Recombinant RquA catalyzes the in vivo conversion of ubiquinone to rhodoquinone in Escherichia coli and Saccharomyces cerevisiae

Terpenoid quinones are liposoluble redox-active compounds that serve as essential electron carriers and antioxidants. One such quinone, rhodoquinone (RQ), couples the respiratory electron transfer chain to the reduction of fumarate to facilitate anaerobic respiration. This mechanism allows RQ-synthesizing organisms to operate their respiratory chain using fumarate as a final electron acceptor. RQ biosynthesis is restricted to a handful of prokaryotic and eukaryotic organisms, and details of this biosynthetic pathway remain enigmatic. One gene, rquA, was discovered to be required for RQ biosynthesis in Rhodospirillum rubrum. However, the function of the gene product, RquA, has remained unclear. Here, using reverse genetics approaches, we demonstrate that RquA converts ubiquinone to RQ directly. We also demonstrate the first in vivo synthetic production of RQ in Escherichia coli and Saccharomyces cerevisiae, two organisms that do not natively produce RQ. These findings help clarify the complete RQ biosynthetic pathway in species which contain RquA homologs.     

Finding elementary flux modes in metabolic networks based on flux balance analysis and flux coupling analysis: application to the analysis of Escherichia coli metabolism

Elementary modes (EMs) are steady-state metabolic flux vectors with minimal set of active reactions. Each EM corresponds to a metabolic pathway. Therefore, studying EMs is helpful for analyzing the production of biotechnologically important metabolites. However, memory requirements for computing EMs may hamper their applicability as, in most genome-scale metabolic models, no EM can be computed due to running out of memory. In this study, we present a method for computing randomly sampled EMs. In this approach, a network reduction algorithm is used for EM computation, which is based on flux balance-based methods. We show that this approach can be used to recover the EMs in the medium- and genome-scale metabolic network models, while the EMs are sampled in an unbiased way. The applicability of such results is shown by computing “estimated” control-effective flux values in Escherichia coli metabolic network.     

Analysis of polyhydroxybutyrate flux limitations by systematic genetic and metabolic perturbations

Poly-3-hydroxybutyrate (PHB) titers in Escherichia coli have benefited from 10+ years of metabolic engineering. In the majority of studies, PHB content, expressed as percent PHB (dry cell weight), is increased, although this increase can be explained by decreases in growth rate or increases in PHB flux. In this study, growth rate and PHB flux were quantified directly in response to systematic manipulation of (1) gene expression in the product-forming pathway and (2) growth rates in a nitrogen-limited chemostat. Gene expression manipulation revealed acetoacetyl-CoA reductase (phaB) limits flux to PHB, although overexpression of the entire pathway pushed the flux even higher. These increases in PHB flux are accompanied by decreases in growth rate, which can be explained by carbon diversion, rather than toxic effects of the PHB pathway. In chemostats, PHB flux was insensitive to growth rate. These results imply that PHB flux is primarily controlled by the expression levels of the product forming pathway and not by the availability of precursors. These results confirm prior in vitro measurements and metabolic models and show expression level is a major affecter of PHB flux.     

Development of bifunctional biosensors for sensing and dynamic control of glycolysis flux in metabolic engineering

Glycolysis is the primary metabolic pathway in all living organisms. Maintaining the balance of glycolysis flux and biosynthetic pathways is the crucial matter involved in the microbial cell factory. Few regulation systems can address the issue of metabolic flux imbalance in glycolysis. Here, we designed and constructed a bifunctional glycolysis flux biosensor that can dynamically regulate glycolysis flux for overproduction of desired biochemicals. A series of positive-and negative-response biosensors were created and modified for varied thresholds and dynamic ranges. These engineered glycolysis flux biosensors were verified to be able to characterize in vivo fructose-1,6-diphosphate concentration. Subsequently, the biosensors were applied for fine-tuning glycolysis flux to effectively balance the biosynthesis of two chemicals: mevalonate and N-acetylglucosamine. A glycolysis flux-dynamically controlled Escherichia coli strain achieved a 111.3 g/L mevalonate titer in a 1L fermenter.     

