State Key Laboratory of Agrobiotechnology and College of Biological Sciences, China Agricultural University, Beijing, 100193, China.
State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China.
Appl Microbiol Biotechnol. 2022 Mar;106(5-6):2191-2205. doi: 10.1007/s00253-022-11854-w. Epub 2022 Mar 8.
Avermectins (AVEs) are economically potent anthelmintic agents produced by Streptomyces avermitilis. Among eight AVE components, B1a exhibits the highest insecticidal activity. The purpose of this study was to enhance B1a production, particularly in the high-yielding industrial strain A229, by a combination strategy involving the following steps. (i) aveC gene was engineered to increase B1a:B2a ratio. Three aveC variants (aveC2m, aveC5m, and aveC8m, respectively encoding two, five, and eight amino acid mutations) were synthesized by fusion PCR. B1a:B2a ratio in A229 derivative having kasOp*-controlled aveC8m reached 1.33 (B1a and B2a titers were 8120 and 6124 μg/mL). Corresponding values in A229 were 0.99 and 6447 and 6480 μg/mL. (ii) β-oxidation pathway genes fadD and fadAB were overexpressed in wild-type (WT) strain and A229 to increase supply of acyl-CoA precursors for AVE production. The resulting strains all showed increased B1a titer. Co-overexpression of pkn5p-driven fadD and fadAB in A229 led to B1a titer of 8537 μg/mL. (iii) Genes bicA and ecaA involved in cyanobacterial CO-concentrating mechanism (CCM) were introduced into WT and A229 to enhance carboxylation velocity of acetyl-CoA and propionyl-CoA carboxylases, leading to increased supply of malonyl- and methylmalonyl-CoA precursors and increased B1a titer. Co-expression of bicA and ecaA in A229 led to B1a titer of 8083 μg/mL. (iv) aveC8m, fadD-fadAB, and bicA-ecaA were co-overexpressed in A229, resulting in maximal B1a titer (9613 μg/mL; 49.1% increase relative to A229). Our findings demonstrate that the combination strategy we provided here is an efficient approach for improving B1a production in industrial strains.Key points• aveC mutation increased avermectin B1a:B2a ratio and B1a titer.• Higher levels of acyl-CoA precursors contributed to enhanced B1a production.• B1a titer in an industrial strain was increased by 49.1% via a combination strategy.
阿维菌素（AVEs）是由链霉菌属阿维链霉菌产生的具有经济潜力的抗蠕虫药物。在八种 AVE 成分中，B1a 表现出最高的杀虫活性。本研究旨在通过以下步骤的组合策略提高 B1a 的产量，特别是在高产的工业菌株 A229 中。（i）aveC 基因被工程化以增加 B1a:B2a 比率。通过融合 PCR 分别合成了三个 aveC 变体（aveC2m、aveC5m 和 aveC8m，分别编码两个、五个和八个氨基酸突变）。具有 kasOp*-控制的 aveC8m 的 A229 衍生物中的 B1a:B2a 比率达到 1.33（B1a 和 B2a 的浓度分别为 8120 和 6124μg/mL）。相应的 A229 值分别为 0.99 和 6447 和 6480μg/mL。（ii）β-氧化途径基因 fadD 和 fadAB 在野生型（WT）菌株和 A229 中过表达，以增加 AVE 产生的酰基辅酶 A 前体的供应。所有产生的菌株均显示 B1a 浓度增加。在 A229 中过表达 pkn5p 驱动的 fadD 和 fadAB 导致 B1a 浓度为 8537μg/mL。（iii）引入参与蓝细菌 CO 浓缩机制（CCM）的 bicA 和 ecaA 基因，以提高乙酰辅酶 A 和丙酰辅酶 A 羧化酶的羧化速度，从而增加丙二酰辅酶 A 和甲基丙二酰辅酶 A 前体的供应，并增加 B1a 浓度。bicA 和 ecaA 在 A229 中的共表达导致 B1a 浓度为 8083μg/mL。（iv）aveC8m、fadD-fadAB 和 bicA-ecaA 在 A229 中共表达，导致 B1a 浓度达到最大值（9613μg/mL；相对于 A229 增加了 49.1%）。我们的研究结果表明，我们提供的这种组合策略是提高工业菌株 B1a 产量的有效方法。关键点• aveC 突变增加了阿维菌素 B1a:B2a 比率和 B1a 浓度。• 更高水平的酰基辅酶 A 前体有助于提高 B1a 产量。• 通过组合策略，工业菌株中的 B1a 浓度增加了 49.1%。
