Dataset Information

Efficient anaerobic consumption of D-xylose by E. coli BL21(DE3) via xylR adaptive mutation.

ABSTRACT:

Background

Microorganisms 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.

Results

When 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.

Conclusions

Anaerobically-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.

SUBMITTER: Heo JM

PROVIDER: S-EPMC8647362 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND:Escherichia coli is one of the most widely used hosts for recombinant protein production in academia and industry. Strain BL21(DE3) is frequently employed due to its advantageous feature of lacking proteases which avoids degradation of target protein. Usually it is used in combination with the T7-pET system where induction is performed by one point addition of IPTG. We recently published a few studies regarding lactose induction in BL21(DE3) strains. BL21(DE3) can only take up the glucose-part of the disaccharide when fed with lactose. However, initially additional glucose has to be supplied as otherwise the ATP-related lactose uptake barely happens. Yet, as lactose is an inexpensive compound compared to glucose and IPTG, a new induction strategy by a lactose-only feed during induction seems attractive. Thus, we investigated this idea in the galactose metabolizing strain HMS174(DE3). RESULTS:We show that strain HMS174(DE3) can be cultivated on lactose as sole carbon source during induction. We demonstrate that strain HMS174(DE3) exhibits higher product and biomass yields compared to BL21(DE3) when cultivated in a lactose fed-batch. More importantly, HMS174(DE3) cultivated on lactose even expresses more product than BL21(DE3) in a standard IPTG induced glucose fed-batch at the same growth rate. Finally, we demonstrate that productivity in HMS174(DE3) lactose-fed batch cultivations can easily be influenced by the specific lactose uptake rate (qs,lac). This is shown for two model proteins, one expressed in soluble form and one as inclusion body. CONCLUSIONS:As strain HMS174(DE3) expresses even slightly higher amounts of target protein in a lactose fed-batch than BL21(DE3) in a standard cultivation, it seems a striking alternative for recombinant protein production. Especially for large scale production of industrial enzymes cheap substrates are essential. Besides cost factors, the strategy allows straight forward adjustment of specific product titers by variation of the lactose feed rate.

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