基于PTP转运蛋白的瓜氨酸利用菌株高通量筛选策略及其应用评估

【目的】为降低发酵体系中氨基甲酸乙酯前体物瓜氨酸的浓度，建立基于PTP (putative transport protein)转运蛋白的瓜氨酸利用菌株高通量筛选策略，对不同样品中的瓜氨酸利用菌株进行分离、筛选和鉴定，并对其胞外瓜氨酸利用能力进行比较和分析。【方法】通过对瓜氨酸转运蛋白PTP的氨基酸序列进行比对分析，确定其保守区域，并通过设计获得简并引物，结合溴甲酚紫变色圈法、简并引物菌落PCR和二乙酰一肟显色法，建立瓜氨酸利用菌株的高通量筛选策略。【结果】设计出一对可用于筛选瓜氨酸利用菌株的简并引物，利用建立的高通量筛选策略从环境中分离获得65株具有瓜氨酸利用能力的菌株，其中Levilactobacillus brevis PC4可在4 h内消耗体系中91.08%的瓜氨酸。对分离菌株PTP编码基因在基因组中的位置进行分析发现，Latilactobacillussakei等4株菌的PTP编码基因都位于arc基因簇内，表明PTP蛋白的功能与分离菌株的瓜氨酸代谢途径密切相关。【结论】PTP蛋白是菌株胞外瓜氨酸利用的重要功能蛋白，基于PTP编码基因设计的高通量筛选策略可用于高效地从环境中分离...

High-throughput screening of citrulline-utilizing strains based on putative transport protein and its application assessmeat

[Objective] Citrulline is a precursor of ethyl carbamate in fermentation systems. To reduce the concentration of citrulline, we established a putative transport protein (PTP)-based high-throughput screening strategy to isolate and identify citrulline-utilizing strains from different samples and then evaluated the extracellular citrulline utilization abilities of the strains. [Methods] We compared the amino acid sequences of PTPs to determine the motif and then designed degeneracy primers to obtain the nucleic acid sequences of PTPs. The citrulline-utilizing strains were isolated by high-throughput screening strategy combined with bromocresol purple chromogenic circle method, colony PCR with degeneracy primers, and diacetyl monoxime colorimetric method. [Results] A pair of degeneracy primers was designed for the screening of citrulline-utilizing strains. Using the high-throughput screening strategy, we isolated 65 citrulline-utilizing strains from different environments. Levilactobacillus brevis PC4 consumed 91.08% citrulline in 4 h. The PTP genes of four strains were located in arc gene cluster, which indicated that the function of PTP was closely related to citrulline metabolic pathway. [Conclusion] PTP is an important functional protein for extracellular citrulline utilization of bacteria. High-throughput screening strategy based on PTP gene is an efficient way to isolate citrulline-utilizing strains from different environments.