Transcription factor shapes chromosomal conformation and regulates gene expression in bacterial adaptation.

Genomic mutations allow bacteria to adapt rapidly to adverse stress environments. The three-dimensional conformation of the genome may also play an important role in transcriptional regulation and environmental adaptation. Here, using chromosome conformation capture, we investigate the high-order architecture of the Zymomonas mobilis chromosome in response to genomic mutation and ambient stimuli (acetic acid and furfural, derived from lignocellulosic hydrolysate). We find that genomic mutation only influences the local chromosome contacts, whereas stress of acetic acid and furfural restrict the long-range contacts and significantly change the chromosome organization at domain scales. Further deciphering the domain feature unveils the important transcription factors, Ferric uptake regulator (Fur) proteins, which act as nucleoid-associated proteins to promote long-range (>200 kb) chromosomal communications and regulate the expression of genes involved in stress response. Our work suggests that ubiquitous transcription factors in prokaryotes mediate chromosome organization and regulate stress-resistance genes in bacterial adaptation.

Molecular mechanism of engineered Zymomonas mobilis to furfural and acetic acid stress.

Acetic acid and furfural (AF) are two major inhibitors of microorganisms during lignocellulosic ethanol production. In our previous study, we successfully engineered Zymomonas mobilis 532 (ZM532) strain by genome shuffling, but the molecular mechanisms of tolerance to inhibitors were still unknown. Therefore, this study investigated the responses of ZM532 and its wild-type Z. mobilis (ZM4) to AF using multi-omics approaches (transcriptomics, genomics, and label free quantitative proteomics). Based on RNA-Seq data, two differentially expressed genes, ZMO_RS02740 (up-regulated) and ZMO_RS06525 (down-regulated) were knocked out and over-expressed through CRISPR-Cas technology to investigate their roles in AF tolerance. Overall, we identified 1865 and 14 novel DEGs in ZM532 and wild-type ZM4. In contrast, 1532 proteins were identified in ZM532 and wild-type ZM4. Among these, we found 96 important genes in ZM532 involving acid resistance mechanisms and survival rates against stressors. Furthermore, our knockout results demonstrated that growth activity and glucose consumption of mutant strains ZM532∆ZMO_RS02740 and ZM4∆ZMO_RS02740 decreased with increased fermentation time from 42 to 55 h and ethanol production up to 58% in ZM532 than that in ZM532∆ZMO_RS02740. Hence, these findings suggest ZMO_RS02740 as a protective strategy for ZM ethanol production under stressful conditions.

Elimination of editing plasmid mediated by theophylline riboswitch in Zymomonas mobilis.

Zymomonas mobilis is regarded as a potential chassis for the production of platform chemicals. Genome editing using the CRISPR-Cas system could meet the need for gene modification in metabolic engineering. However, the low curing efficiency of CRISPR editing plasmid is a common bottleneck in Z. mobilis. In this study, we utilized a theophylline-dependent riboswitch to regulate the expression of the replicase gene of the editing plasmid, thereby promoting the elimination of exogeneous plasmid. The riboswitch D (RSD) with rigorous regulatory ability was identified as the optimal candidate by comparing the transformation efficiency of four theophylline riboswitch-based backbone editing plasmids, and the optimal theophylline concentration for inducing RSD was determined to be 2 mM. A highly effective method for eliminating the editing plasmid, cells with RSD-based editing plasmid which were cultured in liquid and solid RM media in alternating passages at 37 °C without shaking, was established by testing the curing efficiency of backbone editing plasmids pMini and pMini-RSD in RM medium with or without theophylline at 30 °C or 37 °C. Finally, the RSD-based editing plasmid was applied to genome editing, resulting in an increase of more than 10% in plasmid elimination efficiency compared to that of pMini-based editing plasmid. KEY POINTS: • An effective strategy for curing CRISPR editing plasmid has been established in Z. mobilis. • Elimination efficiency of the CRISPR editing plasmid was enhanced by 10% to 20% under the regulation of theophylline-dependent riboswitch RSD.

Enhancing Secretion of Endoglucanase in Zymomonas mobilis by Disturbing Peptidoglycan Synthesis.

