Metabolic Engineering Of Microorganisms Research Articles

Engineering a Lactobacillus Lysine Riboswitch to Dynamically Control Metabolic Pathways for Lysine Production in Corynebacterium glutamicum.

Knock-out of genes of metabolic pathways is conventionally used in the metabolic engineering of microorganisms, but it is not applicable for genes of essential pathways. In order to avoid undesirable effects caused by gene deletion, it is attractive to develop riboswitches to dynamically control the metabolic pathways of microbial cell factories. In this regard, the aim of this study is to utilize the lysine riboswitch to control gene expressions of the biosynthetic pathways and by-pathways and thus improve lysine production in Corynebacterium glutamicum. To achieve this, a natural lysine riboswitch from Lactobacillus plantarum (LPRS) was first detected and then fused with RFP to test its functionality. After that, engineered lysine-activated (Lys-A) and lysine-repressed (Lys-R) riboswitches were successfully screened by dual genetic selection. Furthermore, the optimized A263 and R152 were applied to control the expression of aspartate kinase III and homoserine dehydrogenase in the lysine-producing strain C. glutamicum QW45, respectively. In contrast with QW45, the growth of the resulting A263-lysC mutant QW48 was similar to that of QW45; however, the growth of the resulting R357-hom mutant QW54 was slightly inhibited, indicating an inhibition of threonine biosynthesis caused by the riboswitch upon binding of intracellular lysine. Importantly, the lysine production of QW48 and QW54 was, respectively, 35% and 43% higher than that of the parent strain QW45, implying more metabolic flux directed into the lysine synthesis pathway. Finally, the engineered A263 and R357 were simultaneously applied to the same mutant QW55, which greatly improved lysine production. Thus, the approach demonstrated in this work could be principally used as a powerful tool to dynamically control any other undesired metabolic pathways.

Recent Advancement on Microbe‐Assisted Biohydrogen Production

The requirement for an ecologically sustainable coverage of energy demand is the need of the present day. The major global concern for clean, green, and sustainable energy with environmental protection is rapidly gaining momentum among researchers around the world that leads to the search for alternative energy sources for the transportation and industrial sectors. Among all energy sources, hydrogen generation and utilization have become a promising option as a transportation fuel. Hydrogen is considered as the potential energy source for the future and is therefore also called the fuel of the future. Hydrogen is not only used as fuel source in automobile engines, but also implemented in fuel cells technology. Compared to other fossil-based fuels, hydrogen has a wide array of advantages with more net calorific value. The production of biohydrogen is a global focus to mitigate the wide use of fossil fuels for sustainability. Sustainable production of hydrogen is demanded worldwide due to a wide range of applications for the provision of energy and fuels in various sectors. The biological method, utilizing microbial communities, is recognized as the most promising option for biohydrogen production, as it is an environment-friendly process utilizing renewable waste biomass under acceptable operating conditions. This special issue features articles about hydrogen production in all aspects including algal cells, thermochemical and biochemical methods. Hydrogen production from food waste by biochemical and thermochemical methods is critically described. A complete road map regarding hydrogen-producing cellular machineries, the mechanism of catalysis, and the role of modern genetic engineering for enhancing hydrogen production are also discussed. The potential of different algal cells and their mechanisms are critically explained in this issue. The role of nanotechnology to enhance the rate of hydrogen production from biomass is presented in this issue. Additionally, metabolic pathways such as biophotolysis, photofermentation, and dark fermentation were discussed for biohydrogen production. Therefore, this special issue focuses on recent advancements and approaches on microbe-assisted biohydrogen production by biotechnological interventions like metabolic engineering of microorganisms towards biohydrogen production, fermentation technologies for hydrogen along with nanotechnology application. Dr. Prakash Kumar Sarangi College of Agriculture, Central Agricultural University, Imphal, India Prakash Kumar Sarangi

Metabolic Engineering of Microorganisms Towards the Biomanufacturing of Non-Natural C5 and C6 Chemicals

Metabolic Engineering of Microorganisms Towards the Biomanufacturing of Non-Natural C5 and C6 Chemicals

