Increased 2,3-butanediol production by changing codon usages in Escherichia coli Seo-Young Park 1,2 Borim Kim 1 Soojin Lee 1 Minkyu Oh 3 Jong-In Won 2 Jinwon Lee 1 * 1 Department of Chemical and Biomolecular Engineering, Sogang University, Seoul, Korea 2 Department of Chemical Engineering, Hongik University, Seoul, Korea 3 Department of Chemical Engineering and Biological Engineering, Korea University, Seoul, Korea Abstract The natural microorganism Escherichia coli without modification is not suitable for the efficient production of 2,3-butanediol (2,3-BD) on an industrial scale because of its poor metabolic performance. Metabolic capacities of E. coli have been improved to produce 2,3-BD efficiently, the performance of which is possible for producing such a product. Codon optimization with the ribosome-binding site for the efficient production of target genes (budA and budC) was achieved by molecular engineering, which allowed the metabolic engineering to proceed to the next level. As a result, comparing the productivity in 26 H, where the amount of p18COR was 1.04 g/L and that of p18WTR was 0.41 g/L, represents an approximate 60.6% increase in the productivity of the p18WTR with codon optimization. In other words, p18COR was 2.54-fold greater than p18WTR in the production of 2,3-BD. C  2014 International Union of Biochemistry and Molecular Biology, Inc. Volume 00, Number 00, Pages 1–6, 2014 Keywords: Escherichia coli, codon optimization, ribosome-binding site, 2,3-butanediol, acetolactate decarboxylase, acetoin reductase 1. Introduction The reason why many global companies and governments are trying to seek alternative energy recently is because of the concern about pollution and climate change caused by the use of fossil energy. Due to the shortage of fossil fuel supplies and other environmental problems, the idea of bio-based chemical compounds and bioreineries is receiving signiicant interest. Bioreinery is deined as the sustainable processing of biomass into a spectrum of bio-based products and bioenergy, which has the following features. It can utilize renewable biomass and reduce the greenhouse gas emission while competing with the feedstock for foods and feeds. However, the nonfeedstock Abbreviations: IPTG, isopropyl-β-D-thiogalactoside; HPLC, high-performance liquid chromatography; LB, Luria-Bertani. ∗ Address for correspondence: Professor Jinwon Lee, Department of Chemical and Biomolecular Engineering, Sogang University, Seoul, 121-742, South Korea. Tel.: +82 2 705 8919; Fax: +82 2 711 0439; e-mail: jinwonlee@sogang.ac.kr. Received 13 November 2013; accepted 7 February 2014 DOI: 10.1002/bab.1216 Published online in Wiley Online Library (wileyonlinelibrary.com) carbon source is also the subject of numerous studies; the bioreinery is a promising area in stable chemical development. Lately, there is one chemical concern named 2,3-butanediol (2,3-BD), which still remains to be developed; more efforts are being required to make progress. 2,3-BD is a composition of a four-carbon chemical with three stereoisomers. The physical properties of 2,3-BD offer large-scale industrial applications such as lavor additive, liquid fuel additive, antifreeze sub- stance, cosmetics, and pharmaceuticals [1–3]. Although 2,3-BD can be produced by hydrolyzing petroleum, it is very dificult to produce, and with the cumbersome process, the expenses are costly. As a result, the renewable and petroleum-independent biologically production of 2,3-BD using microorganisms is very promising. Many microorganisms have been identiied to be able to produce 2,3-BD naturally by the fermentation process, such as Klebseiella pneumoniae, Klebseiella oxytoca, Bacillus polymyxa, and Enterobacter aerogenes. However, the pathogenicity and susceptibility to opportunistic infection of the Klebseiella strains, which is the most widely utilized strains, hinder their use in industrial-scale production of 2,3-BD [2, 4]. The harmless strain E. coli, which is most widely studied for the majority of work with recombinant DNA, can be grown easily and inexpensively in laboratories and industrial settings. 1