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Journal of Integrative Agriculture  2024, Vol. 23 Issue (9): 3066-3077    DOI: 10.1016/j.jia.2023.11.047
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A unique role of the pyrimidine de novo synthesis enzyme ODCase in Lysobacter enzymogenes
Mingming Yang1, 2, Yunxiao Tan2, Jiabing Ma2, Yingjia Zhao3, Xia Yan4, Nana Wang3, Pingping Wang5, Jiaqi Tan5, Suilong Ai5, Xiaofei Liang2, Bangshuai Chang3, Obadah E. A. Yousif3, Chao Zhao2, Bo Wang6, Guoliang Qian7, Lili Huang1, 2#

1 State Key Laboratory for Crop Stress Resistance and High-Efficiency Production, Northwest A&F University, Yangling 712100, China

2 College of Plant Protection, Northwest A&F University, Yangling 712100, China

3 College of Life Sciences, Northwest A&F University, Yangling 712100, China

4 College of Natural Resources and Environment, Northwest A&F University, Yangling 712100, China

5 Shaanxi Provincial Tobacco Corporation of China National Tobacco Corporation, Xi’an 710061, China

6 Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China

7 College of Plant Protection, Laboratory of Plant Immunity, Nanjing Agricultural University, Nanjing 210095, China

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摘要  

产酶溶杆菌(Lysobacter enzymogenes)OH11分泌的热稳定性抗真菌因子HSAF具有广谱高效的抑菌活性。目前无法利用化学合成和异源表达等方法生产HSAF。OH11菌株中HSAF原始产量低,是规模化生产的主要瓶颈。从分子层面阐明HSAF生物合成的调控网络可为提升活性代谢物产量,研发绿色高效的新型生防产品奠定理论基础。本研究发现一个编码乳清酸核苷-5'-单磷酸脱羧酶(ODCase)的基因Le0752,该基因在尿嘧啶从头合成途径中催化乳清酸核苷酸(OMP)生成尿嘧啶核苷酸(UMP)。Le0752缺失突变株的HSAF产量及其发酵粗提液对辣椒疫霉(Phytophthora capsici)的皿内抑菌活性均显著降低,而用Le0752缺失突变体发酵液处理过的辣椒疫霉对本氏烟(Nicotiana benthamiana)的毒力较野生型显著提高。Le0752突变体在1/10 TSA平板上生长减缓,但蹭行运动(twitching motility)能力不受影响。qRT-PCR结果表明,Le0752缺失突变后HSAF合成关键基因lafB及其关键调控因子clp的基因表达水平显著降低,遗传学试验证明Le0752通过转录因子Clp调控lafB的转录进而影响HSAF的生物合成。生物信息学分析显示,溶杆菌属中的ODCases归于Group III类群,而与溶杆菌属亲缘关系较近的黄单胞菌属和寡养单胞菌属的ODCases归属Group I类群。基因染色体同源替换实验中,稻黄单胞菌水稻致病变种(Xanthomonas oryzae pv. oryzae)中的ODCase(pyrF)能够完全恢复Le0752缺失突变表型。本研究阐明尿嘧啶从头合成酶ODCase对HSAF合成的关键调控作用,将为改造HSAF高产菌株提供理论支撑,并拓展对细菌ODCase功能与进化的认识。



Abstract  
Bacterial species of the genus Lysobacter are environmentally ubiquitous with strong antifungal biocontrol potential.  Heat-stable antifungal factor (HSAF) secreted by the biocontrol bacterium Lysobacter enzymogenes OH11 has broad-spectrum and highly efficient antifungal activity.  Studying the biosynthetic regulations of HSAF would lay an important foundation for strain engineering toward improved HSAF production.  In this work, we demonstrate that Le0752, an orotidine-5´-phosphate decarboxylase enzyme (ODCase) catalyzing a pivotal step of the UMP de novo biosynthesis pathway, is vital for HSAF-mediated antimicrobial activities and growth of L. enzymogenes OH11, but not for twitching motility.  This gene regulates the production of HSAF by affecting the expression of lafB, a key gene in the HSAF biosynthesis operon, through the transcription factor Clp.  Interestingly, bioinformatics analysis revealed that Le0752 belongs to the Group III ODCases, whereas its homologs in the closely related genera Xanthomonas and Stenotrophomonas belong to Group I, which contains most ODCases from Gram-positive bacteria, Gram-negative bacteria and cyanobacteria.  Moreover, the Group I ODCase PXO_3614 from the Xanthomonas oryzae pv.  oryzae PXO99A strain complemented the Le0752 mutant in regulating HSAF-mediated antagonistic activity.  Together, these results highlight the important requirement of de novo pyrimidine biosynthetic enzymes for antibiotic HSAF production in L. enzymogenes, which lays an important foundation for improving HSAF production via metabolic flow design and for dissecting the regulatory functions of bacterial ODCases.
Keywords:  ODCase       Pyrimidine de novo synthesis       Secondary metabolite HSAF       Lysobacter enzymogenes  
Received: 16 July 2023   Accepted: 17 October 2023
Fund: 
This study was supported by the National Natural Science Foundation of China (32102283 to Mingming Yang), the Science and Technology Major Project of China National Tobacco Corporation (110202101056(LS-16)), the Science and Technology Project of Shaanxi Branch of China National Tobacco Corporation (KJ-2021-02 and KJ-2022-04).
About author:  Mingming Yang, E-mail: yangming862@126.com; #Correspondence Lili Huang, Tel: +86-29-87091312, E-mail: huanglili@nwsuaf.edu.cn

Cite this article: 

Mingming Yang, Yunxiao Tan, Jiabing Ma, Yingjia Zhao, Xia Yan, Nana Wang, Pingping Wang, Jiaqi Tan, Suilong Ai, Xiaofei Liang, Bangshuai Chang, Obadah E. A. Yousif, Chao Zhao, Bo Wang, Guoliang Qian, Lili Huang. 2024. A unique role of the pyrimidine de novo synthesis enzyme ODCase in Lysobacter enzymogenes. Journal of Integrative Agriculture, 23(9): 3066-3077.

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