Growth-coupled enzyme engineering manipulation of redox cofactor regeneration

Growth-coupled enzyme engineering through manipulation of redox cofactor regeneration



Growth-coupled enzyme engineering through manipulation of redox cofactor regeneration

An increasingly wide range of chemicals are now being produced from renewable feedstocks through biotechnological means. Bioprocesses rely heavily on enzymatic catalysis for the efficient production of these compounds. Ensuring that enzymes perform optimally in their required environment is therefore of high interest for sustainable production.

Properties such as substrate specificity, catalytic rate and (thermo)stability are among several critical factors that must be optimized for efficient enzyme-driven bioprocesses. This optimization can be time consuming, costly, and challenging, and therefore effective and cost-efficient ways likely for growth-coupled selection, engineering enzymes with desirable characteristics are highly sought after.

The advantages of using growth-coupled selection as a form of enzyme selection, which involves linking the activity of an enzyme to the growth of a cell. This method can be used as a high-throughput selection strategy, and can be achieved by either ensuring growth is dependent on product synthesis by the target enzyme or by linking the enzyme's activity to the global energy state of the cell. Synthetic biology can be used to engineer strains suitable for growth-coupled selection, and recent advances have focused on engineering strains deficient in the oxidized or reduced states of redox cofactor pairs, which can serve as enzyme engineering platforms. The use of these platforms may accelerate the development of improved biocatalysts and bioprocesses.

Benefits of growth coupling via cofactor auxotrophy

The benefits of using cofactor auxotrophy as a selection method for engineering enzymes involved in the biosynthesis of certain chemicals, such as lipids, biofuels, gasses, organic solvents, or polymeric compounds. This approach offers several unique benefits, including the ability to select for the desired product independently of the substrate or product of interest, making it easier to detect improved enzymatic activity, and offering a readout for detecting improved enzymatic activity. Additionally, the use of cofactor auxotrophs as growth coupling platforms is beneficial because the ubiquitous nature of redox cofactors in microbial metabolism means that engineering strategies can be laterally transferable to other microbes of interest, and enzymes can be directly engineered within the environment of the desired microbial host. (shown as figure 1)


Figure 1: Comparison of several widely used screening/selection techniques for enzyme engineering

Mutants deficient in NADH oxidation

The oxidation of NADH in E. coli can occur through two routes, depending on oxygen availability. Under aerobic conditions, NADH is mainly oxidized through respiration to generate ATP, while under anaerobic conditions, it can be oxidized through fermentation pathways to produce lactate and ethanol. Mutant strains of E. coli, unable to use mixed fermentation pathways for NADH oxidation during anaerobic growth, have been used to drive NADH oxidizing pathways for the anaerobic synthesis of various chemicals, such as 2-methylpropan-1-ol, 2,3-butanediol, 1-butanol, and L-alanine. These mutant strains have also been used to engineer enzymes by exploiting similar redox principles, resulting in improved variants. The resulting strains can be used to engineer other NAD(P)H-dependent enzymes and pathways.

Mutants deficient in NAD+ reduction  

Wenk et al. created an E. coli strain by deleting the dihydrolipoyl dehydrogenase (lpd) gene, which resulted in the strain being unable to generate reducing power (NADH and NADPH) from acetate metabolism due to the absence of pyruvate dehydrogenase activity. This caused the strain to display auxotrophy for reducing power when grown aerobically on acetate as a sole carbon source. (shown as figure 2) The strain was able to grow on acetate when supplemented with upper glycolytic substrates or when expressing NAD+-dependent formate-, ethanol- or methanol dehydrogenases with their respective substrates. The strain was not used for enzyme engineering and was only assessed for redox cofactor auxotrophy.
 


Figure 2: Central Metabolism 

Mutants deficient in NADPH oxidation

There are two different strategies for inducing NADP+ auxotrophy in E. coli, relying on engineering the glycolytic pathway to overproduce NADPH. The first strategy involves deleting the native gapA gene and expressing a heterologous NADP+-dependent GAPDH enzyme, while the second strategy involves redirecting the carbon flux through the pentose phosphate pathway. The resulting strains are unable to grow on glucose but display growth in different conditions, with the first strain grown under anaerobic conditions and the second under aerobic conditions with glycerol as the substrate. These strains are used for engineering enzymes with improved properties, including substrate specificity, catalytic activity, and thermostability.

