Search

Plant-derived secondary metabolites have many applications as fragrances, food additives, dyes, and bioactive medicinal compounds. Production of these compounds in plant cell culture has many advantages, and metabolic engineering strategies have been developed to increase yields. Yet, long-term subculturing can reduce productivity due to epigenetic downregulation of secondary metabolism. Innovative techniques that take advantage of new insights into epigenetic regulation have been developed for a variety of eukaryotes. Applying this epigenetic control to plant cell culture could restore and maintain high productivity, even in cultures that have been in suspension for many years. Here, we highlight current strategies for plant metabolic engineering and review the current understanding of epigenetic regulation in plants, with particular emphasis on applications to secondary metabolic engineering. We then discuss work in the emerging field of epigenetic engineering and postulate how epigenetic engineering techniques developed in mammalian systems could be used to overcome current limitations for industrial production of natural products in plant systems.

Pharmaceutical production hosts may be derived from almost any organism, from Chinese Hamster Ovary (CHO) cell lines to isolated actinomycetes. Each host can be improved, historically only through adaptive evolution. Recently, the maturation of organism engineering has expanded the available models, methods, and tools for altering host phenotypes. New tools like CRISPR-associated endonucleases promise to enable precise cellular reprogramming and to access previously intractable hosts. In this review, we discuss the most recent advances in engineering several types of pharmaceutical production hosts. These include model organisms, potential platform hosts with advantageous metabolism or physiology, specialized producers capable of unique biosynthesis, and CHO, the most widely used recombinant protein production host. To realize improved engineered hosts, an increasing number of approaches involving DNA sequencing and synthesis, host rewriting technologies, computational methods, and organism engineering strategies must be used. Integrative workflows that enable application of the right combination of methods to the right production host could enable economical production solutions for emerging human health treatments.

Metabolic engineering requires multiple rounds of strain construction to evaluate alternative pathways and enzyme concentrations. Optimizing multigene pathways stepwise or by randomly selecting enzymes and expression levels is inefficient. Here, we apply methods from design of experiments (DOE) to guide the construction of strain libraries from which the maximum information can be extracted without sampling every possible combination. We use Saccharomyces cerevisiae as a host for a novel six-gene pathway to itaconic acid, selected by comparing alternative shunt pathways that bypass the mitochondrial TCA cycle. The pathway is distinctive for the use of acetylating acetaldehyde dehydrogenase to increase cytosolic acetyl-CoA pools, a bacterial enzyme to synthesize citrate in the cytosol, and an itaconic acid exporter. Precise control over the expression of each gene is enabled by a set of promoter-terminator pairs that span a 174-fold range. Two large combinatorial libraries (160 variants, 2.4 Mb and 32 variants, 0.6 Mb) are designed where the expression levels are selected by statistical methods (I-optimal response surface methodology, full factorial, or Plackett-Burman) with the intent of extracting different types of guiding information after the screen. This is applied to the design of a third library (24 variants, 0.5 Mb) intended to alleviate a bottleneck in cis-aconitate decarboxylase (CAD) expression. The top strain produces 815 mg/l itaconic acid, a 4-fold improvement over the initial strain achieved by iteratively balancing pathway expression. Including a methylated product in the total, the strain produces 1.3 g/l combined itaconic acids. Further, a regression analysis of the libraries reveals the optimal expression level of CAD as well as pairwise interdependencies between genes that result in increased titer and purity of itaconic acid. This work demonstrates adapting algorithmic design strategies to guide automated yeast strain construction and learn information after each iteration.

Centralized facilities for genetic engineering, or "biofoundries", offer the potential to design organisms to address emerging needs in medicine, agriculture, industry, and defense. The field has seen rapid advances in technology, but it is difficult to gauge current capabilities or identify gaps across projects. To this end, our foundry was assessed via a timed "pressure test", in which 3 months were given to build organisms to produce 10 molecules unknown to us in advance. By applying a diversity of new approaches, we produced the desired molecule or a closely related one for six out of 10 targets during the performance period and made advances toward production of the others as well. Specifically, we increased the titers of 1-hexadecanol, pyrrolnitrin, and pacidamycin D, found novel routes to the enediyne warhead underlying powerful antimicrobials, established a cell-free system for monoterpene production, produced an intermediate toward vincristine biosynthesis, and encoded 7802 individually retrievable pathways to 540 bisindoles in a DNA pool. Pathways to tetrahydrofuran and barbamide were designed and constructed, but toxicity or analytical tools inhibited further progress. In sum, we constructed 1.2 Mb DNA, built 215 strains spanning five species ( Saccharomyces cerevisiae, Escherichia coli, Streptomyces albidoflavus, Streptomyces coelicolor, and Streptomyces albovinaceus), established two cell-free systems, and performed 690 assays developed in-house for the molecules.

