Precise tuning of the glyoxylate cycle in Escherichia coli for efficient tyrosine production from acetate

Abstract

Background: Acetate is one of promising feedstocks owing to its cheap price and great abundance. Considering that tyrosine production is gradually shifting to microbial production method, its production from acetate can be attempted to further improve the economic feasibility of its production.

Results: Here, we engineered a previously reported strain, SCK1, for efficient production of tyrosine from acetate. Initially, the acetate uptake and gluconeogenic pathway were amplified to maximize the flux toward tyrosine. As flux distribution between glyoxylate and TCA cycles is critical for efficient precursor supplementation, the activity of the glyoxylate cycle was precisely controlled by expression of isocitrate lyase gene under different-strength promoters. Consequently, the engineered strain with optimal flux distribution produced 0.70 g/L tyrosine with 20% of the theoretical maximum yield which are 1.6-fold and 1.9-fold increased values of the parental strain.

Conclusions: Tyrosine production from acetate requires precise tuning of the glyoxylate cycle and we obtained substantial improvements in production titer and yield by synthetic promoters and 5' untranslated regions (UTRs). This is the first demonstration of tyrosine production from acetate. Our strategies would be widely applicable to the production of various chemicals from acetate in future.

Keywords: Acetate; Gluconeogenesis; Glyoxylate cycle; Metabolic engineering; Synthetic biology; Tyrosine.

Fig. 1

Overall strategy used in this study. a To produce tyrosine from acetate, the SCK1 strain, with amplification of the pathway from PEP to tyrosine, was used [17]. To further amplify the linear pathway from acetate to PEP, acs (encoding acetyl-CoA synthetase) and pck (encoding phosphoenolpyruvate synthase) were overexpressed. Thereafter, the glyoxylate cycle pathway was precisely controlled by varying the expression of aceA (encoding isocitrate lyase). b A schematic showing the pathway optimization strategy. Acetyl-CoA produced from acetate can be metabolized via two different pathways: oxidation into CO₂ and generation of ATP and NADH via TCA cycle, or assimilation as metabolites and cell biomass via the glyoxylate cycle. Replenishment of precursors (PEP from OAA) and energy consumption for tyrosine production should be considered for efficient tyrosine production. This can be implemented by precise control of the glyoxylate cycle. OAA oxaloacetate, CIT citrate, ICT isocitrate, α-KG α-ketoglutarate, SUC succinate, MAL malate, GLY glyoxylate, PEP phosphoenolpyruvate, G-6-P glucose-6-phosphate, E-4-P erythrose-4-phosphate, DHAP dihydroxyacetone phosphate, TYR tyrosine

Fig. 2

Fermentation profile of the SCK1 strain. The left y-axis shows OD₆₀₀ and the right y-axis indicates concentration of the accumulated tyrosine; the y-offset indicates the concentration of the remaining acetate. The x-axis denotes time. Symbols: black circle, cell biomass (OD₆₀₀); red squares, tyrosine; blue diamonds, acetate. Error bars indicate the standard deviation from three independent cultures

Fig. 3

Effect of the expression of acs and pck. Cell biomass (a) and tyrosine production (b) after 30 h cultivation. Error bars indicate the standard deviation from three independent cultures

Fig. 4

Effect of glyoxylate cycle activation. Comparison of normalized specific isocitrate lyase activity measured at 12 h (a), and cell biomass (b) and tyrosine production (c) after 30 h cultivation, and the fermentation profile of the SCKAPG4 strain (d). The left y-axis shows OD₆₀₀ and the right y-axis indicates the concentration of accumulated tyrosine; the y-offset indicates the concentration of the remaining acetate. The x-axis denotes time. Symbols: black circle, cell biomass (OD₆₀₀); red squares, tyrosine; blue diamonds, acetate. Error bars indicate the standard deviation from three independent cultures