Engineering a Feruloyl-Coenzyme A Synthase for Bioconversion of Phenylpropanoid Acids into High-Value Aromatic Aldehydes.

Aromatic aldehydes find extensive applications in food, perfume, pharmaceutical, and chemical industries. However, a limited natural enzyme selectivity has become the bottleneck of bioconversion of aromatic aldehydes from natural phenylpropanoid acids. Here, based on the original structure of feruloyl-coenzyme A (CoA) synthetase (FCS) from Streptomyces sp. V-1, we engineered five substrate-binding domains to match specific phenylpropanoid acids. FcsCIAE407A/K483L, FcsMAE407R/I481R/K483R, FcsHAE407K/I481K/K483I, FcsCAE407R/I481R/K483T, and FcsFAE407R/I481K/K483R showed 9.96-, 10.58-, 4.25-, 6.49-, and 8.71-fold enhanced catalytic efficiency for degrading CoA thioesters of cinnamic acid, 4-methoxycinnamic acid, 4-hydroxycinnamic acid, caffeic acid, and ferulic acid, respectively. Molecular dynamics simulation illustrated that novel substrate-binding domains formed strong interaction forces with substrates' methoxy/hydroxyl group and provided hydrophobic/alkaline catalytic surfaces. Five recombinant E. coli with FCS mutants were constructed with the maximum benzaldehyde, p-anisaldehyde, p-hydroxybenzaldehyde, protocatechualdehyde, and vanillin productivity of 6.2 ± 0.3, 5.1 ± 0.23, 4.1 ± 0.25, 7.1 ± 0.3, and 8.7 ± 0.2 mM/h, respectively. Hence, our study provided novel and efficient enzymes for the bioconversion of phenylpropanoid acids into aromatic aldehydes.

Construction of an enzymatic shuttling compartment based on reverse micellar for bamboo biomass hydrolysis in ionic liquids.

The enzymatic saccharification of regenerated lignocellulose must occur separately due to the toxicity of ionic liquids to cellulase. Therefore, it is important to develop a biocompatible IL-cellulase system which effectively achieves activation and saccharification of lignocellulose. For this purpose, a dual-phase "enzyme-shuttling compartment" was constructed in this study. Tween 80 was found to form reverse micelles in the isooctane-IL two-liquid phase, acting as a microenvironment that maintains the energetic conformation of the reactive cellulase. The activated bamboo biomass was enzymatically hydrolyzed in 20% (w/v) 1-ethyl-3-methylimidazolium dimethyl phosphate and 50 mM citrate buffer at 50 °C, achieving a high total reducing sugar yield of 71.2% and maintaining an enzymatic activity of 91.2% after 24 h. Thus, an efficient system with the simultaneous activation and saccharification of natural biomass was successfully developed in a one-pot procedure at low temperatures, ensuring large-scale biomass conversion into biofuels and biological products.

Metabolic Engineering of Non-carotenoid-Producing Yeast Yarrowia lipolytica for the Biosynthesis of Zeaxanthin.

