Construction and application of high-quality genome-scale metabolic model of Zymomonas mobilis to guide rational design of microbial cell factories.

High-quality genome-scale metabolic models (GEMs) could play critical roles on rational design of microbial cell factories in the classical Design-Build-Test-Learn cycle of synthetic biology studies. Despite of the constant establishment and update of GEMs for model microorganisms such as Escherichia coli and Saccharomyces cerevisiae, high-quality GEMs for non-model industrial microorganisms are still scarce. Zymomonas mobilis subsp. mobilis ZM4 is a non-model ethanologenic microorganism with many excellent industrial characteristics that has been developing as microbial cell factories for biochemical production. Although five GEMs of Z. mobilis have been constructed, these models are either generating ATP incorrectly, or lacking information of plasmid genes, or not providing standard format file. In this study, a high-quality GEM iZM516 of Z. mobilis ZM4 was constructed. The information from the improved genome annotation, literature, datasets of Biolog Phenotype Microarray studies, and recently updated Gene-Protein-Reaction information was combined for the curation of iZM516. Finally, 516 genes, 1389 reactions, 1437 metabolites, and 3 cell compartments are included in iZM516, which also had the highest MEMOTE score of 91% among all published GEMs of Z. mobilis. Cell growth was then predicted by iZM516, which had 79.4% agreement with the experimental results of the substrate utilization. In addition, the potential endogenous succinate synthesis pathway of Z. mobilis ZM4 was proposed through simulation and analysis using iZM516. Furthermore, metabolic engineering strategies to produce succinate and 1,4-butanediol (1,4-BDO) were designed and then simulated under anaerobic condition using iZM516. The results indicated that 1.68 mol/mol succinate and 1.07 mol/mol 1,4-BDO can be achieved through combinational metabolic engineering strategies, which was comparable to that of the model species E. coli. Our study thus not only established a high-quality GEM iZM516 to help understand and design microbial cell factories for economic biochemical production using Z. mobilis as the chassis, but also provided guidance on building accurate GEMs for other non-model industrial microorganisms.

Neutron Irradiation Effects on Boron Nitride-Based Ceramics for Use in X-ray Detection.

X-ray detectors based on conventional semiconductors with large atomic numbers are suffering from the poor stability under a high dose rate of ionizing irradiation. In this work, we demonstrate that a wide band gap ceramic-boron nitride with small atomic numbers could be used for sensitive X-ray detection. Boron nitride samples showed excellent resistance to ionizing radiation, which have been systematically studied with the neutron- and electron-aging experiments. Then, we fully analyzed the influence of these aging effects on the fundamental properties of boron nitride. Interestingly, we found that the boron nitride samples could maintain relatively good charge transport properties even after large dose of neutron irradiation. The fabricated X-ray detectors showed decent performance metrics, and the neutron-aged boron nitride even showed improved operational stability under continuous X-ray irradiation, suggesting the great potential for real applications.

CAVE: a cloud-based platform for analysis and visualization of metabolic pathways.

Flux balance analysis (FBA) is an important method for calculating optimal pathways to produce industrially important chemicals in genome-scale metabolic models (GEMs). However, for biologists, the requirement of coding skills poses a significant obstacle to using FBA for pathway analysis and engineering target identification. Additionally, a time-consuming manual drawing process is often needed to illustrate the mass flow in an FBA-calculated pathway, making it challenging to detect errors or discover interesting metabolic features. To solve this problem, we developed CAVE, a cloud-based platform for the integrated calculation, visualization, examination and correction of metabolic pathways. CAVE can analyze and visualize pathways for over 100 published GEMs or user-uploaded GEMs, allowing for quicker examination and identification of special metabolic features in a particular GEM. Additionally, CAVE offers model modification functions, such as gene/reaction removal or addition, making it easy for users to correct errors found in pathway analysis and obtain more reliable pathways. With a focus on the design and analysis of optimal pathways for biochemicals, CAVE complements existing visualization tools based on manually drawn global maps and can be applied to a broader range of organisms for rational metabolic engineering. CAVE is available at https://cave.biodesign.ac.cn/.

Preparation Scheme Optimization of Thermosetting Polyurethane Modified Asphalt.

