Huibin Zou

Metabolic engineering of Escherichia coli for biotechnological production of high-value organic acids and alcohols

Confronted with the gradual and inescapable exhaustion of the earth’s fossil energy resources, the bio-based process to produce platform chemicals from renewable carbohydrates is attracting growing interest. Escherichia coli has been chosen as a workhouse for the production of many valuable chemicals due to its clear genetic background, convenient to be genetically modified and good growth properties with low nutrient requirements. Rational strain development of E. coli achieved by metabolic engineering strategies has provided new processes for efficiently biotechnological production of various high-value chemical building blocks. Compared to previous reviews, this review focuses on recent advances in metabolic engineering of the industrial model bacteria E. coli that lead to efficient recombinant biocatalysts for the production of high-value organic acids like succinic acid, lactic acid, 3-hydroxypropanoic acid and glucaric acid as well as alcohols like 1,3-propanediol, xylitol, mannitol, and glycerol with the discussion of the future research in this area. Besides, this review also discusses several platform chemicals, including fumaric acid, aspartic acid, glutamic acid, sorbitol, itaconic acid, and 2,5-furan dicarboxylic acid, which have not been produced by E. coli until now.

Production of extracellular fatty acid using engineered Escherichia coli

BackgroundAs an alternative for economic biodiesel production, the microbial production of extracellular fatty acid from renewable resources is receiving more concerns recently, since the separation of fatty acid from microorganism cells is normally involved in a series of energy-intensive steps. Many attempts have been made to construct fatty acid producing strains by targeting genes in the fatty acid biosynthetic pathway, while few studies focused on the cultivation process and the mass transfer kinetics.ResultsIn this study, both strain improvements and cultivation process strategies were applied to increase extracellular fatty acid production by engineered Escherichia coli. Our results showed overexpressing ‘TesA and the deletion of fadL in E. coli BL21 (DE3) improved extracellular fatty acid production, while deletion of fadD didn’t strengthen the extracellular fatty acid production for an undetermined mechanism. Moreover, the cultivation process controls contributed greatly to extracellular fatty acid production with respect to titer, cell growth and productivity by adjusting the temperature, adding ampicillin and employing on-line extraction. Under optimal conditions, the E. coli strain (pACY-‘tesA-ΔfadL) produced 4.8 g L−1 extracellular fatty acid, with the specific productivity of 0.02 g h−1 g−1dry cell mass, and the yield of 4.4% on glucose, while the ratios of cell-associated fatty acid versus extracellular fatty acid were kept below 0.5 after 15 h of cultivation. The fatty acids included C12:1, C12:0, C14:1, C14:0, C16:1, C16:0, C18:1, C18:0. The composition was dominated by C14 and C16 saturated and unsaturated fatty acids. Using the strain pACY-‘tesA, similar results appeared under the same culture conditions and the titer was also much higher than that ever reported previously, which suggested that the supposedly superior strain did not necessarily perform best for the efficient production of desired product. The strain pACY-‘tesA could also be chosen as the original strain for the next genetic manipulations.ConclusionsThe general strategy of metabolic engineering for the extracellular fatty acid production should be the cyclic optimization between cultivation performance and strain improvements. On the basis of our cultivation process optimization, strain improvements should be further carried out for the effective and cost-effective production process.

Fatty acid from the renewable sources: A promising feedstock for the production of biofuels and biobased chemicals

With the depletion of the nonrenewable petrochemical resources and the increasing concerns of environmental pollution globally, biofuels and biobased chemicals produced from the renewable resources appear to be of great strategic significance. The present review described the progress in the biosynthesis of fatty acid and its derivatives from renewable biomass and emphasized the importance of fatty acid serving as the platform chemical and feedstock for a variety of chemicals. Due to the low efficient conversions of lignocellulosic biomass or carbon dioxide to fatty acid, we also put forward that rational strategies for the production of fatty acid and its derivatives should further derive from the consideration of whole bioprocess (pretreatment, saccharification, fermentation, separation), multiscale analysis and interdisciplinary combinations (omics, kinetics, metabolic engineering, synthetic biology, fermentation and so on).

Microbial production of short chain diols.

Short chain diols (propanediols, butanediols, pentanediols) have been widely used in bulk and fine chemical industries as fuels, solvents, polymer monomers and pharmaceutical precursors. The chemical production of short chain diols from fossil resources has been developed and optimized for decades. Consideration of the exhausting fossil resources and the increasing environment issues, the bio-based process to produce short chain diols is attracting interests. Currently, a variety of biotechnologies have been developed for the microbial production of the short chain diols from renewable feed-stocks. In order to efficiently produce bio-diols, the techniques like metabolically engineering the production strains, optimization of the fermentation processes, and integration of a reasonable downstream recovery processes have been thoroughly investigated. In this review, we summarized the recent development in the whole process of bio-diols production including substrate, microorganism, metabolic pathway, fermentation process and downstream process.

Metabolic engineering of Escherichia coli for the production of xylonate.

Xylonate is a valuable chemical for versatile applications. Although the chemical synthesis route and microbial conversion pathway were established decades ago, no commercial production of xylonate has been obtained so far. In this study, the industrially important microorganism Escherichia coli was engineered to produce xylonate from xylose. Through the coexpression of a xylose dehydrogenase (xdh) and a xylonolactonase (xylC) from Caulobacter crescentus, the recombinant strain could convert 1 g/L xylose to 0.84 g/L xylonate and 0.10 g/L xylonolactone after being induced for 12 h. Furthermore, the competitive pathway for xylose catabolism in E. coli was blocked by disrupting two genes (xylA and xylB) encoding xylose isomerase and xylulose kinase. Under fed-batch conditions, the finally engineered strain produced up to 27.3 g/L xylonate and 1.7 g/L xylonolactone from 30 g/L xylose, about 88% of the theoretical yield. These results suggest that the engineered E. coli strain has a promising perspective for large-scale production of xylonate.

