Arabinose as an overlooked sugar for microbial bioproduction of chemical building blocks.

The circular economy is anticipated to bring a disruptive transformation in manufacturing technologies. Robust and industrial scalable microbial strains that can simultaneously assimilate and valorize multiple carbon substrates are highly desirable, as waste bioresources contain substantial amounts of renewable and fermentable carbon, which is diverse. Lignocellulosic biomass (LCB) is identified as an inexhaustible and alternative resource to reduce global dependence on oil. Glucose, xylose, and arabinose are the major monomeric sugars in LCB. However, primary research has focused on the use of glucose. On the other hand, the valorization of pentose sugars, xylose, and arabinose, has been mainly overlooked, despite possible assimilation by vast microbial communities. The present review highlights the research efforts that have explicitly proven the suitability of arabinose as the starting feedstock for producing various chemical building blocks via biological routes. It begins by analyzing the availability of various arabinose-rich biorenewable sources that can serve as potential feedstocks for biorefineries. The subsequent section outlines the current understanding of arabinose metabolism, biochemical routes prevalent in prokaryotic and eukaryotic systems, and possible products that can be derived from this sugar. Further, currently, exemplar products from arabinose, including arabitol, 2,3-butanediol, 1,2,3-butanetriol, ethanol, lactic acid, and xylitol are discussed, which have been produced by native and non-native microbial strains using metabolic engineering and genome editing tools. The final section deals with the challenges and obstacles associated with arabinose-based production, followed by concluding remarks and prospects.

Design, Analysis, and Implementation of a Novel Biochemical Pathway for Ethylene Glycol Production in Clostridium autoethanogenum.

The platform chemical ethylene glycol (EG) is used to manufacture various commodity chemicals of industrial importance, but largely remains synthesized from fossil fuels. Although several novel metabolic pathways have been reported for its bioproduction in model organisms, none has been reported for gas-fermenting, non-model acetogenic chassis organisms. Here, we describe a novel, synthetic biochemical pathway to convert acetate into EG in the industrially important gas-fermenting acetogen,Clostridium autoethanogenum. We not only developed a computational workflow to design and analyze hundreds of novel biochemical pathways for EG production but also demonstrated a successful pathway construction in the chosen host. The EG production was achieved using a two-plasmid system to bypass unfeasible expression levels and potential toxic enzymatic interactions. Although only a yield of 0.029 g EG/g fructose was achieved and therefore requiring further strain engineering efforts to optimize the designed strain, this work demonstrates an important proof-of-concept approach to computationally design and experimentally implement fully synthetic metabolic pathways in a metabolically highly specific, non-model host organism.

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.

Genetic and metabolic engineering challenges of C1-gas fermenting acetogenic chassis organisms.

Unabated mining and utilisation of petroleum and petroleum resources and their conversion to essential fuels and chemicals have drastic environmental consequences, contributing to global warming and climate change. In addition, fossil fuels are finite resources, with a fast-approaching shortage. Accordingly, research efforts are increasingly focusing on developing sustainable alternatives for chemicals and fuels production. In this context, bioprocesses, relying on microorganisms, have gained particular interest. For example, acetogens use the Wood-Ljungdahl pathway to grow on single carbon C1-gases (CO2 and CO) as their sole carbon source and produce valuable products such as acetate or ethanol. These autotrophs can, therefore, be exploited for large-scale fermentation processes to produce industrially relevant chemicals from abundant greenhouse gases. In addition, genetic tools have recently been developed to improve these chassis organisms through synthetic biology approaches. This review will focus on the challenges of genetically and metabolically modifying acetogens. It will first discuss the physical and biochemical obstacles complicating successful DNA transfer in these organisms. Current genetic tools developed for several acetogens, crucial for strain engineering to consolidate and expand their catalogue of products, will then be described. Recent tool applications for metabolic engineering purposes to allow redirection of metabolic fluxes or production of non-native compounds will lastly be covered.

Sustained Dechlorination of Vinyl Chloride to Ethene in Dehalococcoides-Enriched Cultures Grown without Addition of Exogenous Vitamins and at Low pH.

