Modular polyketide synthases (PKSs) are the largest enzymes known to man and are responsible for synthesizing some of the most important human medicines. Their ability to construct stereochemically-rich carbon chains containing diverse substituents has inspired the biosynthetic community to engineer these factories for the in vitro synthesis of a small library of polyketide compounds. New complex polyketides are discovered every year, yet the lack of compound prohibits characterization and testing of these new compounds for medicinal properties. Smaller polyketide compounds generated in vitro could be organically manipulated to generate larger, more complex polyketide natural products and natural product analogs. Chemoenzymatic approaches like this would be extremely beneficial to the scientific community; however, there are still obstacles that must be overcome before the use of PKS for the preparative synthesis of an in vitro generated polyketide library would prove fruitful: purchasing substrates such as methylmalonyl-CoA is cost-prohibitive, PKSs are often difficult to express and purify, and the products generated are typically nonchromophoric. The use of a malonyl-CoA ligase from Streptomyces coelicolor (MatB) was investigated for the enzymatic synthesis of polyketide extender units such as methylmalonyl-CoA (Chapter 2). MatB synthesized a total of 5 CoA-linked extender units in vitro: malonyl-, methylmalonyl-, ethylmalonyl-, hydroxymalonyl- and methoxymalonyl-CoA. Two ternary complex structures of MatB with bound product and leaving group were also solved to sub-2Å resolution. MatB generated extender units were employed in the module-catalyzed synthesis of a triketide pyrone. The selectivity of a PKS module to incorporate a variety of side chains into triketide pyrones was also investigated (Chapter 3). A total of 10 triketide pyrone compounds were synthesized, 5 produced via modular "stuttering" and one possessing a terminal alkyne chemical handle. Lastly, nonchromphoric polyketide products were made visible upon copper(I)-catalyzed azide alkyne cycloaddition (CuAAC) with fluorescent sulforhodamine B azide revealing insights into in vitro reactivites of a PKS module (Chapter 4). The work described in this dissertation has helped advance the scientific community towards procuring an in vitro synthesized polyketide library for future synthetic applications. / text