Metabolic Engineering Approaches Research Articles

An expanded CRISPRi toolbox for tunable control of gene expression in Pseudomonas putida.

SummaryOwing to its wide metabolic versatility and physiological robustness, together with amenability to genetic manipulations and high resistance to stressful conditions, Pseudomonas putida is increasingly becoming the organism of choice for a range of applications in both industrial and environmental applications. However, a range of applied synthetic biology and metabolic engineering approaches are still limited by the lack of specific genetic tools to effectively and efficiently regulate the expression of target genes. Here, we present a single‐plasmid CRISPR‐interference (CRISPRi) system expressing a nuclease‐deficient cas9 gene under the control of the inducible XylS/Pm expression system, along with the option of adopting constitutively expressed guide RNAs (either sgRNA or crRNA and tracrRNA). We showed that the system enables tunable, tightly controlled gene repression (up to 90%) of chromosomally expressed genes encoding fluorescent proteins, either individually or simultaneously. In addition, we demonstrate that this method allows for suppressing the expression of the essential genes pyrF and ftsZ, resulting in significantly low growth rates or morphological changes respectively. This versatile system expands the capabilities of the current CRISPRi toolbox for efficient, targeted and controllable manipulation of gene expression in P. putida.

Green production of silybin and isosilybin by merging metabolic engineering approaches and enzymatic catalysis

Silymarin extracted from milk thistle seeds, is used for treating hepatic diseases. Silybin and isosilybin are its main components, and synthesized from coupling of taxifolin and coniferyl alcohol. Here, the biosynthetic pathways of taxifolin and coniferyl alcohol were reconstructed in Saccharomyces cerevisiae for the first time. To alleviate substantial burden caused by a great deal of genetic manipulation, expression of the enzymes (e.g. ZWF1, TYR1 and ARO8) playing multiple roles in the relevant biosynthetic pathways was selectively optimized. The strain YT1035 overexpressing seven heterologous enzymes and five native enzymes and the strain YC1053 overexpressing seven heterologous enzymes and four native enzymes, respectively produce 336.8 mg/L taxifolin and 201.1 mg/L coniferyl alcohol. Silybin and isosilybin are synthesized from taxifolin and coniferyl alcohol under catalysis of APX1t (the truncated milk thistle peroxidase), with a yield of 62.5%. This study demonstrates an approach for producing silybin and isosilybin from glucose for the first time.

Metabolic Engineering of Clostridium cellulovorans to Improve Butanol Production by Consolidated Bioprocessing.

Clostridium cellulovorans DSM 743B can produce butyrate when grown on lignocellulose, but it can hardly synthesize butanol. In a previous study, C. cellulovorans was successfully engineered to switch the metabolism from butyryl-CoA to butanol by overexpressing an alcohol aldehyde dehydrogenase gene adhE1 from Clostridium acetobutylicum ATCC 824; however, its full potential in butanol production is still unexplored. In the study, a metabolic engineering approach based on a push-pull strategy was developed to further enhance cellulosic butanol production. In order to accomplish this, the carbon flux from acetyl-CoA to butyryl-CoA was pulled by overexpressing a trans-enoyl-coenzyme A reductase gene (ter), which can irreversibly catalyze crotonyl-CoA to butyryl-CoA. Then an acid reassimilation pathway uncoupled with acetone production was introduced to redirect the carbon flow from butyrate and acetate toward butyryl-CoA. Finally, xylose metabolism engineering was implemented by inactivating xylR (Clocel_0594) and araR (Clocel_1253), as well as overexpressing xylT (CA_C1345), which is expected to supply additional carbon and reducing power for CoA and butanol synthesis pathways. The final engineered strain produced 4.96 g/L of n-butanol from alkali extracted corn cobs (AECC), increasing by 235-fold compared to that of the wild type. It serves as a promising butanol producer by consolidated bioprocessing.