Zymomonas mobilis (Z. mobilis) is a potential candidate strain for consolidated bioprocessing (CBP) in lignocellulosic biorefinery. However, the low-level secretion of cellulases limits this CBP process, and the mechanism of protein secretion that is affected by cell wall peptidoglycan is also not well understood. Here, we constructed several penicillin-binding protein (PBP)-deficient strains derived from Z. mobilis S192 to perturb the cell wall peptidoglycan network and then investigated the effects of peptidoglycan on the endoglucanase secretion. The results showed that extracellular recombinant endoglucanase production was significantly enhanced in PBP mutant strains, notably, Δ1089/0959 (4.09-fold) and Δ0959 (5.76-fold) in comparison to parent strains. For PBP-deficient strains, the growth performance was not significantly inhibited, but cell morphology was altered. In addition, enhanced antibiotic sensitivity and reduced inhibitor tolerance were also detected in our study. The concentration of intracellular soluble peptidoglycan was increased, especially for single-gene deletion. Outer membrane permeability of PBP-deficient strains was also improved, notably, Δ1089/0959 (1.14-fold) and Δ0959 (1.07-fold), which might explain the increased endoglucanase extracellular secretion. Our findings indicated that PBP-deficient Z. mobilis was capable of increasing endoglucanase extracellular secretion via cell wall peptidoglycan disturbance, and it will provide a foundation for the development of CBP technology in Z. mobilis in the future. IMPORTANCE Cell wall peptidoglycan has the function to maintain cell robustness and acts as the barrier to secret recombinant proteins from the cytoplasm to extracellular space in Z. mobilis and other bacteria. Herein, we perturbed the peptidoglycan synthesis network via knocking out PBPs (ZMO0197, ZMO0959, ZMO1089) to enhance recombinant endoglycanase extracellular secretion in Z. mobilis S912. This study could lay the foundation for understanding the regulatory network of cell wall synthesis and guide the construction of CBP strains in Z. mobilis.

The potential use of Zymomonas mobilis for the food industry.

Zymomonas mobilis is a gram-negative facultative anaerobic spore, which is generally recognized as a safe. As a promising ethanologenic organism for large-scale bio-ethanol production, Z. mobilis has also shown a good application prospect in food processing and food additive synthesis for its unique physiological characteristics and excellent industrial characteristics. It not only has obvious advantages in food processing and becomes the biorefinery chassis cell for food additives, but also has a certain healthcare effect on human health. Until to now, most of the research is still in theory and laboratory scale, and further research is also needed to achieve industrial production. This review summarized the physiological characteristics and advantages of Z. mobilis in food industry for the first time and further expounds its research status in food industry from three aspects of food additive synthesis, fermentation applications, and prebiotic efficacy, it will provide a theoretical basis for its development and applications in food industry. This review also discussed the shortcomings of its practical applications in the current food industry, and explored other ways to broaden the applications of Z. mobilis in the food industry, to promote its applications in food processing.

Potential applications of Zymomonas mobilis in food industry summarized for the first time.Research status of Z. mobilis in food additive synthesis, fermentation applications, and probiotics are discussed in details.Future research perspectives of Z. mobilis in food industry further proposed.

High-Efficiency Genome Editing Based on Endogenous CRISPR-Cas System Enhances Cell Growth and Lactic Acid Production in Pediococcus acidilactici.

Pediococcus acidilactici is commonly used for pediocin production and lactic acid fermentation. However, a high-efficiency genome editing tool is unavailable for this species. In this study, we constructed endogenous subtype II-A CRISPR-Cas system-based genome interference plasmids which carried a "repeat-spacer-repeat" cassette in the pMG36e shuttle vector. These plasmids exhibited self-interference activities in P. acidilactici LA412. Then, the genome-editing plasmids were constructed by cloning the upstream/downstream donor DNA into the corresponding interference plasmids to exert high-efficiency markerless gene deletion, gene integration, and point mutation in P. acidilactici LA412. We found that endogenous CRISPR-mediated depletion of the native plasmids enhanced the cell growth and that integration of an l-lactate dehydrogenase gene into the chromosome enhanced both cell growth and lactic acid production. IMPORTANCE A rapid and precise genome editing tool will promote the practical application of Pediococcus acidilactici, one type of lactic acid bacterium with excellent stress tolerance and probiotic characteristics. This study established a high-efficiency endogenous CRISPR-Cas system-based genome editing tool for P. acidilactici and achieved different genetic manipulations, including gene deletion, gene insertion, mononucleotide mutation, and endogenous plasmid depletion. The engineered strain edited by this tool showed significant advantages in cell growth and lactic acid fermentation. Therefore, our tool can satisfy the requirements for genetic manipulations of P. acidilactici, thus making it a sophisticated chassis species for synthetic biology and bioindustry.

