Engineering cell physiology to enhance recombinant protein production in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The advent of recombinant DNA technology has revolutionized the strategies for protein production. Due to the well-characterized genome and a variety of mature tools available for genetic manipulation, Escherichia coli is still the most common workhorse for recombinant protein production. However, the culture for industrial applications often presents E. coli cells with a growth condition that is significantly different from their natural inhabiting environment in the gastrointestinal tract, resulting in deterioration in cell physiology and limitation in cell’s productivity. It has been recognized that innovative design of genetically engineered strains can highly increase the bioprocess yield with minimum investment on the capital and operating costs. Nevertheless, most of these genetic manipulations, by which traits are implanted into the workhorse through recombinant DNA technology, for enhancing recombinant protein productivity often translate into the challenges that deteriorate cell physiology or even jeopardize cell survival. An in-depth understanding of these challenges and their corresponding cellular response at the molecular level becomes crucial for developing superior strains that are more physiologically adaptive to the production environment to improve culture productivity. With the accumulated knowledge in cell physiology, whose importance to gene overexpression was to some extent undervalued previously, this review is intended to focus on the recent biotechnological advancement in engineering cell physiology to enhance recombinant protein production in E. coli.     

Proteome response of Escherichia coli fed-batch culture to temperature downshift

During fed-batch cultivation of Escherichia coli K-12, the proteomic response to a temperature downshift from 37 to 20°C was quantitatively monitored and analyzed by using two-dimensional electrophoresis. When the temperature of exponentially growing E. coli K-12 culture was downshifted to 20°C, the synthesis level of 57 intracellular proteins showed significant changes for a prolonged period of time, compared to the fed-batch culture controlled at 37°C. Thus, these proteins are regarded as important stress proteins responsive to cold shock, which were analyzed by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and identified using the E. coli SWISS-2DPAGE database. Most of the identified proteins were shown to be involved in energy metabolism, several cellular molecule biosynthetic pathways and catabolism, cell processes, flagellar biosynthesis and motility, and protein translation and folding. The systematic approach to the monitoring of proteomic responses and the detailed analysis results reported in this article would be useful in understanding the metabolic adaptation to lowered culture temperature and designing efficient fermentation strategies for the production of recombinant proteins and metabolites using E. coli strains.     

Transient metabolic modeling of Escherichia coli MG1655 and MG1655 ΔackA-pta, ΔpoxB Δpppc ppc-p37 for recombinant β-galactosidase production

Escherichia coli is one of the most widely used hosts for the production of recombinant proteins, among other reasons because its genetics are far better characterized than those of any other microorganism. To improve the understanding of recombinant protein synthesis in E. coli, the production of a model recombinant protein, β-galactosidase, was studied in response to the constitutive overexpression of the anaplerotic reaction afforded by PEP carboxylase. To this end, an IPTG wash-in experiment was performed starting from a well-defined steady-state condition for both the wild-type E. coli and a mutant with a defective acetate pathway and a constitutively overexpressed ppc. In order to compare the dynamics of the fluxes over time during the wash-in experiment, a method referred to as transient metabolic flux analysis, which is based on steady-state metabolic flux analysis, was used. This allowed us to track the intracellular changes/fluxes in both strains. It was observed that the flux towards fermentation products was 3.6 times lower in the ppc overexpression mutant compared to the wild-type E. coli. In the former on the other hand, the PPC flux is in general higher. In addition, the flux towards β-galactosidase was higher (12.4 times), resulting in five times more protein activity. These results indicate that by constitutively overexpressing the anaplerotic ppc gene in E. coli, the TCA cycle intermediates are increasingly replenished. The additional supply of these protein precursors has a positive result on recombinant protein production.     

