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.

Biosensor development for single-cell detection of glucuronate.

Recent work in biosensors has shown promise to enable high throughput searches through large genetic libraries. However, just as physiological limitations and lack of in-depth mechanistic knowledge can prevent us from achieving high titers in microbial systems; similar roadblocks can appear in the application of biosensors. Here, we characterized a previously developed transcription-factor (ExuR) based galacturonate biosensor for its other cognate ligand, glucuronate. Though we saw an ideal response to glucuronate from the biosensor in controlled and ideal experimental circumstances, these results began to deviate from a well-behaved system when we explored the application of the sensor to different MIOX homologs. Through modifications to circuit architecture and culture conditions, we were able to decrease this variation and use these more optimal conditions to apply the biosensor for the separation of two closely related MIOX homologs. ONE-SENTENCE SUMMARY: In this work, a transcription-factor biosensor was investigated for its potential to screen a library of myo -inositol oxygenase variants while seeking to mitigate the impact the production pathway appeared to have on the biosensor.

Optimization of the Isopentenol Utilization Pathway for Isoprenoid Synthesis in Escherichia coli.

Engineering microbes to produce isoprenoids can be limited by the competition between product formation and cell growth because biomass and isoprenoids are naturally derived from central metabolism. Recently, a two-step synthetic pathway was developed to partially decouple isoprenoid formation from central carbon metabolism. The pathway used exogenously added isopentenols as substrates. In the present study, we systematically optimized this isopentenol utilization pathway in Escherichia coli by comparing enzyme variants from different species, tuning enzyme expression levels, and using a two-stage process. Under the optimal conditions found in this study, ∼300 mg/L lycopene was synthesized from 2 g/L isopentenol in 24 h. The strain could be easily modified to synthesize two other isoprenoid molecules efficiently (248 mg/L ß-carotene or 364 mg/L R-(-)-linalool produced from 2 g/L isopentenol). This study lays a solid foundation for producing agri-food isoprenoids at high titer/productivity from cost-effective 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.

Implementation of Synthetic Pathways to Foster Microbe-Based Production of Non-Naturally Occurring Carboxylic Acids and Derivatives.

Microbially produced carboxylic acids (CAs) are considered key players in the implementation of more sustainable industrial processes due to their potential to replace a set of oil-derived commodity chemicals. Most CAs are intermediates of microbial central carbon metabolism, and therefore, a biochemical production pathway is described and can be transferred to a host of choice to enable/improve production at an industrial scale. However, for some CAs, the implementation of this approach is difficult, either because they do not occur naturally (as is the case for levulinic acid) or because the described production pathway cannot be easily ported (as it is the case for adipic, muconic or glucaric acids). Synthetic biology has been reshaping the range of molecules that can be produced by microbial cells by setting new-to-nature pathways that leverage on enzyme arrangements not observed in vivo, often in association with the use of substrates that are not enzymes' natural ones. In this review, we provide an overview of how the establishment of synthetic pathways, assisted by computational tools for metabolic retrobiosynthesis, has been applied to the field of CA production. The translation of these efforts in bridging the gap between the synthesis of CAs and of their more interesting derivatives, often themselves non-naturally occurring molecules, is also reviewed using as case studies the production of methacrylic, methylmethacrylic and poly-lactic acids.

Effective use of biosensors for high-throughput library screening for metabolite production.

The development of fast and affordable microbial production from recombinant pathways is a challenging endeavor, with targeted improvements difficult to predict due to the complex nature of living systems. To address the limitations in biosynthetic pathways, much work has been done to generate large libraries of various genetic parts (promoters, RBSs, enzymes, etc.) to discover library members that bring about significantly improved levels of metabolite production. To evaluate these large libraries, high throughput approaches are necessary, such as those that rely on biosensors. There are various modes of operation to apply biosensors to library screens that are available at different scales of throughput. The effectiveness of each biosensor-based method is dependent on the pathway or strain to which it is applied, and all approaches have strengths and weaknesses to be carefully considered for any high throughput library screen. In this review, we discuss the various approaches used in biosensor screening for improved metabolite production, focusing on transcription factor-based biosensors.

