Effect Of Gene Knockout Research Articles

A role of cellular prion protein in programming T‐cell cytokine responses in disease

The cellular prion protein (PrP(C)) is widely expressed in neural and non-neural tissues, but its function is unknown. Elucidation of the part played by PrP(C) in adaptive immunity has been a particular conundrum: increased expression of cell surface PrP(C) has been documented during T-cell activation, yet the functional significance of this activation remains unclear, with conflicting data on the effects of Prnp gene knockout on various parameters of T-cell immunity. We show here that Prnp mRNA is highly inducible within 8-24 h of T-cell activation, with surface protein levels rising from 24 h. When measured in parallel with CD69 and CD25, PrP(C) is a late activation antigen. Consistent with its up-regulation being a late activation event, PrP deletion did not alter T-cell-antigen presenting cell conjugate formation. Most important, activated PrP(0/0) T cells demonstrated much reduced induction of several T helper (Th) 1, Th2, and Th17 cytokines, whereas others, such as TNF-alpha and IL-9, were unaffected. These changes were investigated in the context of an autoimmune model and a bacterial challenge model. In experimental autoimmune encephalomyelitis, PrP-knockout mice showed enhanced disease in the face of reduced IL-17 responses. In a streptococcal sepsis model, this constrained cytokine program was associated with poorer local control of infection, although with reduced bacteremia. The findings indicate that PrP(C) is a potentially important molecule influencing T-cell activation and effector function.

Adenylate kinase isozyme 2 is essential for growth and development of Drosophila melanogaster

Adenylate kinases are phylogenetically widespread, highly conserved, and involved in energy metabolism and energy transfer. Of these, adenylate kinase (AK) isozyme 2 is uniquely localized in the mitochondrial intermembrane space and its physiological role remains largely unknown. In this study, we selected Drosophila melanogaster to analyze its role in vivo. AK isozyme cDNAs were cloned and their gene expressions were characterized in D. melanogaster. The deduced amino acid sequences contain highly conserved motifs for P-loop, NMP binding, and LID domains of AKs. In addition, the effects of AK2 gene knockout on phenotype of AK2 mutants were examined using P-element technology. Although homozygous AK2 mutated embryos developed without any visible defects, their growth ceased and they died before reaching the third instar larval stage. Maternally provided AK2 mRNA was detected in fertilized eggs, and weak AK2 activity was observed in first and second instar larvae of the homozygous AK2 mutants, suggesting that maternally provided AK2 is sufficient for embryonic development. Disappearance of AK2 activity during larval stages resulted in growth arrest and eventual death. These results demonstrate that AK2 plays a critical role in adenine nucleotide metabolism in the mitochondrial intermembrane space and is essential for growth in D. melanogaster.

Asymmetric relationships between proteins shape genome evolution

An investigation of metabolic networks in E. coli and S. cerevisiae reveals that asymmetric protein interactions affect gene expression, the relative effect of gene-knockouts and genome evolution.

Effect of cra gene knockout together with edd and iclR genes knockout on the metabolism in Escherichia coli

To elucidate the physiological adaptation of Escherichia coli due to cra gene knockout, a total of 3,911 gene expressions were investigated by DNA microarray for continuous culture. About 50 genes were differentially regulated for the cra mutant. TCA cycle and glyoxylate shunt were down-regulated, while pentose phosphate (PP) pathway and Entner Doudoroff (ED) pathway were up-regulated in the cra mutant. The glucose uptake rate and the acetate production rate were increased with less acetate consumption for the cra mutant. To identify the genes controlled by Cra protein, the Cra recognition weight matrix from foot-printing data was developed and used to scan the whole genome. Several new Cra-binding sites were found, and some of the result was consistent with the DNA microarray data. The ED pathway was active in the cra mutant; we constructed cra.edd double genes knockout mutant to block this pathway, where the acetate overflowed due to the down-regulation of aceA,B and icd gene expressions. Then we further constructed cra.edd.iclR triple genes knockout mutant to direct the carbon flow through the glyoxylate pathway. The cra.edd.iclR mutant showed the least acetate production, resulting in the highest cell yield together with the activation of the glycolysis pathway, but the glucose consumption rate could not be improved.