Quantification of metabolic limitations during recombinant protein production in Escherichia coli

Escherichia coli is one of the major microorganisms for recombinant protein production because it has been best characterized in terms of molecular genetics and physiology, and because of the availability of various expression vectors and strains. The synthesis of proteins is one of the most energy consuming processes in the cell, with the result that cellular energy supply may become critical. Indeed, the so called metabolic burden of recombinant protein synthesis was reported to cause alterations in the operation of the host's central carbon metabolism.To quantify these alterations in E. coli metabolism in dependence of the rate of recombinant protein production, ¹³C-tracer-based metabolic flux analysis in differently induced cultures was used. To avoid dilution of the ¹³C-tracer signal by the culture history, the recombinant protein produced was used as a flux probe, i.e., as a read out of intracellular flux distributions. In detail, an increase in the generation rate rising from 36 mmol_ATP g_CDW⁻¹ h⁻¹ for the reference strain to 45 mmol_ATP g_CDW⁻¹ h⁻¹ for the highest yielding strain was observed during batch cultivation. Notably, the flux through the TCA cycle was rather constant at 2.5 ± 0.1 mmol g_CDW⁻¹ h⁻¹, hence was independent of the induced strength for gene expression. E. coli compensated for the additional energy demand of recombinant protein synthesis by reducing the biomass formation to almost 60%, resulting in excess NADPH. Speculative, this excess NADPH was converted to NADH via the soluble transhydrogenase and subsequently used for ATP generation in the electron transport chain. In this study, the metabolic burden was quantified by the biomass yield on ATP, which constantly decreased from 11.7 g_CDW mmol_ATP⁻¹ for the reference strain to 4.9 g_CDW mmol_ATP⁻¹ for the highest yielding strain. The insights into the operation of the metabolism of E. coli during recombinant protein production might guide the optimization of microbial hosts and fermentation conditions.     

Cover Image,Volume 116, Number 6, June 2019

Metabolic engineering consistently demands to produce the maximum carbon and energy flux to target chemicals. To balance metabolic flux, gene expression levels of artificially synthesized pathways usually fine-tuned using multimodular optimization strategy. However, forward construction is an engineering conundrum because a vast number of possible pathway combinations need to be constructed and analyzed. Here, an iterative high-throughput balancing (IHTB) strategy was established to thoroughly fine-tune the (2S)-naringenin biosynthetic pathway. A series of gradient constitutive promoters from Escherichia coli were randomly cloned upstream of pathway genes, and the resulting library was screened using an ultraviolet spectrophotometry–fluorescence spectrophotometry high-throughput method, which was established based on the interactions between AlCl₃ and (2S)-naringenin. The metabolic flux of the screened high-titer strains was analyzed and iterative rounds of screening were performed based on the analysis results. After several rounds, the metabolic flux of the (2S)-naringenin synthetic pathway was balanced, reaching a final titer of 191.9 mg/L with 29.2 mg/L p-coumaric acid accumulation. Chalcone synthase was speculated to be the rate-limiting enzyme because its expression level was closely related to the production of both (2S)-naringenin and p-coumaric acid. The established IHTB strategy can be used to efficiently balance multigene pathways, which will accelerate the development of efficient recombinant strains.     

Extracellular recombinant protein production from <Emphasis Type="Italic">Escherichia coli</Emphasis>

Escherichia coli is the most commonly used host for recombinant protein production and metabolic engineering. Extracellular production of enzymes and proteins is advantageous as it could greatly reduce the complexity of a bioprocess and improve product quality. Extracellular production of proteins is necessary for metabolic engineering applications in which substrates are polymers such as lignocelluloses or xenobiotics since adequate uptake of these substrates is often an issue. The dogma that E. coli secretes no protein has been challenged by the recognition of both its natural ability to secrete protein in common laboratory strains and increased ability to secrete proteins in engineered cells. The very existence of this review dedicated to extracellular production is a testimony for outstanding achievements made collectively by the community in this regard. Four strategies have emerged to engineer E. coli cells to secrete recombinant proteins. In some cases, impressive secretion levels, several grams per liter, were reached. This secretion level is on par with other eukaryotic expression systems. Amid the optimism, it is important to recognize that significant challenges remain, especially when considering the success cannot be predicted a priori and involves much trials and errors. This review provides an overview of recent developments in engineering E. coli for extracellular production of recombinant proteins and an analysis of pros and cons of each strategy.     