Mutants deficient in NADP+ reduction

Three different bacteria, E. coli, P. putida, and C. glutamicum, have been engineered to be NADPH-auxotrophic, meaning they require exogenous NADPH for growth. In the case of E. coli and C. glutamicum, central metabolic enzymes were knocked out to avoid NADP+ reduction when glucose was provided as a carbon source, while in P. putida, CRISPR/nCas9-assisted engineering was used to disrupt sets of target genes sequentially to understand their involvement in redox metabolism. The NADPH-auxotrophic strains were then used for growth-coupled enzyme engineering based on cofactor specificity. A single round of mutagenesis using E. coli yielded the most efficient and specific NADP+-dependent formate dehydrogenase to date, while P. putida and C. glutamicum represent the first strains of their species that can be used for this type of engineering.

Mutants deficient in NMN+ reduction

A growth-coupled selection system was developed to link cofactor cycling and growth based on a deficiency in NMN+ reduction. The E. coli SHuffle strain was used, which carries deletions in two genes involved in the production of reduced glutathione. An NMNH-dependent glutathione reductase was developed through rational mutagenesis to link the process to NMN+/NMNH cycling, and an NMN+-dependent glucose dehydrogenase was used to support NMN+/NMNH cofactor cycling and therefore growth. The study also applied non-canonical redox cofactor auxotrophy for growth-coupled enzyme engineering for the first time, resulting in a thermostable phosphite dehydrogenase variant with improved catalytic efficiency and temporal stability in vitro. The work provides a useful strain for growth-coupled enzyme engineering, dependent on NMN+/NMNH cycling and auxotrophic for the reduced state of a non-canonical redox cofactor.

 

The potential of growth-coupling via redox cofactors worked as a powerful tool to engineer biocatalysts, particularly in the context of sustainable bioproduction. Although considerable progress has been made in the generation of redox cofactor auxotrophic strains, the statement suggests that there are still several unexplored avenues for research. One of these involves engineering cofactor auxotrophic strains of organisms other than E. coli, which could open up new possibilities for growth-coupled enzyme engineering.

Overall, it suggests that the use of redox cofactor-based growth-coupling represents a major opportunity for engineering biocatalysis, particularly for the production of products that cannot satisfy the requirements of other high-throughput enzyme engineering approaches. It emphasizes the need to develop more versatile and efficient biocatalysts for sustainable bioproduction and underscores the importance of continued research in this area.


Reference: Jochem R. Nielsen a, Ruud A. Weusthuis b, Wei E. Huang a, Growth-coupled enzyme engineering through manipulation of redox cofactor regeneration, Biotechnology Advances, 2023. 

Get In Touch


Recommend Read

  • The 24th SupplySide West


    On October 17,2019-18, the 24th western American plant extracts, health and innovation raw materials exhibition (SupplySide West) in Las Vegas Mandela hotel convention and exhibition center, is hosted by the INFORMA, wit

  • Hi & Fi Asia-China 2022


    The 23rd Healthy Natural Raw materials, food ingredients China Exhibition opened in Shanghai National Convention and Exhibition Center Bangtai Biological with the third generation of total enzyme catalytic technology C p

  • The 20th World Pharmaceutical Raw Materials China Exhibition


    From December 16 to 18,2020, the 20th World Pharmaceutical Raw Materials China Exhibition (CPhI China 2020) was successfully held in Shanghai New International Expo Center. Bangtai Biological Engineering (Shenzhen) Co.,

  • Healthy China 2021 Nutrition and Healthy Food Conference


    From July 8th to July 9th, 2021, "Healthy China 2021 Nutrition and Healthy Food Conference" (HFIC) was grandly held in Hangzhou International Expo Center! Bangtai Biological Engineering (Shenzhen) Co., LTD. (he

  • The Importance of NAD Metabolism in White Adipose Tissue


    1. Introduction Nicotinamide adenine dinucleotide (NAD) compartmentalized in adipocytes can modulate adipocyte differentiation and gene expression, in addition to controlling glucose metabolism. White adipose tiss

Leave Your Message