To solve the issue of the poor temperature stability of conventional modified asphalt, polyurethane (PU) was used as a modifier with its corresponding curing agent (CA) to prepare thermosetting PU asphalt. First, the modifying effects of the different types of PU modifiers were evaluated, and the optimal PU modifier was then selected. Second, a three-factor and three-level L9 (33) orthogonal experiment table was designed based on the preparation technology, PU dosage, and CA dosage to prepare the thermosetting PU asphalt and asphalt mixture. Further, the effect of PU dosage, CA dosage, and preparation technology on the 3d, 5d, and 7d splitting tensile strength, freeze-thaw splitting strength, and tensile strength ratio (TSR) of the PU asphalt mixture was analyzed, and a PU-modified asphalt preparation plan was recommended. Finally, a tension test was conducted on the PU-modified asphalt and a split tensile test was performed on the PU asphalt mixture to analyze their mechanical properties. The results show that the content of PU has a significant effect on the splitting tensile strength of PU asphalt mixtures. When the content of the PU modifier is 56.64% and the content of CA is 3.58%, the performance of the PU-modified asphalt and mixture is better when prepared by the prefabricated method. The PU-modified asphalt and mixture have high strength and plastic deformation ability. The modified asphalt mixture has excellent tensile performance, low-temperature performance, and water stability, which meets the requirements of epoxy asphalt and the mixture standards.

Determination of Nucleotide Sequences within Promoter Regions Affecting Promoter Compatibility between Zymomonas mobilis and Escherichia coli.

A promoter plays a crucial role in controlling the expression of the target gene in cells, thus being one of the key biological parts for synthetic biology practices. Although significant efforts have been made to identify and characterize promoters with different strengths in various microorganisms, the compatibility of promoters within different hosts still lacks investigation. In this study, we chose the native Pgap promoter of Zymomonas mobilis to investigate nucleotide sequences within promoter regions affecting promoter compatibility between Escherichia coli and Z. mobilis. Pgap is one of the strongest promotors in Z. mobilis that has many excellent characteristics to be developed as microbial cell factories. Using EGFP as a reporter, a Z. mobilis-derived Pgap mutant library was constructed and sorted in E. coli, with candidate promoters exhibiting high fluorescence intensity collected. A total of 53 variants were finally selected and sequenced by Sanger sequencing. The sequencing results grouped these variants into 12 different Pgap variant types, among which seven types presented higher promoter strength than native Pgap in E. coli. The next-generation sequencing technique was then employed to identify key mutations within the Pgap promoter region that affect the promoter compatibility. Finally, six important sites were identified and confirmed to help increase Pgap strength in E. coli while keeping similar strength of native Pgap in Z. mobilis. Compared to native Pgap, synthetic promoters combining these sites had enhanced strength; especially, Pgap-6M combining all six sites exhibited 20-fold greater strength than native Pgap in E. coli. This study thus not only determined six important sites affecting promoter compatibility but also confirmed a series of Pgap promoter variants with strong promoter activity in both E. coli and Z. mobilis. In addition, a strategy was established in this study to investigate and determine nucleotide sequences in promoter regions affecting promoter compatibility, which can be applied in other microorganisms to help reveal universal factors affecting promoter compatibility and design promoters with desired strengths among different microbial cell factories.

Deciphering Molecular Mechanism Underlying Self-Flocculation of Zymomonas mobilis for Robust Production.

Zymomonas mobilis metabolizes sugar anaerobically through the Entner-Doudoroff pathway with less ATP generated for lower biomass accumulation to direct more sugar for product formation with improved yield, making it a suitable host to be engineered as microbial cell factories for producing bulk commodities with major costs from feedstock consumption. Self-flocculation of the bacterial cells presents many advantages, such as enhanced tolerance to environmental stresses, a prerequisite for achieving high product titers by using concentrated substrates. ZM401, a self-flocculating mutant developed from ZM4, the unicellular model strain of Z. mobilis, was employed in this work to explore the molecular mechanism underlying this self-flocculating phenotype. Comparative studies between ZM401 and ZM4 indicate that a frameshift caused by a single nucleotide deletion in the poly-T tract of ZMO1082 fused the putative gene with the open reading frame of ZMO1083, encoding the catalytic subunit BcsA of the bacterial cellulose synthase to catalyze cellulose biosynthesis. Furthermore, the single nucleotide polymorphism mutation in the open reading frame of ZMO1055, encoding a bifunctional GGDEF-EAL protein with apparent diguanylate cyclase/phosphodiesterase activities, resulted in the Ala526Val substitution, which consequently compromised in vivo specific phosphodiesterase activity for the degradation of cyclic diguanylic acid, leading to intracellular accumulation of the signaling molecule to activate cellulose biosynthesis. These discoveries are significant for engineering other unicellular strains from Z. mobilis with the self-flocculating phenotype for robust production. IMPORTANCE Stress tolerance is a prerequisite for microbial cell factories to be robust in production, particularly for biorefinery of lignocellulosic biomass to produce biofuels, bioenergy, and bio-based chemicals for sustainable socioeconomic development, since various inhibitors are released during the pretreatment to destroy the recalcitrant lignin-carbohydrate complex for sugar production through enzymatic hydrolysis of the cellulose component, and their detoxification is too costly for producing bulk commodities. Although tolerance to individual stress has been intensively studied, the progress seems less significant since microbial cells are inevitably suffering from multiple stresses simultaneously under production conditions. When self-flocculating, microbial cells are more tolerant to multiple stresses through the general stress response due to enhanced quorum sensing associated with the morphological change for physiological and metabolic advantages. Therefore, elucidation of the molecular mechanism underlying such a self-flocculating phenotype is significant for engineering microbial cells with the unique multicellular morphology through rational design to boost their production performance.

