Biocomposite thermoplastic polyurethanes containing evolved bacterial spores as living fillers to facilitate polymer disintegration.

The field of hybrid engineered living materials seeks to pair living organisms with synthetic materials to generate biocomposite materials with augmented function since living systems can provide highly-programmable and complex behavior. Engineered living materials have typically been fabricated using techniques in benign aqueous environments, limiting their application. In this work, biocomposite fabrication is demonstrated in which spores from polymer-degrading bacteria are incorporated into a thermoplastic polyurethane using high-temperature melt extrusion. Bacteria are engineered using adaptive laboratory evolution to improve their heat tolerance to ensure nearly complete cell survivability during manufacturing at 135 °C. Furthermore, the overall tensile properties of spore-filled thermoplastic polyurethanes are substantially improved, resulting in a significant improvement in toughness. The biocomposites facilitate disintegration in compost in the absence of a microbe-rich environment. Finally, embedded spores demonstrate a rationally programmed function, expressing green fluorescent protein. This research provides a scalable method to fabricate advanced biocomposite materials in industrially-compatible processes.

Laboratory evolution reveals general and specific tolerance mechanisms for commodity chemicals.

Although strain tolerance to high product concentrations is a barrier to the economically viable biomanufacturing of industrial chemicals, chemical tolerance mechanisms are often unknown. To reveal tolerance mechanisms, an automated platform was utilized to evolve Escherichia coli to grow optimally in the presence of 11 industrial chemicals (1,2-propanediol, 2,3-butanediol, glutarate, adipate, putrescine, hexamethylenediamine, butanol, isobutyrate, coumarate, octanoate, hexanoate), reaching tolerance at concentrations 60%-400% higher than initial toxic levels. Sequencing genomes of 223 isolates from 89 populations, reverse engineering, and cross-compound tolerance profiling were employed to uncover tolerance mechanisms. We show that: 1) cells are tolerized via frequent mutation of membrane transporters or cell wall-associated proteins (e.g., ProV, KgtP, SapB, NagA, NagC, MreB), transcription and translation machineries (e.g., RpoA, RpoB, RpoC, RpsA, RpsG, NusA, Rho), stress signaling proteins (e.g., RelA, SspA, SpoT, YobF), and for certain chemicals, regulators and enzymes in metabolism (e.g., MetJ, NadR, GudD, PurT); 2) osmotic stress plays a significant role in tolerance when chemical concentrations exceed a general threshold and mutated genes frequently overlap with those enabling chemical tolerance in membrane transporters and cell wall-associated proteins; 3) tolerization to a specific chemical generally improves tolerance to structurally similar compounds whereas a tradeoff can occur on dissimilar chemicals, and 4) using pre-tolerized starting isolates can hugely enhance the subsequent production of chemicals when a production pathway is inserted in many, but not all, evolved tolerized host strains, underpinning the need for evolving multiple parallel populations. Taken as a whole, this study provides a comprehensive genotype-phenotype map based on identified mutations and growth phenotypes for 223 chemical tolerant isolates.

Enzyme activity engineering based on sequence co-evolution analysis.

The utility of engineering enzyme activity is expanding with the development of biotechnology. Conventional methods have limited applicability as they require high-throughput screening or three-dimensional structures to direct target residues of activity control. An alternative method uses sequence evolution of natural selection. A repertoire of mutations was selected for fine-tuning enzyme activities to adapt to varying environments during the evolution. Here, we devised a strategy called sequence co-evolutionary analysis to control the efficiency of enzyme reactions (SCANEER), which scans the evolution of protein sequences and direct mutation strategy to improve enzyme activity. We hypothesized that amino acid pairs for various enzyme activity were encoded in the evolutionary history of protein sequences, whereas loss-of-function mutations were avoided since those are depleted during the evolution. SCANEER successfully predicted the enzyme activities of beta-lactamase and aminoglycoside 3'-phosphotransferase. SCANEER was further experimentally validated to control the activities of three different enzymes of great interest in chemical production: cis-aconitate decarboxylase, α-ketoglutaric semialdehyde dehydrogenase, and inositol oxygenase. Activity-enhancing mutations that improve substrate-binding affinity or turnover rate were found at sites distal from known active sites or ligand-binding pockets. We provide SCANEER to control desired enzyme activity through a user-friendly webserver.

Kinetic compartmentalization by unnatural reaction for itaconate production.

