关键词: Bacterial cellulose Enterobacter sp. FY-07 Fructose-1,6-bisphosphatase I Glycerol λ-Red recombination

Mesh : Enterobacter / metabolism genetics Cellulose / biosynthesis metabolism Metabolic Engineering / methods Glucosyltransferases / genetics metabolism Bacterial Proteins / genetics metabolism Glycerol / metabolism

来  源:   DOI:10.1016/j.ijbiomac.2024.133755

Abstract:
Bacterial cellulose (BC) is an extracellular polysaccharide with myriad unique properties, such as high purity, water-holding capacity and biocompatibility, making it attractive in materials science. However, genetic engineering techniques for BC-producing microorganisms are rare. Herein, the electroporation-based gene transformation and the λ Red-mediated gene knockout method with a nearly 100 % recombination efficiency were established in the fast-growing and BC hyperproducer Enterobacter sp. FY-07. This genetic manipulation toolkit was validated by inactivating the protein subunit BcsA in the cellulose synthase complex. Subsequently, the inducible BC-producing strains from glycerol were constructed through inducible expression of the key gene fbp in the gluconeogenesis pathway, which recovered >80 % of the BC production. Finally, the BC properties analysis results indicated that the induced-synthesized BC pellicles were looser, more porous and reduced crystallinity, which could further broaden the application prospects of BC. To our best knowledge, this is the first attempt to construct the completely inducible BC-producing strains. Our work paves the way for increasing BC productivity by metabolic engineering and broadens the available fabrication methods for BC-based advanced functional materials.
摘要:
细菌纤维素(BC)是一种具有许多独特特性的细胞外多糖,如高纯度,保水能力和生物相容性,使其在材料科学中具有吸引力。然而,用于产生BC的微生物的基因工程技术很少见。在这里,在快速生长和BC高生产者肠杆菌中建立了基于电穿孔的基因转化和λRed介导的基因敲除方法,重组效率接近100%。07财年。通过使纤维素合酶复合物中的蛋白质亚基BcsA失活来验证该遗传操作工具包。随后,通过糖异生途径中关键基因fbp的诱导表达,构建了甘油诱导型BC产生菌株,回收了>80%的BC产量。最后,BC性质分析结果表明,诱导合成的BC膜较松散,更多孔和降低结晶度,可以进一步拓宽BC的应用前景。据我们所知,这是构建完全诱导型BC产生菌株的首次尝试。我们的工作为通过代谢工程提高BC生产率铺平了道路,并拓宽了基于BC的先进功能材料的可用制造方法。
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