Trichloroethene (TCE) bioremediation has been demonstrated at field sites using microbial cultures harboring TCE-respiring Dehalococcoides whose growth is cobalamin (vitamin B12)-dependent. Bioaugmentation cultures grown ex situ with ample exogenous vitamins and at neutral pH may become vitamin-limited or inhibited by acidic pH once injected into field sites, resulting in incomplete TCE dechlorination and accumulation of vinyl chloride (VC). Here, we report growth of the Dehalococcoides-containing bioaugmentation culture KB-1 in a TCE-amended mineral medium devoid of vitamins and in a VC-amended mineral medium at low pH (6.0 and 5.5). In these cultures, Acetobacterium, which can synthesize 5,6-dimethylbenzimidazole (DMB), the lower ligand of cobalamin, and Sporomusa are dominant acetogens. At neutral pH, Acetobacterium supports complete TCE dechlorination by Dehalococcoides at millimolar levels with a substantial increase in cobalamin (∼20-fold). Sustained dechlorination of VC to ethene was achieved at pH as low as 5.5. Below pH 5.0, dechlorination was not stimulated by DMB supplementation but was restored by raising pH to neutral. Cell-extract assays revealed that vinyl chloride reductase activity declines significantly below pH 6.0 and is undetectable below pH 5.0. This study highlights the importance of cobamide-producing populations and pH in microbial dechlorinating communities for successful bioremediation at field sites.

Synergistic substrate cofeeding stimulates reductive metabolism.

Advanced bioproduct synthesis via reductive metabolism requires coordinating carbons, ATP and reducing agents, which are generated with varying efficiencies depending on metabolic pathways. Substrate mixtures with direct access to multiple pathways may optimally satisfy these biosynthetic requirements. However, native regulation favouring preferential use precludes cells from co-metabolizing multiple substrates. Here we explore mixed substrate metabolism and tailor pathway usage to synergistically stimulate carbon reduction. By controlled cofeeding of superior ATP and NADPH generators as 'dopant' substrates to cells primarily using inferior substrates, we circumvent catabolite repression and drive synergy in two divergent organisms. Glucose doping in Moorella thermoacetica stimulates CO2 reduction (2.3 g gCDW-1 h-1) into acetate by augmenting ATP synthesis via pyruvate kinase. Gluconate doping in Yarrowia lipolytica accelerates acetate-driven lipogenesis (0.046 g gCDW-1 h-1) by obligatory NADPH synthesis through the pentose cycle. Together, synergistic cofeeding produces CO2-derived lipids with 38% energy yield and demonstrates the potential to convert CO2 into advanced bioproducts. This work advances the systems-level control of metabolic networks and CO2 use, the most pressing and difficult reduction challenge.

Synthetic biology strategies for improving microbial synthesis of "green" biopolymers.

Polysaccharide-based biopolymers have many material properties relevant to industrial and medical uses, including as drug delivery agents, wound-healing adhesives, and food additives and stabilizers. Traditionally, polysaccharides are obtained from natural sources. Microbial synthesis offers an attractive alternative for sustainable production of tailored biopolymers. Here, we review synthetic biology strategies for select "green" biopolymers: cellulose, alginate, chitin, chitosan, and hyaluronan. Microbial production pathways, opportunities for pathway yield improvements, and advances in microbial engineering of biopolymers in various hosts are discussed. Taken together, microbial engineering has expanded the repertoire of green biological chemistry by increasing the diversity of biobased materials.

Exploring biochemical pathways for mono-ethylene glycol (MEG) synthesis from synthesis gas.

Mono-ethylene glycol (MEG) is an important petrochemical with widespread use in numerous consumer products. The current industrial MEG-production process relies on non-renewable fossil fuel-based feedstocks, such as petroleum, natural gas, and naphtha; hence, it is useful to explore alternative routes of MEG-synthesis from gases as they might provide a greener and more sustainable alternative to the current production methods. Technologies of synthetic biology and metabolic engineering of microorganisms can be deployed for the expression of new biochemical pathways for MEG-synthesis from gases, provided that such promising alternative routes are first identified. We used the BNICE.ch algorithm to develop novel and previously unknown biological pathways to MEG from synthesis gas by leveraging the Wood-Ljungdahl pathway of carbon fixation of acetogenic bacteria. We developed a set of useful pathway pruning and analysis criteria to systematically assess thousands of pathways generated by BNICE.ch. Published genome-scale models of Moorella thermoacetica and Clostridium ljungdahlii were used to perform the pathway yield calculations and in-depth analyses of seven (7) newly developed biological MEG-producing pathways from gases, including CO2, CO, and H2. These analyses helped identify not only better candidate pathways, but also superior chassis organisms that can be used for metabolic engineering of the candidate pathways. The pathway generation, pruning, and detailed analysis procedures described in this study can also be used to develop biochemical pathways for other commodity chemicals from gaseous substrates.

Experimental validation of in silico model-predicted isocitrate dehydrogenase and phosphomannose isomerase from Dehalococcoides mccartyi.

