Metabolic Engineering Mevalonate Pathway Mediated by RNA Scaffolds for Mevalonate and Isoprene Production in Escherichia coli.

Co-localizing biochemical processes is a great strategy when expressing the heterologous metabolic pathway for product biosynthesis. The RNA scaffold is a flexible and efficient synthetic compartmentalization method to co-localize the enzymes involved in the metabolic pathway by binding to the specific RNA, binding domains fused with the engineered enzymes. Herein, we designed two artificial RNA scaffold structures─0D RNA scaffolds and 2D RNA scaffolds─using the reported aptamers PP7 and BIV-Tat and the corresponding RNA-binding domains (RBDs). We verified the interaction of the RBD and RNA aptamer in vitro and in vivo. Then, we determined the efficiencies of these RNA scaffolds by co-localizing fluorescent proteins. We employed the RNA scaffolds combined with the enzyme fusion strategies to increase the metabolic flux involved in the enzymes of the mevalonate pathway for mevalonate and isoprene production. Compared with the no RNA scaffold strain, the mevalonate levels of the 0D RNA scaffolds and 2D RNA scaffolds increased by 84.1% (3.13 ± 0.03 g/L) and 76.5% (3.00 ± 0.09 g/L), respectively. We applied the 0D RNA scaffolds for increasing the isoprene production by localizing the enzymes involved in a heterologous multi-enzyme pathway. When applying the RNA scaffolds for co-localizing the enzymes mvaE and mvaS, the isoprene production reached to 609.3 ± 57.9 mg/L, increasing by 142% compared with the no RNA scaffold strain. Our results indicate that the RNA scaffold is a powerful tool for improving the efficiencies of the reaction process in the metabolic pathway.

Reprogramming of sugar transport pathways in Escherichia coli using a permeabilized SecY protein-translocation channel.

In the initial step of sugar metabolism, sugar-specific transporters play a decisive role in the passage of sugars through plasma membranes into cytoplasm. The SecY complex (SecYEG) in bacteria forms a membrane channel responsible for protein translocation. The present work shows that permeabilized SecY channels can be used as nonspecific sugar transporters in Escherichia coli. SecY with the plug domain deleted allowed the passage of glucose, fructose, mannose, xylose, and arabinose, and, with additional pore-ring mutations, facilitated lactose transport, indicating that sugar passage via permeabilized SecY was independent of sugar stereospecificity. The engineered E. coli showed rapid growth on a wide spectrum of monosaccharides and benefited from the elimination of transport saturation, improvement in sugar tolerance, reduction in competitive inhibition, and prevention of carbon catabolite repression, which are usually encountered with native sugar uptake systems. The SecY channel is widespread in prokaryotes, so other bacteria may also be engineered to utilize this system for sugar uptake. The SecY channel thus provides a unique sugar passageway for future development of robust cell factories for biotechnological applications.

Engineering and manipulation of a mevalonate pathway in Escherichia coli for isoprene production.

Isoprene is a useful phytochemical with high commercial values in many industrial applications including synthetic rubber, elastomers, isoprenoid medicines, and fossil fuel. Currently, isoprene is on large scale produced from petrochemical sources. An efficient biological process for isoprene production utilizing renewable feedstocks would be an important direction of research due to the fossil raw material depletion and air pollution. In this study, we introduced the mevalonate (MVA) pathway genes/acetoacetyl-coenzyme A thiolase (mvaE) and MVA synthase (mvaS) from Enterococcus faecalis (E. faecalis); MVA kinase (mvk) derived from Methanosarcina mazei (M. mazei); and phosphomevalonate kinase (pmk), diphosphomevalonate decarboxylase (mvaD), and isopentenyl diphosphate isomerase (idi) from Streptococcus pneumoniae (S. pneumoniae) to accelerate dimethylallyl diphosphate (DMAPP) accumulation in Escherichia coli (E. coli). Together with a codon-optimized isoprene synthase (ispS) from Populus alba (P. alba), E. coli strain succeeded in formation of isoprene. We then manipulated the heterologous MVA pathway for high-level production of isoprene, by controlling the gene expression levels of the MVA pathway genes. We engineered four E. coli strains which showed different gene expression levels and different isoprene productivities, and we also characterized them with quantitative real-time PCR and metabolite analysis. To further improve the isoprene titers and release the toxicity to cells, we developed the extraction fermentation by adding dodecane in cultures. Finally, strain BL2T7P1TrcP harboring balanced gene expression system produced 587 ± 47 mg/L isoprene, with a 5.2-fold titer improvement in comparison with strain BL7CT7P. This work indicated that a balanced metabolic flux played a significant role to improve the isoprene production via MVA pathway.

