Road to the future of systems biotechnology: CRISPR-Cas-mediated metabolic engineering for recombinant protein production

The rising potential for CRISPR–Cas-mediated genome editing has revolutionized our strategies in basic and practical bioengineering research. It provides a predictable and precise method for genome modification in a robust and reproducible fashion. Emergence of systems biotechnology and synthetic biology approaches coupled with CRISPR–Cas technology could change the future of cell factories to possess some new features which have not been found naturally. We have discussed the possibility and versatile potentials of CRISPR–Cas technology for metabolic engineering of a recombinant host for heterologous protein production. We describe the mechanisms involved in this metabolic engineering approach and present the diverse features of its application in biotechnology and protein production.     

Quantitative physiology of Pichia pastoris during glucose‐limited high‐cell density fed‐batch cultivation for recombinant protein production

Pichia pastoris has become one of the major microorganisms for the production of proteins in recent years. This development was mainly driven by the readily available genetic tools and the ease of high‐cell density cultivations using methanol (or methanol/glycerol mixtures) as inducer and carbon source. To overcome the observed limitations of methanol use such as high heat development, cell lysis, and explosion hazard, we here revisited the possibility to produce proteins with P. pastoris using glucose as sole carbon source. Using a recombinant P. pastoris strain in glucose limited fed‐batch cultivations, very high‐cell densities were reached (more than 200 g_CDW L^?1) resulting in a recombinant protein titer of about 6.5 g L^?1. To investigate the impact of recombinant protein production and high‐cell density fermentation on the metabolism of P. pastoris, we used ¹³C‐tracer‐based metabolic flux analysis in batch and fed‐batch experiments. At a controlled growth rate of 0.12 h^?1 in fed‐batch experiments an increased TCA cycle flux of 1.1 mmol g^?1 h^?1 compared to 0.7 mmol g^?1 h^?1 for the recombinant and reference strains, respectively, suggest a limited but significant flux rerouting of carbon and energy resources. This change in flux is most likely causal to protein synthesis. In summary, the results highlight the potential of glucose as carbon and energy source, enabling high biomass concentrations and protein titers. The insights into the operation of metabolism during recombinant protein production might guide strain design and fermentation development. Biotechnol. Bioeng. 2010;107: 357–368. © 2010 Wiley Periodicals, Inc.     

Modeling with a view to target identification in metabolic engineering: A critical evaluation of the available tools

The state of the art tools for modeling metabolism, typically used in the domain of metabolic engineering, were reviewed. The tools considered are stoichiometric network analysis (elementary modes and extreme pathways), stoichiometric modeling (metabolic flux analysis, flux balance analysis, and carbon modeling), mechanistic and approximative modeling, cybernetic modeling, and multivariate statistics. In the context of metabolic engineering, one should be aware that the usefulness of these tools to optimize microbial metabolism for overproducing a target compound depends predominantly on the characteristic properties of that compound. Because of their shortcomings not all tools are suitable for every kind of optimization; issues like the dependence of the target compound's synthesis on severe (redox) constraints, the characteristics of its formation pathway, and the achievable/desired flux towards the target compound should play a role when choosing the optimization strategy. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

The stimulatory effect of mannitol on levan biosynthesis: Lessons from metabolic systems analysis of Halomonas smyrnensis AAD6T

Halomonas smyrnensis AAD^T is a halophilic, gram‐negative bacterium that can efficiently produce levan from sucrose as carbon source via levansucrase activity. However, systems‐based approaches are required to further enhance its metabolic performance for industrial application. As an important step toward this goal, the genome‐scale metabolic network of Chromohalobacter salexigens DSM3043, which is considered a model organism for halophilic bacteria, has been reconstructed based on its genome annotation, physiological information, and biochemical information. In the present work, the genome‐scale metabolic network of C. salexigens was recruited, and refined via integration of the available biochemical, physiological, and phenotypic features of H. smyrnensis AAD6^T. The generic metabolic model, which comprises 1,393 metabolites and 1,108 reactions, was then systematically analyzed in silico using constraints‐based simulations. To elucidate the relationship between levan biosynthesis and other metabolic processes, an enzyme‐graph representation of the metabolic network and a graph decomposition technique were employed. Using the concept of control effective fluxes, significant links between several metabolic processes and levan biosynthesis were estimated. The major finding was the elucidation of the stimulatory effect of mannitol on levan biosynthesis, which was further verified experimentally via supplementation of mannitol to the fermentation medium. The optimal concentration of 30 g/L mannitol supplemented to the 50 g/L sucrose‐based medium resulted in a twofold increase in levan production in parallel with increased sucrose hydrolysis rate, accumulated extracellular glucose, and decreased fructose uptake rate. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1386–1397, 2013     

Metabolic load of recombinant protein production: inhibition of cellular capacities for glucose uptake and respiration after induction of a heterologous gene in Escherichia coli

The strong expression of recombinant proteins in bacteria affects the primary carbon and energy metabolism resulting in growth inhibition and acetate formation. By applying glucose pulses to fed-batch fermentations performed for production of a heterologous (alpha-glucosidase in Escherichia coli, we show that the induction of the recombinant gene strongly inhibits the maximum specific uptake capacities for glucose and the respiration capacity. The accumulation of glucose in the fermentation medium promotes the growth of plasmid-free cells. These inhibition effects are well described by including the kinetics of product formation into a recently published dynamic model (Lin et al. [2001] Biotechnol Bioeng 73:349-357). The new model also includes the population characteristics and gives a good fit to the measured data describing growth, production, substrate consumption, by-product formation, and respiration.