A tailored series of engineered yeasts for the cell-dependent treatment of inflammatory bowel disease by rational butyrate supplementation.

Intestinal microbiota dysbiosis and metabolic disruption are considered essential characteristics in inflammatory bowel disorders (IBD). Reasonable butyrate supplementation can help patients regulate intestinal flora structure and promote mucosal repair. Here, to restore microbiota homeostasis and butyrate levels in the patient's intestines, we modified the genome of Saccharomyces cerevisiae to produce butyrate. We precisely regulated the relevant metabolic pathways to enable the yeast to produce sufficient butyrate in the intestine with uneven oxygen distribution. A series of engineered strains with different butyrate synthesis abilities was constructed to meet the needs of different patients, and the strongest can reach 1.8 g/L title of butyrate. Next, this series of strains was used to co-cultivate with gut microbiota collected from patients with mild-to-moderate ulcerative colitis. After receiving treatment with engineered strains, the gut microbiota and the butyrate content have been regulated to varying degrees depending on the synthetic ability of the strain. The abundance of probiotics such as Bifidobacterium and Lactobacillus increased, while the abundance of harmful bacteria like Candidatus Bacilloplasma decreased. Meanwhile, the series of butyrate-producing yeast significantly improved trinitrobenzene sulfonic acid (TNBS)-induced colitis in mice by restoring butyrate content. Among the series of engineered yeasts, the strain with the second-highest butyrate synthesis ability showed the most significant regulatory and the best therapeutic effect on the gut microbiota from IBD patients and the colitis mouse model. This study confirmed the existence of a therapeutic window for IBD treatment by supplementing butyrate, and it is necessary to restore butyrate levels according to the actual situation of patients to restore intestinal flora.

Different rapid startups for high-solid anaerobic digestion treating pig manure: Metagenomic insights into antibiotic resistance genes fate and microbial metabolic pathway.

High-solid anaerobic digestion (HSAD), as an emerging disposal technology for swine manure, was commonly hampered by the long lag phase and slow startup, resulting in poor performance. Rapid startups by different leachate reflux forms can solve the problem, but related study was scarcely reported. Therefore, metagenomic analysis was used to exploit the effects of different rapid startups on the biogas performance, antibiotic resistance genes (ARGs) removal and microbial metabolic pathway during HSAD. Compared anaerobic digestion with natural start (T1), three different rapid startups were set, including with autologous leachate reflux (T2), with water reflux (T3) and with exogenous leachate reflux (T4). The results showed that rapid startups (T2-T4) enhanced biogas yield and the cumulative methane yield was increased by 3.7-7.3 times compared with the control. Totally, 922 ARGs were found, most of which belonged to multidrug and MLS ARGs. About 56% of these ARGs could be reduced in T4, while just 32% of ARGs were reduced in T1. Antibiotic efflux pump is the main mechanism of microbial action, which could be decreased largely by these treatments. Moreover, all the rapid startups (T2-T4) made Methanosarcina content (9.59%-75.91%) higher than that in the natural startup of T1 (4.54%-40.27%). This is why these fast-startups helped methane production fast. Network analysis showed that microbial community and environmental factors (pH and VFAs) both contributed to the spread of ARGs. The reconstructed methane metabolic pathway by different identified genes showed that all methanogenesis pathways existed but acetate metabolic pathway was dominant. And the rapid startups made the abundance of acetate metabolic (M00357) higher than the natural startup.

High-Level Production of Hydroxytyrosol in Engineered Saccharomyces cerevisiae.

