Mitochondrial ATP generation is more proteome efficient than glycolysis.

Metabolic efficiency profoundly influences organismal fitness. Nonphotosynthetic organisms, from yeast to mammals, derive usable energy primarily through glycolysis and respiration. Although respiration is more energy efficient, some cells favor glycolysis even when oxygen is available (aerobic glycolysis, Warburg effect). A leading explanation is that glycolysis is more efficient in terms of ATP production per unit mass of protein (that is, faster). Through quantitative flux analysis and proteomics, we find, however, that mitochondrial respiration is actually more proteome efficient than aerobic glycolysis. This is shown across yeast strains, T cells, cancer cells, and tissues and tumors in vivo. Instead of aerobic glycolysis being valuable for fast ATP production, it correlates with high glycolytic protein expression, which promotes hypoxic growth. Aerobic glycolytic yeasts do not excel at aerobic growth but outgrow respiratory cells during oxygen limitation. We accordingly propose that aerobic glycolysis emerges from cells maintaining a proteome conducive to both aerobic and hypoxic growth.

An end-to-end pipeline for succinic acid production at an industrially relevant scale using Issatchenkia orientalis.

Microbial production of succinic acid (SA) at an industrially relevant scale has been hindered by high downstream processing costs arising from neutral pH fermentation for over three decades. Here, we metabolically engineer the acid-tolerant yeast Issatchenkia orientalis for SA production, attaining the highest titers in sugar-based media at low pH (pH 3) in fed-batch fermentations, i.e. 109.5 g/L in minimal medium and 104.6 g/L in sugarcane juice medium. We further perform batch fermentation using sugarcane juice medium in a pilot-scale fermenter (300×) and achieve 63.1 g/L of SA, which can be directly crystallized with a yield of 64.0%. Finally, we simulate an end-to-end low-pH SA production pipeline, and techno-economic analysis and life cycle assessment indicate our process is financially viable and can reduce greenhouse gas emissions by 34-90% relative to fossil-based production processes. We expect I. orientalis can serve as a general industrial platform for production of organic acids.

A landing pad system for multicopy gene integration in Issatchenkia orientalis.

The robust nature of the non-conventional yeast Issatchenkia orientalis allows it to grow under highly acidic conditions and therefore, has gained increasing interest in producing organic acids using a variety of carbon sources. Recently, the development of a genetic toolbox for I. orientalis, including an episomal plasmid, characterization of multiple promoters and terminators, and CRISPR-Cas9 tools, has eased the metabolic engineering efforts in I. orientalis. However, multiplex engineering is still hampered by the lack of efficient multicopy integration tools. To facilitate the construction of large, complex metabolic pathways by multiplex CRISPR-Cas9-mediated genome editing, we developed a bioinformatics pipeline to identify and prioritize genome-wide intergenic loci and characterized 47 gRNAs located in 21 intergenic regions. These loci are screened for guide RNA cutting efficiency, integration efficiency of a gene cassette, the resulting cellular fitness, and GFP expression level. We further developed a landing pad system using components from these well-characterized loci, which can aid in the integration of multiple genes using single guide RNA and multiple repair templates of the user's choice. We have demonstrated the use of the landing pad for simultaneous integrations of 2, 3, 4, or 5 genes to the target loci with efficiencies greater than 80%. As a proof of concept, we showed how the production of 5-aminolevulinic acid can be improved by integrating five copies of genes at multiple sites in one step. We have further demonstrated the efficiency of this tool by constructing a metabolic pathway for succinic acid production by integrating five gene expression cassettes using a single guide RNA along with five different repair templates, leading to the production of 9 g/L of succinic acid in batch fermentations. This study demonstrates the effectiveness of a single gRNA-mediated CRISPR platform to build complex metabolic pathways in a non-conventional yeast. This landing pad system will be a valuable tool for the metabolic engineering of I. orientalis.

Mass Spectrometry-Based High-Throughput Quantification of Bioproducts in Liquid Culture.

