Search

Unfavorable cell heterogeneity is a frequent risk during bioprocess scale-up and characterized by rising frequencies of low-producing cells. Low-producing cells emerge by both non-genetic and genetic variation and will enrich due to their higher specific growth rate during the extended number of cell divisions of large-scale bioproduction. Here, we discuss recent strategies for synthetic stabilization of fermentation populations and argue for their application to make cell factory designs that better suit industrial needs. Genotype-directed strategies leverage DNA-sequencing data to inform strain design. Self-selecting phenotype-directed strategies couple high production with cell proliferation, either by redirected metabolic pathways or synthetic product biosensing to enrich for high-performing cell variants. Evaluating production stability early in new cell factory projects will guide heterogeneity-reducing design choices. As good initial metrics, we propose production half-life from standardized serial-passage stability screens and production load, quantified as production-associated percent-wise growth rate reduction. Incorporating more stable genetic designs will greatly increase scalability of future cell factories through sustaining a high-production phenotype and enabling stable long-term production.

In our previous work, we designed and implemented a synthetic metabolic pathway for 1,2,3-trichloropropane (TCP) biodegradation in Escherichia coli. Significant effects of metabolic burden and toxicity exacerbation were observed on single cell and population levels. Deeper understanding of mechanisms underlying these effects is extremely important for metabolic engineering of efficient microbial cell factories for biotechnological processes. In this paper, we present a novel mathematical model of the pathway. The model addresses for the first time the combined effects of toxicity exacerbation and metabolic burden in the context of bacterial population growth. The model is calibrated with respect to the real data obtained with our original synthetically modified E. coli strain. Using the model, we explore the dynamics of the population growth along with the outcome of the TCP biodegradation pathway considering the toxicity exacerbation and metabolic burden. On the methodological side, we introduce a unique computational workflow utilising algorithmic methods of computer science for the particular modelling problem.

Significance Bioproduction of chemicals offers a sustainable alternative to petrochemical synthesis routes by using genetically engineered microorganisms to convert waste and simple substrates into higher-value products. However, efficient high-yield production commonly introduces a metabolic burden that selects for subpopulations of nonproducing cells in large fermentations. To postpone such detrimental evolution, we have synthetically addicted production cells to production by carefully linking signals of product presence to expression of nonconditionally essential genes. We addict Escherichia coli cells to their engineered biosynthesis of mevalonic acid by fine-tuned control of essential genes using a product-responsive transcription factor. Over the course of a long-term fermentation equivalent to industrial 200-m3 bioreactors such addicted cells remained productive, unlike the control, in which evolution fully terminated production., Bio-production of chemicals is an important driver of the societal transition toward sustainability. However, fermentations with heavily engineered production organisms can be challenging to scale to industrial volumes. Such fermentations are subject to evolutionary pressures that select for a wide range of genetic variants that disrupt the biosynthetic capacity of the engineered organism. Synthetic product addiction that couples high-yield production of a desired metabolite to expression of nonconditionally essential genes could offer a solution to this problem by selectively favoring cells with biosynthetic capacity in the population without constraining the medium. We constructed such synthetic product addiction by controlling the expression of two nonconditionally essential genes with a mevalonic acid biosensor. The product-addicted production organism retained high-yield mevalonic acid production through 95 generations of cultivation, corresponding to the number of cell generations required for >200-m3 industrial-scale production, at which time the nonaddicted strain completely abolished production. Using deep DNA sequencing, we find that the product-addicted populations do not accumulate genetic variants that compromise biosynthetic capacity, highlighting how synthetic networks can be designed to control genetic population heterogeneity. Such synthetic redesign of evolutionary forces with endogenous processes may be a promising concept for realizing complex cellular designs required for sustainable bio-manufacturing.

Chromosomal integration offers a selection-free alternative to DNA plasmids for expression of foreign proteins and metabolic pathways. Episomal plasmid DNA is convenient but has drawbacks including increased metabolic burden and the requirement for selection in the form of antibiotics. E.coli has long been used for the expression of foreign proteins and for the production of valuable metabolites by expression of complete metabolic pathways. The gene encoding the fluorescent reporter protein mCherry was integrated into four genomic loci on the E.coli chromosome to measure protein expression at each site. Expression levels ranged from 25% to 500% compared to the gene expressed on a high-copy plasmid. Modular expression of DNA is one of the most commonly used methods for optimizing metabolite production by metabolic engineering. By combining a recently developed method for integration of large synthetic DNA constructs into the genome, we were able to integrate two foreign pathways into the same four genomic loci. We have demonstrated that only one of the genomic loci resulted in the production of violacein, and that all four loci produced trans-cinnamic acid from the TAL pathway.

