Cloning and expression of Burkholderia polyyne biosynthetic gene clusters in Paraburkholderia hosts provides a strategy for biopesticide development.
Cloning and expression of Burkholderia polyyne biosynthetic gene clusters in Paraburkholderia hosts provides a strategy for biopesticide development.
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DOI：
10.1111/1751-7915.14106
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发表时间：
2022-10
影响因子：
5.7
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中科院分区：
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伯克霍尔德氏菌具有作为生物防治剂的潜力，因为它们编码用于一系列抗菌代谢物的不同生物合成基因簇（BGC）。鉴于与伯克霍尔德菌属物种相关的机会致病性，在非致病性宿主中异源BGC表达是构建安全生物防治菌株的策略。我们构建了具有酵母复制起点2 μ和URA 3选择标记的酵母适应性伯克霍尔德氏菌-埃希氏菌穿梭载体（pMLBAD_yeast），并使用酿酒酵母的体内重组能力对其进行了优化以克隆BGC。将两种多炔伯克霍尔德氏菌BGC，cepacin（13 kb）和caryoynencin（11 kb）作为三个重叠片段进行PCR扩增，克隆到pMLBAD_yeast中pBAD阿拉伯糖启动子的下游，并动员到伯克霍尔德氏菌和副伯克霍尔德氏菌异源宿主中。携带异源多炔构建体的Paraburkholderia phytofirmans显示出与天然多炔生产者类似的针对多种真菌和细菌植物病原体的体外生物活性。还鉴定了13种在30°C下比在37°C下优先生长的副伯克霍尔德氏菌菌株，其中4种适合遗传操作和异源表达核融合蛋白构建体。伯克霍尔德氏菌生物合成基因簇在副伯克霍尔德氏菌内的克隆和成功异源表达，在37°C下限制生长，为工程化非致病性生物防治菌株开辟了途径。
Burkholderia have potential as biocontrol agents because they encode diverse biosynthetic gene clusters (BGCs) for a range of antimicrobial metabolites. Given the opportunistic pathogenicity associated with Burkholderia species, heterologous BGC expression within non‐pathogenic hosts is a strategy to construct safe biocontrol strains. We constructed a yeast‐adapted Burkholderia‐Escherichia shuttle vector (pMLBAD_yeast) with a yeast replication origin 2 μ and URA3 selection marker and optimised it for cloning BGCs using the in vivo recombination ability of Saccharomyces cerevisiae. Two Burkholderia polyyne BGCs, cepacin (13 kb) and caryoynencin (11 kb), were PCR‐amplified as three overlapping fragments, cloned downstream of the pBAD arabinose promoter in pMLBAD_yeast and mobilised into Burkholderia and Paraburkholderia heterologous hosts. Paraburkholderia phytofirmans carrying the heterologous polyyne constructs displayed in vitro bioactivity against a variety of fungal and bacterial plant pathogens similar to the native polyyne producers. Thirteen Paraburkholderia strains with preferential growth at 30°C compared with 37°C were also identified, and four of these were amenable to genetic manipulation and heterologous expression of the caryoynencin construct. The cloning and successful heterologous expression of Burkholderia biosynthetic gene clusters within Paraburkholderia with restricted growth at 37°C opens avenues for engineering non‐pathogenic biocontrol strains.