CRISPRi-Guided Multiplexed Fine-Tuning of Metabolic Flux for Enhanced Lacto-N-neotetraose Production in Bacillus subtilis

Abstract

Lacto-N-neotetraose (LNnT), one of the oligosaccharides in human milk, has many beneficial effects on infant health. In a recent work, we have constructed a recombinant Bacillus subtilis strain for the production of LNnT. Here, we further improved LNnT production with a xylose-induced clustered regularly interspaced short palindromic repeats interference system. In particular, the expressions of pf kA and pyk genes in the Embden−Meyerhof−Parnas pathway module, zwf gene in the pentose phosphate pathway module, and mnaA gene in the teichoic acid synthesis module were downregulated. The LNnT titer was increased from 1.32 to 1.55 g/L. Furthermore, to improve the conversion efficiency of lacto-N-triose II to LNnT, we knocked out tuaD gene in branch pathway and improved the expression of lgtB gene, resulting in the further increase of LNnT titer to 2.01 g/L. Finally, the addition time and amount of inducer xylose were optimized, and LNnT titer reached 2.30 g/L in shake flask and 5.41 g/L in 3 L bioreactor.

Bacillus subtilis: a universal cell factory for industry, agriculture, biomaterials and medicine

“…Furthermore, this CRISPR-Cas9 toolkit was extended to CRISPR interference (CRISPRi) for transcriptional-level regulation [ 21 ]. The CRISPRi system is composed of a deactivated Cas9 (dCas9) protein and gRNA, enabling the targeting of dCsa9 to any target gene on the genome under the guidance of gRNA to inhibit its transcription without inducing a double-strand break, which can be applied to gene repression in metabolic engineering [ 24 ]. So et al developed a CRISPR-derived genome engineering technique to efficiently generate large genomic deletions in B. subtilis without the introduction of counter-selectable markers such as antibiotic-resistance genes, which had previously limited the application of B. subtilis in food engineering [ 25 ].…”

“…So et al developed a CRISPR-derived genome engineering technique to efficiently generate large genomic deletions in B. subtilis without the introduction of counter-selectable markers such as antibiotic-resistance genes, which had previously limited the application of B. subtilis in food engineering [ 25 ]. This method has wide applicability for various types of site-directed mutagenesis in B. subtilis [ 24 , 26 , 27 ]. However, CRISPR/Cas9 has low efficiency in multi-gene editing.…”

Recent advances and challenges in microbial production of human milk oligosaccharides

“…Bacillus subtilis was also reconstructed to produce LNnT. Dong et al constructed and optimized the LNnT biosynthesis by modular pathway engineering and CRISPRi-guided multiplexed fine-tuning of metabolic flux in B. subtilis 168 [59,60]. Apart from strategies mentioned in the reconstructed E. coli, the synthesis of cofactors UDP-galactose and UDP-GlcNac was improved by overexpressing heterologous pathway genes lgtA and lgtB (Fig.…”

“…However, overexpression of multiple genes may reduce the expression of each gene and can also affect the growth of the strain due to the limited carrying capacity of the plasmid. In addition, the overexpression of key enzyme LgtB can significantly affect cell growth, resulting in a low efficiency of LNnT synthesis [60].…”

Metabolic engineering of Escherichia coli for the production of Lacto-N-neotetraose (LNnT)

“…However, the lack of metabolic regulation results a low conversion rate of carbon [14,21]. Recent research has constructed the pathway of LNnT in Bacillus subtilis [22,23]. Engineering of modular pathway of role precursors has improved the titer of LNnT in some degree, but the metabolic regulation and the utilization of substrate still have difficulties.…”