Metabolic engineering of Zymomonas moblis for ethylene production from straw hydrolysate.

Biological ethylene production is a promising sustainable alternative approach for fossil-based ethylene production. The high glucose utilization of Z. mobilis makes it as a promising bioethylene producer. In this study, Zymomonas mobilis has been engineered to produce ethylene through the introduction of the synthetic ethylene-forming enzyme (EFE). We also investigated the effect of systematically knocking out the competitive metabolic pathway of pyruvate in an effort to improve the availability of pyruvate for ethylene production in Z. mobilis expressing EFE. Guided by these results, we tested a number of conjectures that could improve the α-ketoglutarate supply. Optimization of these pathways and different substrate supplies resulted in a greater production of ethylene (from 1.36 to 12.83 nmol/OD600/mL), which may guide future engineering work on ethylene production using other organisms. Meanwhile, we achieved an ethylene production of 5.8 nmol/OD600/mL in the ZM532-efe strain using enzymatic straw hydrolysate of corn straw as the sole carbon source. As a preferred host in biorefinery technologies using lignocellulosic biomass as feedstock, heterologous expression of EFE in Z. mobilis converts the non-ethylene producing strain into an ethylene-producing one using a metabolic engineering approach, which is of great significance for the utilization of cellulosic biomass in the future. KEY POINTS: • Heterologous expression of EFE in Z. mobilis successfully converted the non-ethylene producing strain into an ethylene producer (1.36 nmol/OD600/mL). Targeted modifications of the central carbon metabolism can effectively improve ethylene production (peak production: 8.3 nmol/OD600/mL). • The addition of nutrients to the medium can further increase the production of ethylene (peak production: 12.8 nmol/OD600/mL). • The ZM532-efe strain achieved an ethylene production of 5.8 nmol/OD600/mL when enzymatic hydrolysate of corn straw was used as the sole carbon source.

Novel mutagenesis and screening technologies for food microorganisms: advances and prospects.

Microorganisms are indispensable in the food industry, but wild-type strains hardly meet the current industrial demands due to several undesirable traits. Therefore, microbial strain improvement offers a critical solution to enhance the food industry. Traditional techniques for food microbial improvement, such as the use of chemical mutagens and manual isolation/purification, are inefficient, time-consuming, and laborious, restricting further progress in the area of food fermentation. In this review, the applications of novel mutagenesis and screening technologies used for the improvement of food microbes were summarized, including random mutagenesis based on physical irradiation, microbial screening facilitated by a microtiter plate, fluorescence-activated cell or droplet sorting, and microscaled fermentation in a microtiter plate or microbioreactor. In comparison with conventional methods, these new tools have the potential in accelerating microbial strain improvement and their combined applications could create a new trend for strain development. However, several problems that could affect its potential application may include the following: the lack of specific mutagenesis devices and biosensing systems, the insufficient improvement of the mixed culture system, the low efficiency when using filamentous fungi and flocculating bacteria, and the insufficient safety assessment on harnessing genome-editing technology. Therefore, future works on strain improvement remain challenging for the food industry.

Advances and prospects in metabolic engineering of Zymomonas mobilis.

