Metabolic Engineering Tools Research Articles

Producing Biochemicals in Yarrowia lipolytica from Xylose through a Strain Mating Approach.

Enabling xylose catabolism is challenging, especially for unconventional yeasts and previously engineered background strains. In this study, the efficacy of a yeast mating approach with Yarrowia lipolytica that can combine a previously engineering and evolved xylose phenotype with a metabolite overproduction phenotype is demonstrated. Specifically, several engineered Y. lipolytica strains that produce α-linolenic acid (ALA), riboflavin, and triacetic acid lactone (TAL) with an engineered and adapted xylose-utilizing strain to obtain three diploid strains that rapidly produce these molecules directly from xylose are mated. Titers of 0.52g L-1 ALA, 96.6mg L-1 riboflavin, and 2.9g L-1 TAL, are obtained from xylose in flask cultures and 1.42g L-1 production of ALA is obtained using bioreactor condition. This total production level is generally on par or higher than the parental strain cultivated on glucose, although specific productivities decreased as a result of improved overall cell growth by the diploid strains. In the case of ALA, this lipid content reached similar levels to that of flaxseed oil. This result showcases the first study using strain mating in Y. lipolytica for producing biomolecules from xylose, and thus demonstrates the utility of this approach as a routine tool for metabolic engineering.

Combined genome editing and transcriptional repression for metabolic pathway engineering in Corynebacterium glutamicum using a catalytically active Cas12a.

Corynebacterium glutamicum is a versatile workhorse for producing industrially important commodities. The design of an optimal strain often requires the manipulation of metabolic and regulatory genes to different levels, such as overexpression, downregulation, and deletion. Unfortunately, few tools to achieve multiple functions simultaneously have been reported. Here, a dual-functional clustered regularly interspaced short palindromic repeats (CRISPR) (RE-CRISPR) system that combined genome editing and transcriptional repression was designed using a catalytically active Cas12a (a.k.a. Cpf1) in C. glutamicum. Firstly, gene deletion was achieved using Cas12a under a constitutive promoter. Then, via engineering of the guide RNA sequences, transcriptional repression was successfully achieved using a catalytically active Cas12a with crRNAs containing 15 or 16bp spacer sequences, whose gene repression efficiency was comparable to that of the canonical system (deactivated Cas12a with full-length crRNAs). Finally, RE-CRISPR was developed to achieve genome editing and transcriptional repression simultaneously by transforming a single crRNA plasmid and Cas12a plasmid. The application of RE-CRISPR was demonstrated to increase the production of cysteine and serine for ~ 3.7-fold and 2.5-fold, respectively, in a single step. This study expands the application of CRISPR/Cas12a-based genome engineering and provides a powerful synthetic biology tool for multiplex metabolic engineering of C. glutamicum.

Engineering Pseudomonas putida KT2440 to convert 2,3-butanediol to mevalonate

Biological production of 2,3-butanediol (2,3-BDO), a C4 platform chemical, has been studied recently, but the high cost of separation and purification before chemical conversion is substantial. To overcome this obstacle, we have conducted a study to convert 2,3-BDO to mevalonate, a terpenoid intermediate, using recombinant Pseudomonas putida and this biological process won’t need the separation and purification process of 2,3-BDO. The production of mevalonate when 2,3-BDO was used as a substrate was 6.61 and 8.44 times higher than when glucose and glycerol were used as substrates under the same conditions, respectively. Lower aeration contributed to higher yields of mevalonate in otherwise identical conditions. The maximum mevalonate production on the shaking flask scale was about 2.21 g/L, in this study (product yield was 0.295, 27% of theoretical yield (1.10)). This study was the first successful attempt for mevalonate production by P. putida using 2,3-BDO as the sole carbon source and presented a new metabolic engineering tool and biological process for mevalonate synthesis.

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.

