Metabolic Engineering Strategies Research Articles

Efficient Production of 2'-Fucosyllactose from l-Fucose via Self-Assembling Multienzyme Complexes in Engineered Escherichia coli.

2'-Fucosyllactose (2'-FL) has been widely used as a nutritional additive in infant formula due to its multifarious nutraceutical and pharmaceutical functions in neonate health. As such, it is essential to develop an efficient and extensive microbial fermentation platform to cater to the needs of the 2'-FL market. In this study, a spatial synthetic biology strategy was employed to promote 2'-FL biosynthesis in recombinant Escherichia coli. First, the salvage pathway for 2'-FL production from l-fucose and lactose was constructed by introducing a bifunctional enzyme l-fucokinase/GDP-l-fucose pyrophosphorylase (Fkp) derived from Bacteroides fragilis and an α-1,2-fucosyltransferase (FutC) derived from Helicobacter pylori into engineered E.coli BL21(DE3). Next, the endogenous genes involved in the degradation and shunting of the substrate and key intermediate were inactivated to improve the availability of precursors for 2'-FL biosynthesis. Moreover, to further improve the yield and titer of 2'-FL, a short peptide pair (RIAD-RIDD) was used to form self-assembling multienzyme complexes in vivo. The spatial localization of peptides and stoichiometry of enzyme assemblies were subsequently optimized to further improve 2'-FL production. Finally, cofactor regeneration was also considered to alleviate the potential cofactor deficiency and redox flux imbalance in the biocatalysis process. Fed-batch fermentation of the final WLS20 strain accumulated 30.5 g/L extracellular 2'-FL with the yield and productivity of 0.661 mol/mol fucose and 0.48 g/L/h, respectively. This research has demonstrated that the application of spatial synthetic biology and metabolic engineering strategies can dramatically enlarge the titer and yield of 2'-FL biosynthesis in engineered E.coli.

Obtaining High-Purity Docosahexaenoic Acid Oil in Thraustochytrid Aurantiochytrium through a Combined Metabolic Engineering Strategy.

High-purity docosahexaenoic acid (DHA) resources are insufficient in the pharmaceutical and food industries. Although many efforts have attempted to obtain the high-purity DHA production, few reports have been successful. Here, a combined metabolic engineering strategy was employed to increase the DHA purity in the oleaginous thraustochytrid Aurantiochytrium. The strategy includes both partial deactivation of the competing pathway of DHA biosynthesis, by disrupting one copy of the fatty acid synthase gene, and strengthening of substrate supply and triacylglycerol synthesis, by the overexpression of acetyl-CoA carboxylase and diacylglycerol acyltransferase. With this strategy, a final mutant was obtained with a DHA purity of 61% in total fatty acids and a content of 331 mg/g dry cell weight. This study provides an advanced strategy for sustainable high-purity DHA production and highlights the strategy for producing designer oils in industrial oleaginous microorganisms.

Selective inactivation of Gram-positive bacteria in vitro and in vivo through metabolic labelling

Bacterial infections are grave threats to human health, particularly those caused by the most common Gram-positive bacteria. The massive administration of broad-spectrum antibiotics to treat various bacterial infections has led to the evolution and spread of drug resistance. As a universal antimicrobial technique unapt to induce drug resistance, photothermal therapy (PTT) is attracting extensive attention in recent years. However, its unspecific killing capability and side effects towards adjacent mammalian cells severely impede the practical applications. Herein, we proposed a metabolic engineering strategy to selectively inactivate Gram-positive bacteria by PTT. A bioorthogonal photothermal agent was prepared by the conjugation of IR-780 iodide and dibenzo-cyclooctyne (IR780-DBCO). Upon pre-metabolizing with 3-azido-D-alanine, Gram-positive bacteria rather than Gram-negative ones, such as Staphylococcus aureus and vancomycin-resistant Enterococcus faecalis (VRE), could be specifically tied up by the explosive IR780-DBCO via copper-free click chemistry. Thereafter, they spontaneously detonated under 15 min near-infrared light irradiation and inactivated nearly 100% Gram-positive bacteria in vitro. Moreover, superbug VRE-induced infection was significantly inhibited by this approach in a mouse skin wound model. This metabolic labelling-based photothermal ablation strategy specific to Gram-positive microbes would stimulate the development of precise anti-bacterial candidates for preclinical applications.

