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Novel approaches for in vivo evolution, screening and characterization of enzymes for metabolic engineering of Escherichia coli as hyper L-tryptophan producer
Citation Link: https://doi.org/10.15480/882.3204
Publikationstyp
Doctoral Thesis
Date Issued
2020
Sprache
English
Author(s)
Advisor
Referee
Title Granting Institution
Technische Universität Hamburg
Place of Title Granting Institution
Hamburg
Examination Date
2020-11-27
Institut
TORE-DOI
TORE-URI
Citation
Dr. Hut (2020)
Publisher
Dr. Hut
Nowadays, microbes have been extensively optimized for production of L-tryptophan (Trp) from renewable feedstocks. Numerous strategies have been investigated, including tuning the gene expression levels and alleviation of negative regulations. However, it has become apparent that development of a more efficient Trp producer will be inconceivable without broader optimization of the corresponding enzymes. Thus, the present thesis aims to develop new in vivo evolution, screening and characterization methods for optimization of enzymes for Trp production.
A reliable in vivo screening approach is desired to link the mutations to cell growth or to couple the inconspicuous intracellular molecules with a biomarker, for example, the enhanced green fluorescent protein (eGFP). In the first part of this thesis, a novel enzyme screening approach, namely plasmid-assisted growth-coupled and sensor-guided in vivo screening (PGSS), is developed. This approach combines the advantages of complementary auxotrophy-coupled screening with biosensor-driven in vivo characterization. The efficiency of PGSS was first demonstrated for improving an anthranilate (ANTH)-inhibited enzyme TrpC from E. coli (EcTrpC), which is composed of indole glycerol phosphate synthase and N-(5-phosphoribosyl) anthranilate isomerase. Based on a Trp-auxotrophic strain S028ΔEctrpC, a highly efficient ANTH-resistant candidate EcTrpCS58Q-P59V-S60F-K61Q was identified by using the PGSS approach. Afterwards, the PGSS approach was employed to identify an ANTH-activated TrpC from Aspergillus niger (AnTrpC). As a result, an enzyme variant (AnTrpCR378F) that showed increased ANTH activation was discovered. Fed-batch fermentation demonstrated that the strain S028ΔEctrpC containing AnTrpCR378F was able to produce more Trp (35.36 g/L) than the strain containing AnTrpCWT (31.15 g/L), indicating that the variant AnTrpCR378F is more efficient for the Trp pathway.
To overcome limitations of screening performed under non-representative conditions, PGSS is combined with the CRISPR/Cas9 technique, resulting in a novel strategy called CRISPR/Cas9-facilitated engineering with growth-coupled and sensor-guided in vivo screening (CGSS). The efficiency of this method was demonstrated for the optimization of a key enzyme in the chorismate pathway, namely 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase. E. coli possesses three isoenzymes of DAHP synthase: AroG, AroF, and AroH. The aim was to obtain AroG variants with increased resistance against feedback inhibition by L-phenylalanine (Phe). Starting from a Trp-producing E. coli strain (harboring the reference variant AroGS180F), all the endogenous DAHP synthases were removed and the growth of the subsequent strain exhibited dependence on the activity of introduced AroG variants. The different catalytic efficiencies of AroG variants will lead to different intracellular concentrations of Trp, which can be monitored by a Trp biosensor. Taking cell growth rate and the signal strength of a Trp biosensor as selection criteria, several novel Phe-resistant AroG variants with higher activities were identified. The replacement of AroGS180F with the best variant AroGD6G-D7A in a Trp-producing strain significantly improved the Trp production by 38.50%.
A high glucose conversion yield is a key parameter for cost-effective Trp production. Theoretical analysis suggests that activation of galactose permease/glucokinase (GalP/Glk) in a PTS-defective strain could result in an E. coli strain with significantly increased Trp yield. To explore this possibility, a laboratory adaptive evolution (LAE) approach was applied. To this end, a potentially GalP/Glk-dependent E. coli strain G028 was developed, in which the ptsI gene was deleted and a tandem gene circuit with promoter mutation (ptacMT-galP-pJ23119MT-glk) was integrated. Batch LAE of this strain resulted in a promising candidate B3. However, B3 exhibited similar Trp yield and production as S028. One conceivable explanation is that the PTS-defective strain is forced to strengthen their growth rather than Trp synthesis in conventional LAE. Thus, a continuous LAE system (auto-CGSS) was developed which combines CGSS-facilitated in vivo mutagenesis with real-time measurement of cell growth and online monitoring of fluorescence intensity, leading to a new promising candidate strain D8. Fed-batch fermentation with D8 showed an increase of Trp yield by 23.07% compared with that by B3 (0.16 vs. 0.13 g/g).
Finally, two selected gene variants (aroGD6G-D7A and AntrpCR378F) were integrated into the chromosome of Trp-producing strains S028G and D8 to establish highly producing strains S028AARF and D8AA, respectively. These strains were evaluated in fed-batch fermentations. Remarkably, S028AARF reached a very high Trp concentration (51.19 g/L) after 65h of fermentation, which is 19.20% higher than that of the previously reported strain S028GΔfruR:aroGD6G-D7A (42.95 g/L). Fed-batch cultivations of D8AA clones showed strong variations in growth and Trp production. The reason for the variations is not clear. Nevertheless, one of the clones D8AA-1 exhibited a Trp yield as high as 0.20 g/g (vs. 0.19 g/g with S028AARF), representing the highest Trp yield reported in the literature so far and making it attractive for industrial-scale Trp bioproduction.
