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开发用于在戊糖利用型恶臭假单胞菌环境分离株中异源蛋白表达的遗传工具。

Development of genetic tools for heterologous protein expression in a pentose-utilizing environmental isolate of Pseudomonas putida.

机构信息

The Joint BioEnergy Institute, Emeryville, California, USA.

Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA.

出版信息

Microb Biotechnol. 2023 Mar;16(3):645-661. doi: 10.1111/1751-7915.14205. Epub 2023 Jan 24.


DOI:10.1111/1751-7915.14205
PMID:36691869
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC9948227/
Abstract

Pseudomonas putida has emerged as a promising host for the conversion of biomass-derived sugars and aromatic intermediates into commercially relevant biofuels and bioproducts. Most of the strain development studies previously published have focused on P. putida KT2440, which has been engineered to produce a variety of non-native bioproducts. However, P. putida is not capable of metabolizing pentose sugars, which can constitute up to 25% of biomass hydrolysates. Related P. putida isolates that metabolize a larger fraction of biomass-derived carbon may be attractive as complementary hosts to P. putida KT2440. Here we describe genetic tool development for P. putida M2, a soil isolate that can metabolize pentose sugars. The functionality of five inducible promoter systems and 12 ribosome binding sites was assessed to regulate gene expression. The utility of these expression systems was confirmed by the production of indigoidine from C6 and C5 sugars. Chromosomal integration and expression of non-native genes was achieved by using chassis-independent recombinase-assisted genome engineering (CRAGE) for single-step gene integration of biosynthetic pathways directly into the genome of P. putida M2. These genetic tools provide a foundation to develop hosts complementary to P. putida KT2440 and expand the ability of this versatile microbial group to convert biomass to bioproducts.

摘要

恶臭假单胞菌已成为将生物质衍生糖和芳香族中间体转化为有商业价值的生物燃料和生物制品的有前途的宿主。以前发表的大多数菌株开发研究都集中在恶臭假单胞菌 KT2440 上,该菌株经过工程改造可生产各种非天然生物制品。然而,恶臭假单胞菌不能代谢戊糖,而戊糖可以占到生物质水解物的 25%。能够代谢更多生物质衍生碳的相关恶臭假单胞菌分离株可能是恶臭假单胞菌 KT2440 的互补宿主。在这里,我们描述了用于代谢戊糖的土壤分离株恶臭假单胞菌 M2 的遗传工具开发。评估了五个诱导型启动子系统和 12 个核糖体结合位点的功能,以调节基因表达。通过利用 C6 和 C5 糖生产靛蓝素,证实了这些表达系统的实用性。通过使用无底盘重组酶辅助基因组工程 (CRAGE) ,将生物合成途径的非天然基因直接整合到恶臭假单胞菌 M2 的基因组中,实现了染色体整合和非天然基因的表达。这些遗传工具为开发与恶臭假单胞菌 KT2440 互补的宿主提供了基础,并扩展了这个多功能微生物群将生物质转化为生物制品的能力。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a5af/9948227/c7c92e87d64b/MBT2-16-645-g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a5af/9948227/d7dd86d94388/MBT2-16-645-g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a5af/9948227/e5d49837a5d4/MBT2-16-645-g006.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a5af/9948227/3c6b2ab7fb02/MBT2-16-645-g005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a5af/9948227/209508718cf8/MBT2-16-645-g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a5af/9948227/cbcccc947cf8/MBT2-16-645-g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a5af/9948227/c7c92e87d64b/MBT2-16-645-g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a5af/9948227/d7dd86d94388/MBT2-16-645-g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a5af/9948227/e5d49837a5d4/MBT2-16-645-g006.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a5af/9948227/3c6b2ab7fb02/MBT2-16-645-g005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a5af/9948227/209508718cf8/MBT2-16-645-g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a5af/9948227/cbcccc947cf8/MBT2-16-645-g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a5af/9948227/c7c92e87d64b/MBT2-16-645-g004.jpg

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[2]
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[3]
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本文引用的文献

[1]
Revealing oxidative pentose metabolism in new Pseudomonas putida isolates.

Environ Microbiol. 2023-2

[2]
Chromosomal integration of complex DNA constructs using CRAGE and CRAGE-Duet systems.

STAR Protoc. 2022-9-16

[3]
Complete Genome Sequences of Five Isolated Pseudomonas Strains that Catabolize Pentose Sugars and Aromatic Compounds Obtained from Lignocellulosic Biomass.

Microbiol Resour Announc. 2022-4-21

[4]
Rational orthologous pathway and biochemical process engineering for adipic acid production using Pseudomonas taiwanensis VLB120.

Metab Eng. 2022-3

[5]
Towards robust Pseudomonas cell factories to harbour novel biosynthetic pathways.

Essays Biochem. 2021-7-26

[6]
Rewiring the microbial metabolic network for efficient utilization of mixed carbon sources.

J Ind Microbiol Biotechnol. 2021-12-23

[7]
Development of dual-inducible duet-expression vectors for tunable gene expression control and CRISPR interference-based gene repression in Pseudomonas putida KT2440.

Microb Biotechnol. 2021-11

[8]
Engineering Pseudomonas putida for efficient aromatic conversion to bioproduct using high throughput screening in a bioreactor.

Metab Eng. 2021-7

[9]
Recent Advanced Technologies for the Characterization of Xenobiotic-Degrading Microorganisms and Microbial Communities.

Front Bioeng Biotechnol. 2021-2-10

[10]
Complete Genome Sequences of Four Natural Isolates That Catabolize a Wide Range of Aromatic Compounds Relevant to Lignin Valorization.

Microbiol Resour Announc. 2020-12-3

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