Physical compartmentalization of metabolism using membranous organelles in eukaryotes is helpful for chemical biosynthesis to ensure the availability of substrates from competitive metabolic reactions. Bacterial hosts lack such a membranous system, which is one of the major limitations for efficient metabolic engineering. Here, we employ kinetic compartmentalization with the introduction of an unnatural enzymatic reaction by an engineered enzyme as an alternative strategy to enable substrate availability from competitive reactions through kinetic isolation of metabolic pathways. As a proof of concept, we kinetically isolate the itaconate synthetic pathway from the tricarboxylic acid cycle in Escherichia coli, which is natively separated by mitochondrial membranes in Aspergillus terreus. Specifically, 2-methylcitrate dehydratase is engineered to alternatively catalyze citrate and kinetically secure cis-aconitate for efficient production using a high-throughput screening system. Itaconate production can be significantly improved with kinetic compartmentalization and its strategy has the potential to be widely applicable.

A Vibrio-based microbial platform for accelerated lignocellulosic sugar conversion.

BACKGROUND: Owing to increasing concerns about climate change and the depletion of fossil fuels, the development of efficient microbial processes for biochemical production from lignocellulosic biomass has been a key issue. Because process efficiency is greatly affected by the inherent metabolic activities of host microorganisms, it is essential to utilize a microorganism that can rapidly convert biomass-derived sugars. Here, we report a novel Vibrio-based microbial platform that can rapidly and simultaneously consume three major lignocellulosic sugars (i.e., glucose, xylose, and arabinose) faster than any previously reported microorganisms. RESULTS: The xylose isomerase pathway was constructed in Vibrio sp. dhg, which naturally displays high metabolic activities on glucose and arabinose but lacks xylose catabolism. Subsequent adaptive laboratory evolution significantly improved xylose catabolism of initial strain and led to unprecedently high growth and sugar uptake rate (0.67 h-1 and 2.15 g gdry cell weight-1 h-1, respectively). Furthermore, we achieved co-consumption of the three sugars by deletion of PtsG and introduction of GalP. We validated its superior performance and applicability by demonstrating efficient lactate production with high productivity (1.15 g/L/h) and titer (83 g/L). CONCLUSIONS: In this study, we developed a Vibrio-based microbial platform with rapid and simultaneous utilization of the three major sugars from lignocellulosic biomass by applying an integrated approach of rational and evolutionary engineering. We believe that the developed strain can be broadly utilized to accelerate the production of diverse biochemicals from lignocellulosic biomass.

Multi-level rebalancing of the naringenin pathway using riboswitch-guided high-throughput screening.

Recombinant microbes have emerged as promising alternatives to natural sources of naringenin-a key molecular scaffold for flavonoids. In recombinant strains, expression levels of the pathway genes should be optimized at both transcription and the translation stages to precisely allocate cellular resources and maximize metabolite production. However, the optimization of the expression levels of naringenin generally relies on evaluating a small number of variants from libraries constructed by varying transcription efficiency only. In this study, we introduce a systematic strategy for the multi-level optimization of biosynthetic pathways. We constructed a multi-level combinatorial library covering both transcription and translation stages using synthetic T7 promoter variants and computationally designed 5'-untranslated regions. Furthermore, we identified improved strains through high-throughput screening based on a synthetic naringenin riboswitch. The most-optimized strain obtained using this approach exhibited a 3-fold increase in naringenin production, compared with the parental strain in which only the transcription efficiency was modulated. Furthermore, in a fed-batch bioreactor, the optimized strain produced 260.2 mg/L naringenin, which is the highest concentration reported to date using glycerol and p-coumaric acid as substrates. Collectively, this work provides an efficient strategy for the expression optimization of the biosynthetic pathways.

Plug-in repressor library for precise regulation of metabolic flux in Escherichia coli.

In metabolic engineering, enhanced production of value-added chemicals requires precise flux control between growth-essential competing and production pathways. Although advances in synthetic biology have facilitated the exploitation of a number of genetic elements for precise flux control, their use requires expensive inducers, or more importantly, needs complex and time-consuming processes to design and optimize appropriate regulator components, case-by-case. To overcome this issue, we devised the plug-in repressor libraries for target-specific flux control, in which expression levels of the repressors were diversified using degenerate 5' untranslated region (5' UTR) sequences employing the UTR Library Designer. After we validated a wide expression range of the repressor libraries, they were applied to improve the production of lycopene from glucose and 3-hydroxypropionic acid (3-HP) from acetate in Escherichia coli via precise flux rebalancing to enlarge precursor pools. Consequently, we successfully achieved optimal carbon fluxes around the precursor nodes for efficient production. The most optimized strains were observed to produce 2.59 g/L of 3-HP and 11.66 mg/L of lycopene, which were improved 16.5-fold and 2.82-fold, respectively, compared to those produced by the parental strains. These results indicate that carbon flux rebalancing using the plug-in library is a powerful strategy for efficient production of value-added chemicals in E. coli.

Auxotrophic Selection Strategy for Improved Production of Coenzyme B12 in Escherichia coli.