Membrane water permeability related to antigen-presenting function of dendritic cells

Aquaporin 5 (AQP5) is one of the water channel proteins which participate in a wide array of physiological processes and are primary determinants of membrane osmotic water permeability. The AQP5 gene is located in human chromosome 12q, the same region as the location of the major asthma susceptibility loci. In this study we try to determine whether the AQP5 knock-out has some effect on allergen-induced asthma. With a mouse asthma model induced by ovalbumin (OVA), we found that deletion of AQP5 reduced some major characteristic features of asthma, such as less inflammation cell infiltration in lung tissues, lower cytokine expression and fewer inflammation cells in bronchoalveolar lavage fluids compared with those from wild-type (WT) mice. Because it was found that mice injected intratracheally with OVA-pulsed dendritic cells (DCs), the AQP5 gene knock-out (AQP5(-/-)) ones presented fewer inflammation cells. Because DCs are major antigen-presenting cells that play an important role in antigen-induced asthma, we also probed into the possible effect of gene knock-out on DCs. Surprisingly, reverse transcription-polymerase chain reaction and fluorescence activated cell sorter analysis showed high levels of AQP5 on the surface of DCs from in vivo or bone marrow monocyte-derived DCs (mDC) in vitro. Immature mDC from AQP5 knock-out mice (AQP5(-/-)) showed decreased expression of CD80 and CD86 and endocytosis ability compared with that from WT, but the difference disappeared after mDCs matured with lipopolysaccharide. AQP5-mediated water transmembrane may play some role in the function of DCs. However, the mechanism of the effect of AQP5 on the DCs' function needs to be investigated further.

Altered Neuron Excitability and Synaptic Plasticity in the Cerebellar Granular Layer of Juvenile Prion Protein Knock-Out Mice with Impaired Motor Control

Although the role of abnormal prion protein (PrP) conformation in generating infectious brain diseases (transmissible spongiform encephalopathy) has been recognized, the function of PrP in the normal brain remains mostly unknown. In this investigation, we considered the effect of PrP gene knock-out (PrP(0/0)) on cerebellar neural circuits and in particular on granule cells, which show intense PrP expression during development and selective affinity for PrP. At the third postnatal week, when PrP expression would normally attain mature levels, PrP(0/0) mice showed low performance in the accelerating rotarod and runway tests and the functioning of 40% of granule cells was abnormal. Spikes were slow, nonovershooting, and nonrepetitive in relation with a reduction in transient inward and outward membrane currents, and also the EPSPs and EPSCs had slow kinetics. Overall, these alterations closely resembled an immature phenotype. Moreover, in slow-spiking PrP(0/0) granule cells, theta-burst stimulation was unable to induce any long-term potentiation. This profound impairment in synaptic excitation and plasticity was associated with a protracted proliferation of granule cells and disappeared at P40-P50 along with the recovery of normal motor behavior (Büeler et al., 1992). These results suggest that PrP plays an important role in granule cell development eventually regulating cerebellar network formation and motor control.

Effect of cra gene knockout together with other genes knockouts on the improvement of substrate consumption rate in Escherichia coli under microaerobic condition

The catabolite repressor/activator protein Cra (FruR) is known to repress the glycolysis pathway genes and activate the gluconeogenic pathway genes. Disruption of cra gene may, therefore, activate glycolysis and thus improve the glucose consumption rate, which may in turn lead to the increase of the specific metabolite production rate. However, it was found in our previous investigation that the substrate consumption rate could not be improved for the aerobic cultivation of cra mutant due to activation of Entner–Doudoroff (ED) pathway, and the repression of the TCA cycle caused by the down-regulation of icdA and aceBAK genes. To overcome this problem, we investigated the effects of edd and arcA genes knockout on the glucose consumption rate. The cra.edd mutant showed the higher glucose uptake rate as compared with the wild type under microaerobic condition. Since arcA gene knockout mutant repressed pfl operon under microaerobic condition, the glucose consumption rate was not improved for cra.edd.arcA mutant. In order to show that the increase in the glucose consumption rate can lead to the increase of the metabolite production rate, we considered the lactate production by pflA mutant. It was shown that the substrate consumption rate could be significantly improved for cra.pflA mutant as compared with pflA mutant, and thus the lactate production rate could be improved by this double mutant if fructose was used as a carbon source.

