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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.

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

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

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

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.

The introduction of a new metabolic pathway will affect the host’s other metabolic pathways. Differential metabolomics is a useful method to reveal the relationship between the different metabolic pathways inside a microorganism. Three Klebsiella pneumoniae strains with different 1-butanol production capability were chosen to study the differential metabolomics to reveal the relationship between 1-butanol synthesis and other metabolic pathways. The biosynthesis of Val, Leu, Ile, Met, Gly and Ala were all found to have close relationships with the 1-butanol synthesis. To tuning the synthesis of these amino acids, CRISPRi (Clustered regularly interspaced short palindromic repeats interference) system was adopted. The resulting transcription levels, intracellular amino acid content, and 1-butanol production were significantly affected. In comparison with KLA, the concentrations of intracellular Ile, Leu, Val, Met, and Ala in all the repressed strains was reduced, and the resulting content of intracellular Thr and 1-butanol production was increased. The largest increase in 1-butanol production was obtained with the strain KLA-ilvB3 with a yield increase 154% higher than that of KLA. The results show that CRISPRi is a feasible method to manipulate genes in Klebsiella pneumoniae.

Consolidated bioprocessing (CBP) of cellulose mixed with fermentable sugar(s) is considered as a promising alternative to the use of cellulose as sole substrate for bioethanol production. Our research metabolically engineered Saccharomyces cerevisiae to allow for the co-conversion of cellulose and either sucrose or xylose to bioethanol. Constitutive promoter substitution and xylose metabolic pathway integration were carried out in a strain previously modified to express both bifunctional minicellulosomes by galactose induction and a cellodextrin pathway. Strain EBY101-CC, engineered for the co-fermentation of cellulose and sucrose, produced 4.3 g/L ethanol from 10 g/L carboxymethyl cellulose (CMC) and batch-fed sucrose with an ethanol yield of 0.43 g/g of total sugars. Strains modified for co-fermentation of xylose and cellulose, EBY101-X5CC and EBY101-X5CP were able to produce 2.9 g/L cellulosic ethanol from 8.0 g/L CMC and 1.2 g/L from 3.2 g/L phosphoric acid-swollen cellulose (PASC), respectively, when...

Although many efforts have been made to engineer Escherichia coli for fumaric acid production, the fumarate efflux system has not been investigated as an engineering target to improve fumaric acid production. In this work, we cloned and expressed C4-dicarboxylate transporters of different sources in a previously constructed fumaric-acid-producing strain to study their effects on the production of fumaric acid. In addition, each native C4-dicarboxylate transporter was deleted in separate experiments to investigate their individual effects on fumaric acid production. The results showed that the expression of the genes dcuB-Ec and dcuC-Ec can increase the fumaric acid yield by 48.5% and 53.1%, respectively. Fed-batch cultivations in a 5 L bioreactor of strain A-dcuB-Ec produced 9.42 g L−1 of fumaric acid after 50 hours.

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.

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.

1,3-Propanediol (1,3-PDO) is a monomer for the synthesis of various polyesters. It is widely used in industries including cosmetics, solvents, and lubricants. Here, the multi-modular engineering was used to improve the concentration and tolerance of 1,3-PDO in Klebsiella pneumoniae. Firstly, the concentration of 1,3-PDO was increased by 25 %, while the concentrations of by-products were reduced considerably through one-step evolution which focused on the glycerol pathway. In addition, the 1,3-PDO tolerance was improved to 150 g L−1. Secondly, co-substrate transport system was regulated, and the 1,3-PDO concentration, yield, and productivity of the mutant were improved to 76.4 g L−1, 0.53 mol mol−1, and 2.55 g L−1 h−1, respectively. Finally, NADH regeneration was introduced and the recombinant strain was successfully achieved with a high productivity of 2.69 g L−1 h−1. The concentration and yield of 1,3-PDO were also improved to 86 g L−1 and 0.59 mol mol−1. This strategy described here provides an approach of achieving a superior strain which is able to produce 1,3-PDO with high productivity and yield.

Bioethanol is a renewable biofuel that has a strong inhibitory effect on cells in bioethanol fermentation by yeast. In this study, carbon nanotube (CNT)-mixed polydimethylsiloxane (PDMS) membranes were used for ethanol recovery from model solutions as well as for fermentation by self-flocculating yeast. Imbedding CNTs into the PDMS membrane led to enhanced ethanol recovery, with a maximum total flux of 128.7 g/m2 h and an ethanol titer of 615.1 g/L in permeate. The CNTs can provide a flexible route for ethanol transport through the inner tubes or along the smooth surface. In fed-batch fermentation incorporating pervaporation, 112.3 g/L of ethanol was produced with an overall ethanol productivity and yield of 2.23 g/L h and 0.45 g/g, respectively. The membrane produced a highly concentrated condensate containing 400.3–487.5 g/L of ethanol. Furthermore, as yeast flocs can be throttled down in the bioreactor, self-flocculating yeast can be used to prevent membrane fouling induced by cell adsorption on the membrane. Therefore, the CNT-mixed membrane coupled with ethanol fermentation by self-flocculating yeast not only reduces ethanol-mediated inhibition of cells but also saves the production cost because of the reduced fouling risk. Thus, this combination approach has potential in industrial bioethanol production for long-time operation.

