Metabolic Engineering Research Articles

Combinatorial metabolic engineering strategy of precursor pools for the yield improvement of spinosad in Saccharopolyspora spinosa

Spinosad is an insecticide produced by Saccharopolyspora spinosa, and its larvicidal activity is considered a promising approach to combat crop pests. The aim of this study was to enhance the synthesis of spinosad through increasing the supply of acyl-CoAs precursor by the following steps. (i) Engineering the β-oxidation pathway by overexpressing key genes within the pathway to promote the synthesis of spinosad. The results showed that the overexpression of fadD, fadE, and fadA1 genes, as well as the co-expression of fadA1 and fadE genes, increased the yield of spinosad by 0.36-fold, 0.89-fold, 0.75-fold and 1.25-fold respectively. (ii) Employing combinatorial engineering of the β-oxidation pathway and ACC/PCC pathway to promote the synthesis of spinosad. The results showed that the co-expression of fadE and pccA, as well as accC and fadE, resulted in a 1.77-fold and 1.43-fold increase in spinosad production respectively. (iii) When exogenous triacylglycerol was added to the fermentation medium, the solely engineering of the β-oxidation pathway increased the yield of spinosad by 7.13-fold, reaching 427.23mg/L. While the combinatorial engineering of both the β-oxidation pathway and ACC/PCC pathway increased the yield of spinosad by 9.61-fold, reaching 625.17mg/L, and further optimization of the culture medium resulted in an even higher yield of spinosad, reaching 1293.43mg/L. The results of this study indicate that the above combination strategy can promote the efficient biosynthesis of spinosad.

Metabolic engineering of Mucor circinelloides to improve astaxanthin production.

Astaxanthin is a bioactive natural pigment with antioxidant properties. It has extensive applications within the industrial sector as well as in human and animal health. Mucor circinelloides is a zygomycete fungus that accumulates β-carotene as the main carotenoid compound. M. circinelloides is a well-known model organism among Mucorales for studying carotenogenesis in fungi, which makes it a promising candidate for the biotechnological production of carotenoids. In this study, β-carotene hydroxylase (crtR-B) and ketolase (bkt) genes (codon-optimized) were coexpressed from Haematococcus pluvialis in M. circinelloides using two potent promoters gpd1 and zrt1 respectively to generate an astaxanthin-producing biofactory. Following 72h of cultivation, the recombinant M. circinelloides Mc-57 obtained in this study produced 135 ± 8µg/g of astaxanthin. This is the highest reported amount in M. circinelloides to date. The mRNA levels of crtR-B and bkt in Mc-57 were assayed using RT-qPCR. These levels showed a 5.7-fold increase at 72h and a 5.5-fold increase at 24h, respectively, compared to the control strain. This demonstrated the successful overexpression of both genes, which correlated with the production of astaxanthin in the Mc-57. Moreover, the addition of glutamate (2g/L) and mevalonate (15 mM) resulted in an increase in astaxanthin production in the recombinant strain. The results showed that the combined addition of these metabolic precursors resulted in 281 ± 20µg/g of astaxanthin, which is 2.08-fold higher than the control medium (135 ± 8µg/g). The addition of metabolic precursors also positively impacted the biomass growth of Mc-57, reaching 11.2 ± 0.57g/L compared to 9.1 ± 0.23g/L (control medium). The study successfully addressed the challenge of balancing the accumulation of astaxanthin with biomass growth, which has been regarded as common bottleneck in the metabolic engineering of microbial cells. The development of a recombinant fungal strain of M. circinelloides not only increased astaxanthin content. Additionally, it provided a foundation for further improvement of the biotechnological production of astaxanthin in M. circinelloides.

Markerless deletion of the putative type I and III restriction-modification systems in the cellulolytic bacterium Clostridium cellulovorans using a codBA-based counterselection technique

