Enhancing menaquinone-7 biosynthesis by adaptive evolution of <i>Bacillus natto</i> through chemical modulator

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Abstract

Menaquinone-7 (MK-7) is a kind of vitamin K2 playing an important role in the treatment and prevention of cardiovascular disease, osteoporosis and arterial calcification. The purpose of this study is to establish an adaptive evolution strategy based on a chemical modulator to improve MK-7 biosynthesis in Bacillus natto. The inhibitor of 5-enolpyruvylshikimate-3-phosphate synthase (EPSP synthase), glyphosate, was chosen as the chemical modulator to perform the experiments. The final strain ALE-25–40, which was obtained after 40 cycles in 25 mmol/L glyphosate, showed a maximal MK-7 titer of 62 mg/L and MK-7 productivity of 0.42 mg/(L h), representing 2.5 and 3 times the original strain, respectively. Moreover, ALE-25–40 generated fewer spores and showed a higher NADH and redox potential. Furthermore, the mechanism related to the improved performance of ALE-25–40 was investigated by comparative transcriptomics analysis. Genes related to the sporation formation were down-regulated. In addition, several genes related to NADH formation were also up-regulated. This strategy proposed here may provide a new and alternative directive for the industrial production of vitamin K2.

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Supplementary Information

The online version contains supplementary material available at 10.1186/s40643-022-00609-0.

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The Spo0A regulon of Bacillus subtilis.

Paul Jensen, Songling Liu, J Pastor … (2003)

The master regulator for entry into sporulation in Bacillus subtilis is the DNA-binding protein Spo0A, which has been found to influence, directly or indirectly, the expression of over 500 genes during the early stages of development. To search on a genome-wide basis for genes under the direct control of Spo0A, we used chromatin immunoprecipitation in combination with gene microarray analysis to identify regions of the chromosome at which an activated form of Spo0A binds in vivo. This information in combination with transcriptional profiling using gene microarrays, gel electrophoretic mobility shift assays, using the DNA-binding domain of Spo0A, and bioinformatics enabled us to assign 103 genes to the Spo0A regulon in addition to 18 previously known members. Thus, in total, 121 genes, which are organized as 30 single-gene units and 24 operons, are likely to be under the direct control of Spo0A. Forty of these genes are under the positive control of Spo0A, and 81 are under its negative control. Among newly identified members of the regulon with transcription that was stimulated by Spo0A are genes for metabolic enzymes and genes for efflux pumps. Among members with transcription that was in-hibited by Spo0A are genes encoding components of the DNA replication machinery and genes that govern flagellum biosynthesis and chemotaxis. Also in-cluded in the regulon are many (25) genes with products that are direct or indirect regulators of gene transcription. Spo0A is a master regulator for sporulation, but many of its effects on the global pattern of gene transcription are likely to be mediated indirectly by regulatory genes under its control.

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Adaptive laboratory evolution – principles and applications for biotechnology

Martin Dragosits, Diethard Mattanovich (2013)

Adaptive laboratory evolution is a frequent method in biological studies to gain insights into the basic mechanisms of molecular evolution and adaptive changes that accumulate in microbial populations during long term selection under specified growth conditions. Although regularly performed for more than 25 years, the advent of transcript and cheap next-generation sequencing technologies has resulted in many recent studies, which successfully applied this technique in order to engineer microbial cells for biotechnological applications. Adaptive laboratory evolution has some major benefits as compared with classical genetic engineering but also some inherent limitations. However, recent studies show how some of the limitations may be overcome in order to successfully incorporate adaptive laboratory evolution in microbial cell factory design. Over the last two decades important insights into nutrient and stress metabolism of relevant model species were acquired, whereas some other aspects such as niche-specific differences of non-conventional cell factories are not completely understood. Altogether the current status and its future perspectives highlight the importance and potential of adaptive laboratory evolution as approach in biotechnological engineering.

