Metabolic impact of heterologous protein production in Pseudomonas putida: Insights into carbon and energy flux control.

For engineered microorganisms, the production of heterologous proteins that are often useless to host cells represents a burden on resources, which have to be shared with normal cellular processes. Within a certain metabolic leeway, this competitive process has no impact on growth. However, once this leeway, or free capacity, is fully utilized, the extra load becomes a metabolic burden that inhibits cellular processes and triggers a broad cellular response, reducing cell growth and often hindering the production of heterologous proteins. In this study, we sought to characterize the metabolic rearrangements occurring in the central metabolism of Pseudomonas putida at different levels of metabolic load. To this end, we constructed a P. putida KT2440 strain that expressed two genes encoding fluorescent proteins, one in the genome under constitutive expression to monitor the free capacity, and the other on an inducible plasmid to probe heterologous protein production. We found that metabolic fluxes are considerably reshuffled, especially at the level of periplasmic pathways, as soon as the metabolic load exceeds the free capacity. Heterologous protein production leads to the decoupling of anabolism and catabolism, resulting in large excess energy production relative to the requirements of protein biosynthesis. Finally, heterologous protein production was found to exert a stronger control on carbon fluxes than on energy fluxes, indicating that the flexible nature of P. putida's central metabolic network is solicited to sustain energy production.

Silk fibroin production in Escherichia coli is limited by a positive feedback loop between metabolic burden and toxicity stress.

To investigate the metabolic elasticity and production bottlenecks for recombinant silk proteins in Escherichia coli, we performed a comprehensive characterization of one elastin-like peptide strain (ELP) and two silk protein strains (A5 4mer, A5 16mer). Our approach included 13C metabolic flux analysis, genome-scale modeling, transcription analysis, and 13C-assisted media optimization experiments. Three engineered strains maintained their central flux network during growth, while measurable metabolic flux redistributions (such as the Entner-Doudoroff pathway) were detected. Under metabolic burdens, the reduced TCA fluxes forced the engineered strain to rely more on substrate-level phosphorylation for ATP production, which increased acetate overflow. Acetate (as low as 10 mM) in the media was highly toxic to silk-producing strains, which reduced 4mer production by 43% and 16mer by 84%, respectively. Due to the high toxicity of large-size silk proteins, 16mer's productivity was limited, particularly in the minimal medium. Therefore, metabolic burden, overflow acetate, and toxicity of silk proteins may form a vicious positive feedback loop that fractures the metabolic network. Three solutions could be applied: 1) addition of building block supplements (i.e., eight key amino acids: His, Ile, Phe, Pro, Tyr, Lys, Met, Glu) to reduce metabolic burden; 2) disengagement of growth and production; and 3) use of non-glucose based substrate to reduce acetate overflow. Other reported strategies were also discussed in light of decoupling this positive feedback loop.

Proteome reallocation enables the selective de novo biosynthesis of non-linear, branched-chain acetate esters.

The one-carbon recursive ketoacid elongation pathway is responsible for making various branched-chain amino acids, aldehydes, alcohols, ketoacids, and acetate esters in living cells. Controlling selective microbial biosynthesis of these target molecules at high efficiency is challenging due to enzyme promiscuity, regulation, and metabolic burden. In this study, we present a systematic modular design approach to control proteome reallocation for selective microbial biosynthesis of branched-chain acetate esters. Through pathway modularization, we partitioned the branched-chain ester pathways into four submodules including ketoisovalerate submodule for converting pyruvate to ketoisovalerate, ketoacid elongation submodule for producing longer carbon-chain ketoacids, ketoacid decarboxylase submodule for converting ketoacids to alcohols, and alcohol acyltransferase submodule for producing branched-chain acetate esters by condensing alcohols and acetyl-CoA. By systematic manipulation of pathway gene replication and transcription, enzyme specificity of the first committed steps of these submodules, and downstream competing pathways, we demonstrated selective microbial production of isoamyl acetate over isobutyl acetate. We found that the optimized isoamyl acetate pathway globally redistributed the amino acid fractions in the proteomes and required up to 23-31% proteome reallocation at the expense of other cellular resources, such as those required to generate precursor metabolites and energy for growth and amino acid biosynthesis. From glucose fed-batch fermentation, the engineered strains produced isoamyl acetate up to a titer of 8.8 g/L (>0.25 g/L toxicity limit), a yield of 0.22 g/g (61% of maximal theoretical value), and 86% selectivity, achieving the highest titers, yields and selectivity of isoamyl acetate reported to date.

