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Abstract：
BACKGROUND: Cyanobacteria have the potential to become next-generation cell factories due to their ability to use CO2, light and inorganic nutrients to produce a range of biomolecules of commercial interest. Synechococcus elongatus 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 TfAA10A, a lytic polysaccharide monooxygenase from the Gram-positive bacterium Thermobifida fusca.
RESULTS: Two variations of TfAA10A were successfully expressed in S. elongatus UTEX 2973: One containing the native N-terminal, Sec-targeted, signal peptide and a second with a Tat-targeted signal peptide from the Escherichia coli trimethylamine-N-oxide reductase (TorA). Although the TorA signal peptide correctly targeted the protein to the plasma membrane, the majority of the TorA-TfAA10A 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 TfAA10A into the medium with virtually no protein being found in the cytosol, plasma membrane or periplasm. TfAA10A 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-1 based on densitometric analysis. To our knowledge, this is the highest secretion yield ever registered in cyanobacteria.
CONCLUSIONS: We have shown for the first time high-titer expression and secretion of an industrially relevant and catalytically active enzyme in S. elongatus 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.
RESULTS: Two variations of TfAA10A were successfully expressed in S. elongatus UTEX 2973: One containing the native N-terminal, Sec-targeted, signal peptide and a second with a Tat-targeted signal peptide from the Escherichia coli trimethylamine-N-oxide reductase (TorA). Although the TorA signal peptide correctly targeted the protein to the plasma membrane, the majority of the TorA-TfAA10A 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 TfAA10A into the medium with virtually no protein being found in the cytosol, plasma membrane or periplasm. TfAA10A 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-1 based on densitometric analysis. To our knowledge, this is the highest secretion yield ever registered in cyanobacteria.
CONCLUSIONS: We have shown for the first time high-titer expression and secretion of an industrially relevant and catalytically active enzyme in S. elongatus 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.
摘要：
背景：蓝细菌具有成为下一代细胞工厂的潜力，因为它们能够利用CO2，光和无机营养素生产一系列具有商业价值的生物分子。特别是长毛球藻UTEX2973，是一个快速增长的，基因可处理，蓝细菌作为一种潜在的生物技术底盘而受到关注。为了建立这种独特的菌株作为异源蛋白质生产的宿主，我们旨在证明工业相关的TfAA10A的表达和分泌，来自革兰氏阳性细菌Thermobifidafusca的裂解多糖单加氧酶。
结果：TfAA10A的两种变异在长毛链球菌UTEX2973中成功表达：一种含有天然N端，Sec-targeted,信号肽和第二个具有来自大肠杆菌三甲胺-N-氧化物还原酶（TorA）的Tat靶向信号肽。尽管TorA信号肽正确地将蛋白质靶向质膜，大多数TorA-TfAA10A在质膜中被发现未加工，一小部分成熟蛋白最终易位到周质。天然Sec信号肽允许TfAA10A有效分泌到培养基中，在细胞质中几乎没有发现蛋白质。质膜或周质。TfAA10A被证明在模型底物磷酸溶胀纤维素上是正确切割和有活性的。此外,表达和分泌对细胞生长影响较小。根据光密度分析，分泌产量估计为779±40µgL-1。据我们所知,这是蓝藻有史以来最高的分泌物产量。
结论:我们首次显示了在延伸链球菌UTEX2973中具有工业相关和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在生物修复和生物催化领域开发新的和可持续的应用具有重要意义。
结果：TfAA10A的两种变异在长毛链球菌UTEX2973中成功表达：一种含有天然N端，Sec-targeted,信号肽和第二个具有来自大肠杆菌三甲胺-N-氧化物还原酶（TorA）的Tat靶向信号肽。尽管TorA信号肽正确地将蛋白质靶向质膜，大多数TorA-TfAA10A在质膜中被发现未加工，一小部分成熟蛋白最终易位到周质。天然Sec信号肽允许TfAA10A有效分泌到培养基中，在细胞质中几乎没有发现蛋白质。质膜或周质。TfAA10A被证明在模型底物磷酸溶胀纤维素上是正确切割和有活性的。此外,表达和分泌对细胞生长影响较小。根据光密度分析，分泌产量估计为779±40µgL-1。据我们所知,这是蓝藻有史以来最高的分泌物产量。
结论:我们首次显示了在延伸链球菌UTEX2973中具有工业相关和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在生物修复和生物催化领域开发新的和可持续的应用具有重要意义。
