Kechun Zhang, Adam P. Woodruff, Mingyong Xiong
Aug 22, 2011
Citations
5
Influential Citations
69
Citations
Journal
ChemSusChem
Abstract
Society currently relies on fossil-based resources for energy and chemical feedstocks. Due to the depletion of oil reserves, there is a growing interest in exploring alternatives to petroleum-based products. Biosynthesis is a promising approach that enables the sustainable production of fuels or chemicals from renewable carbon sources. A great challenge is that many useful chemicals are not accessible by natural biological systems. Therefore it is necessary to design or evolve novel metabolic pathways for the production of non-natural metabolites. Here, we report the development of a biosynthetic route to isobutyric acid. Isobutyric acid (1) is a useful platform chemical. It can be converted into methacrylic acid (2) by catalytic oxidative dehydrogenation. The methyl ester of 2 (i.e. , methyl methacrylate) is produced in a quantity of 2.2 million tonnes per year for the synthesis of poly(methyl methacrylate). 1 can also be used to manufacture sucrose acetate isobutyrate (3), an emulsifier that is used in printing inks, automotive paints, and beverage additives with a market size of 100 000 tonnes annually. Another application of 1 is the synthesis of 2,2,4-trimethyl-1,3-pentanediol monoisobutyrate (4) (Texanol) or diisobutyrate (TXIB). TXIB is a non-phthalate plasticizer and Texanol is the most widely used coalescent (produced ca. 50 000 tonnes per year). More applications of 1 include the preparation of isopropyl ketones, such as isobutyrone (5), by decarboxylative coupling. The current manufacturing process of 1 is acid-catalyzed Koch carbonylation of propylene (Scheme 1 a). There are two major concerns for this chemical process: (i) propylene is produced by cracking larger hydrocarbon molecules from non-renewable resources such as petroleum and natural gas, the long-term sustainable supply of which is not guaranteed; and (ii) the usage of carbon monoxide and hydrogen fluoride may cause environmental damage. Such problems could be addressed by replacing chemical synthesis with microbial biosynthesis. While there are some bacteria that can overproduce butyric acid, no natural organism has been discovered to produce a significant amount of isobutyric acid. Retrosynthetic analysis suggests that acids can be prepared by the oxidation of alkanes, alcohols, or aldehydes. Because isobutyraldehyde (10) is a metabolic intermediate in the Ehrlich pathway, we can develop a synthetic metabolic pathway that is composed of a natural metabolic route for generating 10 from glucose and a nonnatural step for oxidizing 10 into 1 (Scheme 1 b). In this pathway, glucose is metabolized to pyruvate (6) through glycolysis. 6 is then converted into 2-ketovaline (9) by valine biosynthetic enzymes AlsS, IlvC and IlvD. 9 can be decarboxylated into 10 by Ehrlich pathway enzyme 2-ketoacid decarboxylase (KIVD) from Lactococcus lactis. The key question for the synthetic pathway is whether or not we can identify an enzyme that could effectively catalyze the conversion of 10 into 1. We speculated that we might discover enzymes capable of catalyzing the oxidation of isobutyraldehyde, despite the fact that these enzymes’ natural functions do not include isobutyric acid biosynthesis. We chose seven aldehyde dehydrogenases as possible candidate enzymes: acetaldehyde dehydrogenase AldB, 3-hydroxypropionaldehyde dehydrogenase AldH, succinate semialdehyde dehydrogenase GabD, phenylacetaldehyde dehydrogenase PadA, g-aminobutyraldehyde dehydrogenase YdcW from E. coli, and a-ketoglutaric semialdehyde dehydrogenase from Burkholderia ambifaria KDHba or from Pseudomonas putida KT2440 KDHpp. [18] These enzymes share little homology and cover a wide range of aldehyde substrates. They (designated X) were individually cloned after KIVD to build an expression cassette kivd-x on a high-copy plasmid (Figure 1 a). Another operon on a medium-copy plasmid in the transcriptional order ilvD-alsS (Figure 1 a) was also constructed to drive the carbon flux towards 2-ketovaline (ilvC was not cloned because the chromosomal copy could be overexpressed upon induction by its substrate acetolactate). The cloned plasmids were transformed into wild-type E. coli strain BW25113. Shake flask fermentation was performed at 30 8C for 48 h. Cultures were grown in M9 minimal medium, containing 40 g L 1 glucose as carbon source, and 0.1 mm IPTG Scheme 1. a) Chemical synthesis of isobutyric acid from petrochemical feedstock, and its representative applications. b) Design of a metabolic pathway for biosynthesis of isobutyric acid from the renewable carbon source glucose. One critical step is to identify an enzyme “X” that can efficiently convert isobutyraldehyde 10 into isobutyric acid.