"Fight-flight-or-freeze" - how Yarrowia lipolytica responds to stress at molecular level?

Yarrowia lipolytica is a popular yeast species employed in multiple biotechnological production processes. High resistance to extreme environmental conditions or metabolic burden triggered by synthetically forced over-synthesis of a target metabolite has its practical consequences. The proud status of an "industrial workhorse" that Y. lipolytica has gained is directly related to such a quality of this species. With the increasing amount of knowledge coming from detailed functional studies and comprehensive omics analyses, it is now possible to start painting the landscape of the molecular background behind stress response and adaptation in Y. lipolytica. This review summarizes the current state-of-art of a global effort in revealing how Y. lipolytica responds to both environmental threats and the intrinsic burden caused by the overproduction of recombinant secretory proteins at the molecular level. Detailed lists of genes, proteins, molecules, and biological processes deregulated upon exposure to external stress factors or affected by over-synthesis of heterologous proteins are provided. Specificities and universalities of Y. lipolytica cellular response to different extrinsic and intrinsic threats are highlighted. KEY POINTS: • Y. lipolytica as an industrial workhorse is subjected to multiple stress factors. • Cellular responses together with involved genes, proteins, and molecules are reviewed. • Native stress response mechanisms are studied and inspire engineering strategies.

Synthesis of Secretory Proteins in Yarrowia lipolytica: Effect of Combined Stress Factors and Metabolic Load.

While overproduction of recombinant secretory proteins (rs-Prots) triggers multiple changes in the physiology of the producer cell, exposure to suboptimal growth conditions may further increase that biological response. The environmental conditions may modulate the efficiency of both the rs-Prot gene transcription and translation but also the polypeptide folding. Insights into responses elicited by different environmental stresses on the rs-Prots synthesis and host yeast physiology might contribute to a better understanding of fundamental biology processes, thus providing some clues to further optimise bioprocesses. Herein, a series of batch cultivations of Yarrowia lipolytica strains differentially metabolically burdened by the rs-Prots overproduction have been conducted. Combinations of different stress factors, namely pH (3/7) and oxygen availability (kLa 28/110 h-1), have been considered for their impact on cell growth and morphology, substrate consumption, metabolic activity, genes expression, and secretion of the rs-Prots. Amongst others, our data demonstrate that a highly metabolically burdened cell has a higher demand for the carbon source, although presenting a compromised cell growth. Moreover, the observed decrease in rs-Prot production under adverse environmental conditions rather results from the emergence of a less-producing cell subpopulation than from the decrease of the synthetic capacity of the whole cell population.

Metabolic compartmentalization in yeast mitochondria: Burden and solution for squalene overproduction.

Harnessing mitochondria is considered as a promising method for biosynthesis of terpenes due to the adequate supply of acetyl-CoA and redox equivalents in mitochondria. However, mitochondrial engineering often causes serious metabolic burden indicated by poor cell growth. Here, we systematically analyzed the metabolic burden caused by the compartmentalization of the MVA pathway in yeast mitochondria for squalene synthesis. The phosphorylated intermediates of the MVA pathway, especially mevalonate-5-P and mevalonate-5-PP, conferred serious toxicity within mitochondria, which significantly compromised its possible advantages for squalene synthesis and was difficult to be significantly improved by routine pathway optimization. These phosphorylated intermediates were converted into ATP analogues, which strongly inhibited ATP-related cell function, such as mitochondrial oxidative respiration. Fortunately, the introduction of a partial MVA pathway from acetyl-CoA to mevalonate in mitochondria as well as the augmentation of the synthesis of mevalonate in cytosol could significantly promote the growth of yeasts. Accordingly, a combinatorial strategy of cytoplasmic and mitochondrial engineering was proposed to alleviate the metabolic burden caused by the compartmentalized MVA pathway in mitochondria and improve cell growth. The strategy also displayed the superimposed effect of cytoplasmic engineering and mitochondrial engineering on squalene production. Through a two-stage fermentation process, the squalene titer reached 21.1 g/L with a specific squalene titer of 437.1 mg/g dcw, which was the highest at present. This provides new insight into the production of squalene and other terpenes in yeasts based on the advantages of mitochondrial engineering.

Cumulative burden of metabolic syndrome and its components on the risk of atrial fibrillation: a nationwide population-based study.

