Dynamic Control Strategy to Produce Riboflavin with Lignocellulose Hydrolysate in the Thermophile Geobacillus thermoglucosidasius

Abstract

The e cient utilization of both glucose and xylose, the two most abundant sugars in biomass hydrolysates, is one of the main objectives of biofermentation with lignocellulosic materials. The utilization of xylose is commonly inhibited by glucose, which is known as glucose catabolite repression (GCR). Here we report a GCR-based dynamic control (GCR-DC) strategy aiming at a better co-utilization of glucose and xylose, by decoupling the cell growth and biosynthesis of ribo avin as a product. Using the thermophilic strain Geobacillus thermoglucosidasius DSM2542 as a host, we constructed extra ribo avin biosynthetic pathways that were activated by xylose but not glucose. The engineered strains showed a two-stage fermentation process. In the rst stage, glucose was preferentially used for cell growth and no production of ribo avin was observed, while in the second stage where glucose was nearly depleted, xylose was effectively utilized for ribo avin biosynthesis. Using the corn cob hydrolysate as a carbon source, the optimized ribo avin yields of strains DSM2542-DCall-MSS (full pathway dynamic control strategy) and DSM2542-DCrib (single module dynamic control strategy) were 5.26 and 2.26 folds higher than that of the control strain DSM2542 Rib-Gtg constitutively producing ribo avin, respectively. This GCR-DC strategy should also be applicable to the construction of cell factories that can e ciently use natural carbon sources with multiple sugar components for the production of high-value chemicals in future.

A highly efficient method for genomic deletion across diverse lengths in thermophilic Parageobacillus thermoglucosidasius

“…To streamline the operation process, we tried to delay gRNA expression by simultaneously adding glucose and xylose to the medium during the inoculation, leveraging the temporal regulation of glucose catabolite repression [ 30 , 31 ]. To assess if the expression of the xylose-inducible promoter could be postponed by co-addition, a plasmid expressing the sfgfp gene under a xylose-inducible promoter was introduced into wild-type strains.…”

A thermostable type I-B CRISPR-Cas system for orthogonal and multiplexed genetic engineering

“…We hypothesized that constitutively expressed crRNA resulted in excessive Cas-mediated cleavage activity that could not be repaired by less efficient DNA recombination mechanisms 34 . To reduce crRNA expression, we used a well-characterized 35 inducible promoter, P xylA (Fig. 4a and Supplementary Table 1 ), which resulted in markedly higher transformation efficiency without inducing crRNA after transformation (Fig.…”

Reprogramming a high robustGeobacillus thermoglucosidasiusfor efficient synthesis of polymer-grade lactic acid under extremely high temperature (60°C)

“…The copyright holder for this preprint this version posted April 14, 2023. ; https://doi.org/10.1101/2023.04.14.536835 doi: bioRxiv preprint promising compounds at elevated temperatures, such as bioethanol 18,47,[52][53][54][55] , isobutanol 56 , 2,3-butanediol 57 , terpenes 32 , and riboflavin 22,58 . However, the production of these compounds is limited to the laboratory and cannot be scaled up for industrial production, mainly because of low product titers.…”

“…Bashir et al engineered G. thermoglucosidasius to produce bioethanol from wheat straw holocellulose 47 . Wang et al explored the utilization of lignocellulose hydrolysates by G. thermoglucosidasius 58 . At present, more studies are needed to improve the utilization of inexpensive carbon sources.…”