On-chip manufacturing of synthetic proteins for point-of-care therapeutics.

Therapeutic proteins have recently received increasing attention because of their clinical potential. Currently, most therapeutic proteins are produced on a large scale using various cell culture systems. However, storing and transporting these therapeutic proteins at low temperatures makes their distribution expensive and problematic, especially for applications in remote locations. To this end, an emerging solution is to use point-of-care technologies that enable immediate and accessible protein production at or near the patient's bedside. Here we present the development of "Therapeutics-On-a-Chip (TOC)", an integrated microfluidic platform that enables point-of-care synthesis and purification of therapeutic proteins. We used fresh and lyophilized materials for cell-free synthesis of therapeutic proteins on microfluidic chips and applied immunoprecipitation for highly efficient, on-chip protein purification. We first demonstrated this approach by expressing and purifying a reporter protein, green fluorescent protein. Next, we used TOC to produce cecropin B, an antimicrobial peptide that is widely used to control biofilm-associated diseases. We successfully synthesized and purified cecropin B at 63 ng/µl within 6 h with a 92% purity, followed by confirming its antimicrobial functionality using a growth inhibition assay. Our TOC technology provides a new platform for point-of-care production of therapeutic proteins at a clinically relevant quantity.

Synergizing 13C Metabolic Flux Analysis and Metabolic Engineering for Biochemical Production.

Metabolic engineering of industrial microorganisms to produce chemicals, fuels, and drugs has attracted increasing interest as it provides an environment-friendly and renewable route that does not depend on depleting petroleum sources. However, the microbial metabolism is so complex that metabolic engineering efforts often have difficulty in achieving a satisfactory yield, titer, or productivity of the target chemical. To overcome this challenge, 13C Metabolic Flux Analysis (13C-MFA) has been developed to investigate rigorously the cell metabolism and quantify the carbon flux distribution in central metabolic pathways. In the past decade, 13C-MFA has been widely used in academic labs and the biotechnology industry to pinpoint the key issues related to microbial-based chemical production and to guide the development of the appropriate metabolic engineering strategies for improving the biochemical production. In this chapter we introduce the basics of 13C-MFA and illustrate how 13C-MFA has been applied to synergize with metabolic engineering to identify and tackle the rate-limiting steps in biochemical production.

Systematic Optimization of Protein Secretory Pathways in Saccharomyces cerevisiae to Increase Expression of Hepatitis B Small Antigen.

Hepatitis B is a major disease that chronically infects millions of people in the world, especially in developing countries. Currently, one of the effective vaccines to prevent Hepatitis B is the Hepatitis B Small Antigen (HBsAg), which is mainly produced by the recombinant yeast Saccharomyces cerevisiae. In order to bring down the price, which is still too high for people in developing countries to afford, it is important to understand key cellular processes that limit protein expression. In this study, we took advantage of yeast knockout collection (YKO) and screened 194 S. cerevisiae strains with single gene knocked out in four major steps of the protein secretory pathway, i.e., endoplasmic-reticulum (ER)-associated protein degradation, protein folding, unfolded protein response (UPR), and translocation and exocytosis. The screening showed that the single deletion of YPT32, SBH1, and HSP42 led to the most significant increase of HBsAg expression over the wild type while the deletion of IRE1 led to a profound decrease of HBsAg expression. The synergistic effects of gene knockout and gene overexpression were next tested. We found that simultaneously deleting YPT32 and overexpressing IRE1 led to a 2.12-fold increase in HBsAg expression over the wild type strain. The results of this study revealed novel genetic targets of protein secretory pathways that could potentially improve the manufacturing of broad scope vaccines in a cost-effective way using recombinant S. cerevisiae.

Compartmentalized metabolic engineering for biochemical and biofuel production.

Sub-cellular compartments create specialized reaction chambers in eukaryotes. These compartments provide favorable micro-environments for many metabolic processes. Recently, metabolic engineers have explored the concept of pathway compartmentalization to enhance the performance of metabolic pathways. This strategy offers many unique advantages, including (i) increased local concentrations of enzymes and substrates, (ii) accessing alternate substrate pools, (iii) separation from competing reactions, and (iv) isolation of harmful intermediates or conditions needed for the pathway. In this review, the method of localizing metabolic pathways into specific organelles as well as the benefits of pathway compartmentalization in terms of enhancing the production of value-added chemicals is discussed.

