Acetate as a Platform for Carbon-Negative Production of Renewable Fuels and Chemicals

Grants and Contracts Details


Project title: Acetate as a Platform for Carbon-Negative Production of Renewable Chemicals UK PIs: Joe Chappell and Chang-Guo Zhan Submitting PI: Brian Pfleger, University of Wisconsin Agency: ARPA-E, due Feb. 2, 2021 I was contacted by Brian Pfleger, a Chemical Engineer at University of Wisconsin and Chad Haynes, lead Scientist at LanzaTech about a joint proposal. LanzaTech has fermentation technology to produce very large amounts of acetate, Dr. Pfleger has fermentation capability to co-culture bugs and especially unusual bugs. They want to co- culture Lanza’s bug to produce acetate and couple that with Cupriavidus necator (which can grow on hydrogen) for the production of fatty acids as an alternative combustible fuel. However, they also would like to complement this work by engineering C. necator with the capability to produce high-value terpene molecules as well. The Chappell lab has worked extensively on the production of 2 triterpene oils, squalene and botryococcene, that fulfill the requirements of having value as alternative feedstocks for fuels as well as other diverse applications. For instance, as adjuvants in vaccine development and as emollients in personal care products. These molecules are not readily available by conventional chemical manufacturing means and instead their availability is dependent upon natural sources. The source most commonly used in shark livers, which is neither sustainable nor humane, and thus there is a desire for the development of alternative sources for these molecules. The most obvious alternative is to engineer fermentable microbe with the genes encoding for this biosynthetic capability. This has been achieved, but this biochemical process is inherently inefficient due to the biophysical properties of the enzymes responsible for this biosynthetic reactions. In the current proposal, we are proposing two objectives to improve the efficiency of these enzymes by using a combination of computational modeling and simulations couple with site-directed mutagenesis to evolve the enzymes responsible for these catalytic activities. Those improved genes would then be deployed into C. necator by the Pfleger group and subsequently co-cultured with the LanzaTech organism to determine quantitative efficiency of converting CO2 to high value terpene molecules. We previously discovered that the biosynthesis of botryococcene from farnesyl diphosphate (FPP) is catalyzed by the combined activity of 2 enzymes. This is in contrast to the biosynthesis of squalene from FPP by squalene synthase. In the past, we engineered platforms for the production of botryococcene by co-expressing a chimeric enzyme, SSL-1,3 – a fusion of Squalene synthase-like 1 enzyme, SSL-1, with SSL-3 into one multi-domain, large polypeptide. However, this complex is even less efficient in the biosynthesis of botryococcene that is the native squalene synthase enzyme for squalene. Therefore, the first objective is to use molecular modeling to predict how to mutate botryococcene biosynthesis into SQS, or how to engineer either SSL-1 and/or SSL-3 with the ability to catalyze the complete conversion of FPP to botryococcene via site-directed mutagenesis. The mutant enzymes will then be constructed and evaluated by both in vivo and in vitro biochemical assays. We also determined in previous work that neither squalene synthase nor botryococcene biosynthesis was particularly efficient in terms of absolute conversion rates of FPP to the final triterpene products. The second objective is therefore to improve the catalytic efficiency of either squalene synthase or botryococcene synthase 3- to 10-fold, which should correspond to an equal increase in the productivity of the microbial host. This work too will rely upon molecular simulations to identify particular residues within the respective enzymes that could be contributing to the catalytic limitations of squalene synthase and botryococcene synthase. We will then mutate these residues and characterize the resulting mutant enzymes by carrying out in vivo and in vitro biochemical assays, similar to those in objective 1.
Effective start/end date10/1/219/30/24


  • University of Wisconsin: $740,115.00


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