Tools and strategies of systems metabolic engineering for the development of microbial cell factories for chemical production.

Sustainable production of chemicals from renewable non-food biomass has become a promising alternative to overcome environmental issues caused by our heavy dependence on fossil resources. Systems metabolic engineering, which integrates traditional metabolic engineering with systems biology, synthetic biology, and evolutionary engineering, is enabling the development of microbial cell factories capable of efficiently producing a myriad of chemicals and materials including biofuels, bulk and fine chemicals, polymers, amino acids, natural products and drugs. In this paper, many tools and strategies of systems metabolic engineering, including in silico genome-scale metabolic simulation, sophisticated enzyme engineering, optimal gene expression modulation, in vivo biosensors, de novo pathway design, and genomic engineering, employed for developing microbial cell factories are reviewed. Also, detailed procedures of systems metabolic engineering used to develop microbial strains producing chemicals and materials are showcased. Finally, future challenges and perspectives in further advancing systems metabolic engineering and establishing biorefineries are discussed.

High-level production of 3-hydroxypropionic acid from glycerol as a sole carbon source using metabolically engineered Escherichia coli.

As climate change is an important environmental issue, the conventional petrochemical-based processes to produce valuable chemicals are being shifted toward eco-friendly biological-based processes. In this study, 3-hydroxypropionic acid (3-HP), an industrially important three carbon (C3) chemical, was overproduced by metabolically engineered Escherichia coli using glycerol as a sole carbon source. As the first step to construct a glycerol-dependent 3-HP biosynthetic pathway, the dhaB1234 and gdrAB genes from Klebsiella pneumoniae encoding glycerol dehydratase and glycerol reactivase, respectively, were introduced into E. coli to convert glycerol into 3-hydroxypropionaldehyde (3-HPA). In addition, the ydcW gene from K. pneumoniae encoding γ-aminobutyraldehyde dehydrogenase, among five aldehyde dehydrogenases examined, was selected to further convert 3-HPA to 3-HP. Increasing the expression level of the ydcW gene enhanced 3-HP production titer and reduced 1,3-propanediol production. To enhance 3-HP production, fed-batch fermentation conditions were optimized by controlling dissolved oxygen (DO) level and employing different feeding strategies including intermittent feeding, pH-stat feeding, and continuous feeding strategies. Fed-batch culture of the final engineered E. coli strain with DO control and continuous feeding strategy produced 76.2 g/L of 3-HP with the yield and productivity of 0.457 g/g glycerol and 1.89 g·L-1 ·h-1 , respectively. To the best of our knowledge, this is the highest 3-HP productivity achieved by any microorganism reported to date.

A Novel Biosynthetic Pathway for the Production of Acrylic Acid through ß-Alanine Route in Escherichia coli.

Acrylic acid (AA) is an important industrial chemical used for several applications including superabsorbent polymers and acrylate esters. Here, we report the development of a new biosynthetic pathway for the production of AA from glucose in metabolically engineered Escherichia coli through the ß-alanine (BA) route. The AA production pathway was partitioned into two modules: an AA forming downstream pathway and a BA forming upstream pathway. We first validated the operation of the downstream pathway in vitro and in vivo, and then constructed the downstream pathway by introducing efficient enzymes (Act, Acl2, and YciA) screened out of various microbial sources and optimizing the expression levels. For the direct fermentative production of AA from glucose, the downstream pathway was introduced into the BA producing E. coli strain. The resulting strain could successfully produce AA from glucose in flask cultivation. AA production was further enhanced by expressing the upstream genes (panD and aspA) under the constitutive BBa_J23100 promoter. Replacement of the native promoter of the acs gene with the BBa_J23100 promoter in the genome increased AA production to 55.7 mg/L in flask. Fed-batch fermentation of the final engineered strain allowed production of 237 mg/L of AA in 57.5 h, representing the highest AA titer reported to date.

Metabolic engineering for the production of dicarboxylic acids and diamines.

