Hi Coja

I PPc I

2CO2 + 2H2

Fumarate

2CO2 + 2H2

A 2Pyruvate —Z^r 2D-lactate

2L-lactate

Succinate <Dl2Acetyl-C°A 4H

2Acetyl-P 2Acetaldehyde

2Acetate 2Ethanol

Figure 5.6 Metabolic engineering of E. coli to produce D- and L-lactate. See Figure 5.1 for explanation of different types of lines/arrows. Genes involved in engineered steps are indicated by italics. Primary fermentation products are indicated in bold. 1- Engineering a homo-D-lactic pathway by introducing mutations in pta and ppc genes; 2- Engineering a homo-D-lactic pathway by introducing mutations in frdBC, ackA, adhE, and pflB genes; 3- Engineering a homo-L-lactic pathway by introducing mutations in pta and ldhA genes and expressing an L-lactate dehydrogenase gene from L. casei. Abbreviations, adhE, gene encoding acetaldehyde/alcohol dehydrogenase; ackA, gene encoding acetate kinase; frdABCD, operon encoding fumarate reductase; ldhA, gene encoding lactate dehydrogenase; PEP, phosphoenolpyruvate; pflB, gene encoding pyruvate-formate lyase; ppc, gene encoding PEP carboxylase; pta, gene encoding phosphotransacetylase.

has no auxotrophic requirement. Glycolytic flux in TC36 (0.3 ijmolmin^mg1 protein) was twice that of the parent. Higher flux was attributed to a deletion of membrane-coupling subunits in (F1F0)H+-ATP synthase that inactivated ATP synthesis while retaining cytoplasmic F1-ATPase activity. The oxygen requirements of this system are energy intensive, so in terms of commercial production of acetate it would be highly desirable to have an anaerobic system able to efficiently produce acetic acid. Our laboratory is currently engineering E. coli to produce acetate as the main fermentation product under anaerobic conditions (data not published). The general strategy is based on first eliminating competing pathways (by inactivating correspondent genes) in order to redirect the carbon flux toward the synthesis of acetate, and then transforming the engineered pathway into a viable alternative for cell growth (by introducing modifications that will balance consumption and production of NADH).

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