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When modifying the metabolism of living organisms with the aim of achieving biosynthesis of useful compounds, it is essential to ensure that it is possible to achieve overall redox balance. We propose a generalized strategy for this, based on fine-tuning of respiration. The strategy was applied on metabolically engineered Lactococcus lactis strains to optimize the production of acetoin and (R,R)-2,3-butanediol (R-BDO). In the absence of an external electron acceptor, a surplus of two NADH per acetoin molecule is produced. We found that a fully activated respiration was able to efficiently regenerate NAD + , and a high titer of 371mM (32g/L) of acetoin was obtained with a yield of 82% of the theoretical maximum. Subsequently, we extended the metabolic pathway from acetoin to R-BDO by introducing the butanediol dehydrogenase gene from Bacillus subtilis. Since one mole of NADH is consumed when acetoin is converted into R-BDO per mole, only the excess of NADH needs to be oxidized via respiration. Either by fine-tuning the respiration capacity or by using a dual-phase fermentation approach involving a switch from fully respiratory to non-respiratory conditions, we obtained 361mM (32g/L) R-BDO with a yield of 81% or 365mM (33g/L) with a yield of 82%, respectively. These results demonstrate the great potential in using finely-tuned respiration machineries for bio-production., (Copyright © 2017 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

The physiological roles of silent genes are still unsolved puzzles and their application potentials are unexplored. Herein, a silent glycerol catabolism pathway encoded by glp system was activated in a Klebsiella pneumoniae mutant, of which the acetoin degradation pathway was blocked. Surprisingly, the activation produced significant effects on cellular metabolism and over 90% of the carbon flux was redirected to acetoin biosynthesis, which was formerly a minor product in this mutant. Transcription analyses suggest that the genes involved in acetoin, 1,3-propanediol and adenosyl-cobalamin biosynthesis were differentially regulated upon the glp system activation, demonstrating the cross-talk between silent and active pathways. Through pathway, cofactor and bioprocess engineering, high-level acetoin production from glycerol (32.2gL -1 , 90.8% of the theoretical value) was achieved. Our findings suggest that some silent genes represent unexplored switches of cellular metabolic status and activating them may be an easy strategy for reprogramming microorganisms into efficient cell factories., (Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Fermentative redox balance has long been utilized as a metabolic evolution platform to improve efficiency of NADH-dependent pathways. However, such system relies on the complete recycling of NADH and may become limited when the target pathway results in excess NADH stoichiometrically. In this study, endogenous capability of Escherichia coli for 2,3-butanediol (2,3-BD) synthesis was explored using the anaerobic selection platform based on redox balance. To address the issue of NADH excess associated with the 2,3-BD pathway, we devised a substrate-decoupled system where a pathway intermediate is externally supplied in addition to the carbon source to decouple NADH recycling ratio from the intrinsic pathway stoichiometry. In this case, feeding of the 2,3-BD precursor acetoin effectively restored anaerobic growth of the mixed-acid fermentation mutant that remained otherwise inhibited even in the presence of a functional 2,3-BD pathway. Using established 2,3-BD dehydrogenases as model enzyme, we verified that the redox-based selection system is responsive to NADPH-dependent reactions but with lower sensitivity. Based on this substrate-decoupled selection scheme, we successfully identified the glycerol/1,2-propanediol dehydrogenase (Ec-GldA) as the major enzyme responsible for the acetoin reducing activity (k cat /K m ≈0.4mM -1 s -1 ) observed in E. coli. Significant shift of 2,3-BD configuration upon withdrawal of the heterologous acetolactate decarboxylase revealed that the endogenous synthesis of acetoin occurs via diacetyl. Among the predicted diacetyl reductase in E. coli, Ec-UcpA displayed the most significant activity towards diacetyl reduction into acetoin (V max ≈6U/mg). The final strain demonstrated a meso-2,3-BD production titer of 3g/L without introduction of foreign genes. The substrate-decoupled selection system allows redox balance regardless of the pathway stoichiometry thus enables segmented optimization of different reductive pathways through enzyme bioprospecting and metabolic evolution., (Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