Effect of adenosine A2A receptors gene knock-out on spatial working memory and reference memory in mice

Objective To investigate the effects and mechanism of adenosine A2A receptors (A2A R)on spatial memory in mice. Methods Mice with adenosine A2A receptors gene knocked out (A2A RKO, n = 13) were compared to their wild type littermates ( WT, n = 15 ). Eight-arm radial maze and Morris water maze were used to measure their spatial reference memory and spatial working memory. Results Compared to the WT littermates,A2A RKO mice displayed significantly improved working memory in both MWM and radial maze performance. However there was no significant difference in spatial reference memory in MWM test between the A2A RKO mice and WT littermate. Conclusion Genetic inactivation of A2A receptors significantly enhances spatial memory in both MWM and radial maze tests, indicating the important role of adenosine A2A receptors in learning spatial memory in mice. This enhancement of spatial memory is particularly evident for spatial working memory by A2A receptor inactivation. Key words: Adenosine A2A receptors; Gene knock out; Spatial memory; Eight-arm radial maze; Water maze

Estimating the size of the solution space of metabolic networks

BackgroundCellular metabolism is one of the most investigated system of biological interactions. While the topological nature of individual reactions and pathways in the network is quite well understood there is still a lack of comprehension regarding the global functional behavior of the system. In the last few years flux-balance analysis (FBA) has been the most successful and widely used technique for studying metabolism at system level. This method strongly relies on the hypothesis that the organism maximizes an objective function. However only under very specific biological conditions (e.g. maximization of biomass for E. coli in reach nutrient medium) the cell seems to obey such optimization law. A more refined analysis not assuming extremization remains an elusive task for large metabolic systems due to algorithmic limitations.ResultsIn this work we propose a novel algorithmic strategy that provides an efficient characterization of the whole set of stable fluxes compatible with the metabolic constraints. Using a technique derived from the fields of statistical physics and information theory we designed a message-passing algorithm to estimate the size of the affine space containing all possible steady-state flux distributions of metabolic networks. The algorithm, based on the well known Bethe approximation, can be used to approximately compute the volume of a non full-dimensional convex polytope in high dimensions. We first compare the accuracy of the predictions with an exact algorithm on small random metabolic networks. We also verify that the predictions of the algorithm match closely those of Monte Carlo based methods in the case of the Red Blood Cell metabolic network. Then we test the effect of gene knock-outs on the size of the solution space in the case of E. coli central metabolism. Finally we analyze the statistical properties of the average fluxes of the reactions in the E. coli metabolic network.ConclusionWe propose a novel efficient distributed algorithmic strategy to estimate the size and shape of the affine space of a non full-dimensional convex polytope in high dimensions. The method is shown to obtain, quantitatively and qualitatively compatible results with the ones of standard algorithms (where this comparison is possible) being still efficient on the analysis of large biological systems, where exact deterministic methods experience an explosion in algorithmic time. The algorithm we propose can be considered as an alternative to Monte Carlo sampling methods.

Reduced body weight is a common effect of gene knockout in mice

BackgroundDuring a search for obesity candidate genes in a small region of the mouse genome, we noticed that many genes when knocked out influence body weight. To determine whether this was a general feature of gene knockout or a chance occurrence, we surveyed the Jackson Laboratory Mouse Genome Database for knockout mouse strains and their phenotypes. Body weights were not available for all strains so we also obtained body weight information by contacting a random sample of investigators responsible for a knockout strain.ResultsWe classified each knockout mouse strain as (1) lighter and smaller, (2) larger and heavier, or (3) the same weight, relative to control mice. We excluded knockout strains that died early in life, even though this type of lethality is often associated with a small embryo or reduced body size. Based on a dataset of 1,977 knockout strains, we found that that 31% of viable knockout mouse strains weighed less and an additional 3% weighed more than did controls.ConclusionBody weight is potentially a latent variable in about a third of experiments that use knockout mice and should be considered in interpreting experimental outcomes, e.g., in studies of hypertension, drug and hormone metabolism, organ development, cell proliferation and apoptosis, digestion, heart rate, or atherosclerosis. If we assume that the knockout genes we surveyed are representative then upward of 6,000 genes are predicted to influence the size of a mouse. Body weight is highly heritable, and numerous quantitative trait loci have been mapped in mice, but "multigenic" is an insufficient term for the thousands of loci that could contribute to this complex trait.