Metabolic engineering of Escherichia coli for the synthesis of polyhydroxyalkanoates using acetate as a main carbon source.

BACKGROUND: High production cost of bioplastics polyhydroxyalkanoates (PHA) is a major obstacle to replace traditional petro-based plastics. To address the challenges, strategies towards upstream metabolic engineering and downstream fermentation optimizations have been continuously pursued. Given that the feedstocks especially carbon sources account up to a large portion of the production cost, it is of great importance to explore low cost substrates to manufacture PHA economically. RESULTS: Escherichia coli was metabolically engineered to synthesize poly-3-hydroxybutyrate (P3HB), poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P3HB4HB), and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) using acetate as a main carbon source. Overexpression of phosphotransacetylase/acetate kinase pathway was shown to be an effective strategy for improving acetate assimilation and biopolymer production. The recombinant strain overexpressing phosphotransacetylase/acetate kinase and P3HB synthesis operon produced 1.27 g/L P3HB when grown on minimal medium supplemented with 10 g/L yeast extract and 5 g/L acetate in shake flask cultures. Further introduction succinate semialdehyde dehydrogenase, 4-hydroxybutyrate dehydrogenase, and CoA transferase lead to the accumulation of P3HB4HB, reaching a titer of 1.71 g/L with a 4-hydroxybutyrate monomer content of 5.79 mol%. When 1 g/L of α-ketoglutarate or citrate was added to the medium, P3HB4HB titer increased to 1.99 and 2.15 g/L, respectively. To achieve PHBV synthesis, acetate and propionate were simultaneously supplied and propionyl-CoA transferase was overexpressed to provide 3-hydroxyvalerate precursor. The resulting strain produced 0.33 g/L PHBV with a 3-hydroxyvalerate monomer content of 6.58 mol%. Further overexpression of propionate permease improved PHBV titer and 3-hydroxyvalerate monomer content to 1.09 g/L and 10.37 mol%, respectively. CONCLUSIONS: The application of acetate as carbon source for microbial fermentation could reduce the consumption of food and agro-based renewable bioresources for biorefineries. Our proposed metabolic engineering strategies illustrate the feasibility for producing polyhydroxyalkanoates using acetate as a main carbon source. Overall, as an abundant and renewable resource, acetate would be developed into a cost-effective feedstock to achieve low cost production of chemicals, materials, and biofuels.

Biosynthesis of D-glucaric acid from sucrose with routed carbon distribution in metabolically engineered Escherichia coli.

D-glucaric acid is a promising platform compound used to synthesize many other value-added or commodity chemicals. The engineering of Escherichia coli for efficiently converting D-glucose to D-glucaric acid has been attempted for several years, with mixed sugar fermentation recently gaining growing interests due to the increased D-glucaric acid yield. Here, we co-expressed cscB, cscA, cscK, ino1, miox, udh, and suhB in E. coli BL21 (DE3), functionally constructing an unreported route from sucrose to D-glucaric acid. Further deletion of chromosomal zwf, pgi, ptsG, uxaC, gudD, over-expression of glk, and use of a D-fructose-dependent translation control system for pgi enabled the strain to use sucrose as the sole carbon source while achieving a high product titer and yield. The titer of D-glucaric acid in M9 medium containing 10 g/L sucrose reached ~1.42 g/L, with a yield of ~0.142 g/g on sucrose.

