Biosynthesis of C4-C8 3-Hydroxycarboxylic Acids from Glucose through the Inverted Fatty Acid ß-Oxidation by Metabolically Engineered Escherichia coli.

Inverted fatty acid ß-oxidation represents a versatile biochemical platform for biosynthesis by the engineered microbial strains of numerous value-added chemicals from convenient and abundant renewable carbon sources, including biomass-derived sugars. Although, in recent years, significant progress has been made in the production through this pathway of n-alcohols, 1,3-diols, and carboxylic acids and its 2,3-unsaturated derivatives, the potential of the pathway for the biosynthesis of 3-hydroxycarboxylic acids remained almost undisclosed. In this study, we demonstrate the microaerobic production of even-chain-length C4-C8 3-hydroxycarboxylic acids from glucose through the inverted fatty acid ß-oxidation by engineered E. coli strains. The notable accumulation of target compounds was achieved upon the strong constitutive expression of the genes atoB, fadA, fadB, fadE/fabI, and tesB, which code for the key enzymes catalysing reactions of aerobic fatty acid ß-oxidation and thioesterase II, in strains devoid of mixed-acid fermentation pathways and lacking nonspecific thioesterase YciA. The best performing recombinants were able to synthesise up to 14.5 mM of 3-hydroxycarboxylic acids from glucose with a total yield of 0.34 mol/mol and a C4/C6/C8 ratio averaging approximately 63/28/9. The results provide a framework for the development of highly efficient strains and processes for the bio-based production of valuable 3-hydroxycarboxylates from renewable raw materials.

Engineering Escherichia coli for efficient aerobic conversion of glucose to fumaric acid.

Escherichia coli was engineered for efficient aerobic conversion of glucose to fumaric acid. A novel design for biosynthesis of the target product through the modified TCA cycle rather than via glyoxylate shunt, implying oxaloacetate formation from pyruvate and artificial channelling of 2-ketoglutarate towards succinic acid via succinate semialdehyde formation, was implemented. The main fumarases were inactivated in the core strain MSG1.0 (∆ackA-pta, ∆poxB, ∆ldhA, ∆adhE, ∆ptsG, PL-glk, Ptac-galP) by the deletion of the fumA, fumB, and fumC genes. The Bacillus subtilis pycA gene was expressed in the strain to ensure pyruvate to oxaloacetate conversion. The Mycobacterium tuberculosis kgd gene was expressed to enable succinate semialdehyde formation. The resulting strain was able to convert glucose to fumaric acid with a yield of 0.86 mol/mol, amounting to 86% of the theoretical maximum. The results demonstrated the high potential of the implemented strategy for development of efficient strains for bio-based fumaric acid production.

Engineering Escherichia coli for respiro-fermentative production of pyruvate from glucose under anoxic conditions.

An Escherichia coli K-12 MG1655-derived strain was engineered for respiro-fermentative production of pyruvate from glucose under anoxic conditions, which is preferred for industrial-scale microbial synthesis of valuable chemicals. The pathways of anaerobic pyruvate dissimilation were blocked in the strain by the deletion of the ackA, pta, poxB, ldhA, adhE, and pflB genes. The phosphoenolpyruvate-dependent phosphotransferase system of glucose transport and phosphorylation was substituted by an alternative ATP-dependent system resulting from the overexpression of galP and glk upon deletion of ptsG. The channelling of pyruvate towards the oxidative branch of the TCA cycle under respiratory conditions was prevented in the strain due to the deletion of aceEF genes, encoding components of pyruvate dehydrogenase, while the operation of the entire reductive branch of the TCA cycle was interrupted by knocking out frdAB and sdhAB. Reoxidation of glycolytic NADH was ensured via anaerobic respiration with nitrate serving as an external electron acceptor. To enforce anaerobic ATP hydrolysis, an ATP-consuming futile cycle of pyruvate-oxaloacetate-malate-pyruvate was established in the strain by expressing the Bacillus subtilis pycA gene, encoding pyruvate carboxylase. In the presence of sufficient amounts of an external electron acceptor and CO2 source, the engineered strain was able to efficiently utilise glucose and convert it to pyruvate anaerobically with a yield of 1.73 mol/mol, amounting to 87% of the theoretical maximum. The implemented strategy offers the potential for the development of highly efficient processes of bio-based pyruvate production.

Biosynthesis of enantiopure (S)-3-hydroxybutyrate from glucose through the inverted fatty acid ß-oxidation pathway by metabolically engineered Escherichia coli.

Enantiomers of 3-hydroxybutyric acid (3-HB) can be used as the chiral precursors for the production of various optically active fine chemicals, including drugs, perfumes, and pheromones. In this study, Escherichia coli was engineered to produce (S)-3-HB from glucose through the inverted reactions of the native aerobic fatty acid ß-oxidation pathway. Expression of only specific genes encoding enzymes responsible for the conversion of acetyl-CoA to acetoacetyl-CoA, reduction of acetoacetyl-CoA to 3-hydroxybutyryl-CoA and subsequent hydrolysis of 3-hydroxybutyryl-CoA to 3-HB was directly upregulated in an engineered strain. The operation of multiple turns of the inverted fatty acid ß-oxidation was precluded by the deletion of gene encoding enzyme that catalyse the terminal stage of the respective cycle. While the overexpression of the C-acetyltransferase gene enabled 3-HB biosynthesis through the inverted fatty acid ß-oxidation, the efficient conversion of glucose to the target product was achieved resulting from the additional overexpression of the gene encoding appropriate termination thioesterase II. The engineered strain synthesised the (S)-stereoisomer of 3-HB with an enantiomeric excess of more than 99%. Under microaerobic conditions, up to 9.58g/L of enantiopure (S)-3-HB was produced from glucose, with a yield of 66% of the theoretical maximum.

Manipulating pyruvate to acetyl-CoA conversion in Escherichia coli for anaerobic succinate biosynthesis from glucose with the yield close to the stoichiometric maximum.

Efficient succinate production in Escherichia coli is attained during anaerobic glucose fermentation in biosynthetic processes combining the reductive branch of the TCA cycle and the glyoxylate bypass. Pyruvate dehydrogenase (PDH) or pyruvate formate lyase (PFL) serves in E. coli as a source of acetyl-CoA, a substrate for the glyoxylate bypass. Depending on enzymes responsible for acetyl-CoA generation, the contribution of the glyoxylate bypass to the anaerobic succinate biosynthesis may vary to support redox balance resulting in diverse maximum achievable yield values. Anaerobic succinate biosynthesis from glucose was studied using E. coli strains with altered expression of genes encoding PFL and PDH. For acetyl-CoA formation by PFL, the yield of 1.32 mol succinate per mole of glucose was achieved with the theoretical value of 1.6 mol/mol. Involvement of PDH in anaerobic acetyl-CoA synthesis increased succinate yield up to 1.49 mol/mol, which is 89.8% of the predicted maximum (1.6(6) mol/mol). The maximum yield of 1.69 mol succinate per mol glucose, amounting to 98.8% of the stoichiometric maximum (1.71 mol/mol), was achieved with the strain possessing PDH as the primary anaerobic source of acetyl-CoA. During high cell density fermentation, the best engineered strain produced high amounts of succinate (570.7 mM) and only small quantities of acetate (11.9 mM).

Comparison of different approaches to activate the glyoxylate bypass in Escherichia coli K-12 for succinate biosynthesis during dual-phase fermentation in minimal glucose media.

Two different approaches to activate the glyoxylate bypass in model Escherichia coli K-12 strains for succinate biosynthesis during dual-phase fermentation in minimal glucose media were examined. Inactivation of IclR and FadR, the transcriptional regulators of the aceBAK operon, were insufficient for the involvement of the glyoxylate bypass in anaerobic succinate biosynthesis by strains grown aerobically under glucose-abundant conditions. In contrast, the strains that constitutively expressed the aceEF-lpdA operon coding for the pyruvate dehydrogenase complex could partially synthesise succinate anaerobically via the glyoxylate bypass, even in the presence of intact regulators. The results suggest that the intensive acetyl-CoA formation in the strains constitutively expressing pyruvate dehydrogenase matches the physiological conditions that favour the activation of the glyoxylate bypass.

Metabolic engineering of Escherichia coli for 1-butanol biosynthesis through the inverted aerobic fatty acid ß-oxidation pathway.

The basic reactions of the clostridial 1-butanol biosynthesis pathway can be regarded to be the inverted reactions of the fatty acid ß-oxidation pathway. A pathway for the biosynthesis of fuels and chemicals was recently engineered by combining enzymes from both aerobic and anaerobic fatty acid ß-oxidation as well as enzymes from other metabolic pathways. In the current study, we demonstrate the inversion of the entire aerobic fatty acid ß-oxidation cycle for 1-butanol biosynthesis. The constructed markerless and plasmidless Escherichia coli strain BOX-3 (MG1655 lacI(Q) attB-P(trc-ideal-4)-SD(φ10)-adhE(Glu568Lys) attB-P(trc-ideal-4)-SD(φ10)-atoB attB-P(trc-ideal-4)-SD(φ10)-fadB attB-P(trc-ideal-4)-SD(φ10)-fadE) synthesises 0.3-1 mg 1-butanol/l in the presence of the specific inducer. No 1-butanol production was detected in the absence of the inducer.

Construction of stably maintained non-mobilizable derivatives of RSF1010 lacking all known elements essential for mobilization.

BACKGROUND: RSF1010 is a well-studied broad-host-range plasmid able to be mobilized to different bacteria and plants. RSF1010-derived plasmid vectors are widely used in both basic research and industrial applications. In the latter case, exploiting of mobilizable plasmids or even the plasmids possessing negligible mobilization frequency, but containing DNA fragments that could promote conjugal transfer, is undesirable because of biosafety considerations. Previously, several mutations significantly decreasing efficiency of RSF1010 mobilization have been selected. Nevertheless, construction of the RSF1010 derivative lacking all known loci involved in the conjugal transfer has not been reported yet. RESULTS: Novel non-mobilizable derivatives of RSF1010 lacking all known DNA sequences involved in the mobilization process have been obtained due to the exploiting of lambdaRed-driven recombination between the plasmid and a constructed in vitro linear DNA fragment. To provide auto-regulated transcription of the essential replication gene, repB, the plasmid loci oriT, mobC and mobA were substituted by the DNA fragment containing PlacUV5-->lacI. Mobilization of the obtained RSFmob plasmid was not detected in standard tests. The derivative of RSFmob with increased copy number has been obtained after lacI elimination. High stability of both constructed plasmids has been demonstrated in Escherichia coli and Pantoea ananatis. Design of RSFmob allows easy substitution of PlacUV5 by any desirable promoter for construction of novel derivatives with changed copy number or host range. CONCLUSION: Novel non-mobilizable derivatives of RSF1010 lacking all known DNA sequences involved in the mobilization process and stably maintained at least in E. coli and P. ananatis have been constructed. The obtained plasmids became the progenitors of new cloning vectors answering all biosafety requirements of genetically modified organisms used in scale-up production.