Hydroxytyrosol (HT) is a valuable aromatic compound with numerous applications. Herein, we enabled the efficient and scalable de novo HT production in engineered Saccharomyces cerevisiae (S. cerevisiae) from glucose. Starting from a tyrosol-overproducing strain, six HpaB/HpaC combinations were investigated, and the best catalytic performance was acquired with HpaB from Pseudomonas aeruginosa (PaHpaB) and HpaC from Escherichia coli (EcHpaC), resulting in 425.7 mg/L HT in shake flasks. Next, weakening the tryptophan biosynthetic pathway through downregulating the expression of TRP2 (encoding anthranilate synthase) further improved the HT titer by 27.2% compared to the base strain. Moreover, the cytosolic NADH supply was improved through introducing the feedback-resistant mutant of the TyrA (the NAD+-dependent chorismate mutase/prephenate dehydrogenase, TyrA*) from E. coli, which further increased the HT titer by 36.9% compared to the base strain. The best performing strain was obtained by optimizing the biosynthesis of HT in S. cerevisiae through a screening for an effective HpaB/HpaC combination, biosynthetic flux rewiring, and cofactor engineering, which enabled the titer of HT reaching 1120.0 mg/L in the shake flask. Finally, the engineered strain produced 6.97 g/L of HT by fed-batch fermentation, which represents the highest titer for de novo HT biosynthesis in microorganisms reported to date.

Core-Shell Three-Dimensional Perovskite Nanocrystals with Chiral-Induced Spin Selectivity for Room-Temperature Spin Light-Emitting Diodes.

We developed type-II core-shell nanocrystals (NCs) with a chiral low-dimensional perovskite shell and an achiral 3D MAPbBr3 core. The core-shell NCs exhibit spin-polarized luminescence at the first excitation band of the achiral core, which is due to the chiral-induced spin selectivity (CISS) effect-governed spin-dependent shell-to-core electron transportation and the subsequent electron-hole recombination in the core. The preferred spin state of the transferred electrons is determined by the handness of the chiral shell. For the core-shell NCs film, a photoluminescence quantum yield (PLQY) of 54% and a circularly polarized luminescence (CPL) with a maximum |glum| of 4.0 × 10-3 are obtained at room temperature. Finally, we achieved a spin-polarized light-emitting diode (spin-LED), affording a circularly polarized electroluminescence (CP-EL) with a |gCP-EL|of 6.0 × 10-3 under ambient conditions.

Comparison of the Unfolded Protein Response in Cellobiose Utilization of Recombinant Angel- and W303-1A-Derived Yeast Expressing ß-Glucosidase.

The unfolded protein response (UPR) is one of the most important protein quality control mechanisms in cells. At least, three factors are predicted to activate the UPR in yeast cells during fermentation. Using UPRE-lacZ as a reporter, we constructed two indicator strains, KZ and WZ, based on Angel-derived K-a and W303-1A strains, respectively, and investigated their UPR response to tunicamycin, ethanol, and acetic acid. Then, four strains carrying plasmids BG-cwp2 and BG were obtained to realize the displaying and secretion of ß-glucosidase, respectively. The results of cellobiose utilization assays indicated interactions between the UPR and the metabolic burden between the strain source, anchoring moiety, oxygen supply, and cellobiose concentration. Meanwhile, as expected, growth (OD600), ß-glucosidase, and ß-galactosidase activities were shown to have a positive inter-relationship, in which the values of the KZ-derived strains were far lower than those of the WZ-derived strains. Additionally, extra metabolic burden by displaying over secreting was also much more serious in strain KZ than in strain WZ. The maximum ethanol titer of the four strains (KZ (BG-cwp2), KZ (BG), WZ (BG-cwp2), and WZ (BG)) in oxygen-limited 10% cellobiose fermentation was 3.173, 5.307, 5.495, and 5.486% (v/v), respectively, and the acetic acid titer ranged from 0.038 to 0.060% (v/v). The corresponding maximum values of the ratio of ß-galactosidase activity to that of the control were 3.30, 5.29, 6.45, and 8.72, respectively. Under aerobic conditions with 2% cellobiose, those values were 3.79, 4.97, 6.99, and 7.67, respectively. A comparison of the results implied that ß-glucosidase expression durably induced the UPR, and the effect of ethanol and acetic acid depended on the titer produced. Further study is necessary to identify ethanol- or acid-specific target gene expression. Taken together, our results indicated that the host strain W303-1A is a better secretory protein producer, and the first step to modify strain K-a for cellulosic ethanol fermentation would be to relieve the bottleneck of UPR capacity. The results of the present study will help to identify candidate host strains and optimize expression and fermentation by quantifying UPR induction.