Engineering Tropane Alkaloid Production Based on Metabolic Characterization of Ornithine Decarboxylase in Atropa belladonna.

Ornithine decarboxylase (ODC) plays an important role in various biological processes; however, its role in plant secondary metabolism, especially in the biosynthesis of tropane alkaloids (TAs) such as pharmaceutical hyoscyamine, anisodamine, and scopolamine, remains largely unknown. In this study, we characterized the physiological and metabolic functions of the ODC gene of Atropa belladonna (AbODC) and determined its role in TA production using metabolic engineering approaches. Feeding assays with enzyme inhibitors indicated that ODC, rather than arginine decarboxylase (ADC), plays a major role in TA biosynthesis. Tissue-specific AbODC expression analysis and β-glucuronidase (GUS) staining assays showed that AbODC was highly expressed in secondary roots, especially in the cylinder tissue. Enzymatic assays indicated that AbODC was able to convert ornithine to putrescine, with the highest activity at pH 8.0 and 30 °C. Additionally, AbODC showed higher catalytic efficiency than other plant ODCs, as evident from the Km, Vmax, and Kcat values of AbODC using ornithine as the substrate. In A.belladonna root cultures, suppression of AbODC greatly reduced the production of putrescine, N-methylputrescine, and TAs, whereas overexpression of AbODC significantly increased the biosynthesis of putrescine, N-methylputrescine, hyoscyamine, and anisodamine. Moreover, transgenic A.belladonna plants overexpressing AbODC showed a significantly higher production of hyoscyamine and anisodamine compared with control plants. These findings indicate that AbODC plays a key role in TA biosynthesis and therefore is a valuable candidate for increasing TA production in A.belladonna.

An Agrobacterium rhizogenes mediated hairy root transformation protocol for fenugreek

•This work describes a protocol for hairy root transformation of the medicinal crop legume fenugreek (Trigonella foenum-graecum L.). Hairy root plant transformation mediated by Agrobacterium rhizogenes is an established method for the rapid genetic transformation of various dicotyledonous plants. We have adapted a hairy root transformation protocol from the model legume Medicago truncatula for use in this metabolically rich non-model crop legume. Considering the great variety and abundance of phytochemicals in fenugreek and its established use in traditional medicine, we aim for this method to become a resource for metabolic pathway identification and for production of valuable specialised metabolites via metabolic engineering approaches.•Development rapid transformation (2.5–3 weeks) of fenugreek roots via A. rhizogenes.•Marker gene cassette with suitable promoter for visual detection of transformed fenugreek roots

Over-expression of LPAAT gene in Phaeodactylum tricornutum enhances fatty acid accumulation and increases fatty acid chain length

To provide a potential method for improving microalgal lipid production, an over expression vector that expresses lysophosphatidic acid acyltransferase gene in Phaeodactylum tricornutum was constructed. Total fatty acid production incresed significantly in over-expression transformants and the length of fatty acids ranged from C14 to C22. The response to both lysophosphatidic acid acyltransferase over- and under-expression over the growth phase was also characterized. This study indicated a potential method to increase fatty acids accumulation in microalgae by employing a metabolic engineering approach.

Construction of artificial membrane transport metabolons - an emerging strategy in metabolic engineering.

The term 'membrane transport metabolon' refers to the physical association of membrane transporters with enzymes that metabolize the transported substrates. In naturally evolved systems, physiological relevance of coupling transport with sequential enzymatic reactions resides, for instance, in faster turnover rates, protection of substrates from competing pathways or shielding the cellular environment from toxic compounds. Such underlying principles offer attractive possibilities for metabolic engineering approaches and concepts for constructing artificial transporter-enzyme complexes are recently being developed. In this minireview, the modes of substrate channeling across biological membranes and design principles for artificial transport metabolons are discussed.

Building cell factories for the production of advanced fuels.

