Enhanced Biosynthesis of d-Allulose from a d-Xylose-Methanol Mixture and Its Self-Inductive Detoxification by Using Antisense RNAs in Escherichia coli.

d-Allulose, a C-3 epimer of d-fructose, has great market potential in food, healthcare, and medicine due to its excellent biochemical and physiological properties. Microbial fermentation for d-allulose production is being developed, which contributes to cost savings and environmental protection. A novel metabolic pathway for the biosynthesis of d-allulose from a d-xylose-methanol mixture has shown potential for industrial application. In this study, an artificial antisense RNA (asRNA) was introduced into engineered Escherichia coli to diminish the flow of pentose phosphate (PP) pathway, while the UDP-glucose-4-epimerase (GalE) was knocked out to prevent the synthesis of byproducts. As a result, the d-allulose yield on d-xylose was increased by 35.1%. Then, we designed a d-xylose-sensitive translation control system to regulate the expression of the formaldehyde detoxification operon (FrmRAB), achieving self-inductive detoxification by cells. Finally, fed-batch fermentation was carried out to improve the productivity of the cell factory. The d-allulose titer reached 98.6 mM, with a yield of 0.615 mM/mM on d-xylose and a productivity of 0.969 mM/h.

Transporter mining and metabolic engineering of Escherichia coli for high-level D-allulose production from D-fructose by thermo-swing fermentation.

D-Allulose is an ultra-low-calorie sweetener with broad market prospects in the fields of food, beverage, health care, and medicine. The fermentative synthesis of D-allulose is still under development and considered as an ideal route to replace enzymatic approaches for large-scale production of D-allulose in the future. Generally, D-allulose is synthesized from D-fructose through Izumoring epimerization. This biological reaction is reversible, and a high temperature is beneficial to the conversion of D-fructose. Mild cell growth conditions seriously limit the efficiency of producing D-allulose through fermentation. FryABC permease was identified to be responsible for the transport of D-allulose in Escherichia coli by comparative transcriptomic analysis. A cell factory was then developed by expression of ptsG-F, dpe, and deletion of fryA, fruA, manXYZ, mak, and galE. The results show that the newly engineered E. coli was able to produce 32.33 ± 1.33 g L-1 of D-allulose through a unique thermo-swing fermentation process, with a yield of 0.94 ± 0.01 g g-1 on D-fructose.

Conversion of acetate and glyoxylate to fumarate by a cell-free synthetic enzymatic biosystem.

Fumarate is a value-added chemical that is widely used in food, medicine, material, and agriculture industries. With the rising attention to the demand for fumarate and sustainable development, many novel alternative ways that can replace the traditional petrochemical routes emerged. The in vitro cell-free multi-enzyme catalysis is an effective method to produce high value chemicals. In this study, a multi-enzyme catalytic pathway comprising three enzymes for fumarate production from low-cost substrates acetate and glyoxylate was designed. The acetyl-CoA synthase, malate synthase, and fumarase from Escherichia coli were selected and the coenzyme A achieved recyclable. The enzymatic properties and optimization of reaction system were investigated, reaching a fumarate yield of 0.34 mM with a conversion rate of 34% after 20 h of reaction. We proposed and realized the conversion of acetate and glyoxylate to fumarate in vitro using a cell-free multi-enzyme catalytic system, thus providing an alternative approach for the production of fumarate.

Engineering Vibrio alginolyticus as a novel chassis for PHB production from starch.

Vibrio alginolyticus LHF01 was engineered to efficiently produce poly-3-hydroxybutyrate (PHB) from starch in this study. Firstly, the ability of Vibrio alginolyticus LHF01 to directly accumulate PHB using soluble starch as the carbon source was explored, and the highest PHB titer of 2.06 g/L was obtained in 18 h shake flask cultivation. Then, with the analysis of genomic information of V. alginolyticus LHF01, the PHB synthesis operon and amylase genes were identified. Subsequently, the effects of overexpressing PHB synthesis operon and amylase on PHB production were studied. Especially, with the co-expression of PHB synthesis operon and amylase, the starch consumption rate was improved and the PHB titer was more than doubled. The addition of 20 g/L insoluble corn starch could be exhausted in 6-7 h cultivation, and the PHB titer was 4.32 g/L. To the best of our knowledge, V. alginolyticus was firstly engineered to produce PHB with the direct utilization of starch, and this stain can be considered as a novel host to produce PHB using starch as the raw material.

