Dataset Information

Engineering microorganisms based on molecular evolutionary analysis: a succinate production case study.

ABSTRACT: Evolution has resulted in thousands of species possessing similar metabolic enzymes with identical functions that are, however, regulated by different mechanisms. It is thus difficult to select optimal gene to engineer novel or manipulated metabolic pathways. Here, we tested the ability of molecular evolutionary analysis to identify appropriate genes from various species. We calculated the fraction of synonymous substitution and the effective number of codons (ENC) for nine genes stemming from glycolysis. Our research indicated that an enzyme gene with a stronger selective constraint in synonymous sites would mainly regulate corresponding reaction flux through altering the concentration of the protein, whereas those with a more relaxed selective constraint would primarily affect corresponding reaction flux by changing kinetic properties of the enzyme. Further, molecular evolutionary analysis was investigated for three types of genes involved in succinate precursor supply by catalysis of pyruvate. In this model, overexpression of Corynebacterium glutamicum pyc should result in greater conversion of pyruvate. Succinate yields in two Escherichia coli strains that overexpressed each of the three types of genes supported the molecular evolutionary analysis. This approach may thus provide an alternative strategy for selecting genes from different species for metabolic engineering and synthetic biology.

SUBMITTER: Ma X

PROVIDER: S-EPMC4211721 | biostudies-literature | 2014 Sep

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Engineering microorganisms based on molecular evolutionary analysis: a succinate production case study.

Ma Xianghui X Zhang Xinbo X Wang Baiyun B Mao Yufeng Y Wang Zhiwen Z Chen Tao T Zhao Xueming X

Evolutionary applications 20140902 8

Evolution has resulted in thousands of species possessing similar metabolic enzymes with identical functions that are, however, regulated by different mechanisms. It is thus difficult to select optimal gene to engineer novel or manipulated metabolic pathways. Here, we tested the ability of molecular evolutionary analysis to identify appropriate genes from various species. We calculated the fraction of synonymous substitution and the effective number of codons (ENC) for nine genes stemming from g ...[more]

PMID: 25469170

Similar Datasets

Project description:BACKGROUND: The recalcitrant nature of hemicellulosic materials and the high cost in depolymerization are the primary obstacles preventing the use of xylan as feedstock for fuel and chemical production. Consolidated bioprocessing, incorporating enzyme-generating, biomass-degrading and bioproduct-producing capabilities into a single microorganism, could potentially avoid the cost of the dedicated enzyme generation in the process of xylan utilization. In this study, we engineered Escherichia coli strains capable of exporting three hemicellulases to the broth for the succinate production directly from beechwood xylan. RESULTS: Xylanases were extracellular environment-directed by fusing with OsmY. Subsequently, twelve variant OsmY fused endoxylanase-xylosidase combinations were characterized and tested. The combination of XynC-A from Fibrobacter succinogenes S85 and XyloA from Fusarium graminearum which appeared to have optimal enzymatic properties was identified as the best choice for xylan hydrolysis (0.18 ± 0.01 g/l protein in the broth with endoxylanase activity of 12.14 ± 0.34 U/mg protein and xylosidase activity of 92 ± 3 mU/mg protein at 8 h after induction). Further improvements of hemicellulases secretion were investigated by lpp deletion, dsbA overexpression and expression level optimization. With co-expression of ?-arabinofuranosidase, the engineered E. coli could hydrolyze beechwood xylan to pentose monosaccharides. The hemicellulolytic capacity was further integrated with a succinate-producing strain to demonstrate the production of succinate directly from xylan without externally supplied hydrolases and any other organic nutrient. The resulting E. coli Z6373 was able to produce 0.37 g/g succinate from xylan anaerobically equivalent to 76% of that from xylan acid hydrolysates. CONCLUSIONS: This report represents a promising step towards the goal of hemicellulosic chemical production. This engineered E. coli expressing and secreting three hemicellulases demonstrated a considerable succinate production on the released monosaccharides from xylan. The ability to use lower-cost crude feedstock will make biological succinate production more economically attractive.

Project description:Background:Succinate has been recognized as one of the most important bio-based building block chemicals due to its numerous potential applications. However, efficient methods for the production of succinate from lignocellulosic feedstock were rarely reported. Nevertheless, Corynebacterium glutamicum was engineered to efficiently produce succinate from glucose in our previous study. Results:In this work, C. glutamicum was engineered for efficient succinate production from lignocellulosic hydrolysate. First, xylose utilization of C. glutamicum was optimized by heterologous expression of xylA and xylB genes from different sources. Next, xylA and xylB from Xanthomonas campestris were selected among four candidates to accelerate xylose consumption and cell growth. Subsequently, the optimal xylA and xylB were co-expressed in C. glutamicum strain SAZ3 (?ldhA?pta?pqo?catPsod-ppcPsod-pyc) along with genes encoding pyruvate carboxylase, citrate synthase, and a succinate exporter to achieve succinate production from xylose in a two-stage fermentation process. Xylose utilization and succinate production were further improved by overexpressing the endogenous tkt and tal genes and introducing araE from Bacillus subtilis. The final strain C. glutamicum CGS5 showed an excellent ability to produce succinate in two-stage fermentations by co-utilizing a glucose-xylose mixture under anaerobic conditions. A succinate titer of 98.6 g L-1 was produced from corn stalk hydrolysate with a yield of 0.87 g/g total substrates and a productivity of 4.29 g L-1 h-1 during the anaerobic stage. Conclusion:This work introduces an efficient process for the bioconversion of biomass into succinate using a thoroughly engineered strain of C. glutamicum. To the best of our knowledge, this is the highest titer of succinate produced from non-food lignocellulosic feedstock, which highlights that the biosafety level 1 microorganism C. glutamicum is a promising platform for the envisioned lignocellulosic biorefinery.

Project description:Enzymes from extremely thermophilic microorganisms have been of technological interest for some time because of their ability to catalyze reactions of industrial significance at elevated temperatures. Thermophilic enzymes are now routinely produced in recombinant mesophilic hosts for use as discrete biocatalysts. Genome and metagenome sequence data for extreme thermophiles provide useful information for putative biocatalysts for a wide range of biotransformations, albeit involving at most a few enzymatic steps. However, in the past several years, unprecedented progress has been made in establishing molecular genetics tools for extreme thermophiles to the point that the use of these microorganisms as metabolic engineering platforms has become possible. While in its early days, complex metabolic pathways have been altered or engineered into recombinant extreme thermophiles, such that the production of fuels and chemicals at elevated temperatures has become possible. Not only does this expand the thermal range for industrial biotechnology, it also potentially provides biodiverse options for specific biotransformations unique to these microorganisms. The list of extreme thermophiles growing optimally between 70 and 100°C with genetic toolkits currently available includes archaea and bacteria, aerobes and anaerobes, coming from genera such as Caldicellulosiruptor, Sulfolobus, Thermotoga, Thermococcus, and Pyrococcus. These organisms exhibit unusual and potentially useful native metabolic capabilities, including cellulose degradation, metal solubilization, and RuBisCO-free carbon fixation. Those looking to design a thermal bioprocess now have a host of potential candidates to choose from, each with its own advantages and challenges that will influence its appropriateness for specific applications. Here, the issues and opportunities for extremely thermophilic metabolic engineering platforms are considered with an eye toward potential technological advantages for high temperature industrial biotechnology.

Dataset Information

Engineering microorganisms based on molecular evolutionary analysis: a succinate production case study.

Publications

Engineering microorganisms based on molecular evolutionary analysis: a succinate production case study.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets