David R. Nielsen

Engineering alternative butanol production platforms in heterologous bacteria

Alternative microbial hosts have been engineered as biocatalysts for butanol biosynthesis. The butanol synthetic pathway of Clostridium acetobutylicum was first re-constructed in Escherichia coli to establish a baseline for comparison to other hosts. Whereas polycistronic expression of the pathway genes resulted in the production of 34 mg/L butanol, individual expression of pathway genes elevated titers to 200 mg/L. Improved titers were achieved by co-expression of Saccharomyces cerevisiae formate dehydrogenase while overexpression of E. coli glyceraldehyde 3-phosphate dehydrogenase to elevate glycolytic flux improved titers to 580 mg/L. Pseudomonas putida and Bacillus subtilis were also explored as alternative production hosts. Polycistronic expression of butanol biosynthetic genes yielded butanol titers of 120 and 24 mg/L from P. putida and B. subtilis, respectively. Production in the obligate aerobe P. putida was dependent upon expression of bcd-etfAB. These results demonstrate the potential of engineering butanol biosynthesis in a variety of heterologous microorganisms, including those cultivated aerobically.

Styrene Biosynthesis from Glucose by Engineered E. coli

Styrene is a large volume, commodity petrochemical with diverse commercial applications, including as a monomer building-block for the synthesis of many useful polymers. Here we demonstrate how, through the de novo design and development of a novel metabolic pathway, styrene can alternatively be synthesized from renewable substrates such as glucose. The conversion of endogenously synthesized l-phenylalanine to styrene was achieved by the co-expression of phenylalanine ammonia lyase and trans-cinnamate decarboxylase. Candidate isoenzymes for each step were screened from bacterial, yeast, and plant genetic sources. Finally, over-expression of PAL2 from Arabidopsis thaliana and FDC1 from Saccharomyces cerevisiae (originally classified as ferulate decarboxylase) in an l-phenylalanine over-producing Escherichia coli host led to the accumulation of up to 260 mg/L in shake flask cultures. Achievable titers already approach the styrene toxicity threshold (determined as ~300 mg/L). To the best of our knowledge, this is the first report of microbial styrene production from sustainable feedstocks.

In Situ Product Recovery of n-Butanol Using Polymeric Resins

Polymeric resins with high n‐butanol adsorption affinities were identified from a candidate pool of commercially available materials representing a wide array of physical and chemical properties. Resin hydrophobicity, which was dictated by the chemical structure of its constituent monomer units, most greatly influenced the resin‐aqueous equilibrium partitioning of n‐butanol whereas ionic functionalization appeared to have no effect. In general, those materials derived from poly(styrene‐co‐divinylbenzene) possessed the greatest n‐butanol affinity, while the adsorption potential of these resins was limited by their specific surface area. Resins were tested for their ability to serve as effective in situ product recovery (ISPR) devices in the n‐butanol fermentation by Clostridium acetobutylicum ATCC 824. In small‐scale batch fermentations, the addition of 0.05 kg/L Dowex® Optipore SD‐2 facilitated achievement of effective n‐butanol titers as high as 2.22% (w/v), well above the inhibitory threshold of C. acetobutylicum ATCC 824, and nearly twice that of traditional, single‐phase fermentations. Retrieval of n‐butanol from resins via thermal treatment was demonstrated with high efficiency and predicted to be economically favorable. Due to its modular nature, the proposed ISPR design exhibits strong potential for compatibility with future n‐butanol fermentation efforts. Biotechnol. Bioeng. 2009; 102: 811–821.