Cellulose from lignocellulosic biomass (LB) is of increasing interest for the production of commodity chemicals. However, its use as substrate for fermentations is a challenge due to its structural complexity. In this context, the highly cellulolytic Clostridium cellulovorans has been considered an interesting microorganism for the breakdown of LB. C. cellulovorans does not naturally produce solvents in useful concentrations, but this could be achieved by metabolic engineering. Unfortunately, this is hampered by the lack of tools for genetic engineering. We describe a genetic system that allows strain engineering by the allelic-coupled exchange method. First, the Gram-positive origin of pUB110 was identified as a suitable clostridial 'pseudo-suicide' origin of replication for the construction of deletion vectors. Second, an efficient counterselection strategy based on a codBA cassette and the use of 5-fluorocytosine as the counterselective compound was employed. Third, since the prevention of DNA transfer by host restriction-modification (RM) systems is a critical barrier to genome engineering, deletion plasmids containing flanking regions for the putative type I (Clocel_1114) and III (Clocel_2651) RM systems were constructed and transferred into C. cellulovorans. The restriction-less strains C. cellulovorans ΔClocel_1114 and C. cellulovorans ΔClocel_2651 exhibit high conjugation efficiency and can be easily used for further metabolic engineering.

Metabolic engineering of Streptomyces roseosporus for increased production of clinically important antibiotic daptomycin.

Daptomycin (DAP), a novel cyclic lipopeptide antibiotic produced by Streptomyces roseosporus, is clinically important for treatment of infections caused by multidrug-resistant Gram-positive pathogens, but the low yield hampers its large-scale industrial production. Here, we describe a combination metabolic engineering strategy for constructing a DAP high-yielding strain. Initially, we enhanced aspartate (Asp) precursor supply in S. roseosporus wild-type (WT) strain by separately inhibiting Asp degradation and competitive pathway genes using CRISPRi and overexpressing Asp synthetic pathway genes using strong promoter kasOp*. The resulting strains all showed increased DAP titre. Combined inhibition of acsA4, pta, pyrB, and pyrC increased DAP titre to 167.4 μg/mL (73.5% higher than WT value). Co-overexpression of aspC, gdhA, ppc, and ecaA led to DAP titre 168 μg/mL (75.7% higher than WT value). Concurrently, we constructed a chassis strain favourable for DAP production by abolishing by-product production (i.e., deleting a 21.1 kb region of the red pigment biosynthetic gene cluster (BGC)) and engineering the DAP BGC (i.e., replacing its native dptEp with kasOp*). Titre for the resulting chassis strain reached 185.8 μg/mL. Application of our Asp precursor supply strategies to the chassis strain further increased DAP titre to 302 μg/mL (2.1-fold higher than WT value). Subsequently, we cloned the engineered DAP BGC and duplicated it in the chassis strain, leading to DAP titre 274.6 μg/mL. The above strategies, in combination, resulted in maximal DAP titre 350.7 μg/mL (2.6-fold higher than WT value), representing the highest reported DAP titre in shake-flask fermentation. These findings provide an efficient combination strategy for increasing DAP production and can also be readily applied in the overproduction of other Asp-related antibiotics.

Diversity of flavonoids in five Torreya grandis cultivars: Integrating metabolome and transcriptome to elucidate potential applications for health and metabolic engineering

Diversity of flavonoids in five Torreya grandis cultivars: Integrating metabolome and transcriptome to elucidate potential applications for health and metabolic engineering

Engineering and evolution of Yarrowia lipolytica for producing lipids from lignocellulosic hydrolysates

Yarrowia lipolytica, an oleaginous yeast, shows promise for industrial fermentation due to its robust acetyl-CoA flux and well-developed genetic engineering tools. However, its lack of an active xylose metabolism restricts the conversion of cellulosic sugars to valuable products. To address this, metabolic engineering, and adaptive laboratory evolution (ALE) were applied to the Y. lipolytica PO1f strain, resulting in an efficient xylose-assimilating strain (XEV). Whole-genome sequencing (WGS) of the XEV followed by reverse engineering revealed that the amplification of the heterologous oxidoreductase pathway and a mutation in the GTPase-activating protein gene (YALI0B12100g) might be the primary reasons for improved xylose assimilation in the XEV strain. When a sorghum hydrolysate was used, the XEV strain showed superior xylose consumption and lipid production compared to its parental strain (X123). This study advances our understanding of xylose metabolism in Y. lipolytica and proposes effective metabolic engineering strategies for optimizing lignocellulosic hydrolysates.

