Carbon flux distribution in the metabolism of Shimwellia blattae (p424IbPSO) for isobutanol production from glucose as function of oxygen availability

Abstract

BACKGROUND: The carbon flux distribution in Shimwellia blattae (p424IbPSO) cultures when glucose is employed as carbon source is studied in a stirred tank bioreactor changing the oxygen availability. The strain has been constructed to produce isobutanol combining the branched-chain amino acids pathway and Ehrlich pathway. The information of many similar genetic modified strains for this bioprocess in the literature presents significant discrepancies on the oxygen influence. RESULTS:The study carried out in this work pointed out the presence of other metabolites formed under aerobic conditions, such as isobutyric acid, 2,3-butanedione, acetoin, 2,3-butanediol and 3-oxo-1-buten-2-yl ether, not previously reported in the literature on the production of isobutanol with S. blattae (p424IbPSO) strain. CONCLUSION: In this work the routes or the chemical reactions involved in the bioprocess as a function of the availability of oxygen are elucidated. Anaerobic conditions are the best for isobutanol production from glucose employing this strain; around 12 g L −1 of isobutanol are obtained under these conditions. Aerobic conditions are not adequate to produce isobutanol, although the need for oxygen, at least for growth, is also demonstrated. Experimental procedure and set-upExperiments were carried out in a stirred tank bioreactor (STBR), BIOSTAT B-PLIS (Sartorius AG, Göttingen, Germany). The above described culture medium M9-2x was employed in all runs.The initial temperature of culture was 37 ∘ C, but after 2 h of growth the temperature was changed to 30 ∘ C and 1 mmol L -1 Isopropyl--D-1-thiogalactopyranoside (IPTG) was added to induce the expression of genes encoding the isobutanol synthetic pathway. The initial pH was fixed at 6.8, decreasing with time during growth; when pH reached a value of 6.0, it was controlled by the addition of 2 mol L -1 NaOH. 39 The airflow rate was set at 1 L L −1 min −1 from the beginning of the run and the stirrer speed was varied from 150 to 600 rpm in the different runs performed in aerobic conditions in order to change the oxygen transfer rate in J Chem Technol Biotechnol 2019; 94: 850-858 Analysis by GC-mass spectroscopyThis analysis was performed to determine the structures of the new compounds present in the S2 sample in comparison with the S1 sample. All the peaks appearing in the GC-MS chromatograms for both samples, S1 and S2, were identified. Table 3 gives the J Chem Technol Biotechnol 2019; 94: 850-858

The pyruvate decarboxylase activity of IpdC is a limitation for isobutanol production by Klebsiella pneumoniae

“…Kp-IpdC had been identified to catalyse the 2-ketoisovalerate decarboxylation reaction in K. pneumoniae . While all artificial isobutanol synthesis pathways using KivD from L. lactis to catalyse this decarboxylation reaction [ 9 , 27 – 30 ]. Initially Kp-IpdC and KivD were both expressed in K. pneumoniae Δ budA –Δ ldhA without induction of IPTG.…”

Kinetic Modeling of the Isobutanol Production from Glucose Using Shimwellia blattae (p424IbPSO) Strain: Effect of Initial Substrate Concentration

“…The runs were carried out at an initial temperature of 37 °C employing an agitation of 250 rpm. After 2 h of bacterial growth, the temperature was changed to 30 °C and IPTG (1 mM) was added to the broth to induce the plasmid expression according to Acedos et al At the same time, the bioreactor was closed, in order to prevent the isobutanol evaporation and to maintain a low oxygen availability, according to the optimal conditions previously determined. , The initial pH value was 6.8, being maintained at a value of 6.0 by adding 2 M NaOH …”

“…This is due to the fact that growth rate only depends on oxygen availability. The DO concentration is practically the same in the five runs performed: At a time of 2 h the reactor is closed and air is not bubbled inside, in order to get the microaerophilic conditions appropriate for isobutanol production as determined elsewhere. ,, According to these results, it is logical to consider that the kinetic parameters related to the growth rate are not affected by the initial glucose concentration.…”

“…According to these previous works, the best systems to produce isobutanol with this strain are in closed recipients, including STBRs, in order to have a low dissolved oxygen concentration in the broth, which after some minutes is totally consumed. If higher concentration of dissolved oxygen is available, the carbon flux is diverted and other compounds are produced . In order to avoid hydrodynamic stress, it has been determined that 600 rpm is the optimum stirring speed to use.…”

Modulating redox metabolism to improve isobutanol production in Shimwellia blattae

“…2-Keto acids then are converted to aldehydes by heterologous broad-substrate-range 2-keto-acid decarboxylases (KDCs) and then to alcohols by alcohol dehydrogenases (ADHs) [7,9]. The main limitations of this route are the NADPH/NADH imbalance [18,23,24] and the synthesis of by-products [25] (Figs. 1 and 2).…”