(303d) A Novel Microbial Platform for the Anaerobic Synthesis of Oxidized Chemicals: Anaerobic Production of Acetic Acid in E. Coli

Current aerobic
methods used to produce oxidized chemicals via fermentation do not compete with
petrochemical routes due to their high capital and operating costs. Fermentation is a leading
expense, which can account for between 50 and 90% of total production costs of
commodity chemicals. Production of acetate, for example, result in a loss of
~50% of the sugar as CO₂ and can not use sugars obtained from
lignocellulosic biomass, which constitute a low-cost (~0.05 $/lb), sustainable
alternative for producing bulk chemicals. We are developing a novel microbial
platform for the anaerobic production of oxidized chemicals from plant biomass
sugars. The proposed E. coli-based platform will use nitrate respiration
to convert 5- and 6-carbon sugars into acetate. This process has lower capital
and operating costs than existing aerobic processes. Furthermore, the reduction
of nitrite obtained from nitrate respiration will generate ammonium and
alkalinize the medium, thus reducing the use of base for pH control and
ammonium salts in the fermentation and purification processes. Metabolic
engineering is being used as a rational approach to obtain these microbial
biocatalysts that will produce acetate via nitrate respiration. The work to be
presented at the meeting includes the engineering of E. coli for the
efficient use of nitrate as electron acceptor and nitrogen source under
anaerobic conditions and the construction of strains for the anaerobic
production of acetate acid as the main fermentation product.

     
Our strategy to maximize the conversion of nitrite into ammonia and avoid its
accumulation in the culture medium is based on preventing the extrusion of
nitrite produced by the more active membrane-associated nitrate reductases
(NarG and NarZ) and avoiding the production of nitrite via the periplasmic
nitrate reductase (NAP). Using this approach, we have disrupted, both
singularly and in combinations, the genes involved in these processes: i.e. napFDAGHBC,
encoding periplasmic nitrate reductase NAP; narK, encoding a
nitrite/nitrate transporter; and narX encoding sensor NarX which is part
of the homologous two-component regulatory system NarX/NarL (in vitro
molecular studies have shown that NarX negatively regulates the NarL protein by
acting as a NarL-phosphate phosphatase). To this end we have created all
single, double, and triple mutants. Wild-type E. coli W3110 and all
recombinant strains have been evaluated for cell growth, sugar consumption and
their capacity for the reduction of nitrate and nitrite. The most robust growth
and sugar consumption behavior (both volumetric and specific rates) were
achieved in the triple mutant strain W3110[DnapFDAGHBC,
DnarK, DnarX], which also exhibited the lowest accumulation of
nitrite in the medium due to the simultaneous reduction of nitrate to nitrite
to ammonium. On the other hand, to achieve the synthesis of acetate at high
yields we inactivated genes involved in the synthesis of competing fermentation
products including lactate (gene ldhA, encoding lactate dehydrogenase)
and succinate (genes frdABCD encoding fumarate reductase). Double mutant
W3110[DldhA, DfrdABCD] produced acetate as the main
fermentation product at a yield exceeding 90% of the theoretical maximum. Our
current efforts focus in the integration of the aforementioned modifications
into a single strain capable of simultaneously reducing nitrate to nitrite to
ammonium and producing acetate at high yield and productivity.