Whole-cell biocomputing.

The ability to manipulate systems on the molecular scale naturally leads to speculation about the rational design of molecular-scale machines. Cells might be the ultimate molecular-scale machines and our ability to engineer them is relatively advanced when compared with our ability to control the synthesis and direct the assembly of man-made materials. Indeed, engineered whole cells deployed in biosensors can be considered one of the practical successes of molecular-scale devices. However, these devices explore only a small portion of cellular functionality. Individual cells or self-organized groups of cells perform extremely complex functions that include sensing, communication, navigation, cooperation and even fabrication of synthetic nanoscopic materials. In natural systems, these capabilities are controlled by complex genetic regulatory circuits, which are only partially understood and not readily accessible for use in engineered systems. Here, we focus on efforts to mimic the functionality of man-made information-processing systems within whole cells.     

Production of β-carotene and acetate in recombinant Escherichia coli with or without mevalonate pathway at different culture temperature or pH

Natural β-carotene has received much attention as consumers have become more health conscious. Its production by various microorganisms including metabolically engineered Escherichia coli or Saccharomyces cerevisiae has been attempted. We successfully created a recombinant E. coli with an engineered whole mevalonate pathway in addition to β-carotene biosynthetic genes and evaluated the engineered cells from the aspects of metabolic balance between central metabolism and β-carotene production by comparison with conventional β-carotene producing recombinant E. coli (control) utilizing a native methylerythritol phosphate (MEP) pathway using bioreactor cultures generated at different temperatures or pHs. Better production of β-carotene was obtained in E. coli cultured at 37°C than at 25°C. A two-fold higher titer and 2.9-fold higher volumetric productivity were obtained in engineered cells compared with control cells. Notably, a marginal amount of acetate was produced in actively growing engineered cells, whereas more than 8 g/L of acetate was produced in control cells with reduced cell growth at 37°C. The data indicated that the artificial operon of the whole mevalonate pathway operated efficiently in redirecting acetyl-CoA into isopentenyl pyrophosphate (IPP), thereby improving production of β-carotene, whereas the native MEP pathway did not convert a sufficient amount of pyruvate into IPP due to endogenous feedback regulation. Engineered cells also produced lycopene with a reduced amount of β-carotene in weak alkaline cultures, consistent with the inhibition of lycopene cyclase.     

Engineering Escherichia coli for the synthesis of short- and medium-chain α,β-unsaturated carboxylic acids

Concerns over sustained availability of fossil resources along with environmental impact of their use have stimulated the development of alternative methods for fuel and chemical production from renewable resources. In this work, we present a new approach to produce α,β-unsaturated carboxylic acids (α,β-UCAs) using an engineered reversal of the β-oxidation (r-BOX) cycle. To increase the availability of both acyl-CoAs and enoyl-CoAs for α,β-UCA production, we use an engineered Escherichia coli strain devoid of mixed-acid fermentation pathways and known thioesterases. Core genes for r-BOX such as thiolase, hydroxyacyl-CoA dehydrogenase, enoyl-CoA hydratase, and enoyl-CoA reductase were chromosomally overexpressed under the control of a cumate inducible phage promoter. Native E. coli thioesterase YdiI was used as the cycle-terminating enzyme, as it was found to have not only the ability to convert trans-enoyl-CoAs to the corresponding α,β-UCAs, but also a very low catalytic efficiency on acetyl-CoA, the primer and extender unit for the r-BOX pathway. Coupling of r-BOX with YdiI led to crotonic acid production at titers reaching 1.5 g/L in flask cultures and 3.2 g/L in a controlled bioreactor. The engineered r-BOX pathway was also used to achieve for the first time the production of 2-hexenoic acid, 2-octenoic acid, and 2-decenoic acid at a final titer of 0.2 g/L. The superior nature of the engineered pathway was further validated through the use of in silico metabolic flux analysis, which showed the ability of r-BOX to support growth-coupled production of α,β-UCAs with a higher ATP efficiency than the widely used fatty acid biosynthesis pathway. Taken together, our findings suggest that r-BOX could be an ideal platform to implement the biological production of α,β-UCAs.     