The production of coenzyme B12 using well-characterized microorganisms, such as Escherichia coli, has recently attracted considerable attention to meet growing demands of coenzyme B12 in various applications. In the present study, we designed an auxotrophic selection strategy and demonstrated the enhanced production of coenzyme B12 using a previously engineered coenzyme B12-producing E. coli strain. To select a high producer, the coenzyme B12-independent methionine synthase (metE) gene was deleted in E. coli, thus limiting its methionine synthesis to only that via coenzyme B12-dependent synthase (encoded by metH). Following the deletion of metE, significantly enhanced production of the specific coenzyme B12 validated the coenzyme B12-dependent auxotrophic growth. Further precise tuning of the auxotrophic system by varying the expression of metH substantially increased the cell biomass and coenzyme B12 production, suggesting that our strategy could be effectively applied to E. coli and other coenzyme B12-producing strains.

Vibrio sp. dhg as a platform for the biorefinery of brown macroalgae.

Although brown macroalgae holds potential as an alternative feedstock, its utilization by conventional microbial platforms has been limited due to the inability to metabolize one of the principal sugars, alginate. Here, we isolate Vibrio sp. dhg, a fast-growing bacterium that can efficiently assimilate alginate. Based on systematic characterization of the genomic information of Vibrio sp. dhg, we establish a genetic toolbox for its engineering. We also demonstrate its ability to rapidly produce ethanol, 2,3-butanediol, and lycopene from brown macroalgae sugar mixture with high productivities and yields. Collectively, Vibrio sp. dhg can be used as a platform for the efficient conversion of brown macroalgae sugars into diverse value-added biochemicals.

Precise tuning of the glyoxylate cycle in Escherichia coli for efficient tyrosine production from acetate.

BACKGROUND: Acetate is one of promising feedstocks owing to its cheap price and great abundance. Considering that tyrosine production is gradually shifting to microbial production method, its production from acetate can be attempted to further improve the economic feasibility of its production. RESULTS: Here, we engineered a previously reported strain, SCK1, for efficient production of tyrosine from acetate. Initially, the acetate uptake and gluconeogenic pathway were amplified to maximize the flux toward tyrosine. As flux distribution between glyoxylate and TCA cycles is critical for efficient precursor supplementation, the activity of the glyoxylate cycle was precisely controlled by expression of isocitrate lyase gene under different-strength promoters. Consequently, the engineered strain with optimal flux distribution produced 0.70 g/L tyrosine with 20% of the theoretical maximum yield which are 1.6-fold and 1.9-fold increased values of the parental strain. CONCLUSIONS: Tyrosine production from acetate requires precise tuning of the glyoxylate cycle and we obtained substantial improvements in production titer and yield by synthetic promoters and 5' untranslated regions (UTRs). This is the first demonstration of tyrosine production from acetate. Our strategies would be widely applicable to the production of various chemicals from acetate in future.

Novel Hybrid Input Part Using Riboswitch and Transcriptional Repressor for Signal Inverting Amplifier.

Genetic circuits are composed of input, logic, and output parts. Construction of complex circuits for practical applications requires numerous tunable genetic parts. However, the limited diversity and complicated tuning methods used for the input parts hinders the scalability of genetic circuits. Therefore, a new type of input part is required that responds to diverse signals and enables easy tuning. Here, we developed RNA-protein hybrid input parts that combine a riboswitch and orthogonal transcriptional repressors. The hybrid inputs successfully regulated the transcription of an output in response to the input signal detected by the riboswitch and resulted in signal inversion because of the expression of transcriptional repressors. Dose-response parameters including fold-change and half-maximal effective concentration were easily modulated and amplified simply by changing the promoter strength. Furthermore, the hybrid input detected both exogenous and endogenous signals, indicating potential applications in metabolite sensing. This hybrid input part could be highly extensible considering the rich variety of components.

Molecular parts and genetic circuits for metabolic engineering of microorganisms.

Microbial conversion of biomass into value-added biochemicals is a highly sustainable process compared to petroleum-based production. In this regard, microorganisms have been engineered via simple overexpression or deletion of metabolic genes to facilitate the production. However, the producer microorganisms require complex regulatory circuits to maximize productivity and performance. To address this issue, diverse genetic circuits have been developed that allow cells to minimize their metabolic burden, overcome metabolic imbalances and respond to a dynamically changing environment. In this review, we briefly explain the basic strategy for constructing genetic circuits by assembling molecular parts such as input, operation and output modules. Next, we describe recent applications of the circuits in the metabolic engineering of microorganisms to improve biochemical production. Beyond those achievements, genetic circuits will facilitate more innovative approaches to future strain development through mining and engineering new genetic elements and improving the complexity of genetic circuit design.

Synthetic auxotrophs for stable and tunable maintenance of plasmid copy number.

