Biosensor-based isolation of amino acid-producing Vibrio natriegens strains

IF 3.7 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
Roberto Giuseppe Stella , Philipp Baumann , Sophia Lorke , Felix Münstermann , Astrid Wirtz , Johanna Wiechert , Jan Marienhagen , Julia Frunzke
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引用次数: 5

Abstract

The marine bacterium Vibrio natriegens has recently been demonstrated to be a promising new host for molecular biology and next generation bioprocesses. V. natriegens is a Gram-negative, non-pathogenic slight-halophilic bacterium, with a high nutrient versatility and a reported doubling time of under 10 min. However, V. natriegens is not an established model organism yet, and further research is required to promote its transformation into a microbial workhorse.

In this work, the potential of V. natriegens as an amino acid producer was investigated. First, the transcription factor-based biosensor LysG, from Corynebacterium glutamicum, was adapted for expression in V. natriegens to facilitate the detection of positively charged amino acids. A set of different biosensor variants were constructed and characterized, using the expression of a fluorescent protein as sensor output. After random mutagenesis, one of the LysG-based sensors was used to screen for amino acid producer strains. Here, fluorescence-activated cell sorting enabled the selective sorting of highly fluorescent cells, i.e. potential producer cells. Using this approach, individual L-lysine, L-arginine and L-histidine producers could be obtained producing up to 1 mM of the effector amino acid, extracellularly. Genome sequencing of the producer strains provided insight into the amino acid production metabolism of V. natriegens.

This work demonstrates the successful expression and application of transcription factor-based biosensors in V. natriegens and provides insight into the underlying physiology, forming a solid basis for further development of this promising microbe.

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基于生物传感器的产氨基酸营养弧菌菌株的分离
海洋细菌营养弧菌最近被证明是分子生物学和下一代生物过程的一个有前途的新宿主。V. natriegens是一种革兰氏阴性、非致病性的轻度嗜盐细菌,具有较高的营养多样性,据报道翻倍时间在10分钟以下。然而,V. natriegens还不是一种确定的模式生物,需要进一步的研究来促进它向微生物的转变。在这项工作中,研究了V. natriegens作为氨基酸生产者的潜力。首先,利用谷氨棒状杆菌转录因子为基础的生物传感器LysG在V. natriegens中表达,以方便检测带正电的氨基酸。利用荧光蛋白的表达作为传感器输出,构建并表征了一组不同的生物传感器变体。随机诱变后,利用一种基于lysg的传感器筛选氨基酸产生菌株。在这里,荧光激活的细胞分选能够选择性地分选高荧光细胞,即潜在的生产者细胞。使用这种方法,可以在细胞外获得单个l -赖氨酸、l -精氨酸和l -组氨酸产生物,产生高达1mm的效应氨基酸。产生菌的基因组测序提供了对弧菌氨基酸生产代谢的深入了解。这项工作证明了基于转录因子的生物传感器在V. natriegens中的成功表达和应用,并提供了对潜在生理学的深入了解,为进一步开发这种有前途的微生物奠定了坚实的基础。
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来源期刊
Metabolic Engineering Communications
Metabolic Engineering Communications Medicine-Endocrinology, Diabetes and Metabolism
CiteScore
13.30
自引率
1.90%
发文量
22
审稿时长
18 weeks
期刊介绍: Metabolic Engineering Communications, a companion title to Metabolic Engineering (MBE), is devoted to publishing original research in the areas of metabolic engineering, synthetic biology, computational biology and systems biology for problems related to metabolism and the engineering of metabolism for the production of fuels, chemicals, and pharmaceuticals. The journal will carry articles on the design, construction, and analysis of biological systems ranging from pathway components to biological complexes and genomes (including genomic, analytical and bioinformatics methods) in suitable host cells to allow them to produce novel compounds of industrial and medical interest. Demonstrations of regulatory designs and synthetic circuits that alter the performance of biochemical pathways and cellular processes will also be presented. Metabolic Engineering Communications complements MBE by publishing articles that are either shorter than those published in the full journal, or which describe key elements of larger metabolic engineering efforts.
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