Biofuels Institute, School of Emergency Management, School of Environment and Safety Engineering, Jiangsu University, Zhenjiang 212013, China.
Biofuels Institute, School of Emergency Management, School of Environment and Safety Engineering, Jiangsu University, Zhenjiang 212013, China.
Microbiol Res. 2023 Sep;274:127416. doi: 10.1016/j.micres.2023.127416. Epub 2023 Jun 2.
Laccases are powerful multi-copper oxidoreductases that have wide applicability as "green" biocatalysts in biotechnological, bioremediation, and industrial applications. Sustainable production of large amounts of functional laccases from original sources is limited by low yields, difficulties in purification, slow growth of the organisms, and high cost of production. Harnessing the full potential of these versatile biocatalysts will require the development of efficient heterologous systems that allow high-yield, scalable, and cost-effective production. We previously cloned a temperature- and pH-stable laccase from Bacillus ligniniphilus L1 (L1-lacc) that demonstrated remarkable activity in the oxidation of lignin and delignification for bioethanol production. However, L1-lacc is limited by low enzyme yields in both the source organism and heterologous systems. Here, to improve production yields and lower the cost of production, we optimized the recombinant E. coli BL21 strain for high-level production of L1-lacc. Several culture medium components and fermentation parameters were optimized using one-factor-at-a-time (OFAT) and Plackett-Burman design (PBD) to screen for important factors that were then optimized using response surface methodology (RSM) and an orthogonal design. The optimized medium composition had compound nitrogen (15.6 g/L), glucose (21.5 g/L), KHPO (0.15 g/L), MgSO (1 g/L), and NaCl (7.5 g/L), which allowed a 3.3-fold yield improvement while subsequent optimization of eight fermentation parameters achieved further improvements to a final volumetric activity titer of 5.94 U/mL in 24 h. This represents a 7-fold yield increase compared to the initial medium and fermentation conditions. This work presents statistically guided optimization strategies for improving heterologous production of a bacterial laccase that resulted in a high-yielding, cost-efficient production system for an enzyme with promising applications in lignin valorization, biomass processing, and generation of novel composite thermoplastics.
漆酶是一种强大的多铜氧化还原酶，作为“绿色”生物催化剂，在生物技术、生物修复和工业应用中具有广泛的适用性。从原始来源可持续生产大量功能漆酶受到产量低、纯化困难、生物生长缓慢和生产成本高的限制。要充分发挥这些多功能生物催化剂的潜力，需要开发高效的异源系统，以实现高产、可扩展和具有成本效益的生产。我们之前从木质素分解菌 Bacillus ligniniphilus L1 中克隆了一种温度和 pH 稳定的漆酶（L1-lacc），该漆酶在木质素氧化和木质素脱除以生产生物乙醇方面表现出显著的活性。然而，L1-lacc 的产量在原始生物和异源系统中都受到限制。在这里，为了提高产量并降低生产成本，我们对用于 L1-lacc 高产的重组大肠杆菌 BL21 菌株进行了优化。使用单因素实验（OFAT）和 Plackett-Burman 设计（PBD）优化了几种培养基成分和发酵参数，以筛选出重要因素，然后使用响应面法（RSM）和正交设计进行优化。优化后的培养基组成为复合氮（15.6 g/L）、葡萄糖（21.5 g/L）、KHPO（0.15 g/L）、MgSO（1 g/L）和 NaCl（7.5 g/L），产量提高了 3.3 倍，随后对 8 个发酵参数进行优化，最终在 24 小时内实现了 5.94 U/mL 的比体积酶活。与初始培养基和发酵条件相比，这代表了 7 倍的产量增加。这项工作提出了一种用于提高细菌漆酶异源生产的统计引导优化策略，该策略为具有广阔应用前景的木质素增值、生物质加工和新型复合热塑性塑料生成的酶提供了高产、经济高效的生产系统。
