(10w) Metabolic Engineering and Synthetic Biology for the Renewable Production of Fuels and Chemicals

Authors: 
Varman, A., Sandia National Laboratories
Tang, Y. J., Washington University
Singh, S., Joint BioEnergy Institute
Research Interests:

Global warming and diminishing fossil fuel reserves have garnered worldwide attention for the renewable production of fuels and chemicals through the metabolic engineering of microbial cell factories. The cyanobacterium Synechocystis sp. strain PCC 6803 was engineered to produce fuels and chemicals from carbon dioxide as carbon substrate and sunlight as energy source. First, metabolic engineering of the cyanobacterium, Synechocystis sp. PCC 6803 was performed for synthesizing isobutanol as a renewable fuel alternative. With the expression of the heterologous genes from the Ehrlich Pathway, by incorporating an in situ isobutanol harvesting system, and also by employing mixotrophic conditions, the engineered Synechocystis 6803 strain accumulated a maximum of ~300 mg/L of isobutanol in a shake flask culture. Second, Synechocystis 6803 was engineered with a novel D-lactate dehydrogenase (encoded by gldA101) for D-lactic acid synthesis. The final titer of D-lactate was improved to 2.2 g/L by employing the following strategies: (i) cofactor balancing, (ii) codon optimization, and (iii) mixotrophic conditions.

Lignin is the only renewable and abundant polymer on the earth with aromatic units as its building blocks; however it remains as an untapped resource. In the first work aimed at lignin valorization, 13C-fingerprinting, 13C metabolic flux analysis (MFA), and RNA-seq differential expression analysis were utilized to uncover the metabolism of Sphingobium sp. SYK-6. SYK-6 is a soil bacterium with a well-studied ligninolytic pathway and an improved understanding of its metabolism will help researchers in the engineering of SYK-6 for lignin valorization. A key finding from this study was the establishment of vanillin catabolic pathway as the major contributor for NAD(P)H synthesis. In the second work, a hybrid promoter engineering approach was employed for the construction of higher strength phenolics inducible promoters. The hybrid promoters were constructed by replacing the spacer region of an endogenous promoter, PemrAB that was naturally inducible by phenolics. In the presence of vanillin, the strains RIF02, RIF03 and RIF04, with the engineered promoters Pvtac, Pvtrc, and Pvtic showed fluorescence increases of 4.6, 3.0, and 1.5 fold respectively in comparison to RIF01 with the native promoter PemrAB. A third study will discuss the research work that was employed for the microbial depolymerization of lignin. We hope that all these different synthetic biology and metabolic engineering efforts will enable the biorefinery to become cost competitive with the petroleum and petrochemical industry.

Teaching Interests: I believe teaching as one of my mentor said, will keep us young in the mind since we will be interacting with younger brains throughout the career. Sharing the knowledge I have gained to others provides joy to my soul and teaching provides a perfect opportunity. As a researcher, it will give me immense pleasure in guiding my students to carry out their individual research and also an opportunity to inspire the younger minds towards research. I will be effective in teaching the core courses of chemical engineering and biotechnology at the undergraduate level. At the graduate level I am interested in developing a course on metabolic engineering and synthetic biology.

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