Repetitive δ-integration of a cellulase-encoding gene into the chromosome of an industrial Angel Yeast-derived strain by URA3 recycling.

Genetic modification of industrial yeast strains often faces more difficulties than that of laboratory strains. Thus, new approaches are still required. In this research, the Angel Yeast-derived haploid strain Kα was genetically modified by multiple rounds of δ-integration, which was achieved via URA3 recycling. Three δ-integrative plasmids, pGδRU, pGδRU-BGL, and pGδRU-EG, were first constructed with two 167 bp δ sequences and a repeat-URA3-repeat fragment. Then, the δ-integrative strains containing the bgl1 or egl2 gene were successfully obtained by one-time transformation of the linearized pGδRU-BGL or pGδRU-EG fragment, respectively. Their counterparts in which the URA3 gene was looped out were also easily isolated by selection for growth on 5´-fluoroorotic acid plates, although the ratio of colonies lacking URA3 to the total number of colonies decreased with increasing copy number of the corresponding integrated cellulase-encoding gene. Similar results were observed during the second round of δ-integration, in which the δ-integration strain Kα(δ::bgl1-repeat) obtained from the first round was transformed with a linearized pGδRU-EG fragment. After 10 rounds of cell growth and transfer to fresh medium, the doubling times and enzyme activities of Kα(δ::bgl1-repeat), Kα(δ::egl2-repeat), and Kα(δ::bgl1-repeat)(δ::egl2-repeat) showed no significant change and were stable. Further, their maximum ethanol concentrations during simultaneous saccharification and fermentation of pretreated corncob over a 7-day period were 46.35, 33.13, and 51.77 g/L, respectively, which were all substantially higher than the parent Kα strain. Thus, repetitive δ-integration with URA3 recycling can be a feasible and valuable method for genetic engineering of Angel Yeast. These results also provide clues about some important issues related to δ-integration, such as the structural stability of δ-integrated genes and the effects of individual integration-site locations on gene expression. Further be elucidation of these issues should help to fully realize the potential of δ-integration-based methods in industrial yeast breeding.

Altering the inhibitory kinetics and molecular conformation of maltase by Tangzhiqing (TZQ), a natural α-glucosidase inhibitor.

BACKGROUND: Tangzhiqing (TZQ), as a potential α-glycosidase inhibitor, possesses postprandial hypoglycaemic effects on maltose in humans. The aim of this study was to investigate the mechanisms by which TZQ attenuates postprandial glucose by interrupting the activity of maltase, including inhibitory kinetics and circular dichroism studies. METHODS: In this study, we determined the inhibitory effect of TZQ on maltase by kinetic analysis to determine the IC50 value and enzyme velocity studies and line weaver-burk plot generation to determine inhibition type. Acarbose was chosen as a standard control drug. After the interaction with TZQ and maltase, secondary structure analysis was conducted with a circular dichroism method. RESULTS: TZQ showed notable inhibition activity on maltase in a reversible and competitive manner with an IC50 value of 1.67 ± 0.09 µg/ml, which was weaker than that of acarbose (IC50 = 0.29 ± 0.01 µg/ml). The circular dichroism spectrum demonstrated that the binding of TZQ to maltase changed the conformation of maltase and varied with the concentration of TZQ in terms of the disappearance of ß-sheets and an increase in the α-helix content of the enzyme, similar to acarbose. CONCLUSIONS: This work provides useful information for the inhibitory effect of TZQ on maltase. TZQ has the potential to be an α-glycosidase inhibitor for the prevention and treatment of prediabetes or mild diabetes mellitus.

Furfural-tolerant Zymomonas mobilis derived from error-prone PCR-based whole genome shuffling and their tolerant mechanism.

