代尔夫特菌PG-8降解青霉素G的特性及分子机制

Characteristics and molecular mechanisms of penicillin G degradation by Delftia sp. PG-8

摘要:

目的 筛选青霉素G (penicillin G, PENG)降解菌，并解析其分解代谢的关键酶，为青霉素菌渣的生物处理提供菌种和基因资源。方法 以青霉素G钾(penicillin G potassium, PGK)为底物，通过富集培养筛选能够利用其为唯一碳源生长的菌株；结合基因组和转录组技术鉴定分解代谢的关键酶并分析其进化起源；表达并纯化关键酶，解析其酶促反应动力学参数；通过基因敲除和回补实验揭示关键基因在细菌利用PGK生长过程中的生理功能。结果 获得的代尔夫特菌属(Delftia sp.) PG-8能够降解并利用PGK作为唯一碳源生长，且在pH 7.0、温度35 ℃、底物浓度为10.00 mmol/L时表现出最佳的底物降解效果和细菌生长状况。PgkA能够催化PGK快速降解[K_m=(99.19±19.45) μmol/L，k_cat/K_m=(1.96±0.55)×10⁵ L/(mol·s)]，并且与已完成功能鉴定的β-内酰胺酶相比PgkA具有独特的进化起源。PgkB也能够催化PGK降解，但其对底物的亲和力仅为PgkA的1/5，且底物催化效率也较低。菌株PG-8-ΔpgkA和PG-8-ΔpgkB降解和利用PGK生长的能力均显著下降，且PG-8-ΔpgkA能力下降更为明显。虽然同时敲除pgkA和pgkB的PG-8-ΔpgkAB仍能降解一定量的底物，但无法利用PGK作为唯一碳源生长。结论 PG-8是代尔夫特菌属中第一株能够利用PGK作为唯一碳源生长的菌株，pgkA和pgkB在PG-8利用PGK作为唯一碳源生长过程中均具有重要的生理功能，但pgkA起主导作用。

Abstract:

Objective To screen out a strain with the ability to degrade penicillin G (PENG) and identify the key enzymes involved in PENG catabolism, providing strain and gene resources for the biological treatment of penicillin waste.Methods Bacterial strains capable of utilizing penicillin G potassium (PGK) as the sole carbon source were screened by enrichment culture. Key enzymes involved in the catabolism of PGK were identified by genome and transcriptome analyses, and their evolutionary origins were examined. The key enzymes were expressed and purified, and their kinetics were analyzed. The physiological roles of the key genes in bacterial growth on PGK were revealed by gene knockout and complementation.Results The obtained strain Delftia sp. PG-8 can degrade PGK and utilize it as the sole carbon source for growth. The strain showed the best performance in PENG degradation and growth at pH 7.0, 35 ℃, and 10.00 mmol/L PGK. PgkA catalyzed the rapid degradation of PGK, with K_m=(99.19±19.45) μmol/L and k_cat/K_m=(1.96±0.55)×10⁵ L/(mol·s). Compared with the functionally characterized β-lactamases, PgkA had a unique evolutionary origin. PgkB also had the ability to catalyze the transformation of PGK, while its substrate affinity was only 1/5 that of PgkA, in addition to the lower catalytic efficiency. The degradation and utilization of PGK for growth by strains PG-8-ΔpgkA and PG-8-ΔpgkB were significantly reduced, with PG-8-ΔpgkA showing a more pronounced decline. Although PG-8-ΔpgkAB, in which both pgkA and pgkB were knocked out, still degraded a certain amount of substrate, it was almost unable to use PGK as the sole carbon source for growth.Conclusion PG-8 is the first strain of Delftia capable of using PGK as the sole carbon source for growth. Both pgkA and pgkB play important physiological roles during PG-8 growth on PGK, with pgkA playing a dominant role.