The development of new antibacterial materials is a constant demand in the manufacturing of personal hygiene products and medical implements. The inhibition of bacterial adhesion onto surfaces is very challenging, and adhesion may cause secondary infection. In this study, we synthesize multiple types of hydroxyl-rich cationic derivatives via a ring-opening reaction of a star-like poly(glycidyl methacrylate) (s-PGMA) for broad-spectrum antibacterial and antifouling coatings. The derivatives containing tertiary amine groups are subsequently quaternized to produce poly(quaternary ammonium). The quaternary and silver-loaded derivatives exhibit potent and broad-spectrum antibacterial activities towards both Gram-negative and Gram-positive bacteria. Particularly, some of the silver-loaded derivatives show a 64-fold improvement in antibacterial activity toward P. aeruginosa compared to their non-silver counterparts, due to the synergistic actions of quaternary ammonium components and silver ions. Moreover, the antibacterial derivatives can be readily coated on substrates such as glass slides via an adhesive layer of polydopamine. The resultant antibacterial coatings with rich hydroxyl groups also endow antifouling capability against killed bacteria, while the release and diffusion of silver ions from the silver-loaded polymer coatings enable the long-range antibacterial abilities. This study offers a new approach for synthesizing effective antibacterial and antifouling coatings that possess promising applications on biomedical devices.

As the initial step in carbohydrate catabolism in cells, the substrate-specific transporters via active transport and facilitated diffusion play a decisive role in passage of sugars through the plasma membrane into the cytoplasm. The SecY complex (SecYEG) in bacteria forms a membrane channel responsible for protein translocation. This work demonstrates that weakening the sealability of the SecY channel allowed free diffusion of sugars, including glucose, fructose, mannose, xylose, arabinose, and lactose, into the engineered cells, facilitating its rapid growth on a wide spectrum of monosaccharides and bypassing/reducing stereospecificity, transport saturation, competitive inhibition, and carbon catabolite repression (CCR), which are usually encountered with the specific sugar transporters. The SecY channel is structurally conserved in prokaryotes, thus it may be engineered to serve as a unique and universal transporter for bacteria to passage sugars as demonstrated inEscherichia coliandClostridium acetobutylicum.

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.

1-Butanol, as a next-generation biofuel, is an important target product of biorefinery research. With the introduction of the CoA-dependent pathway or Ehrlich pathway, many engineered strains have been developed to produce 1-butanol, although cofactor imbalance occurred in engineered strains by the introduction of the 1-butanol synthesis pathway. Several studies have been performed to regenerate NADH by overexpressing NAD+-dependent enzymes. However, the significant role of cofactor regeneration in 1-butanol production and the transcription level of the target genes have seldom been studied. The 1-butanol producer, recombinant Klebsiella pneumoniae (KLA), was constructed by overexpressing the genes kivd, leuABCD, and adhE1 under the control of tac promoter in this study, and several NADH regeneration strategies were adopted to solve the problem of NADH imbalance, including the introduction of NAD+-dependent enzymes (formate dehydrogenase, pyridine nucleotide transhydrogenase, and glucose dehydrogenase) or...

S-adenosylmethionine (SAM) is an essential regulatory compound in many biological reactions and an active small molecule of high biomedical value. Cofactor engineering as an advantageous and emerging strategy was introduced to improve the SAM production. In this work, we constructed two kinds of NADPH regenerators of heterologous NADH kinase (Pos5p) and NADH kinase-like enzymes (combination of transhydrogenase PntAB and NAD kinase YfjB) to increase the availability of NADPH in SAM biosynthesis. Modulating of Pos5p resulted in a superior SAM production and led to approximately 5.30 mg L−1 of SAM in Escherichia coli without L-methionine addition. In addition, the synthetic sRNA-based NADPH regulation strategy was also implemented for enhancing the NADPH supply coupled with decreasing the by-products in the SAM synthesis pathway. The SAM titer of the recombinant strains harboring synthetic sRNA could exceed 1 mg L−1, which increased approximately 70% than that of the control. It was also proposed that the production of SAM is not only dependent on the absolute value of NADPH but also dependent on the NADPH/NADP+ ratio. These NADPH regulation strategies are potentially applicable to various processes for enhancing of NADPH-dependent products.