Engineering yeast for bio-production of food ingredients

The provision of an adequate and high-quality food supply is a challenging issue due to the continued growth of the population and the reduction of arable land resources. To solve these problems, new and efficient food manufacturing processes need to be developed to meet the needs for healthy and nutritious food. Metabolic engineering of microorganisms is a feasible approach to alleviate this problem due to its efficient biosynthesis. For thousands of years, yeast has been used as a cell factory for manufacturing bread, beer and wine. And the development of synthetic biology expands its ability for synthesis of food ingredients, fuels, pharmaceuticals and chemical products. This mini review focuses on metabolic engineering of yeast cell factories to synthesize compounds that have been used as food ingredients with highlighting four food flavors.

Biosynthesis and metabolic engineering of 1-hydroxyphenazine in Pseudomonas chlororaphis H18

Background1-Hydroxyphenazine (1-OH-PHZ) is a phenazine microbial metabolite with broad-spectrum antibacterial activities against a lot of plant pathogens. However, its use is hampered by the low yield all along. Metabolic engineering of microorganisms is an increasingly powerful method for the production of valuable organisms at high levels. Pseudomonas chlororaphis is recognized as a safe and effective plant rhizosphere growth-promoting bacterium, and faster growth rate using glycerol or glucose as a renewable carbon source. Therefore, Pseudomonas chlororaphis is particularly suitable as the chassis cell for the modification and engineering of phenazines.ResultsIn this study, enzyme PhzS (monooxygenase) was heterologously expressed in a phenazine-1-carboxylic acid (PCA) generating strain Pseudomonas chlororaphis H18, and 1-hydroxyphenazine was isolated, characterized in the genetically modified strain. Next, the yield of 1-hydroxyphenazine was systematically engineered by the strategies including (1) semi-rational design remodeling of crucial protein PhzS, (2) blocking intermediate PCA consumption branch pathway, (3) enhancing the precursor pool, (4) engineering regulatory genes, etc. Finally, the titer of 1-hydroxyphenazine reached 3.6 g/L in 5 L fermenter in 54 h.ConclusionsThe 1-OH-PHZ production of Pseudomonas chlororaphis H18 was greatly improved through systematically engineering strategies, which is the highest, reported to date. This work provides a promising platform for 1-hydroxyphenazine engineering and production.Graphical

Current Knowledge on Polyethylene Terephthalate Degradation by Genetically Modified Microorganisms.

The global production of polyethylene terephthalate (PET) is estimated to reach 87.16 million metric tons by 2022. After a single use, a remarkable part of PET is accumulated in the natural environment as plastic waste. Due to high hydrophobicity and high molecular weight, PET is hardly biodegraded by wild-type microorganisms. To solve the global problem of uncontrolled pollution by PET, the degradation of plastic by genetically modified microorganisms has become a promising alternative for the plastic circular economy. In recent years many studies have been conducted to improve the microbial capacity for PET degradation. In this review, we summarize the current knowledge about metabolic engineering of microorganisms and protein engineering for increased biodegradation of PET. The focus is on mutations introduced to the enzymes of the hydrolase class—PETase, MHETase and cutinase—which in the last few years have attracted growing interest for the PET degradation processes. The modifications described in this work summarize the results obtained so far on the hydrolysis of polyethylene terephthalate based on the released degradation products of this polymer.

Promoting the Synthesis of Precursor Substances by Overexpressing Hexokinase (Hxk) and Hydroxymethylglutaryl-CoA Synthase (Erg13) to Elevate β-Carotene Production in Engineered Yarrowia lipolytica.

