Engineering Escherichia coli coculture systems for the production of biochemical products

Abstract

Engineering microbial consortia to express complex biosynthetic pathways efficiently for the production of valuable compounds is a promising approach for metabolic engineering and synthetic biology. Here, we report the design, optimization, and scale-up of an Escherichia coli-E. coli coculture that successfully overcomes fundamental microbial production limitations, such as high-level intermediate secretion and low-efficiency sugar mixture utilization. For the production of the important chemical cis,cis-muconic acid, we show that the coculture approach achieves a production yield of 0.35 g/g from a glucose/xylose mixture, which is significantly higher than reported in previous reports. By efficiently producing another compound, 4-hydroxybenzoic acid, we also demonstrate that the approach is generally applicable for biosynthesis of other important industrial products.metabolic engineering | microbial coculture | muconic acid | 4-hydroxybenzoic acidM etabolic engineering and synthetic biology have made great strides in constructing and optimizing metabolic pathways for biochemical product synthesis in a pure culture (1, 2). There are, however, situations where this approach may be limited, as in the cases where (i) a single host cell cannot provide an optimal environment for the functioning of all pathway enzymes, (ii) biosynthetic efficiency is reduced due to overwhelming metabolic stress from the overexpression of long and complex pathways (3, 4), or (iii) pathway intermediates are secreted yielding undesired byproducts and reducing substrate utilization (5, 6). The above limitations can potentially be overcome through the use of rationally designed microbial coculture systems. Different from previous modular engineering approaches, such coculturebased systems completely modularize and segregate a biosynthetic pathway into two separate cells, each of which carries a portion of the pathway, and thus can be engineered independently to achieve optimal functioning of the combined pathway.The concept of microbial cocultures is not new. However, previous research on microbial consortia was primarily concerned with the study of mixed population stability and dynamic interactions (7-11), although a few recent studies have reported the engineering of microbial consortia for utilization of simple sugars to make small molecules of central carbon metabolism, such as ethanol and lactate (12, 13). Progress was made recently, when a full n-butanol pathway was expressed in two separate E. coli cells to achieve higher production (14) and when a bacterium-yeast coculture was used to address the difficulties of functional reconstitution of a pathway involving prokaryotic and eukaryotic enzymes in a consortium, and thus improved production of complex pharmaceutical molecules (15). Here, we expand the generality of the coculture engineering by demonstrating that microbial cocultures can also be engineered to overcome more universal challenges in metabolic engineering, including high-level intermediate secretion and low-efficiency s...

Co‐culture engineering for microbial biosynthesis of 3‐amino‐benzoic acid in Escherichia coli

“…It has been previously shown that co‐culture engineering can provide important advantages in the production of important biochemicals . Here, we further show that co‐culture engineering can also facilitate effective selection of appropriate E. coli hosts that best accommodate a desired biosynthetic module.…”

“…Both XL2‐Blue and XL10‐Gold have the chloramphenicol resistance genes integrated in the chromosome and were only transformed with pdc‐VS. All 10 transformed strains were co‐cultivated with E. coli P5.2 that had been previously engineered to provide DHS precursor . Plasmid pGro7 contained E. coli chaperone genes, but expression of these genes was completely un‐induced in this study.…”

Requirement of a Functional Flavin Mononucleotide Prenyltransferase for the Activity of a Bacterial Decarboxylase in a Heterologous Muconic Acid Pathway in Saccharomyces cerevisiae

“…One of the homologues (accession no. AAY57855) is organized in a BCD cluster and has been demonstrated to be highly oxygen-sensitive (5), whereas the other homologue (isomeric aroY-C [aroY-C iso ], AB479384) has been successfully utilized for cis,cis-muconic acid production in S. cerevisiae (5)(6)(7)(8) and E. coli (3,4,10,11). Contradictory results exist about the necessity to coexpress B (aroY-B; AAY57854) and D (aroY-D; AAY57856) genes to obtain decarboxylase activity in S. cerevisiae.…”

Emerging tools, enabling technologies, and future opportunities for the bioproduction of aromatic chemicals

“…Finally, Zhang and colleagues demonstrated how, by distributing a pathway across two E. coli modular cells, the natural secretion of a key pathway intermediate (i.e. 3‐dehydroshikimate) could be leveraged to improve the production of both 4‐hydroxybenzoic acid and cis , cis ‐muconic acid . In this case, to ensure stability of the co‐culture, one cell was engineered to import and consume only glucose and the other only xylose.…”