A novel DMAPP-responding genetic circuit sensor for high-throughput screening and evolving isoprene synthase.

High-throughput screening is a popular tool for collating biological data which would otherwise require the use of excessive resources. In this study, an artificial genetic circuit sensor responding to dimethylallyl diphosphate (DMAPP) was constructed based on a modified L-arabinose operon for high-throughput screening and isoprene synthase (ispS) evolution in Escherichia coli (E. coli). As a first step, the DNA sequence of the L-arabinose ligand-binding domain (LBD) was replaced with an ispS gene to enable the AraC operon responding to DMAPP, which is the substrate of the IspS enzyme. Then, an enhanced GFP (eGFP) was also introduced as a reporter for pBAD promoter. The expression level of the reporter was monitored using either of the two tools: flow cytometer (FCM) and microplate reader. Sequentially, we observed that a high DMAPP concentration led to low eGFP fluorescence, and the overexpression of ispS gene, which consumes DMAPP, resulted in a high eGFP expression. These results demonstrated that the artificial genetic circuit sensor responded directly to the intracellular concentration of DMAPP, and the expression of IspS enzyme could be positively correlated to the expression level of eGFP. Finally, we identified two IspS mutants with different activities from an ispS gene library and further validated the screening method.

Engineered yeast with a CO2-fixation pathway to improve the bio-ethanol production from xylose-mixed sugars.

Bio-ethanol production from lignocellulosic raw materials could serve as a sustainable potential for improving the supply of liquid fuels in face of the food-to-fuel competition and the growing energy demand. Xylose is the second abundant sugar of lignocelluloses hydrolysates, but its commercial-scale conversion to ethanol by fermentation is challenged by incomplete and inefficient utilization of xylose. Here, we use a coupled strategy of simultaneous maltose utilization and in-situ carbon dioxide (CO2) fixation to achieve efficient xylose fermentation by the engineered Saccharomyces cerevisiae. Our results showed that the introduction of CO2 as electron acceptor for nicotinamide adenine dinucleotide (NADH) oxidation increased the total ethanol productivity and yield at the expense of simultaneous maltose and xylose utilization. Our achievements present an innovative strategy using CO2 to drive and redistribute the central pathways of xylose to desirable products and demonstrate a possible breakthrough in product yield of sugars.

Co-localization of proteins with defined sequential order and controlled stoichiometric ratio on magnetic nanoparticles.

Co-immobilization of enzymes used in cascade reactions is important for improving the overall catalytic efficiency. In this work, we employed scaffoldins as a bridge and succeeded in a highly-ordered co-localization of multiple proteins on magnetic nanoparticles with a loading capacity of ∼0.831 µmol g-1 supports.

Site-Specific and High-Loading Immobilization of Proteins by Using Cohesin-Dockerin and CBM-Cellulose Interactions.

Immobilization of enzymes enhances their properties for application in industrial processes as reusable and robust biocatalysts. Here, we developed a new immobilization method by mimicking the natural cellulosome system. A group of cohesin and carbohydrate-binding module (CBM)-containing scaffoldins were genetically engineered, and their length was controlled by cohesin number. To use green fluorescent protein (GFP) as an immobilization model, its C-terminus was fused with a dockerin domain. GFP was able to specifically bind to scaffoldin via cohesin-dockerin interaction, while the scaffoldin could attach to cellulose by CBM-cellulose interaction. Our results showed that this mild and convenient approach was able to achieve site-specific immobilization, and the maximum GFP loading capacity reached ∼0.508 µmol/g cellulose.

Engineering yeast with bifunctional minicellulosome and cellodextrin pathway for co-utilization of cellulose-mixed sugars.