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Adaptive evolution of Schizochytrium sp. by continuous high oxygen stimulations to enhance docosahexaenoic acid synthesis.

Xiao-Man Sun, Lu-Jing Ren, Xiao-Jun Ji … (2016)

Adaptive laboratory evolution (ALE) is an effective method in changing the strain characteristics. Here, ALE with high oxygen as a selection pressure was applied to improve the production capacity of Schizochytrium sp. Results showed that cell dry weight (CDW) of endpoint strain was 32.4% higher than that of starting strain. But slight lipid accumulation impairment was observed. These major performance changes were accompanied with enhanced isocitrate dehydrogenase enzyme activity and reduced ATP:citrate lyase enzyme activity. And a serious decrease of 62.6% in SDHA 140rpm→170rpm was observed in the endpoint strain. To further study the docosahexaenoic acid (DHA) production ability of evolved strain, fed-batch strategy was applied and 84.34g/L of cell dry weight and 26.40g/L of DHA yield were observed. In addition, endpoint strain produced greatly less squalene than starting strain. This work demonstrated that ALE may be a promising tool in modifying microalga strains.

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Author and article information

Contributors

Lujing Ren:

ORCID: http://orcid.org/0000-0002-6217-7309

renlujing@njtech.edu.cn

Journal

Journal ID (nlm-ta): Bioresour Bioprocess

Journal ID (iso-abbrev): Bioresour Bioprocess

Title: Bioresources and Bioprocessing

Publisher: Springer Nature Singapore (Singapore )

ISSN (Electronic): 2197-4365

Publication date (Electronic): 22 November 2022

Publication date PMC-release: 22 November 2022

Publication date Collection: December 2022

Volume: 9

Issue: 1

Electronic Location Identifier: 120

Affiliations

[1 ]GRID grid.412022.7, ISNI 0000 0000 9389 5210, College of Biotechnology and Pharmaceutical Engineering, , Nanjing Tech University, ; No. 30 South Puzhu Road, Nanjing, 211816 People’s Republic of China

[2 ]Shanghai JanStar Technology Development Co., Ltd., No. 1288, Huateng Road, Shanghai, 201700 China

Author information

Lujing Ren http://orcid.org/0000-0002-6217-7309

Article

Publisher ID: 609

DOI: 10.1186/s40643-022-00609-0

PMC ID: 10992315

SO-VID: 7fa6cdac-dcfc-40fa-bf38-49ffd84c9ceb

License:

Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

History

Date received : 8 September 2022

Date accepted : 1 November 2022

Funding

Funded by: FundRef http://dx.doi.org/10.13039/501100013140, Jiangsu National Synergistic Innovation Center for Advanced Materials;

Award ID: XTD2213

Award Recipient : Lujing Ren

Funded by: FundRef http://dx.doi.org/10.13039/100017133, National Key Project for Synthetic Biology;

Award ID: No. 2019YFA0905700

Award Recipient : Lujing Ren

Funded by: FundRef http://dx.doi.org/10.13039/501100001809, National Natural Science Foundation of China;

Award ID: No. 21878151

Award Recipient : Lujing Ren

Funded by: FundRef http://dx.doi.org/10.13039/501100004608, Natural Science Foundation of Jiangsu Province;

Award ID: BK20211535

Award Recipient : Lujing Ren

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Keywords: menaquinone-7,bacillus natto,adaptive evolution,glyphosate,epse synthase

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Keywords: menaquinone-7, bacillus natto, adaptive evolution, glyphosate, epse synthase

Enhancing menaquinone-7 biosynthesis by adaptive evolution of Bacillus natto through chemical modulator

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Most cited references 50

The Spo0A regulon of Bacillus subtilis.

Adaptive laboratory evolution – principles and applications for biotechnology

Adaptive evolution of Schizochytrium sp. by continuous high oxygen stimulations to enhance docosahexaenoic acid synthesis.

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