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

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