Electrical-biological hybrid system for carbon efficient isobutanol production.

We have developed an electrical-biological hybrid system wherein an engineered microorganism consumes electrocatalytically produced formate from CO2 to supplement the bioproduction of isobutanol, a valuable fuel chemical. Biological CO2 sequestration is notoriously slow compared to electrochemical CO2 reduction, while electrochemical methods struggle to generate carbon-carbon bonds which readily form in biological systems. A hybrid system provides a promising method for combining the benefits of both biology and electrochemistry. Previously, Escherichia coli was engineered to assimilate formate and CO2 in central metabolism using the reductive glycine pathway. In this work, we have shown that chemical production in E. coli can benefit from single carbon substrates when equipped with the RGP. By installing the RGP and the isobutanol biosynthetic pathway into E. coli and by further genetic modifications, we have generated a strain of E. coli that can consume formate and produce isobutanol at a yield of >100% of theoretical maximum from glucose. Our results demonstrate that carbon produced from electrocatalytically reduced CO2 can bolster chemical production in E. coli. This study shows that E. coli can be engineered towards carbon efficient methods of chemical production.

Fluctuating pH for efficient photomixotrophic succinate production.

Cyanobacteria are attracting increasing attention as a photosynthetic chassis organism for diverse biochemical production, however, photoautotrophic production remains inefficient. Photomixotrophy, a method where sugar is used to supplement baseline autotrophic metabolism in photosynthetic hosts, is becoming increasingly popular for enhancing sustainable bioproduction with multiple input energy streams. In this study, the commercially relevant diacid, succinate, was produced photomixotrophically. Succinate is an important industrial chemical that can be used for the production of a wide array of products, from pharmaceuticals to biopolymers. In this system, the substrate, glucose, is transported by a proton symporter and the product, succinate, is hypothesized to be transported by another proton symporter, but in the opposite direction. Thus, low pH is required for the import of glucose and high pH is required for the export of succinate. Succinate production was initiated in a pH 7 medium containing bicarbonate. Glucose was efficiently imported at around neutral pH. Utilization of bicarbonate by CO2 fixation raised the pH of the medium. As succinate, a diacid, was produced, the pH of the medium dropped. By repeating this cycle with additional pH adjustment, those contradictory requirements for transport were overcome. pH affects a variety of biological factors and by cycling from high pH to neutral pH processes such as CO2 fixation rates and CO2 solubility can vary. In this study the engineered strains produced succinate during fluctuating pH conditions, achieving a titer of 5.0 g L-1 after 10 days under shake flask conditions. These results demonstrate the potential for photomixotrophic production as a viable option for the large-scale production of succinate.

Microbial Production of Human Milk Oligosaccharides.

Human milk oligosaccharides (HMOs) are complex nonnutritive sugars present in human milk. These sugars possess prebiotic, immunomodulatory, and antagonistic properties towards pathogens and therefore are important for the health and well-being of newborn babies. Lower prevalence of breastfeeding around the globe, rising popularity of nutraceuticals, and low availability of HMOs have inspired efforts to develop economically feasible and efficient industrial-scale production platforms for HMOs. Recent progress in synthetic biology and metabolic engineering tools has enabled microbial systems to be a production system of HMOs. In this regard, the model organism Escherichia coli has emerged as the preferred production platform. Herein, we summarize the remarkable progress in the microbial production of HMOs and discuss the challenges and future opportunities in unraveling the scope of production of complex HMOs. We focus on the microbial production of five HMOs that have been approved for their commercialization.

Adaptive laboratory evolution for improved tolerance of isobutyl acetate in Escherichia coli.

Previously, Escherichia coli was engineered to produce isobutyl acetate (IBA). Titers greater than the toxicity threshold (3 g/L) were achieved by using layer-assisted production. To avoid this costly and complex method, adaptive laboratory evolution (ALE) was applied to E. coli for improved IBA tolerance. Over 37 rounds of selective pressure, 22 IBA-tolerant mutants were isolated. Remarkably, these mutants not only tolerate high IBA concentrations, they also produce higher IBA titers. Using whole-genome sequencing followed by CRISPR/Cas9 mediated genome editing, the mutations (SNPs in metH, rho and deletion of arcA) that confer improved tolerance and higher titers were elucidated. The improved IBA titers in the evolved mutants were a result of an increased supply of acetyl-CoA and altered transcriptional machinery. Without the use of phase separation, a strain capable of 3.2-fold greater IBA production than the parent strain was constructed by combing select beneficial mutations. These results highlight the impact improved tolerance has on the production capability of a biosynthetic system.