Engineering of Escherichia coli for D-allose fermentative synthesis from D-glucose through izumoring cascade epimerization.

D-Allose is a potential alternative to sucrose in the food industries and a useful additive for the healthcare products in the future. At present, the methods for large-scale production of D-allose are still under investigation, most of which are based on in vitro enzyme-catalyzed Izumoring epimerization. In contrast, fermentative synthesis of D-allose has never been reported, probably due to the absence of available natural microorganisms. In this work, we co-expressed D-galactose: H+ symporter (GalP), D-glucose isomerase (DGI), D-allulose 3-epimerase (DAE), and ribose-5-phosphate isomerase (RPI) in Escherichia coli, thereby constructing an in vivo Izumoring pathway for yielding D-allose from D-glucose. The carbon fluxes and carbon catabolite repression (CCR) were rationally regulated by knockout of FruA, PtsG, Glk, Mak, PfkA, and PfkB involved in the pathways capable of phosphorylating D-fructose, D-glucose, and fructose-6-phosphate. Moreover, the native D-allose transporter was damaged by inactivation of AlsB, thus driving the reversible Izumoring reactions towards the target product. Fermentation was performed in the M9 medium supplemented with glycerol as a carbon source and D-glucose as a substrate. The results show that the engineered E. coli cell factory was able to produce approximately 127.35 mg/L of D-allose after 84 h. Our achievements in the fermentative production of D-allose in this work may further promote the green manufacturing of rare sugars.

Methanol-Dependent Carbon Fixation for Irreversible Synthesis of d-Allulose from d-Xylose by Engineered Escherichia coli.

d-Allulose is a rare hexose with great application potential, owing to its moderate sweetness, low energy, and unique physiological functions. The current strategies for d-allulose production, whether industrialized or under development, utilize six-carbon sugars such as d-glucose or d-fructose as a substrate and are usually based on the principle of reversible Izumoring epimerization. In this work, we designed a novel route that coupled the pathways of methanol reduction, pentose phosphate (PP), ribulose monophosphate (RuMP), and allulose monophosphate (AuMP) for Escherichia coli to irreversibly synthesize d-allulose from d-xylose and methanol. After improving the expression of AlsE by SUMO fusion and regulating the carbon fluxes by knockout of FrmRAB, RpiA, PfkA, and PfkB, the titer of d-allulose in fed-batch fermentation reached ≈70.7 mM, with a yield of ≈0.471 mM/mM on d-xylose or ≈0.512 mM/mM on methanol.

Metabolically Engineered Escherichia coli for Conversion of D-Fructose to D-Allulose via Phosphorylation-Dephosphorylation.

D-Allulose is an ultra-low calorie sweetener with broad market prospects. As an alternative to Izumoring, phosphorylation-dephosphorylation is a promising method for D-allulose synthesis due to its high conversion of substrate, which has been preliminarily attempted in enzymatic systems. However, in vitro phosphorylation-dephosphorylation requires polyphosphate as a phosphate donor and cannot completely deplete the substrate, which may limit its application in industry. Here, we designed and constructed a metabolic pathway in Escherichia coli for producing D-allulose from D-fructose via in vivo phosphorylation-dephosphorylation. PtsG-F and Mak were used to replace the fructose phosphotransferase systems (PTS) for uptake and phosphorylation of D-fructose to fructose-6-phosphate, which was then converted to D-allulose by AlsE and A6PP. The D-allulose titer reached 0.35 g/L and the yield was 0.16 g/g. Further block of the carbon flux into the Embden-Meyerhof-Parnas (EMP) pathway and introduction of an ATP regeneration system obviously improved fermentation performance, increasing the titer and yield of D-allulose to 1.23 g/L and 0.68 g/g, respectively. The E. coli cell factory cultured in M9 medium with glycerol as a carbon source achieved a D-allulose titer of ≈1.59 g/L and a yield of ≈0.72 g/g on D-fructose.