Versatile Xylose and Arabinose Genetic Switches development for Yeasts

Inducible transcription systems are essential tools in genetic engineering, where tight control, strong inducibility and fast response with cost-effective inducers are highly desired. However, existing systems in yeasts are rarely used in large-scale fermentations due to either cost-prohibitive inducers or incompatible performance. Here, we developed powerful xylose and arabinose induction systems in Saccharomyces cerevisiae, utilizing eukaryotic activators XlnR and AraRA from Aspergillus species and bacterial repressors XylR and AraRR. By integrating these signals into a highly-structured synthetic promoter, we created dual-mode systems with strong outputs and minimal leakiness. These systems demonstrated over 4000- and 300-fold regulation with strong activation and rapid response. The dual-mode xylose system was fully activated by xylose-rich agricultural residues like corncob hydrolysate, outperforming existing systems in terms of leakiness, inducibility, dynamic range, induction rate, and growth impact on host. We validated their utility in metabolic engineering with high-titer linalool production and demonstrated the transferability of the XlnR-based xylose induction system to Pichia pastoris, Candida glabrata and Candida albicans. This work provides robust genetic switches for yeasts and a general strategy for integrating activation-repression signals into synthetic promoters to achieve optimal performance.

Screening, identification, engineering, and characterization of Bacillus-derived α-amylase for effective tobacco starch degradation

In this study, two high-performing α-amylase-producing strains, CK3–5 and A8–1 were successfully isolated and characterized, which were taxonomically confirmed as Bacillus velezensis through whole-genome sequencing and bioinformatics. Bioinformatic sequence analysis and molecular docking revealed the catalytic triad (Asp173-Glu208-Asp274) essential for α-amylase function. Through metabolic engineering, the recombinant strain BAX-5/PT17amy(A8–1)SP002 was developed, which exhibited the highest α-amylase activity of 1440 U/mL upon fermentation optimization, marking a 9.2-fold enhancement over the wild-type strain A8–1, and it successfully degraded 6 % of the starch in the tobacco leaves within 48 h, while the content of 13 harmful substances, including acetamide, pyridine, and acetonitrile, was reduced by 8.6 % to 25.2 %. This study reveals a novel α-amylase gene from B. velezensis and establishes an efficient expression system in B. amyloliquefaciens, offering valuable insights for industrial α-amylase production.

From molasses to purified α-ketoglutarate with engineered Corynebacterium glutamicum

α-ketoglutarate (AKG) is a valuable dicarboxylic acid with multiple applications in the food, pharmaceutical and chemical industries. Its chemical synthesis is associated with toxic by-products, low specificity and high energy input. To create a more environmentally friendly and sustainable alternative, a microbial production process for AKG was developed. Four potential producer strains were generated by metabolic engineering of Corynebacteriumglutamicum and characterized on defined glucose/sucrose media as well as molasses, a side stream from sugar beet processing. While strain C.glutamicum PO6-iolT1Δgdh ΔgltB mscCG’ ΔodhA was not able to grow on defined media it outperformed all predecessor variants on molasses. Successful scale-up into a fed-batch bioreactor process with molasses yielded 96.2 g AKG with a conversion yield of 0.64 gg−1. Finally, downstream processing by liquid–liquid extraction with ethyl acetate enabled product purification with an extraction efficiency of 87 % and an AKG purity of > 93 %.

Xanthan gum production in Xanthomonas campestris is increased by favoring the biosynthesis of its monomers

Current efforts to improve xanthan gum (XG) production by Xanthomonas have focused on the growth medium, operating parameters, and downstream steps. However, a key aspect is the development of optimal strains. The present work aimed to investigate the formation of XG monomers, using kinetic and stoichiometric models to identify possible bottlenecks, and to engineer a recombinant strain to overcome such limitations. The galU and ugd genes involved in thebiosynthesis of the UDP-glucose and UDP-glucuronic acid monomers were overexpressed in Xanthomonas campestris pv. campestris. The strains were cultivated in shake flasks and bioreactor. As predicted by in silico analysis, overexpression of the ugd gene resulted in a significant increase in gum synthesis, up to 50% higher volumetric productivity in thebioreactor. To a lesser extent, galU overexpression was also shown to improve product formation. These findings validated the hypothesis that metabolic engineering of the monomer biosynthesis can enhance XG production.

Efficient 7-Dehydrocholesterol Production by Multiple Metabolic Engineering of Diploid Saccharomyces cerevisiae.

