Application of inorganic phosphate limitation to efficient isoprene production in Pantoea ananatis

Abstract

Aims Establishment of an efficient isoprene fermentation process by adopting inorganic phosphate limitation as the trigger to direct metabolic flux to the isoprene synthetic pathway. Methods and Results We constructed isoprene‐producing strains of Pantoea ananatis (a member of the Enterobacteriaceae family) by integrating a heterologous mevalonate pathway and a metabolic switch that senses external inorganic phosphate (Pi) levels. This metabolic switch enabled dual‐phase isoprene production, where the initial cell growth phase under Pi‐saturating conditions was uncoupled from the subsequent isoprene production phase under Pi‐limiting conditions. In fed‐batch fermentation using our best strain (SWITCH‐PphoC/pIspSM) in a 1‐l bioreactor, isoprene concentration in the off‐gas was maintained between 300 and 460 ppm during the production phase and at 20 ppm during the cell growth phase, respectively. The strain SWITCH‐PphoC/pIspSM produced totally 2·5 g l−1 of isoprene from glucose with a 1·8% volumetric yield in 48 h. Conclusions This proof‐of‐concept study demonstrated that our Pi‐dependent dual‐phase production system using a P. ananatis strain as a producer has potential for industrial‐scale isoprene fermentation. Significance and Impact of the Study This Pi‐dependent dual‐phase fermentation process could be an attractive and economically viable option for the production of various commercially valuable isoprenoids.

Fermentative production of enantiopure (S)-linalool using a metabolically engineered Pantoea ananatis

“…1). This strain also has a metabolic switch to allow redirection of carbon flux to (S)-linalool via the MVA pathway by sensing P i -starvation with the PhoB/PhoR two-component system [23]. As a result of test-tube cultivation under P i -starved conditions using a biphasic fermentation system, SWITCH-PphoC Δgcd/ pAaLINS-ispA* produced 3.4 ± 0.2 g/L of (S)-linalool from 60 ± 0.0 g/L of glucose in 48 h (Table 1), which was greater than the reported linalool titers from yeasts [12,13,29,30] and bacteria [15,31].…”

“…An external P i -dependent dual-phase fed-batch fermentation process was previously established for isoprene production using the SWITCH-PphoC strain [23]. The P i -starvation-inducible metabolic switch enables cells to grow efficiently under the P i -saturated phase and efficiently produce (S)-linalool under the subsequent P i -starved phase.…”

“…In the mid-1990s, a nonpathogenic P. ananatis strain AJ13355 was isolated by specialists from Ajinomoto Co., Inc. and has been demonstrated to be an excellent host for L-glutamate production because of its capability to grow at an acidic pH and resist the effects of high concentrations of L-glutamate [18]. The well-developed genetic tools [19,20] and sequenced complete genome [18] has broadened the attractiveness of P. ananatis as a production host for various bio-based materials such as cysteine [21], dicarboxylic acids [22], and isoprene [23]. We constructed a metabolically engineered P. ananatis strain named SWITCH-PphoC, which contains heterologous genes of the MVA pathway (mvaE and mvaS from Enterococcus faecalis; MVA kinase gene from Methanocella paludicola [mvk]; phosphomevalonate kinase, diphosphomevalonate decarboxylase, and IPP isomerase genes from S. cerevisiae), to supply IPP/DMAPP for isoprene production [23].…”

“…The well-developed genetic tools [19,20] and sequenced complete genome [18] has broadened the attractiveness of P. ananatis as a production host for various bio-based materials such as cysteine [21], dicarboxylic acids [22], and isoprene [23]. We constructed a metabolically engineered P. ananatis strain named SWITCH-PphoC, which contains heterologous genes of the MVA pathway (mvaE and mvaS from Enterococcus faecalis; MVA kinase gene from Methanocella paludicola [mvk]; phosphomevalonate kinase, diphosphomevalonate decarboxylase, and IPP isomerase genes from S. cerevisiae), to supply IPP/DMAPP for isoprene production [23]. This strain was designed to direct carbon flux to the MVA pathway only under external inorganic phosphate (P i )-starved conditions by using the P i -starvation-inducible phoC promoter for driving the expression of the mvaES operon, which encodes the enzymes that catalyze the conversion of acetyl-coenzyme A (CoA) to MVA.…”

“…In this study, we chose (S)-linalool as the target product and aimed to further improve (S)-linalool production through several approaches: (1) by increasing carbon flux to IPP/DMAPP from acetyl-CoA through enhancement of the upper component of the MVA pathway; (2) by increasing intracellular AaLINS activity with N-terminal fusion of a halophilic β-lactamase (BLA) from Chromohalobacter sp. 560 [27] joined to a hexahistidine (6×His); and (3) by adopting an external P i -dependent so-called "dual-phase" fed-batch fermentation [23], which separates the growth-phase from a subsequent production-phase to increase efficiency [28]. Guided by these approaches, the (S)-linalool titer finally reached 10.9 g/L with a 5.1% [w/w] yield from glucose in the biphasic fermentation system, which demonstrates the potential for industrial-scale enantiopure (S)-linalool production using P. ananatis.…”