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Traditional methods like pruning and breeding have historically been used in crop production to divert photoassimilates to harvested organs, but molecular biotechnology is now poised to significantly increase yield by manipulating resource partitioning. It was hypothesized that metabolic engineering in targeted sink tissues can favor resource partitioning to increase harvest. Raffinose Family Oligosaccharides (RFOs) are naturally occurring oligosaccharides that are widespread in plants and are responsible for carbon transport, storage and protection against cold and drought stress. Transgenic plants (GRS47, GRS63) were engineered to generate and transport more RFOs through the phloem than the wild type plants. The transgenic lines produced more RFOs and the RFOs were also detected in their phloem exudates. But the 14CO2 labeling and subsequent thin layer chromatography analysis showed that the RFOs were most likely sequestered in an inactive pool and accumulate over time. Crossing GRS47 and GRS63 lines with MIPS1 plants (that produces more myo-inositol, a substrate in the RFO biosynthetic pathway) did not significantly increase the RFOs in the crossed lines. For future manipulation of RFO degradation in sink organs, the roles of the endogenous α-galactosidases were analyzed. The alkaline α-galactosidases (AtSIP1 and AtSIP2 in Arabidopsis) are most likely responsible for digesting RFOs in the cytoplasm and may influence the ability to manipulate RFO levels in engineered plants. Atsip1/2 (AtSIP1/AtSIP2 double-knockout plants) were generated and phenotypically characterized based on seed germination patterns, flowering time, and sugar content to observe the impact on RFO sugar levels. The observations and analysis from these lines provide a basis for further insight in the manipulation of resource allocation between source and sink tissues in plants for future research.

In an effort to test the feasibility of intracellular expression of enzyme-targeted antibodies to alter metabolism, recombinant antibody fragments in the single-chain format (scFv) were isolated from a phage display library using Arabidopsis chalcone isomerase (CHI) of the flavonoid biosynthetic pathway as the antigen. Each of the genes encoding the scFv's was cloned into a plant transformation vector, which was subsequently used to generate transgenic plants. One transgenic line with low expression of one of the scFv's appeared to have an altered flavonoid metabolism, as evidenced by a reduced capacity for anthocyanin accumulation and a reduction in flavonol glycosides in seedlings. Strong corroborating evidence that implicated the binding of scFv to CHI in the phenotypic alterations was obtained from protein mobility shift assays. Taken together, the results indicate that scFv-mediated metabolic alteration is possible in plants. Thus, we show that intracellular expression of scFv's can be exploited as an additional tool for metabolic engineering. / Ph. D.

In the Paris Agreement from 2015, nations agreed to limit the effects of global warming to well below 2°C. To be able to reach those goals, cheap, abundant and carbon neutral energy alternatives needs to be developed. The microorganisms that several billion years ago oxygenated the atmosphere; cyanobacteria, might hold the key for creating those energy technologies. Due to their capacity for photosynthesis, metabolic engineering of cyanobacteria can reroute the carbon dioxide they fix from the atmosphere into valuable products, thereby converting them into solar powered cell factories. Of the many products bacteria can be engineered to make, the production of terpenoids has gained increasing attention for their attractive properties as fuels, pharmaceuticals, fragrances and food additives. In this thesis, I detail the work I have done on engineering the unicellular cyanobacterium Synechocystis sp. PCC 6803 for terpenoid production. By deleting an enzyme that converts squalene into hopanoids, we could create a strain that accumulates squalene, a molecule with uses as a fuel or chemical feedstock. In another study, we integrated two terpene synthases from the traditional medical plant Coleus forskohlii, into the genome of Synechocystis. Expression of those genes led to the formation of manoyl oxide, a precursor to the pharmaceutically active compound forskolin. Production of manoyl oxide in Synechocystis was further enhanced by engineering in two additional genes from C. forskohlii that boosted the flux to the product. To learn how to increase the production of squalene, manoyl oxide or any other terpenoid, we conducted a detailed investigation of each step in the MEP biosynthesis pathway, which creates the two common building blocks for all terpenoids. Each enzymatic step in the pathway was overexpressed, and increased flux was assayed by using isoprene as a reporter and several potential targets for overexpression were identified. The final part of this thesis details the characterization of native, inducible promoters and ribosomal binding sites in Synechocystis.

The world is hungry for energy. Plant oils in the form of triacylglycerol (TAG) are one of the most reduced storage forms of carbon found in nature and hence represent an excellent source of energy. The myriad of applications for plant oils range across foods, feeds, biofuels, and chemical feedstocks as a unique substitute for petroleum derivatives. Traditionally, plant oils are sourced either from oilseeds or tissues surrounding the seed (mesocarp). Most vegetative tissues, such as leaves and stems, however, accumulate relatively low levels of TAG. Since non-seed tissues constitute the majority of the plant biomass, metabolic engineering to improve their low-intrinsic TAG-biosynthetic capacity has recently attracted significant attention as a novel, sustainable and potentially high-yielding oil production platform. While initial attempts predominantly targeted single genes, recent combinatorial metabolic engineering strategies have focused on the simultaneous optimization of oil synthesis, packaging and degradation pathways (i.e., 'push, pull, package and protect'). This holistic approach has resulted in dramatic, seed-like TAG levels in vegetative tissues. With the first proof of concept hurdle addressed, new challenges and opportunities emerge, including engineering fatty acid profile, translation into agronomic crops, extraction, and downstream processing to deliver accessible and sustainable bioenergy.