BACKGROUND: The metabolic syndrome (MetS) and its components are associated with the development of atrial fibrillation (AF). However, the impact of time-burden of MetS on the risk of AF is unknown. We investigated the effect of the cumulative longitudinal burden of MetS on the development of AF. METHODS: We included 2 885 189 individuals without AF who underwent four annual health examinations during 2009-2013 from the database of the Korean national health insurance service. Metabolic burdens were evaluated in the following three ways: (1) cumulative number of MetS diagnosed at each health examination (0-4 times); (2) cumulative number of each MetS component diagnosed at each health examination (0-4 times per MetS component); and (3) cumulative number of total MetS components diagnosed at each health examination (0 to a maximum of 20). The risk of AF according to the metabolic burden was estimated using Cox proportional-hazards models. RESULTS: Of all individuals, 62.4%, 14.8%, 8.7%, 6.5%, and 7.6% met the MetS diagnostic criteria 0, 1, 2, 3, and 4 times, respectively. During a mean follow-up of 5.3 years, the risk of AF showed a positive association with the cumulative number of MetS diagnosed over four health examinations: adjusted hazard ratios (HRs) with 95% confidence intervals (CIs) of 1, 2, 3, and 4 times compared to 0 times were 1.18 (1.13-1.24), 1.31 (1.25-1.39), 1.46 (1.38-1.55), and 1.72 (1.63-1.82), respectively; P for trend < 0.001. All five components of MetS, when diagnosed repeatedly, were independently associated with an increased risk of AF: adjusted HR (95% CI) from 1.22 (1.15-1.29) for impaired fasting glucose to 1.96 (1.87-2.07) for elevated blood pressure. As metabolic components were accumulated from 0 to 20 counts, the risk of AF also gradually increased up to 3.1-fold (adjusted HR 3.11, 95% CI 2.52-3.83 in those with 20 cumulative components of MetS), however, recovery from MetS was linked to a decreased risk of AF. CONCLUSIONS: Given the positive correlations between the cumulative metabolic burdens and the risk of incident AF, maximal effort to detect and correct metabolic derangements even before MetS development might be important to prevent AF and related cardiovascular diseases.

Recombinant protein production-associated metabolic burden reflects anabolic constraints and reveals similarities to a carbon overfeeding response.

A comparison of the metabolic response of Escherichia coli BL21 (DE3) towards the production of human basic fibroblast growth factor (hFGF-2) or towards carbon overfeeding revealed similarities which point to constraints in anabolic pathways. Contrary to expectations, neither energy generation (e.g., ATP) nor provision of precursor molecules for nucleotides (e.g., uracil) and amino acids (e.g., pyruvate, glutamate) limit host cell and plasmid-encoded functions. Growth inhibition is assumed to occur when hampered anabolic capacities do not match with the ongoing and overwhelming carbon catabolism. Excessive carbon uptake leads to by-product secretion, for example, pyruvate, acetate, glutamate, and energy spillage, for example, accumulation and degradation of adenine nucleotides with concomitant accumulation of extracellular hypoxanthine. The cellular response towards compromised anabolic capacities involves downregulation of cAMP formation, presumably responsible for subsequently better-controlled glucose uptake and resultant accumulation of glucose in the culture medium. Growth inhibition is neglectable under conditions of reduced carbon availability when hampered anabolic capacities also match with catabolic carbon processing. The growth inhibitory effect with accompanying energy spillage, respectively, hypoxanthine secretion and cessation of cAMP formation is not unique to the production of hFGF-2 but observed during the production of other proteins and also during overexpression of genes without transcript translation.

Recombinant protein production provoked accumulation of ATP, fructose-1,6-bisphosphate and pyruvate in E. coli K12 strain TG1.

BACKGROUND: Recently it was shown that production of recombinant proteins in E. coli BL21(DE3) using pET based expression vectors leads to metabolic stress comparable to a carbon overfeeding response. Opposite to original expectations generation of energy as well as catabolic provision of precursor metabolites were excluded as limiting factors for growth and protein production. On the contrary, accumulation of ATP and precursor metabolites revealed their ample formation but insufficient withdrawal as a result of protein production mediated constraints in anabolic pathways. Thus, not limitation but excess of energy and precursor metabolites were identified as being connected to the protein production associated metabolic burden. RESULTS: Here we show that the protein production associated accumulation of energy and catabolic precursor metabolites is not unique to E. coli BL21(DE3) but also occurs in E. coli K12. Most notably, it was demonstrated that the IPTG-induced production of hFGF-2 using a tac-promoter based expression vector in the E. coli K12 strain TG1 was leading to persistent accumulation of key regulatory molecules such as ATP, fructose-1,6-bisphosphate and pyruvate. CONCLUSIONS: Excessive energy generation, respectively, accumulation of ATP during recombinant protein production is not unique to the BL21(DE3)/T7 promoter based expression system but also observed in the E. coli K12 strain TG1 using another promoter/vector combination. These findings confirm that energy is not a limiting factor for recombinant protein production. Moreover, the data also show that an accelerated glycolytic pathway flux aggravates the protein production associated "metabolic burden". Under conditions of compromised anabolic capacities cells are not able to reorganize their metabolic enzyme repertoire as required for reduced carbon processing.