7-Dehydrocholesterol (7-DHC), a direct precursor of vitamin D3, has attracted increasing attention in microbial fermentation recently. In this study, 7-DHC biosynthesis in diploid Saccharomyces cerevisiae with robust ergosterol production was achieved by heterologous 24-dehydrocholesterol reductase expression, generating 44.1 mg/L 7-DHC, whereas the titer of ergosterol decreased by 40.5%. The ergosterol biosynthetic pathway was completely blocked by knocking out ERG6 and ERG5, affording a 4.2-fold increase in the 7-DHC titer. Subsequently, the facilitation of the mevalonate and the postsqualene pathways accompanied by elimination of transcriptional repressors enhanced 7-DHC synthesis, and the 7-DHC titer reached 738.5 mg/L in a shake flask. Further validation in a 50 L fermenter demonstrated that the 7-DHC titer reached 3.80 g/L within just 24 h, with productivity reaching 158.3 mg/L/h, setting a new benchmark as the highest reported to date. This study paves the way toward a large-scale and cost-effective manufacture of 7-DHC.

Metabolic engineering of Escherichia coli for the utilization of methylsuccinate, the product of methane activation via fumarate addition

Methylsuccinate is a branched-chain, 5-carbon (C5) dicarboxylate that can be generated from the O2-independent activation of methane via fumarate addition. However, no established metabolic pathway enables growth and product synthesis from methylsuccinate. Here, we report a synthetic pathway that converts methylsuccinate into two precursor metabolites: pyruvate and acetyl-CoA. The pathway was constructed through rational design and validated both in vitro and in vivo using E. coli as the host. Subsequently, growth on methylsuccinate as the sole carbon source was achieved using two parallel strategies: adaptive laboratory evolution and enzyme mining. Through the latter approach, we identified a heterologous electron transfer pathway mediated by previously uncharacterized enzymes and integrated into E. coli enabling the conversion of methylsuccinyl-CoA to mesaconyl-C4-CoA. The engineered strain demonstrated efficient growth on various C5 dicarboxylates including methylsuccinate, mesaconate, and itaconate, with a specific growth rate of 0.11 h−1 on methylsuccinate. This study represents an important step toward achieving synthetic methanotrophy, as the engineered strain can serve as a platform for screening potential methane activation enzymes and ultimately as a production chassis for the bioconversion of methane into various value-added products.

Metabolic engineering of Corynebacterium glutamicum for the production of pyrone and pyridine dicarboxylic acids

Environmental concerns from plastic waste are driving interest in alternative monomers from bio-based sources. Pseudoaromatic dicarboxylic acids are promising alternatives with chemical structures similar to widely used petroleum-based aromatic dicarboxylic acids. However, their use in polyester synthesis has been limited due to production challenges. Here, we report the fermentative production of five pseudoaromatic dicarboxylic acids, including 2-pyrone-4,6-dicarboxylic acid (PDC) and pyridine dicarboxylic acids (PDCAs: 2,3-, 2,4-, 2,5-, and 2,6-PDCA), from glucose using five engineered Corynebacterium glutamicum strains. A platform C. glutamicum chassis strain was constructed by modulating the expression of nine genes involved in the synthesis and degradation pathways of precursor protocatechuate (PCA) and the glucose-uptake system. Comparative transcriptome analysis of the engineered strain against wild-type C. glutamicum identified iolE (NCgl0160) as a target for PDC production. Optimized fed-batch fermentation conditions enabled the final engineered strain to produce 76.17 ± 1.24 g/L of PDC. Using this platform strain, we constructed 2,3-, 2,4-, and 2,5-PDCA-producing strains by modulating the expression of key enzymes. Additionally, we demonstrated a previously uncharacterized pathway for 2,3-PDCA biosynthesis. The engineered strains produced 2.79 ± 0.005 g/L of 2,3-PDCA, 494.26 ± 2.61 mg/L of 2,4-PDCA, and 1.42 ± 0.02 g/L of 2,5-PDCA through fed-batch fermentation. To complete the portfolio, we introduced the 2,6-PDCA biosynthetic pathway to an L-aspartate pathway-enhanced C. glutamicum strain, producing 15.01 ± 0.03 g/L of 2,6-PDCA in fed-batch fermentation. The metabolic engineering strategies developed here will be useful for the production of pseudoaromatic chemicals.