As an important feedstock in petrochemistry, isoprene is used in a wide range of industrial applications. It is produced almost entirely from petrochemical sources; however, these sources are being progressively depleted. A reliable biological process for isoprene production utilizing renewable feedstocks would be an industry-redefining development. There are two biosynthetic pathways producing isoprene: the mevalonate (MVA) pathway and the methyl erythritol 1-phosphate (MEP) pathway. In this study, the MEP pathway was modified in Escherichia coli BL21 (DE3) to produce isoprene. The isoprene synthase (IspS) gene chemically synthesized from Populus alba after codon optimization for expression in E. coli was heterologously expressed. The endogenous genes of 1-deoxy- d -xylulose-5-phosphate synthase (DXS) and 1-deoxy- d -xylulose-5-phosphate reductoisomerase (DXR) were over-expressed. The isopentenyl pyrophosphate isomerase (Idi) gene from Streptococcus pneumoniae was exogenously over-expressed, and farnesyl diphosphate synthase (ispA) was weakened to enhance the yield. The control strain harboring empty plasmids did not emit any isoprene. The over-expression of the DXR gene only had little impact on the yield of isoprene. Idi from S. pneumoniae played a significant role in the improvement of isoprene production. The highest yield was achieved by an ispA-weakened DXS-IDI-IspS recombinant with 19.9 mg/l isoprene, which resulted in a 33-fold enhancement of the isoprene yield from the IspS recombinant.

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.

To utilize the by-product of biodiesel production, Klebsiella pneumoniae, a well-known glycerol-fermenting microorganism, was engineered to produce 1-butanol. The modified CoA-dependent and 2-keto acid pathways were established by expressing the genes ter-bdhB-bdhA and kivd, respectively. The 1-butanol titer and specific BuOH yield were 15.03 mg L−1 and 27.79 mg-BuOH per g cell in KpTBB (K. pneumoniae overexpressing the genes ter-bdhB-bdhA), and 28.7 mg L−1 and 51.58 mg-BuOH per g cell in Kp-kivd (K. pneumoniae overexpressing the gene kivd), respectively. Moreover, the native products in K. pneumoniae fermentation were down regulated using the antisense RNA strategy. The resulting yield of 1,3-propanediol and 2,3-butanediol was reduced by 81% and 15%, respectively. This work reports a new strain, K. pneumoniae, for 1-butanol production and the application of an antisense RNA strategy as an effective method for reducing the main by-products.

In this study, we expressed lipase 2 from Candida sp. 99-125 in Saccharomyces cerevisiae , and tried direct biodiesel production. Driven by 3-phosphoglycerate kinase promoter, Lip2 showed high expression level in cytoplasm. SDS-PAGE analysis confirmed the successful lipase expression with a 40 kDa molecular weight. The enzyme assay indicated that lipase 2 had a specific activity of 12.12 μmol/min/mg toward p -nitrophenyl palmitate. Gas chromatography showed that the main fatty acids of S. cerevisiae lipids were palmitoleic acid (31.79%) and oleic acid (29.84%). By three-step addition of 4% ethanol to culture broth, the yield of fatty acid ethyl esters by recombinant S. cerevisiae reached 11.4 mg/g dry cell weight. This work proposed a novel pathway for S. cerevisiae that could be applied for producing biodiesel directly.

The present paper reports in vitro strategies for assembly of minicellulosomes with two miniscaffoldins on the Saccharomyces cerevisiae cell surface. It was carried out through incubation of the yeast cells displaying scaffoldins with Escherichia coli lysates containing recombinant cellulases, or using a four-population yeast consortium. The results showed that the display level of miniscaffoldin II was distinctly increased by moving the cellulases production into E. coli or other yeast cells, indicating that the metabolic burden of the yeast host was decreased. The yeast consortium did not show any cellulolytic activity, while the E. coli lysates-treated yeast, whose anchoring miniscaffoldin length was optimized, was able to produce ∼1138 mg/L ethanol from microcrystalline cellulose within 4 days. We also confirmed that the yeast-associated minicellulosome moreover showed both higher thermal stability and lower protease accessibility than free minicellulosome. This research promotes the application of S. cerevisiae as a consolidated bioprocessing (CBP) microorganism in cellulosic bioethanol production.

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 β-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. Electronic supplementary material The online version of this article (doi:10.1186/s13068-016-0554-6) contains supplementary material, which is available to authorized users.

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.

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).

ATP is the limiting precursor and driving force for S-adenosylmethionine (SAM) biosynthesis in Escherichia coli. In contrast to traditional optimization of fermentation processes, the synthetic sRNA-based repression strategy, which was developed as a highly efficient gene knockdown approach, has been applied for the regulation of the intracellular ATP concentration in order to enhance SAM production. In this work, proB, glnA and argB, all involved in the synthesis of ATP-dependent by-products in the S-adenosylmethionine production were selected as candidates for repression. The results show that the S-adenosylmethionine titer and yield in the recombinant strain were doubled compared with the control. The best-performing strain, Anti-argB, produced the highest SAM titer (1.21 mg L(-1)), and strain Anti-glnA gave the highest yield (0.13 mg g(-1), 12 h). Both the concentration of ATP and the ratio of ATP to ADP were shown to have a positive effect on the S-adenosylmethionine synthesis. Overall, the synthetic sRNA-based downregulation strategy has a high potential for cofactor regulation and will be useful for industrial ATP-driven bioprocesses.

Correction for ‘Regulating C4-dicarboxylate transporters for improving fumaric acid production’ by Ting Zhang et al., RSC Adv., 2017, 7, 2897–2904.

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 (β-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.

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).