Cysteine supplementation enhanced inhibitor tolerance of Zymomonas mobilis for economic lignocellulosic bioethanol production.

Inhibitors in lignocellulosic hydrolysates are toxic to Zymomonas mobilis and reduce its bioethanol production. This study revealed cysteine supplementation enhanced furfural tolerance in Z. mobilis with a 2-fold biomass increase. Transcriptomic study illustrated that cysteine biosynthesis pathway was down-regulated while cysteine catabolism was up-regulated with cysteine supplementation. Mutants for genes involved in cysteine metabolism were constructed, and metabolites in cysteine metabolic pathway including methionine, glutathione, NaHS, glutamate, and pyruvate were supplemented into media. Cysteine supplementation boosted glutathione synthesis or H2S release effectively in Z. mobilis leading to the reduced accumulation of reactive oxygen species (ROS) induced by furfural, while pyruvate and glutamate produced in the H2S generation pathway promoted cell growth by serving as the carbon or nitrogen source. Finally, cysteine supplementation was confirmed to enhance Z. mobilis tolerance against ethanol, acetate, and corncob hydrolysate with an enhanced ethanol productivity from 0.38 to 0.55 g-1∙L-1∙h-1.

Molecular mechanism of enhanced ethanol tolerance associated with hfq overexpression in Zymomonas mobilis.

Zymomonas mobilis is a promising microorganism for industrial bioethanol production. However, ethanol produced during fermentation is toxic to Z. mobilis and affects its growth and bioethanol production. Although several reports demonstrated that the RNA-binding protein Hfq in Z. mobilis contributes to the tolerance against multiple lignocellulosic hydrolysate inhibitors, the role of Hfq on ethanol tolerance has not been investigated. In this study, hfq in Z. mobilis was either deleted or overexpressed and their effects on cell growth and ethanol tolerance were examined. Our results demonstrated that hfq overexpression improved ethanol tolerance of Z. mobilis, which is probably due to energy saving by downregulating flagellar biosynthesis and heat stress response proteins, as well as reducing the reactive oxygen species induced by ethanol stress via upregulating the sulfate assimilation and cysteine biosynthesis. To explore proteins potentially interacted with Hfq, the TEV protease mediated Yeast Endoplasmic Reticulum Sequestration Screening system (YESS) was established in Z. mobilis. YESS results suggested that Hfq may modulate the cytoplasmic heat shock response by interacting with the heat shock proteins DnaK and DnaJ to deal with the ethanol inhibition. This study thus not only revealed the underlying mechanism of enhanced ethanol tolerance by hfq overexpression, but also provided an alternative approach to investigate protein-protein interactions in Z. mobilis.

Development and characterization of efficient xylose utilization strains of Zymomonas mobilis.