The Total Population Size of 'Candidatus Liberibacter asiaticus' Inside the Phloem of Citrus Trees and the Corresponding Metabolic Burden Related to Huanglongbing Disease Development.

'Candidatus Liberibacter asiaticus' (CLas) is the predominant causal agent of citrus huanglongbing, the most devastating citrus disease worldwide. CLas colonizes phloem tissue and causes phloem dysfunction. The pathogen population size in local tissues and in the whole plant is critical for the development of disease symptoms by determining the load of pathogenicity factors and metabolic burden to the host. However, the total population size of CLas in a whole plant and the ratio of CLas to citrus cells in local tissues have not been addressed previously. The total CLas population size for 2.5-year-old 'Valencia' sweet orange on 'Kuharske' citrange rootstock trees was quantified using quantitative PCR to be approximately 1.74 × 109 cells/tree, whereas 7- and 20-year-old sweet orange trees were estimated to be 4.3 × 1010 cells/tree, and 6.0 × 1010 cells/tree, respectively. The majority of CLas cells were distributed in leaf tissues (55.58%), followed by those in branch (36.78%), feeder root (4.75%), trunk (2.39%), and structural root (0.51%) tissues. The ratios of citrus cells to CLas cells for branch, leaf, trunk, feeder root, and structural root samples were within approximately 39 to 79, 44 to 124, 153 to 1,355, 191 to 1,054, and 561 to 3,760, respectively, representing the metabolic burden of CLas in different organs. It was estimated that the ratios of phloem cells to CLas cells for branch, leaf, trunk, feeder root, and structural root samples are approximately 0.39 to 0.79, 0.44 to 1.24, 1.53 to 13.55, 1.91 to 10.54, and 5.61 to 37.60, respectively. Approximately 0.01% of the total citrus phloem volume was estimated to be occupied by CLas, explaining the difficulty to observe CLas in most tissues under transmission electron microscopy. The CLas titer inside the leaf was estimated to be approximately 1.64 × 106 cells/leaf or 9.2 × 104 cells cm-2 in leaves, approximately 104 times less than that of typical apoplastic bacterial pathogens. This study provides quantitative estimates of phloem colonization by bacterial pathogens and furthers the understanding of the biology and virulence mechanisms of CLas.

Comprehensive study on Escherichia coli genomic expression: Does position really matter?

As a biorefinery platform host, Escherichia coli has been used extensively to produce metabolites of commercial interest. Integration of foreign DNA onto the bacterial genome allows for stable expression overcoming the need for plasmid expression and its associated instability. Despite the development of numerous tools and genome editing technologies, the question of where to incorporate a synthetic pathway remains unanswered. To address this issue, we studied the genomic expression in E. coli and linked it not only to 26 rationally selected genomic locations, but also to the gene direction in relation to the DNA replication fork, to the carbon and nitrogen source, to DNA folding and supercoiling, and to metabolic burden. To enable these experiments, we have designed a fluorescent expression cassette to eliminate specific local effects on gene expression. Overall it can be concluded that although the expression range obtained by changing the genomic location of a pathway is small compared to the range typically seen in promoter-RBS libraries, the effect of culture medium, environmental stress and metabolic burden can be substantial. The characterization of multiple effects on genomic expression, and the associated libraries of well-characterized strains, will only stimulate and improve the creation of stable production hosts fit for industrial settings.

Independent component analysis of E. coli's transcriptome reveals the cellular processes that respond to heterologous gene expression.