The ultimate goal in the field of metabolic engineering is improving cellular processes in a rational manner using engineering design principles and molecular biology techniques. The syntheses of several industrially useful compounds are cofactor-dependent. The reducing equivalent NADPH is required in several enzymatic reactions leading up to the synthesis of high-value compounds like polymers, chiral alcohols, and antibiotics. However, it’s a highly costly compound with limited intracellular availability. This study focuses on the genetic manipulation of a whole-cell system using the two transhydrogenase isoforms pntAB and udhA. Two model systems are used: 1) the production of (S)-2-chloropropionate and 2) the production of poly(3-hydroxybutyrate). Results suggest that the presence of udhA increases product yield and NADPH availability while the presence of pntAB has the opposite effect. A maximum product yield of 1.4 mole-product/mole-glucose was achieved aerobically in a pntAB-deletion strain with udhA overexpression, a 150% improvement over the wild-type control strain.

Master of Science / Biochemistry and Molecular Biophysics Interdepartmental Program / Timothy P. Durrett / Using vegetable oils directly as an alternative biofuel presents several problems as such oils typically possess poor fuel qualities including high viscosity, low volatility, and poor cold temperature properties. The ornamental shrub Euonymus alatus produces unusual acetyl-1,2-diacyl-sn-glycerols (acetyl-TAGs) that have an acetyl group in the sn-3 position instead of a long chain fatty acid. The presence of this sn-3 acetyl-group give acetyl-TAGs properties desirable for biofuels, such as reduced viscosity, comparted to the normal long chain triacyglycerols found in most vegetable oils. Acetyl-TAGs are synthesized by the Euonymus alatus diacylglycerol acetyltransferase (EaDAcT) and Euonymus fortunei diacylglycerol acetyltransferase (EfDAcT) enzymes. Both enzymes catalyze the transfer of an acetyl group from acetyl-CoA to diaclglycerol (DAG) to produce acetyl-TAGs. Previous work demonstrated that expression of EaDAcT combined with the suppression of a diacylglycerol aceyltransferase (DGAT1) in Camelina sativa led to seeds with 85 mol % acetyl-TAGs. Increasing acetyl-TAG levels further was explored using two strategies. Over expression of citrate lyase to increase the pool of acetyl-CoA to be used as a substrate for the acetyltransferase enzymes failed to increased levels of acetyl-TAGs. A second approach involved expressing EfDAcT in Camelina sativa. EfDAcT has demonstrated higher activity in vitro and in vivo and its expression in yeast leads to approximately 50 % higher levels of acetyl-TAGs compared to EaDAcT. The expression of EfDAcT coupled with the suppression of DGAT1 in Camelina sativa resulted in 90 mol % acetyl-TAGs in the transgenic seeds. Levels of EfDAcT protein analyzed in developing transgenic Camelina sativa seeds across a 40 day time period were highest at 15 and 20 days after flowering. Following these time points acetyl-TAG accumulation increased rapidly, coinciding with the higher enzyme expression levels. The optimization of additional promoters to ensure expression of EfDAcT in the last half of seed development could represent another way to further increase acetyl-TAGs in the future.

Motivated by the surging demand of recombinant proteins and biofuels derived from renewable substrates, increasing attention has been paid to the development of novel strains via metabolic engineering strategies. Pichia pastoris is a eukaryotic platform suitable for protein expression and potentially for biofuel production due to its advantageous traits over Escherichia coli or Saccharomyces cerevisiae. In this thesis, we constructed a xylanase-producing P. pastoris strain. The fungal xylanase Xyn11A was successfully overexpressed under the constitutive GAP promoter. Biochemical characterization of the xylanase revealed that Xyn11A is optimally active at 70 °C and pH 7.4. This xylanase was stable over a wide range of pH ranging from pH 2 to pH 11. Excellent thermal stability was observed at temperature 60 °C. Enhanced production of Xyn11A was achieved by investigating the effect of carbon source and feeding strategies. The highest xylanase activity was detected at 15000 U/mL using high cell density cultivation. Production of optically pure (2R, 3R)-2, 3-BD was achieved by engineering P. pastoris with a heterologous pathway. The pathway genes consisting of Bacillus subtilis alsS, alsD and S. cerevisiae BDH1 were assembled and transformed into P. pastoris. Cultivation conditions were optimized and the highest titer of 2, 3-BD obtained using YPD media was 45 g/L in fed-batch cultivation. To enhance the economic viability of 2, 3-BD production in P. pastoris, statistical medium optimization was performed. It was found that 75 g/L of 2, 3-BD was produced using optimized media in fed-batch cultivation.

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