As a valuable carotenoid, β-carotene is commercially used in food, cosmetics, animal feeds, and other industries. Metabolic engineering of microorganisms has been widely explored to improve the production of β-carotene. Compared with the traditional genetic modifications mainly focused on the pathways of mevalonate (MVA) and β-carotene biosynthesis, this study aims to increase the β-carotene production through promoting the synthesis of precursor substances by overexpressing hexokinase and hydroxymethylglutaryl-CoA synthase in an engineered Yarrowia lipolytica. In this study, we investigated the effect of the unique hexokinase gene (Hxk) overexpression on β-carotene accumulation and glucose consumption. The Hxk gene was introduced into a β-carotene producing strain Y.L-1 to generate strain Y.L-2, and this increased the β-carotene content by 98%. Overexpression of the Hxk gene led to increasing in hexokinase activity (329% higher), glucose-6-phosphate content (92% higher), and improvement of the transcriptional level of Hxk (315% higher) compared to the control Y.L-1 strain. Moreover, Hxk overexpression accelerated the utilization rate of glucose. The gene erg13 encoding hydroxymethylglutaryl-CoA synthase was also overexpressed to increase the precursor supply for β-carotene biosynthesis. Recombinant Y.L-4 harboring two copies of erg13 produced 8.41 mg/g dry cell weight (DCW) of β-carotene, which was 259% higher than Y.L-1. The β-carotene content of 9.56 mg/g DCW was achieved in strain Y.L-6 by integrating erg13 into the chromosome and Hxk overexpression. The 3-Hydroxy-3-Methylglutaryl-CoA content in the cells was increased by overexpressing two copies of the erg13 gene. Finally, the titer of β-carotene reached 2.4 g/L using a 50 L bioreactor by the engineered strain, and the fermentation cycle was shortened from 144 to 120 h. Overall, overexpression of Hxk and erg13 could improve β-carotene production and successfully overcoming the bottleneck of precursor generation to support a more efficient pathway for the production of the target product. Our results revealed a novel strategy to engineer the pathway of β-carotene synthesis.

Development of novel strategies for higher fermentative biohydrogen recovery along with novel metabolites from organic wastes: The present state of the art.

Depletion of fossil fuels and environmental concern has compelled us to search for alternative fuel. Hydrogen is considered as a dream fuel as it has high energy content (142kJg-1 ) and is not chemically bound to carbon. At present, fossil fuel-based methods for producing hydrogen require high-energy input, which makes the processes expensive. The major processes for biohydrogen production are biophotolysis, microbial electrolysis, dark fermentation, and photofermentation. Fermentative hydrogen production has the additional advantages of potentially using various waste streams from different industries as feedstock. Novel strategies to enhance the productivity of fermentative hydrogen production include optimization in pretreatment methods, integrated fermentation systems (sequential and combined fermentation), use of nanoparticles as additives, metabolic engineering of microorganisms, improving the light utilization efficiency, developing more efficient photobioreactors, etc. More focus has been given to produce biohydrogen in a biorefinery approach in which, along with hydrogen gas, other metabolites (ethanol, butyric acid, 1,3-propanediol, etc.) are also produced, which have direct/indirect industrial applications. In present review, various emerging technologies that highlight biohydrogen production methods as effective and sustainable methods on a large scale have been critically reviewed. The possible future developments are also outlined.

Microbial Polyhydroxyalkanoates and Nonnatural Polyesters.

Microorganisms produce diverse polymers for various purposes such as storing genetic information, energy, and reducing power, and serving as structural materials and scaffolds. Among these polymers, polyhydroxyalkanoates (PHAs) are microbial polyesters synthesized and accumulated intracellularly as a storage material of carbon, energy, and reducing power under unfavorable growth conditions in the presence of excess carbon source. PHAs have attracted considerable attention for their wide range of applications in industrial and medical fields. Since the first discovery of PHA accumulating bacteria about 100 years ago, remarkable advances have been made in the understanding of PHA biosynthesis and metabolic engineering of microorganisms toward developing efficient PHA producers. Recently, nonnatural polyesters have also been synthesized by metabolically engineered microorganisms, which opened a new avenue toward sustainable production of more diverse plastics. Herein, the current state of PHAs and nonnatural polyesters is reviewed, covering mechanisms of microbial polyester biosynthesis, metabolic pathways, and enzymes involved in biosynthesis of short-chain-length PHAs, medium-chain-length PHAs, and nonnatural polyesters, especially 2-hydroxyacid-containing polyesters, metabolic engineering strategies to produce novel polymers and enhance production capabilities and fermentation, and downstream processing strategies for cost-effective production of these microbial polyesters. In addition, the applications of PHAs and prospects are discussed.

