Efficient selection of pyruvate decarboxylase sequences from database for high ethanol productivity in Synechocystis sp. PCC 6803.

Ethanol production using the model cyanobacterium Synechocystis sp. PCC 6803 (PCC6803) has garnered considerable attention. A heterologous pyruvate decarboxylase (PDC) is essential for synthesizing ethanol in PCC6803.

Engineering anaerobic electron flow through heterologous rhodoquinone synthesis in model microbial and photosynthetic platforms.

Anaerobic conditions facilitate bioproduction by enabling diverse metabolic pathways; however, they disrupt redox balance due to the accumulation of reduced cofactors, limiting metabolic efficiency. Rhodoquinone (RQ), a low-redox-potential quinone, supports electron transport under anaerobic conditions. Unlike menaquinone, RQ is synthesized from ubiquinone through a single enzymatic reaction catalyzed by rhodoquinone biosynthesis protein A (RquA), making it a simple, adaptable metabolic engineering tool.

Glycolaldehyde affects the growth of and metabolic flux distribution in Synechocystis sp. PCC 6803.

Glycolaldehyde (GA) is widely used as a photosynthetic inhibitor to suppress metabolic reactions. We aimed to evaluate the effects of GA on the growth of and metabolic flux distribution in Synechocystis sp. PCC 6803.

Modification of intracellular metabolism by expression of a C-terminal variant of phosphoribulokinase from Synechocystis sp. PCC 6803.

Phosphoribulokinase (PRK) is a key enzyme in the Calvin cycle of cyanobacteria required for CO2 fixation and enhancing intracellular PRK activity will contribute to altering the metabolic state. In Synechocystis sp. PCC 6803, PRK activity is inhibited by the small protein CP12 and intramolecular disulfide bonds in its C-terminal loop.

Growth inhibition by ppc deletion is rescued by isocitrate dehydrogenase mutations in Escherichia coli.

Phosphoenolpyruvate carboxylase encoded by ppc catalyzes the anaplerotic reaction of oxaloacetate in the tricarboxylic acid (TCA) cycle in Escherichia coli. Deletion of ppc does not prevent the cells from replenishing oxaloacetate via the glyoxylate shunt, but the ppc-deletion strain almost did not grow on glucose. In the present study, we obtained evolved strains by deleting both ppc and mutS to increase the mutation rate and investigated the mechanisms for improving growth by analyzing the mutated genes.

C-metabolic flux analysis of respiratory chain disrupted strain ΔndhF1 of Synechocystis sp. PCC 6803.

Cyanobacteria are advantageous hosts for industrial applications toward achieving sustainable society due to their unique and superior properties such as atmospheric CO fixation via photosynthesis. However, cyanobacterial productivities tend to be weak compared to heterotrophic microbes. To enhance them, it is necessary to understand the fundamental metabolic mechanisms unique to cyanobacteria.

Identification of a novel NADPH generation reaction in the pentose phosphate pathway in using mBFP.

NADPH is a redox cofactor that drives the anabolic reactions. Although major NADPH generation reactions have been identified in , some minor reactions have not been identified. In the present study, we explored novel NADPH generation reactions by monitoring the fluorescence dynamics after the addition of carbon sources to starved cells, using a metagenome-derived blue fluorescent protein (mBFP) as an intracellular NADPH reporter.

A genome-scale metabolic model of a globally disseminated hyperinvasive M1 strain of .

is responsible for a range of diseases in humans contributing significantly to morbidity and mortality. Among more than 200 serotypes of , serotype M1 strains hold the greatest clinical relevance due to their high prevalence in severe human infections. To enhance our understanding of pathogenesis and discovery of potential therapeutic approaches, we have developed the first genome-scale metabolic model (GEM) for a serotype M1 strain, which we name iYH543.

Engineering Escherichia coli for efficient glutathione production.

Glutathione is a tripeptide of excellent value in the pharmaceutical, food, and cosmetic industries that is currently produced during yeast fermentation. In this case, glutathione accumulates intracellularly, which hinders high production. Here, we engineered Escherichia coli for the efficient production of glutathione.

Metabolome analysis of metabolic burden in Escherichia coli caused by overexpression of green fluorescent protein and delta-rhodopsin.

Overexpression of proteins by introducing a DNA vector is among the most important tools for the metabolic engineering of microorganisms such as Escherichia coli. Protein overexpression imposes a burden on metabolism because metabolic pathways must supply building blocks for protein and DNA synthesis. Different E.

Multicolor optogenetics for regulating flux ratio of three glycolytic pathways using EL222 and CcaSR in Escherichia coli.

Optogenetics is an attractive synthetic biology tool for controlling the metabolic flux distribution. Here, we demonstrated optogenetic flux ratio control of glycolytic pathways consisting of the Embden-Meyerhof-Parnas (EMP), pentose phosphate (PP), and Entner-Doudoroff (ED) pathways by illuminating multicolor lights using blue light-responsive EL222 and green/red light-responsive CcaSR in Escherichia coli. EL222 forms a dimer and binds to a particular DNA sequence under blue light; therefore, target gene expression can be reduced or induced by inserting a recognition sequence into its promoter regions.

Effects of n-butanol production on metabolism and the photosystem in Synecococcus elongatus PCC 7942 based on metabolic flux and target proteome analyses.

Although n-butanol (BuOH) is an ideal fuel because of its superior physical properties, it has toxicity to microbes. Previously, a Synechococcus elongatus PCC 7942 derivative strain that produces BuOH from CO was developed by introducing six heterologous genes (BUOH-SE strain). To identify the bottleneck in BuOH production, the effects of BuOH production and its toxicity on central metabolism and the photosystem were investigated.