To meet the ever-increasing need for high-throughput screening in metabolic engineering, information-rich, fast screening methods are needed. Mass spectrometry (MS) provides an efficient and general approach for metabolite screening and offers the capability of characterizing a broad range of analytes in a label-free manner, but often requires a range of sample clean-up and extraction steps. Liquid extraction surface analysis (LESA) coupled MS is an image-guided MS surface analysis approach that directly samples and introduces metabolites from a surface to MS. Here, we combined the advantages of LESA-MS and an acoustic liquid handler with stable isotope-labeled internal standards. This approach provides absolute quantitation of target chemicals from liquid culture-dried droplets and enables high-throughput quantitative screening for microbial metabolites. In this study, LESA-MS was successfully applied to quantify several different metabolites (itaconic acid, triacetic acid lactone, and palmitic acid) from different yeast strains in different mediums, demonstrating its versatility, accuracy, and efficiency across a range of microbial engineering applications.

Comparative study of two Saccharomyces cerevisiae strains with kinetic models at genome-scale.

The parameterization of kinetic models requires measurement of fluxes and/or metabolite levels for a base strain and a few genetic perturbations thereof. Unlike stoichiometric models that are mostly invariant to the specific strain, it remains unclear whether kinetic models constructed for different strains of the same species have similar or significantly different kinetic parameters. This important question underpins the applicability range and prediction limits of kinetic reconstructions. To this end, herein we parameterize two separate large-scale kinetic models using K-FIT with genome-wide coverage corresponding to two distinct strains of Saccharomyces cerevisiae: CEN.PK 113-7D strain (model k-sacce306-CENPK), and growth-deficient BY4741 (isogenic to S288c; model k-sacce306-BY4741). The metabolic network for each model contains 306 reactions, 230 metabolites, and 119 substrate-level regulatory interactions. The two models (for CEN.PK and BY4741) recapitulate, within one standard deviation, 77% and 75% of the fitted dataset fluxes, respectively, determined by 13C metabolic flux analysis for wild-type and eight single-gene knockout mutants of each strain. Strain-specific kinetic parameterization results indicate that key enzymes in the TCA cycle, glycolysis, and arginine and proline metabolism drive the metabolic differences between these two strains of S. cerevisiae. Our results suggest that although kinetic models cannot be readily used across strains as stoichiometric models, they can capture species-specific information through the kinetic parameterization process.

Metabolic Engineering: Methodologies and Applications.

Metabolic engineering aims to improve the production of economically valuable molecules through the genetic manipulation of microbial metabolism. While the discipline is a little over 30 years old, advancements in metabolic engineering have given way to industrial-level molecule production benefitting multiple industries such as chemical, agriculture, food, pharmaceutical, and energy industries. This review describes the design, build, test, and learn steps necessary for leading a successful metabolic engineering campaign. Moreover, we highlight major applications of metabolic engineering, including synthesizing chemicals and fuels, broadening substrate utilization, and improving host robustness with a focus on specific case studies. Finally, we conclude with a discussion on perspectives and future challenges related to metabolic engineering.

Cas9-Based Metabolic Engineering of Issatchenkia orientalis for Enhanced Utilization of Cellulosic Hydrolysates.

Issatchenkia orientalis, exhibiting high tolerance against harsh environmental conditions, is a promising metabolic engineering host for producing fuels and chemicals from cellulosic hydrolysates containing fermentation inhibitors under acidic conditions. Although genetic tools for I. orientalis exist, they require auxotrophic mutants so that the selection of a host strain is limited. We developed a drug resistance gene (cloNAT)-based genome-editing method for engineering any I. orientalis strains and engineered I. orientalis strains isolated from various sources for xylose fermentation. Specifically, xylose reductase, xylitol dehydrogenase, and xylulokinase from Scheffersomyces stipitis were integrated into an intended chromosomal locus in four I. orientalis strains (SD108, IO21, IO45, and IO46) through Cas9-based genome editing. The resulting strains (SD108X, IO21X, IO45X, and IO46X) efficiently produced ethanol from cellulosic and hemicellulosic hydrolysates even though the pH adjustment and nitrogen source were not provided. As they presented different fermenting capacities, selection of a host I. orientalis strain was crucial for producing fuels and chemicals using cellulosic hydrolysates.