BACKGROUND: Consolidated bioprocessing (CBP), integrating cellulase production, cellulose saccharification, and fermentation into one step has been widely considered as the ultimate low-cost configuration for producing second-generation fuel ethanol. However, the requirement of a microbial strain able to hydrolyze cellulosic biomass and convert the resulting sugars into high-titer ethanol limits CBP application. RESULTS: In this work, cellulolytic yeasts were developed by engineering Saccharomyces cerevisiae with a heterologous cellodextrin utilization pathway and bifunctional minicellulosomes. The cell-displayed minicellulosome was two-scaffoldin derived, and contained an endoglucanase and an exoglucanase, while the intracellular cellodextrin pathway consisted of a cellodextrin transporter and a ß-glucosidase, which mimicked the unique cellulose-utilization system in Clostridium thermocellum and allowed S. cerevisiae to degrade and use cellulose without glucose inhibition/repression on cellulases and mixed-sugar uptake. Consequently, only a small inoculation of the non-induced yeast cells was required to efficiently co-convert both cellulose and galactose to ethanol in a single-step co-fermentation process, achieving a high specific productivity of ~62.61 mg cellulosic ethanol/g cell·h from carboxymethyl cellulose and ~56.37 mg cellulosic ethanol/g cell·h from phosphoric acid-swollen cellulose. CONCLUSIONS: Our work provides a versatile engineering strategy for co-conversion of cellulose-mixed sugars to ethanol by S. cerevisiae, and the achievements in this work may further promote cellulosic biofuel production.

Effect of NADH kinase on poly-3-hydroxybutyrate production by recombinant Escherichia coli.

The cofactor NADPH participates in a variety of anabolic reactions and its availability is considered to play a critical role in biotransformation processes. NADH kinase (Pos5) from Saccharomyces cerevisiae catalyzes the phosphorylation of NADH to generate NADPH. To investigate the effect of NADH kinase on poly-3-hydroxybutyrate (PHB) production, pos5 was co-expressed with PHB synthetic operon phbCAB in Escherichia coli. The recombinant strain carrying pos5 and phbCAB co-expression plasmid reached 5.96 g/L cell dry weight with 64.1% PHB accumulation in 72 h shake flask cultivation, while the control strain without pos5 yielded 3.93 g/L cell dry weight with 58.5% PHB content. PHB production titer was enhanced from 2.30 g/L to 3.82 g/L. Intracellular cofactor concentration analysis revealed that the ratio of NADP/NAD in pos5 overexpression strain was two times more compared with that of the control without pos5. The results showed that NADH kinase could be employed as an effective metabolic manipulation target to improve PHB synthesis.

Enhanced production of poly-3-hydroxybutyrate by Escherichia coli over-expressing multiple copies of NAD kinase integrated in the host genome.

OBJECTIVES: With the help of attB-attP recombination technique, multiple copies of yfjB gene encoding the NAD kinase of Escherichia coli were inserted into the host chromosome to promote NADPH-dependent poly-3-hydroxybutyrate (PHB) production. RESULTS: The yfjB integration mutant E. coli T2 harbored a similar metabolic profile to that of the wild type control. When PHB biosynthesis operon was introduced, the yfjB integration mutant produced 3 g PHB l(-1) from 18.2 g glucose l(-1), while the wild type consumed 15.7 g glucose l(-1) to afford 2.34 g PHB l(-1) in 48 h of shake-flask cultivation. Transcriptional analysis showed that the transcription levels of genes within the PHB biosynthesis operon were increased by six to eightfold with yfj Bover-expression, which may be the primary reason for the improved PHB production. CONCLUSION: A practical method is demonstrated to construct genetically-stable strains harboring extra copies of NAD kinase to enhance NADPH-dependent reactions.

Approach for determination of ATP:ADP molar ratio in mixed solution by surface-enhanced Raman scattering.

