Sodium-Ion-Free Fermentative Production Of GABA With Levilactobacillus Brevis And Establishment Of Fusion Primer Driven Racket PCR Genome Walking Method

Gamma-aminobutyric acid（GABA）is a natural amino acid,which has been widely used in food and pharmaceutical fields due to its numerous physiological effects.GABA has been identified as a new resource food.Lactic acid bacteria are generally considered safe organisms.Synthesis of GABA by Lactic acid bacteria is a hot research topic and a future development trend.A Levi Levilactobacillus brevis CD0817 with high GABA yield was isolated in our laboratory in the early stage.In this paper,a sodium ion-free fermentation production of GABA was established for this bacterium.The seeds and fermentation media are Na⁺-free,and the p H of the two media was not adjusted to 5.0.At the same time,in order to further explore and study the genes related to GABA synthesis of this bacterium,this paper also established a genome step method based on fusion primer PCR.The main research contents of this article are as follows:（1）Firstly,the seed preparation scheme was designed and optimized.L-glutamic acid was used as the substrate for the media.The preparation of the seed solution included a one-test tube and a shaker seed medium.Static fermentation was used in the Erlenmeyer flask experiment.The most important factors affecting GABA fermentation were glucose,yeast extract,TW-80,manganese ion,temperature,and species age.The optimal levels were 10.0 g/L,35.0 g/L,1.5 g/L,0.2 m M and 30.0℃,respectively.We used a seed with A₆₀₀value between 3.0–3.5.（2）Based on the optimized data,a sodium ion-free GABA fermentation process was developed using a 10 L fermenter.According to the optimized seed preparation method,the seed bacteria liquid was obtained.The fermentation was started by transferring the inoculum at a size of 10%（v/v）into the 10 L fermenter loading 4.0 L medium and 2600.0 g substrate.The fermentation was hermetically conducted under constant agitation（50 rpm）and 30.0°C for 48 h.During the fermentation,L-glutamate acid power was continuously dissolved to supply the substrate and acid environment essential for GABA synthesis.The current bioprocess accumulated GABA up to 331.0±8.3 g/L after 48 h,having a productivity 6.9 g/L/h and a molar conversion rate 98.1%.The Na⁺-free strategy may be promoted to other lactic acid bacterial strains.（3）In the is paper,we report a new genome walking method based on fusion primer PCR.Because SSP3 can mediate intra-strand annealing to form a racket-like structure,it is named fusion primer driven racket PCR,for reliable retrieving unknown flanking DNA sequences.The function of the fusion primer is to mediate the Genomic walk,selective enrichment,and intra-strand annealing of the target DNA.Four sequence-specific primers（SSP1,SSP2,SSP3,and SSP4）were sequentially selected from known DNA（5’→3’）to complete FPR-PCR.SSP3 is the fragment mediating intra-strand annealing（FISA）.The FISA fragment is attached to the 5’end of the SSP1,generating a fusion primer.The FPR-PCR comprises two rounds of amplification reactions.The primary FPR-PCR initial five moderate-stringency（55℃）cycles only permit the fusion primer 3’-part（SSP1）to hybridize with its complement on the known region,increasing copies of the target first-strand.The subsequent single low-stringency（25℃）cycle allows the fusion primer to be partially annealed to a site on an unknown region of the chain,producing a second target single strand.The target DNA is exponentially amplified in the next thirty high-stringency（65℃）cycles.Since the FISA of the target strand is annealed within the strand with the reverse complementary sequence at the 3’end of it,a racket-like DNA is then formed by loopback extension.The secondary FPR-PCR,a racket-like DNA was amplified exponentially driven by SSP2 and SSP4.The FPR-PCR was validated by identifying the unknown flanks of Levi Levilactobacillus brevis CD0817 glutamic acid decarboxylase genes and rice hygromycin gene.