Zeaxanthin is vital to human health; thus, its production has received much attention, and it is also an essential precursor for the biosynthesis of other critical carotenoids such as astaxanthin and crocetin. Yarrowia lipolytica is one of the most intensively studied non-conventional yeasts and has been genetically engineered as a cell factory to produce carotenoids such as lycopene and ß-carotene. However, zeaxanthin production by Y. lipolytica has not been well investigated. To fill this gap, ß-carotene biosynthesis pathway has been first constructed in this study by the expression of genes, including crtE, crtB, crtI, and carRP. Three crtZ genes encoding ß-carotene hydroxylase from different organisms were individually introduced into ß-carotene-producing Y. lipolytica to evaluate their performance for producing zeaxanthin. The expression of crtZ from the bacterium Pantoea ananatis (formerly Erwinia uredovora, Eu-crtZ) resulted in the highest zeaxanthin titer and content on the basis of dry cell weight (DCW). After verifying the function of Eu-crtZ for producing zeaxanthin, the high-copy-number integration into the ribosomal DNA of Y. lipolytica led to a 4.02-fold increase in the titer of zeaxanthin and a 721% increase in the content of zeaxanthin. The highest zeaxanthin titer achieved 21.98 ± 1.80 mg/L by the strain grown on a yeast extract peptone dextrose (YPD)-rich medium. In contrast, the highest content of DCW reached 3.20 ± 0.11 mg/g using a synthetic yeast nitrogen base (YNB) medium to culture the cells. Over 18.0 g/L of citric acid was detected in the supernatant of the YPD medium at the end of cultivation. Furthermore, the zeaxanthin-producing strains still accumulated a large amount of lycopene and ß-carotene. The results demonstrated the potential of a cell factory for zeaxanthin biosynthesis and opened up an avenue to engineer this host for the overproduction of carotenoids.

Challenges and Potential in Increasing Lutein Content in Microalgae.

Research on enhancing lutein content in microalgae has made significant progress in recent years. However, strategies are needed to address the possible limitations of microalgae as practical lutein producers. The capacity of lutein sequestration may determine the upper limit of cellular lutein content. The preliminary estimation presented in this work suggests that the lutein sequestration capacity of the light-harvesting complex (LHC) of microalgae is most likely below 2% on the basis of dry cell weight (DCW). Due to its nature as a structural pigment, higher lutein content might interfere with the LHC in fulfilling photosynthetic functions. Storing lutein in a lipophilic environment is a mechanism for achieving high lutein content but several critical barriers must be overcome such as lutein degradation and access to lipid droplet to be stored through esterification. Understanding the mechanisms underlying lipid droplet biogenesis in chloroplasts, as well as carotenoid trafficking through chloroplast membranes and carotenoid esterification, may provide insight for new approaches to achieve high lutein contents in algae. In the meantime, building the machinery for esterification and sequestration of lutein and other hydroxyl-carotenoids in model microorganisms, such as yeast, with synthetic biology technology provides a promising option.

Using Unnatural Protein Fusions to Engineer a Coenzyme Self-Sufficiency System for D-Phenyllactic Acid Biosynthesis in Escherichia coli.

The biosynthetic production of D-penyllactic acid (D-PLA) is often affected by insufficient supply and regeneration of cofactors, leading to high production cost, and difficulty in industrialization. In this study, a D-lactate dehydrogenase (D-LDH) and glycerol dehydrogenase (GlyDH) co-expression system was constructed to achieve coenzyme NADH self-sufficiency and sustainable production of D-PLA. Using glycerol and sodium phenylpyruvate (PPA) as co-substrate, the E. coli BL21 (DE3) harboring a plasmid to co-express LfD-LDH and BmGlyDH produced 3.95 g/L D-PLA with a yield of 0.78 g/g PPA, similar to previous studies. Then, flexible linkers were used to construct fusion proteins composing of D-LDH and GlyDH. Under the optimal conditions, 5.87 g/L D-PLA was produced by expressing LfD-LDH-l3-BmGlyDH with a yield of 0.97 g/g PPA, which was 59.3% increased compared to expression of LfD-LDH. In a scaled-up reaction, a productivity of 5.83 g/L/h was reached. In this study, improving the bio-catalytic efficiency by artificial redox self-equilibrium system with a bifunctional fusion protein could reduce the bio-production cost of D-PLA, making this bio-production of D-PLA a more promising industrial technology.

Metabolic engineering of Escherichia coli for polyamides monomer δ-valerolactam production from feedstock lysine.