Microbial production of chemicals and materials from renewable carbon sources is becoming increasingly important to help establish sustainable chemical industry. In this paper, we review current status of metabolic engineering for the bio-based production of linear and saturated dicarboxylic acids and diamines, important platform chemicals used in various industrial applications, especially as monomers for polymer synthesis. Strategies for the bio-based production of various dicarboxylic acids having different carbon numbers including malonic acid (C3), succinic acid (C4), glutaric acid (C5), adipic acid (C6), pimelic acid (C7), suberic acid (C8), azelaic acid (C9), sebacic acid (C10), undecanedioic acid (C11), dodecanedioic acid (C12), brassylic acid (C13), tetradecanedioic acid (C14), and pentadecanedioic acid (C15) are reviewed. Also, strategies for the bio-based production of diamines of different carbon numbers including 1,3-diaminopropane (C3), putrescine (1,4-diaminobutane; C4), cadaverine (1,5-diaminopentane; C5), 1,6-diaminohexane (C6), 1,8-diaminoctane (C8), 1,10-diaminodecane (C10), 1,12-diaminododecane (C12), and 1,14-diaminotetradecane (C14) are revisited. Finally, future challenges are discussed towards more efficient production and commercialization of bio-based dicarboxylic acids and diamines.

Recent advances in systems metabolic engineering tools and strategies.

Metabolic engineering has been playing increasingly important roles in developing microbial cell factories for the production of various chemicals and materials to achieve sustainable chemical industry. Nowadays, many tools and strategies are available for performing systems metabolic engineering that allows systems-level metabolic engineering in more sophisticated and diverse ways by adopting rapidly advancing methodologies and tools of systems biology, synthetic biology and evolutionary engineering. As an outcome, development of more efficient microbial cell factories has become possible. Here, we review recent advances in systems metabolic engineering tools and strategies together with accompanying application examples. In addition, we describe how these tools and strategies work together in simultaneous and synergistic ways to develop novel microbial cell factories.

Biosynthesis of poly(2-hydroxyisovalerate-co-lactate) by metabolically engineered Escherichia coli.

Polyhydroxyalkanoates (PHAs) containing 2-hydroxyacids such as lactate (LA) and 2-hydroxybutyrate (2HB) have recently been produced by metabolically engineered microorganisms. Here, we further expanded 2-hydroxyacid monomer spectrum of PHAs by engineering Escherichia coli to produce PHAs containing 2-hydroxyisovalerate (2HIV). To generate 2HIV in vivo, feedback resistant ilvBNmut genes encoding acetohydroxyacid synthase and ilvCD genes encoding ketol-acid reductoisomerase and dihydroxyacid dehydratase, respectively, and panE gene encoding d-2-hydroxyacid dehydrogenase are overexpressed. Also, pct540 gene encoding evolved propionyl-CoA transferase and phaC1437 gene encoding evolved PHA synthase are overexpressed along with ilvBNmut, ilvCD, and panE genes in E. coli strain for in vivo synthesis of 2HIV containing PHAs. E. coli strain expressing all of these genes can produce poly(13.2 mol% 2HIV-co-7.5 mol% 2HB-co-42.5 mol% 3HB-co-36.8 mol% LA) when it is cultured in a chemically defined medium containing 20 g/L of glucose and 2 g/L of sodium 3-hydroxybutyrate (3HB). To produce PHA containing only 2HIV and LA monomers, poxB, pflB, adhE and frdB genes encoding enzymes involved in competing pathways for pyruvate are deleted so that cells can generate more 2HIV and LA. When this engineered E. coli strain expressing ilvBNmut, ilvCD, panE, pct540 and phaC1437 genes is cultured in the medium containing 20 g/L of glucose and 2 mM l-isoleucine, which can inhibit l-threonine dehydratase responsible for in vivo 2HB generation, poly(20 mol% 2HIV-co-80 mol% LA) can be produced to the polymer content of 9.6% w/w. These results suggest that novel PHAs containing 2HIV can be produced by engineering branched-chain amino acid metabolism.

Metabolic Engineering of Escherichia coli for the Production of 3-Hydroxypropionic Acid and Malonic Acid through ß-Alanine Route.

Escherichia coli was metabolically engineered to produce industrially important platform chemicals, 3-hydroxypropionic acid (3-HP) and malonic acid (MA), through the ß-alanine (BA) route. First, various combinations of downstream enzymes were screened and BA pyruvate transaminase (encoded by pa0132) from P. aeruginosa was selected to generate malonic semialdehyde (MSA) from BA. This platform strain was engineered by introducing E. coli MSA reductase (encoded by ydfG) to reduce MSA to 3-HP. Replacement of native promoter of the sdhC gene with the strong trc promoter in the genome increased 3-HP production to 3.69 g/L in flask culture. Introduction of E. coli semialdehyde dehydrogenase (encoded by yneI) into the platform strain resulted in the production of MA, and additional deletion of the ydfG gene increased MA production to 0.450 g/L in flask culture. Fed-batch cultures of final engineered strains resulted in the production of 31.1 g/L 3-HP or 3.60 g/L MA from glucose.