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Rapid Enabling of Gluconobacter oxydans Resistance to High D-Sorbitol Concentration and High Temperature by Microdroplet-Aided Adaptive Evolution.

ABSTRACT: Gluconobacter oxydans is important in the conversion of D-sorbitol into l-sorbose, which is an essential intermediate for industrial-scale production of vitamin C. In a previous study, the strain G. oxydans WSH-004 could directly produce 2-keto-l-gulonic acid (2-KLG). However, its D-sorbitol tolerance was poor compared with that of other common industrial G. oxydans strains, which grew well in the presence of more than 200 g/L of D-sorbitol. This study aimed to use the microbial microdroplet culture (MMC) system for the adaptive evolution of G. oxydans WSH-004 so as to improve its tolerance to high substrate concentration and high temperature. A series of adaptively evolved strains, G. oxydans MMC1-MMC10, were obtained within 90 days. The results showed that the best strain MMC10 grew in a 300 g/L of D-sorbitol medium at 40°C. The comparative genomic analysis revealed that genetic changes related to increased tolerance were mainly in protein translation genes. Compared with the traditional adaptive evolution method, the application of microdroplet-aided adaptive evolution could improve the efficiency in terms of reducing time and simplifying the procedure for strain evolution. This research indicated that the microdroplet-aided adaptive evolution was an effective tool for improving the phenotypes with undemonstrated genotypes in a short time.

SUBMITTER: Liu L

PROVIDER: S-EPMC8446438 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND:Gluconobacter oxydans is a strictly aerobic Gram-negative acetic acid bacterium used industrially for oxidative biotransformations due to its exceptional type of catabolism. It incompletely oxidizes a wide variety of carbohydrates regio- and stereoselectively in the periplasm using membrane-bound dehydrogenases with accumulation of the products in the medium. As a consequence, only a small fraction of the carbon and energy source enters the cell, resulting in a low biomass yield. Additionally, central carbon metabolism is characterized by the absence of a functional glycolysis and absence of a functional tricarboxylic acid (TCA) cycle. Due to these features, G. oxydans is a highly interesting model organism. Here we analyzed global mRNA decay in G. oxydans to describe its characteristic features and to identify short-lived mRNAs representing potential bottlenecks in the metabolism for further growth improvement by metabolic engineering. RESULTS:Using DNA microarrays we estimated the mRNA half-lives in G. oxydans. Overall, the mRNA half-lives ranged mainly from 3 min to 25 min with a global mean of 5.7 min. The transcripts encoding GroES and GroEL required for proper protein folding ranked at the top among transcripts exhibiting both long half-lives and high abundance. The F-type H+-ATP synthase transcripts involved in energy metabolism ranked among the transcripts with the shortest mRNA half-lives. RNAseq analysis revealed low expression levels for genes of the incomplete TCA cycle and also the mRNA half-lives of several of those were short and below the global mean. The mRNA decay analysis also revealed an apparent instability of full-length 23S rRNA. Further analysis of the ribosome-associated rRNA revealed a 23S rRNA fragmentation pattern exhibiting new cleavage regions in 23S rRNAs which were previously not known. CONCLUSIONS:The very short mRNA half-lives of the H+-ATP synthase, which is likely responsible for the ATP-proton motive force interconversion in G. oxydans under many or most conditions, is notably in contrast to mRNA decay data from other bacteria. Together with the short mRNA half-lives and low expression of some other central metabolic genes it could limit intended improvements of G. oxydans' biomass yield by metabolic engineering. Also, further studies are needed to unravel the multistep process of the 23S rRNA fragmentation in G. oxydans.

Dataset Information

Rapid Enabling of Gluconobacter oxydans Resistance to High D-Sorbitol Concentration and High Temperature by Microdroplet-Aided Adaptive Evolution.

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