Department of Chemical and Biomolecular Engineering, University of California, Berkeley, CA, 94720, USA.
Department of Bioengineering, University of California, Berkeley, CA, 94720, USA.
Microb Cell Fact. 2023 Apr 12;22(1):69. doi: 10.1186/s12934-023-02064-8.
Intracellular biomacromolecules, such as industrial enzymes and biopolymers, represent an important class of bio-derived products obtained from bacterial hosts. A common key step in the downstream separation of these biomolecules is lysis of the bacterial cell wall to effect release of cytoplasmic contents. Cell lysis is typically achieved either through mechanical disruption or reagent-based methods, which introduce issues of energy demand, material needs, high costs, and scaling problems. Osmolysis, a cell lysis method that relies on hypoosmotic downshock upon resuspension of cells in distilled water, has been applied for bioseparation of intracellular products from extreme halophiles and mammalian cells. However, most industrial bacterial strains are non-halotolerant and relatively resistant to hypoosmotic cell lysis.
To overcome this limitation, we developed two strategies to increase the susceptibility of non-halotolerant hosts to osmolysis using Cupriavidus necator, a strain often used in electromicrobial production, as a prototypical strain. In one strategy, C. necator was evolved to increase its halotolerance from 1.5% to 3.25% (w/v) NaCl through adaptive laboratory evolution, and genes potentially responsible for this phenotypic change were identified by whole genome sequencing. The evolved halotolerant strain experienced an osmolytic efficiency of 47% in distilled water following growth in 3% (w/v) NaCl. In a second strategy, the cells were made susceptible to osmolysis by knocking out the large-conductance mechanosensitive channel (mscL) gene in C. necator. When these strategies were combined by knocking out the mscL gene from the evolved halotolerant strain, greater than 90% osmolytic efficiency was observed upon osmotic downshock. A modified version of this strategy was applied to E. coli BL21 by deleting the mscL and mscS (small-conductance mechanosensitive channel) genes. When grown in medium with 4% NaCl and subsequently resuspended in distilled water, this engineered strain experienced 75% cell lysis, although decreases in cell growth rate due to higher salt concentrations were observed.
Our strategy is shown to be a simple and effective way to lyse cells for the purification of intracellular biomacromolecules and may be applicable in many bacteria used for bioproduction.
细胞内生物大分子，如工业酶和生物聚合物，是从细菌宿主中获得的一类重要的生物衍生产品。这些生物分子下游分离的一个共同关键步骤是裂解细菌细胞壁，以释放细胞质内容物。细胞裂解通常通过机械破坏或基于试剂的方法来实现，这会带来能量需求、材料需求、高成本和规模化问题。渗透压休克法是一种通过将细胞重新悬浮在蒸馏水中来实现的细胞裂解方法，已应用于从极端嗜盐菌和哺乳动物细胞中分离细胞内产物。然而，大多数工业细菌菌株不耐盐，对低渗细胞裂解相对具有抗性。
为了克服这一限制，我们使用 Cupriavidus necator 开发了两种策略来增加非耐盐宿主对渗透压休克的敏感性，Cupriavidus necator 是一种常用于电微生物生产的菌株，用作典型菌株。在一种策略中，通过适应性实验室进化将 C. necator 的耐盐性从 1.5%（w/v）NaCl 提高到 3.25%（w/v）NaCl，通过全基因组测序鉴定了可能导致这种表型变化的基因。在 3%（w/v）NaCl 中生长后，进化后的耐盐菌株在蒸馏水中的渗透压休克效率为 47%。在第二种策略中，通过敲除 C. necator 中的大电导机械敏感通道（mscL）基因使细胞对渗透压休克敏感。当将这两种策略结合起来，从进化后的耐盐菌株中敲除 mscL 基因时，渗透压休克后观察到超过 90%的渗透压休克效率。通过敲除 mscL 和 mscS（小电导机械敏感通道）基因对 E. coli BL21 应用了这种策略的修改版本。当在含有 4%NaCl 的培养基中生长并随后重新悬浮在蒸馏水中时，该工程菌株经历了 75%的细胞裂解，尽管由于盐浓度较高而观察到细胞生长速率下降。
我们的策略被证明是一种简单有效的裂解细胞的方法，用于纯化细胞内生物大分子，并且可能适用于许多用于生物生产的细菌。