Mini-review: In vitro Metabolic Engineering for Biomanufacturing of High-value Products.

With the breakthroughs in biomolecular engineering and synthetic biology, many valuable biologically active compound and commodity chemicals have been successfully manufactured using cell-based approaches in the past decade. However, because of the high complexity of cell metabolism, the identification and optimization of rate-limiting metabolic pathways for improving the product yield is often difficult, which represents a significant and unavoidable barrier of traditional in vivo metabolic engineering. Recently, some in vitro engineering approaches were proposed as alternative strategies to solve this problem. In brief, by reconstituting a biosynthetic pathway in a cell-free environment with the supplement of cofactors and substrates, the performance of each biosynthetic pathway could be evaluated and optimized systematically. Several value-added products, including chemicals, nutraceuticals, and drug precursors, have been biosynthesized as proof-of-concept demonstrations of in vitro metabolic engineering. This mini-review summarizes the recent progresses on the emerging topic of in vitro metabolic engineering and comments on the potential application of cell-free technology to speed up the "design-build-test" cycles of biomanufacturing.

Investigate the Metabolic Reprogramming of Saccharomyces cerevisiae for Enhanced Resistance to Mixed Fermentation Inhibitors via 13C Metabolic Flux Analysis.

The fermentation inhibitors from the pretreatment of lignocellulosic materials, e.g., acetic acid and furfural, are notorious due to their negative effects on the cell growth and chemical production. However, the metabolic reprogramming of the cells under these stress conditions, especially metabolic response for resistance to mixed inhibitors, has not been systematically investigated and remains mysterious. Therefore, in this study, 13C metabolic flux analysis (13C-MFA), a powerful tool to elucidate the intracellular carbon flux distributions, has been applied to two Saccharomyces cerevisiae strains with different tolerances to the inhibitors under acetic acid, furfural, and mixed (i.e., acetic acid and furfural) stress conditions to unravel the key metabolic responses. By analyzing the intracellular carbon fluxes as well as the energy and cofactor utilization under different conditions, we uncovered varied metabolic responses to different inhibitors. Under acetate stress, ATP and NADH production was slightly impaired, while NADPH tended towards overproduction. Under furfural stress, ATP and cofactors (including both NADH and NADPH) tended to be overproduced. However, under dual-stress condition, production of ATP and cofactors was severely impaired due to synergistic stress caused by the simultaneous addition of two fermentation inhibitors. Such phenomenon indicated the pivotal role of the energy and cofactor utilization in resisting the mixed inhibitors of acetic acid and furfural. Based on the discoveries, valuable insights are provided to improve the tolerance of S. cerevisiae strain and further enhance lignocellulosic fermentation.

Investigating strain dependency in the production of aromatic compounds in Saccharomyces cerevisiae.

Although Saccharomyces cerevisiae is the most highly domesticated yeast, strain dependency in biotechnological processes still remains as a common, yet poorly understood phenomenon. To investigate this, the entrance to the aromatic amino acid biosynthetic pathway was compared in four commonly used S. cerevisiae laboratory strains. The strains were engineered to accumulate shikimate by overexpressing a mutant version of the pentafunctional ARO1 enzyme with disrupted activity in the shikimate kinase subunit. Carbon tracing and 13 C metabolic flux analysis combined with quantitative PCR, revealed that precursor availability and shikimate production were dramatically different in the four equally engineered strains, which were found to be correlated with the strains' capacity to deal with protein overexpression burden. By implementing a strain-dependent approach, the genetic platform was reformulated, leading to an increase in yield and titer in all strains. The highest producing strain, INVSc1-SA3, produced 358 mg L-1 of shikimate with a yield of 17.9 mg g-1glucose. These results underline the importance of strain selection in developing biological manufacturing processes, demonstrate the first case of high production of shikimate in yeast, and provide an appropriate platform for strain selection for future production of aromatic compounds. Biotechnol. Bioeng. 2016;113: 2676-2685. © 2016 Wiley Periodicals, Inc.

