1株利用乳酸的猪源丁酸梭菌的分离鉴定及基因组序列分析

doi:10.16431/j.cnki.1671-7236.2022.02.018

Abstract

Abstract: 【Objective】 The study was aimed to research the genomic information of a pig-isolate Clostridium butyricum utilizing lactic acid.【Method】 Using anaerobic culture method, strains with fast lactic acid utilization and high butyric acid conversion rate were isolated from the intestinal contents of healthy piglets fed lactic acid bacteria fermented feed through primary screening of lactic acid decomposition medium (LADM) and re-screening of Clostridium proliferating medium (RCM).The screened strains were identified by morphology, 16S rDNA sequence analysis, lactic acid transformation characteristic test, genome re-sequencing and frame map sequencing, and the functional annotation of their coding proteins was carried out.【Result】 A LY33 strain was isolated.The strain LY33 was identified as Clostridium butyricum based on its morphological and 16S rDNA sequence analysis.The strain LY33 had good ability to utilize lactic acid.On the 6th day of growth, 66 mmol lactic acid could be basically transformed into about 17 mmol butyric acid.The re-sequencing of LY33 strain showed that the overall variation of its coding gene was small.Compared with the reference genome, the whole genome heterozygosity ratio of LY33 strain was 0.022‰, the total number of single nucleotide polymorphism (SNP) site variants was 21 590 (0.4666% of the whole genome), the total number of insertion/deletion (Indel) mutations was 594 (0.0128%), the variation of copy number variation (CNV) was 0 (0), and the total number of structure variation (SV) annotated variants was 103 (0.0003%).The mutant genes in the genome of LY33 strain were mainly genes related to strain growth, energy metabolism regulation and vitamin synthesis (especially biotin), which were distributed on multiple sequences of two chromosomes.The frame diagram sequencing of LY33 strain and the functional annotation of its encoded gene protein showed that its genome sequence length was 4 627 127 bp, GC content was 28.60%, contained 4 171 genes, and the total length of coding region accounted for 84.12%, which was involved in the metabolic pathway transformation of various substances such as carbohydrate metabolism, membrane transport and amino acid metabolism.There were many genes resistant to macrolides, bacitracin and VanI glycopeptide in the genome of LY33 strain.【Conclusion】 In this study, a LY33 strain with fast lactic acid utilization and high butyric acid conversion rate was isolated from the intestinal contents of healthy piglets fed with 4% lactic acid bacteria fermented feed, which was identified as Clostridium butyricum.It had a good application prospect and could be used as a new microbial resource for probiotics products.

Key words: Clostridium butyricum; lactic acid; isolation and identification; genome; sequence analysis

CLC Number:

Q939.94

HU Xiaobing, LIN Biaosheng, WANG Zhenwei, ZHENG Lijing. Isolation, Identification and Genomic Sequence Analysis of a Strain of Clostridium butyricum from Pigs Utilizing Lactic Acid[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(2): 569-578.

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Isolation, Identification and Genomic Sequence Analysis of a Strain of Clostridium butyricum from Pigs Utilizing Lactic Acid

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