Furfural-tolerant strain is essential for the fermentative production of biofuels or chemicals from lignocellulosic biomass. In this study, Zymomonas mobilis CP4 was for the first time subjected to error-prone PCR-based whole genome shuffling, and the resulting mutants F211 and F27 that could tolerate 3 g/L furfural were obtained. The mutant F211 under various furfural stress conditions could rapidly grow when the furfural concentration reduced to 1 g/L. Meanwhile, the two mutants also showed higher tolerance to high concentration of glucose than the control strain CP4. Genome resequencing revealed that the F211 and F27 had 12 and 13 single-nucleotide polymorphisms. The activity assay demonstrated that the activity of NADH-dependent furfural reductase in mutant F211 and CP4 was all increased under furfural stress, and the activity peaked earlier in mutant than in control. Also, furfural level in the culture of F211 was also more rapidly decreased. These indicate that the increase in furfural tolerance of the mutants may be resulted from the enhanced NADH-dependent furfural reductase activity during early log phase, which could lead to an accelerated furfural detoxification process in mutants. In all, we obtained Z. mobilis mutants with enhanced furfural and high concentration of glucose tolerance, and provided valuable clues for the mechanism of furfural tolerance and strain development.

Extra metabolic burden by displaying over secreting: Growth, fermentation and enzymatic activity in cellobiose of recombinant yeast expressing ß-glucosidase.

ß-Glucosidase was selected to be a reporter to study metabolic burden imposed by its expression in yeast. Cell growth, fermentation yield and enzymatic activity were used as indicators of the metabolic burden borne by 14 recombinant yeast strains. Various factors were found to affect metabolic burden, including BGLI gene source, gene dose, trafficking of the enzyme (either cell-surface display or secretion), and oxygen supply. While BGLI gene from Aspergillus aculeatus provided better performance for the host cells than that from Saccharomycopsis fibuligera, displaying ß-glucosidase on the cell surface generally led to lower µm, total activity and ethanol titer, and longer lag period, lower (aerobic condition) or higher (anaerobic condition) biomass yield than that of secreting ß-glucosidase. The negative effect on growth increased with gene dose level until a final failure to grow. This growth difference implies that displaying ß-glucosidase on the cell surface imposes an extra metabolic burden. The molecular basis and mechanisms for this phenomenon need to further be investigated in order to develop better strategies for utilizing displayed and secreted enzymes in biotechnology and yeast breeding.

Isolation and characterization of butanol-tolerant Staphylococcus aureus.

OBJECTIVES: A new solvent-tolerant species, Staphylococcus aureus, was isolated and characterized during the screening of butanol-tolerant microorganisms. RESULTS: Three isolates of S. aureus were obtained as contaminants during improvement of butanol tolerance of E. coli K12. Their cell dry weights were 135 % that of K12 in the absence of butanol stress. S. aureus had a growth advantage over K12 when cultured with various concentrations of butanol. It can tolerate up to 3 % (v/v) butanol, while most solventogenic bacteria can tolerate only 2 % (v/v) butanol. The addition of 10-20 g glucose/l enhanced its butanol tolerance. The relative cell biomass of the S. aureus was 71-306 % that of E. coli under 5.5-10 % (v/v) ethanol stress, indicating ethanol resistance. CONCLUSIONS: This is the first study to observe butanol-tolerant S. aureus. As this organism can be genetically manipulated, it could have a wide array of applications.

Mating type and ploidy effect on the ß-glucosidase activity and ethanol-producing performance of Saccharomyces cerevisiae with multiple δ-integrated bgl1 gene.

In order to investigate the effect of mating type and ploidy on enzymatic activity and fermentation performance in yeast with multiple δ-integrated foreign genes, eight ploidy series strains were constructed. The initial haploid strain BGL-a was shown to contain about 19 copies of the bgl1 gene. In rich media containing 2% (w/v) sugar the specific activities of BGL-aα were lower than those of BGL-aa or BGL-αα, which indicates the existence of mating type effects. While the maximum OD660 decreased with rising ploidy, the biomass yield showed no significant difference between the eight strains and the specific activities (expressed as U/mL or U/mg DCW) showed little to no variation. When cellobiose was used as the carbon source and ß-glucosidase substrate, ß-glucosidase was expressed more quickly and at higher levels than in glucose-containing media. The maximum specific activitiy values obtained were 19.07U/mL and 19.39U/mL for BGL-αα and BGL-aa, repsectively. The anaerobic biomass and ethanol-producing performance in rich media containing 10% cellobiose showed no significant difference among the eight strains. Their maximal ethanol concentrations and corresponding yields ranged from 40.27 to 43.46g/L and 77.56 to 83.71%, respectively. When the acid- and alkali-pretreated corncob (10% solids content) was used, the diploid BGL-aα fermented the best. When urea was used as the only supplemented nutrient, the ethanol titer and yield were 35.65g/L and 83.69%, respectively, while a control experiment using industrial Angel yeast with exogenous ß-glucosidase addition gave values of 37.93g/L and 89.04%. The combined effects of δ-integration of bgl1, ploidy and mating type result in BGL-aa or BGL-αα being the optimal choice for enzyme production and BGL-aα being more suitable for cellulosic ethanol fermentation. These results provide valuable information for future yeast breeding and utilization efforts.

