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Xylose, the major component of lignocellulosic biomass, cannot be naturally or efficiently utilized by most microorganisms. Xylose (co)utilization is considered a cornerstone of efficient lignocellulose-based biomanufacturing. We evolved a rapidly xylose-utilizing strain, Cev2-18-5, which showed the highest reported specific growth rate (0.357 h(−1)) on xylose among plasmid-free Corynebacterium glutamicum strains. A genetically clear chassis strain, CGS15, was correspondingly reconstructed with an efficient glucose-xylose coutilization performance based on comparative genomic analysis and mutation reconstruction. With the introduction of a succinate-producing plasmid, the resulting strain, CGS15-SA1, can efficiently produce 97.1 g/L of succinate with an average productivity of 8.09 g/L/h by simultaneously utilizing glucose and xylose from corn stalk hydrolysate. We further revealed a novel xylose regulatory mechanism mediated by the endogenous transcription factor IpsA with global regulatory effects on C. glutamicum. A synergistic effect on carbon metabolism and energy supply, motivated by three genomic mutations (P(sod(C131T))-xylAB, P(tuf(Δ21))-araE, and ipsA(C331T)), was found to endow C. glutamicum with the efficient xylose utilization and rapid growth phenotype. Overall, this work not only provides promising C. glutamicum chassis strains for a lignocellulosic biorefinery but also enriches the understanding of the xylose regulatory mechanism. IMPORTANCE A novel xylose regulatory mechanism mediated by the transcription factor IpsA was revealed. A synergistic effect on carbon metabolism and energy supply was found to endow C. glutamicum with the efficient xylose utilization and rapid growth phenotype. The new xylose regulatory mechanism enriches the understanding of nonnatural substrate metabolism and encourages exploration new engineering targets for rapid xylose utilization. This work also provides a paradigm to understand and engineer the metabolism of nonnatural renewable substrates for sustainable biomanufacturing.

Stoichiometric genome-scale metabolic network models (GEMs) have been widely used to predict metabolic phenotypes. In addition to stoichiometric ratios, other constraints such as enzyme availability and thermodynamic feasibility can also limit the phenotype solution space. Extended GEM models considering either enzymatic or thermodynamic constraints have been shown to improve prediction accuracy. In this paper, we propose a novel method that integrates both enzymatic and thermodynamic constraints in a single Pyomo modeling framework (ETGEMs). We applied this method to construct the EcoETM (E. coli metabolic model with enzymatic and thermodynamic constraints). Using this model, we calculated the optimal pathways for cellular growth and the production of 22 metabolites. When comparing the results with those of iML1515 and models with one of the two constraints, we observed that many thermodynamically unfavorable and/or high enzyme cost pathways were excluded from EcoETM. For example, the synthesis pathway of carbamoyl-phosphate (Cbp) from iML1515 is both thermodynamically unfavorable and enzymatically costly. After introducing the new constraints, the production pathways and yields of several Cbp-derived products (e.g. L-arginine, orotate) calculated using EcoETM were more realistic. The results of this study demonstrate the great application potential of metabolic models with multiple constraints for pathway analysis and phenotype prediction.

Escherichia coli is a model organism with a clear genetic background that is widely used in metabolic engineering and synthetic biology research. To gain a complete picture of the complexly metabolic and regulatory interactions in E. coli, researchers often need to retrieve information from various databases which cover different types of interactions. A central one-stop service integrating various molecular interactions in E. coli would be helpful for the community. We constructed a database called E. coli integrated network (EcoIN) by integrating known molecular interaction information from databases and literature. EcoIN contains nearly 160,000 pairs of interactions and users can easily search the different types of interacting partners for a metabolite, gene or protein, and thus gain access to a more comprehensive interaction map of E. coli. To illustrate the application of EcoIN, we used the full path algorithm to identify metabolic feedback/feedforward regulatory loops having at least two different types of regulatory interactions. Applying this algorithm to analyze the regulatory loops for the amino acid biosynthetic pathways, we found some multi-step regulation loops which may affect the metabolic flux and are potential new engineering targets. The EcoIN database is freely accessible at http://ecoin.ibiodesign.net/ and analysis codes are available at GitHub: https://github.com/maozhitao/EcoIN .

