L. amylovorus CACC736 will aid in the development of functional probiotics in the animal industry.

Keywords: Lactobacillus amylovorus; Swine; Probiotics; Whole-genome sequencing

Lactobacillus spp. are non-pathogenic microorganisms that provide beneficial effects to the host [13]. Lactobacillusamylovorus has been studied as a paraprobiotic (non-viable cells or cell fractions) with the ability to change body adiposity [1]. Additionally, it has been reported that L. amylovorus has probiotic properties such as antiviral and antimicrobial activities through the regulation of the gut microflora [2,3]. In this study, the genomes of L. amylovorus CACC736 are functionally annotated.

L.amylovorus strain CACC736 (KACC22146) was isolated from swine feces in Korea. This strain was inoculated in de Man, Rogosa, and Sharpe (MRS) medium (Difco, Franklin Lakes, NJ, USA) and cultivated at 37°C for 24 h. Genomic DNA (gDNA) of the strain was extracted using the DNeasy UltraClean microbial kit (Qiagen, Hilden, Germany). The complete genome sequence of L.amylovorus strain CACC736 was obtained with single-molecule real-time sequencing technology (SMRT) on the platform of PacBio Sequel (Pacific Biosciences, Menlo Park, CA, USA) at CJ Bioscience, Inc (Seoul, Korea). These gene sequences were performed by de novo genome assembly using the PacBio SMRT Analysis (version 2.3.0, Pacific Biosciences) [4]. All genes were classified by different functional groups using EggNOG 4.5 ( Additionally, functional annotation of the coding sequences (CDSs) was performed by the UBLAST program including the databases of the Swiss-Prot and Kyoto Encyclopedia of Genes and Genomes (KEGG) [5]. Predictions for clustered regularly interspaced short palindromic repeats (CRISPR) were used by CRISPR finder ( [6].

The L.amylovorus CACC736 composed of one circular chromosome (2,057,809 base pair [bp], 38.2% guanine-cytosine [GC] content) along with two plasmids designated as pCACC736-1 (76,480 bp, 36.0% GC content) and pCACC736-2 (20,439 bp, 35.0% GC content) (Table 1 and Fig. 1A). Moreover, the complete genome comprised 2,080 protein- CDSs and 80 non-coding genes (15 rRNA and 65 tRNA genes). A total of 1,848 proteins (88.8%) were classified on a functional categorization by the database of Clusters of Orthologous Groups (COGs) categories (Fig. 1B). The most abundant COGs categories, excluding an ‘unknown function [S]’, were ‘replication, recombination and repair [L]’ (295 genes; 16.0%), ‘carbohydrate transport and metabolism [G]’ (146 genes; 7.9%), ‘translation, ribosomal structure and biogenesis [J]’ (137 genes; 7.4%), and ‘amino acid transport and metabolism [E]’ (126 genes; 6.8%). The genome of the L. amylovorus CACC736 encoded five CRISPR genes/proteins (Cas1, Cas2, Cas3, Cas4, and Cas6) for antiviral-relative mechanisms [7], one bacteriocin class III (helveticin J) for an inhibitory effect against common pathogenic organisms [8], and two potential genes of antimicrobial activity (lysM and qac). In addition, the L.amylovorus CACC736 was confirmed to have genes associated with common probiotic properties, such as lactate synthesis (ldh, L-lactate dehydrogenase), bile salt hydrolases (BSH; cbh) and acid tolerance (atpD, atpH, and grpE) (Table 2). Notably, we revealed the presence of genes involved in vitamin B2 and B12 biosynthesis, including riboflavin (ribB, ribD, ribE, and ribT) and cobalamin (cobC) (Table 2) [9,10]. Taken together, our findings on the complete genome of L.amylovorus CACC736 will provide a scientific improvement for the development of functional probiotics.

Table 1. General features of Lactobacillus amylovorus CACC736 genome
Properties Chromosome Plasmids
CACC736 pCACC736-1 pCACC736-2
BioProject PRJNA881772
BioSample SAMN30915630
Accession No. CP104879 CP104880 CP104881
Genome size (bp) 2,057,809 76,480 20,439
GC content (%) 38.2 36.0 35.0
No. of CDSs 1,989 71 20
No. of CRISPR regions 5
rRNA genes 15
tRNA genes 65

bp, base pair; GC, guanine-cytosine; CDSs, coding sequences; CRISPR, clustered regularly interspaced short palindromic repeats.