Rapid and efficient galactose fermentation by engineered Saccharomyces cerevisiae.

In the important industrial yeast Saccharomyces cerevisiae, galactose metabolism requires energy production by respiration; therefore, this yeast cannot metabolize galactose under strict anaerobic conditions. While the respiratory dependence of galactose metabolism provides benefits in terms of cell growth and population stability, it is not advantageous for producing fuels and chemicals since a substantial fraction of consumed galactose is converted to carbon dioxide. In order to force S. cerevisiae to use galactose without respiration, a subunit (COX9) of a respiratory enzyme was deleted, but the resulting deletion mutant (Δcox9) was impaired in terms of galactose assimilation. Interestingly, after serial sub-cultures on galactose, the mutant evolved rapidly and was able to use galactose via fermentation only. The evolved strain (JQ-G1) produced ethanol from galactose with a 94% increase in yield and 6.9-fold improvement in specific productivity as compared to the wild-type strain. (13)C-metabolic flux analysis demonstrated a three-fold reduction in carbon flux through the TCA cycle of the evolved mutant with redirection of flux toward the fermentation pathway. Genome sequencing of the JQ-G1 strain revealed a loss of function mutation in a master negative regulator of the Leloir pathway (Gal80p). The mutation (Glu348*) in Gal80p was found to act synergistically with deletion of COX9 for efficient galactose fermentation, and thus the double deletion mutant Δcox9Δgal80 produced ethanol 2.4 times faster and with 35% higher yield than a single knockout mutant with deletion of GAL80 alone. When we introduced a functional COX9 cassette back into the JQ-G1 strain, the JQ-G1-COX9 strain showed a 33% reduction in specific galactose uptake rate and a 49% reduction in specific ethanol production rate as compared to JQ-G1. The wild-type strain was also subjected to serial sub-cultures on galactose but we failed to isolate a mutant capable of utilizing galactose without respiration. We concluded that the metabolic "death valley" (i.e. no galactose utilization by the Δcox9 mutant) is a necessary intermediate phenotype to facilitate galactose utilization without respiration in yeast. The results in this study demonstrate a promising approach for directing adaptive evolution toward fermentative metabolism and for generating evolved yeast strains with improved phenotypes under anaerobic conditions.

Pathway Compartmentalization in Peroxisome of Saccharomyces cerevisiae to Produce Versatile Medium Chain Fatty Alcohols.

Fatty alcohols are value-added chemicals and important components of a variety of industries, which have a >3 billion-dollar global market annually. Long chain fatty alcohols (>C12) are mainly used in surfactants, lubricants, detergents, pharmaceuticals and cosmetics while medium chain fatty alcohols (C6-C12) could be used as diesel-like biofuels. Microbial production of fatty alcohols from renewable feedstock stands as a promising strategy to enable sustainable supply of fatty alcohols. In this study, we report, for the first time, that medium chain fatty alcohols could be produced in yeast via targeted expression of a fatty acyl-CoA reductase (TaFAR) in the peroxisome of Saccharomyces cerevisiae. By tagging TaFAR enzyme with peroxisomal targeting signal peptides, the TaFAR could be compartmentalized into the matrix of the peroxisome to hijack the medium chain fatty acyl-CoA generated from the beta-oxidation pathway and convert them to versatile medium chain fatty alcohols (C10 &C12). The overexpression of genes encoding PEX7 and acetyl-CoA carboxylase further improved fatty alcohol production by 1.4-fold. After medium optimization in fed-batch fermentation using glucose as the sole carbon source, fatty alcohols were produced at 1.3 g/L, including 6.9% 1-decanol, 27.5% 1-dodecanol, 2.9% 1-tetradecanol and 62.7% 1-hexadecanol. This work revealed that peroxisome could be engineered as a compartmentalized organelle for producing fatty acid-derived chemicals in S. cerevisiae.

OM-FBA: Integrate Transcriptomics Data with Flux Balance Analysis to Decipher the Cell Metabolism.