Supplementary information is available for this paper at https://doi.org/10.1038/s41587-020-0446-y, Reconstructing metabolic reaction networks enables the development of testable hypotheses of an organisms metabolism under different conditions1. State-of-the-art genome-scale metabolic models (GEMs) can include thousands of metabolites and reactions that are assigned to subcellular locations. Geneproteinreaction (GPR) rules and annotations using database information can add meta-information to GEMs. GEMs with metadata can be built using standard reconstruction protocols2, and guidelines have been put in place for tracking provenance and enabling interoperability, but a standardized means of quality control for GEMs is lacking3. Here we report a community effort to develop a test suite named MEMOTE (for metabolic model tests) to assess GEM quality., We acknowledge D. Dannaher and A. Lopez for their supporting work on the Angular parts of MEMOTE; resources and support from the DTU Computing Center; J. Cardoso, S. Gudmundsson, K. Jensen and D. Lappa for their feedback on conceptual details; and P. D. Karp and I. Thiele for critically reviewing the manuscript. We thank J. Daniel, T. Kristjánsdóttir, J. Saez-Saez, S. Sulheim, and P. Tubergen for being early adopters of MEMOTE and for providing written testimonials. J.O.V. received the Research Council of Norway grants 244164 (GenoSysFat), 248792 (DigiSal) and 248810 (Digital Life Norway); M.Z. received the Research Council of Norway grant 244164 (GenoSysFat); C.L. received funding from the Innovation Fund Denmark (project “Environmentally Friendly Protein Production (EFPro2)”); C.L., A.K., N. S., M.B., M.A., D.M., P.M, B.J.S., P.V., K.R.P. and M.H. received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement 686070 (DD-DeCaF); B.G.O., F.T.B. and A.D. acknowledge funding from the US National Institutes of Health (NIH, grant number 2R01GM070923-13); A.D. was supported by infrastructural funding from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation), Cluster of Excellence EXC 2124 Controlling Microbes to Fight Infections; N.E.L. received funding from NIGMS R35 GM119850, Novo Nordisk Foundation NNF10CC1016517 and the Keck Foundation; A.R. received a Lilly Innovation Fellowship Award; B.G.-J. and J. Nogales received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement no 686585 for the project LIAR, and the Spanish Ministry of Economy and Competitivity through the RobDcode grant (BIO2014-59528-JIN); L.M.B. has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement 633962 for project P4SB; R.F. received funding from the US Department of Energy, Offices of Advanced Scientific Computing Research and the Biological and Environmental Research as part of the Scientific Discovery Through Advanced Computing program, grant DE-SC0010429; A.M., C.Z., S.L. and J. Nielsen received funding from The Knut and Alice Wallenberg Foundation, Advanced Computing program, grant #DE-SC0010429; S.K.’s work was in part supported by the German Federal Ministry of Education and Research (de.NBI partner project “ModSim” (FKZ: 031L104B)); E.K. and J.A.H.W. were supported by the German Federal Ministry of Education and Research (project “SysToxChip”, FKZ 031A303A); M.K. is supported by the Federal Ministry of Education and Research (BMBF, Germany) within the research network Systems Medicine of the Liver (LiSyM, grant number 031L0054); J.A.P. and G.L.M. acknowledge funding from US National Institutes of Health (T32-LM012416, R01-AT010253, R01-GM108501) and the Wagner Foundation; G.L.M. acknowledges funding from a Grand Challenges Exploration Phase I grant (OPP1211869) from the Bill & Melinda Gates Foundation; H.H. and R.S.M.S. received funding from the Biotechnology and Biological Sciences Research Council MultiMod (BB/N019482/1); H.U.K. and S.Y.L. received funding from the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries (grants NRF-2012M1A2A2026556 and NRF-2012M1A2A2026557) from the Ministry of Science and ICT through the National Research Foundation (NRF) of Korea; H.U.K. received funding from the Bio & Medical Technology Development Program of the NRF, the Ministry of Science and ICT (NRF-2018M3A9H3020459); P.B., B.J.S., Z.K., B.O.P., C.L., M.B., N.S., M.H. and A.F. received funding through Novo Nordisk Foundation through the Center for Biosustainability at the Technical University of Denmark (NNF10CC1016517); D.-Y.L. received funding from the Next-Generation BioGreen 21 Program (SSAC, PJ01334605), Rural Development Administration, Republic of Korea; G.F. was supported by the RobustYeast within ERA net project via SystemsX.ch; V.H. received funding from the ETH Domain and Swiss National Science Foundation; M.P. acknowledges Oxford Brookes University; J.C.X. received support via European Research Council (666053) to W.F. Martin; B.E.E. acknowledges funding through the CSIRO-UQ Synthetic Biology Alliance; C.D. is supported by a Washington Research Foundation Distinguished Investigator Award. I.N. received funding from National Institutes of Health (NIH)/National Institute of General Medical Sciences (NIGMS) (grant P20GM125503)., info:eu-repo/semantics/publishedVersion