Synthetic biology-based engineering strategies are being extensively employed for microbial production of advanced fuels. Advanced fuels, being comparable in energy efficiency and properties to conventional fuels, have been increasingly explored as they can be directly incorporated into the current fuel infrastructure without the need for reconstructing the pre-existing set-up rendering them economically viable. Multiple metabolic engineering approaches have been used for rewiring microbes to improve existing or develop newly programmed cells capable of efficient fuel production. The primary challenge in using these approaches is improving the product yield for the feasibility of the commercial processes. Some of the common roadblocks towards enhanced fuel production include - limited availability of flux towards precursors and desired pathways due to presence of competing pathways, limited cofactor and energy supply in cells, the low catalytic activity of pathway enzymes, obstructed product transport, and poor tolerance of host cells for end products. Consequently, despite extensive studies on the engineering of microbial hosts, the costs of industrial-scale production of most of these heterologously produced fuel compounds are still too high. Though considerable progress has been made towards successfully producing some of these biofuels, a substantial amount of work needs to be done for improving the titers of others. In this review, we have summarized the different engineering strategies that have been successfully used for engineering pathways into commercial hosts for the production of advanced fuels and different approaches implemented for tuning host strains and pathway enzymes for scaling up production levels.

Design and Construction of a Synthetic Riboregulator-Based Platform for Metabolic Shunting of Pathways in Lactococcus lactis

Microbial cell factories are subjected to rewiring of basic metabolism to enhance the carbon flux towards the desired product pathway. Conventionally, this metabolic engineering approach often involves over-expression of pathway genes and knocking out genes in the competing pathways. However, these approaches result in severe metabolic burden and eventual poor performance of the cells. Particularly, where biomass formation and product synthesis depend on common precursors, permanent changes like knocking out genes will only result in poor titer. Hence, temporary decoupling of biomass formation and product synthesis is considered to be a potential alternative. In this study, we designed synthetic riboregulators, which are RNA-based genetic switches, to shunt metabolic pathways in Lactococcus lactis bacteria, a GRAS organism employed as a cell factory for many biocompounds. The riboregulators are then subjected to evaluation by tagging the cis-repressive sequence (crRNA) to mCherry, a fluorescent reporter and regulated by the constitutive promoter, [Formula: see text]. The trans-activating RNA (taRNA) that interacts with the crRNA is placed under the control of another inducible promoter, [Formula: see text]. First, we observed that when there is no induction of taRNA, there is negligible fluorescence of mCherry indicating the successful repression of translation by the cis-sequence as expected. This has been further verified by comparing this expression level with the expression level of [Formula: see text]-mCherry without the cis-sequence, using fluorometer. Results from this analysis suggest that there is [Formula: see text]% repression by the designed crRNA sequence. Next, we induced the cells with 2[Formula: see text]ng/mL of nisin in the mid-log phase. Upon induction, there is a maximum of three fold increase in the fluorescence levels when compared to the uninduced cells, suggesting that the trans-activation takes place inside the live cells. However, further studies are necessary to optimize the cis-trans ratio to achieve better dynamic range for expression modulation and time window for operating metabolic shunting of competing pathways successfully in L. lactis.

Conversion of Cytochrome P450 2D6 of Human Into a FRET-Based Tool for Real-Time Monitoring of Ajmalicine in Living Cells.