Escherichia coli minicells with targeted enzymes as bioreactors for producing toxic compounds

Formed by aberrant cell division, minicells possess functional metabolism despite their inability to grow and divide. Minicells exhibit not only superior stability when compared with bacterial cells but also exceptional tolerance—characteristics that are essential for a de novo bioreactor platform. Accordingly, we engineered minicells to accumulate protein, ensuring sufficient production capability. When tested with chemicals regarded as toxic against cells, the engineered minicells produced titers of C6–C10 alcohols and esters, far surpassing the corresponding production from bacterial cells. Additionally, microbial autoinducer production that is limited in expanding bacterial population was conducted in the minicells. Because bacterial population growth was nonexistent, the minicells produced autoinducers in constant amounts, which allowed precise control of the bacterial population having autoinducer-responsive gene circuits. When bacterial population growth was nonexistent, the minicells produced autoinducers in constant amounts, which allowed precise control of the bacterial population having autoinducer-based gene circuits with the minicells. This study demonstrates the potential of minicells as bioreactors suitable for products with known limitations in microbial production, thus providing new possibilities for bioreactor engineering.     

Carotenoid accumulation in bacteria with enhanced supply of isoprenoid precursors by upregulation of exogenous or endogenous pathways

Carotenoids are isoprenoid pigments of industrial and nutritional interest. Although they are produced in non-carotenogenic Escherichia coli engineered with the appropriate biosynthetic genes, only a limited pool of their metabolic precursors is available in these bacteria. We have compared the production of carotenoids (lycopene) in strains in which the supply of precursors was enhanced either by upregulating the endogenous pathway via overexpression of deoxyxylulose 5-phosphate synthase (DXS) or by incorporating an exogenous MVA+ operon. In strains expressing DXS under the control of a leaky IPTG-inducible promoter, lycopene accumulation was increased up to 8-fold in the absence of inducer. Addition of IPTG, however, negatively affected lycopene production. Although induction of too high levels of the MVA+ operon enzymes also appeared to cause interference with cell metabolism, supplementation with mevalonate (to be metabolized into carotenoid precursors) resulted in a 10-fold increase in lycopene levels in cells with a near wild-type background. An additional 2-fold increase (up to 228 mg/l) was obtained using an engineered BL21 strain. These results confirm that the MVA+ pathway is most convenient to upregulate the production of carotenoids (lycopene) production in E. coli but that factors other than precursor supply should be considered for high pigment accumulation levels.     

Characterization of binding proteins that recognize oligoglucoside elicitors of phytoalexin synthesis in soybean

We are studying the cellular signaling pathway leading to pterocarpan phytoalexin biosynthesis in soybean that is induced by a branched hepta-β-glucoside originally isolated from the mycelial walls of the phytopathogenic oomycete Phytophthora sojae. Our research has focused on the specific recognition of the hepta-β-glucoside elicitor by binding proteins in soybean cells. Elicitor-binding proteins with properties expected of physiological receptors for the hepta-β-glucoside elicitor have been identified in soybean root membranes. These elicitor-binding proteins co-migrate with a plasma membrane marker (vanadate-sensitive H⁺-ATPase) on linear sucrose density gradients. Binding of a radio-iodinated derivative of the hepta-β-glucoside elicitor by membrane-localized elicitor-binding proteins is specific, reversible, saturable, and of high affinity (K_d? 1 nM). After solubilization with the nonionic detergent, n-dodecylsucrose, the elicitor-binding proteins retain their high affinity (K_d= 1.8 nM) for the radiolabeled elicitor and their binding specificity for elicitor-active oligoglucosides. A direct correlation is observed between the ability of oligoglucosides to displace labeled elicitor from the elicitor-binding proteins and the elicitor activity of the oligosaccharides. Thus, the elicitor-binding proteins recognize the same structural elements of the hepta-β-glucoside elicitor that are essential for its phytoalexin-inducing activity, suggesting that the binding proteins are physiological receptors for the elicitor. Current research is directed toward the purification of the hepta-β-glucoside elicitor-binding proteins by using ligand affinity chromatography. Purification and characterization of the hepta-β-glucoside binding proteins are among the first steps toward elucidating how the hepta-β-glucoside elicitor triggers the signal transduction pathway that ultimately leads to the synthesis of phytoalexins in soybean.     