Metabolic engineering applications of the Escherichia coli bacterial artificial chromosome

In metabolic engineering and synthetic biology, the number of genes expressed to achieve better production and pathway regulation in each strain is steadily increasing. The method of choice for expression in Escherichia coli is usually one or several multi-copy plasmids. Meanwhile, the industry standard for long-term, robust production is chromosomal integration of the desired genes. Despite recent advances, genetic manipulation of the bacterial chromosome remains more time consuming than plasmid construction. To allow screening of different metabolic engineering strategies at a level closer to industry while maintaining the molecular-biology advantages of plasmid-based expression, we have investigated the single-copy bacterial artificial chromosome (BAC) as a development tool for metabolic engineering. Using (R)-3-hydroxybutyrate as a model product, we show that BAC can outperform multi-copy plasmids in terms of yield, productivity and specific growth rate, with respective increases of 12%, 18%, and 5%. We both show that gene expression by the BAC simplifies pathway optimization and that the phenotype of pathway expression from BAC is very close to that of chromosomal expression. From these results, we conclude that the BAC can provide a simple platform for performing pathway design and optimization.

Case study of xylose conversion to glycolate in Corynebacterium glutamicum: Current limitation and future perspective of the CRISPR-Cas systems

RNA-guided genome engineering technologies have been developed for the advanced metabolic engineering of microbial cells to enhance the production of value-added chemicals in Corynebacterium glutamicum as an industrial host. Here, we described the biotransformation of xylose to glycolate using engineered Corynebacterium glutamicum, a well-known industrial amino acid producer. A synthetic pathway involving heterologous D-tagatose 3-epimerase and L-fuculose kinase/aldolase reactions was introduced in C. glutamicum, resulting in 9.9 ± 0.01 g/L glycolate from 20 g/L xylose at a yield of 0.51 g/g (equal to 1.0 mol/mol). Additional glyoxylate reduction pathway developed by CRISPR-Cas12a recombineering has been introduced and attempted to increase the maximum theoretical molar yield of 2.0 (mol/mol). Due to the limitation of the CRISPR-Cas12a recombineering with TTTV PAM sites, advanced CRISPR-Cas systems were suggested for the next-round metabolic engineering for improving the glycolate yield to overcome the current genome-editing tool for metabolic engineering in C. glutamicum.

Expanding targeting scope, editing window, and base transition capability of base editing in Corynebacterium glutamicum.

CRISPR/Cas9-guided cytidine deaminase enables C:G to T:A base editing in bacterial genome without introduction of lethal double-stranded DNA break, supplement of foreign DNA template, or dependence on inefficient homologous recombination. However, limited by genome-targeting scope, editing window, and base transition capability, the application of base editing in metabolic engineering has not been explored. Herein, four Cas9 variants accepting different protospacer adjacent motif (PAM) sequences were used to increase the genome-targeting scope of bacterial base editing. After a comprehensive evaluation, we demonstrated that PAM requirement of bacterial base editing can be relaxed from NGG to NG using the Cas9 variants, providing 3.9-fold more target loci for gene inactivation in Corynebacterium glutamicum. Truncated or extended guide RNAs were employed to expand the canonical 5-bp editing window to 7-bp. Bacterial adenine base editing was also achieved with Cas9 fused to adenosine deaminase. With these updates, base editing can serve as an enabling tool for fast metabolic engineering. To demonstrate its potential, base editing was used to deregulate feedback inhibition of aspartokinase via amino acid substitution for lysine overproduction. Finally, a user-friendly online tool named gBIG was provided for designing guide RNAs for base editing-mediated inactivation of given genes in any given sequenced genome (www.ibiodesign.net/gBIG).

