Abstract

Milbemycins are used commercially as insect repellents and acaricides; however, their high cost remains a significant challenge to commercial production. Hence, improving the titer of milbemycins for commercial application is an urgent priority. The present study aimed to effectively increase the titer of milbemycins using a combination of genome re-sequencing and metabolic engineering. First, 133 mutation sites were identified by genome re-sequencing in the mutagenized high-yielding strain BC04. Among them, three modifiable candidate genes (sbi_04868 encoding citrate synthase, sbi_06921 and sbi_06922 encoding alpha and beta subunits of acetyl-CoA carboxylase, and sbi_04683 encoding carbon uptake system gluconate transporter) related to primary metabolism were screened and identified. Next, the DNase-deactivated Cpf1–based integrative CRISPRi system was used in S. bingchenggensis to downregulate the transcription level of gene sbi_04868. Then, overexpression of the potential targets sbi_06921-06922 and sbi_04683 further facilitated milbemycin biosynthesis. Finally, those candidate genes were engineered to produce strains with combinatorial downregulation and overexpression, which resulted in the titer of milbemycin A3/A4 increased by 27.6% to 3164.5 mg/L. Our research not only identified three genes in S. bingchenggensis that are closely related to the production of milbemycins, but also offered an efficient engineering strategy to improve the titer of milbemycins using genome re-sequencing.

Key points

• We compared the genomes of two strains with different titers of milbemycins.

• We found three genes belonging to primary metabolism influence milbemycin production.

• We improved titer of milbemycins by a combinatorial engineering of three targets.

中文翻译：

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炳城链霉菌米尔贝霉素效价改善的主要代谢途径工程

摘要

米尔贝霉素在商业上用作驱虫剂和杀螨剂；然而，它们的高成本仍然是商业生产的重大挑战。因此，提高milbemycins的滴度以用于商业应用是当务之急。本研究旨在通过基因组重测序和代谢工程相结合，有效提高米尔贝霉素的效价。首先，在诱变的高产菌株BC04中通过基因组重新测序鉴定了133个突变位点。其中，三个修改候选基因（sbi_04868编码柠檬酸合酶，sbi_06921和sbi_06922乙酰辅酶A羧化酶的编码α和β亚基，和sbi_04683筛选和鉴定了与初级代谢有关的编码碳吸收系统（葡萄糖吸收转运蛋白）。接着，在使用了DNase的失活的基于Cpf1整合型CRISPRi系统S. bingchenggensis下调基因的转录水平sbi_04868。然后，潜在目标表达sbi_06921 - 06922和sbi_04683进一步促进美贝霉素生物合成。最后，这些候选基因经过工程改造，产生了具有组合下调和过度表达的菌株，从而导致米尔贝霉素A3 / A4的效价提高了27.6％，达到3164.5 mg / L。我们的研究不仅鉴定了冰城链球菌的三个基因 与米尔贝霉素的生产密切相关，但也提供了一种有效的工程策略，可通过基因组重测序提高米尔贝霉素的效价。

关键点

•我们比较了具有不同滴度的米尔倍霉素的两种菌株的基因组。

•我们发现属于初级代谢的三个基因影响米尔倍霉素的生产。

•我们通过三个靶标的组合工程改进了米尔贝霉素的效价。