Myceliophthora thermophila, a thermophilic fungus that can degrade and utilize all major polysaccharides in plant biomass, has great potential in biotechnological industries. Here, the first manually curated genome-scale metabolic model iDL1450 for M. thermophila was reconstructed using an autogenerating pipeline with thorough manual curation. The model contains 1450 genes, 2592 reactions, and 1784 unique metabolites. High accuracy was shown in predictions related to carbon and nitrogen source utilization based on data obtained from Biolog experiments. Besides, metabolism profiles were analyzed using iDL1450 integrated with transcriptomics data of M. thermophila at various growth temperatures. The refined model provides new insights into thermophilic fungi metabolism and sheds light on model-driven strain design to improve biotechnological applications of this thermophilic lignocellulosic fungus.

β-Alanine (3-aminopropionic acid) is the only naturally occurring β-type amino acid. Although it is not incorporated into proteins, it has important physiological functions in the metabolism of animals, plants and microorganisms. Furthermore, it has attracted great interest due to its wide usage as a precursor of many significant industrial chemicals for medicine, feed, food, environmental applications and other fields. With the depletion of fossil fuels and concerns regarding environmental issues, biological production of β-alanine has attracted more attention relative to chemical methods. In this review, we first summarize the pathways through which natural microorganisms synthesize β-alanine. Then, the current research progress in the biological synthesis of β-alanine is also elaborated. Finally, we discuss the main problems and challenges in optimizing the biological pathways, offering perspectives on promising new biological approaches.

Background Acetoin, especially the optically pure (3S)- or (3R)-enantiomer, is a high-value-added bio-based platform chemical and important potential pharmaceutical intermediate. Over the past decades, intense efforts have been devoted to the production of acetoin through green biotechniques. However, efficient and economical methods for the production of optically pure acetoin enantiomers are rarely reported. Previously, we systematically engineered the GRAS microorganism Corynebacterium glutamicum to efficiently produce (3R)-acetoin from glucose. Nevertheless, its yield and average productivity were still unsatisfactory for industrial bioprocesses. Results In this study, cellular carbon fluxes in the acetoin producer CGR6 were further redirected toward acetoin synthesis using several metabolic engineering strategies, including blocking anaplerotic pathways, attenuating key genes of the TCA cycle and integrating additional copies of the alsSD operon into the genome. Among them, the combination of attenuation of citrate synthase and inactivation of phosphoenolpyruvate carboxylase showed a significant synergistic effect on acetoin production. Finally, the optimal engineered strain CGS11 produced a titer of 102.45 g/L acetoin with a yield of 0.419 g/g glucose at a rate of 1.86 g/L/h in a 5 L fermenter. The optical purity of the resulting (3R)-acetoin surpassed 95%. Conclusion To the best of our knowledge, this is the highest titer of highly enantiomerically enriched (3R)-acetoin, together with a competitive product yield and productivity, achieved in a simple, green processes without expensive additives or substrates. This process therefore opens the possibility to achieve easy, efficient, economical and environmentally-friendly production of (3R)-acetoin via microbial fermentation in the near future.

Formate can be efficiently produced via electrochemical or photochemical catalytic conversion of CO2, and it can be directly used as an organic carbon source by microorganisms. In theory, formate can be used as the sole carbon source for the microbial production of high-value-added chemicals. Consequently, the construction of efficient formate-assimilation pathways in microorganisms is essential for the utilization of cheap, renewable one-carbon compounds. This paper summarizes new methods of formate synthesis, as well as the natural formate utilization pathways of microorganisms with their advantages and disadvantages. Furthermore, it reviews recent progress in the design of utilization pathways for formate in microbial cells through metabolic engineering and synthetic biology. Besides, we also use the pathway-prediction algorithm comb-FBA to rationally design completely new one-carbon compounds utilization pathways. The pathway with the highest efficiency, named GAA, was corroborated by the in vitro experiments showing a carbon molar yield up to 88%. Finally, it discusses the main problems and challenges presently existing in the pathway design and strain improvement for microbial utilization of formate. KEY POINTS: • Natural and artificial design pathways of formate-assimilation was summarized. • Recent progresses in different hosts and approaches of using one-carbon compounds was reviewed. • Metabolic engineering and synthetic biology methods to improve formate utilization were discussed.

Over the past decade, 5-aminolevulinic acid (5-ALA) has been highlighted as a promising functional feed additive and immunomodulator for improving the general health, immune response, and resistance to disease of livestock and poultry. However, it is very costly to produce 5-ALA using conventional chemical synthesis methods. Classical microbial fermentation fulfills the criteria of environmental friendliness, but the unsatisfactory titers still hinder actual industrial production. This study aimed to develop a solid-state fermentation (SSF) process that can be used to efficiently enrich feed with 5-ALA at a low cost. First, the endogenous 5-ALA synthase was overexpressed in Saccharomyces cerevisiae via integrating a copy of HEM1 gene into the chromosome and introducing a multi-copy plasmid pRS416-HEM1 which constitutively overexpresses HEM1 gene. The resulting strain ScA3 was able to produce 63.82 mg/L 5-ALA in shake-flask fermentation. After process optimization, a titer of 225.63 mg/kg dry materials, exceeding the usual effective dosage reported in animal trials, was achieved within 48 h through SSF of 20 kg feed in a 90-L steel drum. To our knowledge, this is the first report on combining microbial 5-ALA production with SSF in feed processing, which will hopefully promote the application and popularization of 5-ALA in the feed industry.

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Poly-(3-hydroxybutyrate) (P3HB) is a polyester with biodegradable and biocompatible characteristics suitable for bio-plastics and bio-medical use. In order to reduce the raw material cost, cheaper carbon sources such as xylose and glycerol were evaluated for P3HB production. We first conducted genome-scale metabolic network analysis to find the optimal pathways for P3HB production using xylose or glycerol respectively as the sole carbon sources. The results indicated that the non-oxidative glycolysis (NOG) pathway is important to improve the product yields. We then engineered this pathway into E. coli by introducing foreign phophoketolase enzymes. The results showed that the carbon yield improved from 0.19 to 0.24 for xylose and from 0.30 to 0.43 for glycerol. This further proved that the introduction of NOG pathway can be used as a general strategy to improve P3HB production.

To develop an efficient synthetic promoter library for fine-tuned expression of target genes in Corynebacterium glutamicum. A synthetic promoter library for C. glutamicum was developed based on conserved sequences of the − 10 and − 35 regions. The synthetic promoter library covered a wide range of strengths, ranging from 1 to 193% of the tac promoter. 68 promoters were selected and sequenced for correlation analysis between promoter sequence and strength with a statistical model. A new promoter library was further reconstructed with improved promoter strength and coverage based on the results of correlation analysis. Tandem promoter P70 was finally constructed with increased strength by 121% over the tac promoter. The promoter library developed in this study showed a great potential for applications in metabolic engineering and synthetic biology for the optimization of metabolic networks. To the best of our knowledge, this is the first reconstruction of synthetic promoter library based on statistical analysis of C. glutamicum.