Heterologous production of 3-hydroxyvalerate in engineered Escherichia coli

3-Hydroxyacids are a group of valuable fine chemicals with numerous applications, and 3-hydroxybutyrate (3-HB) represents the most common species with acetyl-CoA as a precursor. Due to the lack of propionyl-CoA in most, if not all, microorganisms, bio-based production of 3-hydroxyvalerate (3-HV), a longer-chain 3-hydroxyacid member with both acetyl-CoA and propionyl-CoA as two precursors, is often hindered by high costs associated with the supplementation of related carbon sources, such as propionate or valerate. Here, we report the derivation of engineered Escherichia coli strains for the production of 3-HV from unrelated cheap carbon sources, in particular glucose and glycerol. Activation of the sleeping beauty mutase (Sbm) pathway in E. coli enabled the intracellular formation of non-native propionyl-CoA. A selection of enzymes involved in 3-HV biosynthetic pathway from various microorganisms were explored for investigating their effects on 3-HV biosynthesis in E. coli. Glycerol outperformed glucose as the carbon source, and glycerol dissimilation for 3-HV biosynthesis was primarily mediated through the aerobic GlpK-GlpD route. To further enhance 3-HV production, we developed metabolic engineering strategies to redirect more dissimilated carbon flux from the tricarboxylic acid (TCA) cycle to the Sbm pathway, resulting in an enlarged intracellular pool of propionyl-CoA. Both the presence of succinate/succinyl-CoA and their interconversion step in the TCA cycle were identified to critically limit the carbon flux redirection into the Sbm pathway and, therefore, 3-HV biosynthesis. A selection of E. coli host TCA genes encoding enzymes near the succinate node were targeted for manipulation to evaluate the contribution of the three TCA routes (i.e. oxidative TCA cycle, reductive TCA branch, and glyoxylate shunt) to the redirected carbon flux into the Sbm pathway. Finally, the carbon flux redirection into the Sbm pathway was enhanced by simultaneously deregulating glyoxylate shunt and blocking the oxidative TCA cycle, significantly improving 3-HV biosynthesis. With the implementation of these biotechnological and bioprocessing strategies, our engineered E. coli strains can effectively produce 3-HV up to 3.71 g l⁻¹ with a yield of 24.1% based on the consumed glycerol in shake-flask cultures.     

Elevating 4-hydroxycoumarin production through alleviating thioesterase-mediated salicoyl-CoA degradation

Acyl-CoAs are essential intermediates in the biosynthetic pathways of a number of industrially and pharmaceutically important molecules. When these pathways are reconstituted in a heterologous microbial host for metabolic engineering purposes, the acyl-CoAs may be subject to undesirable hydrolysis by the host's native thioesterases, resulting in a waste of cellular energy and decreased intermediate availability, thus impairing bioconversion efficiency. 4-hydroxycoumarin (4HC) is a direct synthetic precursor to the commonly used oral anticoagulants (e.g. warfarin) and rodenticides. In our previous study, we have established an artificial pathway for 4HC biosynthesis in Escherichia coli, which involves the thioester intermediate salicoyl-CoA. Here, we utilized the 4HC pathway as a demonstration to examine the negative effect of salicoyl-CoA degradaton, identify and inactivate the responsible thioesterase, and eventually improve the 4HC production. We screened a total of 16 E. coli thioesterases and tested their hydrolytic activity towards salicoyl-CoA in vitro. Among all the tested candidate enzymes, YdiI was found to be the dominant contributor to the salicoyl-CoA degradation in E. coli. Remarkably, the ydiI knockout strain carrying the 4HC pathway exhibited an up to 300% increase in 4HC production. An optimized 4HC pathway construct introduced in the ydiI knockout strain led to the accumulation of 935 mg/L of 4HC in shake flasks, which is about 1.5 folds higher than the wild-type strain. This study demonstrates a systematic strategy to alleviate the undesirable hydrolysis of thioester intermediates, allowing production enhancement for other biosynthetic pathways with similar issues.     

The Production of Organic Acids

Abstract

The production of organic acids covers two aspects: first, the metabolic pathways involved in the biosynthesis, and, second, the industrial process strategy adopted. The review seeks to show the underlying biochemical similarities in the biosynthesis of organic acids and the resulting similarities in the commercial processes.

Two groups of acids are defined, those with a “long” biosynthetic path from glucose, involving much of the glycolytic pathway and the tricarboxylic acid cycle, and those acids with a “short pathway”, essentially a biotransformation of glucose. The regulation of the pathways and the future developments in metabolic control theory and genetic manipulations relating to them are considered. The organisms used industrially are also limited, Aspergillus sp. and Candida yeasts; again the underlying metabolic similarities lead to similar strategies for all the acids discussed.     

