School of Biotechnology, Jawaharlal Nehru University, New Delhi, India.
Biotechnology Core Laboratory, National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health, Bethesda, MD, 20892, USA.
Microb Cell Fact. 2020 Jul 23;19(1):148. doi: 10.1186/s12934-020-01407-z.
The expression of recombinant proteins triggers a stress response which downregulates key metabolic pathway genes leading to a decline in cellular health and feedback inhibition of both growth and protein expression. Instead of individually upregulating these downregulated genes or improving transcription rates by better vector design, an innovative strategy would be to block this stress response thereby ensuring a sustained level of protein expression.
We postulated that the genes which are commonly up-regulated post induction may play the role of signalling messengers in mounting the cellular stress response. We identified those genes which have no known downstream regulatees and created knock outs which were then tested for GFP expression. Many of these knock outs showed significantly higher expression levels which was also sustained for longer periods. The highest product yield (Y) was observed in a BW25113ΔcysJ knock out (Y 0.57) and BW25113ΔelaA (Y 0.49), whereas the Y of the control W3110 strain was 0.08 and BW25113 was 0.16. Double knock out combinations were then created from the ten best performing single knock outs leading to a further enhancement in expression levels. Out of 45 double knock outs created, BW25113ΔelaAΔyhbC (Y 0.7) and BW25113ΔcysJΔyhbC (Y 0.64) showed the highest increase in product yield compared to the single gene mutant strains. We confirmed the improved performance of these knock outs by testing and obtaining higher levels of recombinant asparaginase expression, a system better suited for analysing sustained expression since it gets exported to the extracellular medium.
Creating key knock outs to block the CSR and enhance expression is a radically different strategy that can be synergistically combined with traditional methods of improving protein yields thus helping in the design of superior host platforms for protein expression.
表达重组蛋白会引发应激反应，从而下调关键代谢途径基因，导致细胞健康状况下降，并反馈抑制生长和蛋白表达。与其单独上调这些下调的基因，或者通过更好的载体设计来提高转录率，不如采用一种创新策略来阻断这种应激反应，从而确保蛋白表达的持续水平。
我们推测，诱导后普遍上调的基因可能在引发细胞应激反应中发挥信号信使的作用。我们鉴定出那些没有已知下游调控因子的基因，并创建了敲除突变体，然后对其 GFP 表达进行测试。许多敲除突变体表现出显著更高的表达水平，并且持续时间更长。在 BW25113ΔcysJ 敲除突变体（Y0.57）和 BW25113ΔelaA（Y0.49）中观察到最高的产物产率（Y），而对照 W3110 菌株的 Y 为 0.08，BW25113 的 Y 为 0.16。然后从表现最好的十个单敲除突变体中创建双敲除突变体组合，从而进一步提高表达水平。在创建的 45 个双敲除突变体中，BW25113ΔelaAΔyhbC（Y0.7）和 BW25113ΔcysJΔyhbC（Y0.64）与单基因突变株相比，产物产率的提高幅度最大。我们通过测试和获得更高水平的重组天冬酰胺酶表达来验证这些敲除突变体的改进性能，该系统更适合分析持续表达，因为它会被输出到细胞外培养基中。
创建关键敲除突变体来阻断 CSR 并增强表达是一种截然不同的策略，可以与提高蛋白产量的传统方法协同结合，从而有助于设计用于蛋白表达的优越宿主平台。