Light-induced production of isobutanol and 3-methyl-1-butanol by metabolically engineered cyanobacteria.

BACKGROUND: Cyanobacteria are engineered via heterologous biosynthetic pathways to produce value-added chemicals via photosynthesis. Various chemicals have been successfully produced in engineered cyanobacteria. Chemical inducer-dependent promoters are used to induce the expression of target biosynthetic pathway genes. A chemical inducer is not ideal for large-scale reactions owing to its high cost; therefore, it is important to develop scaling-up methods to avoid their use. In this study, we designed a green light-inducible alcohol production system using the CcaS/CcaR green light gene expression system in the cyanobacterium Synechocystis sp. PCC 6803 (PCC 6803). RESULTS: To establish the green light-inducible production of isobutanol and 3-methyl-1-butanol (3MB) in PCC 6803, keto-acid decarboxylase (kdc) and alcohol dehydrogenase (adh) were expressed under the control of the CcaS/CcaR system. Increases in the transcription level were induced by irradiation with red and green light without severe effects on host cell growth. We found that the production of isobutanol and 3MB from carbon dioxide (CO2) was induced under red and green light illumination and was substantially repressed under red light illumination alone. Finally, production titers of isobutanol and 3MB reached 238 mg L-1 and 75 mg L-1, respectively, in 5 days under red and green light illumination, and these values are comparable to those reported in previous studies using chemical inducers. CONCLUSION: A green light-induced alcohol production system was successfully integrated into cyanobacteria to produce value-added chemicals without using expensive chemical inducers. The green light-regulated production of isobutanol and 3MB from CO2 is eco-friendly and cost-effective. This study demonstrates that light regulation is a potential tool for producing chemicals and increases the feasibility of cyanobacterial bioprocesses.

Microbial production of human milk oligosaccharide lactodifucotetraose.

Human milk oligosaccharides (HMOs) are potent bioactive compounds that modulate neonatal health and are of interest for development as potential drug treatments for adult diseases. The potential of these molecules, their limited access from natural sources, and difficulty in large-scale isolation of individual HMOs for studies and applications have motivated the development of chemical syntheses and in vitro enzymatic catalysis strategies. Whole cell biocatalysts are emerging as alternative self-regulating production platforms that have the potential to reduce the cost for enzymatic synthesis of HMOs. Whole cell biocatalysts for the production of short-chained, linear and small monofucosylated HMOs have been reported but those for fucosylated structures with higher complexity have not been explored. In this study, we established a strategy for producing a difucosylated HMO, lactodifucotetraose (LDFT), from lactose and L-fucose in Escherichia coli. We used two bacterial fucosyltransferases with narrow acceptor selectivity to drive the sequential fucosylation of lactose and intermediate 2'-fucosyllactose (2'-FL) to produce LDFT. Deletion of substrate degradation pathways that decoupled cellular growth from LDFT production, enhanced expression of native substrate transporters and modular induction of the genes in the LDFT biosynthetic pathway allowed complete conversion of lactose into LDFT and minor quantities of the side product 3-fucosyllactose (3-FL). Overall, 5.1 g/L of LDFT was produced from 3 g/L lactose and 3 g/L L-fucose in 24 h. Our results demonstrate promising applications of engineered microbial biosystems for the production of multi-fucosylated HMOs for biochemical studies.

Nonphotosynthetic Biological CO2 Reduction.

Alarming changes in environmental conditions have prompted significant research into producing renewable commodities from sources other than fossil fuels. One such alternative is CO2, a determinate greenhouse gas with historically high atmospheric levels. If sequestered, CO2 could be used as a highly renewable feedstock for industrially relevant products and fuels. The vast majority of atmospheric CO2 fixation is accomplished by photosynthetic organisms, which have unfortunately proven difficult to utilize as chassis for industrial production. Nonphotosynthetic CO2 fixing microorganisms and pathways have recently attracted scientific and commercial interest. This Perspective will review promising alternate CO2 fixation strategies and their potential to supply microbially produced fuels and commodity chemicals, such as higher alcohols. Acetogenic fermentation and microbial electrosynthesis are the primary focuses of this review.

Metabolic engineering tools in model cyanobacteria.

