Russo D A, Zedler J A Z, Wittmann D N, Möllers B, Singh R K, Batth T S, van Oort B, Olsen J V, Bjerrum M J, Jensen P E
1Copenhagen Plant Science Centre, Department of Plant and Environmental Sciences, University of Copenhagen, Frederiksberg C, Denmark.
2Department of Geosciences and Natural Resource Management, University of Copenhagen, Frederiksberg C, Denmark.
Biotechnol Biofuels. 2019 Apr 1;12:74. doi: 10.1186/s13068-019-1416-9. eCollection 2019.
Cyanobacteria have the potential to become next-generation cell factories due to their ability to use CO, light and inorganic nutrients to produce a range of biomolecules of commercial interest. UTEX 2973, in particular, is a fast-growing, genetically tractable, cyanobacterium that has garnered attention as a potential biotechnological chassis. To establish this unique strain as a host for heterologous protein production, we aimed to demonstrate expression and secretion of the industrially relevant AA10A, a lytic polysaccharide monooxygenase from the Gram-positive bacterium
Two variations of AA10A were successfully expressed in UTEX 2973: One containing the native N-terminal, Sec-targeted, signal peptide and a second with a Tat-targeted signal peptide from the trimethylamine--oxide reductase (TorA). Although the TorA signal peptide correctly targeted the protein to the plasma membrane, the majority of the TorA-AA10A was found unprocessed in the plasma membrane with a small fraction of the mature protein ultimately translocated to the periplasm. The native Sec signal peptide allowed for efficient secretion of AA10A into the medium with virtually no protein being found in the cytosol, plasma membrane or periplasm. AA10A was demonstrated to be correctly cleaved and active on the model substrate phosphoric acid swollen cellulose. Additionally, expression and secretion only had a minor impact on cell growth. The secretion yield was estimated at 779 ± 40 µg L based on densitometric analysis. To our knowledge, this is the highest secretion yield ever registered in cyanobacteria.
We have shown for the first time high-titer expression and secretion of an industrially relevant and catalytically active enzyme in UTEX 2973. This proof-of-concept study will be valuable for the development of novel and sustainable applications in the fields of bioremediation and biocatalysis.
蓝藻有潜力成为下一代细胞工厂，因为它们能够利用二氧化碳、光和无机营养物来生产一系列具有商业价值的生物分子。特别是UTEX 2973，它是一种生长迅速、遗传上易于操作的蓝藻，作为一种潜在的生物技术底盘已受到关注。为了将这种独特的菌株确立为异源蛋白生产的宿主，我们旨在证明来自革兰氏阳性细菌的工业相关AA10A（一种裂解多糖单加氧酶）的表达和分泌。
AA10A的两种变体在UTEX 2973中成功表达：一种含有天然的N端、Sec靶向信号肽，另一种含有来自三甲胺氧化物还原酶（TorA）的Tat靶向信号肽。尽管TorA信号肽将蛋白质正确靶向到质膜，但发现大多数TorA-AA10A在质膜中未加工，只有一小部分成熟蛋白最终转运到周质。天然的Sec信号肽允许AA10A有效分泌到培养基中，在细胞质、质膜或周质中几乎没有发现蛋白质。已证明AA10A在模型底物磷酸膨胀纤维素上能正确切割并具有活性。此外，表达和分泌对细胞生长只有轻微影响。基于密度分析，分泌产量估计为779±40μg/L。据我们所知，这是蓝藻中记录到的最高分泌产量。
我们首次在UTEX 2973中展示了一种工业相关且具有催化活性的酶的高滴度表达和分泌。这项概念验证研究对于生物修复和生物催化领域新型可持续应用的开发将具有重要价值。