Download Excel Table
Table 2. Predicted CDSs involved in probiotic potency in Lactobacillus amylovorus CACC736
Predicted function L. amylovorus CACC736
Predicted genes Start position End position Length (bp)
 Endonuclease Cas1 c1,507,114 c1,508,103 990
 Endonuclease Cas2 c1,506,827 c1,507,108 282
 Endonuclease/helicase Cas3 c1,508,620 c1,511,049 2,430
 Exonuclease Cas4 c1,508,113 c1,508,604 492
 Endoribonuclease Cas6 c1,515,924 c1,516,679 756
Antimicrobial activity-related
 Lysin motif domain lysM 890,591 891,055 465
 Quaternary ammonium compound-resistance qacC 1,012,642 1,012,962 321
Bacteriocin (Class III) helveticin J c1,995,360 c1,995,992 633
Lactate synthesis ldh 1,795,954 1,796,925 972
Bile salt hydrolases (BSH) cbh 1,052,335 1,053,357 1,023
Acid tolerance-related atpD 690,181 691,692 1,512
atpH 692,699 694,138 1,440
clpB 98,750 100,879 2,130
grpE c1,235,180 c1,235,764 585
Protection or repair-related dnaJ c1,232,063 c1,233,217 1,155
Vitamin B-groups synthesis
 Vitamin B2 ribB c1,025,985 c1,027,160 1,176
ribD c1,027,752 c1,028,810 1,059
ribE c1,027,163 c1,027,759 597
ribT 887,197 887,550 354
 Vitamin B12 cobC c301,776 c302,426 651

CDSs, coding sequences; bp, base pair; CRISPR, clustered regularly interspaced short palindromic repeats; c, complement.

Download Excel Table
Fig. 1. Genome futures of Lactobacillus amylovorus CACC736. (A) Circular genome mapping of Lactobacillus amylovorus CACC736. Circles from the outside to the center denote: (a) forward and (b) reverse strands (colored according to COGs function categories), (c) rRNA and tRNA, (d) GC skew, (e) GC content. (B) Functional classification of COGs. COGs, cluster of orthologous groups of proteins database; GC, guanine-cytosine.
Download Original Figure


The complete genome sequences of L.amylovorus strain CACC736 were deposited at the NCBI GenBank under the accession numbers CP104879 (chromosome) and CP104880-CP104881 (plasmids), respectively.

Competing interests

No potential conflict of interest relevant to this article was reported.

Funding sources

This research was supported by INNOPOLIS FOUNDATION through Science and Technology Project Opens the Future of the Region, which is funded by Ministry of Science and ICT (2022-DD-UP-0333), Korea, and partially supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry (IPET) through Useful Agricultural Life Resources Industry Technology Development Program, funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA) (321094-2).


Not applicable.

Availability of data and material

Upon reasonable request, the datasets of this study can be available from the corresponding author.

Authors’ contributions

Conceptualization: Kim Y.

Data curation: Park S, Jang HJ.

Formal analysis: Park S.

Methodology: Park S, Kim JA.

Software: Park S.

Validation: Park S, Kim JA, Kim DH.

Investigation: Kim Y.

Writing - original draft: Park S.

Writing - review & editing: Park S, Kim JA, Jang HJ, Kim DH, Kim Y.

Ethics approval and consent to participate

This article does not require IRB/IACUC approval because there are no human and animal participants.



Sugawara T, Sawada D, Yanagihara S, Aoki Y, Takehara I, Sugahara H, et al. Daily intake of paraprobiotic Lactobacillus amylovorus CP1563 improves pre-obese conditions and affects the gut microbial community in healthy pre-obese subjects: a double-blind, randomized, placebo-controlled study. Microorganisms. 2020; 8:304


Martinez RCR, Aynaou AE, Albrecht S, Schols HA, de Martinis ECP, Zoetendal EG, et al. In vitro evaluation of gastrointestinal survival of Lactobacillus amylovorus DSM 16698 alone and combined with galactooligosaccharides, milk and/or Bifidobacterium animalis subsp. lactis Bb-12. Int J Food Microbiol. 2011; 149:152-8


Foulquié Moreno MR, Baert B, Denayer S, Cornelis P, De Vuyst L. Characterization of the amylovorin locus of Lactobacillus amylovorus DCE 471, producer of a bacteriocin active against Pseudomonas aeruginosa, in combination with colistin and pyocins. FEMS Microbiol Lett. 2008; 286:199-206


Kim JA, Jung MY, Kim DH, Kim Y. Genome analysis of Bacteroides sp. CACC 737 isolated from feline for its potential application. J Anim Sci Technol. 2020; 62:952-5


Kang S, Li R, Jin H, You HJ, Ji GE. Effects of selenium- and zinc-enriched Lactobacillus plantarum SeZi on antioxidant capacities and gut microbiome in an ICR mouse model. Antioxidants. 2020; 9:1028


Grissa I, Vergnaud G, Pourcel C. CRISPRFinder: a web tool to identify clustered regularly interspaced short palindromic repeats. Nucleic Acids Res. 2007; 35:W52-7


Rath D, Amlinger L, Rath A, Lundgren M. The CRISPR-Cas immune system: biology, mechanisms and applications. Biochimie. 2015; 117:119-28


Ghodhbane H, Elaidi S, Sabatier JM, Achour S, Benhmida J, Regaya I. Bacteriocins active against multi-resistant gram negative bacteria implicated in nosocomial infections. Infect Disord Drug Targets. 2015; 15:2-12


Thakur K, Tomar SK, De S. Lactic acid bacteria as a cell factory for riboflavin production. Microb Biotechnol. 2016; 9:441-51


Capozzi V, Russo P, Dueñas MT, López P, Spano G. Lactic acid bacteria producing B-group vitamins: a great potential for functional cereals products. Appl Microbiol Biotechnol. 2012; 96:1383-94