Rewriting yeast central carbon metabolism for industrial isoprenoid production.

A bio-based economy has the potential to provide sustainable substitutes for petroleum-based products and new chemical building blocks for advanced materials. We previously engineered Saccharomyces cerevisiae for industrial production of the isoprenoid artemisinic acid for use in antimalarial treatments. Adapting these strains for biosynthesis of other isoprenoids such as ß-farnesene (C15H24), a plant sesquiterpene with versatile industrial applications, is straightforward. However, S. cerevisiae uses a chemically inefficient pathway for isoprenoid biosynthesis, resulting in yield and productivity limitations incompatible with commodity-scale production. Here we use four non-native metabolic reactions to rewire central carbon metabolism in S. cerevisiae, enabling biosynthesis of cytosolic acetyl coenzyme A (acetyl-CoA, the two-carbon isoprenoid precursor) with a reduced ATP requirement, reduced loss of carbon to CO2-emitting reactions, and improved pathway redox balance. We show that strains with rewired central metabolism can devote an identical quantity of sugar to farnesene production as control strains, yet produce 25% more farnesene with that sugar while requiring 75% less oxygen. These changes lower feedstock costs and dramatically increase productivity in industrial fermentations which are by necessity oxygen-constrained. Despite altering key regulatory nodes, engineered strains grow robustly under taxing industrial conditions, maintaining stable yield for two weeks in broth that reaches >15% farnesene by volume. This illustrates that rewiring yeast central metabolism is a viable strategy for cost-effective, large-scale production of acetyl-CoA-derived molecules.

Clean manufacturing powered by biology: how Amyris has deployed technology and aims to do it better.

Amyris is a fermentation product company that leverages synthetic biology and has been bringing novel fermentation products to the market since 2009. Driven by breakthroughs in genome editing, strain construction and testing, analytics, automation, data science, and process development, Amyris has commercialized nine separate fermentation products over the last decade. This has been accomplished by partnering with the teams at 17 different manufacturing sites around the world. This paper begins with the technology that drives Amyris, describes some key lessons learned from early scale-up experiences, and summarizes the technology transfer procedures and systems that have been built to enable moving more products to market faster. Finally, the breadth of the Amyris product portfolio continues to expand; thus the steps being taken to overcome current challenges (e.g. automated strain engineering can now outpace the rest of the product commercialization timeline) are described.

Metabolic analysis of wild-type Escherichia coli and a pyruvate dehydrogenase complex (PDHC)-deficient derivative reveals the role of PDHC in the fermentative metabolism of glucose.

Pyruvate is located at a metabolic junction of assimilatory and dissimilatory pathways and represents a switch point between respiratory and fermentative metabolism. In Escherichia coli, the pyruvate dehydrogenase complex (PDHC) and pyruvate formate-lyase are considered the primary routes of pyruvate conversion to acetyl-CoA for aerobic respiration and anaerobic fermentation, respectively. During glucose fermentation, the in vivo activity of PDHC has been reported as either very low or undetectable, and the role of this enzyme remains unknown. In this study, a comprehensive characterization of wild-type E. coli MG1655 and a PDHC-deficient derivative (Pdh) led to the identification of the role of PDHC in the anaerobic fermentation of glucose. The metabolism of these strains was investigated by using a mixture of (13)C-labeled and -unlabeled glucose followed by the analysis of the labeling pattern in protein-bound amino acids via two-dimensional (13)C,(1)H NMR spectroscopy. Metabolite balancing, biosynthetic (13)C labeling of proteinogenic amino acids, and isotopomer balancing all indicated a large increase in the flux of the oxidative branch of the pentose phosphate pathway (ox-PPP) in response to the PDHC deficiency. Because both ox-PPP and PDHC generate CO(2) and the calculated CO(2) evolution rate was significantly reduced in Pdh, it was hypothesized that the role of PDHC is to provide CO(2) for cell growth. The similarly negative impact of either PDHC or ox-PPP deficiencies, and an even more pronounced impairment of cell growth in a strain lacking both ox-PPP and PDHC, provided further support for this hypothesis. The three strains exhibited similar phenotypes in the presence of an external source of CO(2), thus confirming the role of PDHC. Activation of formate hydrogen-lyase (which converts formate to CO(2) and H(2)) rendered the PDHC deficiency silent, but its negative impact reappeared in a strain lacking both PDHC and formate hydrogen-lyase. A stoichiometric analysis of CO(2) generation via PDHC and ox-PPP revealed that the PDHC route is more carbon- and energy-efficient, in agreement with its beneficial role in cell growth.