Gene sequences annotated as proteins of unknown or non-specific function and hypothetical proteins account for a large fraction of most genomes. In the strictly anaerobic and organohalide respiring Dehalococcoides mccartyi, this lack of annotation plagues almost half the genome. Using a combination of bioinformatics analyses and genome-wide metabolic modelling, new or more specific annotations were proposed for about 80 of these poorly annotated genes in previous investigations of D. mccartyi metabolism. Herein, we report the experimental validation of the proposed reannotations for two such genes (KB1_0495 and KB1_0553) from D. mccartyi strains in the KB-1 community. KB1_0495 or DmIDH was originally annotated as an NAD(+)-dependent isocitrate dehydrogenase, but biochemical assays revealed its activity primarily with NADP(+) as a cofactor. KB1_0553, also denoted as DmPMI, was originally annotated as a hypothetical protein/sugar isomerase domain protein. We previously proposed that it was a bifunctional phosphoglucose isomerase/phosphomannose isomerase, but only phosphomannose isomerase activity was identified and confirmed experimentally. Further bioinformatics analyses of these two protein sequences suggest their affiliation to potentially novel enzyme families within their respective larger enzyme super families.

Investigating Moorella thermoacetica metabolism with a genome-scale constraint-based metabolic model.

Moorella thermoacetica is a strictly anaerobic, endospore-forming, and metabolically versatile acetogenic bacterium capable of conserving energy by both autotrophic (acetogenesis) and heterotrophic (homoacetogenesis) modes of metabolism. Its metabolic diversity and the ability to efficiently convert a wide range of compounds, including syngas (CO + H2) into acetyl-CoA have made this thermophilic bacterium a promising host for industrial biotechnology applications. However, lack of detailed information on M. thermoacetica's metabolism is a major impediment to its use as a microbial cell factory. In order to overcome this issue, a genome-scale constraint-based metabolic model of Moorella thermoacetica, iAI558, has been developed using its genome sequence and physiological data from published literature. The reconstructed metabolic network of M. thermoacetica comprises 558 metabolic genes, 705 biochemical reactions, and 698 metabolites. Of the total 705 model reactions, 680 are gene-associated while the rest are non-gene associated reactions. The model, in addition to simulating both autotrophic and heterotrophic growth of M. thermoacetica, revealed degeneracy in its TCA-cycle, a common characteristic of anaerobic metabolism. Furthermore, the model helped elucidate the poorly understood energy conservation mechanism of M. thermoacetica during autotrophy. Thus, in addition to generating experimentally testable hypotheses regarding its physiology, such a detailed model will facilitate rapid strain designing and metabolic engineering of M. thermoacetica for industrial applications.

New insights into Dehalococcoides mccartyi metabolism from a reconstructed metabolic network-based systems-level analysis of D. mccartyi transcriptomes.

Organohalide respiration, mediated by Dehalococcoides mccartyi, is a useful bioremediation process that transforms ground water pollutants and known human carcinogens such as trichloroethene and vinyl chloride into benign ethenes. Successful application of this process depends on the fundamental understanding of the respiration and metabolism of D. mccartyi. Reductive dehalogenases, encoded by rdhA genes of these anaerobic bacteria, exclusively catalyze organohalide respiration and drive metabolism. To better elucidate D. mccartyi metabolism and physiology, we analyzed available transcriptomic data for a pure isolate (Dehalococcoides mccartyi strain 195) and a mixed microbial consortium (KB-1) using the previously developed pan-genome-scale reconstructed metabolic network of D. mccartyi. The transcriptomic data, together with available proteomic data helped confirm transcription and expression of the majority genes in D. mccartyi genomes. A composite genome of two highly similar D. mccartyi strains (KB-1 Dhc) from the KB-1 metagenome sequence was constructed, and operon prediction was conducted for this composite genome and other single genomes. This operon analysis, together with the quality threshold clustering analysis of transcriptomic data helped generate experimentally testable hypotheses regarding the function of a number of hypothetical proteins and the poorly understood mechanism of energy conservation in D. mccartyi. We also identified functionally enriched important clusters (13 for strain 195 and 11 for KB-1 Dhc) of co-expressed metabolic genes using information from the reconstructed metabolic network. This analysis highlighted some metabolic genes and processes, including lipid metabolism, energy metabolism, and transport that potentially play important roles in organohalide respiration. Overall, this study shows the importance of an organism's metabolic reconstruction in analyzing various "omics" data to obtain improved understanding of the metabolism and physiology of the organism.