Yusfi Liza Aulia, Tjong Djong Hon, Chaniago Irawati, Salsabilla Aisyah, Jamsari Jamsari
Pak J Biol Sci. 2022 Nov;25(12):1047-1057. doi: 10.3923/pjbs.2022.1047.1057.
<b>Background and Objective:</b> The optimization of the indole-3-acetic acid (IAA) producing capability of <i>Serratia plymuthica</i> UBCF_13 has been intensively studied. This work tried to reveal the effect of growth phases on IAA production, gene expression and metabolite synthesis related to the IAA biosynthesis pathway. <b>Materials and Methods:</b> The growth curve and IAA production were measured every 3 hrs. The putative IAA biosynthesis pathway was investigated based on the UBCF_13 genome. To identify the possible pathway of IAA biosynthesis in UBCF_13, we applied the Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) and High-Performance Liquid Chromatography (HPLC) analysis to measure the transcript levels of each gene and indole metabolite production based on tryptophan treatment at different times of incubation. <b>Results:</b> The optimal IAA production on colorimetric assay was at 9 hrs of incubation (initial stationary phase). The level expression of <i>puuC</i>, <i>DDC</i>, <i>oxdA</i>, <i>amiE</i>, <i>nthA</i> and <i>nthB</i> have been upregulated maximum in 3 hrs of culture time (lag phase), except <i>tyrB</i> and <i>ipdC</i>. The highest transcript level of the genes was found in nitrile hydratase genes (<i>nthA</i> and <i>nthB</i>) and indole-3- acetamide (IAM) has been detected as the only intermediate in the crude extract of UBCF_13 thus the IAM pathway may be used to produce IAA. The maximum IAA production on HPLC analysis was found at 21 hrs of incubation (late stationary phase). <b>Conclusion:</b> This study gives a new insight that the best time to measure gene expression and intermediates related to the IAA biosynthetic pathway in bacteria was found at a specific growth phase.
背景与目的：对粘质沙雷氏菌UBCF_13产吲哚-3-乙酸（IAA）能力的优化已得到深入研究。本研究试图揭示生长阶段对IAA产生、基因表达以及与IAA生物合成途径相关的代谢物合成的影响。材料与方法：每3小时测量一次生长曲线和IAA产量。基于UBCF_13基因组研究推测的IAA生物合成途径。为确定UBCF_13中IAA生物合成的可能途径，我们应用定量逆转录聚合酶链反应（qRT-PCR）和高效液相色谱（HPLC）分析，在不同培养时间基于色氨酸处理来测量每个基因的转录水平和吲哚代谢物产量。结果：比色法测定的IAA最佳产量出现在培养9小时（初始稳定期）。除tyrB和ipdC外，puuC、DDC、oxdA、amiE、nthA和nthB的表达水平在培养3小时（对数期）时上调至最高。在腈水合酶基因（nthA和nthB）中发现基因的最高转录水平，并且在UBCF_13的粗提物中检测到吲哚-3-乙酰胺（IAM）是唯一的中间体，因此IAM途径可能用于产生IAA。HPLC分析发现IAA的最大产量出现在培养21小时（稳定期末期）。结论：本研究提供了新的见解，即在细菌中测量与IAA生物合成途径相关的基因表达和中间体的最佳时间处于特定的生长阶段。
