Biology subjects

Santala, V.

Publications and source records attributed to Santala, V..

3 recordsLinked to original sources

bioRxiv2018

Synthetic Metabolic Pathway for the Production of 1-Alkenes from Lignin-derived Molecules

Integration of synthetic metabolic pathways to catabolically diverse chassis provides new opportunities for sustainable production. One attractive scenario is the use of abundant waste material to produce readily collectable product, minimizing production costs. Towards that end, we established the production of semivolatile medium-chain -olefins from lignin-derived monomers: we constructed 1-undecene synthesis pathway in Acinetobacter baylyi ADP1 using ferulate as the sole carbon source. In order to overcome the toxicity of ferulate, we first applied adaptive laboratory evolution, resulting in a highly ferulate-tolerant strain. Next, we demonstrated the 1-undecene production from glucose by heterologously expressing a fatty acid decarboxylase UndA and a thioesterase TesA in the wild type strain. Finally, we constructed the alkene synthesis pathway in the ferulate-tolerant strain. We were able to produce 1-undecene from ferulate and collect the product from the culture headspace without downstream processing. This study demonstrates the potential of bacterial lignin upgradation into value-added products.

bioengineering↗

bioRxiv2018

Production of alkanes from CO2 by engineered bacteria

BackgroundMicrobial biosynthesis of alkanes is considered a promising method for the sustainable production of drop-in fuels and chemicals. Carbon dioxide would be an ideal carbon source for these production systems, but efficient production of long carbon chains from CO2 is difficult to achieve in a single organism. A potential solution is to employ acetogenic bacteria for the reduction of CO2 to acetate, and engineer a second organism to convert the acetate into long-chain hydrocarbons.\n\nResultsIn this study, we demonstrate alkane production from CO2 by a system combining the acetogen Acetobacterium woodii and a non-native alkane producer Acinetobacter baylyi ADP1 engineered for alkane production. Nine synthetic two-step alkane biosynthesis pathways consisting of different aldehyde- and alkane-producing enzymes were combinatorically constructed and expressed in A. baylyi. The aldehyde-producing enzymes studied were AAR from Synechococcus elongatus, Acr1 from A. baylyi, and Ramo, a putative dehydrogenase, from Nevskia ramosa. The alkane-producing enzymes were ADOs from S. elongatus and Nostoc punctiforme, and CER1 from Arabidopsis thaliana. The performance of the pathways was evaluated with a twin-layer biosensor, which allowed the monitoring of both the intermediate, fatty aldehyde, as well as the alkane production. The highest alkane production, as indicated by the biosensor, was achieved with a pathway consisting of AAR and ADO from S. elongatus. The performance of this pathway was further improved by balancing the relative expression levels of the enzymes in order to limit the accumulation of the intermediate fatty aldehyde. Finally, the acetogen A. woodii was used to produce acetate from CO2 and H2, and the acetate was used for alkane production by the engineered A. baylyi, thereby leading to the net production of long-chain alkanes from CO2.\n\nConclusionsA modular system for the production of drop-in liquid fuels from CO2 was demonstrated. Among the studied synthetic pathways, the combination of ADO and AAR from S. elongatus was found to be the most efficient in heterologous alkane production in A. baylyi. Furthermore, limiting the accumulation of the fatty aldehyde intermediate was found to be beneficial for the alkane production.

bioengineering↗

bioRxiv2018

Dynamic decoupling of biomass and lipid biosynthesis by autonomously regulated switch

For improving the microbial production of fuels and chemicals, gene knock-outs and overexpression are routinely applied to intensify the carbon flow from substrate to product. However, their possibilities in dynamic control of the flux between the biomass and product synthesis are limited, whereas dynamic metabolic switches can be used for optimizing the distribution of carbon and resources. The production of single cell oils is especially challenging, as the synthesis is strongly regulated, competes directly with biomass, and requires defined conditions, such as nitrogen limitation. Here, we engineered a metabolic switch for redirecting carbon flow from biomass to wax ester production in Acinetobacter baylyi ADP1 using acetate as a carbon source. Isocitrate lyase, an essential enzyme for growth on acetate, was expressed under an arabinose inducible promoter. The autonomous downregulation of the expression is based on the gradual oxidation of the arabinose inducer by a glucose dehydrogenase gcd. The depletion of the inducer, occurring simultaneously to acetate consumption, switches the cells from a biomass mode to a lipid synthesis mode, enabling the efficient channelling of carbon to wax esters in a simple batch culture. In the engineered strain, the yield and titer of wax esters were improved by 3.8 and 3.1 folds, respectively, over the control strain. In addition, the engineered strain accumulated wax esters 19% of cell dry weight, being the highest reported among microbes. The study provides important insights into the dynamic engineering of the biomass-dependent synthesis pathways for the improved production of biocompounds from low-cost, sustainable substrates.\n\nSignificance statementIn the biological production, one of the greatest challenges is to find ways for optimal distribution of resources between cell growth, maintenance, and product synthesis. Robust and reliable circuits are required to allow autonomous switching of cells from biomass mode to lipid synthesis mode. Dynamic production of single cell oils such as triacylglycerols and wax esters is especially challenging due to the strong regulation. We present a dynamic genetic circuit based on conditional knockdown of a glyoxylate shunt enzyme, which is essential for cell growth. By gradual repression of the gene, the cells autonomously switch from biomass mode to product synthesis mode. We demonstrate the functionality of the circuit by using bacterium Acinetobacter baylyi ADP1 for the production of long chain alkyl esters, namely wax esters, with titer and yield improved by over 3-fold using acetate as the carbon source.

synthetic biology↗