Achieving the predictable expression of heterologous genes in a production host has proven difficult. Each heterologous gene expressed in the same host seems to elicit a different host response governed by unknown mechanisms. Historically, most studies have approached this challenge by manipulating the properties of the heterologous gene through methods like codon optimization. Here we approach this challenge from the host side. We express a set of 45 heterologous genes in the same Escherichia coli strain, using the same expression system and culture conditions. We collect a comprehensive RNAseq set to characterize the host's transcriptional response. Independent Component Analysis of the RNAseq data set reveals independently modulated gene sets (iModulons) that characterize the host response to heterologous gene expression. We relate 55% of variation of the host response to: Fear vs Greed (16.5%), Metal Homeostasis (19.0%), Respiration (6.0%), Protein folding (4.5%), and Amino acid and nucleotide biosynthesis (9.0%). If these responses can be controlled, then the success rate with predicting heterologous gene expression should increase.

Amino acid supplementation for enhancing recombinant protein production in E. coli.

Recombinant protein production (RPP) in Escherichia coli (E. coli) often induces metabolic burden to the cells that compromise their overall growth and productivity. Amino acid starvation due to RPP is a major contributor of the metabolic burden on the cells and induces global stress response known as a stringent-like response. In this study, the effect of amino acid supplementation in a chemically defined medium on cellular growth and recombinant pramlintide production was investigated. Based on the consumption profile, few amino acids were categorized as growth-promoting (GP1) and protein production promoting (GP2). Feeding strategies of GP1 and GP2 were tested in shake flasks followed by scale up into the bioreactor. A 40% increase in the recombinant pramlintide (rPramlintide) production (protein concentration of 3.09 ± 0.12 g/L and yield of 227.69 ± 19.72 mg pramlintide per gram dry cell weight) was realized. Furthermore, transcriptomics data indicated the downregulation of several genes associated with global stress response and genes involved in amino acid biosynthesis in test culture, supported by proteomics analysis. These results signify that the external supply of critical amino acids decreases cellular stress during RPP and improves process productivity.

Recombinant protein production associated growth inhibition results mainly from transcription and not from translation.

BACKGROUND: Recombinant protein production can be stressful to the host organism. The extent of stress is determined by the specific properties of the recombinant transcript and protein, by the rates of transcription and translation, and by the environmental conditions encountered during the production process. RESULTS: The impact of the transcription of the T7-promoter controlled genes encoding human basic fibroblast growth factor (hFGF-2) and green fluorescent protein (GFP) as well as the translation into the recombinant protein on the growth properties of the production host E. coli BL21(DE3) were investigated. This was done by using expression vectors where the promoter region or the ribosome binding site(s) or both were removed. It is shown that already transcription without protein translation imposes a metabolic burden on the host cell. Translation of the transcript into large amounts of a properly folded protein does not show any effect on cell growth in the best case, e.g. high-level production of GFP in Luria-Bertani medium. However, translation appears to contribute to the metabolic burden if it is connected to protein folding associated problems, e.g. inclusion body formation. CONCLUSION: The so-called metabolic burden of recombinant protein production is mainly attributed to transcription but can be enhanced through translation and those processes following translation (e.g. protein folding and degradation, heat-shock responses).

The future of self-selecting and stable fermentations.

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.

Understanding the metabolic burden of recombinant antibody production in Saccharomyces cerevisiae using a quantitative metabolomics approach.

The cellular changes induced by heterologous protein expression in the yeast Saccharomyces cerevisiae have been analysed on many levels and found to be significant. However, even though high-level protein production poses a metabolic burden, evaluation of the expression host at the level of the metabolome has often been neglected. We present a comparison of metabolite profiles of a wild-type strain with those of three strains producing recombinant antibody variants of increasing size and complexity: an scFv fragment, an scFv-Fc fusion protein and a full-length IgG molecule. Under producing conditions, all three recombinant strains showed a clear decrease in growth rate compared with the wild-type strain and the severity of the growth phenotype increased with size of the protein. The levels of 76 intracellular metabolites were determined using a targeted (semi) quantitative mass spectrometry based approach. Based on unsupervised and supervised multivariate analysis of metabolite profiles, together with pathway activity profiling, the recombinant strains were found to be significantly different from each other and from the wild-type strain. We observed the most prominent changes in metabolite levels for metabolites involved in amino acid and redox metabolism. Induction of the unfolded protein response was detected in all producing strains and is considered to be a contributing factor to the overall metabolic burden on the cells.

The relationship of metabolic burden to productivity levels in CHO cell lines.