Nylon 5 and nylon 6,5 are recently explored as new commercial polyamides, of which the monomer includes δ-valerolactam. In this study, a novel catalytic activity of lysine 2-monooxygenase (DavB) was explored to produce δ-valerolactam from L-pipecolic acid (L-PA), functioning as oxidative decarboxylase on a cyclic compound. Recombinant Escherichia coli BS01 strain expressing DavB from Pseudomonas putida could synthesize δ-valerolactam from L-pipecolic acid with a concentration of 90.3 mg/L. Through the co-expression of recombinant apoptosis-inducing protein (rAIP) from Scomber japonicus, glucose dehydrogenase (GDH) from Bacillus subtilis, Δ1-piperideine-2-carboxylae reductase (DpkA) from P. putida and lysine permease (LysP) from E. coli with DavB, δ-valerolactam was produced with the highest concentration of 242 mg/L. α-Dioxygenases (αDox) from Oryza sativa could act as a similar catalyst on L-pipecolic acid. A novel δ-valerolactam synthesis pathway was constructed entirely via microbial conversion from feedstock lysine in this study. Our system has great potential in the development of a bio-nylon production process. KEY POINTS: • DavB performs as an oxidative decarboxylase on L-PA with substrate promiscuity. • Strain with rAIP, GDH, DpkA, LysP, and DavB coexpression could produce δ-valerolactam. • This is the first time to obtain valerolactam entirely via biosynthesis from lysine.

Metabolic Engineering of Oleaginous Yeast Yarrowia lipolytica for Overproduction of Fatty Acids.

The oleaginous yeast Yarrowia lipolytica has attracted much attention due to its ability to utilize a wide range of substrates to accumulate high lipid content and its flexibility for genetic manipulation. In this study, intracellular lipid metabolism in Y. lipolytica was tailored to produce fatty acid, a renewable oleochemical and precursor for production of advanced biofuels. Two main strategies, including blocking activation and peroxisomal uptake of fatty acids and elimination of biosynthesis of lipids, were employed to reduce fatty acid consumption by the native pathways in Y. lipolytica. Both genetic modifications improved fatty acid production. However, disruption of the genes responsible for assembly of nonpolar lipid molecules including triacylglycerols (TAGs) and steryl esters resulted in the deleterious effects on the cell growth. The gene tesA encoding thioesterase from Escherichia coli was expressed in the strain with disrupted faa genes encoding fatty acyl-CoA synthetases and pxa1 encoding peroxisomal acyl-CoA transporter, and the titer of fatty acids resulted in 2.3 g/L in shake flask culture, representing 11-fold improvement compared with the parent strain. Expressing the native genes encoding acetyl-CoA carboxylase (ACC) and hexokinase also increased fatty acid production, although the improvement was not as significant as that with tesA expression. Saturated fatty acids including palmitic acid (C16:0) and stearic acid (C18:0) increased remarkably in the fatty acid composition of the recombinant bearing tesA compared with the parent strain. The recombinant expressing tesA gene resulted in high lipid content, indicating the great fatty acid producing potential of Y. lipolytica. The results highlight the achievement of fatty acid overproduction without adverse effect on growth of the strain. Results of this study provided insight into the relationship between fatty acid and lipid metabolism in Y. lipolytica, confirming the avenue to reprogram lipid metabolism of this host for overproduction of renewable fatty acids.

Expanding Toolbox for Genes Expression of Yarrowia lipolytica to Include Novel Inducible, Repressible, and Hybrid Promoters.

Promoters are critical tools to precisely control gene expression for both synthetic biology and metabolic engineering. Although Yarrowia lipolytica has demonstrated many industrially relevant advantages, promoter discovery efforts on this non-conventional yeast are limited due to the challenge in finding suitable inducible and repressible promoters. Six copper-inducible promoters and five repressible promoters were isolated in this work. Especially, Cu2+-repressible promoters showed relatively high activity under non-repressing conditions compared with a constitutive promoter, but the strength could be almost fully repressed by a supplement of a low content of Cu2+. The six Cu2+-inducible promoters were engineered to improve their dynamic regulation range with a tandem upstream activation sequence. An engineered promoter was successfully used to construct a more productive pathway for production of a novel bioproduct, wax ester, than that used for both Cu2+-inducible promoter and constitutive promoter. This study provides effective tools applicable to fine-tune the gene expression in this microbial host.