The ATP:ADP molar ratio is an important physiological factor. However, in previous literatures, ATP and ADP could not be distinguished by Raman spectroscopy due to the high similarity of molecular structure. To challenge this problem, also considering that the γ phosphate group may interact with adenine group and cause a different variation of the Raman spectrum than that of ADP, a highly sensitive, low-cost, environment protecting, flexible and super-hydrophobic Au nanoparticles/cicada wing (Au/CW) substrate with three-dimension structure was fabricated and employed as an active surface-enhanced Raman scattering (SERS) substrate to detect the ATP:ADP molar ratios. The concentration as low as 10(-8)M for ATP and ADP was analyzed to determine the limit of detection. This SERS study on various ATP:ADP molar ratios demonstrates that ATP:ADP could be distinguished and the quantitative determination of ATP content was achieved. Moreover, a principle was speculated based on the molecular structures of ATP and ADP of the Raman peaks centered at ~685 and ~731cm(-1) to explain the linear relationship between the area ratio and the molar ratio. A new method has been developed for quantitative determination of ATP:ADP molar ratio based on Au/CW substrate by the SERS method.

Biobutanol from sweet sorghum bagasse hydrolysate by a hybrid pervaporation process.

In this study, the pervaporation membrane was used not only for the detoxification of sweet sorghum bagasse (SSB) hydrolysate, but also for butanol separation from its fermentation broth. As a result of detoxification, about 94.5% furfural was reduced by the pervaporation method, and 138.25 g/L furfural was obtained in the permeate side. 87.5% phenolic compounds were degradated by further laccase detoxification. As for fermentation part, 12.3±0.1 g/L butanol, 6.1±0.05 g/L acetone and 2.5±0.07 g/L ethanol were obtained. And after 2h of pervaporation separation, 201.9 g/L butanol, 76.2g/L acetone and traces of ethanol were obtained in the permeate. The hybrid pervaporation process shows promising for the industrial production of biofuel butanol and biochemical furfural.

Screening of high α-arbutin producing strains and production of α-arbutin by fermentation.

A mutant Xanthomonas maltophilia BT-112 with high α-anomer-selective glycosylation activity was screened by a series of mutation methods including UV light, N-methyl-N-nitro-N-nitroso-guanidine treatment and quick neutron mutation. The α-arbutin titer increased 15-folds compared with the parent strain. The optimal conditions for culture medium and the operational conditions for lab-scale fermenter were investigated. Under optimized conditions, the maximal hydroquinone (HQ) tolerance of cells and yield of α-arbutin were 120 mM and 30.6 g/l, respectively. The molar conversion yield of α-arbutin based on the amount of HQ supplied reached 93.6 %. The product was identified as α-arbutin by (13)C NMR and (1)H NMR analysis. In conclusion, the results in this work provide a one-step and cost-effective method for the large-scale production of α-arbutin.

Self-surface assembly of cellulosomes with two miniscaffoldins on Saccharomyces cerevisiae for cellulosic ethanol production.

Yeast to directly convert cellulose and, especially, the microcrystalline cellulose into bioethanol, was engineered through display of minicellulosomes on the cell surface of Saccharomyces cerevisiae. The construction and cell surface attachment of cellulosomes were accomplished with two individual miniscaffoldins to increase the display level. All of the cellulases including a celCCA (endoglucanase), a celCCE (cellobiohydrolase), and a Ccel_2454 (ß-glucosidase) were cloned from Clostridium cellulolyticum, ensuring the thermal compatibility between cellulose hydrolysis and yeast fermentation. Cellulases and one of miniscaffoldins were secreted by α-factor; thus, the assembly and attachment to anchoring miniscaffoldin were accomplished extracellularly. Immunofluorescence microscopy, flow cytometric analysis (FACS), and cellulosic ethanol fermentation confirmed the successful display of such complex on the yeast surface. Enzyme-enzyme synergy, enzyme-proximity synergy, and cellulose-enzyme-cell synergy were analyzed, and the length of anchoring miniscaffoldin was optimized. The engineered S. cerevisiae was applied in fermentation of carboxymethyl cellulose (CMC), phosphoric acid-swollen cellulose (PASC), or Avicel. It showed a significant hydrolytic activity toward microcrystalline cellulose, with an ethanol titer of 1,412 mg/L. This indicates that simultaneous saccharification and fermentation of crystalline cellulose to ethanol can be accomplished by the yeast, engineered with minicellulosome.