Metabolic Engineering of Microorganisms for the Production of Natural Compounds

Metabolic Engineering of Microorganisms for the Production of Natural Compounds

State-of-the-Art Genetic Modalities to Engineer Cyanobacteria for Sustainable Biosynthesis of Biofuel and Fine-Chemicals to Meet Bio-Economy Challenges.

In recent years, metabolic engineering of microorganisms has attained much research interest to produce biofuels and industrially pertinent chemicals. Owing to the relatively fast growth rate, genetic malleability, and carbon neutral production process, cyanobacteria has been recognized as a specialized microorganism with a significant biotechnological perspective. Metabolically engineering cyanobacterial strains have shown great potential for the photosynthetic production of an array of valuable native or non-native chemicals and metabolites with profound agricultural and pharmaceutical significance using CO2 as a building block. In recent years, substantial improvements in developing and introducing novel and efficient genetic tools such as genome-scale modeling, high throughput omics analyses, synthetic/system biology tools, metabolic flux analysis and clustered regularly interspaced short palindromic repeats (CRISPR)-associated nuclease (CRISPR/cas) systems have been made for engineering cyanobacterial strains. Use of these tools and technologies has led to a greater understanding of the host metabolism, as well as endogenous and heterologous carbon regulation mechanisms which consequently results in the expansion of maximum productive ability and biochemical diversity. This review summarizes recent advances in engineering cyanobacteria to produce biofuel and industrially relevant fine chemicals of high interest. Moreover, the development and applications of cutting-edge toolboxes such as the CRISPR-cas9 system, synthetic biology, high-throughput “omics”, and metabolic flux analysis to engineer cyanobacteria for large-scale cultivation are also discussed.

Engineering of α-1,3-fucosyltransferases for production of 3-fucosyllactose in Escherichia coli

Fucosyllactoses (FLs), present in human breast milk, have been reported to benefit human health immensely. Especially, 3-fucosyllactose (3-FL) has numerous benefits associated with a healthy gut ecosystem. Metabolic engineering of microorganisms is thought to be currently the only option to provide an economically feasible route for large-scale production of 3-FL. However, engineering principles for α-1,3-fucosyltransferases (1,3-FTs) are not well-known, resulting in the lower productivity of 3-FL than that of 2'-fucosyllactose (2'-FL), although both 2'-FL and 3-FL follow a common pathway to produce GDP-L-fucose. The C-terminus of 1,3-FTs is composed of heptad repeats, responsible for dimerization of the enzymes, and a peripheral membrane anchoring region. It has long been thought that truncation of most heptad repeats, retaining just 1 or 2, helps the soluble expression of 1,3-FTs. However, whether the introduction of truncated version of 1,3-FTs enhances the production of 3-FL in a metabolically engineered strain, is yet to be tested. In this study, the effect of these structural components on the production of 3-FL in Escherichia coli was evaluated through systematic truncation and elongation of the C-terminal regions of three 1,3-FTs from Helicobacter pylori. Although these three 1,3-FTs contained heptad repeats and membrane-anchoring regions of varying lengths, they commonly exhibited an optimal performance when the number of heptad repeats was increased, and membrane-binding region was removed. The production of 3-FL could be increased 10-20-fold through this simple strategy.

