School of Food and Bioengineering, Qilu University of Technology, Jinan 250353, PR China.
Microb Cell Fact. 2014 Jan 16;13:10. doi: 10.1186/1475-2859-13-10.
Transcriptional engineering has presented a strong ability of phenotypic improvement in microorganisms. However, it could not be directly applied to Actinoplanes teichomyceticus L-27 because of the paucity of endogenous transcription factors in the strain. In this study, exogenous transcription factors were rationally selected and transcriptional engineering was carried out to increase the productivity of teicoplanin in L-27.
It was illuminated that the σ(HrdB) molecules shared strong similarity of amino acid sequences among some genera of actinomycetes. Combining this advantage with the ability of transcriptional engineering, exogenous sigma factor σ(HrdB) molecules were rationally selected and engineered to improve L-27. hrdB genes from Actinoplanes missouriensis 431, Micromonospora aurantiaca ATCC 27029 and Salinispora arenicola CNS-205 were selected based on molecular evolutionary analysis. Random mutagenesis, DNA shuffling and point mutation were subsequently performed to generate diversified mutants. A recombinant was identified through screening program, yielding 5.3 mg/ml of teicoplanin, over 2-fold compared to that of L-27. More significantly, the engineered strain presented a good performance in 500-l pilot scale fermentation, which meant its valuable potential application in industry.
Through rational selection and engineering of exogenous transcriptional factor, we have extended the application of transcriptional engineering. To our knowledge, it is the first time to focus on the related issue. In addition, possessing the advantage of efficient metabolic perturbation in transcription level, this strategy could be useful in analyzing metabolic and physiological mechanisms of strains, especially those with the only information on taxonomy.
转录工程在微生物的表型改善方面具有强大的能力。然而，由于该菌株中内源性转录因子的缺乏，无法将其直接应用于游动放线菌 L-27。在本研究中，通过合理选择外源转录因子，对 L-27 进行了转录工程改造，以提高其泰利霉素的产量。
研究表明，σ（HrdB）分子在放线菌的一些属中具有很强的氨基酸序列相似性。结合这一优势和转录工程的能力，合理选择和工程化外源σ（HrdB）因子分子来改善 L-27。基于分子进化分析，从密西西比分枝杆菌 431、橙色小单孢菌 ATCC 27029 和沙雷氏菌 arenicola CNS-205 中选择了 hrdB 基因。随后进行了随机诱变、DNA 改组和定点突变，以产生多样化的突变体。通过筛选程序鉴定出一个重组体，产生的泰利霉素产量为 5.3mg/ml，比 L-27 提高了 2 倍以上。更重要的是，该工程菌株在 500 升中试规模发酵中表现出良好的性能，这意味着其在工业上具有有价值的潜在应用。
通过合理选择和工程化外源转录因子，我们扩展了转录工程的应用。据我们所知，这是首次关注相关问题。此外，该策略在转录水平上具有高效代谢干扰的优势，可用于分析菌株的代谢和生理机制，特别是那些仅具有分类学信息的菌株。