Structural analysis of metabolic networks based on flux centrality

Metabolic reactions are fundamental to living organisms, and a large number of reactions simultaneously occur at a given time in living cells transforming diverse metabolites into each other. There has been an ongoing debate on how to classify metabolites with respect to their importance for metabolic performance, usually based on the analysis of topological properties of genome scale metabolic networks. However, none of these studies have accounted quantitatively for flux in metabolic networks, thus lacking an important component of a cell’s biochemistry.We therefore analyzed a genome scale metabolic network of Escherichia coli by comparing growth under 19 different growth conditions, using flux balance analysis and weighted network centrality investigation. With this novel concept of flux centrality we generated metabolite rankings for each particular growth condition. In contrast to the results of conventional analysis of genome scale metabolic networks, different metabolites were top-ranking dependent on the growth condition. At the same time, several metabolites were consistently among the high ranking ones. Those are associated with pathways that have been described by biochemists as the most central part of metabolism, such as glycolysis, tricarboxylic acid cycle and pentose phosphate pathway. The values for the average path length of the analyzed metabolite networks were between 10.5 and 12.6, supporting recent findings that the metabolic network of E. coli is not a small-world network.     

Cover Image,Volume 116, Number 6, June 2019

Metabolic engineering consistently demands to produce the maximum carbon and energy flux to target chemicals. To balance metabolic flux, gene expression levels of artificially synthesized pathways usually fine-tuned using multimodular optimization strategy. However, forward construction is an engineering conundrum because a vast number of possible pathway combinations need to be constructed and analyzed. Here, an iterative high-throughput balancing (IHTB) strategy was established to thoroughly fine-tune the (2S)-naringenin biosynthetic pathway. A series of gradient constitutive promoters from Escherichia coli were randomly cloned upstream of pathway genes, and the resulting library was screened using an ultraviolet spectrophotometry–fluorescence spectrophotometry high-throughput method, which was established based on the interactions between AlCl₃ and (2S)-naringenin. The metabolic flux of the screened high-titer strains was analyzed and iterative rounds of screening were performed based on the analysis results. After several rounds, the metabolic flux of the (2S)-naringenin synthetic pathway was balanced, reaching a final titer of 191.9 mg/L with 29.2 mg/L p-coumaric acid accumulation. Chalcone synthase was speculated to be the rate-limiting enzyme because its expression level was closely related to the production of both (2S)-naringenin and p-coumaric acid. The established IHTB strategy can be used to efficiently balance multigene pathways, which will accelerate the development of efficient recombinant strains.     

大豆异黄酮代谢途径在大肠杆菌中的构建及表达

自然界异黄酮合成途径主要存在于豆科植物中。以微生物为宿主研究异黄酮代谢,则需要将整个相关代谢途径的多酶体系组装到工程菌种,从而进行表达及代谢研究,这就需要用到多基因的转化和共表达技术。综合应用了多基因单载体和多基因多载体方法,将大豆异黄酮代谢途径中的五个关键酶基因导入到大肠杆菌中,对异黄酮代谢途径在大肠杆菌中的构建和表达进行了研究和探索,获得了含有五个外源基因的重组大肠杆菌;重组菌经IPTG诱导,以L-酪氨酸为底物进行发酵,发酵产物经过HPLC测定,结果表明和空白对照相比有新的代谢产物生成,初步断定为异黄酮类化合物。     

Hypothalamic malonyl-coenzyme A and the control of energy balance

An intermediate in the fatty acid biosynthetic pathway, malonyl-coenzyme A (CoA), has emerged as a major regulator of energy homeostasis not only in peripheral metabolic tissues but also in regions of the central nervous system that control satiety and energy expenditure. Fluctuations in hypothalamic malonyl-CoA lead to changes in food intake and peripheral energy expenditure in a manner consistent with an anorexigenic signaling intermediate. Hypothalamic malonyl-CoA is regulated by nutritional and endocrine cues including glucose and leptin, respectively. That malonyl-CoA is an essential component in the energy homeostatic signaling system of the hypothalamus is supported by convergence of physiological, pharmacological, and genetic evidence. This review will focus on evidence implicating malonyl-CoA as a central player in the control of body weight and adiposity as well as clues to the molecular mechanism by which carbon flux through the fatty acid biosynthetic pathway is linked to the neural control of energy balance.     

Optimal cofactor swapping can increase the theoretical yield for chemical production in Escherichia coli and Saccharomyces cerevisiae

Maintaining cofactor balance is a critical function in microorganisms, but often the native cofactor balance does not match the needs of an engineered metabolic flux state. Here, an optimization procedure is utilized to identify optimal cofactor-specificity “swaps” for oxidoreductase enzymes utilizing NAD(H) or NADP(H) in the genome-scale metabolic models of Escherichia coli and Saccharomyces cerevisiae. The theoretical yields of all native carbon-containing molecules are considered, as well as theoretical yields of twelve heterologous production pathways in E. coli. Swapping the cofactor specificity of central metabolic enzymes (especially GAPD and ALCD2x) is shown to increase NADPH production and increase theoretical yields for native products in E. coli and yeast—including l-aspartate, l-lysine, l-isoleucine, l-proline, l-serine, and putrescine—and non-native products in E. coli—including 1,3-propanediol, 3-hydroxybutyrate, 3-hydroxypropanoate, 3-hydroxyvalerate, and styrene.     

