Abstract:Increased pentose phosphate pathway flux, relative to total substrate uptake flux, is shown to enhance succinic acid (SA) yields under continuous, non-growth conditions of Actinobacillus succinogenes biofilms. Separate fermentations of glucose and xylose were conducted in a custom, continuous biofilm reactor at four different dilution rates. Glucose-6-phosphate dehydrogenase assays were performed on cell extracts derived from in situ removal of biofilm at each steady-state. The results of the assays were coupl… Show more
“…There is also significant opportunity for additional strain engineering; modification of the pentose phosphate pathway, recently shown to be altered under active SA biosynthesis (27), may offer a logical upstream target for flux enhancement. Overexpression of phosphoglucose dehydrogenase in wild-type and pflB mutant backgrounds resulted in limited biosynthetic enhancement (see Fig.…”
, 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, is capnophilic, incorporating CO into SA, making this organism an ideal candidate host for conversion of lignocellulosic sugars and CO to an emerging commodity bioproduct sourced from renewable feedstocks. In this work, we report the development of facile metabolic engineering capabilities in , 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. 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 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 metabolism.
“…There is also significant opportunity for additional strain engineering; modification of the pentose phosphate pathway, recently shown to be altered under active SA biosynthesis (27), may offer a logical upstream target for flux enhancement. Overexpression of phosphoglucose dehydrogenase in wild-type and pflB mutant backgrounds resulted in limited biosynthetic enhancement (see Fig.…”
, 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, is capnophilic, incorporating CO into SA, making this organism an ideal candidate host for conversion of lignocellulosic sugars and CO to an emerging commodity bioproduct sourced from renewable feedstocks. In this work, we report the development of facile metabolic engineering capabilities in , 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. 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 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 metabolism.
“…In contrast, the final stages of the fermentation displayed an over-consumption of NADH (based on measured metabolites), providing a clear indication of an unmeasured NADH producing metabolite. Here, it is thought that the oxidative pentose phosphate pathway (OPPP) is the likely culprit because the pathway results in no additional catabolites other than CO 2 (Bradfield and Nicol, 2016). Because the production of succinic acid entails the incorporation of CO 2 , the reactor was sparged with CO 2 to saturate the environment.…”
Section: Resultsmentioning
confidence: 99%
“…Near homogenous production of succinic acid by A. succinogenes is theoretically possible when considering the redox producing oxidative pentose phosphate pathway (OPPP) [12]. By liberating half a molar equivalent of carbon dioxide, enough redox may by produced to satisfy homogenous succinate production at a theoretical maximum of 1.124 g.g −1 dextrose.…”
A. succinogenes is well known for utilising various catabolic pathways. A multitude of batch fermentation studies confirm flux shifts in the catabolism as time proceeds. It has also been shown that continuous cultures exhibit flux variation as a function of dilution rate. This indicates a direct influence of the external environment on the proteome of the organism. In this work, ATP production efficiency was explored to evaluate the extent of bio-available energy on the production behaviour of A. succinogenes. It was found that the microbe successively utilised its most-to-least efficient energy extraction pathways, providing evidence of an energy optimisation survival strategy. Moreover, data from this study suggest a pyruvate overflow mechanism as a means to throttle acetic and formic acid production, indicating a scenario in which the external concentration of these acids play a role in the energy extraction capabilities of the organism. Data also indicates a fleeting regime where A. succinogenes utilises an oxidised environment to its advantage for ATP production. Here it is postulated that the energy gain and excretion cost of catabolites coupled to the changing environment is a likely mechanism responsible for the proteome alteration and its ensuing carbon flux variation. This offers valuable insights into the microbe’s metabolic logic gates, providing a foundation to understand how to exploit the system.
“…This is most probably because glucose is the most preferable carbon source of the strain and the two pathways harbor the most important steps in the SA production process. Examples of non-glucose-based metabolic modeling studies include xylose [112,113], sugar mixture [113] and glycerol [88,114]. In general, the metabolic modeling study timeline of A. succinogenes could be seen by dividing it into two major separate eras as pre-and post-genomic.…”
Section: Attempts At Metabolic Modeling Of a Succinogenesmentioning
Succinic acid (SA) is one of the top candidate value-added chemicals that can be produced from biomass via microbial fermentation. A considerable number of cell factories have been proposed in the past two decades as native as well as non-native SA producers. Actinobacillus succinogenes is among the best and earliest known natural SA producers. However, its industrial application has not yet been realized due to various underlying challenges. Previous studies revealed that the optimization of environmental conditions alone could not entirely resolve these critical problems. On the other hand, microbial in silico metabolic modeling approaches have lately been the center of attention and have been applied for the efficient production of valuable commodities including SA. Then again, literature survey results indicated the absence of up-to-date reviews assessing this issue, specifically concerning SA production. Hence, this review was designed to discuss accomplishments and future perspectives of in silico studies on the metabolic capabilities of SA producers. Herein, research progress on SA and A. succinogenes, pathways involved in SA production, metabolic models of SA-producing microorganisms, and status, limitations and prospects on in silico studies of A. succinogenes were elaborated. All in all, this review is believed to provide insights to understand the current scenario and to develop efficient mathematical models for designing robust SA-producing microbial strains.
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