Biorefinery of biomass-based biofuels and biochemicals by microorganisms is a competitive alternative of traditional petroleum refineries. Zymomonas mobilis is a natural ethanologen with many desirable characteristics, which makes it an ideal industrial microbial biocatalyst for commercial production of desirable bioproducts through metabolic engineering. In this review, we summarize the metabolic engineering progress achieved in Z. mobilis to expand its substrate and product ranges as well as to enhance its robustness against stressful conditions such as inhibitory compounds within the lignocellulosic hydrolysates and slurries. We also discuss a few metabolic engineering strategies that can be applied in Z. mobilis to further develop it as a robust workhorse for economic lignocellulosic bioproducts. In addition, we briefly review the progress of metabolic engineering in Z. mobilis related to the classical synthetic biology cycle of "Design-Build-Test-Learn", as well as the progress and potential to develop Z. mobilis as a model chassis for biorefinery practices in the synthetic biology era.

Biodetoxification of Phenolic Inhibitors from Lignocellulose Pretreatment using Kurthia huakuii LAM0618T and Subsequent Lactic Acid Fermentation.

Phenolic inhibitors generated during alkaline pretreatment of lignocellulosic biomasses significantly hinder bacterial growth and subsequent biofuel and biochemical production. Water rinsing is an efficient method for removing these compounds. Nevertheless, this method often generates a great amount of wastewater, and leads to the loss of solid fiber particles and fermentable sugars. Kurthia huakuii LAM0618T, a recently identified microorganism, was herein shown to be able to efficiently transform phenolic compounds (syringaldehyde, hydroxybenzaldehyde, and vanillin) into less toxic acids. Taking advantage of these properties, a biodetoxification method was established by inoculating K. huakuii LAM0618T into the NH3/H2O2-pretreated unwashed corn stover to degrade phenolic inhibitors and weak acids generated during the pretreatment. Subsequently, 33.47 and 17.91 g/L lactic acid was produced by Bacillus coagulans LA204 at 50 °C through simultaneous saccharification and fermentation (SSF) from 8% (w/w) of NH3/H2O2-pretreated corn stover with or without K. huakuii LAM0618T-biodetoxification, indicating biodetoxification significantly increased lactic acid titer and yield. Importantly, using 15% (w/w) of the NH3/H2O2-pretreated K. huakuii LAM0618T-biodetoxified corn stover as a substrate through fed-batch simultaneous saccharification and fermentation, high titer and high yield of lactic acid (84.49 g/L and 0.56 g/g corn stover, respectively, with a productivity of 0.88 g/L/h) were produced by Bacillus coagulans LA204. Therefore, this study reported the first study on biodetoxification of alkaline-pretreated lignocellulosic material, and this biodetoxification method could replace water rinsing for removal of phenolic inhibitors and applied in biofuel and biochemical production using the alkaline-pretreated lignocellulosic bioresources.

Brevibacillus halotolerans sp. nov., isolated from saline soil of a paddy field.

Two novel aerobic bacteria, designated strains LAM0312T and LAM0313, were isolated from saline soil samples collected from a paddy field in Dezhou city, Shandong Province, China. Cells of these strains were Gram-stain-positive, sporogenous, rod-shaped and motile with peritrichous flagella. The optimal growth temperature and pH were 30 °C and pH 7.0-8.0. Strain LAM0312T was able to grow in the presence of 12 % (w/v) NaCl. The major fatty acids were anteiso-C15 : 0 and iso-C15 : 0. The dominant polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, glycolipids, five unidentitied lipids and phosphatidylmonomethylethanolamine. The cell-wall peptidoglycan was found to contain meso-diaminopimelic acid. The predominant menaquinone was identified as menaquinone-7. The G+C content of the genomic DNA of strains LAM0312T and LAM0313 was 45.0 and 46.0 mol%, respectively, as determined by the Tm method. 16S rRNA gene sequence similarity analysis indicated that the strains were closely related to Brevibacillus laterosporus DSM 25T and Brevibacillus fluminis JCM 15716T with 98.5 and 96.4 % sequence similarity, respectively. The DNA-DNA hybridization value between strain LAM0312T and LAM0313 was 92±0.6 % (reciprocal 90±0.2 %) and the value between strain LAM0312T and Brevibacillus laterosporus DSM 25T was 48±0.5 % (reciprocal 40±0.4 %). On the basis of the phenotypic, phylogenetic and chemotaxonomic characteristics, the two strains are proposed to represent a novel species of the genus Brevibacillus, for which the name Brevibacillus halotolerans sp. nov. is proposed. The type strain is LAM0312T (=ACCC 06527T=JCM 30849T).