BACKGROUND: Efficient use of glucose and xylose is a key for the economic production of lignocellulosic biofuels and biochemicals, and different recombinant strains have been constructed for xylose utilization including those using Zymomonas mobilis as the host. However, the xylose utilization efficiency still needs to be improved. In this work, the strategy of combining metabolic engineering and adaptive laboratory evolution (ALE) was employed to develop recombinant Z. mobilis strains that can utilize xylose efficiently at high concentrations, and NGS-based genome resequencing and RNA-Seq transcriptomics were performed for strains evolved after serial transfers in different media to understand the impact of xylose and differences among strains with different xylose-utilization capabilities at molecular level. RESULTS: Heterologous genes encoding xylose isomerase and xylulokinase were evaluated, which were then introduced into xylose-utilizing strain Z. mobilis 8b to enhance its capacity of xylose utilization. The results demonstrated that the effect of three xylose isomerases on xylose utilization was different, and the increase of copy number of xylose metabolism genes can improve xylose utilization. Among various recombinant strains constructed, the xylose utilization capacity of the recombinant strain 8b-RsXI-xylB was the best, which was further improved through continuous adaption with 38 transfers over 100 days in 50 g/L xylose media. The fermentation performances of the parental strain 8b, the evolved 8b-S38 strain with the best xylose utilization capability, and the intermediate strain 8b-S8 in different media were compared, and the results showed that only 8b-S38 could completely consume xylose at 50 g/L and 100 g/L concentrations. In addition, the xylose consumption rate of 8b-S38 was faster than that of 8b at different xylose concentrations from 50 to 150 g/L, and the ethanol yield increased by 16 ~ 40%, respectively. The results of the mixed-sugar fermentation also demonstrated that 8b-S38 had a higher xylose consumption rate than 8b, and its maximum ethanol productivity was 1.2 ~ 1.4 times higher than that of 8b and 8b-S8. Whole-genome resequencing identified three common genetic changes in 8b-S38 compared with 8b and 8b-S8. RNA-Seq study demonstrated that the expression levels of genes encoding chaperone proteins, ATP-dependent proteases, phage shock proteins, ribosomal proteins, flagellar operons, and transcriptional regulators were significantly increased in xylose media in 8b-S38. The up-regulated expression of these genes may therefore contribute to the efficient xylose utilization of 8b-S38 by maintaining the normal cell metabolism and growth, repairing cellular damages, and rebalancing cellular energy to help cells resist the stressful environment. CONCLUSIONS: This study provides gene candidates to improve xylose utilization, and the result of expressing an extra copy of xylose isomerase and xylulokinase improved xylose utilization also provides a direction for efficient xylose-utilization strain development in other microorganisms. In addition, this study demonstrated the necessity to combine metabolic engineering and ALE for industrial strain development. The recombinant strain 8b-S38 can efficiently metabolize xylose for ethanol fermentation at high xylose concentrations as well as in mixed sugars of glucose and xylose, which could be further developed as the microbial biocatalyst for the production of lignocellulosic biofuels and biochemicals.

The potential association of polybrominated diphenyl ether concentrations in serum to thyroid function in patients with abnormal thyroids: a pilot study.

BACKGROUND: To explore possible associations between polybrominated diphenyl ether (PBDE) exposure and patients with abnormal thyroid hormone levels whose thyroid function parameters are above normal ranges. METHODS: The serum of 40 patients with thyroid hormone abnormalities was collected in Kunming. triiodothyronine (T3), thyroxine (T4), free triiodothyronine (FT3), free thyroxine (FT4), and thyroid-stimulating hormone (TSH) were detected in serum using chemiluminescence. The PBDE homologs in the patients' serum were quantitatively analyzed by gas chromatography-mass spectrometry. If the detection frequency of the compound exceeded 50%, it was included in the analysis. Spearman's rank correlation coefficient and multiple linear regression were used to evaluate the correlation between PBDE homologs and five thyroid function parameters. RESULTS: A total of 33 PBDE homologs were detected, 7 of which had a more than 50% detection rate. BDE-47 was the main homolog detected. Spearman's correlation showed that no relationship was found between PBDE homologs and thyroid hormones. Multiple linear regression showed that BDE-153 was positively correlated with T4, negatively correlated with T3, while BDE-47 was negatively correlated with FT4 (P<0.05). The correlation between other PBDE homologs and thyroid function parameters was weak (P>0.05). The ß coefficient showed that the increase in the logarithmic unit of ∑7PBDEs was related to an increase in FT4 and T4 levels and decreased TSH, T3, and FT3 levels. CONCLUSIONS: There is a significant correlation between exposure to PBDE and thyroid dysfunction. The increase of total PBDEs was significantly correlated with the increase of FT4 and T4 levels and decreased TSH, T3, and FT3 levels.

Investigation of the impact of a broad range of temperatures on the physiological and transcriptional profiles of Zymomonas mobilis ZM4 for high-temperature-tolerant recombinant strain development.

