Disruption of SMC-related genes promotes recombinant cholesterol esterase production in Burkholderia stabilis,Applied Microbiology and Biotechnology

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Disruption of SMC-related genes promotes recombinant cholesterol esterase production in Burkholderia stabilis
Applied Microbiology and Biotechnology ( IF 3.9 ) Pub Date : 2022-11-18 , DOI: 10.1007/s00253-022-12277-3
Kenji Konishi _{1,

2} , Yoshiaki Yasutake _{3,

4} , Shuji Muramatsu ₁ , Satomi Murata ₁ , Keitaro Yoshida ₃ , Koji Ishiya ₃ , Sachiyo Aburatani ₃ , Shin-Ichi Sakasegawa ₁ , Tomohiro Tamura _{2,

3}

Affiliation

Abstract

Burkholderia stabilis strain FERMP-21014 secretes cholesterol esterase (BsChe), which is used in clinical settings to determine serum cholesterol levels. Previously, we constructed an expression plasmid with an endogenous constitutive promoter to enable the production of recombinant BsChe. In this study, we obtained one mutant strain with 13.1-fold higher BsChe activity than the wild type, using N-methyl-N′-nitro-N-nitrosoguanidine as a mutagen. DNA-sequencing analysis revealed that the strain had lost chromosome 3 (∆Chr3), suggesting that the genes hindering BsChe production may be encoded on Chr3. We also identified common mutations in the functionally unknown BSFP_068720/30 genes in the top 10 active strains generated during transposon mutagenesis. As BSFP_068720/30/40 comprised an operon on Chr3, we created the BSFP_068720/30/40 disruption mutant and confirmed that each disruption mutant containing the expression plasmid exhibited ~ 16.1-fold higher BsChe activity than the wild type. Quantitative PCR showed that each disruption mutant and ΔChr3 had a ~ 9.4-fold higher plasmid copy number than the wild type. Structural prediction models indicate that BSFP_068730/40 is structurally homologous to the structural maintenance of chromosomes (SMC) protein MukBE, which is responsible for chromosome segregation during cell division. Conversely, BSFP_068720/30/40 disruption did not lead to a Chr3 drop-out. These results imply that BSFP_068720/30/40 is not a SMC protein but is involved in destabilizing foreign plasmids to prevent the influx of genetic information from the environment. In conclusion, the disruption of BSFP_068720/30/40 improved plasmid stability and copy number, resulting in exceptionally high BsChe production.

Key points

• Disruption of BSFP_068720/30/40 enabled mass production of Burkholderia Che/Lip.

• BSFP_068730/40 is an SMC protein homolog not involved in chromosome retention.

• BSFP_068720/30/40 is likely responsible for the exclusion of exogenous plasmids.

中文翻译：

SMC 相关基因的破坏促进稳定伯克霍尔德氏菌中重组胆固醇酯酶的产生

摘要

稳定伯克霍尔德菌菌株 FERMP-21014 分泌胆固醇酯酶 (BsChe)，在临床环境中用于确定血清胆固醇水平。以前，我们构建了一个具有内源组成型启动子的表达质粒，以实现重组 BsChe 的生产。在这项研究中，我们使用 N-甲基-N'-硝基-N-亚硝基胍作为诱变剂，获得了一种 BsChe 活性比野生型高 13.1 倍的突变菌株。DNA 测序分析显示该菌株丢失了 3 号染色体 (ΔChr3)，表明阻碍 BsChe 生产的基因可能编码在 Chr3 上。我们还确定了在转座子诱变过程中产生的前 10 个活性菌株中功能未知的 BSFP_068720/30 基因的常见突变。由于 BSFP_068720/30/40 在 Chr3 上包含一个操纵子，我们创建了 BSFP_068720/30/40 中断突变体，并确认每个包含表达质粒的中断突变体表现出比野生型高 16.1 倍的 BsChe 活性。定量 PCR 显示每个破坏突变体和 ΔChr3 的质粒拷贝数比野生型高约 9.4 倍。结构预测模型表明 BSFP_068730/40 在结构上与染色体结构维持 (SMC) 蛋白 MukBE 同源，后者负责细胞分裂过程中的染色体分离。相反，BSFP_068720/30/40 中断并没有导致 Chr3 丢失。这些结果表明 BSFP_068720/30/40 不是 SMC 蛋白，而是参与破坏外来质粒的稳定性以防止遗传信息从环境中流入。综上所述，