TALEN outperforms Cas9 in editing heterochromatin target sites.

Genome editing critically relies on selective recognition of target sites. However, despite recent progress, the underlying search mechanism of genome-editing proteins is not fully understood in the context of cellular chromatin environments. Here, we use single-molecule imaging in live cells to directly study the behavior of CRISPR/Cas9 and TALEN. Our single-molecule imaging of genome-editing proteins reveals that Cas9 is less efficient in heterochromatin than TALEN because Cas9 becomes encumbered by local searches on non-specific sites in these regions. We find up to a fivefold increase in editing efficiency for TALEN compared to Cas9 in heterochromatin regions. Overall, our results show that Cas9 and TALEN use a combination of 3-D and local searches to identify target sites, and the nanoscopic granularity of local search determines the editing outcomes of the genome-editing proteins. Taken together, our results suggest that TALEN is a more efficient gene-editing tool than Cas9 for applications in heterochromatin.

Genome-scale metabolic reconstruction of the non-model yeast Issatchenkia orientalis SD108 and its application to organic acids production.

Many platform chemicals can be produced from renewable biomass by microorganisms, with organic acids making up a large fraction. Intolerance to the resulting low pH growth conditions, however, remains a challenge for the industrial production of organic acids by microorganisms. Issatchenkia orientalis SD108 is a promising host for industrial production because it is tolerant to acidic conditions as low as pH 2.0. With the goal to systematically assess the metabolic capabilities of this non-model yeast, we developed a genome-scale metabolic model for I. orientalis SD108 spanning 850 genes, 1826 reactions, and 1702 metabolites. In order to improve the model's quantitative predictions, organism-specific macromolecular composition and ATP maintenance requirements were determined experimentally and implemented. We examined its network topology, including essential genes and flux coupling analysis and drew comparisons with the Yeast 8.3 model for Saccharomyces cerevisiae. We explored the carbon substrate utilization and examined the organism's production potential for the industrially-relevant succinic acid, making use of the OptKnock framework to identify gene knockouts which couple production of the targeted chemical to biomass production. The genome-scale metabolic model iIsor850 is a data-supported curated model which can inform genetic interventions for overproduction.

Recent advances in domesticating non-model microorganisms.

Non-model microorganisms have been increasingly explored as microbial cell factories for production of chemicals, fuels, and materials owing to their unique physiology and metabolic capabilities. However, these microorganisms often lack facile genetic tools for strain development, which hinders their adoption as production hosts. In this review, we describe recent advances in domestication of non-model microorganisms, including bacteria, actinobacteria, cyanobacteria, yeast, and fungi, with a focus on the development of genetic tools. In addition, we highlight some successful applications of non-model microorganisms as microbial cell factories.

A genetic toolbox for metabolic engineering of Issatchenkia orientalis.

The nonconventional yeast Issatchenkia orientalis can grow under highly acidic conditions and has been explored for production of various organic acids. However, its broader application is hampered by the lack of efficient genetic tools to enable sophisticated metabolic manipulations. We recently constructed an episomal plasmid based on the autonomously replicating sequence (ARS) from Saccharomyces cerevisiae (ScARS) in I. orientalis and developed a CRISPR/Cas9 system for multiplexed gene deletions. Here we report three additional genetic tools including: (1) identification of a 0.8 kb centromere-like (CEN-L) sequence from the I. orientalis genome by using bioinformatics and functional screening; (2) discovery and characterization of a set of constitutive promoters and terminators under different culture conditions by using RNA-Seq analysis and a fluorescent reporter; and (3) development of a rapid and efficient in vivo DNA assembly method in I. orientalis, which exhibited ~100% fidelity when assembling a 7 kb-plasmid from seven DNA fragments ranging from 0.7 kb to 1.7 kb. As proof of concept, we used these genetic tools to rapidly construct a functional xylose utilization pathway in I. orientalis.

