Identifying Energy Extraction Optimisation Strategies of Actinobacillus succinogenes

Lexow, Waldo Gideon; Mokwatlo, Sekgetho Charles; Brink, Hendrik Gideon; Nicol, Willie

doi:10.3390/catal11081016

Cited by 3 publications

(2 citation statements)

References 21 publications

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“…In the initial phase of fermentation, A. succinogenes obtains a brief energ synthesizing acetic acid to initiate growth [33]. The effect of the overexpression growth was not as large as that of the deletion of ackA.…”

Section: Effect Of Acetic Acid Pathwaymentioning

confidence: 98%

New Insights into the Biosynthesis of Succinic Acid by Actinobacillus succinogenes with the Help of Its Engineered Strains

Chen,

Zheng

2023

Fermentation

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Succinic acid (SA), a C4 tricarboxylic acid cycle intermediate, is used as raw material for bulk chemicals and specialty chemicals, such as tetrahydrofuran and 1,4-butanediol, as well as also being used to synthesize the biodegradable biopolymers PBS (polymer poly (butylene succinate)). Actinobacillus succinogenes, which is facultative anaerobic and gram-negative, is one of the most promising natural SA-producing organisms, but genetic engineering of A. succinogenes is rare so far. In this study, a series of engineered strains was constructed using the pLGZ922 expression vector and a cytosine base editor (CBE) based on CRIPSR/Cas9; we found that phosphoenolpyruvate carboxylase (PEPC) was more important for the CO2 fixation pathway than pyruvate carboxylase (PYC) in A. succinogenes, and the annotated oxaloacetic acid decarboxylase (Asuc_0301 and Asuc_0302) had little correlation with the SA synthesis pathway. The by-product pathway was closely related to cell growth, and overexpression of FDH was beneficial to growth, while the knockout of the ackA gene reduced the growth. For the first time, the hypothetic sugars and SA transporters were mined and identified in A. succinogenes, of which Asuc_0914 was responsible for glucose uptake, and Asuc_0715 and Asuc_0716 constituted SA exporters. This deepens the understanding of SA biosynthesis in A. succinogenes and is also valuable for SA production by fermentation.

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Section: Effect Of Acetic Acid Pathwaymentioning

confidence: 98%

New Insights into the Biosynthesis of Succinic Acid by Actinobacillus succinogenes with the Help of Its Engineered Strains

Chen,

Zheng

2023

Fermentation

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“…During all experimental conditions, a light-yellow biofilm formed in varying amounts inside the recycle line and on the pH and DO probe. Since the absorbance measurements were only based on suspended biomass [23], biofilm attachment would lead to an underestimation in yield and productivity. Much of microbial biofilms comprises of extracellular polymeric substances (EPS), attributing to 50-90% of the biofilm's total organic carbon [24].…”

Section: Diazotrophic Growthmentioning

confidence: 99%

Diazotrophic Behaviour in a Non-Sterile Bioreactor: The Effect of O2-Availability

et al. 2021

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The behaviour of a locally isolated diazotrophic consortium was investigated with the prospect of agricultural applications. A repeatable culture was obtained in a non-sterile bioreactor. Metagenomic analysis indicated Chryseobacterium ssp. and Flavobacterium ssp. were the dominant species, making up approximately 50% of the microbial community. The oxygen supply was varied and mass-transfer limited growth was attained under all experimental conditions. Negligible amounts of aqueous metabolites were formed, indicating a high selectivity towards biomass production. High oxygen availability resulted in decreased growth efficiencies i.e., the specific energy requirements for biomass synthesis. This was attributed to reduced electron transport chain efficiencies and nitrogenase protection mechanisms. Mass and energy balances indicated that sessile biomass with a high C:N served as a carbon sink. The most efficient growth was measured at an aeration feed composition of 21% oxygen and 79% nitrogen. The study presents one of the only known investigations of operational conditions on diazotrophic growth in a non-sterile bioreactor. In addition, it provides a strong foundation for the development of a Biological Nitrogen Fixation process with scaling potential.

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Effects of down-regulation of ackA expression by CRISPR-dCpf1 on succinic acid production in Actinobacillus succinogenes

Chen

Zheng

2023

AMB Expr

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Succinic acid (SA), a key intermediate in the cellular tricarboxylic acid cycle (TCA), is a 4-carbon dicarboxylic acid of great industrial value. Actinobacillus succinogenes can ferment various carbon sources and accumulate relatively high concentrations of SA, but few reliable genetic engineering tools exist for A. succinogenes and this has hindered strain improvement to increase SA production for industrial application. Two different repressors, endonuclease-deactivated Cas9 (dCas9) from Streptococcus pyogenes and Cpf1 (dCpf1) from Francisella tularensis, were applied to construct a CRISPRi system in A. succinogenes. Codon-optimized Cas9 and native Cpf1 were successfully expressed in A. succinogenes, and the corresponding sgRNA and crRNA expression elements, promoted by the fumarate reductase promoter, frd, were introduced into the CRISPRi plasmid. The highest repression of the ackA gene (encoding acetate kinase) and thereby acetic acid production (~ eightfold) was achieved by the dCpf1-based CRISPRi system, in which the mutation site, E1006A acted at the start of the coding region of ackA, the gene which regulates acetic acid biosynthesis. Compared with the ackA gene knockout mutant, cell growth was moderately improved and SA production increased by 6.3%. Further, the SA titer and productivity in a 3 L fermenter reached 57.06 g/L and 1.87 g/L/h, and there was less acetic acid production. A dCpf1-based CRISPRi-mediated gene repression system was successfully established for the first time, providing a simple and effective tool for studying functional genomics in A. succinogenes and optimizing SA production.

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Identifying Energy Extraction Optimisation Strategies of Actinobacillus succinogenes

Cited by 3 publications

References 21 publications

New Insights into the Biosynthesis of Succinic Acid by Actinobacillus succinogenes with the Help of Its Engineered Strains

New Insights into the Biosynthesis of Succinic Acid by Actinobacillus succinogenes with the Help of Its Engineered Strains

Diazotrophic Behaviour in a Non-Sterile Bioreactor: The Effect of O2-Availability

Effects of down-regulation of ackA expression by CRISPR-dCpf1 on succinic acid production in Actinobacillus succinogenes

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