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

Abstract

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.

Recent advances in bio-based production of top platform chemical, succinic acid: an alternative to conventional chemistry

“…After five fermentation cycles with a total time of 72 h, 147.6 g/L of glucose was metabolized, leading to a SA concentration of 107 g/L. In the most recent study, Chen and Zheng altered several genes in Actinobacillus strain which were anticipated to play a crucial role in the microbial growth and SA production using pLGZ922 expression vector and a cytosine base editor (CBE) based on CRISPR/Cas9 [ 39 ].Their study revealed that when two of the genes, namely, pyc (pyruvate carboxylase) and pepc (phosphoenolpyruvate carboxylase) from Corynebacterium acetoacidophilum , which were instrumental in CO 2 fixation were individually over-expressed in A. succinogenes , the SA titers increased from 52.35 to 55.66 and 59.47 g/L, respectively. Despite a delayed growth, the SA yields were enhanced from 0.70 g/g to 0.82 and 0.79 g/g, respectively.…”

“…It is the first study wherein data mining of certain sugar and SA transporters was done. When two genes that encoded for two SA exporters, namely, Asuc_0716 and Asuc_0715, were individually knocked out, it had a prominent and deleterious effect on both cell homeostasis and SA biosynthesis [ 39 ]. In the same year Chen et al, developed an efficient, fast and precise gene manipulation toolkit for editing the genes of Actinobacillus by developing series of specific base editors (BE’s) by fusing Cas nuclease and cytidine/adenine deaminase [ 40 ].…”