Constraint-based metabolic modeling such as flux balance analysis (FBA) has been widely used to simulate cell metabolism. Thanks to its simplicity and flexibility, numerous algorithms have been developed based on FBA and successfully predicted the phenotypes of various biological systems. However, their phenotype predictions may not always be accurate in FBA because of using the objective function that is assumed for cell metabolism. To overcome this challenge, we have developed a novel computational framework, namely omFBA, to integrate multi-omics data (e.g. transcriptomics) into FBA to obtain omics-guided objective functions with high accuracy. In general, we first collected transcriptomics data and phenotype data from published database (e.g. GEO database) for different microorganisms such as Saccharomyces cerevisiae. We then developed a "Phenotype Match" algorithm to derive an objective function for FBA that could lead to the most accurate estimation of the known phenotype (e.g. ethanol yield). The derived objective function was next correlated with the transcriptomics data via regression analysis to generate the omics-guided objective function, which was next used to accurately simulate cell metabolism at unknown conditions. We have applied omFBA in studying sugar metabolism of S. cerevisiae and found that the ethanol yield could be accurately predicted in most of the cases tested (>80%) by using transcriptomics data alone, and revealed valuable metabolic insights such as the dynamics of flux ratios. Overall, omFBA presents a novel platform to potentially integrate multi-omics data simultaneously and could be incorporated with other FBA-derived tools by replacing the arbitrary objective function with the omics-guided objective functions.

Molecular basis of 5-hydroxytryptophan synthesis in Saccharomyces cerevisiae.

We report for the first time that 5-hydroxytryptophan can be synthesized in Saccharomyces cerevisiae by heterologously expressing prokaryotic phenylalanine 4-hydroxylase or eukaryotic tryptophan 3/5-hydroxylase, together with enhanced synthesis of MH4 or BH4 cofactors. The innate DFR1 gene in the folate synthesis pathway was found to play pivotal roles in 5-hydroxytryptophan synthesis.

Metabolic engineering of Saccharomyces cerevisiae to produce 1-hexadecanol from xylose.

BACKGROUND: An advantageous but challenging approach to overcome the limited supply of petroleum and relieve the greenhouse effect is to produce bulk chemicals from renewable materials. Fatty alcohols, with a billion-dollar global market, are important raw chemicals for detergents, emulsifiers, lubricants, and cosmetics production. Microbial production of fatty alcohols has been successfully achieved in several industrial microorganisms. However, most of the achievements were using glucose, an edible sugar, as the carbon source. To produce fatty alcohols in a renewable manner, non-edible sugars such as xylose will be a more appropriate feedstock. RESULTS: In this study, we aim to engineer a Saccharomyces cerevisiae strain that can efficiently convert xylose to fatty alcohols. To this end, we first introduced the fungal xylose utilization pathway consisting of xylose reductase (XR), xylitol dehydrogenase (XDH), and xylulose kinase (XKS) into a fatty alcohol-producing S. cerevisiae strain (XF3) that was developed in our previous studies to achieve 1-hexadecanol production from xylose at 0.4 g/L. We next applied promoter engineering on the xylose utilization pathway to optimize the expression levels of XR, XDH, and XKS, and increased the 1-hexadecanol titer by 171 %. To further improve the xylose-based fatty alcohol production, two optimized S. cerevisiae strains from promoter engineering were evolved with the xylose as the sole carbon source. We found that the cell growth rate was improved at the expense of decreased fatty alcohol production, which indicated 1-hexadecanol was mainly produced as a non-growth associated product. Finally, through fed-batch fermentation, we successfully achieved 1-hexadecanol production at over 1.2 g/L using xylose as the sole carbon source, which represents the highest titer of xylose-based 1-hexadecanol reported in microbes to date. CONCLUSIONS: A fatty alcohol-producing S. cerevisiae strain was engineered in this study to produce 1-hexadecanol from xylose. Although the xylose pathway we developed in this study could be further improved, this proof-of-concept study, for the first time to our best knowledge, demonstrated that the xylose-based fatty alcohol could be produced in S. cerevisiae with potential applications in developing consolidated bioprocessing for producing other fatty acid-derived chemicals.

¹³C Pathway Analysis for the Role of Formate in Electricity Generation by Shewanella Oneidensis MR-1 Using Lactate in Microbial Fuel Cells.