Engineering levoglucosan metabolic pathway in Rhodococcus jostii RHA1 for lipid production.

Oleaginous strains of Rhodococcus including R. jostii RHA1 have attracted considerable attention due to their ability to accumulate triacylglycerols (TAGs), robust growth properties and genetic tractability. In this study, a novel metabolic pathway was introduced into R. jostii by heterogenous expression of the well-characterized gene, lgk encoding levoglucosan kinase from Lipomyces starkeyi YZ-215. This enables the recombinant R. jostii RHA1 to produce TAGs from the anhydrous sugar, levoglucosan, which can be generated efficiently as the major molecule from the pyrolysis of cellulose. The recombinant R. jostii RHA1 could grow on levoglucosan as the sole carbon source, and the consumption rate of levoglucosan was determined. Furthermore, expression of one more copy of lgk increased the enzymatic activity of LGK in the recombinant. However, the growth performance of the recombinant bearing two copies of lgk on levoglucosan was not improved. Although expression of lgk in the recombinants was not repressed by the glucose present in the media, glucose in the sugar mixture still affected consumption of levoglucosan. Under nitrogen limiting conditions, lipid produced from levoglucosan by the recombinant bearing lgk was up to 43.54 % of the cell dry weight, which was comparable to the content of lipid accumulated from glucose. This work demonstrated the technical feasibility of producing lipid from levoglucosan, an anhydrosugar derived from the pyrolysis of lignocellulosic materials, by the genetically modified rhodococci strains.

Engineering of an L-arabinose metabolic pathway in Rhodococcus jostii RHA1 for biofuel production.

The oleaginous bacterium, Rhodococcus jostii RHA1 has attracted considerable attention due to its capability to accumulate significant levels of triacylglycerol as renewable hydrocarbon. To enable the strain to utilize arabinose derived from lignocellulosic biomass, the metabolic pathway of L-arabinose utilization was introduced into R. jostii RHA1 by heterogenous expression of the operon, araBAD from Escherichia coli. The results showed that recombinant bearing araBAD could grow on L-arabinose as the sole carbon source, and additional expression of araFGH encoding the arabinose transporter from E. coli could improve the cell biomass yield from high contents of arabinose. We further increased the content of lipid produced from arabinose in the recombinants from 47.9 to 56.8 % of the cell dry weight (CDW) by overexpression of a gene, atf1 encoding a diglyceride acyltransferase from R. opacus PD630. This work demonstrated the feasibility of producing lipid from arabinose by genetic modification of the rhodococci strain.

Exploring fatty alcohol-producing capability of Yarrowia lipolytica.

BACKGROUND: Fatty alcohols are important oleochemicals widely used in detergents, surfactants and personal care products. Bio-synthesized fatty alcohol provides a promising alternative to traditional fatty alcohol industry. Harnessing oleaginous microorganisms for fatty alcohol production may offer a new strategy to achieve a commercially viable yield that currently still seems to be a remote target. RESULTS: In this study, we introduced functional fatty acyl-CoA reductase (FAR), TaFAR1 to direct the conversion from fatty acyl-CoA to fatty alcohol in Yarrowia lipolytica (Y. lipolytica), an oleaginous non-conventional yeast showing great lipid-producing capability. Tri-module optimizations including eliminating fatty alcohol degradation pathway, enhancing TaFAR1 expression, and increasing fatty acyl-CoA supply were furtherly conducted, resulting in 63-fold increase in intracellular fatty alcohol-producing capability compared to the starting strain. Thus, this work demonstrated successful construction of first generation of Y. lipolytica fatty alcohol-producing cell factory. Through the study of effect of environmental nutrition on fatty alcohol production, up to 636.89 mg/L intracellular hexadecanol (high fatty alcohol-retaining capability) and 53.32 mg/L extracellular hexadecanol were produced by this cell factory through batch fermentation, which was comparable to the highest production of Saccharomyces cerevisiae under the similar condition. CONCLUSION: This work preliminarily explored fatty alcohol-producing capability through mobilization of FAR and fatty acid metabolism, maximizing the intracellular fatty alcohol-producing capability, suggesting that Y. lipolytica cell factory potentially offers a promising platform for fatty alcohol production.

