Brian Pereira

Engineering Escherichia coli coculture systems for the production of biochemical products

Significance Production of industrial compounds by using engineered microorganisms is a robust method to reduce our reliance on nonrenewable petroleum resources and increase the utility of renewable resources, such as lignocellulose. Because there are major limitations for engineering a single microbial cell to achieve high-yield production, we developed microbial coculture systems consisting of two different microbial cell types to use sugar mixtures that can be derived from lignocellulose efficiently. We demonstrate that this approach is successful for achieving high-level production of two important value-added molecules, cis,cis-muconic acid and 4-hydroxybenzoic acid. This accomplishment establishes a previously unidentified technology for advancing future research in metabolic engineering and synthetic biology. 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.

Efficient utilization of pentoses for bioproduction of the renewable two-carbon compounds ethylene glycol and glycolate.

The development of lignocellulose as a sustainable resource for the production of fuels and chemicals will rely on technology capable of converting the raw materials into useful compounds; some such transformations can be achieved by biological processes employing engineered microorganisms. Towards the goal of valorizing the hemicellulose fraction of lignocellulose, we designed and validated a set of pathways that enable efficient utilization of pentoses for the biosynthesis of notable two-carbon products. These pathways were incorporated into Escherichia coli, and engineered strains produced ethylene glycol from various pentoses, including simultaneously from D-xylose and L-arabinose; one strain achieved the greatest reported titer of ethylene glycol, 40 g/L, from D-xylose at a yield of 0.35 g/g. The strategy was then extended to another compound, glycolate. Using D-xylose as the substrate, an engineered strain produced 40 g/L glycolate at a yield of 0.63 g/g, which is the greatest reported yield to date.

Engineering a novel biosynthetic pathway in Escherichia coli for production of renewable ethylene glycol

Ethylene glycol (EG) is an important commodity chemical with broad industrial applications. It is presently produced from petroleum or natural gas feedstocks in processes requiring consumption of significant quantities of non‐renewable resources. Here, we report a novel pathway for biosynthesis of EG from the renewable sugar glucose in metabolically engineered Escherichia coli. Serine‐to‐EG conversion was first achieved through a pathway comprising serine decarboxylase, ethanolamine oxidase, and glycolaldehyde reductase. Serine provision in E. coli was then enhanced by overexpression of the serine‐biosynthesis pathway. The integration of these two parts into the complete EG‐biosynthesis pathway in E. coli allowed for production of 4.1 g/L EG at a cumulative yield of 0.14 g‐EG/g‐glucose, establishing a foundation for a promising biotechnology. Biotechnol. Bioeng. 2016;113: 376–383.

Biosynthesis of poly(glycolate-co-lactate-co-3-hydroxybutyrate) from glucose by metabolically engineered Escherichia coli

Metabolically engineered Escherichia coli strains were constructed to effectively produce novel glycolate-containing biopolymers from glucose. First, the glyoxylate bypass pathway and glyoxylate reductase were engineered such as to generate glycolate. Second, glycolate and lactate were activated by the Megasphaera elsdenii propionyl-CoA transferase to synthesize glycolyl-CoA and lactyl-CoA, respectively. Third, β-ketothiolase and acetoacetyl-CoA reductase from Ralstonia eutropha were introduced to synthesize 3-hydroxybutyryl-CoA from acetyl-CoA. At last, the Ser325Thr/Gln481Lys mutant of polyhydroxyalkanoate (PHA) synthase from Pseudomonas sp. 61-3 was over-expressed to polymerize glycolyl-CoA, lactyl-CoA and 3-hydroxybutyryl-CoA to produce poly(glycolate-co-lactate-co-3-hydroxybutyrate). The recombinant E. coli was able to accumulate the novel terpolymer with a titer of 3.90g/l in shake flask cultures. The structure of the resulting polymer was chemically characterized by proton NMR analysis. Assessment of thermal and mechanical properties demonstrated that the produced terpolymer possessed decreased crystallinity and improved toughness, in comparison to poly(3-hydroxybutyrate) homopolymer. This is the first study reporting efficient microbial production of poly(glycolate-co-lactate-co-3-hydroxybutyrate) from glucose.