CRISPRi allows optimal temporal control of N-acetylglucosamine bioproduction by a dynamic coordination of glucose and xylose metabolism in Bacillus subtilis

Abstract

Glucose and xylose are the two most abundant sugars in renewable lignocellulose sources; however, typically they cannot be simultaneously utilized due to carbon catabolite repression. N-acetylglucosamine (GlcNAc) is a typical nutraceutical and has many applications in the field of healthcare. Here, we have developed a gene repressor system based on xylose-induced CRISPR interference (CRISPRi) in Bacillus subtilis, aimed at downregulating the expression of three genes (zwf, pfkA, glmM) that control the major competing reactions of GlcNAc synthesis (pentose phosphate pathway (HMP), glycolysis, and peptidoglycan synthesis pathway (PSP)), with the potential to relieve glucose repression and allow the co-utilization of both glucose and xylose. Simultaneous repression of these three genes by CRISPRi improved GlcNAc titer by 13.2% to 17.4 ± 0.47 g/L, with the GlcNAc yield on glucose and xylose showing an 84.1% improvement, reaching 0.42 ± 0.036 g/g. In order to further engineer the synergetic utilization of glucose and xylose, a combinatorial approach was developed based on 27 arrays containing sgRNAs with different repression capacities targeting the three genes. We further optimized the temporal control of the system and found that when 15 g/L xylose was added 6 h after inoculation, the most efficient strain, BNX122, synthesized 20.5 ± 0.85 g/L GlcNAc with a yield of 0.46 ± 0.010 g/g glucose and xylose in shake flask culture. Finally, the GlcNAc titer and productivity in a 3-L fed-batch bioreactor reached 103.1 ± 2.11 g/L and 1.17 ± 0.024 g/L/h, which were 5.0-fold and 2.7-fold of that in shake flask culture, respectively. Taken together, these findings suggest that a CRISPRi-enabled regulation method provides a simple, efficient, and universal way to promote the synergetic utilization of multiple carbon sources by microbial cell factories.

Systems metabolic engineering of Bacillus subtilis for efficient biosynthesis of 5‐methyltetrahydrofolate

“…CRISPRi has been demonstrated to be effective in repressing the expression of target genes in B. subtilis (Wu et al, 2018). As sgRNA has different repression effects at different positions in the gene sequence, it is necessary to select the appropriate repression intensity in the gene sequence (Wu et al, 2018). Presently, the sgRNAs were selected that targeted the 5′ end, middle, and 3′ end positions of each gene sequence (Table S4).…”

“…The gene sequence used for CRISPRi plasmid construction is listed in the Supporting Information. The plasmids used to express the dCas9 protein were previously described (Wu et al, 2018). To construct the recombinant plasmid pHT01_sgRNA, primers psga_sg_F and psga_sg_R were used to amplify the sgRNA expression cassette in the psga plasmid (Wu et al, 2018) and integrated into the pHT01 plasmid using primers pHT01_sg_F and pTH01_sg_R between Sac I and Xba I restriction sites by Gibson Assembly.…”

“…Plasmid pHT01_ pyk 1 containing a sgRNA targeting the non‐template strand of pyk was generated by inverse PCR of pHT01_sgRNA using primers sg‐F and sg‐ pyk 1‐R, and plasmids containing sgRNAs targeting other genes were similarly constructed. Construction of the plasmid containing two or three sgRNAs was performed using Golden Gate assembly (Engler, Gruetzner, Kandzia, & Marillonnet, 2009), as detailed elsewhere (Wu et al, 2018).…”

Secretory Expression Fine-Tuning and Directed Evolution of Diacetylchitobiose Deacetylase by Bacillus subtilis

“…qRT-PCR was performed by using iQ SYBR green Supermix (Bio-Rad, Hercules, CA, USA) and CFX Connect (Bio-Rad). During that, 0.5 ng of cDNA served as the template, and the 16S rRNA gene and hbsU gene served as the internal standard using primer pairs of qPCR16S-F, qPCR16S-R, qPCRhbsU-F, and qPCRhbsU-R. At the same time, the negative control was the reaction without template (34,35), and the conditions were 95°C for 3 min followed by 50 three-step cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s.…”

Impact of CRISPR interference on strain development in biotechnology

“…1). CRISPRi has been developed in a diverse group of bacteria, including E. coli [5] , Corynebacterium glutamicum [7] , Lactococcus lactis [18] , Rhodococcus opacus [19] , Burkholderia [20] , Clostridia [21–25] , Bacillus subtilis [26–29] , Bacillus methanolicus [30] , Streptomyces [31,32], and Mycobacteria [33–36], and applied for gene repression to interrogate their physiology or to identify gene targets for biotech applications (see Sections 3 and 4).…”