Acinetobacter larvae sp. nov., isolated from the larval gut of Omphisa fuscidentalis.

A Gram-stain-negative, non-spore-forming, non-motile and aerobic coccobacilli-shaped strain, designated BRTC-1T, was isolated from the gut of Omphisa fuscidentalis Hampson, which is a larva of a moth and was collected from Xishuangbanna Dai Autonomous Prefecture in China. The isolate was found to grow at NaCl concentrations of 0-5 % (w/v) (optimum: 0 %), 10-45 °C (optimum: 30-35 °C) and pH 5.0-9.0 (optimum: pH 6.0) on tryptic soy agar. Analysis of the 16S rRNA gene sequence indicated that the isolate belonged to the genus Acinetobacter and was most closely related to Acinetobacter rudis LMG 26107T, Acinetobacter guillouiae LMG 988T and Acinetobacter bereziniae LMG 1003T with 96.4, 96.3 and 96.3 % sequence similarity, respectively. The comparative sequence analyses of the rpoB and gyrB genes showed that BRTC-1T is distant from the species of the genus Acinetobacter with validly published names (≤84.0 and ≤82.0 % similarity, respectively). The average nucleotide identity and digital DNA-DNA hybridization values (≤77.0 and ≤22.8 %, respectively) between the whole-genome sequence of BRTC-1T and those of the known taxa were far below the thresholds used to discriminate bacterial species. The major fatty acids were determined to be C16 : 0, C18 : 1ω9c and C16 : 1ω7c/iso-C15 : 0 2-OH. The respiratory quinone was Q-9. The polar lipids were found to be diphosphatidyglycerol, phosphatidylglycerol, phosphatidylethanolamine, five phospholipids and one phosphatidylcholine. Based on its phenotypic and chemotaxonomic characteristics from this study, the isolate is concluded to represent a novel species of the genus Acinetobacter, for which the name Acinetobacter larvae sp. nov. is proposed. The type strain is BRTC-1T (=ACCC 19936T=JCM 31367T).

Calcium-rich biochar from the pyrolysis of crab shell for phosphorus removal.

Calcium-rich biochars (CRB) prepared through pyrolysis of crab shell at various temperatures were characterized for physicochemical properties and P removal potential. Elemental analysis showed that CRB was rich in calcium (22.91%-36.14%), while poor in carbon (25.21%-9.08%). FTIR, XRD and TG analyses showed that calcite-based CRB was prepared at temperature ≤600 °C, while lime-based CRB was prepared at temperature ≥700 °C. Phosphorus removal experiment showed that P removal efficiencies in 80 mg P/L phosphate solution and biogas effluent ranged from 26% to 11%, respectively, to about 100% and 63%, respectively, depending on the pyrolysis temperature of the resulting biochar. Specifically, compared to common used CaCO3 and Ca(OH)2, P removal potential of calcite-based CRB was much higher than that of CaCO3; while that of lime-based CRB was close to that of Ca(OH)2. These results suggested that CRB was competent for P removal/recovery from wastewater.

Bacillus vini sp. nov. isolated from alcohol fermentation pit mud.

A novel aerobic, Gram-stain-positive, sporogenous, rod-shaped bacterium, designated LAM0415(T), was isolated from an alcohol fermentation pit mud sample collected from Sichuan Luzhou-flavour liquor enterprise in China. The isolate was found to be able to grow at NaCl concentrations of 0-10 % (w/v) (optimum: 1.0 %), 10-50 °C (optimum: 30-35 °C) and pH 3.0-10.0 (optimum: 7.0-8.0). Phylogenetic analysis of 16S rRNA gene sequences indicated that the new isolate belonged to the genus Bacillus and was closely related to Bacillus sporothermodurans DSM 10599(T) and Bacillus oleronius DSM 9356(T), with 98.4 and 97.2 % sequence similarity, respectively. The DNA-DNA hybridization values between strain LAM0415(T) and the two reference strains were 33.3 ± 1.2 and 42.8 ± 0.8 %, respectively. The genomic DNA G+C content was 35.2 mol% as determined by the T m method. The major fatty acids were determined to be iso-C15:0, anteiso-C15:0 and anteiso-C17:0. The predominant menaquinones were identified as MK7 and MK8. The major polar lipids were found to be diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, one unidentified phospholipid and four unidentified glycolipids. The diagnostic amino acid of the cell wall peptidoglycan was determined to be meso-diaminopimelic acid. On the basis of its phenotypic, phylogenetic and chemotaxonomic characteristics, strain LAM0415(T) (=ACCC 06413(T) = JCM 19841(T)) represents the type strain of a novel species of the genus Bacillus, for which the name Bacillus vini sp. nov. is proposed.