Models from experiments: combinatorial drug perturbations of cancer cells

We present a novel method for deriving network models from molecular profiles of perturbed cellular systems. The network models aim to predict quantitative outcomes of combinatorial perturbations, such as drug pair treatments or multiple genetic alterations. Mathematically, we represent the system by a set of nodes, representing molecular concentrations or cellular processes, a perturbation vector and an interaction matrix. After perturbation, the system evolves in time according to differential equations with built-in nonlinearity, similar to Hopfield networks, capable of representing epistasis and saturation effects. For a particular set of experiments, we derive the interaction matrix by minimizing a composite error function, aiming at accuracy of prediction and simplicity of network structure. To evaluate the predictive potential of the method, we performed 21 drug pair treatment experiments in a human breast cancer cell line (MCF7) with observation of phospho-proteins and cell cycle markers. The best derived network model rediscovered known interactions and contained interesting predictions. Possible applications include the discovery of regulatory interactions, the design of targeted combination therapies and the engineering of molecular biological networks.

Comparative Genomic Hybridization Detects Secondary Chromosomal Deletions in Escherichia coli K-12 MG1655 Mutants and Highlights Instability in the flhDC Region

The use of whole-genome microarrays for monitoring mutagenized or otherwise engineered genetic derivatives is a potentially powerful tool for checking genomic integrity. Using comparative genomic hybridization of a number of unrelated, directed deletion mutants in Escherichia coli K-12 MG1655, we identified unintended secondary genomic deletions in the flhDC region in delta fnr, delta crp, and delta creB mutants. These deletions were confirmed by PCR and phenotypic tests. Our findings show that nonmotile progeny are found in some MG1655 directed deletion mutants, and studies on the effects of gene knockouts should be viewed with caution when the mutants have not been screened for the presence of secondary deletions or confirmed by other methods.

SPINE: a framework for signaling-regulatory pathway inference from cause-effect experiments

The complex program of gene expression allows the cell to cope with changing genetic, developmental and environmental conditions. The accumulating large-scale measurements of gene knockout effects and molecular interactions allow us to begin to uncover regulatory and signaling pathways within the cell that connect causal to affected genes on a network of physical interactions. We present a novel framework, SPINE, for Signaling-regulatory Pathway INferencE. The framework aims at explaining gene expression experiments in which a gene is knocked out and as a result multiple genes change their expression levels. To this end, an integrated network of protein-protein and protein-DNA interactions is constructed, and signaling pathways connecting the causal gene to the affected genes are searched for in this network. The reconstruction problem is translated into that of assigning an activation/repression attribute with each protein so as to explain (in expectation) a maximum number of the knockout effects observed. We provide an integer programming formulation for the latter problem and solve it using a commercial solver. We validate the method by applying it to a yeast subnetwork that is involved in mating. In cross-validation tests, SPINE obtains very high accuracy in predicting knockout effects (99%). Next, we apply SPINE to the entire yeast network to predict protein effects and reconstruct signaling and regulatory pathways. Overall, we are able to infer 861 paths with confidence and assign effects to 183 genes. The predicted effects are found to be in high agreement with current biological knowledge. The algorithm and data are available at http://cs.tau.ac.il/~roded/SPINE.html.

Multiple interactions among the competence proteins of Bacillus subtilis

Proteins required for transformation of Bacillus subtilis and other competent bacteria are associated with the membrane or reside in the cytosol. Previous work has shown that RecA, ComGA, ComFA and SsbB are directed to the cell poles in competent cells, and that the uptake of transforming DNA occurs preferentially at the poles. We show that ComGA, ComFA, DprA (Smf), SsbB (YwpH), RecA and YjbF (CoiA) are located at the cell poles, where they appear to colocalize. Using fluorescence resonance energy transfer, we have shown that these six competent (Com) proteins reside in close proximity to one another. This conclusion was supported by the effects of com gene knockouts on the stabilities of Com proteins. Data obtained from the com gene knockout studies, as well as information from other sources, extend the list of proteins in the transformation complex to include ComEC and ComEA. Because ComGA and ComFA are membrane-associated, while DprA, SsbB, RecA and YjbF are soluble, a picture emerges of a large multiprotein polar complex, involving both cytosolic and membrane proteins. This complex mediates the binding and uptake of single-stranded DNA, the protection of this DNA from cellular nucleases and its recombination with the recipient chromosome.

