Engineering Biology Research Center, Kobe University, 1-1 Rokkodai, Nada, Kobe, 657-8501, Japan.
Graduate School of Science, Technology and Innovation, Kobe University, 1-1 Rokkodai, Nada, Kobe, 657-8501, Japan.
Microb Cell Fact. 2024 Oct 8;23(1):268. doi: 10.1186/s12934-024-02543-6.
Marine cyanobacteria such as Picosynechococcus sp. (formerly called Synechococcus sp.) PCC 7002 are promising chassis for photosynthetic production of commodity chemicals with low environmental burdens. Genetic engineering of cyanobacteria conventionally employs antibiotic resistance markers. However, limited availability of antibiotic-resistant markers is a problem for highly multigenic strain engineering. Although several markerless genetic manipulation methods have been developed for PCC 7002, they often lack versatility due to the requirement of gene disruption in the host strain. To achieve markerless transformation in Synechococcus sp. with no requirements for the host strain, this study developed a method in which temporarily introduces a mutated phenylalanyl-tRNA synthetase gene (pheS) into the genome for counter selection. Amino acid substitutions in the PheS that cause high susceptibility of PCC 7002 to the phenylalanine analog p-chlorophenylalanine were examined, and the combination of T261A and A303G was determined as the most suitable mutation. The mutated PheS-based selection was utilized for the markerless knockout of the nblA gene in PCC 7002. In addition, the genetic construct containing the lldD and lldP genes from Escherichia coli was introduced into the ldhA gene site using the counter selection strategy, resulting in a markerless recombinant strain. The repeatability of this method was demonstrated by the double markerless knockin recombinant strain, suggesting it will be a powerful tool for multigenic strain engineering of cyanobacteria.
海洋蓝藻，如 Picosynechococcus sp.（以前称为 Synechococcus sp.）PCC 7002，是一种很有前途的底盘，可以用于生产环境负担低的商品化学品。蓝藻的遗传工程传统上使用抗生素抗性标记。然而，抗生素抗性标记的有限可用性是高度多基因工程菌株工程的一个问题。尽管已经为 PCC 7002 开发了几种无标记遗传操作方法，但由于宿主菌株中基因缺失的要求，它们往往缺乏通用性。为了在 Synechococcus sp. 中实现无标记转化，而无需宿主菌株的要求，本研究开发了一种方法，该方法暂时将突变的苯丙氨酸-tRNA 合成酶基因（pheS）引入基因组中进行反选择。研究了导致 PCC 7002 对苯丙氨酸类似物对氯苯丙氨酸高度敏感的 PheS 中的氨基酸取代，并确定 T261A 和 A303G 的组合是最合适的突变。基于突变的 PheS 的选择用于 PCC 7002 中 nblA 基因的无标记敲除。此外，使用反选择策略将包含大肠杆菌 lldD 和 lldP 基因的遗传构建体引入 ldhA 基因位点，从而产生无标记重组菌株。该方法的可重复性通过双无标记敲入重组菌株得到证明，表明它将成为蓝藻多基因工程菌株的有力工具。