L-valine production in Corynebacterium glutamicum based on systematic metabolic engineering: progress and prospects.

L-valine is an essential branched-chain amino acid that cannot be synthesized by the human body and has a wide range of applications in food, medicine and feed. Market demand has stimulated people's interest in the industrial production of L-valine. At present, the mutagenized or engineered Corynebacterium glutamicum is an effective microbial cell factory for producing L-valine. Because the biosynthetic pathway and metabolic network of L-valine are intricate and strictly regulated by a variety of key enzymes and genes, highly targeted metabolic engineering can no longer meet the demand for efficient biosynthesis of L-valine. In recent years, the development of omics technology has promoted the upgrading of traditional metabolic engineering to systematic metabolic engineering. This whole-cell-scale transformation strategy has become a productive method for developing L-valine producing strains. This review provides an overview of the biosynthesis and regulation mechanism of L-valine, and summarizes the current metabolic engineering techniques and strategies for constructing L-valine high-producing strains. Finally, the opinion of constructing a cell factory for efficiently biosynthesizing L-valine was proposed.

Production of (−)-α-bisabolol in metabolically engineered Saccharomyces cerevisiae

(−)-α-Bisabolol is a natural monocyclic sesquiterpene alcohol present in German chamomile and has been used as an ingredient of functional foods, cosmetics and pharmaceuticals. In this study, metabolic engineering strategies were attempted to produce (−)-α-bisabolol in Saccharomyces cerevisiae. The codon-optimized MrBBS gene coding for (−)-α-bisabolol synthase from Matricaria recutita was expressed in S. cerevisiae for (−)-α-bisabolol production. The resulting strain (DM) produced 9.5 mg/L of (−)-α-bisabolol in 24 h of batch culture. Additionally, the mevalonate pathway was intensified by introducing a truncated HMG1 gene coding for HMG-CoA reductase and ERG10 encoding acetyl-CoA thiolase. The resulting strain (DtEM) produced a 2.9-fold increased concentration of (−)-α-bisabolol than the DM strain. To increase the acetyl-CoA pool, the ACS1 gene coding for acetyl-CoA synthetase was also overexpressed in the DtEM strain. Finally, the DtEMA strain produced 124 mg/L of (−)-α-bisabolol with 2.7 mg/L-h of productivity in a fed-batch fermentation, which were 13 and 6.8 times higher than the DM strain in batch culture, respectively. Conclusively, these metabolically-engineered approaches might pave the way for the sustainable production of other sesquiterpenes in engineered S. cerevisiae.

Plant Metabolic Gene Clusters: Evolution, Organization, and Their Applications in Synthetic Biology.

Plants are a remarkable source of high-value specialized metabolites having significant physiological and ecological functions. Genes responsible for synthesizing specialized metabolites are often clustered together for a coordinated expression, which is commonly observed in bacteria and filamentous fungi. Similar to prokaryotic gene clustering, plants do have gene clusters encoding enzymes involved in the biosynthesis of specialized metabolites. More than 20 gene clusters involved in the biosynthesis of diverse metabolites have been identified across the plant kingdom. Recent studies demonstrate that gene clusters are evolved through gene duplications and neofunctionalization of primary metabolic pathway genes. Often, these clusters are tightly regulated at nucleosome level. The prevalence of gene clusters related to specialized metabolites offers an attractive possibility of an untapped source of highly useful biomolecules. Accordingly, the identification and functional characterization of novel biosynthetic pathways in plants need to be worked out. In this review, we summarize insights into the evolution of gene clusters and discuss the organization and importance of specific gene clusters in the biosynthesis of specialized metabolites. Regulatory mechanisms which operate in some of the important gene clusters have also been briefly described. Finally, we highlight the importance of gene clusters to develop future metabolic engineering or synthetic biology strategies for the heterologous production of novel metabolites.

Engineering Plant Sesquiterpene Synthesis into Yeasts: A Review.

