Enzyme fusions have been widely used as a tool in metabolic engineering to increase pathway efficiency by reducing substrate loss and accumulation of toxic intermediates. Alternatively, enzymes can be colocalized through attachment to a synthetic scaffold via noncovalent interactions. Here we describe the use of affibodies for enzyme tagging and scaffolding. The scaffolding is based on the recognition of affibodies to their anti-idiotypic partners in vivo, and was first employed for colocalization of farnesyl diphosphate synthase and farnesene synthase in S. cerevisiae. Different parameters were modulated to improve the system, and the enzyme:scaffold ratio was most critical for its functionality. Ultimately, the yield of farnesene on glucose Y could be improved by 135% in fed-batch cultivations using a 2-site affibody scaffold. The scaffolding strategy was then extended to a three-enzyme polyhydroxybutyrate (PHB) pathway, heterologously expressed in E. coli. Within a narrow range of enzyme and scaffold induction, the affibody tagging and scaffolding increased PHB production 7-fold. This work demonstrates how the versatile affibody can be used for metabolic engineering purposes.
Terpenes have various applications as fragrances, cosmetics and fuels. One of the most prominent examples is the sesquiterpene farnesene, which can be used as diesel substitute in its hydrogenated form farnesane. Recent metabolic engineering efforts have enabled efficient production of several terpenes in Saccharomyces cerevisiae and Escherichia coli. Plant terpene synthases take on an essential function for sesquiterpene production as they catalyze the specific conversion of the universal precursor farnesyl diphosphate (FPP) to the sesquiterpene of interest and thereby impose limitations on the overall productivity. Using farnesene as a case study, we chose three terpene synthases with distinct plant origins and compared their applicability for farnesene production in the yeast S. cerevisiae. Differences regarding the efficiency of these enzymes were observed in shake flask cultivation with maximal final titers of 4 mg/L using α-farnesene synthase from Malus domestica. By employing two existing platform strains optimized for sesquiterpene production, final titers could be raised up 170 mg/L in fed-batch fermentations with RQ-controlled exponential feeding. Based on these experiments, the difference between the selected synthases was not significant. Lastly, the same fermentation setup was used to compare these results to production of the fragrance sesquiterpene santalene, and almost equivalent titers were obtained with 163 mg/L, using the highest producing strain expressing a santalene synthase from Clausena lansium. However, a reduction of the product yield on biomass by 50% could indicate a higher catalytic efficiency of the farnesene synthase.
Isoprenoids denote the largest group of chemicals in the plant kingdom and are employed for a wide range of applications in the food and pharmaceutical industry. In recent years, isoprenoids have additionally been recognized as suitable replacements for petroleum‐derived fuels and could thus promote the transition towards a more sustainable society. To realize the biofuel potential of isoprenoids, a very efficient production system is required. While complex chemical structures as well as the low abundance in nature demonstrate the shortcomings of chemical synthesis and plant extraction, isoprenoids can be produced by genetically engineered microorganisms from renewable carbon sources. In this article, we summarize the development of isoprenoid applications from flavors and pharmaceuticals to advanced biofuels and review the strategies to design microbial cell factories, focusing on Saccharomyces cerevisiae for the production of these compounds. While the high complexity of biosynthetic pathways and the toxicity of certain isoprenoids still denote challenges that need to be addressed, metabolic engineering has enabled large‐scale production of several terpenoids and thus, the utilization of these compounds is likely to expand in the future.
Efficient production of sesquiterpenes in Saccharomyces cerevisiae requires a high flux through the mevalonate pathway. To achieve this, the supply of acetyl-CoA plays a crucial role, partially because nine moles of acetyl-CoA are necessary to produce one mole of farnesyl diphosphate, but also to overcome the thermodynamic constraint imposed on the first reaction, in which acetoacetyl-CoA is produced from two moles of acetyl-CoA by acetoacetyl-CoA thiolase. Recently, a novel acetoacetyl-CoA synthase (nphT7) has been identified from Streptomyces sp. strain CL190, which catalyzes the irreversible condensation of malonyl-CoA and acetyl-CoA to acetoacetyl-CoA and, therefore, represents a potential target to increase the flux through the mevalonate pathway. This study investigates the effect of acetoacetyl-CoA synthase on growth as well as the production of farnesene and compares different homologs regarding their efficiency. While plasmid-based expression of nphT7 did not improve final farnesene titers, the construction of an alternative pathway, which exclusively relies on the malonyl-CoA bypass, was detrimental for growth and farnesene production. The presented results indicate that the overall functionality of the bypass was limited by the efficiency of acetoacetyl-CoA synthase (nphT7). Besides modulation of the expression level, which could be used as a means to partially restore the phenotype, nphT7 from Streptomyces glaucescens showed clearly higher efficiency compared to Streptomyces sp. strain CL190.Electronic supplementary materialThe online version of this article (doi:10.1007/s10295-017-1911-6) contains supplementary material, which is available to authorized users.
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