Optimization strategies for CO2 biological fixation.

Global sustainable development faces a significant challenge in effectively utilizing CO2. Meanwhile, CO2 biological fixation offers a promising solution. CO2 has the highest oxidation state (+4 valence state), whereas typical multi­carbon chemicals have lower valence states. The Gibbs free energy (ΔG) changes of CO2 reductive reactions are generally positive and this renders it necessary to input different forms of energy. Although biological carbon fixation processes are friendly to operate, the thermodynamic obstacles must be overcome. To make this reaction occur favorably and efficiently, diverse strategies to enhance CO2 biological fixation efficiency have been proposed by numerous researchers. This article reviews recent advances in optimizing CO2 biological fixation and intends to provide new insights into achieving efficient biological utilization of CO2. It first outlines the thermodynamic characteristics of diverse carbon fixation reactions and proposes optimization directions for CO2 biological fixation. A comprehensive overview of the catalytic mechanisms, optimization strategies, and challenges encountered by common carbon-fixing enzymes is then provided. Subsequently, potential routes for improving the efficiency of biological carbon fixation are discussed, including the ATP supply, reducing power supply, energy supply, reactor design, and carbon enrichment system modules. In addition, effective artificial carbon fixation pathways were summarized and analyzed. Finally, prospects are made for the research direction of continuously improving the efficiency of biological carbon fixation.

Biosynthetic Pathway and Metabolic Engineering of Succinic Acid.

Succinic acid, a dicarboxylic acid produced as an intermediate of the tricarboxylic acid (TCA) cycle, is one of the most important platform chemicals for the production of various high value-added derivatives. As traditional chemical synthesis processes suffer from nonrenewable resources and environment pollution, succinic acid biosynthesis has drawn increasing attention as a viable, more environmentally friendly alternative. To date, several metabolic engineering approaches have been utilized for constructing and optimizing succinic acid cell factories. In this review, different succinic acid biosynthesis pathways are summarized, with a focus on the key enzymes and metabolic engineering approaches, which mainly include redirecting carbon flux, balancing NADH/NAD+ ratios, and optimizing CO2 supplementation. Finally, future perspectives on the microbial production of succinic acid are discussed.

Characterization and directed evolution of propionyl-CoA carboxylase and its application in succinate biosynthetic pathway with two CO2 fixation reactions.

Propionyl-CoA carboxylase (PCC) is a promising enzyme in the fields of biological CO2 utilization, synthesis of natrual products, and so on. The activity and substrate specificity of PCC are dependent on its key subunit carboxyltransferase (CT). To obtain PCC with high enzyme activity, seven pccB genes encoding CT subunit from diverse microorganisms were expressed in recombinant E. coli, and PccB from Bacillus subtilis showed the highest activity in vitro. To further optimize this protein using directed evolution, a genetic screening system based on oxaloacetate availability was designed to enrich the active variants of PccBBs. Four amino acid substitutions (D46G, L97Q, N220I and I391T) proved of great assistance in PccBBs activity improvement, and a double mutant of PccBBs (N220I/I391T) showed a 94-fold increase of overall catalytic efficiency indicated by kcat/Km. Moreover, this PccBBs double mutant was applied in construction of new succinate biosynthetic pathway. This new pathway produces succinate from acetyl-CoA with fixation of two CO2 molecules, which was confirmed by isotope labeling experiment with NaH13CO3. Compared with previous succinate production based on carboxylation of phosphoenolpyruvate or pyruvate, this new pathway showed some advantages including higher CO2 fixation potentiality and availability under aerobic conditions. In summary, this study developed a PCC with high enzyme activity which can be widely used in biotechnology field, and also demonstrated the feasibility of new succinate biosynthetic pathway with two CO2 fixation reactions.

Biosynthesis of acetylacetone inspired by its biodegradation.

BACKGROUND: Acetylacetone is a commercially bulk chemical with diverse applications. However, the traditional manufacturing methods suffer from many drawbacks such as multiple steps, harsh conditions, low yield, and environmental problems, which hamper further applications of petrochemical-based acetylacetone. Compared to conventional chemical methods, biosynthetic methods possess advantages such as being eco-friendly, and having mild conditions, high selectivity and low potential costs. It is urgent to develop biosynthetic route for acetylacetone to avoid the present problems. RESULTS: The biosynthetic pathway of acetylacetone was constructed by reversing its biodegradation route, and the acetylacetone was successfully produced by engineered Escherichia coli (E. coli) by overexpression of acetylacetone-cleaving enzyme (Dke1) from Acinetobacter johnsonii. Several promising amino acid residues were selected for enzyme improvement based on sequence alignment and structure analysis, and the acetylacetone production was improved by site-directed mutagenesis of Dke1. The double-mutant (K15Q/A60D) strain presented the highest acetylacetone-producing capacity which is 3.6-fold higher than that of the wild-type protein. Finally, the strain accumulated 556.3 ± 15.2 mg/L acetylacetone in fed-batch fermentation under anaerobic conditions. CONCLUSIONS: This study presents the first intuitive biosynthetic pathway for acetylacetone inspired by its biodegradation, and shows the potential for large-scale production.

Coupled biosynthesis and esterification of 1,2,4-butanetriol to simplify its separation from fermentation broth.

1,2,4-Butanetriol (BT) is a valuable chemical with versatile applications in many fields and can be produced through biosynthetic pathways. As a trihydric alcohol, BT possesses good water solubility and is very difficult to separate from fermentation broth, which does complicate the production process and increase the cost. To develop a novel method for BT separation, a biosynthetic pathway for 1,2,4-butanetriol esters with poor water solubility was constructed. Wax ester synthase/acyl-coenzyme A: diacylglycerol acyltransferase (Atf) from Acinetobacter baylyi, Mycobacterium smegmatis, and Escherichia coli were screened, and the acyltransferase from A. baylyi (AtfA) was found to have higher capability. The BT producing strain with AtfA overexpression produced 49.5 mg/L BT oleate in flask cultivation. Through enhancement of acyl-CoA production by overexpression of the acyl-CoA synthetase gene fadD and deleting the acyl coenzyme A dehydrogenase gene fadE, the production was improved to 64.4 mg/L. Under fed-batch fermentation, the resulting strain produced up to 1.1 g/L BT oleate. This is the first time showed that engineered E. coli strains can successfully produce BT esters from xylose and free fatty acids.

Production of D-lactate from glucose using Klebsiella pneumoniae mutants.

BACKGROUND: D-Lactate is a valued chemical which can be produced by some bacteria including Klebsiella pneumoniae. However, only a few studies have focused on K. pneumoniae for D-lactate production with a significant amount of by-products, which complicated the purification process and decreased the yield of D-lactate. RESULTS: Based on the redirection of carbon towards by-product formation, the effects of single-gene and multiple-gene deletions in K. pneumoniae on D-lactate production from glucose via acetolactate synthase (budB), acetate kinase (ackA), and alcohol dehydrogenase (adhE) were tested. Klebsiella pneumoniae mutants had different production behaviours. The accumulation of the main by-products was decreased in the mutants. The triple mutant strain had the most powerful ability to produce optically pure D-lactate from glucose, and was tested with xylose and arabinose as carbon sources. Fed-batch fermentation was also carried out under various aeration rates, and the strain accumulated 125.1 g/L D-lactate with a yield of 0.91 g/g glucose at 2.5 vvm. CONCLUSIONS: Knocking out by-product synthesis genes had a remarkable influence on the production and yield of D-lactate. This study demonstrated, for the first time, that K. pneumoniae has great potential to convert monosaccharides into D-lactate. The results provide new insights for industrial production of D-lactate by K. pneumoniae.