Synergy between methylerythritol phosphate pathway and mevalonate pathway for isoprene production in Escherichia coli

Isoprene, a key building block of synthetic rubber, is currently produced entirely from petrochemical sources. In this work, we engineered both the methylerythritol phosphate (MEP) pathway and the mevalonate (MVA) pathway for isoprene production in E. coli. The synergy between the MEP pathway and the MVA pathway was demonstrated by the production experiment, in which overexpression of both pathways improved the isoprene yield about 20-fold and 3-fold, respectively, compared to overexpression of the MEP pathway or the MVA pathway alone. The ¹³C metabolic flux analysis revealed that simultaneous utilization of the two pathways resulted in a 4.8-fold increase in the MEP pathway flux and a 1.5-fold increase in the MVA pathway flux. The synergy of the dual pathway was further verified by quantifying intracellular flux responses of the MEP pathway and the MVA pathway to fosmidomycin treatment and mevalonate supplementation. Our results strongly suggest that coupling of the complementary reducing equivalent demand and ATP requirement plays an important role in the synergy of the dual pathway. Fed-batch cultivation of the engineered strain overexpressing the dual pathway resulted in production of 24.0 g/L isoprene with a yield of 0.267 g/g of glucose. The synergy of the MEP pathway and the MVA pathway also successfully increased the lycopene productivity in E. coli, which demonstrates that it can be used to improve the production of a broad range of terpenoids in microorganisms.     

Cellular biosensors for drug discovery.

Recent advances in cell biology, fluorescent probe chemistry, miniaturization and automation have allowed the use of mammalian cells in a variety of medical and industrial applications. Here we describe the generation of cell-based biosensors, engineered to optically report specific biological activity. Cellular biosensors are comprised of living cells and can be used in various applications, including screening chemical libraries for drug discovery and environmental sensing. Panels of biosensors may also be useful for elucidating the function of novel genes. Here we describe two examples of the construction and use of engineered cell lines as biosensors for drug discovery.     

基于2-C-甲基-D-赤藓糖醇-4磷酸途径的产紫槐二烯大肠杆菌构建及其补糖策略优化

2-C-甲基-D-赤藻糖醇-4-磷酸(2-methyl-D-erythritol-4-phosphate, MEP) 途径是大肠杆菌Escherichiacoli 唯一的萜类前体合成途径,研究表明它比甲羟戊酸(Mevalonate, MVA)途径具有更高的理论产率。但目前有关MEP 途径的调控所知非常有限,故单独强化MEP 途径对萜类异源合成产量的提高效果并不理想。研究中通过引入外源MEP 途径基因强化E. coli 萜类合成的遗传改造策略和发酵过程补糖控制优化,尝试更有效地释放MEP 途径的潜力,建立青蒿素前体——紫槐二烯的高密度发酵过程。研究结果表明共表达阿维链霉菌Streptomyces avermitilis dxs2 基因和枯草芽胞杆菌Bacillus subtilis idi 基因可使紫槐二烯的摇瓶发酵产量比野生菌株提高12.2 倍。随后针对该菌株建立了高密度发酵过程,发现稳定期的中前期(24?72 h) 是产物合成的关键期,通过稳定期补糖速率的调整,明显改善了产物合成速度,使紫槐二烯的产量从2.5 g/L 提高到了4.85 g/L,但不影响产物积累的周期。考虑到72 h 后菌体老化可能会影响产物合成,进一步采取了调整对数期的补糖速率控制菌体生长的策略,使紫槐二烯的产量达到6.1 g/L。研究结果为基于MEP 途径的萜类异源合成工程菌构建及其发酵工艺的建立奠定了基础。     

Controlling the Metabolic Flux through the Carotenoid Pathway Using Directed mRNA Processing and Stabilization