CRISPR/Cas9-guided cytidine deaminase enables C:G to T:A base editing in bacterial genome without introduction of lethal double-stranded DNA break, supplement of foreign DNA template, or dependence on inefficient homologous recombination. However, limited by genome-targeting scope, editing window, and base transition capability, the application of base editing in metabolic engineering has not been explored. Herein, four Cas9 variants accepting different protospacer adjacent motif (PAM) sequences were used to increase the genome-targeting scope of bacterial base editing. After a comprehensive evaluation, we demonstrated that PAM requirement of bacterial base editing can be relaxed from NGG to NG using the Cas9 variants, providing 3.9-fold more target loci for gene inactivation in Corynebacterium glutamicum. Truncated or extended guide RNAs were employed to expand the canonical 5-bp editing window to 7-bp. Bacterial adenine base editing was also achieved with Cas9 fused to adenosine deaminase. With these updates, base editing can serve as an enabling tool for fast metabolic engineering. To demonstrate its potential, base editing was used to deregulate feedback inhibition of aspartokinase via amino acid substitution for lysine overproduction. Finally, a user-friendly online tool named gBIG was provided for designing guide RNAs for base editing-mediated inactivation of given genes in any given sequenced genome (www.ibiodesign.net/gBIG).

The utilization of one-carbon (C1) assimilation pathways to produce chemicals and fuels from low-cost C1 compounds could greatly reduce the substrate-related production costs, and would also alleviate the pressure of the resource supply for bio-manufacturing. However, the natural C1 assimilation pathways normally involve ATP consumption or the loss of carbon resources as CO2, resulting in low product yields, making the design of novel pathways highly pertinent. Here we present several new ATP-independent and carbon-conserving C1 assimilation cycles with 100% theoretical carbon yield, which were discovered by computational analysis of metabolic reaction set with 6578 natural reactions from MetaCyc database and 73 computationally predicted aldolase reactions from ATLAS database. Then, kinetic evaluation of these cycles was conducted and the cycles without kinetic traps were chosen for further experimental verification. Finally, we used the two engineered enzymes Gals and TalBF178Y for the artificial reactions to construct a novel C1 assimilation pathway in vitro and optimized the pathway to achieve 88% carbon yield. These results demonstrate the usefulness of computational design in finding novel metabolic pathways for the efficient utilization of C1 compounds and shedding light on other promising pathways.

To find new metabolic engineering strategies to improve the yield of acetone in Escherichia coli.Results of flux balance analysis from a modified Escherichia coli genome-scale metabolic network suggested that the introduction of a non-oxidative glycolysis (NOG) pathway would improve the theoretical acetone yield from 1 to 1.5 mol acetone/mol glucose. By inserting the fxpk gene encoding phosphoketolase from Bifidobacterium adolescentis into the genome, we constructed a NOG pathway in E.coli. The resulting strain produced 47 mM acetone from glucose under aerobic conditions in shake-flasks. The yield of acetone was improved from 0.38 to 0.47 mol acetone/mol glucose which is a significant over the parent strain.Guided by computational analysis of metabolic networks, we introduced a NOG pathway into E. coli and increased the yield of acetone, which demonstrates the importance of modeling analysis for the novel metabolic engineering strategies.

5-Aminolevulinic acid (ALA) is the precursor for the biosynthesis of tetrapyrroles and has broad agricultural and medical applications. Currently ALA is mainly produced by chemical synthesis and microbial fermentation. Cell free multi-enzyme catalysis is a promising method for producing high value chemicals. Here we reported our work on developing a cell free process for ALA production using thermostable enzymes. Cheap substrates (succinate and glycine) were used for ALA synthesis by two enzymes: 5-aminolevulinic acid synthase (ALAS) from Laceyella sacchari (LS-ALAS) and succinyl-CoA synthase (Suc) from Escherichia coli. ATP was regenerated by polyphosphate kinase (Ppk) using polyphosphate as the substrate. Succinate was added into the reaction system in a fed-batch mode to avoid its inhibition effect on Suc. After reaction for 160min, ALA concentration was increased to 5.4mM. This is the first reported work on developing the cell free process for ALA production. Through further process and enzyme optimization the cell free process could be an effective and economic way for ALA production.

