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Kakumanu, Ramu

Publications and source records attributed to Kakumanu, Ramu.

8 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2026

Evaluating isoprenol production using the IPP-bypass pathway in the oleaginous yeast Rhodosporidium toruloides

Background To strengthen the national energy supply, there is an increasing demand for domestically generated aviation fuels. Bio-derived advanced aviation fuels offer the opportunity to meet this domestic need while presenting a unique opportunity to investigate the production of novel aviation fuels. Isoprenol, a chemical precursor to such novel fuels, has been shown to be a biologically producible compound in model organisms, but its bio-producibility needs to be further explored in organisms more compatible with industrial bioproduction. Results In this work, we evaluate isoprenol production using the promising bioproduction yeast, Rhodosporidium toruloides. First, we show successful isoprenol production using the IPP-bypass pathways most successful in laboratory strains of E. coli and S. cerevisiae. Next, we demonstrate that increased flux through the mevalonate pathway only modestly increases isoprenol titers. Using proteomics, we identified a potential bottleneck in production at the final step in the IPP-bypass pathway and explored alternative enzymes for this step. Finally, the top three strains of R. toruloides were evaluated in sorghum hydrolysates generated using cholinium lysinate. Through this work, 93.1 mg/L of isoprenol was produced in mock medium and 27.3 mg/L in sorghum hydrolysates. Conclusion Together these results lay the foundation for future work for the production of isoprenol from bioproduction crops.

Advanced aviation fuel↗
DOE OSTI2024

Characterization of lignin-degrading enzyme PmdC, which catalyzes a key step in the synthesis of polymer precursor 2-pyrone-4,6-dicarboxylic acid

Pyrone-2,4-dicarboxylic acid (PDC) is a valuable polymer precursor that can be derived from the microbial degradation of lignin. The key enzyme in the microbial production of PDC is 4-carboxy-2-hydroxymuconate-6-semialdehyde (CHMS) dehydrogenase, which acts on the substrate CHMS. We present the crystal structure of CHMS dehydrogenase (PmdC from Comamonas testosteroni) bound to the cofactor NADP, shedding light on its three-dimensional architecture, and revealing residues responsible for binding NADP. Using a combination of structural homology, molecular docking, and quantum chemistry calculations, we have predicted the binding site of CHMS. Key histidine residues in a conserved sequence are identified as crucial for binding the hydroxyl group of CHMS and facilitating dehydrogenation with NADP. Mutating these histidine residues results in a loss of enzyme activity, leading to a proposed model for the enzyme's mechanism. These findings are expected to help guide efforts in protein and metabolic engineering to enhance PDC yields in biological routes to polymer feedstock synthesis.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2024

Complete biosynthesis of QS-21 in engineered yeast

QS-21 is a potent vaccine adjuvant and remains the only saponin-based adjuvant that has been clinically approved for use in humans. However, owing to the complex structure of QS-21, its availability is limited. Today, the supply depends on laborious extraction from the Chilean soapbark tree or on low-yielding total chemical synthesis. Here we demonstrate the complete biosynthesis of QS-21 and its precursors, as well as structural derivatives, in engineered yeast strains. The successful biosynthesis in yeast requires fine-tuning of the host’s native pathway fluxes, as well as the functional and balanced expression of 38 heterologous enzymes. The required biosynthetic pathway spans seven enzyme families—a terpene synthase, P450s, nucleotide sugar synthases, glycosyltransferases, a coenzyme A ligase, acyl transferases and polyketide synthases—from six organisms, and mimics in yeast the subcellular compartmentalization of plants from the endoplasmic reticulum membrane to the cytosol. Finally, by taking advantage of the promiscuity of certain pathway enzymes, we produced structural analogues of QS-21 using this biosynthetic platform. This microbial production scheme will allow for the future establishment of a structure–activity relationship, and will thus enable the rational design of potent vaccine adjuvants.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Edible mycelium bioengineered for enhanced nutritional value and sensory appeal using a modular synthetic biology toolkit

Filamentous fungi are critical in the transition to a more sustainable food system. While genetic modification of these organisms has promise for enhancing the nutritional value, sensory appeal, and scalability of fungal foods, genetic tools and demonstrated use cases for bioengineered food production by edible strains are lacking. Here, we develop a modular synthetic biology toolkit for Aspergillus oryzae, an edible fungus used in fermented foods, protein production, and meat alternatives. Our toolkit includes a CRISPR-Cas9 method for gene integration, neutral loci, and tunable promoters. We use these tools to elevate intracellular levels of the nutraceutical ergothioneine and the flavor-and color molecule heme in the edible biomass. The strain overproducing heme is red in color and is readily formulated into imitation meat patties with minimal processing. These findings highlight the promise of synthetic biology to enhance fungal foods and provide useful genetic tools for applications in food production and beyond.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Biomass pretreatment with distillable ionic liquids for an effective recycling and recovery approach

Ionic liquid (IL) pretreatment methods show incredible promise for the efficient conversion of lignocellulosic feedstocks to fuels and chemicals. Given their low vapor pressures, distillation-based methods of extracting ionic liquids out of biomass post-pretreatment have historically been ignored in favor of alternative methods. We demonstrate a process to distill four acetate-based ionic liquids ([EthA][OAc], [PropA][OAc], [MAEthA][OAc], and [DMAEthA][OAc]) at low pressure and high purity that overcome some disadvantages of “water washing” and “one pot” recovery methods. Out of four tested ILs, ethanolamine acetate ([EthA][OAc]) is shown to have the most agreeable conversion metrics for commercial bioconversion processes achieving 73.6% and 51.4% of theoretical glucose and xylose yields respectively and >85 % recovery rates. Our process metrics are factored into a techno-economic analysis where [EthA][OAc] distillation is compared to other recovery methods as well as ethanolamine pretreatment at both milliliter and liter scales. In conclusion, although our TEA shows [EthA][OAc] distillation underperforming against other processes, we show a step-by-step avenue to reduce sugar production cost below the wholesale dextrose price at scale.

09 BIOMASS FUELS↗
DOE OSTI2023

Complete integration of carbene-transfer chemistry into biosynthesis

Biosynthesis is an environmentally benign and renewable approach that can be used to produce a broad range of natural and, in some cases, new-to-nature products. However, biology lacks many of the reactions that are available to synthetic chemists, resulting in a narrower scope of accessible products when using biosynthesis rather than synthetic chemistry. A prime example of such chemistry is carbene-transfer reactions. Although it was recently shown that carbene-transfer reactions can be performed in a cell and used for biosynthesis, carbene donors and unnatural cofactors needed to be added exogenously and transported into cells to effect the desired reactions, precluding cost-effective scale-up of the biosynthesis process with these reactions. Here, in this paper, we report the access to a diazo ester carbene precursor by cellular metabolism and a microbial platform for introducing unnatural carbene-transfer reactions into biosynthesis. The α-diazoester azaserine was produced by expressing a biosynthetic gene cluster in Streptomyces albus. The intracellularly produced azaserine was used as a carbene donor to cyclopropanate another intracellularly produced molecule—styrene. The reaction was catalysed by engineered P450 mutants containing a native cofactor with excellent diastereoselectivity and a moderate yield. Our study establishes a scalable, microbial platform for conducting intracellular abiological carbene-transfer reactions to functionalize a range of natural and new-to-nature products and expands the scope of organic products that can be produced by cellular metabolism.

59 BASIC BIOLOGICAL SCIENCES↗