Functional evaluation of non-oxidative glycolysis in Escherichia coli in the stationary phase under microaerobic conditions.

In microbial bioproduction, CO emissions via pyruvate dehydrogenase in the Embden-Meyerhof pathway, which converts glucose to acetyl-CoA, is one of the challenges for enhancing carbon yield. The synthetic non-oxidative glycolysis (NOG) pathway transforms glucose into three acetyl-CoA molecules without CO emission, making it an attractive module for metabolic engineering. Because the NOG pathway generates no ATP and NADH, it is expected to use a resting cell reaction.

Effect of light fluctuations on photosynthesis and metabolic flux in Synechocystis sp. PCC 6803.

In nature, photosynthetic organisms are exposed to fluctuating light, and their physiological systems must adapt to this fluctuation. To maintain homeostasis, these organisms have a light fluctuation photoprotective mechanism, which functions in both photosystems and metabolism. Although the photoprotective mechanisms functioning in the photosystem have been studied, it is unclear how metabolism responds to light fluctuations within a few seconds.

Conversion of Mevalonate to Isoprenol Using Light Energy in without Consuming Sugars for ATP Supply.

Bioconversion of key intermediate metabolites such as mevalonate into various useful chemicals is a promising strategy for microbial production. However, the conversion of mevalonate into isoprenoids requires a supply of adenosine triphosphate (ATP). Light-driven ATP regeneration using microbial rhodopsin is an attractive module for improving the intracellular ATP supply.

Logistic Regression-Guided Identification of Cofactor Specificity-Contributing Residues in Enzyme with Sequence Datasets Partitioned by Catalytic Properties.

Changing the substrate/cofactor specificity of an enzyme requires multiple mutations at spatially adjacent positions around the substrate pocket. However, this is challenging when solely based on crystal structure information because enzymes undergo dynamic conformational changes during the reaction process. Herein, we proposed a method for estimating the contribution of each amino acid residue to substrate specificity by deploying a phylogenetic analysis with logistic regression.

Evolutionary approach for improved proton pumping activity of heterologous rhodopsin expressed in Escherichia coli.

A light-driven ATP regeneration system using rhodopsin has been utilized as a method to improve the production of useful substances by microorganisms. To enable the industrial use of this system, the proton pumping rate of rhodopsin needs to be enhanced. Nonetheless, a method for this enhancement has not been established.

Metabolic pathway design for growth-associated phenylalanine production using synthetically designed mutualism.

Combination of growth-associated pathway engineering based on flux balance analysis (FBA) and adaptive laboratory evolution (ALE) is a powerful approach to enhance the production of useful compounds. However, the feasibility of such growth-associated pathway designs depends on the type of target compound. In the present study, FBA predicted a set of gene deletions (pykA, pykF, ppc, zwf, and adhE) that leads to growth-associated phenylalanine production in Escherichia coli.

Constitutive expression of the global regulator AbrB restores the growth defect of a genome-reduced Bacillus subtilis strain and improves its metabolite production.

Partial bacterial genome reduction by genome engineering can improve the productivity of various metabolites, possibly via deletion of non-essential genome regions involved in undesirable metabolic pathways competing with pathways for the desired end products. However, such reduction may cause growth defects. Genome reduction of Bacillus subtilis MGB874 increases the productivity of cellulases and proteases but reduces their growth rate.

Metabolic pathway engineering for the non-growth-associated succinate production in Escherichia coli based on flux solution space.

Non-growth-associated bio-production using microorganisms has the potential to achieve a higher target yield than growth-associated production since the latter approach does not waste the substrate for cell growth. We previously proposed a metabolic pathway engineering method (SSDesign) for non-growth-associated target production based on metabolic flux solution space using elementary mode analysis. SSDesign predicts gene knockout combinations for enforcing cells to produce a target compound under non-growing conditions.

Optogenetic reprogramming of carbon metabolism using light-powering microbial proton pump systems.

In microbial fermentative production, ATP regeneration, while crucial for cellular processes, conflicts with efficient target chemical production because ATP regeneration exhausts essential carbon sources also required for target chemical biosynthesis. To wrestle with this dilemma, we harnessed the power of microbial rhodopsins with light-driven proton pumping activity to supplement with ATP, thereby facilitating the bioproduction of various chemicals. We first demonstrated a photo-driven ATP supply and redistribution of metabolic carbon flows to target chemical synthesis by installing already-known delta rhodopsin (dR) in Escherichia coli.

Acceleration of target production in co-culture by enhancing intermediate consumption through adaptive laboratory evolution.

Co-culture is a promising way to alleviate metabolic burden by dividing the metabolic pathways into several modules and sharing the conversion processes with multiple strains. Since an intermediate is passed from the donor to the recipient via the extracellular environment, it is inevitably diluted. Therefore, enhancing the intermediate consumption rate is important for increasing target productivity.

Reactor control system in bacterial co-culture based on fluorescent proteins using an Arduino-based home-made device.

Background: Co-culture, fermentation with more than two microbial strains, is a potential flexible method for optimizing the metabolic conversion process in bio-production. However, maintaining an ideal population throughout the fermentation process remains a challenge.

Methods And Results: In this study, we developed a proportional control system for controlling the population ratio of Escherichia coli strains to a set value during continuous co-culture.

Recent advances in metabolic engineering-integration of in silico design and experimental analysis of metabolic pathways.

Microorganisms are widely used to produce valuable compounds. Because thousands of metabolic reactions occur simultaneously and many metabolic reactions are related to target production and cell growth, the development of a rational design method for metabolic pathway modification to optimize target production is needed. In this paper, recent advances in metabolic engineering are reviewed, specifically considering computational pathway modification design and experimental evaluation of metabolic fluxes by C-metabolic flux analysis.