This tutorial review covers tools, strategies, and procedures of systems metabolic engineering facilitating the development of microbial cell factories efficiently producing chemicals and materials.
Escherichia coli was metabolically engineered to produce industrially important platform chemicals, 3-hydroxypropionic acid (3-HP) and malonic acid (MA), through the β-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.
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 β-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.
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.
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.
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