Microbial fuel cell (MFC) is a promising technology for direct electricity generation from organics by microorganisms. The type of electron donors fed into MFCs affects the electrical performance, and mechanistic understanding of such effects is important to optimize the MFC performance. In this study, we used a model organism in MFCs, Shewanella oneidensis MR-1, and (13)C pathway analysis to investigate the role of formate in electricity generation and the related microbial metabolism. Our results indicated a synergistic effect of formate and lactate on electricity generation, and extra formate addition on the original lactate resulted in more electrical output than using formate or lactate as a sole electron donor. Based on the (13)C tracer analysis, we discovered decoupled cell growth and electricity generation in S. oneidensis MR-1 during co-utilization of lactate and formate (i.e., while the lactate was mainly metabolized to support the cell growth, the formate was oxidized to release electrons for higher electricity generation). To our best knowledge, this is the first time that (13)C tracer analysis was applied to study microbial metabolism in MFCs and it was demonstrated to be a valuable tool to understand the metabolic pathways affected by electron donors in the selected electrochemically-active microorganisms.

Transcriptional profiling reveals molecular basis and novel genetic targets for improved resistance to multiple fermentation inhibitors in Saccharomyces cerevisiae.

BACKGROUND: Lignocellulosic biomass is a promising source of renewable biofuels. However, pretreatment of lignocellulosic biomass generates fermentation inhibitors that adversely affect the growth of industrial microorganisms such as Saccharomyces cerevisiae and prevent economic production of lignocellulosic biofuels. A critical challenge on developing S. cerevisiae with improved inhibitor resistance lies in incomplete understanding of molecular basis for inhibitor stress response and limited information on effective genetic targets for increasing yeast resistance to mixed fermentation inhibitors. In this study, we applied comparative transcriptomic analysis to determine the molecular basis for acetic acid and/or furfural resistance in S. cerevisiae. RESULTS: We recently developed a yeast strain YC1 with superior resistance to acetic acid, furfural, and their mixture through inverse metabolic engineering. In this study, we first determined transcriptional changes through RNA sequencing in YC1 versus the wild-type strain S-C1 under three different inhibitor conditions, including acetic acid alone, furfural alone, and mixture of acetic acid and furfural. The genes associated with stress responses of S. cerevisiae to single and mixed inhibitors were revealed. Specifically, we identified 184 consensus genes that were differentially regulated in response to the distinct inhibitor resistance between YC1 and S-C1. Bioinformatic analysis next revealed key transcription factors (TFs) that regulate these consensus genes. The top TFs identified, Sfp1p and Ace2p, were experimentally tested as overexpression targets for strain optimization. Overexpression of the SFP1 gene improved specific ethanol productivity by nearly four times, while overexpression of the ACE2 gene enhanced the rate by three times in the presence of acetic acid and furfural. Overexpression of SFP1 gene in the resistant strain YC1 further resulted in 42 % increase in ethanol productivity in the presence of acetic acid and furfural, suggesting the effect of Sfp1p in optimizing the yeast strain for improved tolerance to mixed fermentation inhibitor. CONCLUSIONS: Transcriptional regulation underlying yeast resistance to acetic acid and furfural was determined. Two transcription factors, Sfp1p and Ace2p, were uncovered for the first time for their functions in improving yeast resistance to mixed fermentation inhibitors. The study demonstrated an omics-guided metabolic engineering framework, which could be developed as a promising strategy to improve complex microbial phenotypes.

iTAP: integrated transcriptomics and phenotype database for stress response of Escherichia coli and Saccharomyces cerevisiae.