Metabolic engineering of enhanced glycerol-3-phosphate synthesis to increase lipid production in Synechocystis sp. PCC 6803.

With the growing attention to global warming and energy sustainability, biosynthesis of lipids by photosynthetic microorganisms has attracted more interest for the production of renewable transportation fuels. Recently, the cyanobacterium Synechocystis sp. PCC 6803 has been widely used for biofuel production through metabolic engineering because of its efficient photosynthesis and well-developed genetic tools. In lipid biosynthesis, glycerol-3-phosphate (G3P) is a key node for both CO2 fixation and lipid metabolism in cyanobacteria. However, few studies have explored the use of G3P synthesis to improve photosynthetic lipid production. In this study, metabolic engineering combined with flux balance analysis (FBA) was conducted to reveal the effect of G3P synthesis on lipid production. Heterologous genes that encoded glycerol-3-phosphate dehydrogenase (GPD) and diacylglycerol acyltransferase (DGAT) were engineered into Synechocystis sp. PCC 6803 to enhance G3P supply and lipid production. The resultant recombinant Synechocystis produced higher levels of lipids without a significant reduction in cell growth. Compared with the wild-type strain, lipid content and productivity of the engineered cyanobacteria increased by up to 36 and 31 %, respectively, under autotrophic conditions. Lipid production under mixotrophic conditions of the engineered cyanobacteria was also investigated. This work demonstrated that enhanced G3P synthesis was an important factor in photosynthetic lipid production and that introducing heterologous GPD and DGAT genes was an effective strategy to increase lipid production in Synechocystis sp. PCC 6803.

Mitochondria thioredoxin's backup role in oxidative stress resistance in Trichoderma reesei.

Microorganisms often suffer from oxidative stress created from nutrient starvation and environmental changes. Thioredoxin (Trx) and glutathione (GSH) pathways are believed critical in related protective functions. The roles of Trx in improving abiotic stress resistance in Trichoderma reesei are still unclear. In this study, we identified a Trx-encoding gene, Trtrx1. The protein expressed located specifically in the mitochondria as verified by the fluorescence signals of TrTRX1-EGFP. TrTRX1 can catalyze the reduction of insulin disulfides by dithiothreitol (DTT). Loss of Trtrx1 however, did not lead to either significant morphology abnormality under normal and oxidative stress condition, or detectable difference in reactive oxygen species (ROS) resistance. The unchanged GSH amount in Trtrx1 deletion strain under normal condition and slight increase under oxidative stress condition, as well as the interplay between Trx and GSH systems suggested that GSH system was dominant and sufficient to maintain the mitochondrial redox state in T. reesei, where TrTRX1 played a role as the backup oxidative stress resistance.

Induction of D-xylose uptake and expression of NAD(P)H-linked xylose reductase and NADP + -linked xylitol dehydrogenase in the oleaginous microalga Chlorella sorokiniana.

