Metabolic Engineering of Actinobacillus succinogenes Provides Insights into Succinic Acid Biosynthesis.

Actinobacillus succinogenes, a Gram-negative facultative anaerobe, exhibits the native capacity to convert pentose and hexose sugars to succinic acid (SA) with high yield as a tricarboxylic acid (TCA) cycle intermediate. In addition, A. succinogenes is capnophilic, incorporating CO2 into SA, making this organism an ideal candidate host for conversion of lignocellulosic sugars and CO2 to an emerging commodity bioproduct sourced from renewable feedstocks. In this work, we report the development of facile metabolic engineering capabilities in A. succinogenes, enabling examination of SA flux determinants via knockout of the primary competing pathways-namely, acetate and formate production-and overexpression of the key enzymes in the reductive branch of the TCA cycle leading to SA. Batch fermentation experiments with the wild-type and engineered strains using pentose-rich sugar streams demonstrate that the overexpression of the SA biosynthetic machinery (in particular, the enzyme malate dehydrogenase) enhances flux to SA. Additionally, removal of competitive carbon pathways leads to higher-purity SA but also triggers the generation of by-products not previously described from this organism (e.g., lactic acid). The resultant engineered strains also lend insight into energetic and redox balance and elucidate mechanisms governing organic acid biosynthesis in this important natural SA-producing microbe.IMPORTANCE Succinic acid production from lignocellulosic residues is a potential route for enhancing the economic feasibility of modern biorefineries. Here, we employ facile genetic tools to systematically manipulate competing acid production pathways and overexpress the succinic acid-producing machinery in Actinobacillus succinogenes Furthermore, the resulting strains are evaluated via fermentation on relevant pentose-rich sugar streams representative of those from corn stover. Overall, this work demonstrates genetic modifications that can lead to succinic acid production improvements and identifies key flux determinants and new bottlenecks and energetic needs when removing by-product pathways in A. succinogenes metabolism.

Methods for Metabolic Engineering of a Filamentous Trichoderma reesei.

In this work, we describe genetic tools and techniques for engineering Trichoderma reesei for the production of farnesene. Enhanced production of farnesene was used as an example of this methodology; as were the overexpression of a key enzyme, HMGS, in the MVA pathway.

Transcriptomic Profiles of Zymomonas mobilis 8b to Furfural Acute and Long-Term Stress in Both Glucose and Xylose Conditions.

Zymomonas mobilis 8b is an ethanologenic bacterium engineered to utilize both glucose and xylose. The impacts of lignocellulosic hydrolyzate inhibitors on the growth of Zymomonas mobilis 8b have been investigated. However, the molecular responses of these inhibitors have not been completely elucidated yet. In this study, molecular responses to furfural were investigated using transcriptomic approaches of both chip-based microarray and a directional mRNA-Seq. Furfural acute shock time-course experiment with 3 g/L furfural supplemented when cells reached exponential phase and stress response experiment in the presence of 2 g/L furfural from the beginning of fermentation were carried out to study the physiological and transcriptional profiles of short-term and long-term effects of furfural on 8b. Furfural negatively affected 8b growth in terms of final biomass and the fermentation time. Transcriptomic studies indicated that the response of 8b to furfural was dynamic and complex, and differences existed between short-term shock and long-term stress responses. However, the gene function categories were similar with most down-regulated genes related to translation and biosynthesis, while the furfural up-regulated genes were mostly related to general stress responses. Several gene candidates have been identified and genetic studies indicated that expression of ZMO0465 and cysteine synthase operon ZMO0003-0006 driven by its native promoter in a shuttle vector enhanced the furfural tolerance of 8b. In addition, the relationship between microarray and mRNA-Seq was compared with good correlations. The directional mRNA-Seq data not only provided the gene expression profiling, but also can be applied for transcriptional architecture improvement to identify and confirm operons, novel transcripts, hypothetical gene functions, transcriptional start sites, and promoters with different strength.

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.

Identification of Inhibitors in Lignocellulosic Slurries and Determination of Their Effect on Hydrocarbon-Producing Microorganisms.

The aim of this work was to identify inhibitors in pretreated lignocellulosic slurries, evaluate high-throughput screening strategies, and investigate the impact of inhibitors on potential hydrocarbon-producing microorganisms. Compounds present in slurries that could inhibit microbial growth were identified through a detailed analysis of saccharified slurries by applying a combination of approaches of high-performance liquid chromatography, GC-MS, LC-DAD-MS, and ICP-MS. Several high-throughput assays were then evaluated to generate toxicity profiles. Our results demonstrated that Bioscreen C was useful for analyzing bacterial toxicity but not for yeast. AlamarBlue reduction assay can be a useful high-throughput assay for both bacterial and yeast strains as long as medium components do not interfere with fluorescence measurements. In addition, this work identified two major inhibitors (furfural and ammonium acetate) for three potential hydrocarbon-producing bacterial species that include Escherichia coli, Cupriavidus necator, and Rhodococcus opacus PD630, which are also the primary inhibitors for ethanologens. This study was strived to establish a pipeline to quantify inhibitory compounds in biomass slurries and high-throughput approaches to investigate the effect of inhibitors on microbial biocatalysts, which can be applied for various biomass slurries or hydrolyzates generated through different pretreatment and enzymatic hydrolysis processes or different microbial candidates.

Metabolic engineering of Zymomonas mobilis for 2,3-butanediol production from lignocellulosic biomass sugars.