Chronic hypoxia (CH)‐induced inflammation and the effect of interleukin‐6 (IL‐6) gene deletion on chemoreceptor adaptation in mouse carotid body (CB)

Exposure to CH elicits a marked increase in chemosensory excitability in the mammalian CB. In chronic inflammatory pain unique hyperexcitable phenotypes emerge in primary afferent neurons as a consequence of invasive activated immune cells and their secretory cytokine products. The present study examines the effect of CH on the expression of inflammatory cytokines in mouse CB, and the effect of IL-6 gene knockout (KO) on CH-induced chemoreceptor hyperexcitability. Real-time quantitative PCR showed that 1, 3 or 7 days of hypobaric hypoxia (BP = 380 Torr) elicited significant increases (4–12 fold) in the expression of interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). In addition, the chemokine, monocyte chemoattractant protein-1 (MCP-1), was increased 6-, 31- and 41-fold, on days 1, 3, and 7 of CH, respectively. In vitro recordings of carotid sinus nerve (CSN) activity showed that following 10–12 days of CH, responses evoked by an acute hypoxic challenge were increased by approximately 30% in normal mice. Responses to acute hypoxia were similar to normal in IL-6 KO preparations, but following CH they were not elevated. The data are in agreement with our previous findings that CH elicits an inflammatory condition in rat CB, and they further suggest that increased cytokine production is critical for adaptation and the development of hypersensitivity. USPHS Grants NS 12636 and NS 07938.

Effect of poxB gene knockout on metabolism in Escherichia coli based on growth characteristics and enzyme activities

The effect of poxB gene knockout on metabolism in Escherichia coli was investigated in the present paper based on the growth characteristics and the activities of the enzymes involved in the central metabolic pathways. The absence of pyruvate oxidase reduced the glucose uptake rate and cell growth rate, and increased O2 consumption and CO2 evolution. The enzyme assay results showed that although glucokinase activity increased, the flux through glycolysis was reduced due to the down-regulation of the other glycolytic enzymes such as 6-phosphofructosekinase and fructose bisphosphate aldolase in the poxB mutant. TCA cycle enzymes such as citrate synthase and malate dehydrogenase were repressed in the poxB mutant when the cells were cultivated in LB medium. The pyruvate oxidase mutation also resulted in the activation of glucose-6-phosphate dehydrogenase and acetyl-CoA synthetase. All these results suggest that pyruvate oxidase is not only a stationary-phase enzyme as previously known, and that the removal of the poxB gene affects the central metabolism at the enzyme level in E. coli.

Effect of sucA or sucC gene knockout on the metabolism in Escherichia coli based on gene expressions, enzyme activities, intracellular metabolite concentrations and metabolic fluxes by 13C-labeling experiments

The effect of sucA or sucC gene knockout on the metabolism in Escherichia coli was investigated for the aerobic cell growth in batch and continuous cultivations based on gene expressions, enzyme activities, intracellular metabolite concentrations and metabolic flux analysis. In the batch cultivation, the cell growth rate and the glucose uptake rate were lower for sucA mutant as compared with the parent strain, while it was not the case for sucC mutant. A significantly higher amount of acetate was produced, and it was not utilized in s ucC mutant, while a little less acetate was produced in sucA mutant as compared with the parent strain. Unlike the parent strain and sucC mutant, sucA mutant excreted a little amount of l-glutamate. Enzyme activity results show that some of the glycolytic enzymes such as Tpi and Pgk were up-regulated, while Pfk, Fba and Pyk activities were down-regulated for sucA mutant as compared with the parent strain. For sucC mutant, the activities of Pfk, Fba, Tpi, GAPDH, Pgk and Pyk activities were down-regulated. As for the TCA cycle enzymes, the activities of CS and ICDH were down-regulated, while those of Icl, MS, Fum and MDH were up-regulated for sucA mutant. The activities of the oxidative pentose phosphate (PP) pathway enzymes such as G6PDH and 6PGDH and the gluconeogenic pathway enzyme such as Mez were up-regulated in sucA mutant. The Ack activity was down-regulated for sucA mutant, but not for sucC mutant. In continuous cultivation, the gene expression results indicate that the global regulatory genes such as fadR and iclR were slightly down-regulated in sucA mutant, which enhanced the expression of aceA gene and caused the up-regulation of the isocitrate lyase activity in sucA mutant, while fadR and iclR of sucC mutant changed little and no isocitrate lyase activation was observed for sucC mutant. Some other global regulatory genes such as arcA and fnr genes were down-regulated in both mutants, which caused some of the TCA cycle genes to be up-regulated. The effect of the sucA gene knockout on the metabolic flux distributions was investigated based on 1H– 13C NMR spectra and GC–MS signals obtained from 13C-labeling experiments. Flux analysis results indicate that the knockout of sucA gene caused the activation of PP pathway and the glyoxylate shunt. The fluxes through glycolysis and the TCA cycle were down-regulated in the sucA mutant. On the other hand, the fluxes through PP pathway and the anaplerotic reactions of Ppc-Pck and Mez increased.