In vivo Quantification of Alkanes in Escherichia coli.

Microbial production of alkanes employing synthetic biology tools has gained tremendous attention owing to the high energy density and similarity of alkanes to existing petroleum fuels. One of the most commonly studied pathways includes the production of alkanes by AAR (acyl-ACP (acyl carrier protein) reductase)-ADO (aldehyde deformylating oxygenase) pathway. Here, the intermediates of fatty acid synthesis pathway are used as substrate by the AAR enzyme to make fatty aldehyde, which is then deformylated by ADO to make linear chain alkane. However, the variation in substrate availability to the first enzyme of the pathway, i.e., AAR, via fatty acid synthesis pathway and low turnover of the ADO enzyme make calculation of yields and titers under in vivo conditions extremely difficult. In vivo assay employing external addition of defined substrates for ADO enzyme into the medium helps to monitor the influx of substrate hence providing a more accurate measurement of the product yields. In this protocol, we include a detailed guide for implementing the in vivo assay for monitoring alkane production in E. coli.

Development of a CRISPR/Cas9-Based Tool for Gene Deletion in Issatchenkia orientalis.

The nonconventional yeast Issatchenkia orientalis has emerged as a potential platform microorganism for production of organic acids due to its ability to grow robustly under highly acidic conditions. However, lack of efficient genetic tools remains a major bottleneck in metabolic engineering of this organism. Here we report that the autonomously replicating sequence (ARS) from Saccharomyces cerevisiae (ScARS) was functional for plasmid replication in I. orientalis, and the resulting episomal plasmid enabled efficient genome editing by the CRISPR/Cas9 system. The optimized CRISPR/Cas9-based system employed a fusion RPR1'-tRNA promoter for single guide RNA (sgRNA) expression and could attain greater than 97% gene disruption efficiency for various gene targets. Additionally, we demonstrated multiplexed gene deletion with disruption efficiencies of 90% and 47% for double gene and triple gene knockouts, respectively. This genome editing tool can be used for rapid strain development and metabolic engineering of this organism for production of biofuels and chemicals.IMPORTANCE Microbial production of fuels and chemicals from renewable and readily available biomass is a sustainable and economically attractive alternative to petroleum-based production. Because of its unusual tolerance to highly acidic conditions, I. orientalis is a promising potential candidate for the manufacture of valued organic acids. Nevertheless, reliable and efficient genetic engineering tools in I. orientalis are limited. The results outlined in this paper describe a stable episomal ARS-containing plasmid and the first CRISPR/Cas9-based system for gene disruptions in I. orientalis, paving the way for applying genome engineering and metabolic engineering strategies and tools in this microorganism for production of fuels and chemicals.

A consensus-guided approach yields a heat-stable alkane-producing enzyme and identifies residues promoting thermostability.

Aldehyde-deformylating oxygenase (ADO) is an essential enzyme for production of long-chain alkanes as drop-in biofuels, which are compatible with existing fuel systems. The most active ADOs are present in mesophilic cyanobacteria, especially Nostoc punctiforme Given the potential applications of thermostable enzymes in biorefineries, here we generated a thermostable (Cts)-ADO based on a consensus of ADO sequences from several thermophilic cyanobacterial strains. Using an in silico design pipeline and a metagenome library containing 41 hot-spring microbial communities, we created Cts-ADO. Cts-ADO displayed a 3.8-fold increase in pentadecane production on raising the temperature from 30 to 42 °C, whereas ADO from N. punctiforme (Np-ADO) exhibited a 1.7-fold decline. 3D structure modeling and molecular dynamics simulations of Cts- and Np-ADO at different temperatures revealed differences between the two enzymes in residues clustered on exposed loops of these variants, which affected the conformation of helices involved in forming the ADO catalytic core. In Cts-ADO, this conformational change promoted ligand binding to its preferred iron, Fe2, in the di-iron cluster at higher temperature, but the reverse was observed in Np-ADO. Detailed mapping of residues conferring Cts-ADO thermostability identified four amino acids, which we substituted individually and together in Np-ADO. Among these substitution variants, A161E was remarkably similar to Cts-ADO in terms of activity optima, kinetic parameters, and structure at higher temperature. A161E was located in loop L6, which connects helices H5 and H6, and supported ligand binding to Fe2 at higher temperatures, thereby promoting optimal activity at these temperatures and explaining the increased thermostability of Cts-ADO.