A synthetic operon containing the crtI and crtY genes, encoding the phytoene desaturase and the lycopene cyclase, respectively, was placed under the control of the araBAD promoter. DNA cassettes encoding mRNA secondary structures were placed at the 5' and 3' ends of the genes and a putative RNase E site was placed between the genes. This construct was transformed into Escherichia coli cells harboring the genes for phytoene production. By varying the mRNA secondary structures, we were able to modulate the flux through the carotenoid pathway, resulting in a 300-fold variation in the production of beta-carotene relative to lycopene. In addition, intermediates in the pathway from phytoene to beta-carotene production that are not observed in cells expressing the recombinant operon were observed when the engineered operons were used, indicating that changes in levels of the enzymes affected the formation of intermediates. These results indicate that it is possible to coordinately regulate the genes encoding the enzymes of a metabolic pathway and balance the production of the intermediates.     

Increased ethanol production from glycerol by Saccharomyces cerevisiae strains with enhanced stress tolerance from the overexpression of SAGA complex components

During the industrial production of ethanol using yeast, the cells are exposed to stresses that affect their growth and productivity; therefore, stress-tolerant yeast strains are highly desirable. To increase ethanol production from glycerol, a greater tolerance to osmotic and ethanol stress was engineered in yeast strains that were impaired in endogenous glycerol production by the overexpression of both SPT3 and SPT15, components of the SAGA (Spt-Ada-Gcn5-acetyltransferase) complex. The engineered strain YPH499fps1Δgpd2Δ (pGcyaDak, pGupSpt3.15Cas) formed significantly more biomass compared to the strain YPH499fps1Δgpd2Δ (pGcyaDak, pGupCas), and both engineered strains displayed increased biomass when compared to the control YPH499 fps1Δgpd2Δ (pESC-TRP) strain. The trehalose accumulation and ergosterol content of these strains were 2.3-fold and 1.6-fold higher, respectively, than the parent strains, suggesting that levels of cellular membrane components were correlated with the enhanced stress tolerance of the engineered strains. Consequently, the ethanol production of the engineered strain YPH499fps1Δgpd2Δ (pGcyaDak, pGupSpt3.15Cas) was 1.8-fold more than that of strain YPH499fps1Δgpd2Δ (pGcyaDak, pGupCas), with about 8.1g/L ethanol produced. In conclusion, we successfully established that the co-expression of SPT3 and SPT15 that improved the fermentation performance of the engineered yeast strains which produced higher ethanol yields than stress-sensitive yeast strains.     

Protein design for pathway engineering

Design and construction of biochemical pathways has increased the complexity of biosynthetically-produced compounds when compared to single enzyme biocatalysis. However, the coordination of multiple enzymes can introduce a complicated set of obstacles to overcome in order to achieve a high titer and yield of the desired compound. Metabolic engineering has made great strides in developing tools to optimize the flux through a target pathway, but the inherent characteristics of a particular enzyme within the pathway can still limit the productivity. Thus, judicious protein design is critical for metabolic and pathway engineering. This review will describe various strategies and examples of applying protein design to pathway engineering to optimize the flux through the pathway. The proteins can be engineered for altered substrate specificity/selectivity, increased catalytic activity, reduced mass transfer limitations through specific protein localization, and reduced substrate/product inhibition. Protein engineering can also be expanded to design biosensors to enable high through-put screening and to customize cell signaling networks. These strategies have successfully engineered pathways for significantly increased productivity of the desired product or in the production of novel compounds.     

Unlocking the biosynthesis of sesquiterpenoids from methane via the methylerythritol phosphate pathway in methanotrophic bacteria,using α-humulene as a model compound