The model ethanologenic bacterium Zymomonas mobilis has many advantages for diverse biochemical production. Although the impact of temperature especially high temperature on the growth and ethanol production of Z. mobilis has been reported, the transcriptional profiles of Z. mobilis grown at different temperatures have not been systematically investigated. In this study, Z. mobilis wild-type strain ZM4 was used to study the effect of a broad range of temperatures of 24, 30, 36, 40, and 45 °C on cell growth and morphology, glucose utilization and ethanol production, as well as the corresponding global gene expression profiles using RNA-Seq-based transcriptomics. In addition, a recombinant Z. mobilis strain expressing reporter gene EGFP (ZM4_EGFP) was constructed to study the effect of temperature on heterologous protein expression at different temperatures. Our result demonstrated that the effect of temperature on the growth and morphology of ZM4 and ZM4_EGFP were similar. The biomass of these two strains decreased along with the temperature increase, and an optimal temperature range is needed for efficient glucose utilization and ethanol production. Temperatures lower or higher than normal temperature investigated in this work was not favorable for the glucose utilization and ethanol production as well as the expression of exogenous protein EGFP based on the results of flow cytometry and Western blot. Temperature also affected the transcriptional profiles of Z. mobilis especially under high temperature. Compared with ZM4 cultured at 30 °C, 478 genes were up-regulated and 481 genes were down-regulated at 45 °C. The number of differentially expressed genes of ZM4 cultured at other temperatures (24, 36 or 40 °C) was relatively small though compared with those at 30 °C. Since temperature usually increases during the fermentation process, and heat tolerance is one of the important robustness traits of industrial strains, candidate genes related to heat resistance based on our RNA-Seq result and literature report were then selected for genetics study using the strategies of plasmid overexpression of candidate gene or replacement of the native promoter of candidate gene by an inducible Ptet promoter. The genetics studies indicated that ZMO0236, ZMO1335, ZMO0994, operon groESL, and cspL, which encodes Mrp family chromosome partitioning ATPase, flavoprotein WrbA, an uncharacterized protein, chaperonin Cpn10 and GroEL, and an exogenous cold shock protein, respectively, were associated with heat tolerance, and recombinant strains over-expressing these genes can improve their heat tolerance. Our work thus not only explored the effects of temperature on the expression of exogenous gene EGFP and endogenous genes, but also selected and confirmed several genes associated with heat tolerance in Z. mobilis, which provided a guidance on identifying candidate genes associated with phenotypic improvement through systems biology strategy and genetics studies for other microorganisms.

[Progress and perspective on development of non-model industrial bacteria as chassis cells for biochemical production in the synthetic biology era].

The development and implement of microbial chassis cells can provide excellent cell factories for diverse industrial applications, which help achieve the goal of environmental protection and sustainable bioeconomy. The synthetic biology strategy of Design-Build-Test-Learn (DBTL) plays a crucial role on rational and/or semi-rational construction or modification of chassis cells to achieve the goals of "Building to Understand" and "Building for Applications". In this review, we briefly comment on the technical development of the DBTL cycle and the research progress of a few model microorganisms. We mainly focuse on non-model bacterial cell factories with potential industrial applications, which possess unique physiological and biochemical characteristics, capabilities of utilizing one-carbon compounds or of producing platform compounds efficiently. We also propose strategies for the efficient and effective construction and application of synthetic microbial cell factories securely in the synthetic biology era, which are to discover and integrate the advantages of model and non-model industrial microorganisms, to develop and deploy intelligent automated equipment for cost-effective high-throughput screening and characterization of chassis cells as well as big-data platforms for storing, retrieving, analyzing, simulating, integrating, and visualizing omics datasets at both molecular and phenotypic levels, so that we can build both high-quality digital cell models and optimized chassis cells to guide the rational design and construction of microbial cell factories for diverse industrial applications.

Development and characterization of acidic-pH-tolerant mutants of Zymomonas mobilis through adaptation and next-generation sequencing-based genome resequencing and RNA-Seq.

