Abstract
BackgroundMicroorganisms can prioritize the uptake of different sugars depending on their metabolic needs and preferences. When both D-glucose and D-xylose are present in growth media, E. coli cells typically consume D-glucose first and then D-xylose. Similarly, when E. coli BL21(DE3) is provided with both D-glucose and D-xylose under anaerobic conditions, glucose is consumed first, whereas D-xylose is consumed very slowly.ResultsWhen BL21(DE3) was adaptively evolved via subculture, the consumption rate of D-xylose increased gradually. Strains JH001 and JH019, whose D-xylose consumption rate was faster, were isolated after subculture. Genome analysis of the JH001 and JH019 strains revealed that C91A (Q31K) and C740T (A247V) missense mutations in the xylR gene (which encodes the XylR transcriptional activator), respectively, controlled the expression of the xyl operon. RT-qPCR analyses demonstrated that the XylR mutation caused a 10.9-fold and 3.5-fold increase in the expression of the xylA (xylose isomerase) and xylF (xylose transporter) genes, respectively, in the adaptively evolved JH001 and JH019 strains. A C91A adaptive mutation was introduced into a new BL21(DE3) background via single-base genome editing, resulting in immediate and efficient D-xylose consumption.ConclusionsAnaerobically-adapted BL21(DE3) cells were obtained through short-term adaptive evolution and xylR mutations responsible for faster D-xylose consumption were identified, which may aid in the improvement of microbial fermentation technology.
Highlights
Microorganisms can prioritize the uptake of different sugars depending on their metabolic needs and preferences
Different D‐xylose consumption rates in E. coli strains under anaerobic conditions E. coli BL21(DE3), BW25113, C strain, and MG1655 strains were anaerobically grown in a fermentation medium supplemented with D-glucose (12.5 mM) and D-xylose (12.5 mM)
Cells began to uptake D-xylose after depleting the D-glucose; the D-xylose consumption rate varied in a strain-dependent manner
Summary
Different D‐xylose consumption rates in E. coli strains under anaerobic conditions E. coli BL21(DE3), BW25113, C strain, and MG1655 strains were anaerobically grown in a fermentation medium supplemented with D-glucose (12.5 mM) and D-xylose (12.5 mM). When each strain was grown in fermentation media containing only D-xylose (25 mM), the JH001 and JH019 cells exhibited significantly elevated transcript levels of the xylA and xylF genes, which were at least 5 times higher than those in the wild type BL21(DE3) strain (Fig. 4B). These results suggest that D-xylose transporting and metabolizing enzymes are highly expressed in adapted BL21(DE3) cells carrying xylR adaptive mutations. These results indicated that the carB mutation enhanced anaerobic cell growth in the D-xylose medium
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