Adaptive Evolution in Producing Microtiter Cultivations Generates Genetically Stable Escherichia coli Production Hosts for Continuous Bioprocessing

The production of recombinant proteins usually reduces cell fitness and the growth rate of producing cells. The growth disadvantage favors faster-growing non-producer mutants. Therefore, continuous bioprocessing is hardly feasible in Escherichia coli due to the high escape rate. The stability of E. coli expression systems under long-term production conditions and how metabolic load triggered by recombinant gene expression influences the characteristics of mutations are investigated. Iterated fed-batch-like microbioreactor cultivations are conducted under production conditions. The easy-to-produce green fluorescent protein (GFP) and a challenging antigen-binding fragment (Fab) are used as model proteins, and BL21(DE3) and BL21^Q strains as expression hosts. In comparative whole-genome sequencing analyses, mutations that allowed cells to grow unhindered despite recombinant protein production are identified. A T7 RNA polymerase expression system is only conditionally suitable for long-term cultivation under production conditions. Mutations leading to non-producers occur in either the T7 RNA polymerase gene or the T7 promoter. The host RNA polymerase-based BL21^Q expression system remains stable in the production of GFP in long-term cultivations. For the production of Fab, mutations in lacI of the BL21^Q derivatives have positive effects on long-term stability. The results indicate that adaptive evolution carried out with genome-integrated E. coli expression systems in microtiter cultivations under industrial-relevant production conditions is an efficient strain development tool for production hosts.     

Physiological and environmental effects on metabolic flux change caused by heterologous gene expression inEscherichia coli

Expression of theZymomonas mobilis pyruvate decarboxylase (pdc) and alcohol dehydrogenase (adh) genes inEscherichia coli has been known to reduce acetate accumulation by shifting carbon flow from acetate to ethanol. In this study, we investigated the effects of physiological and environmental conditions on the metabolic flux alteration caused by the expression of thepdc andadh genes. In the batch cultures, no significant differences, regardless of medium composition, were found in growth rate and glucose uptake rate between the host strains and the recombinant strains expressing thepdc andadh genes. In the continuous cultures performed with glucose minimal medium, however, the recombinant strains gave more biomass than the host strains at the same specific growth rates. On the contrary, in the continuous cultures with complex medium, the host strains yielded more biomass than the recombinant strains. Analysis of the culture supernatants revealed that the effect of thepdc andadh expression on byproduct formation was more significant at low specific growth rates than at high specific growth rates. This study suggests that physiological and environmental conditions should be carefully considered and precisely defined in assessing the effects of heterologous gene expression on metabolic activities of recombinantE. coli.     

Assessing glycolytic flux alterations resulting from genetic perturbations in E. coli using a biosensor

We describe the development of an optimized glycolytic flux biosensor and its application in detecting altered flux in a production strain and in a mutant library. The glycolytic flux biosensor is based on the Cra-regulated ppsA promoter of E. coli controlling fluorescent protein synthesis. We validated the glycolytic flux dependency of the biosensor in a range of different carbon sources in six different E. coli strains and during mevalonate production. Furthermore, we studied the flux-altering effects of genome-wide single gene knock-outs in E. coli in a multiplex FlowSeq experiment. From a library consisting of 2126 knock-out mutants, we identified 3 mutants with high-flux and 95 mutants with low-flux phenotypes that did not have severe growth defects. This approach can improve our understanding of glycolytic flux regulation improving metabolic models and engineering efforts.     

