Molecular Biotechnology ( IF 2.4 ) Pub Date : 2022-06-14 , DOI: 10.1007/s12033-022-00520-1 Vimmy Khetrapal 1 , Patrick Dussault 1 , Liangcheng Du 1
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Fatty dicarboxylic acids (FDCA) are useful as starting materials or components for plastics, polyesters, nylons, and fragrances. Most of the commercially available FDCA contain an even number of carbons, and there remain few sustainable methods for production of FDCA with an odd number of carbons (o-FDCA). In this work, we explored a novel biosynthetic route to unsaturated o-FDCA. The approach was based on genetic modifications of hsaf pks-nrps, encoding a hybrid polyketide synthase–nonribosomal peptide synthetase (PKS–NRPS) in Lysobacter enzymogenes, an environmental bacterium emerging as a new biocontrol agent. This single-module PKS–NRPS catalyzes the biosynthesis of lysobacterene A, a polyene-containing precursor of the antifungal natural product Heat-Stable Antifungal Factor (HSAF). We genetically removed the NRPS module from this gene and generated a new strain of L. enzymogenes, in which the PKS module was fused to the thioesterase domain of hsaf pks–nrps. The chimeric gene was verified by DNA sequencing, and its expression in L. enzymogenes was confirmed by reverse transcription-polymerase chain reaction (RT-PCR). The total fatty acids were extracted, esterified, and analyzed by GC–MS. The results showed that the engineered strain produced new fatty acids that were absent in the wild type. The main product was identified as hepta-2,4-dienedioic acid, an unsaturated o-FDCA. This work sets the foundation to explore a sustainable and environment-friendly approach toward unsaturated o-FDCA, which could be used as precursors for new compounds that can serve as versatile feedstock for industrial materials.
Graphical Abstract
中文翻译：
通过改造溶菌酶原中的 HSAF 生物合成基因生物合成奇数碳不饱和脂肪酸二羧酸
脂肪二羧酸 (FDCA) 可用作塑料、聚酯、尼龙和香料的起始材料或组分。大多数市售的 FDCA 含有偶数碳，并且几乎没有可持续的生产奇数碳 FDCA (o-FDCA) 的方法。在这项工作中，我们探索了一种新的生物合成不饱和 o-FDCA 的途径。该方法基于 hsaf pks-nrps 的遗传修饰，编码溶菌酶基因中的杂合聚酮化合物合酶-非核糖体肽合酶 (PKS-NRPS)，一种环境细菌，作为一种新的生物防治剂出现。这种单模块 PKS-NRPS 催化溶菌素 A 的生物合成，溶菌素 A 是抗真菌天然产物热稳定抗真菌因子 (HSAF) 的含多烯前体。我们从该基因中遗传去除了 NRPS 模块，并产生了一种新的L. enzymogenes菌株，其中 PKS 模块与hsaf pks-nrps的硫酯酶结构域融合。嵌合基因通过 DNA 测序验证，其在L. enzymogenes中的表达通过逆转录聚合酶链反应（RT-PCR）证实。通过 GC-MS 提取、酯化和分析总脂肪酸。结果表明，工程菌株产生了野生型中不存在的新脂肪酸。主要产物被鉴定为七-2,4-二烯二酸，一种不饱和的o-FDCA。这项工作为探索可持续和环境友好的不饱和 o-FDCA 方法奠定了基础，该方法可用作新化合物的前体，可用作工业材料的多功能原料。
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