Model-assisted metabolic engineering of Escherichia coli for long chain alkane and alcohol production.

Biologically-derived hydrocarbons are considered to have great potential as next-generation biofuels owing to the similarity of their chemical properties to contemporary diesel and jet fuels. However, the low yield of these hydrocarbons in biotechnological production is a major obstacle for commercialization. Several genetic and process engineering approaches have been adopted to increase the yield of hydrocarbon, but a model driven approach has not been implemented so far. Here, we applied a constraint-based metabolic modeling approach in which a variable demand for alkane biosynthesis was imposed, and co-varying reactions were considered as potential targets for further engineering of an E. coli strain already expressing cyanobacterial enzymes towards higher chain alkane production. The reactions that co-varied with the imposed alkane production were found to be mainly associated with the pentose phosphate pathway (PPP) and the lower half of glycolysis. An optimal modeling solution was achieved by imposing increased flux through the reaction catalyzed by glucose-6-phosphate dehydrogenase (zwf) and iteratively removing 7 reactions from the network, leading to an alkane yield of 94.2% of the theoretical maximum conversion determined by in silico analysis at a given biomass rate. To validate the in silico findings, we first performed pathway optimization of the cyanobacterial enzymes in E. coli via different dosages of genes, promoting substrate channelling through protein fusion and inducing substantial equivalent protein expression, which led to a 36-fold increase in alka(e)ne production from 2.8 mg/L to 102 mg/L. Further, engineering of E. coli based on in silico findings, including biomass constraint, led to an increase in the alka(e)ne titer to 425 mg/L (major components being 249 mg/L pentadecane and 160 mg/L heptadecene), a 148.6-fold improvement over the initial strain, respectively; with a yield of 34.2% of the theoretical maximum. The impact of model-assisted engineering was also tested for the production of long chain fatty alcohol, another commercially important molecule sharing the same pathway while differing only at the terminal reaction, and a titer of 1506 mg/L was achieved with a yield of 86.4% of the theoretical maximum. Moreover, the model assisted engineered strains had produced 2.54 g/L and 12.5 g/L of long chain alkane and fatty alcohol, respectively, in the bioreactor under fed-batch cultivation condition. Our study demonstrated successful implementation of a combined in silico modeling approach along with the pathway and process optimization in achieving the highest reported titers of long chain hydrocarbons in E. coli.

Engineered Production of Short Chain Fatty Acid in Escherichia coli Using Fatty Acid Synthesis Pathway.

Short-chain fatty acids (SCFAs), such as butyric acid, have a broad range of applications in chemical and fuel industries. Worldwide demand of sustainable fuels and chemicals has encouraged researchers for microbial synthesis of SCFAs. In this study we compared three thioesterases, i.e., TesAT from Anaerococcus tetradius, TesBF from Bryantella formatexigens and TesBT from Bacteroides thetaiotaomicron, for production of SCFAs in Escherichia coli utilizing native fatty acid synthesis (FASII) pathway and modulated the genetic and bioprocess parameters to improve its yield and productivity. E. coli strain expressing tesBT gene yielded maximum butyric acid titer at 1.46 g L-1, followed by tesBF at 0.85 g L-1 and tesAT at 0.12 g L-1. The titer of butyric acid varied significantly depending upon the plasmid copy number and strain genotype. The modulation of genetic factors that are known to influence long chain fatty acid production, such as deletion of the fadD and fadE that initiates the fatty acid degradation cycle and overexpression of fadR that is a global transcriptional activator of fatty acid biosynthesis and repressor of degradation cycle, did not improve the butyric acid titer significantly. Use of chemical inhibitor cerulenin, which restricts the fatty acid elongation cycle, increased the butyric acid titer by 1.7-fold in case of TesBF, while it had adverse impact in case of TesBT. In vitro enzyme assay indicated that cerulenin also inhibited short chain specific thioesterase, though inhibitory concentration varied according to the type of thioesterase used. Further process optimization followed by fed-batch cultivation under phosphorous limited condition led to production of 14.3 g L-1 butyric acid and 17.5 g L-1 total free fatty acid at 28% of theoretical yield. This study expands our understanding of SCFAs production in E. coli through FASII pathway and highlights role of genetic and process optimization to enhance the desired product.