Brassica Vegetables - An Undervalued Nutritional Goldmine

Abstract The genus Brassica includes six species and over 15 types of vegetables that are widely cultivated and consumed globally. This group of vegetables is rich in bioactive compounds, including glucosinolates, vitamins (such as vitamin C, folate, tocopherol, and phylloquinone), carotenoids, phenols, and minerals, which are crucial for enriching diets and maintaining human health. However, the full extent of these phytonutrients and their significant health benefits remain to be fully elucidated. This review highlights the nutrient compositions and health advantages of Brassica vegetables and discusses the impacts of various processing methods on their nutritional value. Additionally, we discuss potential strategies for enhancing the nutrition of Brassica crops through agronomic biofortification, conventional breeding, and biotechnological or metabolic engineering approaches. This review lays the foundation for the nutritional improvement of Brassica crops.

Production of L-lactic acid from methanol by engineered yeast Pichia pastoris

Lactic acid (LA) serves as a widely used platform compound and has received significant attention as a raw material for synthesis of biodegradable polylactic acid. Currently, LA is mainly produced through microbial fermentation, but its high costs undermine its competitive advantage against other materials, necessitating the development of novel production routes. Methanol bioconversion represents an emerging low-carbon circular economy, where LA could become an outstanding representative product. This study successfully established an efficient methanol-based LA synthesis route in Pichia pastoris. Through systematic metabolic engineering strategies, including screening lactate dehydrogenase, modification of cofactor preference, blocking LA consumption pathway, and mitochondrial LA synthesis compartmentalization, 4.2 g/L L-LA was produced in fed-batch fermentation by using methanol as the sole carbon source. Through multi-dimensional and spatial engineering of enzyme, a cell factory was developed for efficient synthesis of L-LA, highlights the significant potential of the low-carbon synthesis route for L-LA via methanol bioconversion.

Sustainable production of the drug precursor tyramine by engineered Corynebacterium glutamicum.

Tyramine has attracted considerable interest due to recent findings that it is an excellent starting material for the production of high-performance thermoplastics and hydrogels. Furthermore, tyramine is a precursor of a diversity of pharmaceutically relevant compounds, contributing to its growing importance. Given the limitations of chemical synthesis, including lack of selectivity and laborious processes with harsh conditions, the biosynthesis of tyramine by decarboxylation of L-tyrosine represents a promising sustainable alternative. In this study, the de novo production of tyramine from simple nitrogen and sustainable carbon sources was successfully established by metabolic engineering of theL-tyrosine overproducing Corynebacterium glutamicum strain AROM3. A phylogenetic analysis of aromatic-L-amino acid decarboxylases (AADCs) revealed potential candidate enzymes for the decarboxylation of tyramine. The heterologous overexpression of the respective AADC genes resulted in successful tyramine production, with the highest tyramine titer of 1.9gL-1 obtained for AROM3 overexpressing the tyrosine decarboxylase gene of Levilactobacillus brevis. Further metabolic engineering of this tyramine-producing strain enabled tyramine production from the alternative carbon sources ribose and xylose. Additionally, up-scaling of tyramine production from xylose to a 1.5L bioreactor batch fermentation was demonstrated to be stable, highlighting the potential for sustainable tyramine production. KEY POINTS: • Phylogenetic analysis revealed candidate l-tyrosine decarboxylases • C. glutamicum was engineered for de novo production of tyramine • Tyramine production from alternative carbon substrates was enabled.

Creating a Genetic Toolbox for the Carbon-Fixing, Nitrogen-Fixing and Dehalogenating Bacterium Xanthobacter autotrophicus.

Xanthobacter autotrophicus is a metabolically flexible microorganism with two key features: (1) The organism has adapted to grow on a wide variety of carbon sources including CO2, methanol, formate, propylene, haloalkanes and haloacids; and (2) X. autotrophicus was the first chemoautotroph identified that could also simultaneously fix N2, meaning the organism can utilize CO2, N2, and H2 for growth. This metabolic flexibility has enabled use of X. autotrophicus for gas fixation, the creation of fertilizers and foods from gases, and the dehalogenation of environmental contaminants. Despite the wide variety of applications that have already been demonstrated for this organism, there are few genetic tools available to explore and exploit its metabolism. Here, we report a genetic toolbox for use in X. autotrophicus. We first identified suitable origins of replication and quantified their copy number, and identified antibiotic resistance cassettes that could be used as selectable markers. We then tested several constitutive and inducible promoters and terminators and quantified their promoter strengths and termination efficiencies. Finally, we demonstrated that gene expression tools remain effective under both autotrophic and dehalogenative metabolic conditions to show that these tools can be used in the environments that make X. autotrophicus unique. Our extensive characterization of these tools in X. autotrophicus will enable genetic and metabolic engineering to optimize production of fertilizers and foods from gases, and enable bioremediation of halogenated environmental contaminants.