BACKGROUND: Organisms are subject to various stress conditions, which affect both the organism's gene expression and phenotype. It is critical to understand microbial responses to stress conditions and uncover the underlying molecular mechanisms. To this end, it is necessary to build a database that collects transcriptomics and phenotypic data of microbes growing under various stress factors for in-depth systems biology analysis. Despite of numerous databases that collect gene expression profiles, to our best knowledge, there are few, if any, databases that collect both transcriptomics and phenotype data simultaneously. In light of this, we have developed an open source, web-based database, namely integrated transcriptomics and phenotype (iTAP) database, that records and links the transcriptomics and phenotype data for two model microorganisms, Escherichia coli and Saccharomyces cerevisiae in response to exposure of various stress conditions. RESULTS: To collect the data, we chose relevant research papers from the PubMed database containing all the necessary information for data curation including experimental conditions, transcriptomics data, and phenotype data. The transcriptomics data, including the p value and fold change, were obtained through the comparison of test strains against control strains using Gene Expression Omnibus's GEO2R analyzer. The phenotype data, including the cell growth rate and the productivity, volumetric rate, and mass-based yield of byproducts, were calculated independently from charts or graphs within the reference papers. Since the phenotype data was never reported in a standardized format, the curation of correlated transcriptomics-phenotype datasets became extremely tedious and time-consuming. Despite the challenges, till now, we successfully correlated 57 and 143 datasets of transcriptomics and phenotype for E. coli and S. cerevisiae, respectively, and applied a regression model within the iTAP database to accurately predict over 93 and 73 % of the growth rates of E. coli and S. cerevisiae, respectively, directly from the transcriptomics data. CONCLUSION: This is the first time that transcriptomics and phenotype data are categorized and correlated in an open-source database. This allows biologists to access the database and utilize it to predict the phenotype of microorganisms from their transcriptomics data. The iTAP database is freely available at https://sites.google.com/a/vt.edu/biomolecular-engineering-lab/software .

Two pathways for glutamate biosynthesis in the syntrophic bacterium Syntrophus aciditrophicus.

The anaerobic metabolism of crotonate, benzoate, and cyclohexane carboxylate by Syntrophus aciditrophicus grown syntrophically with Methanospirillum hungatei provides a model to study syntrophic cooperation. Recent studies revealed that S. aciditrophicus contains Re-citrate synthase but lacks the common Si-citrate synthase. To establish whether the Re-citrate synthase is involved in glutamate synthesis via the oxidative branch of the Krebs cycle, we have used [1-(13)C]acetate and [1-(14)C]acetate as well as [(13)C]bicarbonate as additional carbon sources during axenic growth of S. aciditrophicus on crotonate. Our analyses showed that labeled carbons were detected in at least 14 amino acids, indicating the global utilization of acetate and bicarbonate. The labeling patterns of alanine and aspartate verified that pyruvate and oxaloacetate were synthesized by consecutive carboxylations of acetyl coenzyme A (acetyl-CoA). The isotopomer profile and (13)C nuclear magnetic resonance (NMR) spectroscopy of the obtained [(13)C]glutamate, as well as decarboxylation of [(14)C]glutamate, revealed that this amino acid was synthesized by two pathways. Unexpectedly, only the minor route used Re-citrate synthase (30 to 40%), whereas the majority of glutamate was synthesized via the reductive carboxylation of succinate. This symmetrical intermediate could have been formed from two acetates via hydration of crotonyl-CoA to 4-hydroxybutyryl-CoA. 4-Hydroxybutyrate was detected in the medium of S. aciditrophicus when grown on crotonate, but an active hydratase could not be measured in cell extracts, and the annotated 4-hydroxybutyryl-CoA dehydratase (SYN_02445) lacks key amino acids needed to catalyze the hydration of crotonyl-CoA. Besides Clostridium kluyveri, this study reveals the second example of a microbial species to employ two pathways for glutamate synthesis.

Metabolic engineering of yeast to produce fatty acid-derived biofuels: bottlenecks and solutions.

Fatty acid-derived biofuels can be a better solution than bioethanol to replace petroleum fuel, since they have similar energy content and combustion properties as current transportation fuels. The environmentally friendly microbial fermentation process has been used to synthesize advanced biofuels from renewable feedstock. Due to their robustness as well as the high tolerance to fermentation inhibitors and phage contamination, yeast strains such as Saccharomyces cerevisiae and Yarrowia lipolytica have attracted tremendous attention in recent studies regarding the production of fatty acid-derived biofuels, including fatty acids, fatty acid ethyl esters, fatty alcohols, and fatty alkanes. However, the native yeast strains cannot produce fatty acids and fatty acid-derived biofuels in large quantities. To this end, we have summarized recent publications in this review on metabolic engineering of yeast strains to improve the production of fatty acid-derived biofuels, identified the bottlenecks that limit the productivity of biofuels, and categorized the appropriate approaches to overcome these obstacles.