BACKGROUND: Acid pretreatment is a common strategy used to break down the hemicellulose component of the lignocellulosic biomass to release pentoses, and a subsequent enzymatic hydrolysis step is usually applied to release hexoses from the cellulose. The hydrolysate after pretreatment and enzymatic hydrolysis containing both hexoses and pentoses can then be used as substrates for biochemical production. However, the acid-pretreated liquor can also be directly used as the substrate for microbial fermentation, which has an acidic pH and contains inhibitory compounds generated during pretreatment. Although the natural ethanologenic bacterium Zymomonas mobilis can grow in a broad range of pH 3.5 ~ 7.5, cell growth and ethanol fermentation are still affected under acidic-pH conditions below pH 4.0. RESULTS: In this study, adaptive laboratory evolution (ALE) strategy was applied to adapt Z. mobilis under acidic-pH conditions. Two mutant strains named 3.6M and 3.5M with enhanced acidic pH tolerance were selected and confirmed, of which 3.5M grew better than ZM4 but worse than 3.6M in acidic-pH conditions that is served as a reference strain between 3.6M and ZM4 to help unravel the acidic-pH tolerance mechanism. Mutant strains 3.5M and 3.6M exhibited 50 ~ 130% enhancement on growth rate, 4 ~ 9 h reduction on fermentation time to consume glucose, and 20 ~ 63% improvement on ethanol productivity than wild-type ZM4 at pH 3.8. Next-generation sequencing (NGS)-based whole-genome resequencing (WGR) and RNA-Seq technologies were applied to unravel the acidic-pH tolerance mechanism of mutant strains. WGR result indicated that compared to wild-type ZM4, 3.5M and 3.6M have seven and five single nucleotide polymorphisms (SNPs), respectively, among which four are shared in common. Additionally, RNA-Seq result showed that the upregulation of genes involved in glycolysis and the downregulation of flagellar and mobility related genes would help generate and redistribute cellular energy to resist acidic pH while keeping normal biological processes in Z. mobilis. Moreover, genes involved in RND efflux pump, ATP-binding cassette (ABC) transporter, proton consumption, and alkaline metabolite production were significantly upregulated in mutants under the acidic-pH condition compared with ZM4, which could help maintain the pH homeostasis in mutant strains for acidic-pH resistance. Furthermore, our results demonstrated that in mutant 3.6M, genes encoding F1F0 ATPase to pump excess protons out of cells were upregulated under pH 3.8 compared to pH 6.2. This difference might help mutant 3.6M manage acidic conditions better than ZM4 and 3.5M. A few gene targets were then selected for genetics study to explore their role in acidic pH tolerance, and our results demonstrated that the expression of two operons in the shuttle plasmids, ZMO0956-ZMO0958 encoding cytochrome bc1 complex and ZMO1428-ZMO1432 encoding RND efflux pump, could help Z. mobilis tolerate acidic-pH conditions. CONCLUSION: An acidic-pH-tolerant mutant 3.6M obtained through this study can be used for commercial bioethanol production under acidic fermentation conditions. In addition, the molecular mechanism of acidic pH tolerance of Z. mobilis was further proposed, which can facilitate future research on rational design of synthetic microorganisms with enhanced tolerance against acidic-pH conditions. Moreover, the strategy developed in this study combining approaches of ALE, genome resequencing, RNA-Seq, and classical genetics study for mutant evolution and characterization can be applied in other industrial microorganisms.

Connecting Microbial Genotype with Phenotype in the Omics Era.

Although rational design-based metabolic engineering has been applied widely to obtain promising microbial biocatalysts, conventional strategies such as adaptive laboratory evolution (ALE) and mutagenesis are still efficient approaches to improve microorganisms for exceptional features such as a broad spectrum of substrate utilization, robustness of cell growth, as well as high titer, yield, and productivity of the target products. In this chapter, we describe the procedure to generate mutant strains with desired phenotypes using ALE and a new mutagenesis approach of Atmosphere and Room Temperature Plasma (ARTP). In addition, we discuss the methodology to combine next-generation sequencing (NGS)-based genome-resequencing and RNA-Seq transcriptomics approaches to characterize the mutant strains and connect the phenotypes with their corresponding genotypic changes.

Molecular Mechanism Associated With the Impact of Methane/Oxygen Gas Supply Ratios on Cell Growth of Methylomicrobium buryatense 5GB1 Through RNA-Seq.

The methane (CH4)/oxygen (O2) gas supply ratios significantly affect the cell growth and metabolic pathways of aerobic obligate methanotrophs. However, few studies have explored the CH4/O2 ratios of the inlet gas, especially for the CH4 concentrations within the explosion range (5∼15% of CH4 in air). This study thoroughly investigated the molecular mechanisms associated with the impact of different CH4/O2 ratios on cell growth of a model type I methanotroph Methylomicrobium buryatense 5GB1 cultured at five different CH4/O2 supply molar ratios from 0.28 to 5.24, corresponding to CH4 content in gas mixture from 5% to 50%, using RNA-Seq transcriptomics approach. In the batch cultivation, the highest growth rate of 0.287 h-1 was achieved when the CH4/O2 supply molar ratio was 0.93 (15% CH4 in air), and it is crucial to keep the availability of carbon and oxygen levels balanced for optimal growth. At this ratio, genes related to methane metabolism, phosphate uptake system, and nitrogen fixation were significantly upregulated. The results indicated that the optimal CH4/O2 ratio prompted cell growth by increasing genes involved in metabolic pathways of carbon, nitrogen and phosphate utilization in M. buryatense 5GB1. Our findings provided an effective gas supply strategy for methanotrophs, which could enhance the production of key intermediates and enzymes to improve the performance of bioconversion processes using CH4 as the only carbon and energy source. This research also helps identify genes associated with the optimal CH4/O2 ratio for balancing energy metabolism and carbon flux, which could be candidate targets for future metabolic engineering practice.