Rapid in vitro prototyping of O-methyltransferases for pathway applications in Escherichia coli.

O-Methyltransferases are ubiquitous enzymes involved in biosynthetic pathways for secondary metabolites such as bacterial antibiotics, human catecholamine neurotransmitters, and plant phenylpropanoids. While thousands of putative O-methyltransferases are found in sequence databases, few examples are functionally characterized. From a pathway engineering perspective, however, it is crucial to know the substrate and product ranges of the respective enzymes to fully exploit their catalytic power. In this study, we developed an in vitro prototyping workflow that allowed us to screen ∼30 enzymes against five substrates in 3 days with high reproducibility. We combined in vitro transcription/translation of the genes of interest with a microliter-scale enzymatic assay in 96-well plates. The substrate conversion was indirectly measured by quantifying the consumption of the S-adenosyl-L-methionine co-factor by time-resolved fluorescence resonance energy transfer rather than time-consuming product analysis by chromatography. This workflow allowed us to rapidly prototype thus far uncharacterized O-methyltransferases for future use as biocatalysts.

Transcription factor allosteric regulation through substrate coordination to zinc.

The development of new synthetic biology circuits for biotechnology and medicine requires deeper mechanistic insight into allosteric transcription factors (aTFs). Here we studied the aTF UxuR, a homodimer of two domains connected by a highly flexible linker region. To explore how ligand binding to UxuR affects protein dynamics we performed molecular dynamics simulations in the free protein, the aTF bound to the inducer D-fructuronate or the structural isomer D-glucuronate. We then validated our results by constructing a sensor plasmid for D-fructuronate in Escherichia coli and performed site-directed mutagenesis. Our results show that zinc coordination is necessary for UxuR function since mutation to alanines prevents expression de-repression by D-fructuronate. Analyzing the different complexes, we found that the disordered linker regions allow the N-terminal domains to display fast and large movements. When the inducer is bound, UxuR can sample an open conformation with a more pronounced negative charge at the surface of the N-terminal DNA binding domains. In opposition, in the free and D-glucuronate bond forms the protein samples closed conformations, with a more positive character at the surface of the DNA binding regions. These molecular insights provide a new basis to harness these systems for biological systems engineering.

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.

Natural combinatorial genetics and prolific polyamine production enable siderophore diversification in Serratia plymuthica.

BACKGROUND: Iron is essential for bacterial survival. Bacterial siderophores are small molecules with unmatched capacity to scavenge iron from proteins and the extracellular milieu, where it mostly occurs as insoluble Fe3+. Siderophores chelate Fe3+ for uptake into the cell, where it is reduced to soluble Fe2+. Siderophores are key molecules in low soluble iron conditions. The ability of bacteria to synthesize proprietary siderophores may have increased bacterial evolutionary fitness; one way that bacteria diversify siderophore structure is by incorporating different polyamine backbones while maintaining the catechol moieties. RESULTS: We report that Serratia plymuthica V4 produces a variety of siderophores, which we term the siderome, and which are assembled by the concerted action of enzymes encoded in two independent gene clusters. Besides assembling serratiochelin A and B with diaminopropane, S. plymuthica utilizes putrescine and the same set of enzymes to assemble photobactin, a siderophore found in the bacterium Photorhabdus luminescens. The enzymes encoded by one of the gene clusters can independently assemble enterobactin. A third, independent operon is responsible for biosynthesis of the hydroxamate siderophore aerobactin, initially described in Enterobacter aerogenes. Mutant strains not synthesizing polyamine-siderophores significantly increased enterobactin production levels, though lack of enterobactin did not impact the production of serratiochelins. Knocking out SchF0, an enzyme involved in the assembly of enterobactin alone, significantly reduced bacterial fitness. CONCLUSIONS: This study shows the natural occurrence of serratiochelins, photobactin, enterobactin, and aerobactin in a single bacterial species and illuminates the interplay between siderophore biosynthetic pathways and polyamine production, indicating routes of molecular diversification. Given its natural yields of diaminopropane (97.75 µmol/g DW) and putrescine (30.83 µmol/g DW), S. plymuthica can be exploited for the industrial production of these compounds.