Sesquiterpenes are natural compounds composed of three isoprene units. They represent the largest class of terpene compounds found in plants, and many have remarkable biological activities. Furthermore, sesquiterpenes have broad applications in the flavor, pharmaceutical and biofuel industries due to their complex structures. With the development of metabolic engineering and synthetic biology, the production of different sesquiterpenes has been realized in various chassis microbes. The microbial production of sesquiterpenes provides a promising alternative to plant extraction and chemical synthesis, enabling us to meet the increasing market demand. In this review, we summarized the heterologous production of different plant sesquiterpenes using the eukaryotic yeasts Saccharomyces cerevisiae and Yarrowia lipolytica, followed by a discussion of common metabolic engineering strategies used in this field.

Development and optimization of a microbial co-culture system for heterologous indigo biosynthesis

BackgroundIndigo is a color molecule with a long history of being used as a textile dye. The conventional production methods are facing increasing economy, sustainability and environmental challenges. Therefore, developing a green synthesis method converting renewable feedstocks to indigo using engineered microbes is of great research and application interest. However, the efficiency of the indigo microbial biosynthesis is still low and needs to be improved by proper metabolic engineering strategies.ResultsIn the present study, we adopted several metabolic engineering strategies to establish an efficient microbial biosynthesis system for converting renewable carbon substrates to indigo. First, a microbial co-culture was developed using two individually engineered E. coli strains to accommodate the indigo biosynthesis pathway, and the balancing of the overall pathway was achieved by manipulating the ratio of co-culture strains harboring different pathway modules. Through carbon source optimization and application of biosensor-assisted cell selection circuit, the indigo production was improved significantly. In addition, the global transcription machinery engineering (gTME) approach was utilized to establish a high-performance co-culture variant to further enhance the indigo production. Through the step-wise modification of the established system, the indigo bioproduction reached 104.3 mg/L, which was 11.4-fold higher than the parental indigo producing strain.ConclusionThis work combines modular co-culture engineering, biosensing, and gTME for addressing the challenges of the indigo biosynthesis, which has not been explored before. The findings of this study confirm the effectiveness of the developed approach and offer a new perspective for efficient indigo bioproduction. More broadly, this innovative approach has the potential for wider application in future studies of other valuable biochemicals’ biosynthesis.

A glance at the chemodiversity of Ocimum species: Trends, implications, and strategies for the quality and yield improvement of essential oil.

Ocimum species represent commercially important medicinal and aromatic plants. The essential oil biosynthesized by Ocimum species is enriched with specialized metabolites specifically, terpenoids and phenylpropanoids. Interestingly, various Ocimum species are known to exhibit diverse chemical profiles, and this chemical diversity has been at the center of many studies to identify commercially important chemotypes. Here, we present various chemotypes from the Ocimum species and emphasize trends, implications, and strategies for the quality and yield improvement of essential oil. Globally, many Ocimum species have been analyzed for their essential oil composition in over 50 countries. Asia represents the highest number of chemotypes, followed by Africa, South America, and Europe. Ocimum basilicum L. has been the most widespread and well-studied species, followed by O. gratissimum L., O. tenuiflorum L., O. canum Sims, O. americanum and O. kilimandscharicum Gürke. Moreover, various molecular reasons, benefits, adverse health effects and mechanisms behind this vast chemodiversity have been discussed. Different strategies of plant breeding, metabolic engineering, transgenic, and tissue-culture, along with anatomical modifications, are surveyed to enhance specific chemotypic profiles and essential oil yield in numerous Ocimum species. Consequently, chemical characterization of the essential oil obtained from Ocimum species has become indispensable for its proper utilization. The present chemodiversity knowledge from Ocimum species will help to exploit various applications in the industrial, agriculture, biopharmaceutical, and food sectors.Supplementary InformationThe online version contains supplementary material available at 10.1007/s11101-021-09767-z.

Improving squalene production by blocking the competitive branched pathways and expressing rate-limiting enzymes in Rhodopseudomonas palustris.