Ethanol production from acid- and alkali-pretreated corncob by endoglucanase and ß-glucosidase co-expressing Saccharomyces cerevisiae subject to the expression of heterologous genes and nutrition added.

Low-cost technologies to overcome the recalcitrance of cellulose are the key to widespread utilization of lignocellulosic biomass for ethanol production. Efficient enzymatic hydrolysis of cellulose requires the synergism of various cellulases, and the ratios of each cellulase are required to be regulated to achieve the maximum hydrolysis. On the other hand, engineering of cellulolytic Saccharomyces cerevisiae strains is a promising strategy for lignocellulosic ethanol production. The expression of cellulase-encoding genes in yeast would affect the synergism of cellulases and thus the fermentation ability of strains with exogenous enzyme addition. However, such researches are rarely reported. In this study, ten endoglucanase and ß-glucosidase co-expressing S. cerevisiae strains were constructed and evaluated by enzyme assay and fermentation performance measurement. The results showed that: (1) maximum ethanol titers of recombinant strains exhibited high variability in YPSC medium (20 g/l peptone, 10 g/l yeast extract, 100 g/l acid- and alkali-pretreated corncob) within 10 days. However, they had relatively little difference in USC medium (100 g/l acid- and alkali-pretreated corncob, 0.33 g/l urea, pH 5.0). (2) Strains 17# and 19#, with ratio (CMCase to ß-glucosidase) of 7.04 ± 0.61 and 7.40 ± 0.71 respectively, had the highest fermentation performance in YPSC. However, strains 11# and 3# with the highest titers in USC medium had a higher ratio of CMCase to ß-glucosidase, and CMCase activities. These results indicated that nutrition, enzyme activities and the ratio of heterologous enzymes had notable influence on the fermentation ability of cellulase-expressing yeast.

Expression optimization and biochemical properties of two glycosyl hydrolase family 3 beta-glucosidases.

The ß-glucosidases from Saccharomycopsis fibuligera (SfBGL1) and Trichoderma reesei (TrBGL1) were cloned and expressed in Pichia pastoris. Methanol concentration and pH significantly affected the production. The combined effects of the two factors were optimized by using the response surface method, resulting in a 137% and 84% increase in rTrBGL1 and rSfBGL1 yield compared to single-factor experiment. Structure and biochemical properties of the two enzyme were investigated and compared. They belong to glycosyl hydrolase family 3 and exhibit significant hydrolysis activity and low-level transglycosylation activity. The two enzymes show similar substrate affinity and ion-tolerance, and both of them can be activated by Cr(6+), Mn(2+) and Fe(2+). The rSfBGL1 has greater catalytic speed, higher specific activity and acid-tolerance than rTrBGL1, but rTrBGL1 is more thermostable and has higher optimal temperature than rSfBGL1. This study provides a useful and quick optimal method for recombinant enzyme production and makes a valuable comparison of biochemical properties, which opens important avenues of exploration for relationship between structure and function and further practical applications.

Reducing ß-glucosidase supplementation during cellulase recovery using engineered strain for successive lignocellulose bioconversion.