Prospecting Biochemical Pathways to Implement Microbe-Based Production of the New-to-Nature Platform Chemical Levulinic Acid.

Levulinic acid is a versatile platform molecule with potential to be used as an intermediate in the synthesis of many value-added products used across different industries, from cosmetics to fuels. Thus far, microbial biosynthetic pathways having levulinic acid as a product or an intermediate are not known, which restrains the development and optimization of a microbe-based process envisaging the sustainable bioproduction of this chemical. One of the doors opened by synthetic biology in the design of microbial systems is the implementation of new-to-nature pathways, that is, the assembly of combinations of enzymes not observed in vivo, where the enzymes can use not only their native substrates but also non-native ones, creating synthetic steps that enable the production of novel compounds. Resorting to a combined approach involving complementary computational tools and extensive manual curation, in this work, we provide a thorough prospect of candidate biosynthetic pathways that can be assembled for the production of levulinic acid in Escherichia coli or Saccharomyces cerevisiae. Out of the hundreds of combinations screened, five pathways were selected as best candidates on the basis of the availability of substrates and of candidate enzymes to catalyze the synthetic steps (that is, those steps that involve conversions not previously described). Genome-scale metabolic modeling was used to assess the performance of these pathways in the two selected hosts and to anticipate possible bottlenecks. Not only does the herein described approach offer a platform for the future implementation of the microbial production of levulinic acid but also it provides an organized research strategy that can be used as a framework for the implementation of other new-to-nature biosynthetic pathways for the production of value-added chemicals, thus fostering the emerging field of synthetic industrial microbiotechnology.

Production of D-Glyceric acid from D-Galacturonate in Escherichia coli.

A microbial production platform has been developed in Escherichia coli to synthesize D-glyceric acid from D-galacturonate. The expression of uronate dehydrogenase (udh) from Pseudomonas syringae and galactarolactone isomerase (gli) from Agrobacterium fabrum, along with the inactivation of garK, encoding for glycerate kinase, enables D-glyceric acid accumulation by utilizing the endogenous expression of galactarate dehydratase (garD), 5-keto-4-deoxy-D-glucarate aldolase (garL), and 2-hydroxy-3-oxopropionate reductase (garR). Optimization of carbon flux through the elimination of competing metabolic pathways led to the development of a ΔgarKΔhyiΔglxKΔuxaC mutant strain that produced 4.8 g/l of D-glyceric acid from D-galacturonate, with an 83% molar yield. Cultivation in a minimal medium produced similar yields and demonstrated that galactose or glycerol serve as possible carbon co-feeds for industrial production. This novel platform represents an alternative for the production of D-glyceric acid, an industrially relevant chemical, that addresses current challenges in using acetic acid bacteria for its synthesis: increasing yield, enantio-purity and biological stability.

Sequence-based bioprospecting of myo-inositol oxygenase (Miox) reveals new homologues that increase glucaric acid production in Saccharomyces cerevisiae.

myo-Inositol oxygenase (Miox) is a rate-limiting enzyme for glucaric acid production via microbial fermentation. The enzyme converts myo-inositol to glucuronate, which is further converted to glucaric acid, a natural compound with industrial uses that range from detergents to pharmaceutical synthesis to polymeric materials. More than 2,000 Miox sequences are available in the Uniprot database but only thirteen are classified as reviewed in Swiss-Prot (August 2019). In this study, sequence similarity networks were used to identify new homologues to be expressed in Saccharomyces cerevisiae for glucaric acid production. The expression of four homologues did not lead to product formation. Some of these enzymes may have a defective "dynamic lid" - a structural feature important to close the reaction site - which might explain the lack of activity. Thirty-one selected Miox sequences did allow for product formation, of which twenty-five were characterized for the first time. Expression of Talaromyces marneffei Miox led to the accumulation of 1.76 ±â€¯0.33 g glucaric acid/L from 20 g glucose/L and 10 g/L myo-inositol. Specific glucaric acid titer with TmMiox increased 44 % compared to the often-used Arabidopsis thaliana variant AtMiox4 (0.258 vs. 0.179 g glucaric acid/g biomass). AtMiox4 activity decreased from 12.47 to 0.40 nmol/min/mg protein when cells exited exponential phase during growth on glucose, highlighting the importance of future research on Miox stability in order to further improve microbial production of glucaric acid.

