Homofermentative production of d- or l-lactate in metabolically engineered escherichia coli rr1

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 1999, p. 1384–1389 0099-2240/99/$04.00 0 Copyright © 1999, American Society for Microbiology. All Rights Reserved.

Vol. 65, No. 4

Homofermentative Production of D- or L-Lactate in Metabolically Engineered Escherichia coli RR1
DONG-EUN CHANG,1,2 HEUNG-CHAE JUNG,1 JOON-SHICK RHEE,2
AND

JAE-GU PAN1*

Bioprocess Engineering Division, KoreaResearch Institute of Bioscience and Biotechnology, Yusong, Taejon 305-600,1 and Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Yusong, Taejon 305-701,2 Korea
Received 16 September 1998/Accepted 3 January 1999

We investigated metabolic engineering of fermentation pathways in Escherichia coli for production of optically pure D- or L-lactate. Several ptamutant strains were examined, and a pta mutant of E. coli RR1 which was deficient in the phosphotransacetylase of the Pta-AckA pathway was found to metabolize glucose to D-lactate and to produce a small amount of succinate by-product under anaerobic conditions. An additional mutation in ppc made the mutant produce D-lactate like a homofermentative lactic acid bacterium. When the pta ppc double mutantwas grown to higher biomass concentrations under aerobic conditions before it shifted to the anaerobic phase of D-lactate production, more than 62.2 g of D-lactate per liter was produced in 60 h, and the volumetric productivity was 1.04 g/liter/h. To examine whether the blocked acetate flux could be reoriented to a nonindigenous L-lactate pathway, an L-lactate dehydrogenase gene from Lactobacilluscasei was introduced into a pta ldhA strain which lacked phosphotransacetylase and D-lactate dehydrogenase. This recombinant strain was able to metabolize glucose to L-lactate as the major fermentation product, and up to 45 g of L-lactate per liter was produced in 67 h. These results demonstrate that the central fermentation metabolism of E. coli can be reoriented to the production of D-lactate, anindigenous fermentation product, or to the production of L-lactate, a nonindigenous fermentation product. Lactate and its derivatives have been used in a wide range of food-processing and industrial applications (8, 27). Because lactate can be easily converted to strong, highly transparent, and readily biodegradable polyesters, it is emerging as a potential material for environmentally friendlyplastics. As the physical properties of polylactate depend on the isomeric composition of lactate (28), production of optically pure lactate is a prerequisite for polymer synthesis in which lactate is used. Lactate has been produced commercially either by chemical synthesis or by fermentation (8). In contrast to chemical processes, the fermentation process is able to produce the desiredstereoisomer. Many microorganisms produce D-lactate, and some lactic acid bacteria, such as Lactobacillus bulgaricus, produce highly pure D-lactate (2). L-Lactate also has been produced by using lactic acid bacteria, such as Lactobacillus helveticus, Lactobacillus amylophilus, and Lactobacillus delbruekii (27). It has also been proposed that a mutant of the racemic mixture producer L. helveticus defective inD-lactate dehydrogenase (D-LDH) could be used for production of optically pure L-lactate (3). As lactic acid bacteria have complex nutritional requirements and very low growth rates (24), Rhizopus oryzae and Bacillus laevolacticus have been proposed as alternative producers (9, 25). Escherichia coli has many advantageous characteristics as a production host, such as rapid growth under aerobic andanaerobic conditions and simple nutritional requirements. Moreover, well-established protocols for genetic manipulation and a large physiological knowledge base should enable the development of E. coli as a host for production of optically pure D- or L-lactate by metabolic engineering. E. coli, a facultative anaerobe, carries out mixed-acid fer* Corresponding author. Mailing address: Bioprocess...