Traditional metabolic pathway engineering rarely considers the influence of molecular transport. Here, we describe the directed evolution of two heterologous transporters, Candida intermedia GXS1 and Scheffersomyces stipitis XUT3. Growth rate on xylose was improved up to 70% by mutant transporter expression. Most mutants were found to exhibit vastly improved V max values and display an increase in high cell density sugar consumption rates. Mixed glucose and xylose fermentations reveal that mutant transporters can alter the diauxic shift dynamics and the simultaneous sugar utilization capacity of the host strain. Analysis of mutations highlights several important residues influencing transporter function including point mutations at F40 of C. intermedia GXS1 and at E538 of S. stipitis XUT3. This work is the first to demonstrate that molecular transporter proteins can be improved for biotechnological applications through directed evolution in yeast.

Yeasts serve as an attractive host organism for the production of biofuels from biomass. These organisms are well suited for industrial fermentation and have many advantages in biomass conversion processes. However, many challenges still remain that limit overall production efficiency, especially for the case of converting lignocellulose. This chapter first highlights advances in metabolic engineering, systems biology, and synthetic biology that support the rewiring of yeasts for fuels applications. Next, we address challenges and advances in moving beyond glucose as the sole carbon source. Finally, we address opportunities for the future by describing advances in moving beyond bioethanol as the fuel and describe methodologies to move beyond current limits of rate, titer, and yield.

Utilization of exogenous sugars found in lignocellulosic biomass hydrolysates, such as xylose, must be improved before yeast can serve as an efficient biofuel and biochemical production platform. In particular, the first step in this process, the molecular transport of xylose into the cell, can serve as a significant flux bottleneck and is highly inhibited by other sugars. Here we demonstrate that sugar transport preference and kinetics can be rewired through the programming of a sequence motif of the general form G-G/F-XXX-G found in the first transmembrane span. By evaluating 46 different heterologously expressed transporters, we find that this motif is conserved among functional transporters and highly enriched in transporters that confer growth on xylose. Through saturation mutagenesis and subsequent rational mutagenesis, four transporter mutants unable to confer growth on glucose but able to sustain growth on xylose were engineered. Specifically, Candida intermedia gxs1 Phe(38)Ile(39)Met(40), Scheffersomyces stipitis rgt2 Phe(38) and Met(40), and Saccharomyces cerevisiae hxt7 Ile(39)Met(40)Met(340) all exhibit this phenotype. In these cases, primary hexose transporters were rewired into xylose transporters. These xylose transporters nevertheless remained inhibited by glucose. Furthermore, in the course of identifying this motif, novel wild-type transporters with superior monosaccharide growth profiles were discovered, namely S. stipitis RGT2 and Debaryomyces hansenii 2D01474. These findings build toward the engineering of efficient pentose utilization in yeast and provide a blueprint for reprogramming transporter properties.

Molecular transport is a key process in cellular metabolism. This step is often limiting when using a nonnative carbon source, as exemplified by xylose catabolism in Saccharomyces cerevisiae . As a step toward addressing this limitation, this study seeks to characterize monosaccharide transport preference and efficiency. A group of 26 known and putative monosaccharide transport proteins was expressed in a recombinant Saccharomyces cerevisiae host unable to transport several monosaccharides. A growth-based assay was used to detect transport capacity across six different carbon sources (glucose, xylose, galactose, fructose, mannose, and ribose). A mixed glucose-and-xylose cofermentation was performed to determine substrate preference. These experiments identified 10 transporter proteins that function as transporters of one or more of these sugars. Most of these proteins exhibited broad substrate ranges, and glucose was preferred in all cases. The broadest transporters confer the highest growth rates and strongly prefer glucose. This study reports the first molecular characterization of the annotated XUT genes of Scheffersomyces stipitis and open reading frames from the yeasts Yarrowia lipolytica and Debaryomyces hansenii. Finally, a phylogenetic analysis demonstrates that transporter function clusters into three distinct groups. One particular group comprised of D. hansenii XylHP and S. stipitis XUT1 and XUT3 demonstrated moderate transport efficiency and higher xylose preferences.