Frigoribacterium salinisoli sp. nov., isolated from saline soil, transfer of Frigoribacterium mesophilum to Parafrigoribacterium gen. nov. as Parafrigoribacterium mesophilum comb. nov.

A Gram-stain-positive, short-rod, aerobic bacterium, designated as strain LAM9155T, was isolated from saline soil sample collected from Lingxian County, Shandong Province, PR China. The strain grew optimally at 25-30 °C, pH 7.0 and 0.5 % (w/v) NaCl. The 16S rRNA gene sequence analysis revealed that strain LAM9155T belonged to the genus Frigoribacterium and was closely related to Frigoribacteriumendophyticum EGI 6500707T (99.4 %), Frigoribacteriumfaeni 801T (98.6 %) and Frigoribacteriummesophilum MSL-08T (96.2 %). The DNA-DNA hybridization values between strain LAM9155T and F. endophyticum JCM 30093T and between strain LAM9155T and F. faeni DSM 10309T were 40.2±2.1 and 32.8±1.6 %, respectively. The major fatty acids of LAM9155T were anteiso-C15 : 0, anteiso-C17 : 0 and iso-C16 : 0. The cell-wall analysis showed the B-type peptidoglycan containing alanine, glutamate, glycine, serine and lysine and that the cell wall contained the sugars galactose and ribose. The genomic DNA G+C content of strain LAM9155T was 68.2 mol%. The predominant menaquinone was MK-9. The main polar lipids were diphosphatidylglycerol, phosphatidylglycerol, one unknown glycolipid and four unknown lipids. Based on the DNA-DNA hybridization and phenotypic, phylogenetic and chemotaxonomic properties, strain LAM9155T could be distinguished from the recognized species of the genus Frigoribacterium and was suggested to represent a novel species, for which the name Frigoribacterium salinisoli sp. nov. is proposed. The type strain is LAM9155T (=ACCC 19902T=JCM 30848T). Moreover, the transfer of F. mesophilum Dastager et al. 2008 to Parafrigoribacterium gen. nov. as Parafrigoribacterium mesophilum comb. nov. (type strain MSL-08T=DSM 19442T=KCTC 19311T) is also proposed.

Chromobacterium rhizoryzae sp. nov., isolated from rice roots.

A novel facultatively anaerobic, rod-shaped bacterium, designated LAM1188T, was isolated from the roots of rice (Oryzasativa) in Hubei Province. Cells of LAM1188T were Gram-stain-negative and motile. The temperature and pH ranges for growth were 15-40 °C (optimum: 30 °C) and pH 5-10 (optimum: pH 7), respectively. The strain did not require NaCl for growth but tolerated up to 3.5 % NaCl (w/v). Analysis of the 16S rRNA gene sequence indicated that the isolate represented a member of the genus Chromobacterium, and was most closely related to Chromobacterium haemolyticum MDA0585T and Chromobacterium aquaticum CC-SEYA-1T with 98.7 % and 97.3 % sequence similarity, respectively. The values of DNA-DNA hybridization between LAM1188T and C. haemolyticum JCM 14163T and C. aquaticum CCUG 55175T were 54.0±2.1 % and 44.0±1.2 %, respectively. The major cellular fatty acids were C16 : 0 and summed feature 3 (C16 : 1ω6c and/or C16 : 1ω7c). The main polar lipids were phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, four unidentified aminolipids and four unidentified lipids. The respiratory quinone was ubiquinone Q-8. The DNA G+C content was 64.1 mol% as determined by the Tm method. On the basis of its phenotypic, chemotaxonomic and phylogenetic characteristics, strain LAM1188T is suggested to represent a novel species of the genus Chromobacterium, for which the name Chromobacte riumrhizoryzae sp. nov. is proposed. The type strain is LAM1188T (=ACCC 19900T=JCM 31180T).