Although plasmid-based expression systems have advantages in multi-copy expression of genes, heterogeneity of plasmid copy number (PCN) in individual cells is inevitable even with the addition of antibiotics. Here, we developed a synthetic auxotrophic system for stable and tunable maintenance of the PCN in Escherichia coli without addition of antibiotics. This auxotroph expresses infA, one of the essential genes encoding a translation initiation factor, on a plasmid instead of on the chromosome. With this system, the gene expression was stably maintained for 40 generations with minimized cell-to-cell variation under antibiotic-free conditions. Moreover, varying the expression level of infA enabled us to rationally tune the PCN by more than 5.6-fold. This antibiotic-free PCN control system significantly improved the production of itaconic acid and lycopene compared to the conventional system based on antibiotics (2-fold). Collectively, the developed strategy could be a platform for the production of value-added products in antibiotic-free cultivation.

Rediscovering Acetate Metabolism: Its Potential Sources and Utilization for Biobased Transformation into Value-Added Chemicals.

One of the great advantages of microbial fermentation is the capacity to convert various carbon compounds into value-added chemicals. In this regard, there have been many efforts to engineer microorganisms to facilitate utilization of abundant carbon sources. Recently, the potential of acetate as a feedstock has been discovered; efforts have been made to produce various biochemicals from acetate based on understanding of its metabolism. In this review, we discuss the potential sources of acetate and summarized the recent progress to improve acetate utilization with microorganisms. Furthermore, we also describe representative studies that engineered microorganisms for the production of biochemicals from acetate.

Production of itaconic acid from acetate by engineering acid-tolerant Escherichia coli W.

Utilization of abundant and cheap carbon sources can effectively reduce the production cost and enhance the economic feasibility. Acetate is a promising carbon source to achieve cost-effective microbial processes. In this study, we engineered an Escherichia coli strain to produce itaconic acid from acetate. As acetate is known to inhibit cell growth, we initially screened for a strain with a high tolerance to 10 g/L of acetate in the medium, and the W strain was selected as the host. Subsequently, the WC strain was obtained by overexpression of cad (encoding cis-aconitate decarboxylase) using a synthetic promoter and 5' UTR. However, the WC strain produced only 0.13 g/L itaconic acid because of low acetate uptake. To improve the production, the acetate assimilating pathway and glyoxylate shunt pathway were amplified by overexpression of pathway genes as well as its deregulation. The resulting strain, WCIAG4 produced 3.57 g/L itaconic acid (16.1% of theoretical maximum yield) after 88 hr of fermentation with rapid acetate assimilation. These efforts support that acetate can be a potential feedstock for biochemical production with engineered E. coli.

Precise flux redistribution to glyoxylate cycle for 5-aminolevulinic acid production in Escherichia coli.

Microbial production of 5-aminolevulinic acid (ALA) has received much attention because of its potential in clinical applications. Overexpression along with the deciphering of regulation of the related enzymes and an analogue transporter yielded remarkable achievements in ALA production. Nonetheless, there is significant room for carbon flux optimization to enhance ALA production. The aim of this study was precise carbon flux optimization for high ALA production in Escherichia coli expressing the ALA biosynthetic pathway. Initially, genes hemA and hemL were overexpressed with strong promoters and synthetic 5'-untranslated regions (5'-UTRs). Then, the tricarboxylic acid (TCA) cycle was blocked to force carbon flux toward the ALA production pathway by deletion of sucA. Although the resulting strain showed a severe metabolic imbalance and low ALA production, further precise tuning of carbon flux to the glyoxylate cycle by varying the transcriptional strength of aceA led to substantially improved cell growth and ALA production. Thus, this precise tuning of the glyoxylate cycle in a quantitative manner should also enable efficient production of other value-added products derived from the TCA cycle.

Optimum Rebalancing of the 3-Hydroxypropionic Acid Production Pathway from Glycerol in Escherichia coli.

3-Hydroxypropionic acid (3-HP) can be biologically produced from glycerol by two consecutive enzymatic reactions, dehydration of glycerol to 3-hydroxypropionaldehyde (3-HPA) and oxidation of 3-HPA. The pathway has been proved efficient, but imbalance between the rates of the two enzymatic reactions often results in the accumulation of the toxic 3-HPA, which severely reduces cell viability and 3-HP production. In this study, we used UTR engineering to maximally increase the activities of glycerol dehydratase (GDHt) and aldehyde dehydrogenase (ALDH) for the high conversion of glycerol to 3-HP. Thereafter, the activity of GDHt was precisely controlled to avoid the accumulation of 3-HPA by varying expression of dhaB1, a gene encoding a main subunit of GDHt. The optimally balanced E. coli HGL_DBK4 showed a substantially enhanced 3-HP titer and productivity compared with the parental strain. The yield on glycerol, 0.97 g 3-HP/g glycerol, in a fed-batch experiment, was the highest ever reported.