Ajmalicine is naturally present in the root bark of Catharanthus roseus L. and Rauvolfia serpentina (L.) Benth ex.Kurz. It has been extensively utilized in the treatment of hypertension across the world. The increased demand, overconsumption, and low content of the alkaloid in the plants have raised the issue of the depletion of natural sources. The metabolic engineering approach has not been successful in improving the content of the ajmalicine because the metabolic regulation of this metabolite is not known. The regulation of a metabolite in the metabolic pathway requires a tool that can carry out real-time measurement of the flux of the metabolite in living system. Given this, the present study was conducted to develop a genetically encoded FRET-based nanosensor by engineering human Cytochrome P450-2D6, an ajmalicine binding protein. The Cytochrome P450-2D6 was sandwiched between two FRET fluorophores. The design of the nanosensor brings two fluorescent proteins in conjunction with the ajmalicine binding protein, such that it undergoes FRET (Fluorescence Resonance Energy Transfer) upon binding of the ligand. The nanosensor, named as FLIP-Ajn (Fluorescence Indicator Protein for Ajmalicine), was pH stable and ajmalicine specific. The affinity of the FLIP-Ajn was 582 μM. The FLIP-Ajn successfully performed real-time measurement of ajmalicine in prokaryotic (bacteria) and eukaryotic systems (yeast, animal cell line, and plant suspension culture), thereby, establishing its biocompatibility in monitoring of ajmalicine in living cells. Besides, several affinity mutants of the nanosensor were generated through mutations in the ajmalicine binding protein to increase the detection range of the nanosensor at varying physiological scales. The non-invasiveness and high spatial and temporal resolution of the tool holds a great significance in the bio-imaging of a highly compartmentalized metabolic pathway. The flux study of ajmalicine will help in identifying the regulatory steps involved in the synthesis of the alkaloids and, hence, will improve the production rate of ajmalicine from its natural sources.

Establishment and application of multiplexed CRISPR interference system in Bacillus licheniformis

Bacillus licheniformis has been regarded as an outstanding microbial cell factory for the production of biochemicals and enzymes. Due to lack of genetic tools to repress gene expression, metabolic engineering and gene function elucidation are limited in this microbe. In this study, an integrated CRISPR interference (CRISPRi) system was constructed in B. licheniformis. Several endogenous genes, including yvmC, cypX, alsD, pta, ldh, and essential gene rpsC, were severed as the targets to test this CRISPRi system, and the repression efficiencies were ranged from 45.02 to 94.00%. Moreover, the multiple genes were simultaneously repressed with high efficiency using this CRISPRi system. As a case study, the genes involved in by-product synthetic and L-valine degradation pathways were selected as the silence targets to redivert metabolic flux toward L-valine synthesis. Repression of acetolactate decarboxylase (alsD) and leucine dehydrogenase (bcd) led to 90.48% and 80.09 % increases in L-valine titer, respectively. Compared with the control strain DW9i△leuA (1.47 g/L and 1.79 g/L), the L-valine titers of combinatorial strain DW9i△leuA/pHYi-alsD-bcd were increased by 1.27-fold and 2.89-fold, respectively, in flask and bioreactor. Collectively, this work provides a feasible approach for multiplex metabolic engineering and functional genome studies of B. licheniformis.

Metabolic Engineering of Bacterial Respiration: High vs. Low P/O and the Case of Zymomonas mobilis.

Respiratory chain plays a pivotal role in the energy and redox balance of aerobic bacteria. By engineering respiration, it is possible to alter the efficiency of energy generation and intracellular redox state, and thus affect the key bioprocess parameters: cell yield, productivity and stress resistance. Here we summarize the current metabolic engineering and synthetic biology approaches to bacterial respiratory metabolism, with a special focus on the respiratory chain of the ethanologenic bacterium Zymomonas mobilis. Electron transport in Z. mobilis can serve as a model system of bacterial respiration with low oxidative phosphorylation efficiency. Its application for redox balancing and relevance for improvement of stress tolerance are analyzed.

Recent systems biology approaches for probiotics use in health aspects: a review.

The market of probiotics is growing dynamically for the food and supplements, which provides better health to an individual. Probiotics are used as dietary management for diseases, but it varies between regions and persons. Systems biology can help in resolving the strain specificity of probiotics by studying their genome level organization. In this review, we have compiled facets of systems biology and next-generation omics methods such as metagenomics, proteomics and metabolomics. These tools are crucial for the optimization of the metabolic processes in probiotics and hence, their use for human health. The limitations and challenges associated with the development of probiotics involve their stability and function in different individuals. Systems biology facilitates emerging metabolic engineering approaches to improve probiotics strain for their broader application. This review provides comprehensive and updated knowledge of engineered probiotics as therapeutics and various challenges in the development of engineered probiotics.