Squalene is a medically valuable bioactive compound that can be used as a raw material for fuels. Microbial fermentation is the preferred method for the squalene production. In this study, we employed several metabolic engineering strategies to increase squalene yield in Rhodopseudomonas palustris. A 57% increase in squalene titer was achieved by blocking the carotenoid pathway, thus directing more FPP into the squalene biosynthetic pathway. In order to cut down the conversion of squalene to haponoids, a recombinant strain R. palustris [Δshc, ΔcrtB] in which both carotenoid and haponoid pathways were blocked was then constructed, resulting in a 50-fold increase in squalene titer. Based on the expression of rate-limiting enzymes involved in the squalene pathway, the final squalene content reached 23.3mg/g DCW, which was 178-times higher than that of the wild-type strain. In this study, several methods effective in improving squalene yield have been described and the potential of R. palustris for producing squalene has been demonstrated.

Exploring metal ion metabolisms to improve xylose fermentation in Saccharomycescerevisiae.

SummaryThe development of high‐performance xylose‐fermenting yeast is essential to achieve feasible conversion of biomass‐derived sugars in lignocellulose‐based biorefineries. However, engineered C5‐strains of Saccharomyces cerevisiae still present low xylose consumption rates under anaerobic conditions. Here, we explore alternative metabolisms involved in metal homeostasis, which positively affect C5 fermentation and analyse the non‐obvious regulatory network connection of both metabolisms using time‐course transcriptome analysis. Our results indicated the vacuolar Fe2+/Mn2+ transporter CCC1, and the protein involved in heavy metal ion homeostasis BSD2, as promising new targets for rational metabolic engineering strategies, enhancing xylose consumption in nine and 2.3‐fold compared with control. Notably, intracellular metal concentration levels were affected differently by mutations and the results were compared with positive controls isu1Δ, a Fe‐S cluster scaffold protein, and ssk2Δ, a component of HOG pathway. Temporal expression profiles indicate a metabolic remodelling in response to xylose, demonstrating changes in the main sugar sensing signalling pathways.

Production of proteins and commodity chemicals using engineered Bacillus subtilis platform strain.

Currently, increasing demand of biochemicals produced from renewable resources has motivated researchers to seek microbial production strategies instead of traditional chemical methods. As a microbial platform, Bacillus subtilis possesses many advantages including the generally recognized safe status, clear metabolic networks, short growth cycle, mature genetic editing methods and efficient protein secretion systems. Engineered B. subtilis strains are being increasingly used in laboratory research and in industry for the production of valuable proteins and other chemicals. In this review, we first describe the recent advances of bioinformatics strategies during the research and applications of B. subtilis. Secondly, the applications of B. subtilis in enzymes and recombinant proteins production are summarized. Further, the recent progress in employing metabolic engineering and synthetic biology strategies in B. subtilis platform strain to produce commodity chemicals is systematically introduced and compared. Finally, the major limitations for the further development of B. subtilis platform strain and possible future directions for its research are also discussed.

Metabolic engineering strategies to enable microbial utilization of C1 feedstocks.

One-carbon (C1) substrates are preferred feedstocks for the biomanufacturing industry and have recently gained attention owing to their natural abundance, low production cost and availability as industrial by-products. However, native pathways to utilize these substrates are absent in most biotechnologically relevant microorganisms. Recent advances in synthetic biology, genome engineering and laboratory evolution are enabling the first steps towards the creation of synthetic C1-utilizing microorganisms. Here, we briefly review the native metabolism of methane, methanol, CO2, CO and formate, and how these C1-utilizing pathways can be engineered into heterologous hosts. In addition, this review analyses the potential, the challenges and the perspectives of C1-based biomanufacturing.

Metabolic engineering of non-pathogenic microorganisms for 2,3-butanediol production.

2,3-Butanediol (2,3-BDO) is a promising commodity chemical with various industrial applications. While petroleum-based chemical processes currently dominate the industrial production of 2,3-BDO, fermentation-based production of 2,3-BDO provides an attractive alternative to chemical-based processes with regards to economic and environmental sustainability. The achievement of high 2,3-BDO titer, yield, and productivity in microbial fermentation is a prerequisite for the production of 2,3-BDO at large scales. Also, enantiopure production of 2,3-BDO production is desirable because 2,3-BDO stereoisomers have unique physicochemical properties. Pursuant to these goals, many metabolic engineering strategies to improve 2,3-BDO production from inexpensive sugars by Klebsiella oxytoca, Bacillus species, and Saccharomyces cerevisiae have been developed. This review summarizes the recent advances in metabolic engineering of non-pathogenic microorganisms to enable efficient and enantiopure production of 2,3-BDO. KEY POINTS: • K. oxytoca, Bacillus species, and S. cerevisiae have been engineered to achieve efficient 2,3-BDO production. • Metabolic engineering of non-pathogenic microorganisms enabled enantiopure production of 2,3-BDO. • Cost-effective 2,3-BDO production can be feasible by using renewable biomass.

