随着对可持续可再生资源的需求不断增长,能够生产生物产品如生物塑料的微生物是有吸引力的。虽然许多生物生产系统在模型生物中得到了很好的研究,研究非模式生物对于扩大该领域和利用代谢上通用的菌株至关重要。这项研究的重点是沼泽红假单胞菌TIE-1,一种能够生产生物塑料的紫色非硫细菌。为了增加生物塑料产量,编码推定的调节蛋白PhaR和聚羟基链烷酸(PHA)生物合成途径的解聚酶PhaZ的基因被删除。与可能与PHA生产竞争的途径相关的基因,特别是那些与糖原产生和固氮有关的,已删除。此外,RuBisCOI型和II型基因通过噬菌体整合系统整合到TIE-1的基因组中,在这项研究中发展。我们的结果表明,当TIE-1与丁酸盐和氯化铵(NH4Cl)进行光异养生长时,phaR的缺失会增加PHA的产生。无法产生糖原或固定氮的突变体在氢和NH4Cl的光合自养生长下显示出增加的PHA产量。当RuBisCOI型和I型和II型基因过表达时,观察到PHA产生的最显著增加,在丁酸盐的光营养下五次,用氢气和NH4Cl两次,并在N2下进行两次光电营养生长。总之,将RuBisCO基因的拷贝插入TIE-1基因组是比删除竞争途径以增加TIE-1中PHA产量更有效的策略。噬菌体整合系统的成功使用为TIE-1中的合成生物学开辟了许多机会。在过去的几十年中,由于广泛使用石油衍生塑料而造成的污染给我们的星球带来了负担。自从发现可生物降解的塑料替代品以来,已经做出了一致的努力来提高他们的生物生产。多才多艺的微生物沼泽红假单胞菌TIE-1(TIE-1)是生物塑料合成的有希望的候选者,由于它能够使用多个电子源,解决温室气体CO2,并使用光作为能源。从TIE-1野生型精心设计了两类菌株,以增加聚羟基链烷酸酯(PHA)的生产,一种这样的生物塑料生产。第一组包括在PHA途径中携带phaR或phaZ基因缺失的突变体,以及那些缺乏潜在的竞争性碳和能源汇入PHA途径的人(即,糖原生物合成和固氮)。第二组包含TIE-1菌株,其过表达通过噬菌体整合系统插入的RuBisCO形式I或形式I&II基因。通过研究大量的代谢突变体和过表达菌株,我们得出结论,环境微生物TIE-1中的遗传修饰可以提高PHA的产量。当与其他方法(如反应堆设计,使用微生物聚生体,和不同的原料),TIE-1等紫色非硫细菌的遗传和代谢操作对于用PHA等生物降解塑料代替石油衍生塑料至关重要。
With the rising demand for sustainable renewable resources, microorganisms capable of producing bioproducts such as bioplastics are attractive. While many bioproduction systems are well-studied in model organisms, investigating non-model organisms is essential to expand the field and utilize metabolically versatile strains. This investigation centers on Rhodopseudomonas palustris TIE-1, a purple non-sulfur bacterium capable of producing bioplastics. To increase bioplastic production, genes encoding the putative regulatory protein PhaR and the depolymerase PhaZ of the polyhydroxyalkanoate (PHA) biosynthesis pathway were deleted. Genes associated with pathways that might compete with PHA production, specifically those linked to glycogen production and nitrogen fixation, were deleted. Additionally,
RuBisCO form I and II genes were integrated into TIE-1\'s genome by a phage integration system, developed in this study. Our results show that deletion of phaR increases PHA production when TIE-1 is grown photoheterotrophically with butyrate and ammonium chloride (NH4Cl). Mutants unable to produce glycogen or fix nitrogen show increased PHA production under photoautotrophic growth with hydrogen and NH4Cl. The most significant increase in PHA production was observed when
RuBisCO form I and form I & II genes were overexpressed, five times under photoheterotrophy with butyrate, two times with hydrogen and NH4Cl, and two times under photoelectrotrophic growth with N2 . In summary, inserting copies of
RuBisCO genes into the TIE-1 genome is a more effective strategy than deleting competing pathways to increase PHA production in TIE-1. The successful use of the phage integration system opens numerous opportunities for synthetic biology in TIE-1.IMPORTANCEOur planet has been burdened by pollution resulting from the extensive use of petroleum-derived plastics for the last few decades. Since the discovery of biodegradable plastic alternatives, concerted efforts have been made to enhance their bioproduction. The versatile microorganism Rhodopseudomonas palustris TIE-1 (TIE-1) stands out as a promising candidate for bioplastic synthesis, owing to its ability to use multiple electron sources, fix the greenhouse gas CO2, and use light as an energy source. Two categories of strains were meticulously designed from the TIE-1 wild-type to augment the production of polyhydroxyalkanoate (PHA), one such bioplastic produced. The first group includes mutants carrying a deletion of the phaR or phaZ genes in the PHA pathway, and those lacking potential competitive carbon and energy sinks to the PHA pathway (namely, glycogen biosynthesis and nitrogen fixation). The second group comprises TIE-1 strains that overexpress
RuBisCO form I or form I & II genes inserted via a phage integration system. By studying numerous metabolic mutants and overexpression strains, we conclude that genetic modifications in the environmental microbe TIE-1 can improve PHA production. When combined with other approaches (such as reactor design, use of microbial consortia, and different feedstocks), genetic and metabolic manipulations of purple nonsulfur bacteria like TIE-1 are essential for replacing petroleum-derived plastics with biodegradable plastics like PHA.