Expanding Metabolic Capabilities Using Novel Pathway Designs: Computational Tools and Case Studies

Design and selection of efficient metabolic pathways is critical for the success of metabolic engineering endeavors. Convenient pathways should not only produce the target metabolite in high yields but also are required to be thermodynamically feasible under production conditions, and to prefer efficient enzymes. To support the design and selection of such pathways, different computational approaches have been proposed for exploring the feasible pathway space under many of the above constraints. In this review, an overview of recent constraint-based optimization frameworks for metabolic pathway prediction, as well as relevant pathway engineering case studies that highlight the importance of rational metabolic designs is presented. Despite the availability and suitability of in silico design tools for metabolic pathway engineering, scarce-although increasing-application of computational outcomes is found. Finally, challenges and limitations hindering the broad adoption and successful application of these tools in metabolic engineering projects are discussed.

Development of a CRISPR/Cas9-Based Tool for Gene Deletion in Issatchenkia orientalis

The nonconventional yeast Issatchenkia orientalis has emerged as a potential platform microorganism for production of organic acids due to its ability to grow robustly under highly acidic conditions. However, lack of efficient genetic tools remains a major bottleneck in metabolic engineering of this organism. Here we report that the autonomously replicating sequence (ARS) from Saccharomyces cerevisiae (ScARS) was functional for plasmid replication in I. orientalis, and the resulting episomal plasmid enabled efficient genome editing by the CRISPR/Cas9 system. The optimized CRISPR/Cas9-based system employed a fusion RPR1'-tRNA promoter for single guide RNA (sgRNA) expression and could attain greater than 97% gene disruption efficiency for various gene targets. Additionally, we demonstrated multiplexed gene deletion with disruption efficiencies of 90% and 47% for double gene and triple gene knockouts, respectively. This genome editing tool can be used for rapid strain development and metabolic engineering of this organism for production of biofuels and chemicals.IMPORTANCE Microbial production of fuels and chemicals from renewable and readily available biomass is a sustainable and economically attractive alternative to petroleum-based production. Because of its unusual tolerance to highly acidic conditions, I. orientalis is a promising potential candidate for the manufacture of valued organic acids. Nevertheless, reliable and efficient genetic engineering tools in I. orientalis are limited. The results outlined in this paper describe a stable episomal ARS-containing plasmid and the first CRISPR/Cas9-based system for gene disruptions in I. orientalis, paving the way for applying genome engineering and metabolic engineering strategies and tools in this microorganism for production of fuels and chemicals.

Broadening the Scope of Enforced ATP Wasting as a Tool for Metabolic Engineering in Escherichia coli.

The targeted increase of cellular adenosine triphosphate (ATP) turnover (enforced ATP wasting) has recently been recognized as a promising tool for metabolic engineering when product synthesis is coupled with net ATP formation. The goal of the present study is to further examine and to further develop the concept of enforced ATP wasting and to broaden its scope for potential applications. In particular, considering the fermentation products synthesized byEscherichia coli under anaerobic conditions as a proxy for target chemical(s), i) a new genetic module for dynamic and gradual induction of the F1 -part of the ATPase is developed and it is found that ii) induction of the ATPase leads to higher metabolic activity and increased product formation in E. coli under anaerobic conditions, and that iii) ATP wasting significantly increases substrate uptake and productivity of growth-arrested cells, which is vital for its use in two-stage processes. To the best of the authors' knowledge, the glucose uptake rate of 6.49 mmol gCDW-1 h-1 achieved with enforced ATP wasting is the highest value reported for nongrowing E. coli cells. In summary, this study shows that enforced ATP wasting can be used to improve yield and titer (in growth-coupled processes) as well as volumetric productivity (in two-stage processes) depending on which of the performance measures is more crucial for the process and product of interest.

A Heterocyst-Specific Antisense RNA Contributes to Metabolic Reprogramming in Nostoc sp. PCC 7120.

Upon nitrogen deficiency, some filamentous cyanobacteria differentiate specialized cells, called heterocysts, devoted to N2 fixation. Heterocysts appear regularly spaced along the filaments and exhibit structural and metabolic adaptations, such as loss of photosynthetic CO2 fixation or increased respiration, to provide a proper microaerobic environment for its specialized function. Heterocyst development is under transcriptional control of the global nitrogen regulator NtcA and the specific regulator HetR. Transcription of a large number of genes is induced or repressed upon nitrogen deficiency specifically in cells undergoing differentiation. In recent years, the HetR regulon has been described to include heterocyst-specific trans-acting small RNAs and antisense RNAs (asRNAs), suggesting that there is an additional layer of post-transcriptional regulation involved in heterocyst development. Here, we characterize in the cyanobacterium Nostoc (Anabaena) sp. PCC 7120 an asRNA, that we call as_glpX, transcribed within the glpX gene encoding the Calvin cycle bifunctional enzyme sedoheptulose-1,7-bisphosphatase/fructose-1,6-bisphosphatase (SBPase). Transcription of as_glpX is restricted to heterocysts and is induced very early during the process of differentiation. Expression of as_glpX RNA promotes the cleavage of the glpX mRNA by RNase III, resulting in a reduced amount of SBPase. Therefore, the early expression of this asRNA could contribute to the quick shut-down of CO2 fixation in those cells in the filament that are undergoing differentiation into heterocysts. In summary, as_glpX is the first naturally occurring asRNA shown to rapidly and dynamically regulate metabolic transformation in Nostoc heterocysts. The use of antisense transcripts to manipulate gene expression specifically in heterocysts could became a useful tool for metabolic engineering in cyanobacteria.

Integrating Enzyme and Metabolic Engineering Tools for Enhanced α-Ionone Production.

Metabolic engineering aims to balance intracellular pathways and increase the precursor supply. However, some heterologous enzymes are not evolved to support high flux. To remove the limitation, the catalytic properties of rate-limiting enzymes must be enhanced. Here, we engineered carotenoid cleavage dioxygenase 1 (CCD1), whose intrinsic promiscuity and low activity limited the production of α-ionone in Escherichia coli. Site-directed mutagenesis was carried out to mutate three structural elements of CCD1: an active site loop, η-helices, and α-helices. Furthermore, mutated CCD1 was fused with lycopene ε-cyclase to facilitate substrate channelling. Collectively, these methods improved the α-ionone concentration by >2.5-fold compared to our previously optimized strain. Lastly, the engineered enzyme was used in conjunction with the metabolic engineering strategy to further boost the α-ionone concentration by another 20%. This work deepens our understanding of CCD1 catalytic properties and proves that integrating enzyme and metabolic engineering can be synergistic for a higher microbial production yield.

Artificial Caprolactam-Specific Riboswitch as an Intracellular Metabolite Sensor.

Caprolactam is a monomer used for the synthesis of nylon-6, and a recombinant microbial strain for biobased production of nylon-6 was recently developed. An intracellular biosensor for caprolactam can facilitate high-throughput metabolic engineering of recombinant microbial strains. Because of the mixed production of caprolactam and valerolactam in the recombinant strain, a caprolactam biosensor should be highly specific for caprolactam. However, a highly specific caprolactam sensor has not been reported. Here, we developed an artificial riboswitch that specifically responds to caprolactam. This riboswitch was prepared using a coupled in vitro- in vivo selection strategy with a heterogeneous pool of RNA aptamers obtained from in vitro selection to construct a riboswitch library used in in vivo selection. The caprolactam riboswitch successfully discriminated caprolactam from valerolactam. Moreover, the riboswitch was activated by 3.36-fold in the presence of 50 mM caprolactam. This riboswitch enabled caprolactam-dependent control of cell growth, which will be useful for improving caprolactam production and is a valuable tool for metabolic engineering.

Improvement of phytochemical production by plant cells and organ culture and by genetic engineering

Plants display an amazing ability to synthesize a vast array of secondary metabolites that are an inexhaustible source of phytochemicals, bioactive molecules some of which impact the human health. Phytochemicals present in medicinal herbs and spices have long been used as natural remedies against illness. Plant tissue culture represents an alternative to whole plants as a source of phytochemicals. This approach spares agricultural land that can be used for producing food and other raw materials, thus favoring standardized phytochemical production regardless of climatic adversities and political events. Over the past 20years, different strategies have been developed to increase the synthesis and the extraction of phytochemicals from tissue culture often obtaining remarkable results. Moreover, the availability of genomics and metabolomics tools, along with improved recombinant methods related to the ability to overexpress, silence or disrupt one or more genes of the pathway of interest promise to open new exciting possibilities of metabolic engineering. This review provides a general framework of the cellular and molecular tools developed so far to enhance the yield of phytochemicals. Additionally, some emerging topics such as the culture of cambial meristemoid cells, the selection of plant cell following the expression of genes encoding human target proteins, and the bioextraction of phytochemicals from plant material have been addressed. Altogether, the herein described techniques and results are expected to improve metabolic engineering tools aiming at improving the production of phytochemicals of pharmaceutical and nutraceutical interest.

Metabolic engineering of Clostridium carboxidivorans for enhanced ethanol and butanol production from syngas and glucose

Clostridium carboxidivorans can convert CO2, CO and H2 to ethanol and n-butanol; however, its industrial application is limited by the lack of tools for metabolic pathway engineering. In this study, C. carboxidivorans was successfully engineered to overexpress aor, adhE2, and fnr together with adhE2 or aor. In glucose fermentation, all engineered strains showed higher alcohol yields compared to the wild-type. Strains overexpressing aor showed CO2 re-assimilation during heterotrophic growth. In syngas fermentation, compared to the wild-type, the strain overexpressing adhE2 produced ∼50% more ethanol and the strain overexpressing adhE2 and fnr produced ∼18% more butanol and ∼22% more ethanol. Interestingly, both strains showed obvious acid re-assimilation, with <0.15 g/L total acid remaining at the end of fermentation. Overexpressing fnr with adhE2 enhanced butanol production compared to only adhE2. This is the first report of overexpressing homologous and heterologous genes in C. carboxidivorans for enhancing alcohols production from syngas and glucose.

New Applications of Synthetic Biology Tools for Cyanobacterial Metabolic Engineering.

Cyanobacteria are promising microorganisms for sustainable biotechnologies, yet unlocking their potential requires radical re-engineering and application of cutting-edge synthetic biology techniques. In recent years, the available devices and strategies for modifying cyanobacteria have been increasing, including advances in the design of genetic promoters, ribosome binding sites, riboswitches, reporter proteins, modular vector systems, and markerless selection systems. Because of these new toolkits, cyanobacteria have been successfully engineered to express heterologous pathways for the production of a wide variety of valuable compounds. Cyanobacterial strains with the potential to be used in real-world applications will require the refinement of genetic circuits used to express the heterologous pathways and development of accurate models that predict how these pathways can be best integrated into the larger cellular metabolic network. Herein, we review advances that have been made to translate synthetic biology tools into cyanobacterial model organisms and summarize experimental and in silico strategies that have been employed to increase their bioproduction potential. Despite the advances in synthetic biology and metabolic engineering during the last years, it is clear that still further improvements are required if cyanobacteria are to be competitive with heterotrophic microorganisms for the bioproduction of added-value compounds.

Tandem Mass Spectrometry for 13C Metabolic Flux Analysis: Methods and Algorithms Based on EMU Framework.

In the past two decades, 13C metabolic flux analysis (13C-MFA) has matured into a powerful and widely used scientific tool in metabolic engineering and systems biology. Traditionally, metabolic fluxes have been determined from measurements of isotopic labeling by means of mass spectrometry (MS) or nuclear magnetic resonance (NMR). In recent years, tandem MS has emerged as a new analytical technique that can provide additional information for high-resolution quantification of metabolic fluxes in complex biological systems. In this paper, we present recent advances in methods and algorithms for incorporating tandem MS measurements into existing 13C-MFA approaches that are based on the elementary metabolite units (EMU) framework. Specifically, efficient EMU-based algorithms are presented for simulating tandem MS data, tracing isotopic labeling in biochemical network models and for correcting tandem MS data for natural isotope abundances.

