Combinatorial optimization of CO₂ transport and fixation to improve succinate production by promoter engineering

Abstract

To balance the flux of an engineered metabolic pathway to achieve high yield of target product is a major challenge in metabolic engineering. In previous work, the collaborative regulation of CO2 transport and fixation was investigated with co-overexpressing exogenous genes regulating both CO2 transport (sbtA and bicA) and PEP carboxylation (phosphoenolpyruvate (PEP) carboxylase (ppc) and carboxykinase (pck)) under trc promoter in Escherichia coli for succinate biosynthesis. For balancing metabolic flux to maximize succinate titer, a combinatorial optimization strategy to fine-tuning CO2 transport and fixation process was implemented by promoter engineering in this study. Firstly, based on the energy matrix a synthetic promoter library containing 20 rationally designed promoters with strengths ranging from 0.8% to 100% compared with the widely used trc promoter was generated. Evaluations of rfp and cat reporter genes provided evidence that the synthetic promoters were stably and had certain applicability. Secondly, four designed promoters with different strengths were used for combinatorial assembly of single CO2 transport gene (sbtA or bicA) and single CO2 fixation gene (ppc or pck) expression. Three combinations, such as Tang1519 (P4 -bicA + pP19 -pck), Tang1522 (P4 -sbtA + P4 -ppc), Tang1523 (P4 -sbtA + P17 -ppc) with a more than 10% increase in succinate production were screened in bioreactor. Finally, based on the above results, co-expression of the four transport and fixation genes were further investigated. Co-expression of sbtA, bicA, and ppc with weak promoter P4 and pck with strong promoter P19 (AFP111/pT-P4 -bicA-P4 -sbtA + pACYC-P19 -pck-P4 -ppc) provided the best succinate production among all the combinations. The highest succinate production of 89.4 g/L was 37.5% higher than that obtained with empty vector control. This work significantly enhanced succinate production through combinatorial optimization of CO2 transport and fixation. The promoter engineering and combinatorial optimization strategies used herein represents a powerful approach to tailor-making metabolic pathways for the production of other industrially important chemicals. Biotechnol. Bioeng. 2016;113: 1531-1541. © 2016 Wiley Periodicals, Inc.

Deletingpckimproves growth and suppresses by-product formation during 1,3-propanediol fermentation byKlebsiella pneumoniae

“…pneumoniae when glycerol is used as the carbon source. Although succinate formation mainly occurs through the PEP carboxylation pathway in other bacteria (Sauer et al 1999;Yu et al 2016), the role of PEP carboxylation in the cell physiology and succinate formation of Kl. pneumoniae remains unclear.…”

Bio‐based succinic acid: an overview of strain development, substrate utilization, and downstream purification

“…Overexpression of the ppc gene in the engineered E. coli strain SD121 ( △pflB , △ldhA and △ptsG ) led to 116.2 g L −1 of succinic acid with a yield as high as 1.13 g g −1 of glucose within 75 h . A combinative modification of introducing ppc, pck, bicA and sbtA genes as well as deletion of genes including pflB, ldhA and ptsG could increase succinic acid production effectively . It was also found that the disruption of ldhA and pflB genes (NZN111) would prevent the regeneration of NAD + from NADH, resulting in the loss of capability to grow anaerobically on glucose.…”

Short-Term Adaptation Modulates Anaerobic Metabolic Flux to Succinate by Activating ExuT, a Novel D-Glucose Transporter in Escherichia coli

“…Therefore, the PEP pool is very important in cellular succinate fermentation. The overexpression of endogenous or heterologous carbon dioxide fixing enzymes such as PEP carboxylase (encoded by the ppc), PEP carboxykinase (pck), pyruvate carboxylase (pyc), and malic enzymes (maeA and maeB) has been used for the overproduction of succinic acid (Millard et al, 1996;Lin et al, 2005b;Tan et al, 2013;Yu et al, 2016). PEP carboxykinase (encoded by the pck) of the rumen bacterium is critical in succinate production and anaerobic growth because it catalyzes the carbon dioxide fixation as well as the ATP formation (Lee et al, 2006).…”