In Vitro Assembly of Multiple DNA Fragments Using Successive Hybridization

Construction of recombinant DNA from multiple fragments is widely required in molecular biology, especially for synthetic biology purposes. Here we describe a new method, successive hybridization assembling (SHA) which can rapidly do this in a single reaction in vitro. In SHA, DNA fragments are prepared to overlap one after another, so after simple denaturation-renaturation treatment they hybridize in a successive manner and thereby assemble into a recombinant molecule. In contrast to traditional methods, SHA eliminates the need for restriction enzymes, DNA ligases and recombinases, and is sequence-independent. We first demonstrated its feasibility by constructing plasmids from 4, 6 and 8 fragments with high efficiencies, and then applied it to constructing a customized vector and two artificial pathways. As SHA is robust, easy to use and can tolerate repeat sequences, we expect it to be a powerful tool in synthetic biology.

Not only osmoprotectant: Betaine increased lactate dehydrogenase activity and L-lactate production in lactobacilli

Lactobacilli are commonly used for industrial production of polymer-grade L-lactic acid. The present study tested the Tween 80 alternative betaine in L-lactate production by several industrial lactobacilli. In flask fermentation of Lactobacillus casei, Lactobacillus buchneri, Lactobacillus lactis and Lactobacillus rhamnosus, the betaine addition (2g/l) had similar osmoprotectant effect with Tween 80 but had increased the lactate dehydrogenase activities and L-lactate production than Tween 80 control. In fed-batch fermentation of L. casei, betaine supplementation improved the L-lactic acid titer to 190 g/l, the yield to 95.5% (g L-lactic acid/g glucose), the productivity to 2.6g/lh, and the optical purity to 97.0%. The results demonstrated that supplementation of Tween 80 alternative - betaine in the fermentation medium is feasible for industrial l-lactic acid fermentation by lactobacilli, which will improve the lactate production but will not increase the process costs and modify any process conditions.

The metabolism and biotechnological application of betaine in microorganism

Glycine betaine (betaine) is widely distributed in nature and can be found in many microorganisms, including bacteria, archaea, and fungi. Due to its particular functions, many microorganisms utilize betaine as a functional chemical and have evolved different metabolic pathways for the biosynthesis and catabolism of betaine. As in animals and plants, the principle role of betaine is to protect microbial cells against drought, osmotic stress, and temperature stress. In addition, the role of betaine in methyl group metabolism has been observed in a variety of microorganisms. Recent studies have shown that betaine supplementation can improve the performance of microbial strains used for the fermentation of lactate, ethanol, lysine, pyruvate, and vitamin B12, during which betaine can act as stress protectant or methyl donor for the biosynthesis of structurally complex compounds. In this review, we summarize the transport, synthesis, catabolism, and functions of betaine in microorganisms and discuss potential engineering strategies that employ betaine as a methyl donor for the biosynthesis of complex secondary metabolites such as a variety of vitamins, coenzymes, and antibiotics. In conclusion, the biocompatibility, C/N ratio, abundance, and comprehensive metabolic information of betaine collectively indicate that this molecule has great potential for broad applications in microbial biotechnology.

Biodegradation-inspired bioproduction of methylacetoin and 2-methyl-2,3-butanediol

Methylacetoin (3-hydroxy-3-methylbutan-2-one) and 2-methyl-2,3-butanediol are currently obtained exclusively via chemical synthesis. Here, we report, to the best of our knowledge, the first alternative route, using engineered Escherichia coli. The biological synthesis of methylacetoin was first accomplished by reversing its biodegradation, which involved modifying the enzyme complex involved, switching the reaction substrate, and coupling the process to an exothermic reaction. 2-Methyl-2,3-butanediol was then obtained by reducing methylacetoin by exploiting the substrate promiscuity of acetoin reductase. A complete biosynthetic pathway from renewable glucose and acetone was then established and optimized via in vivo enzyme screening and host metabolic engineering, which led to titers of 3.4 and 3.2 g l−1 for methylacetoin and 2-methyl-2,3-butanediol, respectively. This work presents a biodegradation-inspired approach to creating new biosynthetic pathways for small molecules with no available natural biosynthetic pathway.

The base-free and selective oxidative transformation of 1,3-propanediol into methyl esters by different Au/CeO2 catalysts

Different Au/CeO2 catalysts, prepared by depositing gold on different facets of ceria nanocubes ({100}), nanorods ({110} and {100}) and nanopolyhedra ({111} and {100}), were separately characterized by means of XRD, N2 sorption, TPD and TPR. It was found that certain types of Au/CeO2 could selectively catalyze the oxidative transformation of 1,3-propanediol in methanol to methyl 3-hydroxypropionate, methyl 3-methoxypropionate, methyl acrylate or dimethyl malonate by molecular oxygen in the absence of any base. The selectivities of these Au/CeO2 catalysts depended on the shapes of the supporting CeO2 and the reaction temperature. The Au/CeO2 cube catalyst with less acidic and basic sites exhibited high selectivity towards methyl 3-hydroxypropionate (93.1% at 21.6% conversion). Comparatively, selectivities towards methyl acrylate (41.6% at 92.3% conversion) and methyl 3-methoxypropionate (40.2% at 92% conversion) increased using Au/CeO2 rod and polyhedron catalysts, which contained more acidic and basic sites than the cube catalyst. Moreover, we found the Au/CeO2 cube catalyst could be recycled without losing the gold nanoparticles.