Dataset Information

Eliminating host-guest incompatibility via enzyme mining enables the high-temperature production of N-acetylglucosamine.

ABSTRACT: The host-guest incompatibility between a production host and non-native enzymes has posed an arduous challenge for synthetic biology, particularly between mesophile-derived enzymes and a thermophilic chassis. In the present study, we develop a thermophilic enzyme mining strategy comprising an automated co-evolution-based screening pipeline (http://cem.sjtu.edu.cn), computation-based enzyme characterization, and gene synthesis-based function validation. Using glucosamine-6-phosphate acetyltransferase (GNA1) as an example, we successfully mined four novel GNA1s with excellent thermostabilities and catalytic performances. Calculation and analysis based on AlphaFold2-generated structures were also conducted to uncover the mechanism underlying their excellent properties. Finally, our mined GNA1s were used to enable the high-temperature N-acetylglucosamine (GlcNAc) production with high titers of up to 119.3 g/L, with the aid of systems metabolic engineering and temperature programming. This study demonstrates the effectiveness of the enzyme mining strategy, highlighting the application prospects of mining new enzymes from massive databases and providing an effective solution for tackling host-guest incompatibility.

SUBMITTER: Wu Y

PROVIDER: S-EPMC9829697 | biostudies-literature | 2023 Jan

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Eliminating host-guest incompatibility via enzyme mining enables the high-temperature production of <i>N</i>-acetylglucosamine.

Wu Yutong Y Liu Jiongqin J Han Xiao X Meng Xuanlin X Li Mengke M Wang Jing J Xue Hongsong H Yang Yuhan Y Xu Ping P Tao Fei F

iScience 20221209 1

The host-guest incompatibility between a production host and non-native enzymes has posed an arduous challenge for synthetic biology, particularly between mesophile-derived enzymes and a thermophilic chassis. In the present study, we develop a thermophilic enzyme mining strategy comprising an automated co-evolution-based screening pipeline (http://cem.sjtu.edu.cn), computation-based enzyme characterization, and gene synthesis-based function validation. Using glucosamine-6-phosphate acetyltransfe ...[more]

PMID: 36636338

Similar Datasets

Project description:BackgroundGlucosamine-6-phosphate N-acetyltransferase (GNA1) is the key enzyme that causes overproduction of N-acetylglucosamine in Bacillus subtilis. Previously, we increased GlcNAc production by promoting the expression of GNA1 from Caenorhabditis elegans (CeGNA1) in an engineered B. subtilis strain BSGN12. In this strain overflow metabolism to by-products acetoin and acetate had been blocked by mutations, however pyruvate accumulated as an overflow metabolite. Although overexpression of CeGNA1 drove carbon flux from pyruvate to the GlcNAc synthesis pathway and decreased pyruvate accumulation, the residual pyruvate reduced the intracellular pH, resulting in inhibited CeGNA1 activity and limited GlcNAc production.ResultsIn this study, we attempted to further overcome pyruvate overflow by enzyme engineering and host engineering for enhanced GlcNAc production. To this end, the key enzyme CeGNA1 was evolved through error-prone PCR under pyruvate stress to enhance its catalytic activity. Then, the urease from Bacillus paralicheniformis was expressed intracellularly to neutralize the intracellular pH, making it more robust in growth and more efficient in GlcNAc production. It was found that the activity of mutant CeGNA1 increased by 11.5% at pH 6.5-7.5, with the catalytic efficiency increasing by 27.5% to 1.25 s-1 µM-1. Modulated expression of urease increased the intracellular pH from 6.0 to 6.8. The final engineered strain BSGN13 overcame pyruvate overflow, produced 25.6 g/L GlcNAc with a yield of 0.43 g GlcNAc/g glucose in a shake flask fermentation and produced 82.5 g/L GlcNAc with a yield of 0.39 g GlcNAc/g glucose by fed-batch fermentation, which was 1.7- and 1.2-times, respectively, of the yield achieved previously.ConclusionsThis study highlights a strategy that combines pathway enzyme engineering and host engineering to resolve overflow metabolism in B. subtilis for the overproduction of GlcNAc. By means of modulated expression of urease reduced pyruvate burden, conferred bacterial survival fitness, and enhanced GlcNAc production, all of which improved our understanding of co-regulation of cell growth and metabolism to construct more efficient B. subtilis cell factories.

Dataset Information

Eliminating host-guest incompatibility via enzyme mining enables the high-temperature production of N-acetylglucosamine.

Publications

Eliminating host-guest incompatibility via enzyme mining enables the high-temperature production of <i>N</i>-acetylglucosamine.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets