Metabolic Pathway Assembly Research Articles

Phase-Separated Synthetic Organelles Based on Intrinsically Disordered Protein Domain for Metabolic Pathway Assembly in Escherichia coli.

Extensive research efforts have been focused on spatial organization of biocatalytic cascades or catalytic networks in confined cellular environments. Inspired by the natural metabolic systems that spatially regulate pathways via sequestration into subcellular compartments, formation of artificial membraneless organelles through expressing intrinsically disordered proteins in host strains has been proven to be a feasible strategy. Here we report the engineering of a synthetic membraneless organelle platform, which can be used to extend compartmentalization and spatially organize pathway sequential enzymes. We show that heterologous overexpression of the RGG domain derived from the disordered P granule protein LAF-1 in an Escherichia coli strain can form intracellular protein condensates via liquid-liquid phase separation. We further demonstrate that different clients can be recruited to the synthetic compartments via directly fusing with the RGG domain or cooperating with different protein interaction motifs. Using the 2'-fucosyllactose de novo biosynthesis pathway as a model system, we show that clustering sequential enzymes into synthetic compartments can effectively increase the titer and yield of the target product compared to strains with free-floating pathway enzymes. The synthetic membraneless organelle system constructed here gives a promising approach in the development of microbial cell factories, wherein it could be used for the compartmentalization of pathway enzymes to streamline metabolic flux.

A CRISPR–Cas12a system for multi-gene editing (CCMGE) and metabolic pathway assembly in Starmerella bombicola

The traditional homologous recombination (HR) gene-editing method faces problems such as low editing efficiency and absence of marker genes. CRISPR–Cas9-editing efficiency is high and has been widely used in bacteria and yeast. In comparison with CRISPR–Cas9, CRISPR–Cas12a has many outstanding advantages. Here, we report an Acidaminococcus sp. BV3L6 (As) Cas12a-based genome-editing method used for Starmerella bombicola. To demonstrate the high efficiency of the CCMGE system, we verified a counter-selectable marker in S. bombicola, orotidine 5’-phosphate decarboxylase (100% for URA3). We also tested the common gene UDP-glucosyltransferase (100% for UGTA) using a 300 bp donor containing hygromycin expression cassette. This toolkit was further extended to simultaneously edit two genes (18% for UGTA and leu) and three genes (13.8% for UGTA, leu and URA3). The system greatly reduces the screening time for such multi-site editing. Based on the CCMGE system, the PHA (polyhydroxyalkanoate)-producing strain was constructed by increasing the copy number of the PHA synthase (PHAC). The PHA content and DCW reached 11.8% and 30.1 g/L, respectively. The yield of PHA was about three times higher than that of the single-copy strain using the same fermentation method.

Bottom-Up Synthesis of Artificial Cells: Recent Highlights and Future Challenges.

The bottom-up approach in synthetic biology aims to create molecular ensembles that reproduce the organization and functions of living organisms and strives to integrate them in a modular and hierarchical fashion toward the basic unit of life-the cell-and beyond. This young field stands on the shoulders of fundamental research in molecular biology and biochemistry, next to synthetic chemistry, and, augmented by an engineering framework, has seen tremendous progress in recent years thanks to multiple technological and scientific advancements. In this timely review of the research over the past decade, we focus on three essential features of living cells: the ability to self-reproduce via recursive cycles of growth and division, the harnessing of energy to drive cellular processes, and the assembly of metabolic pathways. In addition, we cover the increasing efforts to establish multicellular systems via different communication strategies and critically evaluate the potential applications.

Construction of Escherichia coli cell factories

As an important model industrial microorganism, Escherichia coli has been widely used in pharmaceutical, chemical industry and agriculture. In the past 30 years, a variety of new strategies and techniques, including artificial intelligence, gene editing, metabolic pathway assembly, and dynamic regulation have been used to design, construct, and optimize E. coli cell factories, which remarkably improved the efficiency for biotechnological production of chemicals. In this review, three key aspects for constructing E. coli cell factories, including pathway design, pathway assembly and regulation, and optimization of global cellular performance, are summarized. The technologies that have played important roles in metabolic engineering of E. coli, as well as their future applications, are discussed.

Application of serine integrases for secondary metabolite pathway assembly in Streptomyces

Serine integrases have been shown to be efficient tools for metabolic pathway assembly. To further improve the flexibility and efficiency of pathway engineering via serine integrases, we explored how multiple orthogonally active serine integrases can be applied for use in vitro for the heterologous expression of complex biosynthesis pathways in Streptomyces spp., the major producers of useful bioactive natural products. The results show that multiple orthogonal serine integrases efficiently assemble the genes from a complex biosynthesis pathway in a single in vitro recombination reaction, potentially permitting a versatile combinatorial assembly approach. Furthermore, the assembly strategy also permitted the incorporation of a well-characterised promoter upstream of each gene for expression in a heterologous host. The results demonstrate how site-specific recombination based on orthogonal serine integrases can be applied in Streptomyces spp.

Synthetic metabolic channel by functional membrane microdomains for compartmentalized flux control

The anchoring of metabolic pathway enzymes to spatial scaffolds can significantly improve their reaction efficiency. Here, we successfully constructed a multi-enzyme complex assembly system able to enhance bioproduction in bacteria by using the endogenous spatial scaffolds─functional membrane microdomains (FMMs). First, using VA-TIRFM and SPT analysis, we reveal that FMMs possess high temporal and spatial stability at the plasma membrane and can be used as endogenous spatial scaffolds to organize enzyme pathways. Then, taking the synthesis of N-acetylglucosamine (GlcNAc) in Bacillus subtilis as a proof-of-concept demonstration, we found that anchoring of various enzymes required for GlcNAc synthesis onto FMMs to obtain the FMMs-multi-enzyme complex system resulted in a significant increase in GlcNAc titer and an effectively alleviate in cell lysis at the later stage of fermentation compared to that in control strains expressing the related enzymes in the cytoplasm. Combining with metabolic model and kinetics analysis, the existence of a constructed substrate channel that maximizes the reaction efficiency is verified. In summary, we propose a novel metabolic pathway assembly model which allowed improved titers and compartmentalized flux control with high spatial resolution in bacterial metabolism.

Variability in Assembly of Degradation Operons for Naphthalene and its derivative, Carbaryl, Suggests Mobilization through Horizontal Gene Transfer.

In the biosphere, the largest biological laboratory, increased anthropogenic activities have led microbes to evolve and adapt to the changes occurring in the environment. Compounds, specifically xenobiotics, released due to such activities persist in nature and undergo bio-magnification in the food web. Some of these compounds act as potent endocrine disrupters, mutagens or carcinogens, and therefore their removal from the environment is essential. Due to their persistence, microbial communities have evolved to metabolize them partially or completely. Diverse biochemical pathways have evolved or been assembled by exchange of genetic material (horizontal gene transfer) through various mobile genetic elements like conjugative and non-conjugative plasmids, transposons, phages and prophages, genomic islands and integrative conjugative elements. These elements provide an unlimited opportunity for genetic material to be exchanged across various genera, thus accelerating the evolution of a new xenobiotic degrading phenotype. In this article, we illustrate examples of the assembly of metabolic pathways involved in the degradation of naphthalene and its derivative, Carbaryl, which are speculated to have evolved or adapted through the above-mentioned processes.

Engineered bidirectional promoters enable rapid multi-gene co-expression optimization

Numerous synthetic biology endeavors require well-tuned co-expression of functional components for success. Classically, monodirectional promoters (MDPs) have been used for such applications, but MDPs are limited in terms of multi-gene co-expression capabilities. Consequently, there is a pressing need for new tools with improved flexibility in terms of genetic circuit design, metabolic pathway assembly, and optimization. Here, motivated by nature’s use of bidirectional promoters (BDPs) as a solution for efficient gene co-expression, we generate a library of 168 synthetic BDPs in the yeast Komagataella phaffii (syn. Pichia pastoris), leveraging naturally occurring BDPs as a parts repository. This library of synthetic BDPs allows for rapid screening of diverse expression profiles and ratios to optimize gene co-expression, including for metabolic pathways (taxadiene, β-carotene). The modular design strategies applied for creating the BDP library could be relevant in other eukaryotic hosts, enabling a myriad of metabolic engineering and synthetic biology applications.

Identification of parallel and divergent optimization solutions for homologous metabolic enzymes

Metabolic pathway assembly typically involves the expression of enzymes from multiple organisms in a single heterologous host. Ensuring that each enzyme functions effectively can be challenging, since many potential factors can disrupt proper pathway flux. Here, we compared the performance of two enzyme homologs in a pathway engineered to allow Escherichia coli to grow on 4-hydroxybenzoate (4-HB), a byproduct of lignocellulosic biomass deconstruction. Single chromosomal copies of the 4-HB 3-monooxygenase genes pobA and praI, from Pseudomonas putida KT2440 and Paenibacillus sp. JJ-1B, respectively, were introduced into a strain able to metabolize protocatechuate (PCA), the oxidation product of 4-HB. Neither enzyme initially supported consistent growth on 4-HB. Experimental evolution was used to identify mutations that improved pathway activity. For both enzymes, silent mRNA mutations were identified that increased enzyme expression. With pobA, duplication of the genes for PCA metabolism allowed growth on 4-HB. However, with praI, growth required a mutation in the 4-HB/PCA transporter pcaK that increased intracellular concentrations of 4-HB, suggesting that flux through PraI was limiting. These findings demonstrate the value of directed evolution strategies to rapidly identify and overcome diverse factors limiting enzyme activity.

Combinatorial metabolic pathway assembly in the yeast genome with RNA-guided Cas9.

The yeast Saccharomyces cerevisiae is an important industrial platform for the production of grain and cellulosic ethanol, isobutanol, butanediol, isoprenoids, and other chemicals. The construction of a successful production strain usually involves multiple gene knockouts and chromosomal integration of expression cassettes to redirect the metabolic fluxes for the conversion of sugars and other feed stocks into the desired product. RNA-guided Cas9 based genome editing has been demonstrated in many prokaryotic and eukaryotic hosts including S. cerevisiae, in which it has been additionally exploited as a tool for metabolic engineering. To extend the utilization of RNA-guided Cas9 as a metabolic pathway building tool, we demonstrated the direct assembly and chromosomal integration of up to 17 overlapping DNA fragments encoding the beta-carotene biosynthetic pathway. Furthermore, we generated a combinatorial strain library for the beta-carotene biosynthetic pathway, directly integrated into the yeast genome to create a diverse library of strains. This enabled the screening of combinatorial libraries in stable chromosomally integrated strains for rapid improvements of product titers. This combinatorial approach for pathway assembly will significantly accelerate the current speed of metabolic engineering for S. cerevisiae as an industrial platform, and increase the number of strains that can be simultaneously evaluated for enzyme screening, expression optimization and protein engineering to achieve the titer, rate and yield necessary for the commercialization of new industrial fermentation products.

Yeast Pathway Kit: A Method for Metabolic Pathway Assembly with Automatically Simulated Executable Documentation.

We have developed the Yeast Pathway Kit (YPK) for rational and random metabolic pathway assembly in Saccharomyces cerevisiae using reusable and redistributable genetic elements. Genetic elements are cloned in a suicide vector in a rapid process that omits PCR product purification. Single-gene expression cassettes are assembled in vivo using genetic elements that are both promoters and terminators (TP). Cassettes sharing genetic elements are assembled by recombination into multigene pathways. A wide selection of prefabricated TP elements makes assembly both rapid and inexpensive. An innovative software tool automatically produces detailed self-contained executable documentation in the form of pydna code in the narrative Jupyter notebook format to facilitate planning and sharing YPK projects. A d-xylose catabolic pathway was created using YPK with four or eight genes that resulted in one of the highest growth rates reported on d-xylose (0.18 h(-1)) for recombinant S. cerevisiae without adaptation. The two-step assembly of single-gene expression cassettes into multigene pathways may improve the yield of correct pathways at the cost of adding overall complexity, which is offset by the supplied software tool.

Designing overall stoichiometric conversions and intervening metabolic reactions.

Existing computational tools for de novo metabolic pathway assembly, either based on mixed integer linear programming techniques or graph-search applications, generally only find linear pathways connecting the source to the target metabolite. The overall stoichiometry of conversion along with alternate co-reactant (or co-product) combinations is not part of the pathway design. Therefore, global carbon and energy efficiency is in essence fixed with no opportunities to identify more efficient routes for recycling carbon flux closer to the thermodynamic limit. Here, we introduce a two-stage computational procedure that both identifies the optimum overall stoichiometry (i.e., optStoic) and selects for (non-)native reactions (i.e., minRxn/minFlux) that maximize carbon, energy or price efficiency while satisfying thermodynamic feasibility requirements. Implementation for recent pathway design studies identified non-intuitive designs with improved efficiencies. Specifically, multiple alternatives for non-oxidative glycolysis are generated and non-intuitive ways of co-utilizing carbon dioxide with methanol are revealed for the production of C2+ metabolites with higher carbon efficiency.