Consistent biosynthesis of D-glycerate from variable mixed substrates.

The use of waste streams and other renewable feedstocks in microbial biosynthesis has long been a goal for metabolic engineers. Microbes can utilize the substrate mixtures found in waste streams, though they are more technically challenging to convert to useful products compared to the single substrates of standard practice. It is difficult to achieve consistent biosynthesis in the face of the temporally changing nature of waste streams. Furthermore, the expression of all the enzymes necessary to convert mixed substrates into a product likely presents significant metabolic burden, which already plagues processes that utilize a single substrate. We developed an approach to utilize mixed feedstocks for production by activating expression of each biosynthetic pathway in the presence of its substrate. This expression control was used for two novel pathways that converted two substrates, galacturonate and gluconate, into a single product, D-glycerate. A production strain harboring both pathway plasmids produced 1.8 ± 0.3 and 1.64 ± 0.09 g L-1 of D-glycerate from galacturonate and gluconate alone, respectively. Fermentations that were fed a mixture of the two substrates, at different ratios, resulted in product titers between 1.48 ± 0.03 and 1.8 ± 0.1 g L-1. All fermentations were fed a total of 10 g L-1 substrate and there was no statistically significant difference in D-glycerate titer from the single or mixed substrate fermentations. We thus demonstrated consistent D-glycerate biosynthesis from single and mixed substrates as an example of robust conversion of complex feedstocks.

Substrate-activated expression of a biosynthetic pathway in Escherichia coli.

Microbes can facilitate production of valuable chemicals more sustainably than traditional chemical processes in many cases: they utilize renewable feedstocks, require less energy intensive process conditions, and perform a variety of chemical reactions using endogenous or heterologous enzymes. In response to the metabolic burden imposed by production pathways, chemical inducers are frequently used to initiate gene expression after the cells have reached sufficient density. While chemically inducible promoters are a common research tool used for pathway expression, they introduce a compound extrinsic to the process along with the associated costs. We developed an expression control system for a biosynthetic pathway for the production of d-glyceric acid that utilizes galacturonate as both the inducer and the substrate, thereby eliminating the need for an extrinsic chemical inducer. Activation of expression in response to the feed is actuated by a galacturonate-responsive transcription factor biosensor. We constructed variants of the galacturonate biosensor with a heterologous transcription factor and cognate hybrid promoter, and selected for the best performer through fluorescence characterization. We showed that native E. coli regulatory systems do not interact with our biosensor and favorable biosensor response exists in the presence and absence of galacturonate consumption. We then employed the control circuit to regulate the expression of the heterologous genes of a biosynthetic pathway for the production d-glyceric acid that was previously developed in our lab. Productivity via substrate-induction with our control circuit was comparable to IPTG-controlled induction and significantly outperformed a constitutive expression control, producing 2.13 ± 0.03 g L-1  d-glyceric acid within 6 h of galacturonate substrate addition. This work demonstrated feed-activated pathway expression to be an attractive control strategy for more readily scalable microbial biosynthesis.

Dynamic Control of Metabolism.

Metabolic engineering reprograms cells to synthesize value-added products. In doing so, endogenous genes are altered and heterologous genes can be introduced to achieve the necessary enzymatic reactions. Dynamic regulation of metabolic flux is a powerful control scheme to alleviate and overcome the competing cellular objectives that arise from the introduction of these production pathways. This review explores dynamic regulation strategies that have demonstrated significant production benefits by targeting the metabolic node corresponding to a specific challenge. We summarize the stimulus-responsive control circuits employed in these strategies that determine the criterion for actuating a dynamic response and then examine the points of control that couple the stimulus-responsive circuit to a shift in metabolic flux.

Alginate core-shell beads for simplified three-dimensional tumor spheroid culture and drug screening.

We demonstrate that when using cell-laden core-shell hydrogel beads to support the generation of tumor spheroids, the shell structure reduces the out-of-bead and monolayer cell proliferation that occurs during long-term culture of tumor cells within core-only alginate beads. We fabricate core-shell beads in a two-step process using simple, one-layer microfluidic devices. Tumor cells encapsulated within the bead core will proliferate to form multicellular aggregates which can serve as three-dimensional (3-D) models of tumors in drug screening. Encapsulation in an alginate shell increased the time that cells could be maintained in three-dimensional culture for MCF-7 breast cancer cells prior to out-of-bead proliferation, permitting formation of spheroids over a period of 14 days without the need move the cell-laden beads to clean culture flasks to separate beads from underlying monolayers. Tamoxifen and docetaxel dose response shows decreased toxicity for multicellular aggregates in three-dimensional core-shell bead culture compared to monolayer. Using simple core-only beads gives mixed monolayer and 3-D culture during drug screening, and alters the treatment result compared to the 3-D core-shell or the 2-D monolayer groups, as measured by standard proliferation assay. By preventing the out-of-bead proliferation and subsequent monolayer formation that is observed with core-only beads, the core-shell structure can obviate the requirement to transfer the beads to a new culture flask during drug screening, an important consideration for cell-based drug screening and drugs which have high multicellular resistance index.