Industrial production of recombinant therapeutics in <Emphasis Type="Italic">Escherichia coli</Emphasis> and its recent advancements

Nearly 30% of currently approved recombinant therapeutic proteins are produced in Escherichia coli. Due to its well-characterized genetics, rapid growth and high-yield production, E. coli has been a preferred choice and a workhorse for expression of non-glycosylated proteins in the biotech industry. There is a wealth of knowledge and comprehensive tools for E. coli systems, such as expression vectors, production strains, protein folding and fermentation technologies, that are well tailored for industrial applications. Advancement of the systems continues to meet the current industry needs, which are best illustrated by the recent drug approval of E. coli produced antibody fragments and Fc-fusion proteins by the FDA. Even more, recent progress in expression of complex proteins such as full-length aglycosylated antibodies, novel strain engineering, bacterial N-glycosylation and cell-free systems further suggests that complex proteins and humanized glycoproteins may be produced in E. coli in large quantities. This review summarizes the current technology used for commercial production of recombinant therapeutics in E. coli and recent advances that can potentially expand the use of this system toward more sophisticated protein therapeutics.     

Rationally Engineered Synthetic Coculture for Improved Biomass and Product Formation

In microbial ecosystems, bacteria are dependent on dynamic interspecific interactions related to carbon and energy flow. Substrates and end-metabolites are rapidly converted to other compounds, which protects the community from high concentrations of inhibitory molecules. In biotechnological applications, pure cultures are preferred because of the more straight-forward metabolic engineering and bioprocess control. However, the accumulation of unwanted side products can limit the cell growth and process efficiency. In this study, a rationally engineered coculture with a carbon channeling system was constructed using two well-characterized model strains Escherichia coli K12 and Acinetobacter baylyi ADP1. The directed carbon flow resulted in efficient acetate removal, and the coculture showed symbiotic nature in terms of substrate utilization and growth. Recombinant protein production was used as a proof-of-principle example to demonstrate the coculture utility and the effects on product formation. As a result, the biomass and recombinant protein titers of E. coli were enhanced in both minimal and rich medium simple batch cocultures. Finally, harnessing both the strains to the production resulted in enhanced recombinant protein titers. The study demonstrates the potential of rationally engineered cocultures for synthetic biology applications.     

Application of dynamic flux balance analysis to an industrial Escherichia coli fermentation

We have developed a reactor-scale model of Escherichia coli metabolism and growth in a 1000L process for the production of a recombinant therapeutic protein. The model consists of two distinct parts: (1) a dynamic, process specific portion that describes the time evolution of 37 process variables of relevance and (2) a flux balance based, 123-reaction metabolic model of E. coli metabolism. This model combines several previously reported modeling approaches including a growth rate-dependent biomass composition, maximum growth rate objective function, and dynamic flux balancing. In addition, we introduce concentration-dependent boundary conditions of transport fluxes, dynamic maintenance demands, and a state-dependent cellular objective. This formulation was able to describe specific runs with high-fidelity over process conditions including rich media, simultaneous acetate and glucose consumption, glucose minimal media, and phosphate depleted media. Furthermore, the model accurately describes the effect of process perturbations—such as glucose overbatching and insufficient aeration—on growth, metabolism, and titer.     

An antiphage Escherichia coli mutant for higher production of L-threonine obtained by atmospheric and room temperature plasma mutagenesis