Multiple Small RNAs Interact to Co-regulate Ethanol Tolerance in Zymomonas mobilis.

sRNAs represent a powerful class of regulators that influences multiple mRNA targets in response to environmental changes. However, very few direct sRNA-sRNA interactions have been deeply studied in any organism. Zymomonas mobilis is a bacterium with unique ethanol-producing metabolic pathways in which multiple small RNAs (sRNAs) have recently been identified, some of which show differential expression in ethanol stress. In this study, we show that two sRNAs (Zms4 and Zms6) are upregulated under ethanol stress and have significant impacts on ethanol tolerance and production in Z. mobilis. We conducted multi-omics analysis (combining transcriptomics and sRNA-immunoprecipitation) to map gene networks under the influence of their regulation. We confirmed that Zms4 and Zms6 bind multiple RNA targets and regulate their expressions, influencing many downstream pathways important to ethanol tolerance and production. In particular, Zms4 and Zms6 interact with each other as well as many other sRNAs, forming a novel sRNA-sRNA direct interaction network. This study thus uncovers a sRNA network that co-orchestrates multiple ethanol related pathways through a diverse set of mRNA targets and a large number of sRNAs. To our knowledge, this study represents one of the largest sRNA-sRNA direct interactions uncovered so far.

Identification and characterization of ethanol-inducible promoters of Zymomonas mobilis based on omics data and dual reporter-gene system.

Zymomonas mobilis is a model bacterial ethanologen and has been engineered to produce lignocellulosic biofuels and biochemicals such as 2,3-butanediol. We have previously identified promoters of different strengths using systems biology datasets and characterized them using the flow cytometry-based dual reporter-gene system. Here, we further demonstrated the capability of applying the dual reporter-gene system and omics datasets on discovering inducible promoters. Ten candidate ethanol-inducible promoters were identified through omics datasets mining and clustering. Using the dual reporter-gene system, these promoters were characterized under natural growth, ethanol stress, and ethanol-induced condition to investigate the transcriptional strength and ethanol inducibility. The results demonstrated that three promoters of P0405, P0435, and P0038 driving the expression of native genes of ZMO0405, ZMO0435, and ZMO0038, correspondingly, are potential ethanol-responsive promoters and may be growth related. This study not only identified and verified three ethanol-inducible promoters as biological parts, which can be used to synchronize the expression of heterologous pathway genes with the ethanol production process of Z. mobilis, but also demonstrated the power of combining omics datasets and dual reporter-gene system to identify biological parts for metabolic engineering and synthetic biology applications in Z. mobilis and related microorganisms.

Discovery of potential pathways for biological conversion of poplar wood into lipids by co-fermentation of Rhodococci strains.

BACKGROUND: Biological routes for utilizing both carbohydrates and lignin are important to reach the ultimate goal of bioconversion of full carbon in biomass into biofuels and biochemicals. Recent biotechnology advances have shown promises toward facilitating biological transformation of lignin into lipids. Natural and engineered Rhodococcus strains (e.g., R. opacus PD630, R. jostii RHA1, and R. jostii RHA1 VanA-) have been demonstrated to utilize lignin for lipid production, and co-culture of them can promote lipid production from lignin. RESULTS: In this study, a co-fermentation module of natural and engineered Rhodococcus strains with significant improved lignin degradation and/or lipid biosynthesis capacities was established, which enabled simultaneous conversion of glucose, lignin, and its derivatives into lipids. Although Rhodococci sp. showed preference to glucose over lignin, nearly half of the lignin was quickly depolymerized to monomers by these strains for cell growth and lipid synthesis after glucose was nearly consumed up. Profiles of metabolites produced by Rhodococcus strains growing on different carbon sources (e.g., glucose, alkali lignin, and dilute acid flowthrough-pretreated poplar wood slurry) confirmed lignin conversion during co-fermentation, and indicated novel metabolic capacities and unexplored metabolic pathways in these organisms. Proteome profiles suggested that lignin depolymerization by Rhodococci sp. involved multiple peroxidases with accessory oxidases. Besides the ß-ketoadipate pathway, the phenylacetic acid (PAA) pathway was another potential route for the in vivo ring cleavage activity. In addition, deficiency of reducing power and cellular oxidative stress probably led to lower lipid production using lignin as the sole carbon source than that using glucose. CONCLUSIONS: This work demonstrated a potential strategy for efficient bioconversion of both lignin and glucose into lipids by co-culture of multiple natural and engineered Rhodococcus strains. In addition, the involvement of PAA pathway in lignin degradation can help to further improve lignin utilization, and the combinatory proteomics and bioinformatics strategies used in this study can also be applied into other systems to reveal the metabolic and regulatory pathways for balanced cellular metabolism and to select genetic targets for efficient conversion of both lignin and carbohydrates into biofuels.