Enzyme recycling by re-adsorption is one of the primary methods for reducing enzyme usage in lignocellulose conversion. This work proposes the combination of an engineered yeast strain that expresses ß-glucosidase with enzyme recycling to reduce the amount of supplemented ß-glucosidase in enzyme recycling experiments. Using the engineered strain, a slight increase in ethanol concentration was obtained after a 96-h fermentation of pretreated corncobs. Ethanol concentrations increased by 34.7% and 62.7% in the following two recycle rounds using the engineered strain compared with those using its parental strain without ß-glucosidase addition. Furthermore, with the addition of ß-glucosidase at 30CBU/g cellulose, the ethanol concentration after two recycle rounds exceeded 90% of that observed in the first SSF round with the engineered strain at a high initial cellulase loading of 45FPU/g cellulose.

Development of a cellulolytic Saccharomyces cerevisiae strain with enhanced cellobiohydrolase activity.

Consolidated bioprocessing (CBP) is a promising technology for lignocellulosic ethanol production, and the key is the engineering of a microorganism that can efficiently utilize cellulose. Development of Saccharomyces cerevisiae for CBP requires high level expression of cellulases, particularly cellobiohydrolases (CBH). In this study, to construct a CBP-enabling yeast with enhanced CBH activity, three cassettes containing constitutively expressed CBH-encoding genes (cbh1 from Aspergillus aculeatus, cbh1 and cbh2 from Trichoderma reesei) were constructed. T. reesei eg2, A. aculeatus bgl1, and the three CBH-encoding genes were then sequentially integrated into the S. cerevisiae W303-1A chromosome via δ-sequence-mediated integration. The resultant strains W1, W2, and W3, expressing uni-, bi-, and trifunctional cellulases, respectively, exhibited corresponding cellulase activities. Furthermore, both the activities and glucose producing activity ascended. The growth test on cellulose containing plates indicated that CBH was a necessary component for successful utilization of crystalline cellulose. The three recombinant strains and the control strains W303-1A and AADY were evaluated in acid- and alkali-pretreated corncob containing media with 5 FPU exogenous cellulase/g biomass loading. The highest ethanol titer (g/l) within 7 days was 5.92 ± 0.51, 18.60 ± 0.81, 28.20 ± 0.84, 1.40 ± 0.12, and 2.12 ± 0.35, respectively. Compared with the control strains, W3 efficiently fermented pretreated corncob to ethanol. To our knowledge, this is the first study aimed at creating cellulolytic yeast with enhanced CBH activity by integrating three types of CBH-encoding gene with a strong constitutive promoter Ptpi.

Improving bgl1 gene expression in Saccharomyces cerevisiae through meiosis in an isogenic triploid.

Introducing large numbers of target genes into the chromosome of Saccharomyces cerevisiae via δ-sequence-mediated integration is a good strategy for exploring the effects of gene dosage on expression and secretion of heterologous proteins. The expression of exogenous genes might be further improved through meiosis in an isogenic triploid. Here, a stable strain A-8 was screened from 35 sexual spore colonies obtained from an isogenic triploid integratively expressing bgl1 from Aspergillus aculeatus. The corresponding ß-glucosidase activity in this strain was increased by ~120 % compared with the parent strain BGL-a. Measurement of doubling time, flow cytometry, and mating experiments further confirmed that A-8 was a spore-forming strain obtained from a triploid parent. Thus, combining δ-integration and meiosis in an isogenic triploid is a promising approach for improving the expression of exogenous proteins in S. cerevisiae.

Comparison of process configurations for ethanol production from acid- and alkali-pretreated corncob by Saccharomyces cerevisiae strains with and without ß-glucosidase expression.

ß-Glucosidase was shown to have synergistic effects with commercial cellulase in the hydrolysis of acid- and alkali-pretreated corncob, especially at the dose of 5 U/g biomass and 5 or 10 FPU/g biomass. An integrating yeast strain 45# expressing ß-glucosidase was constructed that utilized cellobiose quickly and efficiently. Process configurations were compared under conditions of 10% solid content, 10 FPU cellulase/g biomass, 5 U ß-glucosidase/g biomass (only used for parental strain W303-1A), 1g/kg yeast loading and 3.3g/kg urea supplementation. While separate hydrolysis and fermentation was optimal for W303-1A and the ethanol titer and yield reached 3.22 g/100g and 75.6% (expressed as a percentage of the theoretical yield), respectively, simultaneous saccharification and fermentation was optimal for strain 45# and the ethanol titer and yield reached 3.31 g/100g and 77.7%, respectively. These results are valuable in optimization of the process configuration and improving the yeast strain selected for cellulosic ethanol production.