Isoprenoids are an abundant and diverse class of natural products with various applications in the pharmaceutical, cosmetics and biofuel industries. A methanotroph-based biorefinery is an attractive scenario for the production of a variety of value-added compounds from methane, because methane is a promising alternative feedstock for industrial biomanufacturing. In this study, we metabolically engineered Methylotuvimicrobium alcaliphilum 20Z for de novo synthesis of a sesquiterpenoid from methane, using α-humulene as a model compound, via optimization of the native methylerythritol phosphate (MEP) pathway. Expression of codon-optimized α-humulene synthase from Zingiber zerumbet in M. alcaliphilum 20Z resulted in an initial yield of 0.04 mg/g dry cell weight. Overexpressing key enzymes (IspA, IspG, and Dxs) for debottlenecking of the MEP pathway increased α-humulene production 5.2-fold compared with the initial strain. Subsequently, redirecting the carbon flux through the Embden–Meyerhof–Parnas pathway resulted in an additional 3-fold increase in α-humulene production. Additionally, a genome-scale model using flux scanning based on enforced objective flux method was used to identify potential overexpression targets to increase flux towards isoprenoid production. Several target reactions from cofactor synthesis pathways were probed and evaluated for their effects on α-humulene synthesis, resulting in α-humulene yield up to 0.75 mg/g DCW with 18.8-fold enhancement from initial yield. This study first demonstrates production of a sesquiterpenoid from methane using methanotrophs as the biocatalyst and proposes potential strategies to enhance production of sesquiterpenoid and related isoprenoid products in engineered methanotrophic bacteria.     

Metabolic engineering of type II methanotroph,Methylosinus trichosporium OB3b,for production of 3-hydroxypropionic acid from methane via a malonyl-CoA reductase-dependent pathway

We engineered a type II methanotroph, Methylosinus trichosporium OB3b, for 3-hydroxypropionic acid (3HP) production by reconstructing malonyl-CoA pathway through heterologous expression of Chloroflexus aurantiacus malonyl-CoA reductase (MCR), a bifunctional enzyme. Two strategies were designed and implemented to increase the malonyl-CoA pool and thus, increase in 3HP production. First, we engineered the supply of malonyl-CoA precursors by overexpressing endogenous acetyl-CoA carboxylase (ACC), substantially enhancing the production of 3HP. Overexpression of biotin protein ligase (BPL) and malic enzyme (NADP⁺-ME) led to a ∼22.7% and ∼34.5% increase, respectively, in 3HP titer in ACC-overexpressing cells. Also, the acetyl-CoA carboxylation bypass route was reconstructed to improve 3HP productivity. Co-expression of methylmalonyl-CoA carboxyltransferase (MMC) of Propionibacterium freudenreichii and phosphoenolpyruvate carboxylase (PEPC), which provides the MMC precursor, further improved the 3HP titer. The highest 3HP production of 49 mg/L in the OB3b-MCRMP strain overexpressing MCR, MMC and PEPC resulted in a 2.4-fold improvement of titer compared with that in the only MCR-overexpressing strain. Finally, we could obtain 60.59 mg/L of 3HP in 42 h using the OB3b-MCRMP strain through bioreactor operation, with a 6.36-fold increase of volumetric productivity compared than that in the flask cultures. This work demonstrates metabolic engineering of type II methanotrophs, opening the door for using type II methanotrophs as cell factories for biochemical production along with mitigation of greenhouse gases.     

Enhanced lycopene productivity by manipulation of carbon flow to isopentenyl diphosphate in Escherichia coli

Lycopene is a useful phytochemical that holds great commercial value. In our study the lycopene production pathway in E. coli originating from the precursor isopentenyl diphosphate (IPP) of the non-mevalonate pathway was reconstructed. This engineered strain of E. coli accumulated lycopene intracellularly under aerobic conditions. As a next step, the production of lycopene was enhanced through metabolic engineering methodologies. Various competing pathways at the pyruvate and acetyl-CoA nodes were inactivated to divert more carbon flux to IPP and subsequently to lycopene. It was found that the ackA-pta, nuo mutant produced a higher amount of lycopene compared to the parent strain. To further enhance lycopene production, a novel mevalonate pathway, in addition to the already existing non-mevalonate pathway, was engineered. This pathway utilizes acetyl-CoA as precursor, condensing it to form acetoacetyl-CoA and subsequently leading to formation of IPP. Upon the introduction of this new pathway, lycopene production increased by over 2-fold compared to the ackA-pta, nuo mutant strain.     