Metabolic engineering of Pseudomonas mendocina NK-01 for enhanced production of medium-chain-length polyhydroxyalkanoates with enriched content of the dominant monomer

In this study, six genes involved in β-oxidation pathway of P. mendocina NK-01 were deleted to construct mutant strains NKU-∆β1 and NKU-∆β5. Compared with the wild strain NKU, the mcl-PHA titers of NKU-∆β5 were respectively increased by 5.58- and 4.85-fold for culturing with sodium octanoate and sodium decanoate. And the mcl-PHA titers of NKU-∆β1 was increased by 10.02-fold for culturing with dodecanoic acid. The contents of dominant monomers 3-hydroxydecanoate (3HD) and 3-hydroxydodecanoate (3HDD) of the mcl-PHA synthesized by NKU-∆β5 were obviously increased to 90.01 and 58.60 mol%, respectively. Further deletion of genes phaG and phaZ, the 3HD and 3HDD contents were further improved to 94.71 and 68.67 mol%, respectively. The highest molecular weight of mcl-PHA obtained in this study was 80.79 × 104 Da, which was higher than the previously reported mcl-PHA. With the increase of dominant monomer contents, the synthesized mcl-PHA showed better thermal properties, mechanical properties and crystallization properties. Interestingly, the cell size of NKU-∆β5 was larger than that of NKU due to the accumulation of more PHA granules. This study indicated that a systematically metabolic engineering approach for P. mendocina NK-01 could significantly improve the mcl-PHA titer, dominant monomer contents and physical properties of mcl-PHA.

Dual pathway for metabolic engineering of Escherichia coli to produce the highly valuable hydroxytyrosol.

One of the most abundant phenolic compounds traced in olive tissues is hydroxytyrosol (HT), a molecule that has been attributed with a pile of beneficial effects, well documented by many epidemiological studies and thus adding value to products containing it. Strong antioxidant capacity and protection from cancer are only some of its exceptional features making it ideal as a potential supplement or preservative to be employed in the nutraceutical, agrochemical, cosmeceutical, and food industry. The HT biosynthetic pathway in plants (e.g. olive fruit tissues) is not well apprehended yet. In this contribution we employed a metabolic engineering strategy by constructing a dual pathway introduced in Escherichia coli and proofing its significant functionality leading it to produce HT. Our primary target was to investigate whether such a metabolic engineering approach could benefit the metabolic flow of tyrosine introduced to the conceived dual pathway, leading to the maximalization of the HT productivity. Various gene combinations derived from plants or bacteria were used to form a newly inspired, artificial biosynthetic dual pathway managing to redirect the carbon flow towards the production of HT directly from glucose. Various biosynthetic bottlenecks faced due to feaB gene function, resolved through the overexpression of a functional aldehyde reductase. Currently, we have achieved equimolar concentration of HT to tyrosine as precursor when overproduced straight from glucose, reaching the level of 1.76 mM (270.8 mg/L) analyzed by LC-HRMS. This work realizes the existing bottlenecks of the metabolic engineering process that was dependent on the utilized host strain, growth medium as well as to other factors studied in this work.

Location, location! cellular relocalization primes specialized metabolic diversification.

Specialized metabolites are structurally diverse and cell- or tissue-specific molecules produced in restricted plant lineages. In contrast, primary metabolic pathways are highly conserved in plants and produce metabolites essential for all of life, such as amino acids and nucleotides. Substrate promiscuity - the capacity to accept non-native substrates - is a common characteristic of enzymes, and its impact is especially apparent in generating specialized metabolite variation. However, promiscuity only leads to metabolic diversity when alternative substrates are available; thus, enzyme cellular and subcellular localization directly influence chemical phenotypes. We review a variety of mechanisms that modulate substrate availability for promiscuous plant enzymes. We focus on examples where evolution led to modification of the 'cellular context' through changes in cell-type expression, subcellular relocalization, pathway sequestration, and cellular mixing via tissue damage. These varied mechanisms contributed to the emergence of structurally diverse plant specialized metabolites and inform future metabolic engineering approaches.