Expression of cellulase genes in Saccharomyces cerevisiae via δ-integration subject to auxotrophic markers.

δ-Integration can improve the expression stability and increase the copy number of exogenous genes in Saccharomyces cerevisiae. Generally, δ-integration vectors employ auxotrophic markers, such as LEU2-d, HIS3, TRP1, and URA3, to screen transformants. In this study, two cellulase genes (ß-glucosidase and endoglucanase) were integrated into the S. cerevisiae W303-1A chromosome as reporters, by δ-integration with the aforementioned auxotrophic markers. Eight strains, L-BGL, H-BGL, T-BGL, U-BGL, L-EG, H-EG, T-EG, and U-EG, were selected and tested. After 5 days growth, cellulase activities (U ml(-1)) were 3, 2.3, 1.8, 1.5, 29, 12, 8, and 1.5, respectively. The results showed that auxotrophic markers influenced the expression of cellulase genes.

Hydrogen sulfide formation as well as ethanol production in different media by cysND- and/or cysIJ-inactivated mutant strains of Zymomonas mobilis ZM4.

Many bacteria reduce inorganic sulfate to sulfide to satisfy their need for sulfur, one of the most important elements for biological life. But little is known about the metabolic pathways involving hydrogen sulfide (H2S) in mesophilic bacteria. By genomic sequence analysis, a complete set of genes for the assimilatory sulfate reduction pathway has been identified in the ethanologen Zymomonas mobilis. In this study, the first ATP sulfurylase- and final sulfite reductase-encoding genes cysND and cysIJ, respectively, in the putative pathway from sulfate to sulfite in Z. mobilis ZM4 was singly or doubly inactivated by homologous recombination and a site-specific FLP-FRT recombination. The resultant mutants, ∆cysND, ∆cysIJ and ∆cysND-cat∆cysIJ, were unable to produce detectable H2S in glucose or sucrose-containing rich medium and sweet sorghum juice, in which the wild-type ZM4 produced detectable H2S. While adding sulfite (SO3²â») into media impaired the growth of the mutants and ZM4 to varying degrees, the sulfite restored the H2S formation in the ∆cysND in the above media, but not in the ∆cysIJ and ∆cysND-cat∆cysIJ mutants. Although it seemed that the inactivation of cysND and cysIJ did not exert a significant negative effect on the cell growth at least in glucose or sucrose medium, the ethanol production of all mutants was inferior to that of ZM4 in sucrose medium and sweet sorghum juice. In addition, adding L-cysteine to glucose-containing rich media restored H2S formation of all mutants, indicating the existence of another pathway for producing H2S in Z. mobilis. All these results would help to further elucidate the metabolic pathways involving H2S in Z. mobilis and exploit the biotechnological applications of this industrially important bacterium.

Comparison of glucose/xylose co-fermentation by recombinant Zymomonas mobilis under different genetic and environmental conditions.

Three xylose-fermenting recombinant Zymomonas mobilis strains containing different Peno-talB/tktA operon terminators were engineered. Each showed similar levels of foreign protein expression and xylose fermentation performance. Strain CP4-P2-1 was further used to compare the glucose/xylose co-fermentation under various cultivation environments to improve the efficiency of the process. Optimal co-fermentation was achieved at 30-34 °C and pH 5.5 using xylose-grown preculture cells giving 20.5 g ethanol/l, ethanol productivity of 0.43 g/l h and ethanol yield of 0.44 g/g at 48 h. Adverse culture conditions mainly influenced the efficiency of xylose fermentation but not glucose fermentation. The key factors affecting co-fermentation were also explored at the molecular level. This study provides valuable insights into the effective harnessing of biomass resources.