Identification and Characterization of Five Cold Stress-Related Rhododendron Dehydrin Genes: Spotlight on a FSK-Type Dehydrin With Multiple F-Segments.

Dehydrins are a family of plant proteins that accumulate in response to dehydration stresses, such as low temperature, drought, high salinity, or during seed maturation. We have previously constructed cDNA libraries from Rhododendron catawbiense leaves of naturally non-acclimated (NA; leaf LT50, temperature that results in 50% injury of maximum, approximately -7°C) and cold-acclimated (CA; leaf LT50 approximately -50°C) plants and analyzed expressed sequence tags (ESTs). Five ESTs were identified as dehydrin genes. Their full-length cDNA sequences were obtained and designated as RcDhn 1-5. To explore their functionality vis-à-vis winter hardiness, their seasonal expression kinetics was studied at two levels. Firstly, in leaves of R. catawbiense collected from the NA, CA, and de-acclimated (DA) plants corresponding to summer, winter and spring, respectively. Secondly, in leaves collected monthly from August through February, which progressively increased freezing tolerance from summer through mid-winter. The expression pattern data indicated that RcDhn 1-5 had 6- to 15-fold up-regulation during the cold acclimation process, followed by substantial down-regulation during deacclimation (even back to NA levels for some). Interestingly, our data shows RcDhn 5 contains a histidine-rich motif near N-terminus, a characteristic of metal-binding dehydrins. Equally important, RcDhn 2 contains a consensus 18 amino acid sequence (i.e., ETKDRGLFDFLGKKEEEE) near the N-terminus, with two additional copies upstream, and it is the most acidic (pI of 4.8) among the five RcDhns found. The core of this consensus 18 amino acid sequence is a 11-residue amino acid sequence (DRGLFDFLGKK), recently designated in the literature as the F-segment (based on the pair of hydrophobic F residues it contains). Furthermore, the 208 orthologs of F-segment-containing RcDhn 2 were identified across a broad range of species in GenBank database. This study expands our knowledge about the types of F-segment from the literature-reported single F-segment dehydrins (FSKn) to two or three F-segment dehydrins: Camelina sativa dehydrin ERD14 as F2S2Kn type; and RcDhn 2 as F3SKn type identified here. Our results also indicate some consensus amino acid sequences flanking the core F-segment in dehydrins. Implications for these cold-responsive RcDhn genes in future genetic engineering efforts to improve plant cold hardiness are discussed.

Prediction and characterization of promoters and ribosomal binding sites of Zymomonas mobilis in system biology era.

BACKGROUND: Zymomonas mobilis is a model bacterial ethanologen with many systems biology studies reported. Besides lignocellulosic ethanol production, Z. mobilis has been developed as a platform for biochemical production through metabolic engineering. However, identification and rigorous understanding of the genetic origins of cellular function, especially those based in non-coding region of DNA, such as promoters and ribosomal binding sites (RBSs), are still in its infancy. This knowledge is crucial for the effective application of Z. mobilis to new industrial applications of biotechnology for fuels and chemicals production. RESULTS: In this study, we explored the possibility to systematically predict the strength of promoters based on systems biology datasets. The promoter strength was clustered based on the expression values of downstream genes (or proteins) from systems biology studies including microarray, RNA-Seq and proteomics. Candidate promoters with different strengths were selected for further characterization, which include 19 strong, nine medium, and ten weak ones. A dual reporter-gene system was developed which included appropriate reporter genes. These are the opmCherry reporter gene driven by the constitutive PlacUV5 promoter for calibration, and EGFP reporter gene driven by candidate promoters for quantification. This dual reporter-gene system was confirmed using the inducible promoter, Ptet, which was used to determine the strength of these predicted promoters with different strengths. In addition, the dual reporter-gene system was applied to determine four synthetic RBSs with different translation initiation rates based on the prediction from bioinformatics server RBS calculator. Our results showed that the correlations between the prediction and experimental results for the promoter and RBS strength are relatively high, with R 2 values more than 0.7 and 0.9, respectively. CONCLUSIONS: This study not only identified and characterized 38 promoters and four RBSs with different strengths for future metabolic engineering in Z. mobilis, but also established a flow cytometry-based dual reporter-gene system to characterize genetic elements including, but not limited to the promoters and RBSs studied in this work. This study also suggested the feasibility of predicting and selecting candidate genetic elements based on omics datasets and bioinformatics tools. Moreover, the dual reporter-gene system developed in this study can be utilized to characterize other genetic elements of Z. mobilis, which can also be applied to other microorganisms.