Marine ammonia-oxidizing archaea (AOA) play an important role in the global nitrogen cycle by obtaining energy for biomass production from CO2 via oxidation of ammonium. The isolation of Candidatus “Nitrosopumilus maritimus” strain SCM1, which represents the globally distributed AOA in the ocean, provided an opportunity for uncovering the contributions of those AOA to carbon and nitrogen cycles in ocean. Although several ammonia oxidation pathways have been proposed for SCM1, little is known about its ATP production efficiency. Here, based on the published genome of Nitrosopumilus maritimus SCM1, a genome-scale metabolic model named NmrFL413 was reconstructed. Based on the model NmrFL413, the estimated ATP/NH4+ yield (0.149–0.276 ATP/NH4+) is tenfold lower than the calculated theoretical yield of the proposed ammonia oxidation pathways in marine AOA (1.5–1.75 ATP/NH4+), indicating a low energy production efficiency of SCM1. Our model also suggested the minor contribution of marine AOA to carbon cycle comparing with their significant contribution to nitrogen cycle in the ocean. Electronic supplementary material The online version of this article (10.1186/s13568-018-0635-y) contains supplementary material, which is available to authorized users.

Poly-(3-hydroxybutyrate) (P3HB) is a promising biodegradable plastic synthesized from acetyl-CoA. One important factor affecting the P3HB production cost is the P3HB yield. Through flux balance analysis of an extended genome-scale metabolic network of E. coli, we found that the introduction of non-oxidative glycolysis pathway (NOG), a previously reported pathway enabling complete carbon conservation, can increase the theoretical carbon yield from 67% to 89%, equivalent to the theoretical mass yield from 0.48g P3HB/g glucose to 0.64g P3HB/g glucose. Based on this analysis result, we introduced phosphoketolase and enhanced the NOG pathway in E. coli. The mass yield in the engineered strain was increased from 0.16g P3HB/g glucose to 0.24g P3HB/g glucose. We further overexpressed pntAB to enhance the NADPH availability and down-regulated TCA cycle to divert more acetyl-CoA toward P3HB. The final construct accumulated 5.7g/L P3HB and reached a carbon yield of 0.43 (a mass yield of 0.31g P3HB/g glucose) in shake flask cultures in shake flask cultures. The introduction of NOG pathway could also be useful for improving yields of many other biochemicals derived from acetyl-coA.

Background 2,3-Butanediol (2,3-BD) with low toxicity to microbes, could be a promising alternative for biofuel production. However, most of the 2,3-BD producers are opportunistic pathogens that are not suitable for industrial-scale fermentation. In our previous study, wild-type Bacillus subtilis 168, as a class I microorganism, was first found to generate only d-(−)-2,3-BD (purity >99 %) under low oxygen conditions. Results In this work, B. subtilis was engineered to produce chiral pure meso-2,3-BD. First, d-(−)-2,3-BD production was abolished by deleting d-(−)-2,3-BD dehydrogenase coding gene bdhA, and acoA gene was knocked out to prevent the degradation of acetoin (AC), the immediate precursor of 2,3-BD. Next, both pta and ldh gene were deleted to decrease the accumulation of the byproducts, acetate and l-lactate. We further introduced the meso-2,3-BD dehydrogenase coding gene budC from Klebsiellapneumoniae CICC10011, as well as overexpressed alsSD in the tetra-mutant (ΔacoAΔbdhAΔptaΔldh) to achieve the efficient production of chiral meso-2,3-BD. Finally, the pool of NADH availability was further increased to facilitate the conversion of meso-2,3-BD from AC by overexpressing udhA gene (coding a soluble transhydrogenase) and low dissolved oxygen control during the cultivation. Under microaerobic oxygen conditions, the best strain BSF9 produced 103.7 g/L meso-2,3-BD with a yield of 0.487 g/g glucose in the 5-L batch fermenter, and the titer of the main byproduct AC was no more than 1.1 g/L. Conclusion This work offered a novel strategy for the production of chiral pure meso-2,3-BD in B. subtilis. To our knowledge, this is the first report indicating that metabolic engineered B. subtilis could produce chiral meso-2,3-BD with high purity under limited oxygen conditions. These results further demonstrated that B. subtilis as a class I microorganism is a competitive industrial-level meso-2,3-BD producer. Electronic supplementary material The online version of this article (doi:10.1186/s13068-016-0502-5) contains supplementary material, which is available to authorized users.