Developing sustainable routes for producing chemicals and fuels is one of the most important challenges in metabolic engineering. Photoautotrophic hosts are particularly attractive because of their potential to utilize light as an energy source and CO2 as a carbon substrate through photosynthesis. Cyanobacteria are unicellular organisms capable of photosynthesis and CO2 fixation. While engineering in heterotrophs, such as Escherichia coli, has result in a plethora of tools for strain development and hosts capable of producing valuable chemicals efficiently, these techniques are not always directly transferable to cyanobacteria. However, recent efforts have led to an increase in the scope and scale of chemicals that cyanobacteria can produce. Adaptations of important metabolic engineering tools have also been optimized to function in photoautotrophic hosts, which include Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9, 13C Metabolic Flux Analysis (MFA), and Genome-Scale Modeling (GSM). This review explores innovations in cyanobacterial metabolic engineering, and highlights how photoautotrophic metabolism has shaped their development.

Electrical-biological hybrid system for CO2 reduction.

Here we have developed an electrochemical-biological hybrid system to fix CO2. Natural biological CO2 fixation processes are relatively slow. To increase the speed of fixation we applied electrocatalysts to reduce CO2 to formate. We chose a user-friendly organism, Escherichia coli, as host. Overall, the newly constructed CO2 and formate fixation pathway converts two formate and one CO2 to one pyruvate via glycine and L-serine in E. coli. First, one formate and one CO2 are converted to one glycine. Second, L-serine is produced from one glycine and one formate. Lastly, L-serine is converted to pyruvate. E. coli's genetic tractability allowed us to balance various parameters of the pathway. The carbon flux of the pathway was sufficient to compensate L-serine auxotrophy in the strain. In total, we integrated both electrocatalysis and biological systems into a single pot to support E. coli growth with CO2 and electricity. Results show promise for using this hybrid system for chemical production from CO2 and electricity.

2,3 Butanediol production in an obligate photoautotrophic cyanobacterium in dark conditions via diverse sugar consumption.

Cyanobacteria are under investigation as a means to utilize light energy to directly recycle CO2 into chemical compounds currently derived from petroleum. Any large-scale photosynthetic production scheme must rely on natural sunlight for energy, thereby limiting production time to only lighted hours during the day. Here, an obligate photoautotrophic cyanobacterium was engineered for enhanced production of 2,3-butanediol (23BD) in continuous light, 12h:12h light-dark diurnal, and continuous dark conditions via supplementation with glucose or xylose. This study achieved 23BD production under diurnal conditions comparable to production under continuous light conditions. The maximum 23BD titer was 3.0gL(-1) in 10d. Also achieving chemical production under dark conditions, this work enhances the feasibility of using cyanobacteria as industrial chemical-producing microbes.

Biological conversion of gaseous alkenes to liquid chemicals.

Industrial gas-to-liquid (GTL) technologies are well developed. They generally employ syngas, require complex infrastructure, and need high capital investment to be economically viable. Alternatively, biological conversion has the potential to be more efficient, and easily deployed to remote areas on relatively small scales for the utilization of otherwise stranded resources. The present study demonstrates a novel biological GTL process in which engineered Escherichia coli converts C2-C4 gaseous alkenes into liquid diols. Diols are versatile industrially important chemicals, used routinely as antifreeze agents, polymer precursors amongst many other applications. Heterologous co-expression of a monooxygenase and an epoxide hydrolase in E. coli allows whole cell conversion of C2-C4 alkenes for the formation of ethylene glycol, 1,2-propanediol, 1,2-butanediol, and 2,3-butanediol at ambient temperature and pressure in one pot. Increasing intracellular NADH supply via addition of formate and a formate dehydrogenase increases ethylene glycol production titers, resulting in an improved productivity of 9mg/L/h and a final titer of 250mg/L. This represents a novel biological method for GTL conversion of alkenes to industrially valuable diols.

Expanding ester biosynthesis in Escherichia coli.

To expand the capabilities of whole-cell biocatalysis, we have engineered Escherichia coli to produce various esters. The alcohol O-acyltransferase (ATF) class of enzyme uses acyl-CoA units for ester formation. The release of free CoA upon esterification with an alcohol provides the free energy to facilitate ester formation. The diversity of CoA molecules found in nature in combination with various alcohol biosynthetic pathways allows for the biosynthesis of a multitude of esters. Small to medium volatile esters have extensive applications in the flavor, fragrance, cosmetic, solvent, paint and coating industries. The present work enables the production of these compounds by designing several ester pathways in E. coli. The engineered pathways generated acetate esters of ethyl, propyl, isobutyl, 2-methyl-1-butyl, 3-methyl-1-butyl and 2-phenylethyl alcohols. In particular, we achieved high-level production of isobutyl acetate from glucose (17.2 g l(-1)). This strategy was expanded to realize pathways for tetradecyl acetate and several isobutyrate esters.