The hyperthermophilic archaeon, Pyrococcus furiosus, grows optimally near 100°C by fermenting sugars to acetate, carbon dioxide and molecular hydrogen as the major end products. The organism has recently been exploited to produce biofuels using a temperature-dependent metabolic switch using genes from microorganisms that grow near 70°C. However, little is known about its metabolism at the lower temperatures. We show here that P. furiosus produces acetoin (3-hydroxybutanone) as a major product at temperatures below 80°C. A novel type of acetolactate synthase (ALS), which is involved in branched-chain amino acid biosynthesis, is responsible and deletion of the als gene abolishes acetoin production. Accordingly, deletion of als in a strain of P. furiosus containing a novel pathway for ethanol production significantly improved the yield of ethanol. These results also demonstrate that P. furiosus is a potential platform for the biological production of acetoin at temperatures in the 70-80°C range., (Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Acetoin, a valuable compound, has high potential as a biochemical building block. In this study, subcellular metabolic engineering was applied to engineer the mitochondrion of Candida glabrata for acetoin production. With the aid of mitochondrial targeting sequences, a heterologous acetoin pathway was targeted into the mitochondria to increase the enzyme concentrations and level of intermediate, followed by coupling with the mitochondrial pyruvate carrier (MPC) to increase the availability of mitochondrial pyruvate. As a result, the strain comprising the combination of the mitochondrial pathway and MPC could yield approximately 3.26 g/L of acetoin, which was about 59.8% higher than that produced by the cytoplasmic pathway. These results provided a new insight into the metabolic engineering of C. glabrata for acetoin production, and offered a potential platform to improve the performance of engineered pathways., (Copyright © 2014. Published by Elsevier Inc.)

Biobutanol is a potential fuel substitute and has been receiving increased attention in recent years. However, the economics of biobutanol production have been hampered by a number of bottlenecks such as high cost of raw material and low yield of solvent. Co-production of value-added products is a possible way to improve the economics of biobutanol production. Here, we present metabolic engineering strategies to substitute the major by-product acetone for a value-added product acetoin during butanol fermentation. By overexpressing the α-acetolactate decarboxylase gene alsD in Clostridium acetobutylicum B3, the acetoin yield was markedly increased while acetone formation was reduced. Subsequent disruption of adc gene effectively abolished acetone formation and further increased acetoin yield. After optimization of fermentation conditions, the alsD-overexpressing adc mutant generated butanol (13.8g/L), acetoin (4.3g/L), and ethanol (3.9g/L), but no acetone. Thus, acetone was completely substituted for acetoin, and both mass yield and product value were improved. This study provides valuable insights into the regulation of acetoin synthesis and should be highly useful for the development of acetoin-derived products like 2,3-butanediol and 2-butanol in C. acetobutylicum., (Copyright © 2014 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

