Lactic acid is a major organic acid and precursor of ethyl lactate, which confers the important flavor of Chinese Maotai-flavor liquor. Moreover, lactic acid helps modulate the microbiome pH, which coordinates the microbial community. Lactobacillus panis L7 is the major lactic acid producer, however, the whole genome sequence is not available and the important genes for lactic acid biosynthesis have not been unidentified, which limits understanding of lactic acid biosynthesis. Whole genome sequencing of L. panis L7 was undertaken, obtaining 127 genome sequence scaffolds with an N50 of 33,743 bp. The genome total length was 2,026,099 bp encoding 1932 genes with a GC content of 47.0%. Heterolactic acid fermentation pathways and the involved genes were identified, including multiple copies of lactate dehydrogenase encoding genes. To identify important genes for lactic acid production, L. panis L7 was genetically perturbed using atmospheric and room temperature plasma followed by high-throughput screening of mutants with improved or reduced lactic acid production. Comparative genomic analysis of L. panis L7 and its mutants identified genes encoding for glutamate racemase, methylated-DNA-protein-cysteine methyltransferase, fumarate reductase flavoprotein subunit, and amidophosphoribosyltransferase as potentially important factors to improve lactic acid biosynthesis; genes encoding for ribokinase, fructokinase, argininosuccinate lyase, and large subunit ribosomal protein L30 as potentially important factors for reduced lactic acid biosynthesis. The results improved the understanding of the mechanism of lactic acid metabolism by L. panis for the benefit of the Chinese liquor brewing industry, and will, hopefully, help develop effective measures to modulate lactic acid content during brewing.