核糖体结合位点间隔序列的长度控制枯草芽孢杆菌对细胞内和分泌蛋白的产量。,Microbial Cell Factories

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核糖体结合位点间隔序列的长度控制枯草芽孢杆菌对细胞内和分泌蛋白的产量。
Microbial Cell Factories ( IF 4.3 ) Pub Date : 2020-07-29 , DOI: 10.1186/s12934-020-01404-2
Kristina Volkenborn _{1,

2} , Laura Kuschmierz _{1,

3} , Nuka Benz _{1,

2} , Patrick Lenz ₁ , Andreas Knapp _{1,

2} , Karl-Erich Jaeger _{1,

2,

4}

Affiliation

枯草芽孢杆菌广泛用于重组蛋白的工业生产，这主要是由于其分泌能力高，但是只有消除瓶颈，才能实现更高的产量。为此，关键的过程是翻译起始，其发生在围绕Shine Dalgarno序列的核糖体结合位点，靶基因的起始密码子和之间的短间隔序列。在这里，我们研究了体内不同间隔区序列长度对不同细胞内和细胞外蛋白产量的影响。含有强组成型启动子PHpaII和最佳Shine Dalgarno序列TAAGGAGG的穿梭载体pBSMul1被用作模板来构建一系列间隔长度从4到12个腺苷不等的载体。对于细胞内蛋白GFPmut3和β-葡萄糖醛酸苷酶，间隔区长度从4个核苷酸增加到7-9个核苷酸会导致产物产量逐渐增加，最高可达27倍，甚至更长的间隔区达到平台。用角质酶Cut和swollenin EXLX1测试分泌的蛋白质的产生，它们在N末端与Sec依赖性信号肽SPPel，SPEpr或SPBsn之一融合。同样，更长的间隔序列导致细胞外蛋白的产量增加多达十倍。与信号肽SPPel或SPBsn融合后，间隔长度为7-10nt的片段显示出最高的产量。值得注意的是，与SPEpr的融合导致6或7nt间隔基的产量降低了两倍，而10-12nt间隔基的产量最高。对于与SPEpr融合的两种分泌蛋白均观察到该模式，表明编码相应信号肽以用于翻译起始的核苷酸序列也起主要作用。RT qPCR证实了这一结论，仅揭示了少量的转录本。同样，可以排除推定的替代翻译起始位点的影响。我们的结果证实了靶基因的5'末端序列对于翻译起始的重要性。因此，优化生产产量可能需要筛选最佳间隔子序列长度。在分泌蛋白质的情况下，还应考虑编码信号肽的5'序列，用于Sec依赖性分泌。RT qPCR证实了这一结论，仅揭示了少量的转录本。同样，可以排除推定的替代翻译起始位点的影响。我们的结果证实了靶基因的5'末端序列对于翻译起始的重要性。因此，优化生产产量可能需要筛选最佳间隔区序列长度。在分泌蛋白质的情况下，还应考虑编码信号肽的5'序列，用于Sec依赖性分泌。RT qPCR证实了这一结论，仅揭示了少量的转录本。同样，可以排除推定的替代翻译起始位点的影响。我们的结果证实了靶基因的5'末端序列对于翻译起始的重要性。因此，优化生产产量可能需要筛选最佳间隔子序列长度。在分泌蛋白质的情况下，还应考虑编码信号肽的5'序列，用于Sec依赖性分泌。

"点击查看英文标题和摘要"

The length of ribosomal binding site spacer sequence controls the production yield for intracellular and secreted proteins by Bacillus subtilis.

Bacillus subtilis is widely used for the industrial production of recombinant proteins, mainly due to its high secretion capacity, but higher production yields can be achieved only if bottlenecks are removed. To this end, a crucial process is translation initiation which takes place at the ribosome binding site enclosing the Shine Dalgarno sequence, the start codon of the target gene and a short spacer sequence in between. Here, we have studied the effects of varying spacer sequence lengths in vivo on the production yield of different intra- and extracellular proteins. The shuttle vector pBSMul1 containing the strong constitutive promoter PHpaII and the optimal Shine Dalgarno sequence TAAGGAGG was used as a template to construct a series of vectors with spacer lengths varying from 4 to 12 adenosines. For the intracellular proteins GFPmut3 and β-glucuronidase, an increase of spacer lengths from 4 to 7–9 nucleotides resulted in a gradual increase of product yields up to 27-fold reaching a plateau for even longer spacers. The production of secreted proteins was tested with cutinase Cut and swollenin EXLX1 which were N-terminally fused to one of the Sec-dependent signal peptides SPPel, SPEpr or SPBsn. Again, longer spacer sequences resulted in up to tenfold increased yields of extracellular proteins. Fusions with signal peptides SPPel or SPBsn revealed the highest production yields with spacers of 7–10nt length. Remarkably, fusions with SPEpr resulted in a twofold lower production yield with 6 or 7nt spacers reaching a maximum with 10–12nt spacers. This pattern was observed for both secreted proteins fused to SPEpr indicating a dominant role also of the nucleotide sequence encoding the respective signal peptide for translation initiation. This conclusion was corroborated by RT qPCR revealing only slightly different amounts of transcript. Also, the effect of a putative alternative translation initiation site could be ruled out. Our results confirm the importance of the 5′ end sequence of a target gene for translation initiation. Optimizing production yields thus may require screenings for optimal spacer sequence lengths. In case of secreted proteins, the 5′ sequence encoding the signal peptide for Sec-depended secretion should also be considered.

更新日期：2020-07-29

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