Model-Driven Redox Pathway Manipulation for Improved Isobutanol Production in Bacillus subtilis Complemented with Experimental Validation and Metabolic Profiling Analysis

Abstract

To rationally guide the improvement of isobutanol production, metabolic network and metabolic profiling analysis were performed to provide global and profound insights into cell metabolism of isobutanol-producing Bacillus subtilis. The metabolic flux distribution of strains with different isobutanol production capacity (BSUL03, BSUL04 and BSUL05) drops a hint of the importance of NADPH on isobutanol biosynthesis. Therefore, the redox pathways were redesigned in this study. To increase NADPH concentration, glucose-6-phosphate isomerase was inactivated (BSUL06) and glucose-6-phosphate dehydrogenase was overexpressed (BSUL07) successively. As expected, NADPH pool size in BSUL07 was 4.4-fold higher than that in parental strain BSUL05. However, cell growth, isobutanol yield and production were decreased by 46%, 22%, and 80%, respectively. Metabolic profiling analysis suggested that the severely imbalanced redox status might be the primary reason. To solve this problem, gene udhA of Escherichia coli encoding transhydrogenase was further overexpressed (BSUL08), which not only well balanced the cellular ratio of NAD(P)H/NAD(P)+, but also increased NADH and ATP concentration. In addition, a straightforward engineering approach for improving NADPH concentrations was employed in BSUL05 by overexpressing exogenous gene pntAB and obtained BSUL09. The performance for isobutanol production by BSUL09 was poorer than BSUL08 but better than other engineered strains. Furthermore, in fed-batch fermentation the isobutanol production and yield of BSUL08 increased by 11% and 19%, up to the value of 6.12 g/L and 0.37 C-mol isobutanol/C-mol glucose (63% of the theoretical value), respectively, compared with parental strain BSUL05. These results demonstrated that model-driven complemented with metabolic profiling analysis could serve as a useful approach in the strain improvement for higher bio-productivity in further application.

NADPH-generating systems in bacteria and archaea

“…Various studies have shown that several bacterial species require STH for growth under metabolic conditions with excess NADPH formation (Canonaco et al, 2001 ; Hua et al, 2003 ; Sauer et al, 2004 ; Zhao et al, 2008 ; Fuhrer and Sauer, 2009 ). Moreover, mutant bacteria suffering from a disturbed redox balance due to an increased NADPH concentration often benefit from the heterologous expression of STH (Boonstra et al, 2000 ; Angermayr et al, 2012 ; Qi et al, 2014 ; Reddy et al, 2015 ). These observations support the idea that the physiological role of STH is to convert NADPH into NADH to prevent an excess of NADPH (Voordouw et al, 1983 ), a notion that is generally accepted and is supported by actual NAD + /NADH and NADP + /NADPH ratios (see above), which indicate that the formation of NADPH from NADH will generally require energy input.…”

Bacillus subtilis SalA is a phosphorylation‐dependent transcription regulator that represses scoC and activates the production of the exoprotease AprE

“…Next, we repeated the electrophoretic mobility-shift assay in the presence of 10 mM ATP. We chose this concentration as representative of high physiological ATP concentration in exponentially growing B. subtilis, based on several recent studies (Kleijn et al, 2010;Qi et al, 2014). In the presence of 10 mM ATP, SalA bound efficiently to the target DNA ( Fig.…”

Solid‐state fermentation of Moringa oleifera leaf meal using Bacillus pumilusCICC 10440

“…Distilled water was then added to obtain a 1:1 leaf powder moisture ratio (w/v). Sealed fermentation was conducted at 30 °C for 4 days, and the Moringa proteins were then extracted …”

“…Sealed fermentation was conducted at 30 ∘ C for 4 days, and the Moringa proteins were then extracted. 25,26 Analytical methods 2 g of fermentation matter was used to determine the protein concentration of the samples at 24 h time points. With this approach, the proteins were extracted from the fermentation matter by adding approximately 10 volumes of distilled water (w/v, 20 mL) to 2 g of fermentation matter and then extracting the samples for 1 h at 60 rpm in the shaker incubator.…”