Clostridium cellulovorans Proteomic Responses to Butanol Stress.

Combination of butanol-hyperproducing and hypertolerant phenotypes is essential for developing microbial strains suitable for industrial production of bio-butanol, one of the most promising liquid biofuels. Clostridium cellulovorans is among the microbial strains with the highest potential for direct production of n-butanol from lignocellulosic wastes, a process that would significantly reduce the cost of bio-butanol. However, butanol exhibits higher toxicity compared to ethanol and C. cellulovorans tolerance to this solvent is low. In the present investigation, comparative gel-free proteomics was used to study the response of C. cellulovorans to butanol challenge and understand the tolerance mechanisms activated in this condition. Sequential Window Acquisition of all Theoretical fragment ion spectra Mass Spectrometry (SWATH-MS) analysis allowed identification and quantification of differentially expressed soluble proteins. The study data are available via ProteomeXchange with the identifier PXD024183. The most important response concerned modulation of protein biosynthesis, folding and degradation. Coherent with previous studies on other bacteria, several heat shock proteins (HSPs), involved in protein quality control, were up-regulated such as the chaperones GroES (Cpn10), Hsp90, and DnaJ. Globally, our data indicate that protein biosynthesis is reduced, likely not to overload HSPs. Several additional metabolic adaptations were triggered by butanol exposure such as the up-regulation of V- and F-type ATPases (involved in ATP synthesis/generation of proton motive force), enzymes involved in amino acid (e.g., arginine, lysine, methionine, and branched chain amino acids) biosynthesis and proteins involved in cell envelope re-arrangement (e.g., the products of Clocel_4136, Clocel_4137, Clocel_4144, Clocel_4162 and Clocel_4352, involved in the biosynthesis of saturated fatty acids) and a redistribution of carbon flux through fermentative pathways (acetate and formate yields were increased and decreased, respectively). Based on these experimental findings, several potential gene targets for metabolic engineering strategies aimed at improving butanol tolerance in C. cellulovorans are suggested. This includes overexpression of HSPs (e.g., GroES, Hsp90, DnaJ, ClpC), RNA chaperone Hfq, V- and F-type ATPases and a number of genes whose function in C. cellulovorans is currently unknown.

Metabolic Engineering of Escherichia coli for High-Yield Production of (R)-1,3-Butanediol.

1,3-Butanediol (1,3-BDO) is an important C4 platform chemical widely used as a solvent in cosmetics and a key intermediate for the synthesis of fragrances, pheromones, and pharmaceuticals. The development of sustainable bioprocesses to produce enantiopure 1,3-BDO from renewable bioresources by fermentation is a promising alternative to conventional chemical routes and has aroused great interest in recent years. Although two metabolic pathways have been previously established for the biosynthesis of (R)-1,3-PDO, the reported titer and yield are too low for cost-competitive production. In this study, we report the combination of different metabolic engineering strategies to improve the production of (R)-1,3-BDO by Escherichia coli, including (1) screening of key pathway enzymes; (2) increasing NADPH supply by cofactor engineering; (3) optimization of fermentation conditions to divert more flux into 1,3-BDO pathway; (4) reduction of byproducts formation by pathway engineering. With these efforts, the best engineered E. coli strain can efficiently produce (R)-1,3-BDO with a yield of 0.6 mol/mol glucose, corresponding to 60% of the theoretical yield. Besides, we also showed the feasibility of aerobically producing 1,3-BDO via a new pathway using 3-hydroxybutyrate as an intermediate.

Research progress in bioremediation of petroleum pollution.