Cyanobacterial conversion of carbon dioxide to 2,3-butanediol.

Conversion of CO(2) for the synthesis of chemicals by photosynthetic organisms is an attractive target for establishing independence from fossil reserves. However, synthetic pathway construction in cyanobacteria is still in its infancy compared with model fermentative organisms. Here we systematically developed the 2,3-butanediol (23BD) biosynthetic pathway in Synechococcus elongatus PCC7942 as a model system to establish design methods for efficient exogenous chemical production in cyanobacteria. We identified 23BD as a target chemical with low host toxicity, and designed an oxygen-insensitive, cofactor-matched biosynthetic pathway coupled with irreversible enzymatic steps to create a driving force toward the target. Production of 23BD from CO(2) reached 2.38 g/L, which is a significant increase for chemical production from exogenous pathways in cyanobacteria. This work demonstrates that developing strong design methods can continue to increase chemical production in cyanobacteria.

A carbon sink pathway increases carbon productivity in cyanobacteria.

The burning of fossil reserves, and subsequent release of carbon into the atmosphere is depleting the supply of carbon-based molecules used for synthetic materials including plastics, oils, medicines, and glues. To provide for future society, innovations are needed for the conversion of waste carbon (CO2) into organic carbon useful for materials. Chemical production directly from photosynthesis is a nascent technology, with great promise for capture of CO2 using sunlight. To improve low yields, it has been proposed that photosynthetic capacity can be increased by a relaxation of bottlenecks inherent to growth. The limits of carbon partitioning away from growth within the cell and the effect of partitioning on carbon fixation are not well known. Here we show that expressing genes in a pathway between carbon fixation and pyruvate increases partitioning to 2,3-butanediol (23BD) and leads to a 1.8-fold increase in total carbon yield in the cyanobacterium Synechococcus elongatus PCC 7942. Specific 2,3-butanediol production increases 2.4-fold. As partitioning increases beyond 30%, it leads to a steep decline in total carbon yield. The data suggests a local maximum for carbon partitioning from the Calvin Benson cycle that is scalable with light intensity.

Isobutanol production from cellobionic acid in Escherichia coli.

BACKGROUND: Liquid fuels needed for the global transportation industry can be produced from sugars derived from plant-based lignocellulosics. Lignocellulosics contain a range of sugars, only some of which (such as cellulose) have been shown to be utilizable by microorganisms capable of producing biofuels. Cellobionic acid makes up a small but significant portion of lignocellulosic degradation products, and had not previously been investigated as an utilizable substrate. However, aldonic acids such as cellobionic acid are the primary products of a promising new group of lignocellulosic-degrading enzymes, which makes this compound group worthy of study. Cellobionic acid doesn't inhibit cellulose degradation enzymes and so its inclusion would increase lignocellulosic degradation efficiency. Also, its use would increase overall product yield from lignocellulose substrate. For these reasons, cellobionic acid has gained increased attention for cellulosic biofuel production. RESULTS: This study describes the discovery that Escherichia coli are naturally able to utilize cellobionic acid as a sole carbon source with efficiency comparable to that of glucose and the construction of an E. coli strain able to produce the drop-in biofuel candidate isobutanol from cellobionic acid. The gene primarily responsible for growth of E. coli on cellobionic acid is ascB, a gene previously thought to be cryptic (expressed only after incurring specific mutations in nearby regulatory genes). In addition to AscB, the ascB knockout strain can be complemented by the cellobionic acid phosphorylase from the fungus Neurospora crassa. An E. coli strain engineered to express the isobutanol production pathway was successfully able to convert cellobionic acid into isobutanol. Furthermore, to demonstrate potential application of this strain in a sequential two-step bioprocessing system, E. coli was grown on hydrolysate (that was degraded by a fungus) and was successfully able to produce isobutanol. CONCLUSIONS: These results demonstrate that cellobionic acid is a viable carbon source for biofuel production. This work suggests that with further optimization, a bacteria-fungus co-culture could be used in decreased-cost biomass-based biofuel production systems.

2-Keto acids based biosynthesis pathways for renewable fuels and chemicals.