Using global transcription machinery engineering (gTME) to improve ethanol tolerance of Zymomonas mobilis.

BACKGROUND: With the increasing global crude oil crisis and resulting environmental concerns, the production of biofuels from renewable resources has become increasingly important. One of the major challenges faced during the process of biofuel production is the low tolerance of the microbial host towards increasing biofuel concentrations. RESULTS: Here, we demonstrate that the ethanol tolerance of Zymomonas mobilis can be greatly enhanced through the random mutagenesis of global transcription factor RpoD protein, (σ(70)). Using an enrichment screening, four mutants with elevated ethanol tolerance were isolated from error-prone PCR libraries. All mutants showed significant growth improvement in the presence of ethanol stress when compared to the control strain. After an ethanol (9 %) stress exposure lasting 22 h, the rate of glucose consumption was approximately 1.77, 1.78 and 1.39 g L(-1) h(-1) in the best ethanol-tolerant strain ZM4-mrpoD4, its rebuilt mutant strain ZM4-imrpoD and the control strain, respectively. Our results indicated that both ZM4-mrpoD4 and ZM4-imrpoD consumed glucose at a faster rate after the initial 9 % (v/v) ethanol stress, as nearly 0.64 % of the initial glucose remained after 54 h incubation versus approximately 5.43 % for the control strain. At 9 % ethanol stress, the net ethanol productions by ZM4-mrpoD4 and ZM4-imrpoD during the 30-54 h were 13.0-14.1 g/l versus only 6.6-7.7 g/l for the control strain. The pyruvate decarboxylase activity of ZM4-mrpoD4 was 62.23 and 68.42 U/g at 24 and 48 h, respectively, which were 2.6 and 1.6 times higher than the control strain. After 24 and 48 h of 9 % ethanol stress, the alcohol dehydrogenase activities of ZM4-mrpoD4 were also augmented, showing an approximate 1.4 and 1.3 times increase, respectively, when compared to the control strain. Subsequent quantitative real-time PCR analysis under these stress conditions revealed that the relative expression of pdc in cultured (6 and 24 h) ZM4-mrpoD4 increased by 9.0- and 12.7-fold when compared to control strain. CONCLUSIONS: Collectively, these results demonstrate that the RpoD mutation can enhance ethanol tolerance in Z. mobilis. Our results also suggested that RpoD may play an important role in resisting high ethanol concentration in Z. mobilis and manipulating RpoD via global transcription machinery engineering (gTME) can provide an alternative and useful approach for strain improvement for complex phenotypes.

Engineered Zymomonas mobilis for salt tolerance using EZ-Tn5-based transposon insertion mutagenesis system.

BACKGROUND: The cell growth and ethanol yield of Zymomonas mobilis may be detrimentally affected by salt stress frequently present in some biomass-based fermentation systems, leading to a decrease in the rate of sugar conversion to ethanol or other bioproducts. To address this problem, improving the salt tolerance of Z. mobilis is a desirable way. However, limited progress has been made in development of Z. mobilis with higher salt tolerance for some technical challenges in the past decades. Recently, transposon insertion mutant system has been widely used as a novel genetic tool in many organisms to develop mutant strains. In this study, Tn5-based transposon insertion mutagenesis system firstly used for construction of higher salt tolerance strain in Z. mobilis. RESULTS: Approximately 200 Z. mobilis ZM4 mutants were generated by using Tn5-based transposon mutagenesis system. The mutant strain ZMT2 with improved salt tolerance phenotype was obtained by screening on RM agar plates with additional 1 % NaCl. Strain ZMT2 was confirmed to exhibit better fermentation performance under NaCl stress than wild type of strain ZM4. The transposon insertion was located in ZMO1122 (himA) by genome walking. Discruption of himA gene showed that himA may play an important role in response to salt tolerance in Z. mobils. CONCLUSIONS: The mutant strain ZMT2 with a transposon insertion in himA gene of the genome showed obviously higher sugar conversion rate to ethonal under up to 2 % NaCl stress than did the wild ZM4 strain. Besides, ZMT2 exhibited shared fermentative capabilities with wild ZM4 strain under no or low NaCl stress. This report firstly showed that himA played a role in responding to NaCl stress. Furthermore, the result indicated that Tn5-based transposon mutagenesis system was a feasible tool not only for genetic engineering in Z. mobilis strain improvement, but also in tapping resistent genes.

