Efficient Biofilm-Based Fermentation Strategies for L-Threonine Production by Escherichia coli

Abstract

Biofilms provide cells favorable growth conditions, which have been exploited in industrial biotechnological processes. However, industrial application of the biofilm has not yet been reported in Escherichia coli, one of the most important platform strains, though the biofilm has been extensively studied for pathogenic reasons. Here, we engineered E. coli by overexpressing the fimH gene, which successfully enhanced its biofilm formation under industrial aerobic cultivation conditions. Subsequently, a biofilm-based immobilized fermentation strategy was developed. L-threonine production was increased from 10.5 to 14.1 g/L during batch fermentations and further to 17.5 g/L during continuous (repeated-batch) fermentations with enhanced productivities. Molecular basis for the enhanced biofilm formation and L-threonine biosynthesis was also studied by transcriptome analysis. This study goes beyond the conventional research focusing on pathogenic aspects of E. coli biofilm and represents a successful application case of engineered E. coli biofilm to industrial processes.

Keywords: Escherichia coli; L-threonine; biofilm; fimH gene; transcriptome analysis.

FIGURE 1

A schematic illustration of constructions for the overexpression or knockout of fimH in Escherichia coli W1688 and the effects on the biofilm formation.

FIGURE 2

Quantitative analysis for biofilm formation in the three strains with different characterization methods. (A) Crystal violet staining for LB medium and fermentation medium; (B) Scanning electron microscope (SEM) images of biofilm formation and cell adhesion under different magnification levels; and (C) Fluorescence microscope images of biofilm and cell adhesion under different magnification levels. I: E. coli W1688, II: E. coli W1688-fimH^*, and III: E. coli W1688-ΔfimH.

FIGURE 3

L-threonine production and glucose consumption in batch fermentation by E. coli W1688, E. coli W1688-fimH^*, and E. coli W1688-ΔfimH.

FIGURE 4

(A) L-threonine production by E. coli W1688-fimH^* in continuous immobilized fermentation. (B) Comparison of L-threonine productivities in continuous (repeated batch) immobilized fermentation with those in free-cell fermentation.

FIGURE 5

Scanning electron microscopy images of carrier in the immobilized fermentation by three different strains. I: fresh carrier, II: E. coli W1688, III: E. coli W1688-fimH^*, IV: E. coli W1688-ΔfimH.

FIGURE 6

(A) Transcriptome analysis of genes involved in biofilm biosynthesis pathway in E. coli W1688, E. coli W1688-fimH^*, and E. coli W1688-ΔfimH. Red and blue indicate up- and down-regulated genes, respectively. (B) qRT-PCR verification of the genes related to biofilm biosynthesis. Values and error bars represent the mean and the s.d. (n = 3). ^∗∗∗p < 0.001, ^∗∗p < 0.01, ^*p < 0.05 as determined by two-tailed t-test.

FIGURE 7

(A) Transcriptome analysis of L-threonine biosynthesis pathway genes in three different strains and the expression levels of these genes in E. coli W1688-fimH^*. Red and blue indicate up- and down-regulated genes, respectively. (B) qRT-PCR verification of the genes related to L-threonine biosynthesis and transportation. Values and error bars represent the mean and the s.d. (n = 3). ^∗∗∗p < 0.001, ^∗∗p < 0.01, ^*p < 0.05 as determined by two-tailed t-test.