Global energy and environmental concerns have driven the development of biological chemical production from renewable sources. Biological processes using microorganisms are efficient and have been traditionally utilized to convert biomass (i.e., glucose) to useful chemicals such as amino acids. To produce desired fuels and chemicals with high yield and rate, metabolic pathways have been enhanced and expanded with metabolic engineering and synthetic biology approaches. 2-Keto acids, which are key intermediates in amino acid biosynthesis, can be converted to a wide range of chemicals. 2-Keto acid pathways were engineered in previous research efforts and these studies demonstrated that 2-keto acid pathways have high potential for novel metabolic routes with high productivity. In this review, we discuss recently developed 2-keto acid-based pathways.

Toward aldehyde and alkane production by removing aldehyde reductase activity in Escherichia coli.

Advances in synthetic biology and metabolic engineering have enabled the construction of novel biological routes to valuable chemicals using suitable microbial hosts. Aldehydes serve as chemical feedstocks in the synthesis of rubbers, plastics, and other larger molecules. Microbial production of alkanes is dependent on the formation of a fatty aldehyde intermediate which is converted to an alkane by an aldehyde deformylating oxygenase (ADO). However, microbial hosts such as Escherichia coli are plagued by many highly active endogenous aldehyde reductases (ALRs) that convert aldehydes to alcohols, which greatly complicates strain engineering for aldehyde and alkane production. It has been shown that the endogenous ALR activity outcompetes the ADO enzyme for fatty aldehyde substrate. The large degree of ALR redundancy coupled with an incomplete database of ALRs represents a significant obstacle in engineering E. coli for either aldehyde or alkane production. In this study, we identified 44 ALR candidates encoded in the E. coli genome using bioinformatics tools, and undertook a comprehensive screening by measuring the ability of these enzymes to produce isobutanol. From the pool of 44 candidates, we found five new ALRs using this screening method (YahK, DkgA, GldA, YbbO, and YghA). Combined deletions of all 13 known ALRs resulted in a 90-99% reduction in endogenous ALR activity for a wide range of aldehyde substrates (C2-C12). Elucidation of the ALRs found in E. coli could guide one in reducing competing alcohol formation during alkane or aldehyde production.

Combinatorial optimization of cyanobacterial 2,3-butanediol production.

A vital goal of renewable technology is the capture and re-energizing of exhausted CO2 into usable carbon products. Cyanobacteria fix CO2 more efficiently than plants, and can be engineered to produce carbon feedstocks useful for making plastics, solvents, and medicines. However, fitness of this technology in the economy is threatened by low yields in engineered strains. Robust engineering of photosynthetic microorganisms is lagging behind model microorganisms that rely on energetic carbon, such as Escherichia coli, due in part to slower growth rates and increased metabolic complexity. In this work we show that protein expression from characterized parts is unpredictable in Synechococcus elongatus sp. strain PCC 7942, and may contribute to slow development. To overcome this, we apply a combinatorial approach and show that modulation of the 5'-untranslated region (UTR) can produce a range of protein expression sufficient to optimize chemical feedstock production from CO2.

Metabolic engineering for higher alcohol production.

Engineering microbial hosts for the production of higher alcohols looks to combine the benefits of renewable biological production with the useful chemical properties of larger alcohols. In this review we outline the array of metabolic engineering strategies employed for the efficient diversion of carbon flux from native biosynthetic pathways to the overproduction of a target alcohol. Strategies for pathway design from amino acid biosynthesis through 2-keto acids, from isoprenoid biosynthesis through pyrophosphate intermediates, from fatty acid biosynthesis and degradation by tailoring chain length specificity, and the use and expansion of natural solvent production pathways will be covered.

Metabolic design for cyanobacterial chemical synthesis.

Photosynthetic chemical production in cyanobacteria is a promising technology for renewable energy, CO2 mitigation, and fossil fuel replacement. Metabolic engineering has enabled a direct biosynthetic process from CO2 fixation to chemical feedstocks and biofuels, without requiring costly production, storage, and breakdown of cellulose or sugars. However, direct production technology is challenged by a need to achieve high-carbon partitioning to products in order to be competitive. This review discusses principles for the design of biosynthetic pathways in cyanobacteria and describes recent advances in relevant genetic tools. This field is at a critical juncture in assessing the strength and feasibility of carbon partitioning. To address this, we have included the stoichiometry of reducing equivalents and carbon conservation for heterologous pathways, and a method for calculating product yields against a theoretical maximum.