Construction and Optimization of a Yeast Cell Factory for Producing Active Unnatural Ginsenoside 3β-O-Glc2-DM.

Ginsenosides are major active components of Panax ginseng, which are generally glycosylated at C3-OH and/or C20-OH of protopanaxadiol (PPD) and C6-OH and/or C20-OH of protopanaxatriol. However, the glucosides of dammarenediol-II (DM), which is the direct precursor of PPD, have scarcely been separated from P. ginseng. Because different positions and numbers of the hydroxyl and glycosyl groups lead to a diversity of structure and function of the ginsenosides, it can be inferred that DM glucosides may have different pharmacological activities compared with natural ginsenosides. Herein, we first constructed the cell factory for de novo biosynthesis of 3-O-(β-D-glucopyranosyl-(1→2)-β-D-glucopyranosyl)-dammar-24-ene-3β,20S-diol (3β-O-Glc2-DM) by introducing the codon-optimized genes encoding dammarenediol-II synthase, two UDP-glycosyltransferases (UGTs) including UGT74AC1-M7 from Siraitia grosvenorii and UGTPg29 from P. ginseng in Saccharomyces cerevisiae via the CRISPR/Cas9 system. The titer of 3β-O-Glc2-DM was then increased from 18.9 to 148.0 mg/L by several metabolic engineering strategies including overexpressing the rate-limiting enzymes of triterpenoid biosynthesis, balancing carbon flux of biosynthetic pathways of triterpenoid and ergosterol, and engineering endoplasmic reticulum. Furthermore, the 3β-O-Glc2-DM titer of 766.3 mg/L was achieved through fed-batch fermentation in a 3-L bioreactor. Finally, in vitro assays demonstrated that 3β-O-Glc2-DM exhibited a protective effect on H/R-induced cardiomyocyte damage. This work provides a feasible approach for production of 3β-O-Glc2-DM as a potential cardioprotective drug candidate.

Deciphering the Cell Surface Sugar-Coating via Biochemical Pathways.

Cell surface components, specifically glycans, play a significant role in several biological functions like cell structure, crosstalk between cells, and eventual target recognition of the cells for therapeutics. The dense layer of glycans, i. e., glycocalyx, could differ in taxon, species, and cell type. Glycans are coupled with lipids and proteins to form glycolipids, glycoproteins, proteoglycans, and glycosylphosphatidylinositol-anchored proteins, making their study challenging. However, understanding glycosylation at the cellular level is vital for fundamental research and advancing glycan-targeted therapy. Among different pathways, metabolic glycan labelling uses the natural metabolic processes of the cell to introduce abiotic functionality into glycan residues. The Bertozzi group pioneered metabolic oligosaccharide engineering using glycan salvage pathways to convert monosaccharides with unnatural modifications. This eventually results in the probe becoming part of the complex cellular glycan structures via click chemistry using copper. On the other hand, the boronic acid-based probe can recognise carbohydrates in a single step without any chemical modification of the surface. This review discusses the significance of glycans as biomarkers for different diseases and the necessity to evaluate them in situ within the physiological environment. The review also discusses the prospect of this field and its potential applications.

Link Between Individual Codon Frequencies and Protein Expression: Going Beyond Codon Adaptation Index.

An important role of a particular synonymous codon composition of a gene in its expression level is well known. There are a number of algorithms optimizing codon usage of recombinant genes to maximize their expression in host cells. Nevertheless, the underlying mechanism remains unsolved and is of significant relevance. In the realm of modern biotechnology, directing protein production to a specific level is crucial for metabolic engineering, genome rewriting and a growing number of other applications. In this study, we propose two new simple statistical and empirical methods for predicting the protein expression level from the nucleotide sequence of the corresponding gene: Codon Expression Index Score (CEIS) and Codon Productivity Score (CPS). Both of these methods are based on the influence of each individual codon in the gene on the overall expression level of the encoded protein and the frequencies of isoacceptors in the species. Our predictions achieve a correlation level of up to r = 0.7 with experimentally measured quantitative proteome data of Escherichia coli, which is superior to any previously proposed methods. Our work helps understand how codons determine protein abundances. Based on these methods, it is possible to design proteins optimized for expression in a particular organism.