Metabolic engineering of the 2-ketobutyrate biosynthetic pathway for 1-propanol production in Saccharomyces cerevisiae

BackgroundTo produce 1-propanol as a potential biofuel, metabolic engineering of microorganisms, such as E. coli, has been studied. However, 1-propanol production using metabolically engineered Saccharomyces cerevisiae, which has an amazing ability to produce ethanol and is thus alcohol-tolerant, has infrequently been reported. Therefore, in this study, we aimed to engineer S. cerevisiae strains capable of producing 1-propanol at high levels.ResultsWe found that the activity of endogenous 2-keto acid decarboxylase and alcohol/aldehyde dehydrogenase is sufficient to convert 2-ketobutyrate (2 KB) to 500 mg/L 1-propanol in yeast. Production of 1-propanol could be increased by: (i) the construction of an artificial 2 KB biosynthetic pathway from pyruvate via citramalate (cimA); (ii) overexpression of threonine dehydratase (tdcB); (iii) enhancement of threonine biosynthesis from aspartate (thrA, thrB and thrC); and (iv) deletion of the GLY1 gene that regulates a competing pathway converting threonine to glycine. With high-density anaerobic fermentation of the engineered S. cerevisiae strain YG5C4231, we succeeded in producing 180 mg/L 1-propanol from glucose.ConclusionThese results indicate that the engineering of a citramalate-mediated pathway as a production method for 1-propanol in S. cerevisiae is effective. Although optimization of the carbon flux in S. cerevisiae is necessary to harness this pathway, it is a promising candidate for the large-scale production of 1-propanol.

Advanced Biosystems: Looking Back on our First Year and Welcome to 2018

<i>Advanced Biosystems</i>: Looking Back on our First Year and Welcome to 2018

Exploring biochemical pathways for mono-ethylene glycol (MEG) synthesis from synthesis gas

Mono-ethylene glycol (MEG) is an important petrochemical with widespread use in numerous consumer products. The current industrial MEG-production process relies on non-renewable fossil fuel-based feedstocks, such as petroleum, natural gas, and naphtha; hence, it is useful to explore alternative routes of MEG-synthesis from gases as they might provide a greener and more sustainable alternative to the current production methods. Technologies of synthetic biology and metabolic engineering of microorganisms can be deployed for the expression of new biochemical pathways for MEG-synthesis from gases, provided that such promising alternative routes are first identified. We used the BNICE.ch algorithm to develop novel and previously unknown biological pathways to MEG from synthesis gas by leveraging the Wood-Ljungdahl pathway of carbon fixation of acetogenic bacteria. We developed a set of useful pathway pruning and analysis criteria to systematically assess thousands of pathways generated by BNICE.ch. Published genome-scale models of Moorella thermoacetica and Clostridium ljungdahlii were used to perform the pathway yield calculations and in-depth analyses of seven (7) newly developed biological MEG-producing pathways from gases, including CO2, CO, and H2. These analyses helped identify not only better candidate pathways, but also superior chassis organisms that can be used for metabolic engineering of the candidate pathways. The pathway generation, pruning, and detailed analysis procedures described in this study can also be used to develop biochemical pathways for other commodity chemicals from gaseous substrates.

Metabolic Engineering of microorganisms for the production of 3-hydroxypropionic acid from glycerol

Metabolic Engineering of microorganisms for the production of 3-hydroxypropionic acid from glycerol

Production of Fatty Acid-derived valuable chemicals in synthetic microbes.

Fatty acid derivatives, such as hydroxy fatty acids, fatty alcohols, fatty acid methyl/ethyl esters, and fatty alka(e)nes, have a wide range of industrial applications including plastics, lubricants, and fuels. Currently, these chemicals are obtained mainly through chemical synthesis, which is complex and costly, and their availability from natural biological sources is extremely limited. Metabolic engineering of microorganisms has provided a platform for effective production of these valuable biochemicals. Notably, synthetic biology-based metabolic engineering strategies have been extensively applied to refactor microorganisms for improved biochemical production. Here, we reviewed: (i) the current status of metabolic engineering of microbes that produce fatty acid-derived valuable chemicals, and (ii) the recent progress of synthetic biology approaches that assist metabolic engineering, such as mRNA secondary structure engineering, sensor-regulator system, regulatable expression system, ultrasensitive input/output control system, and computer science-based design of complex gene circuits. Furthermore, key challenges and strategies were discussed. Finally, we concluded that synthetic biology provides useful metabolic engineering strategies for economically viable production of fatty acid-derived valuable chemicals in engineered microbes.