Synapsin Utilization Differs among Functional Classes of Synapses on Thalamocortical Cells

Several proteins in nerve terminals participate in synaptic transmission between neurons. The synapsins, which are synaptic vesicle-associated proteins, have widespread distribution in the brain and are assumed essential for sustained recruitment of vesicles during high rates of synaptic transmission. We compared the role of synapsins in two types of glutamatergic synapses on thalamocortical cells in the dorsal lateral geniculate nucleus of mice: retinogeniculate synapses, which transmit primary afferent input at high frequencies and show synaptic depression, and corticogeniculate synapses, which provide modulatory feedback at lower frequencies and show synaptic facilitation. We used electrophysiological methods to determine effects of gene knock-out of synapsin I and II on short-term synaptic plasticity in paired-pulse, pulse-train, and posttetanic potentiation paradigms. The gene inactivation changed the plasticity properties in corticogeniculate, but not in retinogeniculate, synapses. Immunostaining with antibodies against synapsins in wild-type mice demonstrated that neither synapsin I nor II occurred in retinogeniculate terminals, whereas both occurred in corticogeniculate terminals. In GABAergic terminals, only synapsin I occurred. In corticogeniculate terminals of knock-out mice, the density of synaptic vesicles was reduced because of increased terminal size rather than reduced number of vesicles and the intervesicle distance was increased compared with wild-type mice. In the retinogeniculate terminals, no significant morphometric differences occurred between knock-out and wild-type mice. Together, this indicates that synapsin I and II are not present in the retinogeniculate terminals and therefore are not essential for sustained, high-rate synaptic transmission.

Wound Healing Is Defective in Mice Lacking Tetraspanin CD151

The tetraspanin CD151 forms complexes in epithelial cell membranes with laminin-binding integrins alpha6beta4, alpha3beta1, and alpha6beta1, and modifies integrin-mediated cell migration in vitro. We demonstrate in this study that CD151 expression is upregulated in a distinct temporal and spatial pattern during wound healing, particularly in the migrating epidermal tongue at the wound edge, suggesting a role for CD151 in keratinocyte migration. We show that healing is significantly impaired in CD151-null mice, with wounds gaping wider at 7 days post-injury. The rate of re-epithelialization of the CD151-null wounds is adversely affected, with significantly less wound area being covered by migrating epidermal cells. Our studies reveal that although laminin levels are similar in wild-type and CD151-null wounds, the organization of the laminin in the basement membrane is impaired. Furthermore, upregulation of alpha6 and beta4 integrin expression is adversely affected in CD151-null mice wounds. In contrast, we find no significant effect of CD151 gene knockout on alpha3 and beta1 integrin expression in wound repair. We suggest that mice lacking the CD151 gene are defective in wound healing, primarily owing to impairment of the re-epithelialization process. This may be due to defective basement membrane formation and epithelial cell adhesion and migration.

Effects of methylmalonyl-CoA mutase gene knockouts on erythromycin production in carbohydrate-based and oil-based fermentations of Saccharopolyspora erythraea

In carbohydrate-based fermentations of Saccharopolyspora erythraea, a polar knockout of the methylmalonyl-CoA mutase (MCM) gene, mutB, improved erythromycin production an average of 126% (within the range of 102-153% for a 0.95 confidence interval). In oil-based fermentations, where erythromycin production by the wild-type strain averages 184% higher (141-236%, 0.95 CI) than in carbohydrate-based fermentations, the same polar knockout in mutB surprisingly reduced erythromycin production by 66% (53-76%, 0.95 CI). A metabolic model is proposed where in carbohydrate-based fermentations MCM acts as a drain on the methylmalonyl-CoA metabolite pool, and in oil-based fermentations, MCM acts in the reverse direction to fill the methylmalonyl-CoA pool. Therefore, the model explains, in part, how the well-known oil-based process improvement for erythromycin production operates at the biochemical level; furthermore, it illustrates how the mutB erythromycin strain improvement mutation operates at the genetic level in carbohydrate-based fermentations.