Genome Sequence of the Thermophilic Strain Bacillus coagulans2-6, an Efficient Producer of High-Optical-Purity l-Lactic Acid

Abstract

Bacillus coagulans2-6 is an efficient producer of lactic acid. The genome of B. coagulans2-6 has the smallest genome among the members of the genus Bacillusknown to date. The frameshift mutation at the start of the d-lactate dehydrogenase sequence might be responsible for the production of high-optical-purity l-lactic acid.

GENOME ANNOUNCEMENT

Bacillus coagulans, from spoiled canned milk, was first described in 1915 by Hammer (8). Because of stable high performance in the utilization of renewable resources and nonsterilization fermentation at high temperature, the thermophilic B. coagulansstrains have been suggested to be superior producers of lactic acid (11, 13). In addition to the production of lactic acid, B. coagulanshas also been found to be a source of many other commercially valuable products, such as thermostable enzymes and coagulin, an antimicrobial peptide (6). Compared with other probiotic bacteria such as Lactobacillusspecies, some strains of B. coagulansare able to survive in the environment of extremes of heat, acidity of the stomach, and bile acids (3). However, little genetic information is known. Here, we present the genome sequence of B. coagulansstrain 2-6, which is an efficient producer of high-optical-purity l-lactic acid with the advantages of high carbon efficiency, less by-product formation, and thermotolerance (13).

The whole genome of B. coagulans2-6 was sequenced using the Illumina GA system performed by the Helmholtz Center for Infection Research in Germany with a combination of paired-end library and mate pair. Reads were assembled with Velvet (14). According to the draft sequence of B. coagulans36D1 and contigs from different assembly softwares (Edena [5], Euler-SR [2], and SOAPdenovo [7]), the complete genome sequence of strain 2-6 was completed. Closure of the gaps was finished by Bubble PCR primer walking using the routine Sanger method and edited in the Phred/Phrap/Consed (4) package. Finally, Illumina data were used to correct potential base errors and increase consensus quality by mapping the reads to the genome. The genome sequence of B. coagulans2-6 was annotated with the NCBI Prokaryotic Genomes Automatic Annotation Pipeline (12) and functional annotation using Clusters of Orthologous Genes and KEGG (9).

The genome of B. coagulans2-6, which is the smallest of the known Bacillusgenomes, is composed of a 3,073,079-bp single circular chromosome with a mean GC content of 47.3% and a 9,910-bp plasmid whose mean GC content is 38.0%. We identified 2,975 protein-coding sequences (CDS) in the chromosome and 10 CDS in the plasmid. No putative biological functions were predicted for the plasmid. The CDS in the chromosome constitute 79.9% of the genome. Putative biological functions were assigned to 2,332 (78.4%) predicted proteins based on BLAST (1) results. The frameshift mutation at the start of the d-lactate dehydrogenase sequence might be responsible for the production of high-optical-purity l-lactic acid (optical purity, >99%) by strain 2-6 (13). Only a fragment of pyrophosphokinase in the phosphoketolase pathway was predicted, which suggested the pentose mainly lost in the transaldolase/transketolase pathway. Compared with the phosphoketolase pathway, the transaldolase/transketolase pathway could produce 1.67 mol of lactic acid per mol pentose, whereas the phosphoketolase pathway produces only 1 mol of lactic acid in addition to 1 mol acetate (10).

Nucleotide sequence accession number.

The complete genome sequence of B. coagulans2-6 has been submitted to GenBank under accession number CP002472.