Metabolic Engineering of Escherichia coli for Enhanced Production of (R)- and (S)-3-Hydroxybutyrate

ABSTRACT Synthetic metabolic pathways have been constructed for the production of enantiopure (R)- and (S)-3-hydroxybutyrate (3HB) from glucose in recombinant Escherichia coli strains. To promote maximal activity, we profiled three thiolase homologs (BktB, Thl, and PhaA) and two coenzyme A (CoA) removal mechanisms (Ptb-Buk and TesB). Two enantioselective 3HB-CoA dehydrogenases, PhaB, producing the (R)-enantiomer, and Hbd, producing the (S)-enantiomer, were utilized to control the 3HB chirality across two E. coli backgrounds, BL21Star(DE3) and MG1655(DE3), representing E. coli B- and K-12-derived strains, respectively. MG1655(DE3) was found to be superior for the production of each 3HB stereoisomer, although the recombinant enzymes exhibited lower in vitro specific activities than BL21Star(DE3). Hbd in vitro activity was significantly higher than PhaB activity in both strains. The engineered strains achieved titers of enantiopure (R)-3HB and (S)-3HB as high as 2.92 g liter−1 and 2.08 g liter−1, respectively, in shake flask cultures within 2 days. The NADPH/NADP+ ratio was found to be two- to three-fold higher than the NADH/NAD+ ratio under the culture conditions examined, presumably affecting in vivo activities of PhaB and Hbd and resulting in greater production of (R)-3HB than (S)-3HB. To the best of our knowledge, this study reports the highest (S)-3HB titer achieved in shake flask E. coli cultures to date.

Engineering cyanobacteria for photosynthetic production of 3-hydroxybutyrate directly from CO2.

(S)- and (R)-3-hydroxybutyrate (3HB) are precursors to synthesize the biodegradable plastics polyhydroxyalkanoates (PHAs) and many fine chemicals. To date, however, their production has been restricted to petroleum-based chemical industry and sugar-based microbial fermentation, limiting its sustainability and economical feasibility. With the ability to fix CO2 photosynthetically, cyanobacteria have attracted increasing interest as a biosynthesis platform to produce fuels and chemicals from alternative renewable resources. To this end, synthesis metabolic pathways have been constructed and optimized in cyanobacterium Synechocystis sp. PCC 6803 to photosynthetically produce (S)- and (R)-3HB directly from CO2. Both types of 3HB molecules were produced and readily secreted from Synechocystis cells without over-expression of transporters. Additional inactivation of the competing pathway by deleting slr1829 and slr1830 (encoding PHB polymerase) from the Synechocystis genome further promoted the 3HB production. Up to 533.4mg/L 3HB has been produced after photosynthetic cultivation of the engineered cyanobacterium Synechocystis TABd for 21 days. Further analysis indicated that the phosphate consumption during the photoautrophic growth and the concomitant elevated acetyl-CoA pool acted as a key driving force for 3HB biosynthesis in Synechocystis. For the first time, the study has demonstrated the feasibility of photosynthetic production of (S)- and (R)-3HB directly from sunlight and CO2.

Metabolic engineering of acetoin and meso‐2, 3‐butanediol biosynthesis in E. coli

The functional reconstruction of acetoin and meso‐2,3‐butanediol (meso‐2,3‐BD) biosynthetic pathways in Escherichia coli have been explored systematically. Pathway construction involved the in vsivo screening of prospective pathway isozymes of yeast and bacterial origin. After substantial engineering of the host background to increase pyruvate availability, E. coli YYC202(DE3) ldhA( ilvC( expressing ilvBN from E. coli and aldB from L. lactis (encoding acetolactate synthase and acetolactate decarboxylase activities, respectively) was able to produce up to 870 mg/L acetoin, with no coproduction of diacetyl observed. These strains were also found to produce small quantities of meso‐2,3‐BD, suggesting the existence of endogenous 2,3‐BD dehydrogenase activity. Finally, the coexpression of bdh1 from S. cerevisiae, encoding 2,3‐BD dehydrogenase, in this strain resulted in the production of up to 1120 mg/L meso‐2,3‐BD, with glucose a yield of 0.29 g/g. While disruption of the native lactate biosynthesis pathway increased pyruvate precursor availability to this strain, increased availability of NADH for acetoin reduction to meso‐2,3‐BD was found to be the most important consequence of ldhA deletion.