BACKGROUND: The heterotrophic and mixotrophic culture of oleaginous microalgae is a promising process to produce biofuel feedstock due to the advantage of fast growth. Various organic carbons have been explored for this application. However, despite being one of the most abundant and economical sugar resources in nature, D-xylose has never been demonstrated as a carbon source for wild-type microalgae. The purpose of the present work was to identify the feasibility of D-xylose utilization by the oleaginous microalga Chlorella sorokiniana. RESULTS: The sugar uptake kinetic analysis was performed with (14)C-labeled sugars and the data showed that the D-glucose induced algal cells (the alga was heterotrophically grown on D-glucose and then harvested as D-glucose induced cells) exhibited a remarkably increased D-xylose uptake rate. The maximum D-xylose transport rate was 3.8 nmol min(-1) mg(-1) dry cell weight (DCW) with K m value of 6.8 mM. D-xylose uptake was suppressed in the presence of D-glucose, D-galactose and D-fructose but not L-arabinose and D-ribose. The uptake of D-xylose activated the related metabolic pathway, and the activities of a NAD(P)H-linked xylose reductase (XR) and a unique NADP(+)-linked xylitol dehydrogenase (XDH) were detected in C. sorokiniana. Compared with the culture in the dark, the consumption of D-xylose increased 2 fold under light but decreased to the same level with addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), indicating that extra chemical energy from the light-dependent reaction contributed the catabolism of D-xylose for C. sorokiniana. CONCLUSIONS: An inducible D-xylose transportation system and a related metabolic pathway were discovered for microalga for the first time. The transportation of D-xylose across the cell membrane of C. sorokiniana could be realized by an inducible hexose symporter. The uptake of D-xylose subsequently activated the expression of key catalytic enzymes that enabled D-xylose entering central metabolism. Results of this research are useful to better understand the D-xylose metabolic pathway in the microalga C. sorokiniana and provide a target for genetic engineering to improve D-xylose utilization for microalgal lipid production.

Location and contribution of individual ß-glucosidase from Neurospora crassa to total ß-glucosidase activity.

This study investigated the cellular location and the contribution of individual ß-glucosidase (BGL) to total BGL activity in Neurospora crassa. Among the seven bgl genes, bgl3, bgl5, and bgl7 were transcribed at basal levels, whereas bgl1, bgl2, bgl4, and bgl6 were significantly up-regulated when the wild-type strain was induced with cellulose (Avicel). BGL1 and BGL4 were found to be contributors to intracellular BGL activity, whereas the activities of BGL2 and BGL6 were mainly extracellular. Sextuple bgl deletion strains expressing one of the three basally transcribed bgls did not produce any detectable BGL activity when they were grown on Avicel. BGL6 is the major contributor to overall BGL activity, and most of its activity resides cell-bound. The sextuple bgl deletion strain containing only bgl6 utilized cellobiose at a rate similar to that of the wild type, while the strain with only bgl6 deleted utilized cellobiose much slower than that of the wild type.

Direct cellobiose production from cellulose using sextuple beta-glucosidase gene deletion Neurospora crassa mutants.

Direct cellobiose production from cellulose by a genetically modified fungus-Neurospora crassa, was explored in this study. A library of N. crassa sextuple beta-glucosidase (bgl) gene deletion strains was constructed. Various concentrations of cellobiose were detected in the culture broth of the N. crassa sextuple beta-glucosidase (bgl) gene deletion strains when grown on Avicel without exogenous cellulase addition. The sextuple bgl deletion strains expressing one of the three basally transcribed bgl genes are the best cellobiose producers. For most sextuple strains, the multiple bgl gene deletion has no negative effect on the production of other cellulases. The induction of major endoglucanases and exoglucanases on Avicel in most of the sextuple bgl deletions strains was as fast as or faster than that of the wild type, except for strain F4. The best cellobiose producing strain, F5, produced 7.7 g/L of cellobiose from 20 g/L of Avicel in four days and utilized the Avicel as fast as did the wild type (even in the presence of high cellobiose concentration). The cellobiose yield from cellulose was about 48.3%.

Engineering of a xylose metabolic pathway in Rhodococcus strains.