With the enhancement of environmental protection awareness, research on the bioremediation of petroleum hydrocarbon environmental pollution has intensified. Bioremediation has received more attention due to its high efficiency, environmentally friendly by-products, and low cost compared with the commonly used physical and chemical restoration methods. In recent years, bacterium engineered by systems biology strategies have achieved biodegrading of many types of petroleum pollutants. Those successful cases show that systems biology has great potential in strengthening petroleum pollutant degradation bacterium and accelerating bioremediation. Systems biology represented by metabolic engineering, enzyme engineering, omics technology, etc., developed rapidly in the twentieth century. Optimizing the metabolic network of petroleum hydrocarbon degrading bacterium could achieve more concise and precise bioremediation by metabolic engineering strategies; biocatalysts with more stable and excellent catalytic activity could accelerate the process of biodegradation by enzyme engineering; omics technology not only could provide more optional components for constructions of engineered bacterium, but also could obtain the structure and composition of the microbial community in polluted environments. Comprehensive microbial community information lays a certain theoretical foundation for the construction of artificial mixed microbial communities for bioremediation of petroleum pollution. This article reviews the application of systems biology in the enforce of petroleum hydrocarbon degradation bacteria and the construction of a hybrid-microbial degradation system. Then the challenges encountered in the process and the application prospects of bioremediation are discussed. Finally, we provide certain guidance for the bioremediation of petroleum hydrocarbon-polluted environment.

Research advancement and commercialization of microalgae edible oil: a review.

The global food crisis has led to a great deal of attention being given to microalgal oil as a sustainable natural food source. This article provides an overview of the progress and future directions in promoting the commercialization of microalgal edible oils, including microalgal triglyceride accumulation, suitable edible oil culture strategies for high nutritional value, metabolic engineering, production, and downstream technologies. The integration of the production process, biosafety, and the economic sustainability of microalgal oil production are analyzed for their critical roles in the commercialization of microalgal edible oil to provide a theoretical and scientific basis for the comprehensive development and utilization of microalgal edible oil. © 2021 Society of Chemical Industry.

Rewiring the microbial metabolic network for efficient utilization of mixed carbon sources.

Carbon sources represent the most dominant cost factor in the industrial biomanufacturing of products. Thus, it has attracted much attention to seek cheap and renewable feedstocks, such as lignocellulose, crude glycerol, methanol, and carbon dioxide, for biosynthesis of value-added compounds. Co-utilization of these carbon sources by microorganisms not only can reduce the production cost but also serves as a promising approach to improve the carbon yield. However, co-utilization of mixed carbon sources usually suffers from a low utilization rate. In the past few years, the development of metabolic engineering strategies to enhance carbon source co-utilization efficiency by inactivation of carbon catabolite repression has made significant progress. In this article, we provide informative and comprehensive insights into the co-utilization of two or more carbon sources including glucose, xylose, arabinose, glycerol, and C1 compounds, and we put our focus on parallel utilization, synergetic utilization, and complementary utilization of different carbon sources. Our goal is not only to summarize strategies of co-utilization of carbon sources, but also to discuss how to improve the carbon yield and the titer of target products.

One stone two birds: Biosynthesis of 3-hydroxypropionic acid from CO2 and syngas-derived acetic acid in Escherichia coli

Syngas, which contains large amount of CO2 as well as H2 and CO, can be convert to acetic acid chemically or biologically. Nowadays, acetic acid become a cost-effective nonfood-based carbon source for value-added biochemical production. In this study, acetic acid and CO2 were used as substrates for the biosynthesis of 3-hydroxypropionic acid (3-HP) in metabolically engineered Escherichia coli carrying heterogeneous acetyl-CoA carboxylase (Acc) from Corynebacterium glutamicum and codon-optimized malonyl-CoA reductase (MCR) from Chloroflexus aurantiacus. Strategies of metabolic engineering included promoting glyoxylate shunt pathway, inhibiting fatty acid synthesis, dynamic regulating of TCA cycle, and enhancing the assimilation of acetic acid. The engineered strain LNY07(M*DA) accumulated 15.8 g/L of 3-HP with the yield of 0.71 g/g in 48 h by whole-cell biocatalysis. Then, syngas-derived acetic acid was used as substrate instead of pure acetic acid. The concentration of 3-HP reached 11.2 g/L with the yield of 0.55 g/g in LNY07(M*DA). The results could potentially contribute to the future development of an industrial bioprocess of 3-HP production from syngas-derived acetic acid.