Engineering microbes with synthetic biology frameworks

Typically, the outcome of biologically engineered unit operations cannot be controlled a priori due to the incorporation of ad hoc design into complex natural systems. To mitigate this problem, synthetic biology presents a systematic approach to standardizing biological components for the purpose of increasing their programmability and robustness when assembled with the aim to achieve novel biological functions. A complex engineered biological system using only standardized biological components is yet to exist. Nevertheless, current attempts to create and to implement modular, standardized biological components pave the way for the future creation of highly predictable artificial biological systems. Although synthetic biology frameworks can be applied to any biological engineering endeavor, this article will focus on providing a brief overview of advances in the field and its recent utilization for the engineering of microbes.

Synthetic Metabolism : Engineering Biology at the Protein and Pathway Scales

Biocatalysis has become a powerful tool for the synthesis of high-value compounds, particularly so in the case of highly functionalized and/or stereoactive products. Nature has supplied thousands of enzymes and assembled them into numerous metabolic pathways. Although these native pathways can be use to produce natural bioproducts, there are many valuable and useful compounds that have no known natural biochemical route. Consequently, there is a need for both unnatural metabolic pathways and novel enzymatic activities upon which these pathways can be built. Here, we review the theoretical and experimental strategies for engineering synthetic metabolic pathways at the protein and pathway scales, and highlight the challenges that this subfield of synthetic biology currently faces.

Engineering microbial chemical factories to produce renewable “biomonomers”

By applying metabolic engineering tools and strategies to engineer synthetic enzyme pathways, the number and diversity of commodity and specialty chemicals that can be derived directly from renewable feedstocks is rapidly and continually expanding. This of course includes a number of monomer building-block chemicals that can be used to produce replacements to many conventional plastic materials. This review aims to highlight numerous recent and important advancements in the microbial production of these so-called “biomonomers.” Relative to naturally-occurring renewable bioplastics, biomonomers offer several important advantages, including improved control over the final polymer structure and purity, the ability to synthesize non-natural copolymers, and allowing products to be excreted from cells which ultimately streamlines downstream recovery and purification. To highlight these features, a handful of biomonomers have been selected as illustrative examples of recent works, including polyamide monomers, styrenic vinyls, hydroxyacids, and diols. Where appropriate, examples of their industrial penetration to date and end-product uses are also highlighted. Novel biomonomers such as these are ultimately paving the way toward new classes of renewable bioplastics that possess a broader diversity of properties than ever before possible.

Engineering Escherichia coli for renewable production of the 5‐carbon polyamide building‐blocks 5‐aminovalerate and glutarate

Through metabolic pathway engineering, novel microbial biocatalysts can be engineered to convert renewable resources into useful chemicals, including monomer building‐blocks for bioplastics production. Here we describe the systematic engineering of Escherichia coli to produce, as individual products, two 5‐carbon polyamide building blocks, namely 5‐aminovalerate (AMV) and glutarate. The modular pathways were derived using “parts” from the natural lysine degradation pathway of Pseudomonas putida KT2440. Endogenous over‐production of the required precursor, lysine, was first achieved through metabolic deregulation of its biosynthesis pathway by introducing feedback resistant mutants of aspartate kinase III and dihydrodipicolinate synthase. Further disruption of native lysine decarboxylase activity (by deleting cadA and ldcC) limited cadaverine by‐product formation, enabling lysine production to 2.25 g/L at a glucose yield of 138 mmol/mol (18% of theoretical). Co‐expression of lysine monooxygenase and 5‐aminovaleramide amidohydrolase (encoded by davBA) then resulted in the production of 0.86 g/L AMV in 48 h. Finally, the additional co‐expression of glutaric semialdehyde dehydrogenase and 5‐aminovalerate aminotransferase (encoded by davDT) led to the production of 0.82 g/L glutarate under the same conditions. At this output, yields on glucose were 71 and 68 mmol/mol for AMV and glutarate (9.5 and 9.1% of theoretical), respectively. These findings further expand the number and diversity of polyamide monomers that can be derived directly from renewable resources. Biotechnol. Bioeng. 2013; 110: 1726–1734.