The two metabolically versatile actinobacteria Rhodococcus opacus PD630 and R. jostii RHA1 can efficiently convert diverse organic substrates into neutral lipids mainly consisting of triacylglycerol (TAG), the precursor of energy-rich hydrocarbon. Neither, however, is able to utilize xylose, the important component present in lignocellulosic biomass, as the carbon source for growth and lipid accumulation. In order to broaden their substrate utilization range, the metabolic pathway of d-xylose utilization was introduced into these two strains. This was accomplished by heterogenous expression of two well-selected genes, xylA, encoding xylose isomerase, and xylB, encoding xylulokinase from Streptomyces lividans TK23, under the control of the tac promoter with an Escherichia coli-Rhodococcus shuttle vector. The recombinant R. jostii RHA1 bearing xylA could grow on xylose as the sole carbon source, and additional expression of xylB further improved the biomass yield. The recombinant could consume both glucose and xylose in the sugar mixture, although xylose metabolism was still affected by the presence of glucose. The xylose metabolic pathway was also introduced into the high-lipid-producing strain R. opacus PD630 by expression of xylA and xylB. Under nitrogen-limited conditions, the fatty acid composition was determined, and lipid produced from xylose by recombinants of R. jostii RHA1 and R. opacus PD630 carrying xylA and xylB represented up to 52.5% and 68.3% of the cell dry weight (CDW), respectively. This work demonstrates that it is feasible to produce lipid from the sugars, including xylose, derived from renewable feedstock by genetic modification of rhodococcus strains.

A novel biochemical route for fuels and chemicals production from cellulosic biomass.

The conventional biochemical platform featuring enzymatic hydrolysis involves five key steps: pretreatment, cellulase production, enzymatic hydrolysis, fermentation, and product recovery. Sugars are produced as reactive intermediates for subsequent fermentation to fuels and chemicals. Herein, an alternative biochemical route is proposed. Pretreatment, enzymatic hydrolysis and cellulase production is consolidated into one single step, referred to as consolidated aerobic processing, and sugar aldonates are produced as the reactive intermediates for biofuels production by fermentation. In this study, we demonstrate the viability of consolidation of the enzymatic hydrolysis and cellulase production steps in the new route using Neurospora crassa as the model microorganism and the conversion of cellulose to ethanol as the model system. We intended to prove the two hypotheses: 1) cellulose can be directed to produce cellobionate by reducing ß-glucosidase production and by enhancing cellobiose dehydrogenase production; and 2) both of the two hydrolysis products of cellobionate--glucose and gluconate--can be used as carbon sources for ethanol and other chemical production. Our results showed that knocking out multiple copies of ß-glucosidase genes led to cellobionate production from cellulose, without jeopardizing the cellulose hydrolysis rate. Simulating cellobiose dehydrogenase over-expression by addition of exogenous cellobiose dehydrogenase led to more cellobionate production. Both of the two hydrolysis products of cellobionate: glucose and gluconate can be used by Escherichia coli KO 11 for efficient ethanol production. They were utilized simultaneously in glucose and gluconate co-fermentation. Gluconate was used even faster than glucose. The results support the viability of the two hypotheses that lay the foundation for the proposed new route.

[Expression of Vitreoscilla hemoglobin gene in Pseudomonas delafieldii R-8 and its application to diesel desulfurization].

OBJECTIVE: To construct an engineering strain of higher desulfurization activity with Vitreoscilla hemoglobin gene (vgb) for desulfurization of diesel. METHODS: We constructed the vgb expressing plasmid, pPR-Pdsz-vgb and transformed by electroporation to Pseudomonas delafieldii R-8, then obtained a genetic engineering strain R-8-2. RESULTS: The results of CO-difference spectrum analysis indicated that R-8-2 expressed Vitreoscilla hemoglobin (VHb) having biological activity expressed in strain R-8-2. Compared with the wild-type R-8, the density of strain R-8-2 increased by 20%, and its desulfurization activity is also higher in the lower deliquescence oxygen environment. In the end, about 69.9% of total sulfur in diesel oil was removed by strain R-8-2, whereas only 57.2% of sulfur was removed by strain R-8. CONCLUSION: The activity of strain R-8-2 was enhanced and furthermore, the work is helpful for further development in biodesulfurization.