Classifying DNA assembly protocols for devising cellular architectures.

DNA assembly is one of the most fundamental techniques in synthetic biology. Efficient methods can turn traditional DNA cloning into time-saving and higher efficiency practice, which is a foundation to accomplish the dreams of synthetic biologists for devising cellular architectures, reprogramming cellular behaviors, or creating synthetic cells. In this review, typical strategies of DNA assembly are discussed with special emphasis on the assembly of long and multiple DNA fragments into intact plasmids or assembled compositions. Constructively, all reported strategies were categorized into in vivo and in vitro types, and protocols are presented in a functional and practice-oriented way in order to portray the general nature of DNA assembly applications. Significantly, a five-step blueprint is proposed for devising cell architectures that produce valuable chemicals.

Esterification synthesis of ethyl oleate in solvent-free system catalyzed by lipase membrane from fermentation broth.

In this study, the immobilized lipase was prepared by fabric membrane adsorption in fermentation broth. The lipase immobilization method in fermentation broth was optimized on broth activity units and pH adjustments. The viscose fermentation broth can be used with a certain percentage of dilution based on the original broth activity units. The fermentation broth can be processed directly without pH adjustment. In addition, the oleic acid ethyl ester production in solvent-free system catalyzed by the immobilized lipase was optimized. The molar ratio of ethanol to oil acid, the enzyme amount, the molecular amount, and the temperature were 1:1, 12% (w/w), 9% (w/w)(based the total amount of reaction mixture), and 30 °C, respectively. Finally, the optimal condition afforded at least 19 reuse numbers with esterification rate above 80% under stepwise addition of ethanol. Due to simple lipase immobilization preparation, acceptable esterification result during long-time batch reactions and lower cost; the whole process was suitable for industrial ethyl oleate production.

Preparation and properties of a novel biodegradable polyester elastomer with functional groups.

A novel biodegradable poly(sebacate-glycerol-citrate) (PGSC) elastomer with functional groups was prepared in this study. First, moldable mixtures were obtained by mixing citric acid with the poly(glycerol-sebacate) (PGS) pre-polymers synthesized in our lab. The PGSC elastomers were obtained from moldable mixtures that were thermally cured in the moulds. Then, the structures, compositions and properties of the elastomers were studied by Fourier transformation infrared spectroscopy (FT-IR), swelling test, differential scanning calorimeter (DSC), tensile test, water contact angle measurement, water absorption experiments and a in vitro degradation test. It showed that the hydroxyl groups remained in the elastomers which would endow the polymer chains with functionality such as good surface modification. By controlling the thermal curing time, the compositions of the PGSC elastomers were adjusted for different mechanical and biodegradable properties. Therefore, PGSC elastomers might be used as anti-conglutination films in surgery, guided tissue regeneration membranes and drug-delivery matrices.

One-step rapid determination and purification of puerarin from Radix puerariae by n-octylamine-modified poly(methacrylate-co-ethylene dimethacrylate) monolith.

n-Octylamine-modified poly(methacrylate-co-ethylene dimethacrylate) monoliths were prepared for rapid screening, determination and one-step purification of puerarin from Radix puerariae (a crude extract of the root of Pueraria lobata). The modified monolith showed a specific surface area of 17.8 m(2) g(-1), an average pore size of 0.76 microm and a total porosity of 60.8%. Fast separation of R. puerariae crude extract was achieved within 5 min at a flow velocity of 722 cm h(-1) resulting in a puerarin purity of 97%, with a recovery of 85%. This demonstrates the potential of n-octylamine-modified poly(methacrylate-co-ethylene dimethacrylate) monolith for the rapid analysis and separation of isoflavonoids. Preparative scale sample loading (12 mg in 2 mL) resulted in a purity of 95%, and a recovery of about 69%. HPLC, FTIR, MS and (1)H NMR spectroscopy were used for the characterization and quantification of puerarin in isolated fraction.