High-throughput enrichment of temperature-sensitive argininosuccinate synthetase for two-stage citrulline production in E. coli

Abstract

Controlling metabolism of engineered microbes is important to modulate cell growth and production during a bioprocess. For example, external parameters such as light, chemical inducers, or temperature can act on metabolism of production strains by changing the abundance or activity of enzymes. Here, we created temperature-sensitive variants of an essential enzyme in arginine biosynthesis of Escherichia coli (argininosuccinate synthetase, ArgG) and used them to dynamically control citrulline overproduction and growth of E. coli. We show a method for high-throughput enrichment of temperature-sensitive ArgG variants with a fluorescent TIMER protein and flow cytometry. With 90 of the thus derived ArgG variants, we complemented an ArgG deletion strain showing that 90% of the strains exhibit temperature-sensitive growth and 69% of the strains are auxotrophic for arginine at 42 °C and prototrophic at 30 °C. The best temperature-sensitive ArgG variant enabled precise and tunable control of cell growth by temperature changes. Expressing this variant in a feedback-dysregulated E. coli strain allowed us to realize a two-stage bioprocess: a 33 °C growth-phase for biomass accumulation and a 39 °C stationary-phase for citrulline production. With this two-stage strategy, we produced 3 g/L citrulline during 45 h cultivation in a 1-L bioreactor. These results show that temperature-sensitive enzymes can be created en masse and that they may function as metabolic valves in engineered bacteria.

Keywords: Citrulline overproduction; Flow cytometry; Single-cell growth; Temperature-sensitive enzymes; Two-stage bioprocess.

Graphical abstract

Fig. 1

High-throughput enrichment of temperature-sensitive ArgG variants. (a) ArgG catalyzes the seventh reaction in the arginine biosynthesis pathway and converts citrulline into argininosuccinate (argsucc). asp, aspartate. (b) A plasmid library with mutagenized argG was generated with error-prone-PCR and used to complement an argG knockout strain of E. coli BW25113. The strain also carried a plasmid with the single-cell growth rate reporter TIMER. The pooled strain library was first incubated at 30 °C to enrich ArgG variants that are catalytically active and support growth. Subsequently, the culture was shifted to 42 °C to select non-growing cells with FACS based on the green/red signal of the TIMER protein. (c) Red and green fluorescence (top) of cells after 6 h culturing at 42 °C. FACS was used to isolate the fraction with a low green/red ratio (shown in blue). Histogram (bottom) showing the distribution of cells according to the green to red ratio. (d) Growth at 30 °C and 42 °C of 90 single strains isolated from the fraction with a low green/red ratio (blue fraction in Fig. 1c). Strains that did not reach an OD of 0.5 at 42 °C are shown in blue. A control strain expressing wild-type ArgG is shown in red. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

Temperature-dependent arginine auxotrophy and mutations of nine ArgG variants. (a) Growth of nine strains with ArgG variants at 42 °C with supplementation of arginine (black lines) and without (blue lines). Lines show means and shades the standard deviation of n = 3 plate reader cultures. (b) Mutations of the nine ArgG variants. Binding sites of ATP, citrulline, and aspartate are shown in green. Non-synonymous mutations are red, synonymous are blue. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

Overproduction of citrulline in an argG deletion strain. (a) Arginine biosynthesis in E. coli is feedback regulated by arginine at the level of transcription (ArgR) and allosteric control of the first enzyme (ArgA). The transcription factor ArgR regulates the expression of all genes in the pathway. The activity of the protein ArgA, which catalyzes the first reaction in the pathway, is directly regulated by allosteric interaction with arginine. Engineering targets are shown in red: deletion of argG, deletion of argR, H15Y mutation removes allosteric inhibition of ArgA. glu, L-glutamate; acglu, N-acetyl-L-glutamate; acgluP, N-acetylglutamyl-phosphate; acglu5s, N-acetyl-L-glutamate 5-semialdehyde; acorn, N-acetyl-L-ornithine; orn, L-ornithine; argsucc, L-arginino-succinate; acCoA, acetyl-coenzyme-A; (b) Citrulline concentration in the whole cultivation broth of three argG deletion strains after removing arginine from the cultivation medium. Blue: a strain with only argG deletion, Orange: a strain with argG deletion and additional deletion of the transcriptional repressor argR. Green: a strain with deletion of argG, argR and a point mutation (H15Y) in argA that removes inhibition of ArgA by arginine. Specific citrulline production rates were calculated by regression analysis in the three time intervals. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

Growth and citrulline production of the doubly dysregulated citrulline producer in combination with the nine ArgG variants. (a) Schematic of a temperature-sensitive metabolic valve at ArgG. (b) Schematic of the experimental setup to screen citrulline production and growth of nine ArgG variants at different temperatures. (c) The nine ArgG variants were expressed in the doubly dysregulated citrulline producer (ΔargG ΔargR argA^H15Y). Shown is the OD (top) after 7 h cultivation in minimal medium at 30 °C and 42 °C. All cultures started at an OD of 0.05. Error bars show the standard deviation of n = 3 cultures. Biomass specific citrulline concentration (bottom) after 7 h cultivation in minimal medium at 30 °C and 42 °C. Error bars show the standard deviation of n = 3 cultures. (d) In vitro enzymatic assays with purified ArgG variant G9 (top) and wild-type ArgG (bottom) at different temperatures (n = 2 enzyme assays, proteins purified 2 times). Shown is the formation of the reaction product (argininosuccinate) after starting the reaction at t = 0 min. Specific enzyme activities were calculated with linear regression. (e) Growth of the doubly dysregulated citrulline producer (ΔargG ΔargR argA^H15Y) expressing the ArgG variant G9 (top), and wild-type ArgG (bottom). Colors indicate different temperatures. Dots are means, and error bars show the difference between n = 2 cultures. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

Proteome data of the doubly dysregulated citrulline producer (ΔargG ΔargR argA^H15Y) expressing the ArgG variant G9 at different temperatures (33 °C, 39 °C, 42 °C). Data is normalized to the proteome of exponentially growing wild-type cells at 37 °C. Dots are the mean of independent replicates (n = 3). (a) Relative abundance of heat-shock proteins: IbpB, IbpA, DnaJ, GroS, DnaK, FxsA, GroL, ClpB, HtpX, HtpG, GrpE, Lon, YcjF, PrlC, HslV, MutM, HslU, YbbN, YbeZ, RpoD, YbeD, YcjX, LdhA, ClpP, ClpX, HslJ. Red lines indicate the medians. Boxes indicate the 25th and 75th percentiles. (b) Relative abundance of enzymes in the arginine biosynthesis pathway as well as CarA and CarB. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 6

Two-stage production of citrulline with the ArgG variant G9. (a) The doubly dysregulated citrulline producer (ΔargG ΔargR argA^H15Y) expressing the ArgG variant G9 was cultivated in two independent 1-L bioreactors. OD and the temperature are shown for bioreactor 1 (orange) and bioreactor 2 (green). t₁ and t₂ indicate the time window when temperature was increased from 33 °C to 39 °C. t₃ indicates the time when glucose and ammonium was fed. blue area: growth phase, red area: production phase. (b) Glucose concentration in the supernatant of the two bioreactors. Dots are the mean, and error bars are the standard deviation of n = 4 analytical replicates per bioreactor. (c) Citrulline concentration in the whole cultivation broth of the two bioreactors. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)