A reliable in vivo screening approach is desired to link the mutations to cell growth or to couple the inconspicuous intracellular molecules with a biomarker, for example, the enhanced green fluorescent protein (eGFP). In the first part of this thesis, a novel enzyme screening approach, namely plasmid-assisted growth-coupled and sensor-guided in vivo screening (PGSS), is developed. This approach combines the advantages of complementary auxotrophy-coupled screening with biosensor-driven in vivo characterization. The efficiency of PGSS was first demonstrated for improving an anthranilate (ANTH)-inhibited enzyme TrpC from E. coli (EcTrpC), which is composed of indole glycerol phosphate synthase and N-(5-phosphoribosyl) anthranilate isomerase. Based on a Trp-auxotrophic strain S028ΔEctrpC, a highly efficient ANTH-resistant candidate EcTrpCS58Q-P59V-S60F-K61Q was identified by using the PGSS approach. Afterwards, the PGSS approach was employed to identify an ANTH-activated TrpC from Aspergillus niger (AnTrpC). As a result, an enzyme variant (AnTrpCR378F) that showed increased ANTH activation was discovered. Fed-batch fermentation demonstrated that the strain S028ΔEctrpC containing AnTrpCR378F was able to produce more Trp (35.36 g/L) than the strain containing AnTrpCWT (31.15 g/L), indicating that the variant AnTrpCR378F is more efficient for the Trp pathway.
To overcome limitations of screening performed under non-representative conditions, PGSS is combined with the CRISPR/Cas9 technique, resulting in a novel strategy called CRISPR/Cas9-facilitated engineering with growth-coupled and sensor-guided in vivo screening (CGSS). The efficiency of this method was demonstrated for the optimization of a key enzyme in the chorismate pathway, namely 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase. E. coli possesses three isoenzymes of DAHP synthase: AroG, AroF, and AroH. The aim was to obtain AroG variants with increased resistance against feedback inhibition by L-phenylalanine (Phe). Starting from a Trp-producing E. coli strain (harboring the reference variant AroGS180F), all the endogenous DAHP synthases were removed and the growth of the subsequent strain exhibited dependence on the activity of introduced AroG variants. The different catalytic efficiencies of AroG variants will lead to different intracellular concentrations of Trp, which can be monitored by a Trp biosensor. Taking cell growth rate and the signal strength of a Trp biosensor as selection criteria, several novel Phe-resistant AroG variants with higher activities were identified. The replacement of AroGS180F with the best variant AroGD6G-D7A in a Trp-producing strain significantly improved the Trp production by 38.50%.
A high glucose conversion yield is a key parameter for cost-effective Trp production. Theoretical analysis suggests that activation of galactose permease/glucokinase (GalP/Glk) in a PTS-defective strain could result in an E. coli strain with significantly increased Trp yield. To explore this possibility, a laboratory adaptive evolution (LAE) approach was applied. To this end, a potentially GalP/Glk-dependent E. coli strain G028 was developed, in which the ptsI gene was deleted and a tandem gene circuit with promoter mutation (ptacMT-galP-pJ23119MT-glk) was integrated. Batch LAE of this strain resulted in a promising candidate B3. However, B3 exhibited similar Trp yield and production as S028. One conceivable explanation is that the PTS-defective strain is forced to strengthen their growth rather than Trp synthesis in conventional LAE. Thus, a continuous LAE system (auto-CGSS) was developed which combines CGSS-facilitated in vivo mutagenesis with real-time measurement of cell growth and online monitoring of fluorescence intensity, leading to a new promising candidate strain D8. Fed-batch fermentation with D8 showed an increase of Trp yield by 23.07% compared with that by B3 (0.16 vs. 0.13 g/g).
Finally, two selected gene variants (aroGD6G-D7A and AntrpCR378F) were integrated into the chromosome of Trp-producing strains S028G and D8 to establish highly producing strains S028AARF and D8AA, respectively. These strains were evaluated in fed-batch fermentations. Remarkably, S028AARF reached a very high Trp concentration (51.19 g/L) after 65h of fermentation, which is 19.20% higher than that of the previously reported strain S028GΔfruR:aroGD6G-D7A (42.95 g/L). Fed-batch cultivations of D8AA clones showed strong variations in growth and Trp production. The reason for the variations is not clear. Nevertheless, one of the clones D8AA-1 exhibited a Trp yield as high as 0.20 g/g (vs. 0.19 g/g with S028AARF), representing the highest Trp yield reported in the literature so far and making it attractive for industrial-scale Trp bioproduction.
Subjects
L-tryptophan biosynthesis
Protein engineering
Laboratory adaptive evolution
Library screening and characterization
CRISPR/Cas9 technique
Metabolic engineering
DDC Class
500: Naturwissenschaften
600: Technik
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