Dataset Information

Modular pathway engineering of key precursor supply pathways for lacto-N-neotetraose production in Bacillus subtilis.

ABSTRACT: Background:Lacto-N-neotetraose (LNnT) is one of the important ingredients of human milk oligosaccharides, which can enhance immunity, regulate intestinal bacteria and promote cell maturation. Results:In this study, the synthetic pathway of LNnT was constructed by co-expressing the lactose permease (LacY) β-1,3-N-acetylglucosaminyltransferase (LgtA) and β-1,4-galactostltransferase (LgtB) in Bacillus subtilis, resulting in an LNnT titer of 0.61 g/L. Then, by fine-tuning the expression level of LgtB, the growth inhibition was reduced and the LNnT titer was increased to 1.31 g/L. In addition, by modular pathway engineering, the positive-acting enzymes of the UDP-GlcNAc and UDP-Gal pathways were strengthened to balance the two key precursors supply, and the LNnT titer was improved to 1.95 g/L. Finally, the LNnT titer reached 4.52 g/L in a 3-L bioreactor with an optimal glucose and lactose feeding strategy. Conclusions:In general, this study showed that the LNnT biosynthesis could be significantly increased by optimizing enzymes expression levels and modular pathway engineering for balancing the precursors supply in B. subtilis.

SUBMITTER: Dong X

PROVIDER: S-EPMC6732834 | biostudies-other | 2019

REPOSITORIES: biostudies-other

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Publications

Modular pathway engineering of key precursor supply pathways for lacto-<i>N</i>-neotetraose production in <i>Bacillus subtilis</i>.

Dong Xiaomin X Li Nan N Liu Zhenmin Z Lv Xueqin X Li Jianghua J Du Guocheng G Wang Miao M Liu Long L

Biotechnology for biofuels 20190909

<h4>Background</h4>Lacto-<i>N</i>-neotetraose (LNnT) is one of the important ingredients of human milk oligosaccharides, which can enhance immunity, regulate intestinal bacteria and promote cell maturation.<h4>Results</h4>In this study, the synthetic pathway of LNnT was constructed by co-expressing the lactose permease (LacY) β-1,3-<i>N</i>-acetylglucosaminyltransferase (LgtA) and β-1,4-galactostltransferase (LgtB) in <i>Bacillus subtilis</i>, resulting in an LNnT titer of 0.61 g/L. Then, by fin ...[more]

PMID: 31516551

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Project description:There is a growing need for a highly stable system to allow the production of biologics for diagnoses and therapeutic interventions on demand that could be used in extreme environments. Among the variety of biologics, nanobodies (Nbs) derived from single-chain variable antibody fragments from camelids have attracted great attention in recent years due to their small size and great stability with translational potentials in whole-body imaging and the development of new drugs. Intracellular expression using the bacterium Escherichia coli has been the predominant system to produce Nbs, and this requires lengthy steps for releasing intracellular proteins for purification as well as removal of endotoxins. Lyophilized, translationally competent cell extracts have also been explored as offering portability and long shelf life, but such extracts may be difficult to scale up and suffer from batch-to-batch variability. To address these problems, we present a new system to do the following: (i) engineer the spore-forming bacterium Bacillus subtilis to secrete Nbs that can target small molecules or protein antigens on mammalian cells, (ii) immobilize Nbs containing a cellulose-binding domain on a cellulose matrix for long-term storage and small-molecule capturing, (iii) directly use Nb-containing bacterial supernatant fluid to perform protein detection on cell surfaces, and (iv) convert engineered B. subtilis to spores that are resistant to most environmental extremes. In summary, our work may open a new paradigm for using B. subtilis as an extremely stable microbial factory to produce Nbs with applications in extreme environments on demand.IMPORTANCE It is highly desirable to produce biologics for diagnoses and therapeutic interventions on demand that could be used in a variety of settings. Among the many biologics, Nbs have attracted attention due to their small size, thermal stability, and broad utility in diagnoses, therapies, and fundamental research. Nbs originate from antibodies found in camelids, and >10 companies have invested in Nbs as potential drugs. Here, we present a system using cells of the bacterium Bacillus subtilis as a versatile platform for production of Nbs and then antigen detection via customized affinity columns. Importantly, B. subtilis carrying engineered genes for Nbs can form spores, which survive for years in a desiccated state. However, upon rehydration and exposure to nutrients, spores rapidly transition to growing cells which secrete encoded Nbs, thus allowing their manufacture and purification.

Dataset Information

Modular pathway engineering of key precursor supply pathways for lacto-N-neotetraose production in Bacillus subtilis.

Publications

Modular pathway engineering of key precursor supply pathways for lacto-<i>N</i>-neotetraose production in <i>Bacillus subtilis</i>.

Similar Datasets

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