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

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

摘要:

【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，Latilactobacillus sakei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瓜氨酸利用菌株，从而降低发酵体系中的瓜氨酸浓度。

关键词:氨基甲酸乙酯;瓜氨酸;高通量筛选;精氨酸脱亚氨基途径;PTP转运蛋白

Abstract:

[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.

Key words:ethyl carbamate;citrulline;high-throughput screening;arginine deiminase pathway;putative transport protein