Identification of integration site library for the development of plasmid-free microbial cell factory in <i>Bacillus subtilis</i>.
The integration site library for Bacillus subtilis significantly enhances gene expression and product yields, offering potential for improved microbial cell factories.
Where it sits
this study against the rest of the sermorelin corpusSummary and findings
A library of integration sites was developed for Bacillus subtilis to enhance gene expression and cell growth. The highest expression site, spxA, showed 1.89 times the expression level compared to the control site amyE. The integration site library increased the production of lacto-N-triose II and lycopene by 95% and 83%, respectively.
Abstract
<i>Bacillus subtilis</i> was widely used for enzyme production and was gradually engineered to biosynthesize value-added chemicals with the development of genetic parts for it. Compared to other genetic parts for expression, the identified integration sites were fewer, which limits the development of <i>B. subtilis</i>. Here, a library of integration sites was developed for <i>B. subtilis</i>. All candidate sites were selected at the 3'-untranslated region of two opposite nonessential genes and separated by essential genes among genome to avoid the destruction for coding sequence of genes and eliminate the homologously recombined strains with the loss of essential genes. The expression of GFP and cell growth were detected for candidate sites to evaluate the gene expression strength and the influence on cell growth. As a result, 12 loci revealed higher gene expression level and cell growth compared with control site <i>amyE</i>, the highest expression site <i>spxA</i> was 1.89 times as high as <i>amyE</i>. Using the developed integration sites library, threefold gene expression range could be achieved without the replacement of promoter and RBS. When the integration site library was used to construct cell factories, the production of lacto-<i>N</i>-triose II and lycopene was increased by 95% and 83%, respectively. In addition, integration site library was also successfully used to increase the enzymatic activity of secretory β-galactosidase by 101% when the strain using <i>spxA</i> locus compared with that using <i>amyE.</i> The developed integration site library could accelerate the construction of stable and plasmid-free cell factories for <i>B. subtilis</i> in the future.
Background
Bacillus subtilis is widely used for enzyme production and has been engineered to biosynthesize value-added chemicals. However, the limited number of integration sites for gene expression has constrained its development. This study addresses the need for more integration sites to enhance the capabilities of B. subtilis as a microbial cell factory.
Methods
The study developed a library of integration sites in Bacillus subtilis, selecting candidate sites at the 3'-untranslated region of two opposite nonessential genes. The integration sites were evaluated based on GFP expression and cell growth to assess gene expression strength and impact on cell growth.
Results
Twelve loci demonstrated higher gene expression and cell growth compared to the control site amyE. The spxA site showed 1.89 times the expression level of amyE. The integration site library increased the production of lacto-N-triose II and lycopene by 95% and 83%, respectively. Additionally, the enzymatic activity of secretory β-galactosidase was increased by 101% using the spxA locus.
Interpretation
The study provides a significant advancement in the genetic engineering of Bacillus subtilis by expanding the available integration sites. The observed increases in gene expression and product yields suggest potential for more efficient and stable microbial cell factories. However, the clinical or commercial relevance of these findings would require further validation in practical applications.
Key findings
- 12 loci revealed higher gene expression level and cell growth compared with control site amyE.
- The highest expression site spxA was 1.89 times as high as amyE.
- Production of lacto-N-triose II increased by 95%.
- Production of lycopene increased by 83%.
- Enzymatic activity of secretory β-galactosidase increased by 101% using spxA locus.
Limitations
- Not reported in abstract.