Intermediate-sensor assisted push–pull strategy and its application in heterologous deoxyviolacein production in Escherichia coli

Because high-throughput screening tools are typically unavailable when using the pathway-engineering approach, we developed a new strategy, named intermediate sensor-assisted push–pull strategy, which enables sequential pathway optimization by incorporating a biosensor targeting a key pathway intermediate. As proof of concept, we constructed an l-Trp biosensor and used it to optimize the deoxyviolacein biosynthetic pathway, which we divided into two modules with l-Trp being the product of the upstream and the substrate of the downstream module for deoxyviolacein synthesis. Using the biosensor and fluorescence-activated cell sorting, the activities of the two modules were sequentially and independently optimized in Escherichia coli to achieve the desired phenotypes. By this means, we increased the deoxyviolacein titer 4.4-fold (1.92 g/L), which represents the greatest deoxyviolacein production reported. This work suggests that a biosynthetic pathway can be enhanced to produce a value-added secondary metabolite(s) without available end-product screening method by using a central metabolic junction molecule biosensor(s).     

Metabolic engineering of Escherichia coli for the production of benzoic acid from glucose

Benzoic acid (BA) is an important platform aromatic compound in chemical industry and is widely used as food preservatives in its salt forms. Yet, current manufacture of BA is dependent on petrochemical processes under harsh conditions. Here we report the de novo production of BA from glucose using metabolically engineered Escherichia coli strains harboring a plant-like β-oxidation pathway or a newly designed synthetic pathway. First, three different natural BA biosynthetic pathways originated from plants and one synthetically designed pathway were systemically assessed for BA production from glucose by in silico flux response analyses. The selected plant-like β-oxidation pathway and the synthetic pathway were separately established in E. coli by expressing the genes encoding the necessary enzymes and screened heterologous enzymes under optimal plasmid configurations. BA production was further optimized by applying several metabolic engineering strategies to the engineered E. coli strains harboring each metabolic pathway, which included enhancement of the precursor availability, removal of competitive reactions, transporter engineering, and reduction of byproduct formation. Lastly, fed-batch fermentations of the final engineered strain harboring the β-oxidation pathway and the strain harboring the synthetic pathway were conducted, which resulted in the production of 2.37 ± 0.02 g/L and 181.0 ± 5.8 mg/L of BA from glucose, respectively; the former being the highest titer reported by microbial fermentation. The metabolic engineering strategies developed here will be useful for the production of related aromatics of high industrial interest.     

Reconstructing a recycling and nonauxotroph biosynthetic pathway in Escherichia coli toward highly efficient production of L-citrulline

L-citrulline is a high-value amino acid with promising application in medicinal and food industries. Construction of highly efficient microbial cell factories for L-citrulline production is still an open issue due to complex metabolic flux distribution and L-arginine auxotrophy. In this study, we constructed a nonauxotrophic cell factory in Escherichia coli for high-titer L-citrulline production by coupling modular engineering strategies with dynamic pathway regulation. First, the biosynthetic pathway of L-citrulline was enhanced after blockage of the degradation pathway and introduction of heterologous biosynthetic genes from Corynebacterium glutamicum. Specifically, a superior recycling biosynthetic pathway was designed to replace the native linear pathway by deleting native acetylornithine deacetylase. Next, the carbamoyl phosphate and L-glutamate biosynthetic modules, the NADPH generation module, and the efflux module were modified to increase L-citrulline titer further. Finally, a toggle switch that responded to cell density was designed to dynamically control the expression of the argG gene and reconstruct a nonauxotrophic pathway. Without extra supplement of L-arginine during fermentation, the final CIT24 strain produced 82.1 g/L L-citrulline in a 5-L bioreactor with a yield of 0.34 g/g glucose and a productivity of 1.71 g/(L ⋅ h), which were the highest values reported by microbial fermentation. Our study not only demonstrated the successful design of cell factory for high-level L-citrulline production but also provided references of coupling the rational module engineering strategies and dynamic regulation strategies to produce high-value intermediate metabolites.     

