Engineering a hyperthermophilic archaeon for temperature-dependent product formation

Abstract

Microorganisms growing near the boiling point have enormous biotechnological potential but only recently have molecular engineering tools become available for them. We have engineered the hyperthermophilic archaeon Pyrococcus furiosus, which grows optimally at 100°C, to switch its end products of fermentation in a temperature-controlled fashion without the need for chemical inducers. The recombinant strain (LAC) expresses a gene (ldh) encoding lactate dehydrogenase from the moderately thermophilic Caldicellulosiruptor bescii (optimal growth temperature [T(opt)] of 78°C) controlled by a "cold shock" promoter that is upregulated when cells are transferred from 98°C to 72°C. At 98°C, the LAC strain fermented sugar to produce acetate and hydrogen as end products, and lactate was not detected. When the LAC strain was grown at 72°C, up to 3 mM lactate was produced instead. Expression of a gene from a moderately thermophilic bacterium in a hyperthermophilic archaeon at temperatures at which the hyperthermophile has low metabolic activity provides a new perspective to engineering microorganisms for bioproduct and biofuel formation.

Importance: Extremely thermostable enzymes from microorganisms that grow near or above the boiling point of water are already used in biotechnology. However, the use of hyperthermophilic microorganisms themselves for biotechnological applications has been limited by the lack of their genetic accessibility. Recently, a genetic system for Pyrococcus furiosus, which grows optimally near 100°C, was developed in our laboratory. In this study, we present the first heterologous protein expression system for a microorganism that grows optimally at 100°C, a first step towards the potential expression of genes involved in biomass degradation or biofuel production in hyperthermophiles. Moreover, we developed the first system for specific gene induction in P. furiosus. As the cold shock promoter for protein expression used in this study is activated at suboptimal growth temperatures of P. furiosus, it is a powerful genetic tool for protein expression with minimal interference of the host's metabolism and without the need for chemical inducers.

FIG 1

Recombinant expression of lactate dehydrogenase (LDH) in P. furiosus strain LAC changes its fermentation pattern. (A) Concept of temperature-dependent switch in end product formation by P. furiosus. Abbreviations: GAPOR, glyceraldehyde-3-phosphate ferredoxin oxidoreductase; POR, pyruvate ferredoxin oxidoreductase; Fd, ferredoxin; acetyl-CoA, acetyl coenzyme A; Cbes LDH, C. bescii LDH. (B) Specific activity of lactate dehydrogenase in the protein extract of C. bescii DSM 6725, P. furiosus DSM 3638 (wild type), P. furiosus ΔpdaD host strain, and P. furiosus LAC obtained from 400-ml batch cultures. (C) Lactate production in the same P. furiosus cultures. Values given are averages ± standard deviations (SD) (error bars) of three independent biological cultures.

FIG 2

Plasmid vector pMPF301 containing the pdaD P_cipACbes-ldh cassette, 1-kb upstream and downstream flanking regions of the pdaD gene and the apr gene as a selective marker in Escherichia coli (apramycin resistance). Plasmid diagrams were constructed using Vector NTI software (Invitrogen).

FIG 3

Cloning strategy for the mutant strain P. furiosus LAC. The fusion product P_cipACbes-ldh was obtained by overlapping PCR and integrated into vector pSPF300 (11). The new vector, pMPF301 (Fig. 2), additionally carried the pdaD gene essential for agmatine biosynthesis and 1-kb upstream and downstream flanking regions of the pdaD gene. Linearized DNA was used for transformation of the P. furiosus ΔpdaD host strain. The pdaD P_cipACbes-ldh cassette integrated into the genome by homologous recombination, replacing the P_gdhpyrF cassette. Therefore, the resulting new strain, P. furiosus LAC, exhibits a uracil auxotrophy, but does not, in contrast to the host, require agmatine for growth.

FIG 4

(A and B) Lactate production (blue squares), acetate production (green triangles), cell density (red circles), and relative mRNA fold expression levels (broken lines) in 15-liter fermentor cultures of P. furiosus LAC. One culture was grown at 72°C (A), while another culture was grown at 94°C and rapidly cooled to 72°C after a cell density of 1.5 × 10⁸ was reached (indicated by the black arrow) (B). After the temperature switch, higher mRNA levels for the heterologous gene Cbes-ldh, high specific activity of lactate dehydrogenase, and a high rate of lactate formation were observed.

FIG 5

Recombinant expression and activity of C. bescii lactate dehydrogenase in P. furiosus at different temperatures. (A and B) Cell density (A) and relative Cbes-ldh mRNA level and specific activity of lactate dehydrogenase (LDH) (B) in cell extracts of P. furiosus LAC grown at different temperatures (for 72°C, n = 2; for 98°C, n = 1). Although growth was negligible at 72°C and 75°C, the highest ldh mRNA level and lactate dehydrogenase activities were observed at these growth temperatures. (C and D) Thermostability (C) and temperature dependence of lactate dehydrogenase activity (D) in protein extracts of C. bescii DSM 6725 (native LDH) and P. furiosus strain LAC (recombinant LDH) grown at 75°C and harvested in the stationary phase. Values given are averages ± SD of three independent biological cultures (B) or three independent enzymatic measurements (D), unless denoted otherwise. n.d., not determined.