Improving bioplastic production by Rhodopseudomonas palustris TIE-1 using synthetic biology and metabolic engineering
Improving bioplastic production by Rhodopseudomonas palustris TIE-1 using synthetic biology and metabolic engineering
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DOI：
10.1101/2023.05.17.541174
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发表时间：
2023-05
期刊：
影响因子：
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通讯作者：
T. Ranaivoarisoa;W. Bai;K. Rengasamy;Hope Steele;Miriam Silberman;Jennifer Olabode;A. Bose
中科院分区：
文献类型：
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作者：
T. Ranaivoarisoa;W. Bai;K. Rengasamy;Hope Steele;Miriam Silberman;Jennifer Olabode;A. Bose
随着对可持续生产的可再生资源的需求不断增加，重要的是要寻找能够生产生物产品的微生物，如生物燃料和生物塑料。尽管许多生物产品生产系统都有很好的文件记录，并在模式生物中进行了测试，但必须超越非模式生物，以扩大领域并利用代谢多功能性的菌株。这项研究以沼泽红假单胞菌Tie-1为中心，它是一种紫色、非硫自养和厌氧细菌，能够产生与石油产品相当的生物产品。为了诱导生物塑料的过量生产，可能在PHB生物合成中具有潜在作用的基因，如调节剂Phar和以其降解PHB颗粒的能力而闻名的phaZ被用无标记缺失来删除。还测试了可能与聚羟基丁酸酯(PHB)生产竞争的途径中的突变体，例如先前为增加Tie-1的正丁醇产量而创造的糖原和固氮。此外，还开发了一个噬菌体整合系统，将由组成型启动子PphII驱动的Rubisco(Rubisco形式I和II基因)插入到Tie-1基因组中。我们的结果表明，当Tie-1与丁酸盐和氯化铵(NH4Cl)光异养生长时，PHB途径的Phar基因的缺失提高了PHB的产量。不能制造糖原或固定氮源气体的突变株在有氢的光自养生长条件下表现出PHB产量的增加。此外，高效表达Rubisco形式I和形式II的工程化Tie-1在丁酸光养和氢光自养下产生的多羟基丁酸显著高于野生型。在Tie-1基因组中插入Rubisco基因是一种比删除竞争途径更有效的策略来增加Tie-1中PHB的产量。因此，为Tie-1开发的噬菌体整合系统为Tie-1的合成生物学创造了大量机会。
With the increasing demand for sustainably produced renewable resources, it is important to look towards microorganisms capable of producing bioproducts such as biofuels and bioplastics. Though many systems for bioproduct production are well documented and tested in model organisms, it is essential to look beyond to non-model organisms to expand the field and take advantage of metabolically versatile strains. This investigation centers on Rhodopseudomonas palustris TIE-1, a purple, non-sulfur autotrophic, and anaerobic bacterium capable of producing bioproducts that are comparable to their petroleum-based counterparts. To induce bioplastic overproduction, genes that might have a potential role in the PHB biosynthesis such as the regulator, phaR, and phaZ known for its ability to degrade PHB granules were deleted using markerless deletion. Mutants in pathways that might compete with polyhydroxybutyrate (PHB) production such as glycogen and nitrogen fixation previously created to increase n-butanol production by TIE-1 were also tested. In addition, a phage integration system was developed to insert RuBisCO (RuBisCO form I and II genes) driven by a constitutive promoter PaphII into TIE- 1 genome. Our results show that deletion of the phaR gene of the PHB pathway increases PHB productivity when TIE-1 was grown photoheterotrophically with butyrate and ammonium chloride (NH4Cl). Mutants unable to make glycogen or fix dinitrogen gas show an increase in PHB productivity under photoautotrophic growth conditions with hydrogen. In addition, the engineered TIE-1 overexpressing RuBisCO form I and form II produces significantly more polyhydroxybutyrate than the wild type under photoheterotrophy with butyrate and photoautotrophy with hydrogen. Inserting RuBisCO genes into TIE-1 genome is a more effective strategy than deleting competitive pathways to increase PHB production in TIE-1. The phage integration system developed for TIE-1 thus creates numerous opportunities for synthetic biology in TIE-1.