The importance and future of biochemical engineering.

Today's Biochemical Engineer may contribute to advances in a wide range of technical areas. The recent Biochemical and Molecular Engineering XXI conference focused on "The Next Generation of Biochemical and Molecular Engineering: The role of emerging technologies in tomorrow's products and processes". On the basis of topical discussions at this conference, this perspective synthesizes one vision on where investment in research areas is needed for biotechnology to continue contributing to some of the world's grand challenges.

Development of a Quorum-Sensing Based Circuit for Control of Coculture Population Composition in a Naringenin Production System.

As synthetic biology and metabolic engineering tools improve, it is feasible to construct more complex microbial synthesis systems that may be limited by the machinery and resources available in an individual cell. Coculture fermentation is a promising strategy for overcoming these constraints by distributing objectives between subpopulations, but the primary method for controlling the composition of the coculture of production systems has been limited to control of the inoculum composition. We have developed a quorum sensing (QS)-based growth-regulation circuit that provides an additional parameter for regulating the composition of a coculture over the course of the fermentation. Implementation of this tool in a naringenin-producing coculture resulted in a 60% titer increase over a system that was optimized by varying inoculation ratios only. We additionally demonstrated that the growth control circuit can be implemented in combination with a communication module that couples transcription in one subpopulation to the cell-density of the other population for coordination of behavior, resulting in an additional 60% improvement in naringenin titer.

Heterologous caffeic acid biosynthesis in Escherichia coli is affected by choice of tyrosine ammonia lyase and redox partners for bacterial Cytochrome P450.

BACKGROUND: Caffeic acid is industrially recognized for its antioxidant activity and therefore its potential to be used as an anti-inflammatory, anticancer, antiviral, antidiabetic and antidepressive agent. It is traditionally isolated from lignified plant material under energy-intensive and harsh chemical extraction conditions. However, over the last decade bottom-up biosynthesis approaches in microbial cell factories have been established, that have the potential to allow for a more tailored and sustainable production. One of these approaches has been implemented in Escherichia coli and only requires a two-step conversion of supplemented L-tyrosine by the actions of a tyrosine ammonia lyase and a bacterial Cytochrome P450 monooxygenase. Although the feeding of intermediates demonstrated the great potential of this combination of heterologous enzymes compared to others, no de novo synthesis of caffeic acid from glucose has been achieved utilizing the bacterial Cytochrome P450 thus far. RESULTS: The herein described work aimed at improving the efficiency of this two-step conversion in order to establish de novo caffeic acid formation from glucose. We implemented alternative tyrosine ammonia lyases that were reported to display superior substrate binding affinity and selectivity, and increased the efficiency of the Cytochrome P450 by altering the electron-donating redox system. With this strategy we were able to achieve final titers of more than 300 µM or 47 mg/L caffeic acid over 96 h in an otherwise wild type E. coli MG1655(DE3) strain with glucose as the only carbon source. We observed that the choice and gene dose of the redox system strongly influenced the Cytochrome P450 catalysis. In addition, we were successful in applying a tethering strategy that rendered even a virtually unproductive Cytochrome P450/redox system combination productive. CONCLUSIONS: The caffeic acid titer achieved in this study is about 10% higher than titers reported for other heterologous caffeic acid pathways in wildtype E. coli without L-tyrosine supplementation. The tethering strategy applied to the Cytochrome P450 appears to be particularly useful for non-natural Cytochrome P450/redox partner combinations and could be useful for other recombinant pathways utilizing bacterial Cytochromes P450.