Design and application of an in vivo reporter assay for phenylalanine ammonia-lyase

Phenylalanine ammonia-lyase (PAL) is an important enzyme that links primary metabolism to secondary metabolism. Its efficiency is often a critical factor that affects the overall flux of a related metabolic pathway, the titer of the final products, and the efficacy of PAL-based therapies. Thus, PAL is a common target for metabolic engineering, and it is of significant interest to screen efficient PALs for industrial and medical applications. In this study, a novel and efficient visible reporter assay for screening of PAL efficiency in Escherichia coli was established based on a plant type III polyketide biosynthetic pathway. The candidate PALs were co-expressed with a 4-coumarate:CoA ligase 4CL1 from Arabidopsis thaliana and curcuminoid synthase (CUS) from Oryza sativa in E. coli BL21(DE3) to form a dicinnamoylmethane biosynthetic pathway. Taking advantage of the yellow color of the product, a microplate-based assay was designed to measure the titer of dicinnamoylmethane, which was validated by HPLC analysis. The different titers of the product reflect the overall performance (expression level and enzymatic activity) of the individual PALs in E. coli. Using this system, we have screened three PALs (PAL1, PAL3, and PAL4) from Trifolium pratense, among which PAL1 showed the best performance in E. coli. The engineered E. coli strain containing PAL1, 4CL1, and CUS led to the production of dicinnamoylmethane at a high level of 0.36 g/l. Supplement of 2-fluoro-phenylalanine yielded two fluorinated dicinnamoylmethane derivatives, 6,6′-difluoro-dicinnamoylmethane and 6-fluoro-dicinnamoylmethane, of which the latter is a new curcuminoid.     

Isopentenyl diphosphate (IPP)-bypass mevalonate pathways for isopentenol production

Branched C₅ alcohols are promising biofuels with favorable combustion properties. A mevalonate (MVA)-based isoprenoid biosynthetic pathway for C₅ alcohols was constructed in Escherichia coli using genes from several organisms, and the pathway was optimized to achieve over 50% theoretical yield. Although the MVA pathway is energetically less efficient than the native methylerythritol 4-phosphate (MEP) pathway, implementing the MVA pathway in bacterial hosts such as E. coli is advantageous due to its lack of endogenous regulation. The MVA and MEP pathways intersect at isopentenyl diphosphate (IPP), the direct precursor to isoprenoid-derived C₅ alcohols and initial precursor to longer chain terpenes, which makes independent regulation of the pathways difficult. In pursuit of the complete “decoupling” of the MVA pathway from native cellular regulation, we designed novel IPP-bypass MVA pathways for C₅ alcohol production by utilizing promiscuous activities of two enzymes, phosphomevalonate decarboxylase (PMD) and an E. coli-endogenous phosphatase (AphA). These bypass pathways have reduced energetic requirements, are further decoupled from intrinsic regulation, and are free from IPP-related toxicity. In addition to these benefits, we demonstrate that reduced aeration rate has less impact on the bypass pathway than the original MVA pathway. Finally, we showed that performance of the bypass pathway was primarily determined by the activity of PMD. We designed PMD mutants with improved activity and demonstrated titer increases in the mutant strains. These modified pathways would be a good platform for industrial production of isopentenol and related chemicals such as isoprene.     

Chemical investigation of different extracts and essential oil from the tubers of (Tunisian)Cyperus rotundus. Correlation with their antiradical and antimutagenic properties

The mutagenic potential of aqueous, Total Oligomers Flavonoids (TOF), ethyl acetate, and methanol extracts as well as essential oil (EO) obtained from tubers ofCyperus rotundus L. was assessed by “Ames assay”, usingSalmonella tester strains TA98 and TA100, and “SOS chromotest” usingEscherichia coli PQ37 strain with and without an exogenous metabolic activation system (S9). None of the different extracts showed a mutagenic effect. Likewise, the antimutagenicity of the same extracts was tested using the “Ames test” and the “SOS chromotest”. Our results showed thatC. rotundus extracts have antimutagenic effects withSalmonella typhimurium TA98 and TA100 strains towards the mutagen Aflatoxin B1 (AFB1), as well as withE. coli PQ37 strain against AFB1 and nifuroxazide mutagens. A free radical scavenging test was used in order to explore the antioxidant capacity of the extracts obtained from the tubers ofC. rotundus. TOF, ethyl acetate and methanol extracts showed an important free radical scavenging activity towards the 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical. These extracts showed IC50 values of respectively 5, 20 and 65 μg/ml. The beneficial effects of TOF, ethyl acetate, methanol and essential oil extracts ofC. rotundus have been assessed by antioxidant and antimutagenic activities.