Rational design of a synthetic Entner–Doudoroff pathway for improved and controllable NADPH regeneration

NADPH is an essential cofactor for the biosynthesis of several high-value chemicals, including isoprenoids, fatty acid-based fuels, and biopolymers. Tunable control over all potentially rate-limiting steps, including the NADPH regeneration rate, is crucial to maximizing production titers. We have rationally engineered a synthetic version of the Entner–Doudoroff pathway from Zymomonas mobilis that increased the NADPH regeneration rate in Escherichia coli MG1655 by 25-fold. To do this, we combined systematic design rules, biophysical models, and computational optimization to design synthetic bacterial operons expressing the 5-enzyme pathway, while eliminating undesired genetic elements for maximum expression control. NADPH regeneration rates from genome-integrated pathways were estimated using a NADPH-binding fluorescent reporter and by the productivity of a NADPH-dependent terpenoid biosynthesis pathway. We designed and constructed improved pathway variants by employing the RBS Library Calculator to efficiently search the 5-dimensional enzyme expression space and by performing 40 cycles of MAGE for site-directed genome mutagenesis. 624 pathway variants were screened using a NADPH-dependent blue fluorescent protein, and 22 were further characterized to determine the relationship between enzyme expression levels and NADPH regeneration rates. The best variant exhibited 25-fold higher normalized mBFP levels when compared to wild-type strain. Combining the synthetic Entner–Doudoroff pathway with an optimized terpenoid pathway further increased the terpenoid titer by 97%.     

TGF-β1 diminishes collagen production during long-term cyclic stretching of engineered connective tissue: implication of decreased ERK signaling

Cyclic stretching and growth factors like TGF-β have been used to enhance extracellular matrix (ECM) production by cells in engineered tissue to achieve requisite mechanical properties. In this study, the effects of TGF-β1 were evaluated during long-term cyclic stretching of fibrin-based tubular constructs seeded with neonatal human dermal fibroblasts. Samples were evaluated at 2, 5, and 7 weeks for tensile mechanical properties and ECM deposition. At 2 weeks, +TGF-β1 samples had 101% higher collagen concentration but no difference in ultimate tensile strength (UTS) or modulus compared to -TGF-β1 samples. However, at weeks 5 and 7, -TGF-β1 samples had higher UTS/modulus and collagen concentration, but lower elastin concentration compared to +TGF-β1 samples. The collagen was better organized in -TGF-β1 samples based on picrosirius red staining. Western blot analysis at weeks 5 and 7 showed increased phosphorylation of ERK in -TGF-β1 samples, which correlated with higher collagen deposition. The TGF-β1 effects were further evaluated by western blot for αSMA and SMAD2/3 expression, which were 16-fold and 10-fold higher in +TGF-β1 samples, respectively. The role of TGF-β1 activated p38 in inhibiting phosphorylation of ERK was evaluated by treating samples with SB203580, an inhibitor of p38 activation. SB203580-treated cells showed increased phosphorylation of ERK after 1 hour of stretching and increased collagen production after 1 week of stretching, demonstrating an inhibitory role of activated p38 via TGF-β1 signaling during cyclic stretching. One advantage of TGF-β1 treatment was the 4-fold higher elastin deposition in samples at 7 weeks. Further cyclic stretching experiments were thus conducted with constructs cultured for 5 weeks without TGF-β1 to obtain improved tensile properties followed by TGF-β1 supplementation for 2 weeks to obtain increased elastin content, which correlated with a reduction in loss of pre-stress during preconditioning for tensile testing, indicating functional elastin. This study shows that a sequential stimulus approach - cyclic stretching with delayed TGF-β1 supplementation - can be used to engineer tissue with desirable tensile and elastic properties.     

Synthetic scaffolds increased resveratrol biosynthesis in engineered yeast cells

Resveratrol is a polyphenolic compound produced by a few higher plants when under attack by pathogens such as bacteria or fungi. Besides antioxidant benefits to humans, this health-promoting compound has been reported to extend longevity in yeasts, flies, worms, fishes and obesity mice. Here we utilized the synthetic scaffolds strategy to improve resveratrol production in Saccharomyces cerevisiae. We observed a 5.0-fold improvement over the non-scaffolded control, and a 2.7-fold increase over the previous reported with fusion protein. This work demonstrated the synthetic scaffolds can be used for the optimization of engineered metabolic pathway.