Improvement of dicarboxylic acid production with Methylorubrum extorquens by reduction of product reuptake

In the past, two dicarboxylic acid products, namely mesaconic acid and 2-methylsuccinic acid, were successfully produced by Methylorubrum extorquens AM1 with heterologous thioesterase YciA, but the yield was reduced by product reuptake. In our study we conducted comprehensive research on the uptake mechanism of those dicarboxylic acid products. To identify potential import factors, we performed transcriptome analysis of a strain harboring plasmid pCM160_RBS_yciAHI and an empty vector control strain. Samples were taken and analyzed at different timepoints throughout cultivation. However, we could not identify genes that were clearly upregulated in stationary growth phase compared to early sampling points in the exponential growth phase and which were annotated as potential transporters. When specifically analyzing the time-dependent expression levels of dctA homologs and kgtP (suggested as targets in previous studies) and comparing them with a control strain, we found slightly increased levels only for dctA1. After concluding that the transcriptome analysis was obviously no appropriate approach to identify relevant transport factors, we chose a straightforward mutant selection approach using a cytotoxic dicarboxylic acid. Overall design: Examination of M. extorquens AM1 harboring pCM160_RBS_YciAHI (="P") and M. extorquens AM1 harboring pCM160 (empty vector control="L") in three replicates, respectively

既往研究中，研究人员借助异源硫酯酶YciA（thioesterase YciA），利用甲基营养菌Methylorubrum extorquens AM1成功合成了中康酸（mesaconic acid）与2-甲基琥珀酸（2-methylsuccinic acid）两种二羧酸产物，但产物的细胞重摄取现象导致目标产物得率下降。本研究针对上述两类二羧酸产物的重摄取机制开展了系统性探究。为筛选潜在的底物转运因子，我们对携带质粒pCM160_RBS_yciAHI的工程菌株及空载质粒对照菌株进行了转录组分析：在菌株培养的不同时间点取样并开展检测分析。然而，与指数生长期早期取样的样本相比，我们未能在稳定生长期显著上调的基因集合中，筛选到被注释为潜在转运蛋白的候选基因。随后我们针对性分析了dctA同源基因与kgtP（既往研究提示的潜在转运靶标）的时序表达水平，并与对照菌株进行比对，结果仅发现dctA1的表达水平出现小幅上调。鉴于转录组分析显然并非筛选相关转运因子的合适策略，我们转而采用基于细胞毒性二羧酸的简便突变体筛选方案。整体实验设计：分别对携带pCM160_RBS_YciAHI（记为"P"组）的M. extorquens AM1菌株，以及携带空载质粒pCM160（记为"L"组，即空载对照）的M. extorquens AM1菌株设置三组生物学重复，开展相关检测分析。