Phage infection is common during the production of L-threonine by E. coli, and low L-threonine production and glucose conversion percentage are bottlenecks for the efficient commercial production of L-threonine. In this study, 20 antiphage mutants producing high concentration of L-threonine were obtained by atmospheric and room temperature plasma (ARTP) mutagenesis, and an antiphage E. coli variant was characterized that exhibited the highest production of L-threonine Escherichia coli ([E. coli] TRFC-AP). The elimination of fhuA expression in E. coli TRFC-AP was responsible for phage resistance. The biomass and cell growth of E. coli TRFC-AP showed no significant differences from those of the parent strain (E. coli TRFC), and the production of L-threonine (159.3 g L⁻¹) and glucose conversion percentage (51.4%) were increased by 10.9% and 9.1%, respectively, compared with those of E. coli TRFC. During threonine production (culture time of 20 h), E. coli TRFC-AP exhibited higher activities of key enzymes for glucose utilization (hexokinase, glucose phosphate dehydrogenase, phosphofructokinase, phosphoenolpyruvate carboxylase, and PYK) and threonine synthesis (glutamate synthase, aspartokinase, homoserine dehydrogenase, homoserine kinase and threonine synthase) compared to those of E. coli TRFC. The analysis of metabolic flux distribution indicated that the flux of threonine with E. coli TRFC-AP reached 69.8%, an increase of 16.0% compared with that of E. coli TRFC. Overall, higher L-threonine production and glucose conversion percentage were obtained with E. coli TRFC-AP due to increased activities of key enzymes and improved carbon flux for threonine synthesis.     

Construction and performance of heterologous polyketide‐producing K‐12‐ and B‐derived Escherichia coli

Aims: Escherichia coli has emerged as a viable heterologous host for the production of complex, polyketide natural compounds. In this study, polyketide biosynthesis was compared between different E. coli strains for the purpose of better understanding and improving heterologous production. Methods and Results: Both B and K‐12 E. coli strains were genetically modified to support heterologous polyketide biosynthesis [specifically, 6‐deoxyerythronolide B (6dEB)]. Polyketide production was analysed using a helper plasmid designed to overcome rare codon usage within E. coli. Each strain was analysed for recombinant protein production, precursor consumption, by‐product production, and 6dEB biosynthesis. Of the strains tested for biosynthesis, 6dEB production was greatest for E. coli B strains. When comparing biosynthetic improvements as a function of mRNA stability vs codon bias, increased 6dEB titres were observed when additional rare codon tRNA molecules were provided. Conclusions: Escherichia coli B strains and the use of tRNA supplementation led to improved 6dEB polyketide titres. Significance and Impact of the Study: Given the medicinal potential and growing field of polyketide heterologous biosynthesis, the current study provides insight into host‐specific genetic backgrounds and gene expression parameters aiding polyketide production through E. coli.     

Improvement of <Emphasis Type="Italic">Escherichia coli</Emphasis> production strains by modification of the phosphoenolpyruvate:sugar phosphotransferase system

The application of metabolic engineering in Escherichia coli has resulted in the generation of strains with the capacity to produce metabolites of commercial interest. Biotechnological processes with these engineered strains frequently employ culture media containing glucose as the carbon and energy source. In E. coli, the phosphoenolpyruvate:sugar phosphotransferase system (PTS) transports glucose when this sugar is present at concentrations like those used in production fermentations. This protein system is involved in phosphoenolpyruvate-dependent sugar transport, therefore, its activity has an important impact on carbon flux distribution in the phosphoenolpyruvate and pyruvate nodes. Furthermore, PTS has a very important role in carbon catabolite repression. The properties of PTS impose metabolic and regulatory constraints that can hinder strain productivity. For this reason, PTS has been a target for modification with the purpose of strain improvement. In this review, PTS characteristics most relevant to strain performance and the different strategies of PTS modification for strain improvement are discussed. Functional replacement of PTS by alternative phosphoenolpyruvate-independent uptake and phosphorylation activities has resulted in significant improvements in product yield from glucose and productivity for several classes of metabolites. In addition, inactivation of PTS components has been applied successfully as a strategy to abolish carbon catabolite repression, resulting in E. coli strains that use more efficiently sugar mixtures, such as those obtained from lignocellulosic hydrolysates.     