Development of an autonomous and bifunctional quorum-sensing circuit for metabolic flux control in engineered Escherichia coli.

Metabolic engineering seeks to reprogram microbial cells to efficiently and sustainably produce value-added compounds. Since chemical production can be at odds with the cell's natural objectives, strategies have been developed to balance conflicting goals. For example, dynamic regulation modulates gene expression to favor biomass and metabolite accumulation at low cell densities before diverting key metabolic fluxes toward product formation. To trigger changes in gene expression in a pathway-independent manner without the need for exogenous inducers, researchers have coupled gene expression to quorum-sensing (QS) circuits, which regulate transcription based on cell density. While effective, studies thus far have been limited to one control point. More challenging pathways may require layered dynamic regulation strategies, motivating the development of a generalizable tool for regulating multiple sets of genes. We have developed a QS-based regulation tool that combines components of the lux and esa QS systems to simultaneously and dynamically up- and down-regulate expression of 2 sets of genes. Characterization of the circuit revealed that varying the expression level of 2 QS components leads to predictable changes in switching dynamics and that using components from 2 QS systems allows for independent tuning capability. We applied the regulation tool to successfully address challenges in both the naringenin and salicylic acid synthesis pathways. Through these case studies, we confirmed the benefit of having multiple control points, predictable tuning capabilities, and independently tunable regulation modules.

Development of a Vanillate Biosensor for the Vanillin Biosynthesis Pathway in E. coli.

The engineered de novo vanillin biosynthesis pathway constructed in Escherichia coli is industrially relevant but limited by the reaction catalyzed by catechol O-methyltransferase, which is intended to catalyze the conversion of protocatechuate to vanillate. To identify alternative O-methyltransferases, we constructed a vanillate sensor based on the Caulobacter crescentus VanR-VanO system. Using an E. coli promoter library, we achieved greater than 14-fold dynamic range in our best rationally constructed sensor. We found that this construct and an evolved variant demonstrate remarkable substrate selectivity, exhibiting no detectable response to the regioisomer byproduct isovanillate and minimal response to structurally similar pathway intermediates. We then harnessed the evolved biosensor to conduct rapid bioprospecting of natural catechol O-methyltransferases and identified three previously uncharacterized but active O-methyltransferases. Collectively, these efforts enrich our knowledge of how biosensing can aid metabolic engineering and constitute the foundation for future improvements in vanillin pathway productivity.

From lignocellulosic residues to market: Production and commercial potential of xylooligosaccharides.

The updated definition of prebiotic expands the range of potential applications in which emerging xylooligosaccharides (XOS) can be used. It has been demonstrated that XOS exhibit prebiotic effects at lower amounts compared to others, making them competitively priced prebiotics. As a result, the industry is focused on developing alternative approaches to improve processes efficiency that can meet the increasing demand while reducing costs. Recent advances have been made towards greener and more efficient processes, by applying process integration strategies to produce XOS from costless lignocellulosic residues and using genetic engineering to create microorganisms that convert these residues to XOS. In addition, collecting more in vivo data on their performance will be key to achieve regulatory claims, greatly increasing XOS commercial value.

Engineered microbial biofuel production and recovery under supercritical carbon dioxide.

Culture contamination, end-product toxicity, and energy efficient product recovery are long-standing bioprocess challenges. To solve these problems, we propose a high-pressure fermentation strategy, coupled with in situ extraction using the abundant and renewable solvent supercritical carbon dioxide (scCO2), which is also known for its broad microbial lethality. Towards this goal, we report the domestication and engineering of a scCO2-tolerant strain of Bacillus megaterium, previously isolated from formation waters from the McElmo Dome CO2 field, to produce branched alcohols that have potential use as biofuels. After establishing induced-expression under scCO2, isobutanol production from 2-ketoisovalerate is observed with greater than 40% yield with co-produced isopentanol. Finally, we present a process model to compare the energy required for our process to other in situ extraction methods, such as gas stripping, finding scCO2 extraction to be potentially competitive, if not superior.