Metabolic flux analysis of wild-type Escherichia coli and mutants deficient in pyruvate-dissimilating enzymes during the fermentative metabolism of glucuronate.

The fermentative metabolism of d-glucuronic acid (glucuronate) in Escherichia coli was investigated with emphasis on the dissimilation of pyruvate via pyruvate formate-lyase (PFL) and pyruvate dehydrogenase (PDH). In silico and in vivo metabolic flux analysis (MFA) revealed that PFL and PDH share the dissimilation of pyruvate in wild-type MG1655. Surprisingly, it was found that PDH supports fermentative growth on glucuronate in the absence of PFL. The PDH-deficient strain (Pdh-) exhibited a slower transition into the exponential phase and a decrease in specific rates of growth and glucuronate utilization. Moreover, a significant redistribution of metabolic fluxes was found in PDH- and PFL-deficient strains. Since no role had been proposed for PDH in the fermentative metabolism of E. coli, the metabolic differences between MG1655 and Pdh- were further investigated. An increase in the oxidative pentose phosphate pathway (ox-PPP) flux was observed in response to PDH deficiency. A comparison of the ox-PPP and PDH pathways led to the hypothesis that the role of PDH is the supply of reducing equivalents. The finding that a PDH deficiency lowers the NADH : NAD(+) ratio supported the proposed role of PDH. Moreover, the NADH : NAD(+) ratio in a strain deficient in both PDH and the ox-PPP (Pdh-Zwf-) was even lower than that observed for Pdh-. Strain Pdh-Zwf- also exhibited a slower transition into the exponential phase and a lower growth rate than Pdh-. Finally, a transhydrogenase-deficient strain grew more slowly than wild-type but did not show the slower transition into the exponential phase characteristic of Pdh- mutants. It is proposed that PDH fulfils two metabolic functions. First, by creating the appropriate internal redox state (i.e. appropriate NADH : NAD(+) ratio), PDH ensures the functioning of the glucuronate utilization pathway. Secondly, the NADH generated by PDH can be converted to NADPH by the action of transhydrogenases, thus serving as biosynthetic reducing power in the synthesis of building blocks and macromolecules.

Anaerobic fermentation of glycerol in Paenibacillus macerans: metabolic pathways and environmental determinants.

Paenibacillus macerans is one of the species with the broadest metabolic capabilities in the genus Paenibacillus, able to ferment hexoses, deoxyhexoses, pentoses, cellulose, and hemicellulose. However, little is known about glycerol metabolism in this organism, and some studies have reported that glycerol is not fermented. Despite these reports, we found that several P. macerans strains are capable of anaerobic fermentation of glycerol. One of these strains, P. macerans N234A, grew fermentatively on glycerol at a maximum specific growth rate of 0.40 h(-1) and was chosen for further characterization. The use of [U-13C]glycerol and further analysis of extracellular metabolites and proteinogenic amino acids via nuclear magnetic resonance (NMR) spectroscopy allowed identification of ethanol, formate, acetate, succinate, and 1,2-propanediol (1,2-PDO) as fermentation products and demonstrated that glycerol is incorporated into cellular components. A medium formulation with low concentrations of potassium and phosphate, cultivation at acidic pH, and the use of a CO2-enriched atmosphere stimulated glycerol fermentation and are proposed to be environmental determinants of this process. The pathways involved in glycerol utilization and synthesis of fermentation products were identified using NMR spectroscopy in combination with enzyme assays. Based on these studies, the synthesis of ethanol and 1,2-PDO is proposed to be a metabolic determinant of glycerol fermentation in P. macerans N234A. Conversion of glycerol to ethanol fulfills energy requirements by generating one molecule of ATP per molecule of ethanol synthesized. Conversion of glycerol to 1,2-PDO results in the consumption of reducing equivalents, thus facilitating redox balance. Given the availability, low price, and high degree of reduction of glycerol, the high metabolic rates exhibited by P. macerans N234A are of paramount importance for the production of fuels and chemicals.