In this study, we describe a novel method for producing valuable chemicals from glucose and xylose in Escherichia coli. The notable features in our method are avoidance of plasmids and expensive inducers for foreign gene expression to reduce production costs; foreign genes are knocked into the chromosome, and their expression is induced with xylose that is present in most biomass feedstock. As loci for the gene knock-in, lacZYA and some pseudogenes are chosen to minimize unexpected effects of the knock-in on cell physiology. The promoter of xylF is inducible with xylose and is combined with the T7 RNA polymerase-T7 promoter system to ensure strong gene expression. This expression system was named BICES (biomass-inducible chromosome-based expression system). As examples of BICES application, 2,3-butanediol and acetoin were successfully produced from glucose and xylose, and the maximal concentrations reached 54gL(-1) [99.6% in (R,S)-form] and 31gL(-1), respectively. 2,3-Butanediol and acetoin are industrially important chemicals that are, at present, produced primarily through petrochemical processes. To demonstrate usability of BICES in practical situations, we produced these chemicals from a saccharified cedar solution. From these results, we can conclude that BICES is suitable for practical production of valuable chemicals from biomass., (Copyright © 2014 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Bacillus subtilis produces acetoin as a major extracellular product. However, the by-products of 2,3-butanediol, lactic acid and ethanol were accompanied in the NADH-dependent pathways. In this work, metabolic engineering strategies were proposed to redistribute the carbon flux to acetoin by manipulation the NADH levels. We first knocked out the acetoin reductase gene bdhA to block the main flux from acetoin to 2,3-butanediol. Then, among four putative candidates, we successfully screened an active water-forming NADH oxidase, YODC. Moderate-expression of YODC in the bdhA disrupted B. subtilis weakened the NADH-linked pathways to by-product pools of acetoin. Through these strategies, acetoin production was improved to 56.7g/l with an increase of 35.3%, while the production of 2,3-butanediol, lactic acid and ethanol were decreased by 92.3%, 70.1% and 75.0%, respectively, simultaneously the fermentation duration was decreased 1.7-fold. Acetoin productivity by B. subtilis was improved to 0.639g/(lh)., (Copyright © 2014 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Utilization of Candida glabrata overproducing pyruvate is a promising strategy for high-level acetoin production. Based on the known regulatory and metabolic information, acetaldehyde and thiamine were fed to identify the key nodes of carboligase activity reaction (CAR) pathway and provide a direction for engineering C. glabrata. Accordingly, alcohol dehydrogenase, acetaldehyde dehydrogenase, pyruvate decarboxylase, and butanediol dehydrogenase were selected to be manipulated for strengthening the CAR pathway. Following the rational metabolic engineering, the engineered strain exhibited increased acetoin biosynthesis (2.24 g/L). In addition, through in silico simulation and redox balance analysis, NADH was identified as the key factor restricting higher acetoin production. Correspondingly, after introduction of NADH oxidase, the final acetoin production was further increased to 7.33 g/L. By combining the rational metabolic engineering and cofactor engineering, the acetoin-producing C. glabrata was improved stepwise, opening a novel pathway for rational development of microorganisms for bioproduction., (Copyright © 2013. Published by Elsevier Inc.)

Controlling the amounts of redox cofactors to manipulate metabolic fluxes is emerging as a useful approach to optimizing byproduct yields in yeast biotechnological processes. Redox cofactors are extensively interconnected metabolites, so predicting metabolite patterns is challenging and requires in-depth knowledge of how the metabolic network responds to a redox perturbation. Our aim was to analyze comprehensively the metabolic consequences of increased cytosolic NADPH oxidation during yeast fermentation. Using a genetic device based on the overexpression of a modified 2,3-butanediol dehydrogenase catalyzing the NADPH-dependent reduction of acetoin into 2,3-butanediol, we increased the NADPH demand to between 8 and 40-fold the anabolic demand. We developed (i) a dedicated constraint-based model of yeast fermentation and (ii) a constraint-based modeling method based on the dynamical analysis of mass distribution to quantify the in vivo contribution of pathways producing NADPH to the maintenance of redox homeostasis. We report that yeast responds to NADPH oxidation through a gradual increase in the flux through the PP and acetate pathways, providing 80% and 20% of the NADPH demand, respectively. However, for the highest NADPH demand, the model reveals a saturation of the PP pathway and predicts an exchange between NADH and NADPH in the cytosol that may be mediated by the glycerol-DHA futile cycle. We also reveal the contribution of mitochondrial shuttles, resulting in a net production of NADH in the cytosol, to fine-tune the NADH/NAD(+) balance. This systems level study helps elucidate the physiological adaptation of yeast to NADPH perturbation. Our findings emphasize the robustness of yeast to alterations in NADPH metabolism and highlight the role of the glycerol-DHA cycle as a redox valve, providing additional NADPH from NADH under conditions of very high demand., (Copyright © 2012 Elsevier Inc. All rights reserved.)