Poly(3-hydroxybutyrate) (PHB), have been considered to be good candidates for completely biodegradable polymers due to their similar mechanical properties to petroleum-derived polymers and complete biodegradability. Escherichia coli has been used to simulate the distribution of metabolic fluxes in recombinant E. coli producing poly(3-hydroxybutyrate) (PHB). Genome-scale metabolic network analysis can reveal unexpected metabolic engineering strategies to improve the production of biochemicals and biofuels. In this study, we reported the discovery of a new pathway called threonine bypass by flux balance analysis of the genome-scale metabolic model of E. coli. This pathway, mainly containing the reactions for threonine synthesis and degradation, can potentially increase the yield of PHB and other acetyl-CoA derived products by reutilizing the CO2 released at the pyruvate dehydrogenase step. To implement the threonine bypass for PHB production in E. coli, we deregulated the threonine and serine degradation pathway and enhanced the threonine synthesis, resulting in 2.23-fold improvement of PHB titer. Then, we overexpressed glyA to enhance the conversion of glycine to serine and activated transhydrogenase to generate NADPH required in the threonine bypass. The result strain TB17 (pBHR68) produced 6.82 g/L PHB with the yield of 0.36 g/g glucose in the shake flask fermentation and 35.92 g/L PHB with the yield of 0.23 g/g glucose in the fed-batch fermentation, which was almost 3.3-fold higher than the parent strain. The work outlined here shows that genome-scale metabolic network analysis can reveal novel metabolic engineering strategies for developing efficient microbial cell factories.

As 5-aminolevulinic acid synthase (ALAS), the key enzyme for 5-aminolevulinic acid (ALA) synthesis, is unstable, we have sought to find thermostable ALASs from thermophilic organisms. Three ALASs from thermophiles Geobacillus thermoglucosidasius (GT-ALAS), Laceyella sacchari (LS-ALAS) and Pseudomonas alcaliphila (PA-ALAS) were purified and characterized. All enzymes were more stable than two previously studied ALASs from Rhodopseudomonas palustris and Rhodobacter sphaeroides. There was almost no activity change after 60 h at 37 °C for the three thermostable enzymes. This contrasts with the other two enzymes which lost over 90 % activities in just 1 h. Furthermore, the specific activity of LS-ALAS (7.8 U mg−1) was also higher than any previously studied ALASs. Thermostable ALASs were found in thermophilic organisms and this paves the way for developing cell free processes for enzymatic production of ALA from bulk chemicals succinate and glycine.