Background Recombinant protein production using Escherichia coli as expression host is highly efficient, however, it also induces strong host cell metabolic burden. Energy and biomass precursors are withdrawn from the host’s metabolism as they are required for plasmid replication, heterologous gene expression and protein production. Rare codons in a heterologous gene may be a further drawback. This study aims to investigate the influence of particular silent codon exchanges within a heterologous gene on host cell metabolic activity. Silent mutations were introduced into the coding sequence of a model protein to introduce all synonymous arginine or leucine codons at two randomly defined positions, as well as substitutions leading to identical amino acid exchanges with different synonymous codons. The respective E. coli clones were compared during cultivation in a mineral autoinduction medium using specialized online and offline measuring techniques to quantitatively analyze effects on respiration, biomass and protein production, as well as on carbon source consumption, plasmid copy number, intracellular nucleobases and mRNA content of each clone. Results Host stain metabolic burden correlates with recombinant protein production. Upon heterologous gene expression, tremendous differences in respiration, biomass and protein production were observed. According to their different respiration activity the E. coli clones could be classified into two groups, Type A and Type B. Type A clones tended to higher product formation, Type B clones showed stronger biomass formation. Whereas codon usage and intracellular nucleobases had no influence on the Type-A–Type-B-behavior, plasmid copy number, mRNA content and carbon source consumption strongly differed between the two groups. Conclusions Particular silent codon exchanges in a heterologous gene sequence led to differences in initial growth of Type A and Type B clones. Thus, the biomass concentration at the time point of induction varied. In consequence, not only plasmid copy number and expression levels differed between the two groups, but also the kinetics of lactose and glycerol consumption. Even though the underlying molecular mechanisms are not yet identified we observed the astonishing phenomenon that particular silent codon exchanges within a heterologous gene tremendously affect host cell metabolism and recombinant protein production. This could have great impact on codon optimization of heterologous genes, screening procedures for improved variants, and biotechnological protein production processes. Electronic supplementary material The online version of this article (doi:10.1186/s12934-015-0348-8) contains supplementary material, which is available to authorized users.

Background Heterologous expression systems based on promoters inducible with isopropyl-β-D-1-thiogalactopyranoside (IPTG), e.g., Escherichia coli BL21(DE3) and cognate LacIQ/PlacUV5-T7 vectors, are commonly used for production of recombinant proteins and metabolic pathways. The applicability of such cell factories is limited by the complex physiological burden imposed by overexpression of the exogenous genes during a bioprocess. This burden originates from a combination of stresses that may include competition for the expression machinery, side-reactions due to the activity of the recombinant proteins, or the toxicity of their substrates, products and intermediates. However, the physiological impact of IPTG-induced conditional expression on the recombinant host under such harsh conditions is often overlooked. Results The physiological responses to IPTG of the E. coli BL21(DE3) strain and three different recombinants carrying a synthetic metabolic pathway for biodegradation of the toxic anthropogenic pollutant 1,2,3-trichloropropane (TCP) were investigated using plating, flow cytometry, and electron microscopy. Collected data revealed unexpected negative synergistic effect of inducer of the expression system and toxic substrate resulting in pronounced physiological stress. Replacing IPTG with the natural sugar effector lactose greatly reduced such stress, demonstrating that the effect was due to the original inducer’s chemical properties. Conclusions IPTG is not an innocuous inducer; instead, it exacerbates the toxicity of haloalkane substrate and causes appreciable damage to the E. coli BL21(DE3) host, which is already bearing a metabolic burden due to its content of plasmids carrying the genes of the synthetic metabolic pathway. The concentration of IPTG can be effectively tuned to mitigate this negative effect. Importantly, we show that induction with lactose, the natural inducer of Plac, dramatically lightens the burden without reducing the efficiency of the synthetic TCP degradation pathway. This suggests that lactose may be a better inducer than IPTG for the expression of heterologous pathways in E. coli BL21(DE3). Electronic supplementary material The online version of this article (doi:10.1186/s12934-015-0393-3) contains supplementary material, which is available to authorized users.