Identification of long chain specific aldehyde reductase and its use in enhanced fatty alcohol production in E. coli.

Long chain fatty alcohols have wide application in chemical industries and transportation sector. There is no direct natural reservoir for long chain fatty alcohol production, thus many groups explored metabolic engineering approaches for its microbial production. Escherichia coli has been the major microbial platform for this effort, however, terminal endogenous enzyme responsible for converting fatty aldehydes of chain length C14-C18 to corresponding fatty alcohols is still been elusive. Through our in silico analysis we selected 35 endogenous enzymes of E. coli having potential of converting long chain fatty aldehydes to fatty alcohols and studied their role under in vivo condition. We found that deletion of ybbO gene, which encodes NADP(+) dependent aldehyde reductase, led to >90% reduction in long chain fatty alcohol production. This feature was found to be strain transcending and reinstalling ybbO gene via plasmid retained the ability of mutant to produce long chain fatty alcohols. Enzyme kinetic study revealed that YbbO has wide substrate specificity ranging from C6 to C18 aldehyde, with maximum affinity and efficiency for C18 and C16 chain length aldehyde, respectively. Along with endogenous production of fatty aldehyde via optimized heterologous expression of cyanobaterial acyl-ACP reductase (AAR), YbbO overexpression resulted in 169mg/L of long chain fatty alcohols. Further engineering involving modulation of fatty acid as well as of phospholipid biosynthesis pathway improved fatty alcohol production by 60%. Finally, the engineered strain produced 1989mg/L of long chain fatty alcohol in bioreactor under fed-batch cultivation condition. Our study shows for the first time a predominant role of a single enzyme in production of long chain fatty alcohols from fatty aldehydes as well as of modulation of phospholipid pathway in increasing the fatty alcohol production.

Heterogeneity of alkane chain length in freshwater and marine cyanobacteria.

The potential utilization of cyanobacteria for the biological production of alkanes represents an exceptional system for the next generation of biofuels. Here, we analyzed a diverse group of freshwater and marine cyanobacterial isolates from Indian culture collections for their ability to produce both alkanes and alkenes. Among the 50 cyanobacterial isolates screened, 32 isolates; 14 freshwater and 18 marine isolates; produced predominantly alkanes. The GC-MS/MS profiles revealed a higher percentage of pentadecane and heptadecane production for marine and freshwater strains, respectively. Oscillatoria species were found to be the highest producers of alkanes. Among the freshwater isolates, Oscillatoria CCC305 produced the maximum alkane level with 0.43 µg/mg dry cell weight, while Oscillatoria formosa BDU30603 was the highest producer among the marine isolates with 0.13 µg/mg dry cell weight. Culturing these strains under different media compositions showed that the alkane chain length was not influenced by the growth medium but was rather an inherent property of the strains. Analysis of the cellular fatty acid content indicated the presence of predominantly C16 chain length fatty acids in marine strains, while the proportion of C18 chain length fatty acids increased in the majority of freshwater strains. These results correlated with alkane chain length specificity of marine and freshwater isolates indicating that alkane chain lengths may be primarily determined by the fatty acid synthesis pathway. Moreover, the phylogenetic analysis showed clustering of pentadecane-producing marine strains that was distinct from heptadecane-producing freshwater strains strongly suggesting a close association between alkane chain length and the cyanobacteria habitat.