Enzymes in lipid modification: Past achievements and current trends

In this minireview, the application of enzymes for the modification of fats and oils is covered with special emphasis on lipase‐catalyzed synthesis of structured triglycerides. Furthermore, protein engineering methods to alter the fatty acid chain‐length or the trans‐fatty acid selectivity of lipase A from Candida antarctica and an enzymatic two‐step method to remove the contaminant 3‐monochloro‐1,2‐propane diol are presented. Also the combination of several enzymes enables cascade reactions to obtain products such as ω‐hydroxycarboxylic acids useful for oleochemistry and recently published achievements to produce valuable compounds by metabolic engineering of microorganisms as exemplified for PUFA or fatty acid esters starting from sugars as renewable resources are covered. Special emphasis is given on achievements made in the authors group.This review highlights developments in the field of enzymatic lipid modification covering the use of commercial enzymes, the design of lipases via protein engineering as well as recently established enzyme cascade reactions and examples for metabolic engineering to yield fatty acids and their derivatives.

Assessment of Zymomonas mobilis biotechnological potential in ethanol production by flux variability analysis

Metabolic engineering of microorganisms is a promising direction in industrial biotechnology. Engineering approach requests finding the best possible alternative of metabolic pathway fluxes, with preset limitations for productivity, yield, set of by- products, robustness and other features. In case of industrial applications the steady state precondition has to be taken into ac- count, thus enabling application of stoichiometric modeling. Spectrum of solutions maximizing ethanol or succinate production from glucose or xylose is analyzed at fixed maximal production rate of products and for 80-100% range of maximal production rate to assess the robustness of yield to disturbances in the performance of reactions. Maximal production of desired components from glucose and xylose turned out to be very similar by the flux distribution with very limited ability to compensate throughput limitations of important reactions thus causing reduction of yield. Requesting the network performance at 80-100% of yield leads to increase of robustness, and majority of reactions can be compensated by others to maintain the steady state at the desired yield level.

Production of miltiradiene by metabolically engineered Saccharomyces cerevisiae

Metabolic engineering of microorganisms is an alternative and attractive route for production of valuable terpenoids that are usually extracted from plant sources. Tanshinones are the bioactive components of Salvia miltiorrhizha Bunge, which is a well-known traditional Chinese medicine widely used for treatment of many cardiovascular diseases. As a step toward microbial production of tanshinones, copalyl diphosphate (CPP) synthase, and normal CPP kaurene synthase-like genes, which convert the universal diterpenoid precursor geranylgeranyl diphosphate (GGPP) to miltiradiene (an important intermediate of the tanshinones synthetic pathway), was introduced into Saccharomyces cerevisiae, resulting in production of 4.2 mg/L miltiradiene. Improving supplies of isoprenoid precursors was then investigated for increasing miltiradiene production. Although over-expression of a truncated 3-hydroxyl-3-methylglutaryl-CoA reductase (tHMGR) and a mutated global regulatory factor (upc2.1) gene did improve supply of farnesyl diphosphate (FPP), production of miltiradiene was not increased while large amounts of squalene (78 mg/L) were accumulated. In contrast, miltiradiene production increased to 8.8 mg/L by improving supply of GGPP through over-expression of a fusion gene of FPP synthase (ERG20) and endogenous GGPP synthase (BTS1) together with a heterologous GGPP synthase from Sulfolobus acidocaldarius (SaGGPS). Auxotrophic markers in the episomal plasmids were then replaced by antibiotic markers, so that engineered yeast strains could use rich medium to obtain better cell growth while keeping plasmid stabilities. Over-expressing ERG20-BTS1 and SaGGPS genes increased miltiradiene production from 5.4 to 28.2 mg/L. Combinatorial over-expression of tHMGR-upc2.1 and ERG20-BTS1-SaGGPS genes had a synergetic effects on miltiradiene production, increasing titer to 61.8 mg/L. Finally, fed-batch fermentation was performed, and 488 mg/L miltiradiene was produced. The yeast strains engineered in this work provide a basis for creating an alternative way for production of tanshinones in place of extraction from plant sources.