Lentibacillus amyloliquefaciens sp. nov., a halophilic bacterium isolated from saline sediment sample.

A Gram-stain positive, non-motile, non-sporogenous, aerobic, rod-shaped and halophilic bacterium, designated LAM0015(T), was isolated from a saline sediment sample collected from Yantai City in China. The isolate was found to be able to grow at NaCl concentrations of 5-25 % (w/v) (optimum: 7-12 %), 15-45 °C (optimum: 35 °C) and pH 5.0-9.0 (optimum: 7.0). The major fatty acids were determined to be anteiso-C15:0 and anteiso-C17:0. The predominant respiratory quinone was identified as MK-7. The cell wall peptidoglycan was determined to contain meso-diaminopimelic acid. The polar lipids were found to be diphosphatidyglycerol, phosphatidylglycerol, five phospholipids and one glycolipid. The DNA G+C content was 43.1 mol% as determined by the T m method. Analysis of the 16S rRNA gene sequence indicated that the isolate belongs within the genus Lentibacillus and is closely related to Lentibacillus persicus DSM 22530(T), Lentibacillus salicampi JCM 11462(T) and Lentibacillus jeotgali JCM 15795(T) with 97.3, 96.7 and 96.4 % sequence similarity, respectively. The DNA-DNA hybridization value between LAM0015(T) and L. persicus DSM 22530(T) was 51.2 ± 1.4 %. Based on its phenotypic, phylogenetic and chemotaxonomic characteristics, strain LAM0015(T) is concluded to represent a novel species of the genus Lentibacillus, for which the name Lentibacillus amyloliquefaciens sp. nov. is proposed. The type strain is LAM0015(T) (=ACCC 06401(T) = JCM 19838(T)).

Paenibacillus salinicaeni sp. nov., isolated from saline silt sample.

A novel facultatively anaerobic bacterium, designated strain LAM0A28(T), was isolated from a saline silt sample collected from the Chinese Sea of Death located in Suining city, Sichuan province, China. Cells of strain LAM0A28(T) were observed to be Gram-stain positive, motile, endospore-forming and straight-rod shaped. Strain LAM0A28(T) was found to be able to grow at 15-45 °C (optimum: 30-35 °C), pH 5.0-10.0 (optimum: 7.5) and 0-5 % NaCl (w/v) (optimum: 0.5 %). The 16S rRNA gene sequence similarity analysis showed that strain LAM0A28(T) is closely related to Paenibacillus jilunlii DSM 23019(T) (97.5 %) and Paenibacillus graminis DSM 15220(T) (97.2 %). The DNA-DNA hybridization values between the isolate and P. jilunlii DSM 23019(T), P. graminis DSM 15220(T) were 30.2 ± 1.6 % and 44.7 ± 2.1 %, respectively. The DNA G+C content was found to be 51.2 mol% as determined by the T m method. The major cellular fatty acids were identified as anteiso-C15:0, C16:0, iso-C16:0 and C14:0. The major isoprenoid quinone was identified as MK-7. The cell wall peptidoglycan was found to contain meso-diaminopimelic acid. The major polar lipids were found to be diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, two aminophospholipids and six unidentified lipids. Based on the phylogenetic, phenotypic and chemotaxonomic characteristics, strain LAM0A28(T) is concluded to represent a novel species within the genus Paenibacillus, for which the name Paenibacillus salinicaeni sp. nov. is proposed. The type strain is LAM0A28(T) (=ACCC 00741(T) = JCM 30850(T)).