Production of recombinant protein in Escherichia coli cultured in extract from waste product alga,Ulva lactuca

This study examined the potential for waste product alga, Ulva lactuca, to serve as a media component for recombinant protein production in Escherichia coli. To facilitate this investigation, U. lactuca harvested from Jamaica Bay was dried, and nutrients acid extracted for use as a growth media. The E. coli cell line BL21(DE3) was used to assess the effects on growth and production of recombinant green fluorescent protein (GFP). This study showed that media composed of acid extracts without further nutrient addition maintained E. coli growth and recombinant protein production. Extracts made from dried algae lots less than six‐months‐old were able to produce two‐fold more GFP protein than traditional Lysogeny Broth media. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:784–789, 2014     

The Escherichia coli Proteome: Past, Present, and Future Prospects

Proteomics has emerged as an indispensable methodology for large-scale protein analysis in functional genomics. The Escherichia coli proteome has been extensively studied and is well defined in terms of biochemical, biological, and biotechnological data. Even before the entire E. coli proteome was fully elucidated, the largest available data set had been integrated to decipher regulatory circuits and metabolic pathways, providing valuable insights into global cellular physiology and the development of metabolic and cellular engineering strategies. With the recent advent of advanced proteomic technologies, the E. coli proteome has been used for the validation of new technologies and methodologies such as sample prefractionation, protein enrichment, two-dimensional gel electrophoresis, protein detection, mass spectrometry (MS), combinatorial assays with n-dimensional chromatographies and MS, and image analysis software. These important technologies will not only provide a great amount of additional information on the E. coli proteome but also synergistically contribute to other proteomic studies. Here, we review the past development and current status of E. coli proteome research in terms of its biological, biotechnological, and methodological significance and suggest future prospects.     

In silico and in vivo stability analysis of a heterologous biosynthetic pathway for 1,4-butanediol production in metabolically engineered E. coli

Recently, several approaches have been published in order to develop a functional biosynthesis route for the non-natural compound 1,4-butanediol (BDO) in E. coli using glucose as a sole carbon source or starting from xylose. Among these studies, there was reported as high as 18 g/L product concentration achieved by industrial strains, however BDO production varies greatly in case of the reviewed studies. Our motivation was to build a simple heterologous pathway for this compound in E. coli and to design an appropriate cellular chassis based on a systemic biology approach, using constraint-based flux balance analysis and bi-level optimization for gene knock-out prediction. Thus, the present study reports, at the “proof-of concept” level, our findings related to model-driven development of a metabolically engineered E. coli strain lacking key genes for ethanol, lactate and formate production (ΔpflB, ΔldhA and ΔadhE), with a three-step biosynthetic pathway. We found this strain to produce a limited quantity of 1,4-BDO (.89 mg/L BDO under microaerobic conditions and .82 mg/L under anaerobic conditions). Using glycerol as carbon source, an approach, which to our knowledge has not been tackled before, our results suggest that further metabolic optimization is needed (gene-introductions or knock-outs, promoter fine-tuning) to address the redox potential imbalance problem and to achieve development of an industrially sustainable strain. Our experimental data on culture conditions, growth dynamics and fermentation parameters can consist a base for ongoing research on gene expression profiles and genetic stability of such metabolically engineered E. coli strains.     

Metabolic costs of amino acid and protein production in Escherichia coli

Escherichia coli is the most popular microorganism for the production of recombinant proteins and is gaining increasing importance for the production of low-molecular weight compounds such as amino acids. The metabolic cost associated with the production of amino acids and (recombinant) proteins from glucose, glycerol and acetate was determined using three different computational techniques to identify those amino acids that put the highest burden on the biosynthetic machinery of E. coli. Comparing the costs of individual amino acids, we find that methionine is the most expensive amino acid in terms of consumed mol of ATP per molecule produced, while leucine is the most expensive amino acid when taking into account the cellular abundances of amino acids. Moreover, we show that the biosynthesis of a large number of amino acids from glucose and particularly from glycerol provides a surplus of energy, which can be used to balance the high energetic cost of amino acid polymerization.