Enhanced theanine production with reduced ATP supply by alginate entrapped Escherichia coli co-expressing γ-glutamylmethylamide synthetase and polyphosphate kinase

IF 3.4 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Enzyme and Microbial Technology Pub Date : 2024-01-15 DOI:10.1016/j.enzmictec.2024.110394

Do Hyun Cho , Suwon Kim , Yeda Lee , Yuni Shin , Suhye Choi , Jinok Oh , Hee Taek Kim , See-Hyoung Park , Kyungmoon Park , Shashi Kant Bhatia , Yung-Hun Yang

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引用次数: 0

Abstract

L-theanine is an amino acid with a unique flavor and many therapeutic effects. Its enzymatic synthesis has been actively studied and γ-Glutamylmethylamide synthetase (GMAS) is one of the promising enzymes in the biological synthesis of theanine. However, the theanine biosynthetic pathway with GMAS is highly ATP-dependent and the supply of external ATP was needed to achieve high concentration of theanine production. As a result, this study aimed to investigate polyphosphate kinase 2 (PPK2) as ATP regeneration system with hexametaphosphate. Furthermore, the alginate entrapment method was employed to immobilize whole cells containing both gmas and ppk2 together resulting in enhanced reusability of the theanine production system with reduced supply of ATP. After immobilization, theanine production was increased to 239 mM (41.6 g/L) with a conversion rate of 79.7% using 15 mM ATP and the reusability was enhanced, maintaining a 100% conversion rate up to the fifth cycles and 60% of conversion up to eighth cycles. It could increase long-term storage property for future uses up to 35 days with 75% activity of initial activity. Overall, immobilization of both production and cofactor regeneration system could increase the stability and reusability of theanine production system.

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藻酸盐包裹的大肠杆菌共同表达 γ-谷氨酰甲酰胺合成酶和多磷酸激酶，在减少 ATP 供应的情况下提高了茶氨酸产量

L -茶氨酸是一种具有独特风味和多种治疗效果的氨基酸。人们对它的酶法合成进行了积极的研究，γ-谷氨酰甲酰胺合成酶（GMAS）是生物合成茶氨酸的有前途的酶之一。然而，GMAS 的茶氨酸生物合成途径高度依赖 ATP，需要外部 ATP 的供应才能实现高浓度茶氨酸的生产。因此，本研究旨在利用六偏磷酸钠研究多磷酸激酶 2（PPK2）作为 ATP 再生系统。此外，本研究还采用藻酸盐包埋法将含有 gmas 和 PPK2 的全细胞固定在一起，从而在减少 ATP 供应的情况下提高了丙氨酸生产系统的可再利用性。固定化后，丙氨酸产量增加到 239 mM（41.6 g/L），在使用 15 mM ATP 的情况下转化率为 79.7%，而且可重复使用性也得到了提高，在第五次循环之前保持了 100%的转化率，在第八次循环之前保持了 60%的转化率。它还能提高长期储存性能，供未来使用长达 35 天，活性为初始活性的 75%。总之，生产和辅助因子再生系统的固定化可提高丙氨酸生产系统的稳定性和可重复使用性。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Enzyme and Microbial Technology 生物-生物工程与应用微生物

CiteScore

7.60

自引率

5.90%

发文量

142

审稿时长

38 days

期刊介绍： Enzyme and Microbial Technology is an international, peer-reviewed journal publishing original research and reviews, of biotechnological significance and novelty, on basic and applied aspects of the science and technology of processes involving the use of enzymes, micro-organisms, animal cells and plant cells. We especially encourage submissions on: Biocatalysis and the use of Directed Evolution in Synthetic Biology and Biotechnology Biotechnological Production of New Bioactive Molecules, Biomaterials, Biopharmaceuticals, and Biofuels New Imaging Techniques and Biosensors, especially as applicable to Healthcare and Systems Biology New Biotechnological Approaches in Genomics, Proteomics and Metabolomics Metabolic Engineering, Biomolecular Engineering and Nanobiotechnology Manuscripts which report isolation, purification, immobilization or utilization of organisms or enzymes which are already well-described in the literature are not suitable for publication in EMT, unless their primary purpose is to report significant new findings or approaches which are of broad biotechnological importance. Similarly, manuscripts which report optimization studies on well-established processes are inappropriate. EMT does not accept papers dealing with mathematical modeling unless they report significant, new experimental data.