Trends in Systems Biology for the Analysis and Engineering of Clostridium acetobutylicum Metabolism.

Clostridium acetobutylicum has received renewed interest worldwide as a promising producer of biofuels and bulk chemicals such as n-butanol, 1,3-propanediol, 1,3-butanediol, isopropanol, and butyrate. To develop commercial processes for the production of bulk chemicals via a metabolic engineering approach it is necessary to better characterize both the primary metabolism and metabolic regulation of C.acetobutylicum. Here, we review the history of the development of omics studies of C.acetobutylicum, summarize the recent application of quantitative/integrated omics approaches to the physiological analysis and metabolic engineering of this bacterium, and provide directions for future studies to address current challenges.

Efficient engineered probiotics using synthetic biology approaches: A review.

The uses of probiotics-based food supplements are getting emphasis due to their power to ensure better health conditions. Probiotics have diverse and significant applications in the health sector, so probiotic strains require an understanding of the genome level organizations. Probiotics elucidate various functional parameters that control their metabolic functions. In this review, we have compiled aspects of synthetic biology, which are used for the optimization of metabolic processes in probiotics for their use as a supplement in allopathic medicines. Synthetic biology approaches provide information about diverse biosynthetic pathways and also facilitate the novel metabolic engineering approaches for probiotics strain improvement. We have discussed the synthetic biology approaches for producing engineered probiotics via genetic circuits, expression systems, and genome editing tools like CRISPR-Cas and PEVLAB. This review also enlightens future challenges in the development of engineered probiotics.

Engineering the metabolic pathways of lipid biosynthesis to develop robust microalgal strains for biodiesel production.

Algal lipids have shown promising feedstock to produce biodiesel due to higher energy content, higher cetane number, and renewable nature. However, at present, the lipid productivity is too low to meet the commercial needs. Various approaches can be employed to enhance the lipid content and lipid productivity in microalgae. Stress manipulation is an attractive option to modify the algal lipid content, but it faces the drawback of time-consuming production processing and lack of information about molecular mechanisms related to triacylglycerides production in response to stress. Developing the robust hyper lipid accumulating algal strains has gained momentum due to advances in metabolic engineering and synthetic biology tools. Understanding the molecular basis of lipid biosynthesis followed by reorienting the related pathways through genomic modification is an alluring strategy that is believed to achieve the industrial and economic robustness. This review portrays the use of integrated OMIC approaches to elucidate the molecular mechanisms of strain adaptability in response to stress conditions, and identification of molecular pathways that should become novel targets to develop novel algal strains. Moreover, an update on the metabolic engineering approaches to improve the lipid production in microalgae is also provided.

Emerging Species and Genome Editing Tools: Future Prospects in Cyanobacterial Synthetic Biology.

Recent advances in synthetic biology and an emerging algal biotechnology market have spurred a prolific increase in the availability of molecular tools for cyanobacterial research. Nevertheless, work to date has focused primarily on only a small subset of model species, which arguably limits fundamental discovery and applied research towards wider commercialisation. Here, we review the requirements for uptake of new strains, including several recently characterised fast-growing species and promising non-model species. Furthermore, we discuss the potential applications of new techniques available for transformation, genetic engineering and regulation, including an up-to-date appraisal of current Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein (CRISPR/Cas) and CRISPR interference (CRISPRi) research in cyanobacteria. We also provide an overview of several exciting molecular tools that could be ported to cyanobacteria for more advanced metabolic engineering approaches (e.g., genetic circuit design). Lastly, we introduce a forthcoming mutant library for the model species Synechocystis sp. PCC 6803 that promises to provide a further powerful resource for the cyanobacterial research community.