Assembly of Complex Pathways Using Type IIs Restriction Enzymes.

Efficient DNA assembly methods are essential tools for synthetic biology and metabolic engineering. Among several recently developed methods that allow assembly of multiple DNA fragments in a single step, DNA assembly using type IIS enzymes provides many advantages for complex pathway engineering. In particular, it provides the ability for the user to quickly assemble multigene constructs using a series of simple one-pot assembly steps starting from libraries of cloned and sequenced parts. We describe here a protocol for assembly of multigene constructs using the modular cloning system (MoClo). Making constructs using the MoClo system requires to first define the structure of the final construct to identify all basic parts and vectors required for the construction strategy. Basic parts that are not yet available need to be made. Multigene constructs are then assembled using a series of one-pot assembly steps with the set of identified parts and vectors.

Biosensor-based enzyme engineering approach applied to psicose biosynthesis.

Bioproduction of chemical compounds is of great interest for modern industries, as it reduces their production costs and ecological impact. With the use of synthetic biology, metabolic engineering and enzyme engineering tools, the yield of production can be improved to reach mass production and cost-effectiveness expectations. In this study, we explore the bioproduction of D-psicose, also known as D-allulose, a rare non-toxic sugar and a sweetener present in nature in low amounts. D-psicose has interesting properties and seemingly the ability to fight against obesity and type 2 diabetes. We developed a biosensor-based enzyme screening approach as a tool for enzyme selection that we benchmarked with the Clostridium cellulolyticum D-psicose 3-epimerase for the production of D-psicose from D-fructose. For this purpose, we constructed and characterized seven psicose responsive biosensors based on previously uncharacterized transcription factors and either their predicted promoters or an engineered promoter. In order to standardize our system, we created the Universal Biosensor Chassis, a construct with a highly modular architecture that allows rapid engineering of any transcription factor-based biosensor. Among the seven biosensors, we chose the one displaying the most linear behavior and the highest increase in fluorescence fold change. Next, we generated a library of D-psicose 3-epimerase mutants by error-prone PCR and screened it using the biosensor to select gain of function enzyme mutants, thus demonstrating the framework’s efficiency.

Monoamine Biosynthesis via a Noncanonical Calcium-Activatable Aromatic Amino Acid Decarboxylase in Psilocybin Mushroom

Aromatic l-amino acid decarboxylases (AAADs) are a phylogenetically diverse group of enzymes responsible for the decarboxylation of aromatic amino acid substrates into their corresponding aromatic arylalkylamines. AAADs have been extensively studied in mammals and plants as they catalyze the first step in the production of neurotransmitters and bioactive phytochemicals, respectively. Unlike mammals and plants, the hallucinogenic psilocybin mushroom Psilocybe cubensis reportedly employs an unrelated phosphatidylserine-decarboxylase-like enzyme to catalyze l-tryptophan decarboxylation, the first step in psilocybin biosynthesis. To explore the origin of this chemistry in psilocybin mushroom, we generated the first de novo transcriptomes of P. cubensis and investigated several putative l-tryptophan-decarboxylase-like enzymes. We report the biochemical characterization of a noncanonical AAAD from P. cubensis ( PcncAAAD) that exhibits substrate permissiveness toward l-phenylalanine, l-tyrosine, and l-tryptophan, as well as chloro-tryptophan derivatives. The crystal structure of PcncAAAD revealed the presence of a unique C-terminal appendage domain featuring a novel double-β-barrel fold. This domain is required for PcncAAAD activity and regulates catalytic rate and thermal stability through calcium binding. PcncAAAD likely plays a role in psilocybin production in P. cubensis and offers a new tool for metabolic engineering of aromatic-amino-acid-derived natural products.