Intelligent self-control of carbon metabolic flux in SecY-engineered Escherichia coli for xylitol biosynthesis from xylose-glucose mixtures.

Xylitol is a salutary sugar substitute that has been widely used in the food, pharmaceutical, and chemical industries. Co-fermentation of xylose and glucose by metabolically engineered cell factories is a promising alternative to chemical hydrogenation of xylose for commercial production of xylitol. Here, we engineered a mutant of SecY protein-translocation channel (SecY [ΔP]) in xylitol-producing Escherichia coli JM109 (DE3) as a passageway for xylose uptake. It was found that SecY (ΔP) channel could rapidly transport xylose without being interfered by XylB-catalyzed synthesis of xylitol-phosphate, which is impossible for native XylFGH and XylE transporters. More importantly, with the coaction of SecY (ΔP) channel and carbon catabolite repression (CCR), the flux of xylose to the pentose phosphate (PP) pathway and the xylitol synthesis pathway in E. coli could be automatically controlled in response to glucose, thereby ensuring that the mutant cells were able to fully utilize sugars with high xylitol yields. The E. coli cell factory developed in this study has been proven to be applicable to a broad range of xylose-glucose mixtures, which is conducive to simplifying the mixed-sugar fermentation process for efficient and economical production of xylitol.

Engineering Escherichia coli for d-Allulose Production from d-Fructose by Fermentation.

d-Allulose is considered an ideal alternative to sucrose and has shown tremendous application potential in many fields. Recently, most efforts on production of d-allulose have focused on in vitro enzyme-catalyzed epimerization of cheap hexoses. Here, we proposed an approach to efficiently produce d-allulose through fermentation using metabolically engineered Escherichia coli JM109 (DE3), in which a SecY (ΔP) channel and a d-allulose 3-epimerase (DPEase) were co-expressed, ensuring that d-fructose could be transported in its nonphosphorylated form and then converted into d-allulose by cells. Further deletion of fruA, manXYZ, mak, galE, and fruK and the use of Ni2+ in a medium limited the carbon flux flowing into the byproduct-generating pathways and the Embden-Meyerhof-Parnas (EMP) pathway, achieving a ≈ 0.95 g/g yield of d-allulose on d-fructose using E. coli (DPEase, SecY [ΔP], ΔFruA, ΔManXYZ, ΔMak, ΔGalE, ΔFruK) and 8 µM Ni2+. In fed-batch fermentation, the titer of d-allulose reached ≈23.3 g/L.

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.

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.

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.

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.

Cell surface display of carbonic anhydrase on Escherichia coli using ice nucleation protein for CO2 sequestration.

Carbonic anhydrase (CA) has recently gained renewed interests for its potential as a mass-transfer facilitator for CO(2) sequestration. However, the low stability and high price severely limit its applications. In this work, the expression of α-CA from Helicobacter pylori on the outer membrane of Escherichia coli using a surface-anchoring system derived from ice nucleation protein (INP) from Pseudomonas syringae was developed. To find the best surface anchoring motif, full-length INP (114 kDa), truncated INP (INP-NC, 33 kDa), and INP's N-domain with first two subunits (INP-N, 22 kDa) were evaluated. Two vectors, pKK223-3 and pET22b(+), with different promoters (T7 and Tac) were used to construct the fusion genes, and for each vector, three recombinant strains, each expressing a different length of the fusion protein, were obtained. SDS-PAGE, Western blot, immunofluorescence microscopy, FACS, and whole-cell ELISA confirmed the expression of fusion proteins on the surface of E. coli. The smallest fusion protein with INP-N as the anchoring motif had the highest expression level and CA activity, suggesting that INP-N is the best carrying protein due to its smaller size. Also, the T7 promoter in pET22b(+) induced with 0.2 mM IPTG gave high protein expression levels, whereas the Tac promoter in pKK223-3 gave low expression levels. The surface displayed CA was at least twofold more stable than that of the free form, and did not show any adverse effect on cell growth and outer membrane integrity. Cells with surface displayed CA were successfully used to facilitate CO(2) sequestration in contained liquid membrane (CLM).