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xi CHAPTER 1. General Introduction 1 I. Dependence on fossil fuels 1 II. Reducing our dependence through biofuels 3 III. Alternative biofuels 6 CHAPTER 2. Literature Review 8 Introduction 8 Alternative biofuels production 9 I. Butanol 9 II. Microbial hydrocarbon biosynthesis 17 III. Fatty acid biosynthesis 17 IV. Alkane biosynthesis 20 V. Ethylene 21 VI. Isobutene 22 Optimizing enzyme function 25 I. Rational design 25 II. Directed enzyme evolution 26 Mevalonate Decarboxylase 35 I. Mevalonate diphosphate decarboxylase (MDD) 35 II. Putative mevalonate monophosphate decarboxylase (MPD) 41

Isobutene formation from 3-hydroxy-3-methylbutyrate (3-HMB) by the Saccharomyces cerevisiae diphosphomevalonate decarboxylase (ScMDD) and directed enzyme evolution to improve enzyme function