13C-Metabolic Flux Analysis: An Accurate Approach to Demystify Microbial Metabolism for Biochemical Production.

Metabolic engineering of various industrial microorganisms to produce chemicals, fuels, and drugs has raised interest since it is environmentally friendly, sustainable, and independent of nonrenewable resources. However, microbial metabolism is so complex that only a few metabolic engineering efforts have been able to achieve a satisfactory yield, titer or productivity of the target chemicals for industrial commercialization. In order to overcome this challenge, 13C Metabolic Flux Analysis (13C-MFA) has been continuously developed and widely applied to rigorously investigate cell metabolism and quantify the carbon flux distribution in central metabolic pathways. In the past decade, many 13C-MFA studies have been performed in academic labs and biotechnology industries to pinpoint key issues related to microbe-based chemical production. Insightful information about the metabolic rewiring has been provided to guide the development of the appropriate metabolic engineering strategies for improving the biochemical production. In this review, we will introduce the basics of 13C-MFA and illustrate how 13C-MFA has been applied via integration with metabolic engineering to identify and tackle the rate-limiting steps in biochemical production for various host microorganisms.

Metabolic engineering of Saccharomyces cerevisiae to improve 1-hexadecanol production.

Fatty alcohols are important components of a vast array of surfactants, lubricants, detergents, pharmaceuticals and cosmetics. We have engineered Saccharomyces cerevisiae to produce 1-hexadecanol by expressing a fatty acyl-CoA reductase (FAR) from barn owl (Tyto alba). In order to improve fatty alcohol production, we have manipulated both the structural genes and the regulatory genes in yeast lipid metabolism. The acetyl-CoA carboxylase gene (ACC1) was over-expressed, which improved 1-hexadecanol production by 56% (from 45mg/L to 71mg/L). Knocking out the negative regulator of the INO1 gene in phospholipid metabolism, RPD3, further enhanced 1-hexadecanol production by 98% (from 71mg/L to 140mg/L). The cytosolic acetyl-CoA supply was next engineered by expressing a heterologous ATP-dependent citrate lyase, which increased the production of 1-hexadecanol by an additional 136% (from 140mg/L to 330mg/L). Through fed-batch fermentation using resting cells, over 1.1g/L 1-hexadecanol can be produced in glucose minimal medium, which represents the highest titer reported in yeast to date.

Incomplete Wood-Ljungdahl pathway facilitates one-carbon metabolism in organohalide-respiring Dehalococcoides mccartyi.

The acetyl-CoA "Wood-Ljungdahl" pathway couples the folate-mediated one-carbon (C1) metabolism to either CO2 reduction or acetate oxidation via acetyl-CoA. This pathway is distributed in diverse anaerobes and is used for both energy conservation and assimilation of C1 compounds. Genome annotations for all sequenced strains of Dehalococcoides mccartyi, an important bacterium involved in the bioremediation of chlorinated solvents, reveal homologous genes encoding an incomplete Wood-Ljungdahl pathway. Because this pathway lacks key enzymes for both C1 metabolism and CO2 reduction, its cellular functions remain elusive. Here we used D. mccartyi strain 195 as a model organism to investigate the metabolic function of this pathway and its impacts on the growth of strain 195. Surprisingly, this pathway cleaves acetyl-CoA to donate a methyl group for production of methyl-tetrahydrofolate (CH3-THF) for methionine biosynthesis, representing an unconventional strategy for generating CH3-THF in organisms without methylene-tetrahydrofolate reductase. Carbon monoxide (CO) was found to accumulate as an obligate by-product from the acetyl-CoA cleavage because of the lack of a CO dehydrogenase in strain 195. CO accumulation inhibits the sustainable growth and dechlorination of strain 195 maintained in pure cultures, but can be prevented by CO-metabolizing anaerobes that coexist with D. mccartyi, resulting in an unusual syntrophic association. We also found that this pathway incorporates exogenous formate to support serine biosynthesis. This study of the incomplete Wood-Ljungdahl pathway in D. mccartyi indicates a unique bacterial C1 metabolism that is critical for D. mccartyi growth and interactions in dechlorinating communities and may play a role in other anaerobic communities.