A new model for the anaerobic fermentation of glycerol in enteric bacteria: trunk and auxiliary pathways in Escherichia coli.

Anaerobic fermentation of glycerol in the Enterobacteriaceae family has long been considered a unique property of species that synthesize 1,3-propanediol (1,3-PDO). However, we have discovered that Escherichia coli can ferment glycerol in a 1,3-PDO-independent manner. We identified 1,2-propanediol (1,2-PDO) as a fermentation product and established the pathway that mediates its synthesis as well as its role in the metabolism of glycerol. We also showed that the trunk pathway responsible for the conversion of glycerol into glycolytic intermediates is composed of two enzymes: a type II glycerol dehydrogenase (glyDH-II) and a dihydroxyacetone kinase (DHAK), the former of previously unknown physiological role. Based on our findings, we propose a new model for glycerol fermentation in enteric bacteria in which: (i) the production of 1,2-PDO provides a means to consume reducing equivalents generated in the synthesis of cell mass, thus facilitating redox balance, and (ii) the conversion of glycerol to ethanol, through a redox-balanced pathway, fulfills energy requirements by generating ATP via substrate-level phosphorylation. The activity of the formate hydrogen-lyase and F(0)F(1)-ATPase systems were also found to facilitate the fermentative metabolism of glycerol, and along with the ethanol and 1,2-PDO pathways, were considered auxiliary or enabling. We demonstrated that glycerol fermentation in E. coli was not previously observed due to the use of medium formulations and culture conditions that impair the aforementioned pathways. These include high concentrations of potassium and phosphate, low concentrations of glycerol, alkaline pH, and closed cultivation systems that promote the accumulation of hydrogen gas.

Fermentative utilization of glycerol by Escherichia coli and its implications for the production of fuels and chemicals.

Availability, low prices, and a high degree of reduction make glycerol an ideal feedstock to produce reduced chemicals and fuels via anaerobic fermentation. Although glycerol metabolism in Escherichia coli had been thought to be restricted to respiratory conditions, we report here the utilization of this carbon source in the absence of electron acceptors. Cells grew fermentatively on glycerol and exhibited exponential growth at a maximum specific growth rate of 0.040 +/- 0.003 h(-1). The fermentative nature of glycerol metabolism was demonstrated through studies in which cell growth and glycerol utilization were observed despite blocking several respiratory processes. The incorporation of glycerol in cellular biomass was also investigated via nuclear magnetic resonance analysis of cultures in which either 50% U-13C-labeled or 100% unlabeled glycerol was used. These studies demonstrated that about 20% of the carbon incorporated into the protein fraction of biomass originated from glycerol. The use of U-13C-labeled glycerol also allowed the unambiguous identification of ethanol and succinic, acetic, and formic acids as the products of glycerol fermentation. The synthesis of ethanol was identified as a metabolic determinant of glycerol fermentation; this pathway fulfills energy requirements by generating, in a redox-balanced manner, 1 mol of ATP per mol of glycerol converted to ethanol. A fermentation balance analysis revealed an excellent closure of both carbon (approximately 95%) and redox (approximately 96%) balances. On the other hand, cultivation conditions that prevent H2 accumulation were shown to be an environmental determinant of glycerol fermentation. The negative effect of H2 is related to its metabolic recycling, which in turn generates an unfavorable internal redox state. The implications of our findings for the production of reduced chemicals and fuels were illustrated by coproducing ethanol plus formic acid and ethanol plus hydrogen from glycerol at yields approaching their theoretical maximum.

Approaches and Recent Developments for the Commercial Production of Semi-synthetic Artemisinin.

