Engineering NAD+ availability for Escherichia coli whole-cell biocatalysis: a case study for dihydroxyacetone production

Abstract

Background: Whole-cell redox biocatalysis has been intensively explored for the production of valuable compounds because excellent selectivity is routinely achieved. Although the cellular cofactor level, redox state and the corresponding enzymatic activity are expected to have major effects on the performance of the biocatalysts, our ability remains limited to predict the outcome upon variation of those factors as well as the relationship among them.

Results: In order to investigate the effects of cofactor availability on whole-cell redox biocatalysis, we devised recombinant Escherichia coli strains for the production of dihydroxyacetone (DHA) catalyzed by the NAD+-dependent glycerol dehydrogenase (GldA). In this model system, a water-forming NAD+ oxidase (NOX) and a NAD+ transporter (NTT4) were also co-expressed for cofactor regeneration and extracellular NAD+ uptake, respectively. We found that cellular cofactor level, NAD+/NADH ratio and NOX activity were not only strain-dependent, but also growth condition-dependent, leading to significant differences in specific DHA titer among different whole-cell biocatalysts. The host E. coli DH5α had the highest DHA specific titer of 0.81 g/gDCW with the highest NAD+/NADH ratio of 6.7 and NOX activity of 3900 U. The biocatalyst had a higher activity when induced with IPTG at 37°C for 8 h compared with those at 30°C for 8 h and 18 h. When cells were transformed with the ntt4 gene, feeding NAD+ during the cell culture stage increased cellular NAD(H) level by 1.44 fold and DHA specific titer by 1.58 fold to 2.13 g/gDCW. Supplementing NAD+ during the biotransformation stage was also beneficial to cellular NAD(H) level and DHA production, and the highest DHA productivity reached 0.76 g/gDCW/h. Cellular NAD(H) level, NAD+/NADH ratio, and NOX and GldA activity dropped over time during the biotransformation process.

Conclusions: High NAD+/NADH ratio driving by NOX was very important for DHA production. Once cofactor was efficiently cycled, high cellular NAD(H) level was also beneficial for whole-cell redox biocatalysis. Our results indicated that NAD+ transporter could be applied to manipulate redox cofactor level for biocatalysis. Moreover, we suggested that genetically designed redox transformation should be carefully profiled for further optimizing whole-cell biocatalysis.

Figure 1

Construction of recombinant E. coli strains for DHA production. (A) Schematic representation of the engineered E. coli for DHA production by oxidizing glycerol with NAD⁺ regeneration and uptaking system; (B) Genetic arrangement of plasmids used for DHA production. Trc P, Trc promoter; gnt105 P, gluconate transporter promoter 105 mutation; RBS, ribosome binding site; rrnB T, rrnB terminator; B0015, synthetic artificial terminator B0015 from IGEM.

Figure 2

DHA production by different E. coli strains harboring the plasmid pTrc99A-gldA-nox. Cells were induced by IPTG for 20 h at 30°C, and collected for biotransformation experiments in 5 mL of reaction buffer with 20 g/L glycerol in 50-mL test tubes at 37°C, 200 rpm for 10 h. (A) DHA specific titer; (B) Initial enzymatic activity of GldA and NOX; (C) Initial NAD⁺/NADH ratio; (D) Initial intracellular NAD(H) level. The data represent the averages ± standard deviations (SDs) from three independent clones.

Figure 3

DHA production by E. coli DH5α cells harboring the plasmid pTrc99A-gldA-nox. Cells were cultivated at 30°C or 37°C for 8 h or 18 h after being induced by IPTG, and collected for biotransformation experiments in 5 mL of reaction buffer with 20 g/L glycerol at 37°C, 200 rpm for 10 h. (A) DHA specific titer; (B) Initial enzymatic activity of GldA and NOX; (C) Initial NAD⁺/NADH ratio. All data represent the averages ± SDs for three independent samples.

Figure 4

DHA production by E. coli YJE005 and YJE006. Cells were cultivated at 37°C for 8 h after being induced by IPTG, and collected for biotransformation experiments with 20 g/L glycerol and 0.2 mM NAD⁺ in 5 mL of reaction buffer in 50-mL test tubes at 37°C, 200 rpm for 10 h. (A) DHA specific titer; (B) Initial and end-point enzymatic activity of GldA and NOX; (C) Initial and end-point cellular NAD(H) level. All data represent the averages ± SDs for three independent clones.

Figure 5

DHA production by E. coli YJE006 whole cells in 20 mL of reaction buffer in 500-mL shake-flasks at 37°C, 200 rpm. (A) The time course of DHA formation; (B) Initial and end-point enzymatic activity of GldA and NOX; (C) Initial and end-point cellular NAD(H) level. The data represent the averages standard deviations for three independent samples. All data represent the averages ± SDs for three independent samples.