Background Poly(3-hydroxybutyrate) (PHB), a biodegradable bio-plastic, is one of the most common homopolymer of polyhydroxyalkanoates (PHAs). PHB is synthesized by a variety of microorganisms as intracellular carbon and energy storage compounds in response to environmental stresses. Bio-based production of PHB from renewable feedstock is a promising and sustainable alternative to the petroleum-based chemical synthesis of plastics. In this study, a novel strategy was applied to improve the PHB biosynthesis from different carbon sources. Results In this research, we have constructed E. coli strains to produce PHB by engineering the Serine-Deamination (SD) pathway, the Entner-Doudoroff (ED) pathway, and the pyruvate dehydrogenase (PDH) complex. Firstly, co-overexpression of sdaA (encodes L-serine deaminase), L-serine biosynthesis genes and pgk (encodes phosphoglycerate kinase) activated the SD Pathway, and the resulting strain SD02 (pBHR68), harboring the PHB biosynthesis genes from Ralstonia eutropha, produced 4.86 g/L PHB using glucose as the sole carbon source, representing a 2.34-fold increase compared to the reference strain. In addition, activating the ED pathway together with overexpressing the PDH complex further increased the PHB production to 5.54 g/L with content of 81.1% CDW. The intracellular acetyl-CoA concentration and the [NADPH]/[NADP+] ratio were enhanced after the modification of SD pathway, ED pathway and the PDH complex. Meanwhile, these engineering strains also had a significant increase in PHB concentration and content when xylose or glycerol was used as carbon source. Conclusions Significant levels of PHB biosynthesis from different kinds of carbon sources can be achieved by engineering the Serine-Deamination pathway, Entner-Doudoroff pathway and pyruvate dehydrogenase complex in E. coli JM109 harboring the PHB biosynthesis genes from Ralstonia eutropha. This work demonstrates a novel strategy for improving PHB production in E. coli. The strategy reported here should be useful for the bio-based production of PHB from renewable resources.

The authors wish to notify that the part of the nitrogen assimilationmodel dealing with AmtB-mediated transport of ammonium across the cytoplasmic membrane contains an error. The set of reactions constituting ammonium transport (reactions rAN4, rAN3, rN3 and the external NH3/NH + 4 balance reaction: NH3ex +Hex = NH4ex) does not comply with the detailed balance principle. The net reaction of these four reactions is NH3ex =NH3in. In particular, the product of the forward reaction constants should have equalled that of the backward reaction constants, that is, their ratio should equal 1, while it actually amounts to 3.6 in the model. Exactly for this reason, the model allows for intracellular NH3 accumulation (at most 3.6-fold) and the concomitant occurrence of negative NH3 diffusion. Thus, although the transport process was modelled according to what is known about the mechanism (without explicit reference to the driving force that is still a controversial issue), the reaction constants chosen implicitly caused ammonium transport to become a case of active transport. Consequently, at a kAmtB value of 2000 s−1, the steady-state ratio of the internal/external NH3 concentration in the model was 1.3 when besides active transport also nitrogen assimilation, passive NH3 permeation, G lnK activity, and pH were taken into account (see Table 3). The authors apologize for any confusion this error might have caused and thank Dr D. Molenaar for his critical alertness.

Background The overexpression of key enzymes in a metabolic pathway is a frequently used genetic engineering strategy for strain improvement. Metabolic control analysis has been proposed to quantitatively determine key enzymes. However, the lack of quality data often makes it difficult to correctly identify key enzymes through control analysis. Here, we proposed a method combining in vitro metabolic pathway analysis and proteomics measurement to find the key enzymes in threonine synthesis pathway. Results All enzymes in the threonine synthesis pathway were purified for the reconstruction and perturbation of the in vitro pathway. Label-free proteomics technology combined with APEX (absolute protein expression measurements) data analysis method were employed to determine the absolute enzyme concentrations in the crude enzyme extract obtained from a threonine production strain during the fastest threonine production period. The flux control coefficient of each enzyme in the pathway was then calculated by measuring the flux changes after titration of the corresponding enzyme. The isoenzyme LysC catalyzing the first step in the pathway has the largest flux control coefficient, and thus its concentration change has the biggest impact on pathway flux. To verify that the key enzyme identified through in vitro pathway analysis is also the key enzyme in vivo, we overexpressed LysC in the original threonine production strain. Fermentation results showed that the threonine concentration was increased 30% and the yield was increased 20%. Conclusions In vitro metabolic pathways simulating in vivo cells can be built based on precise measurement of enzyme concentrations through proteomics technology and used for the determination of key enzymes through metabolic control analysis. This provides a new way to find gene overexpression targets for industrial strain improvement. Electronic supplementary material The online version of this article (doi:10.1186/s12934-015-0275-8) contains supplementary material, which is available to authorized users.