The growing demand for recombinant therapeutics has driven biotechnologists to develop new production strategies. One such strategy for increasing the expression of heterologous proteins has focused on enhancing cell-specific productivity through environmental perturbations. In this work, the effects of hypothermia, hyperosmolarity, high shear stress, and sodium butyrate treatment on growth and productivity were studied using three (low, medium, and high producing) CHO cell lines that differed in their specific productivities of monoclonal antibody. In all three cell lines, the inhibitory effect of these parameters on proliferation was demonstrated. Additionally, compared to the control, specific productivity was enhanced under all conditions and exhibited a consistent cell line specific pattern, with maximum increases (50-290%) in the low producer, and minimum increases (7-20%) in the high producer. Thus, the high-producing cell line was less responsive to environmental perturbations than the low-producing cell line. We hypothesize that this difference is most likely due to the bottleneck associated with a higher metabolic burden caused by higher antibody expression. Increased recombinant mRNA levels and pyruvate carboxylase activities due to low temperature and hyperosmotic stress were found to be positively associated with the metabolic burden.

Enhancing fatty acid production in Escherichia coli by Vitreoscilla hemoglobin overexpression.

Our recent 13 C-metabolic flux analysis (13 C-MFA) study indicates that energy metabolism becomes a rate-limiting factor for fatty acid overproduction in E. coli strains (after "Push-Pull-Block" based genetic modifications). To resolve this bottleneck, Vitreoscilla hemoglobin (VHb, a membrane protein facilitating O2 transport) was introduced into a fatty-acid-producing strain to promote oxygen supply and energy metabolism. The resulting strain, FAV50, achieved 70% percent higher fatty acid titer than the parent strain in micro-aerobic shake tube cultures. In high cell-density bioreactor fermentations, FAV50 achieved free fatty acids at a titer of 7.02 g/L (51% of the theoretical yield). In addition to "Push-Pull-Block-Power" strategies, our experiments and flux balance analysis also revealed the fatty acid over-producing strain is sensitive to metabolic burden and oxygen influx, and thus a careful evaluation of the cost-benefit tradeoff with the guidance of fluxome analysis will be fundamental for the rational design of synthetic biology strains. Biotechnol. Bioeng. 2017;114: 463-467. © 2016 Wiley Periodicals, Inc.

Overcoming the metabolic burden of protein secretion in Schizosaccharomyces pombe--a quantitative approach using 13C-based metabolic flux analysis.

Protein secretion in yeast is generally associated with a burden to cellular metabolism. To investigate this metabolic burden in Schizosaccharomyces pombe, we constructed a set of strains secreting the model protein maltase in different amounts. We quantified the influence of protein secretion on the metabolism applying (13)C-based metabolic flux analysis in chemostat cultures. Analysis of the macromolecular biomass composition revealed an increase in cellular lipid content at elevated levels of protein secretion and we observed altered metabolic fluxes in the pentose phosphate pathway, the TCA cycle, and around the pyruvate node including mitochondrial NADPH supply. Supplementing acetate to glucose or glycerol minimal media was found to improve protein secretion, accompanied by an increased cellular lipid content and carbon flux through the TCA cycle as well as increased mitochondrial NADPH production. Thus, systematic metabolic analyses can assist in identifying factors limiting protein secretion and in deriving strategies to overcome these limitations.

Engineering E. coli for the biosynthesis of 3-hydroxy-γ-butyrolactone (3HBL) and 3,4-dihydroxybutyric acid (3,4-DHBA) as value-added chemicals from glucose as a sole carbon source.

3-hydroxy-γ-butyrolactone (3HBL) is a versatile chiral synthon, deemed a top value-added chemical from biomass by the DOE. We recently reported the first biosynthetic pathway towards 3HBL and its hydrolyzed form, 3,4-dihydroxybutyric acid (3,4-DHBA) in recombinant Escherichia coli using glucose and glycolic acid as feedstocks and briefly described their synthesis solely from glucose. Synthesis from glucose requires integration of the endogenous glyoxylate shunt with the 3,4-DHBA/3HBL pathway and co-overexpression of seven genes, posing challenges with respect to expression, repression of the glyoxylate shunt and optimal carbon distribution between the two pathways. Here we discuss engineering this integration. While appropriate media and over-expression of glyoxylate shunt enzymes helped overcome repression, two orthogonal expression systems were employed to address the expression and carbon distribution challenge. Synthesis of up to 0.3g/L of 3HBL and 0.7g/L of 3,4-DHBA solely from glucose was demonstrated, amounting to 24% of the theoretical maximum.