The antimalarial drug artemisinin is a natural product produced by the plant Artemisia annua. Extracts of A. annua have been used in Chinese herbal medicine for over two millennia. Following the re-discovery of A. annua extract as an effective antimalarial, and the isolation and structural elucidation of artemisinin as the active agent, it was recommended as the first-line treatment for uncomplicated malaria in combination with another effective antimalarial drug (Artemisinin Combination Therapy) by the World Health Organization (WHO) in 2002. Following the WHO recommendation, the availability and price of artemisinin fluctuated greatly, ranging from supply shortfalls in some years to oversupply in others. To alleviate these supply and price issues, a second source of artemisinin was sought, resulting in an effort to produce artemisinic acid, a late-stage chemical precursor of artemisinin, by yeast fermentation, followed by chemical conversion to artemisinin (i.e., semi-synthesis). Engineering to enable production of artemisinic acid in yeast relied on the discovery of A. annua genes encoding artemisinic acid biosynthetic enzymes, and synthetic biology to engineer yeast metabolism. The progress of this effort, which resulted in semi-synthetic artemisinin entering commercial production in 2013, is reviewed with an emphasis on recent publications and opportunities for further development. Aspects of both the biology of artemisinin production in A. annua, and yeast strain engineering are discussed, as are recent developments in the chemical conversion of artemisinic acid to artemisinin.

A novel data mining method to identify assay-specific signatures in functional genomic studies.

BACKGROUND: The highly dimensional data produced by functional genomic (FG) studies makes it difficult to visualize relationships between gene products and experimental conditions (i.e., assays). Although dimensionality reduction methods such as principal component analysis (PCA) have been very useful, their application to identify assay-specific signatures has been limited by the lack of appropriate methodologies. This article proposes a new and powerful PCA-based method for the identification of assay-specific gene signatures in FG studies. RESULTS: The proposed method (PM) is unique for several reasons. First, it is the only one, to our knowledge, that uses gene contribution, a product of the loading and expression level, to obtain assay signatures. The PM develops and exploits two types of assay-specific contribution plots, which are new to the application of PCA in the FG area. The first type plots the assay-specific gene contribution against the given order of the genes and reveals variations in distribution between assay-specific gene signatures as well as outliers within assay groups indicating the degree of importance of the most dominant genes. The second type plots the contribution of each gene in ascending or descending order against a constantly increasing index. This type of plots reveals assay-specific gene signatures defined by the inflection points in the curve. In addition, sharp regions within the signature define the genes that contribute the most to the signature. We proposed and used the curvature as an appropriate metric to characterize these sharp regions, thus identifying the subset of genes contributing the most to the signature. Finally, the PM uses the full dataset to determine the final gene signature, thus eliminating the chance of gene exclusion by poor screening in earlier steps. The strengths of the PM are demonstrated using a simulation study, and two studies of real DNA microarray data--a study of classification of human tissue samples and a study of E. coli cultures with different medium formulations. CONCLUSION: We have developed a PCA-based method that effectively identifies assay-specific signatures in ranked groups of genes from the full data set in a more efficient and simplistic procedure than current approaches. Although this work demonstrates the ability of the PM to identify assay-specific signatures in DNA microarray experiments, this approach could be useful in areas such as proteomics and metabolomics.

Anaerobic fermentation of glycerol by Escherichia coli: a new platform for metabolic engineering.

The worldwide surplus of glycerol generated as inevitable byproduct of biodiesel fuel and oleochemical production is resulting in the shutdown of traditional glycerol-producing/refining plants and new applications are needed for this now abundant carbon source. In this article we report our finding that Escherichia coli can ferment glycerol in a pH-dependent manner. We hypothesize that glycerol fermentation is linked to the availability of CO(2), which under acidic conditions is produced by the oxidation of formate by the enzyme formate hydrogen lyase (FHL). In agreement with this hypothesis, glycerol fermentation was severely impaired by blocking the activity of FHL. We demonstrated that, unlike CO(2), hydrogen (the other product of FHL-mediated formate oxidation) had a negative impact on cell growth and glycerol fermentation. In addition, supplementation of the medium with CO(2) partially restored the ability of an FHL-deficient strain to ferment glycerol. High pH resulted in low CO(2) generation (low activity of FHL) and availability (most CO(2) is converted to bicarbonate), and consequently very inefficient fermentation of glycerol. Most of the fermented glycerol was recovered in the reduced compounds ethanol and succinate (93% of the product mixture), which reflects the highly reduced state of glycerol and confirms the fermentative nature of this process. Since glycerol is a cheap, abundant, and highly reduced carbon source, our findings should enable the development of an E. coli-based platform for the anaerobic production of reduced chemicals from glycerol at yields higher than those obtained from common sugars, such as glucose.