BACKGROUND: To develop pathways for advanced biofuel production, and to understand the impact of host metabolism and environmental conditions on heterologous pathway engineering for economic advanced biofuels production from biomass, we seek to redirect the carbon flow of the model ethanologen Zymomonas mobilis to produce desirable hydrocarbon intermediate 2,3-butanediol (2,3-BDO). 2,3-BDO is a bulk chemical building block, and can be upgraded in high yields to gasoline, diesel, and jet fuel. RESULTS: 2,3-BDO biosynthesis pathways from various bacterial species were examined, which include three genes encoding acetolactate synthase, acetolactate decarboxylase, and butanediol dehydrogenase. Bioinformatics analysis was carried out to pinpoint potential bottlenecks for high 2,3-BDO production. Different combinations of 2,3-BDO biosynthesis metabolic pathways using genes from different bacterial species have been constructed. Our results demonstrated that carbon flux can be deviated from ethanol production into 2,3-BDO biosynthesis, and all three heterologous genes are essential to efficiently redirect pyruvate from ethanol production for high 2,3-BDO production in Z. mobilis. The down-selection of best gene combinations up to now enabled Z. mobilis to reach the 2,3-BDO production of more than 10 g/L from glucose and xylose, as well as mixed C6/C5 sugar streams derived from the deacetylation and mechanical refining process. CONCLUSIONS: This study confirms the value of integrating bioinformatics analysis and systems biology data during metabolic engineering endeavors, provides guidance for value-added chemical production in Z. mobilis, and reveals the interactions between host metabolism, oxygen levels, and a heterologous 2,3-BDO biosynthesis pathway. Taken together, this work provides guidance for future metabolic engineering efforts aimed at boosting 2,3-BDO titer anaerobically.

Cofermentation of glucose, xylose, and arabinose by genomic DNA-integrated xylose/arabinose fermenting strain of Zymomonas mobilis AX101.

Cofermentation of glucose, xylose, and arabinose is critical for complete bioconversion of lignocellulosic biomass, such as agricultural residues and herbaceous energy crops, to ethanol. We have previously developed a plasmid-bearing strain of Zymomonas mobilis (206C[pZB301]) capable of cofermenting glucose, xylose, and arabinose to ethanol. To enhance its genetic stability, several genomic DNA-integrated strains of Z. mobilis have been developed through the insertion of all seven genes necessay for xylose and arabinose fermentation into the Zymomonas genome. From all the integrants developed, four were selected for further evaluation. The integrants were tested for stability by repeated transfer in a nonselective medium (containing only glucose). Based on the stability test, one of the integrants (AX101) was selected for further evaluation. A series of batch and continuous fermentations was designed to evaluate the cofermentation of glucose, xylose, and L-arabinose with the strain AX101. The pH range of study was 4.5, 5.0, and 5.5 at 30 degrees C. The cofermentation process yield was about 84%, which is about the same as that of plasmid-bearing strain 206C(pZB301). Although cofermentation of all three sugars was achieved, there was a preferential order of sugar utilization: glucose first, then xylose, and arabinose last.

Insights into acetate toxicity in Zymomonas mobilis 8b using different substrates.

BACKGROUND: Lignocellulosic biomass is a promising renewable feedstock for biofuel production. Acetate is one of the major inhibitors liberated from hemicelluloses during hydrolysis. An understanding of the toxic effects of acetate on the fermentation microorganism and the efficient utilization of mixed sugars of glucose and xylose in the presence of hydrolysate inhibitors is crucial for economic biofuel production. RESULTS: A new microarray was designed including both coding sequences and intergenic regions to investigate the acetate stress responses of Zymomonas mobilis 8b when using single carbon sources of glucose or xylose, or mixed sugars of both glucose and xylose. With the supplementation of exogenous acetate, 8b can utilize all the glucose with a similar ethanol yield, although the growth, final biomass, and ethanol production rate were reduced. However, xylose utilization was inhibited in both media containing xylose or a mixed sugar of glucose and xylose, although the performance of 8b was better in mixed sugar than xylose-only media. The presence of acetate caused genes related to biosynthesis, the flagellar system, and glycolysis to be downregulated, and genes related to stress responses and energy metabolism to be upregulated. Unexpectedly, xylose seems to pose more stress on 8b, recruiting more genes for xylose utilization, than does acetate. Several gene candidates based on transcriptome results were selected for genetic manipulation, and a TonB-dependent receptor knockout mutant was confirmed to have a slight advantage regarding acetate tolerance. CONCLUSIONS: Our results indicate Z. mobilis utilized a different mechanism for xylose utilization, with an even more severe impact on Z. mobilis than that caused by acetate treatment. Our study also suggests redox imbalance caused by stressful conditions may trigger a metabolic reaction leading to the accumulation of toxic intermediates such as xylitol, but Z. mobilis manages its carbon and energy metabolism through the control of individual reactions to mitigate the stressful conditions. We have thus provided extensive transcriptomic datasets and gained insights into the molecular responses of Z. mobilis to the inhibitor acetate when grown in different sugar sources, which will facilitate future metabolic modeling studies and strain improvement efforts for better xylose utilization and acetate tolerance.

Performance of a newly developed integrant of Zymomonas mobilis for ethanol production on corn stover hydrolysate.

Efficient conversion of lignocellulosic biomass requires biocatalysts able to tolerate inhibitors produced by many pretreatment processes. Recombinant Zymomonas mobilis 8b, a recently developed integrant of Zymomonas mobilis 31821(pZB5), tolerated acetic acid up to 16 g l(-1) and achieved 82%-87% (w/w) ethanol yields from pure glucose/xylose solutions at pH 6 and temperatures of 30 degrees C and 37 degrees C. An ethanol yield of 85% (w/w) was achieved on glucose/xylose from hydrolysate produced by dilute sulfuric acid pretreatment of corn stover after an overliming' process was used to improve hydrolysate fermentability.