The general central dogma frames the emergent properties of life, which make biology both necessary and difficult to engineer. In a process engineering paradigm, each biological process stream and process unit is heavily influenced by regulatory interactions and interactions with the surrounding environment. Synthetic biology is developing the tools and methods that will increase control over these interactions, eventually resulting in an integrative synthetic biology that will allow ground-up cellular optimization. In this review, we attempt to contextualize the areas of synthetic biology into three tiers: (1) the process units and associated streams of the central dogma, (2) the intrinsic regulatory mechanisms, and (3) the extrinsic physical and chemical environment. Efforts at each of these three tiers attempt to control cellular systems and take advantage of emerging tools and approaches. Ultimately, it will be possible to integrate these approaches and realize the vision of integrative synthetic biology when cells are completely rewired for biotechnological goals. This review will highlight progress towards this goal as well as areas requiring further research.

Variation in scion physiology caused by rootstock families, scion clones, and the rootstock–scion interactions were studied in loblolly pine (Pinustaeda L.). Ten full-sib families were used as rootstocks, and six scion clones were grafted on the rootstocks in all combinations. The trees were measured during and after the 2nd year after grafting; the traits measured were needle carbohydrate (starch, sucrose, and hexoses) concentrations and needle N, P, K, Ca, and Mg concentrations. These traits were measured in early fall and early spring. There were highly significant differences among scion clones for all traits. Rootstocks significantly affected hexoses in fall, total sugars in spring, P, K, Ca, and Mg in fall, and Ca and Mg in spring. For most traits the largest variance component was within each rootstock-scion combination; next largest were those for scion clones. Variance components for rootstocks were small or negative for most traits, indicating that the scion has more control than the rootstock on scion physiology of young grafted loblolly pine.

Hexose and pentose cofermentation is regarded as one of the chief obstacles impeding economical conversion of lignocellulosic biomass to biofuels. Over time, successful application of traditional metabolic engineering strategy has produced yeast strains capable of utilizing the pentose sugars (especially xylose and arabinose) as sole carbon sources, yet major difficulties still remain for engineering simultaneous, exogenous sugar metabolism. Beyond catabolic pathways, the focus must shift towards non-traditional aspects of cellular engineering such as host molecular transport capability, catabolite sensing and stress response mechanisms. This review highlights the need for an approach termed 'panmetabolic engineering', a new paradigm for integrating new carbon sources into host metabolic pathways. This approach will concurrently optimize the interdependent processes of transport and metabolism using novel combinatorial techniques and global cellular engineering. As a result, panmetabolic engineering is a whole pathway approach emphasizing better pathways, reduced glucose-induced repression and increased product tolerance. In this paper, recent publications are reviewed in light of this approach and their potential to expand metabolic engineering tools. Collectively, traditional approaches and panmetabolic engineering enable the reprogramming of extant biological complexity and incorporation of exogenous carbon catabolism.

Blue spruce (Piceapungens Engelm.) seedlings grown in a nursery for 1 to 5 years and seedlings grown from seed in a greenhouse under continuous light for 2 to 6 months were studied to determine (1) time to bud set on transfer to short days, (2) time to bud-break on subsequent transfer to long days, and (3) the anatomy of the dormant shoot apex. Seasonal development of the shoot apex of a single 50-year-old blue spruce was also monitored.Time to but set on transfer to short days decreased after long periods under continuous light. Time to budbreak on subsequent transfer to long days increased with increasing age in nursery- and greenhouse-grown seedlings. The dormant shoot apex became more highly differentiated as the nursery-grown seedlings aged from 1 to 3 years, then showed no significant difference after 3 years of age.The 50-year-old blue spruce initiated many new needles in the current bud before bud scale formation, which began in mid-May. Needle primordia initiation in the new bud began in late June and slowed down in late August. Apical dome diameter increased and decreased concurrently with the increase and decrease in rate of needle primordia initiation.