Practical genetic control strategies for industrial bioprocesses.

Optimization of metabolism to maximize production of bio-based chemicals must consistently balance cellular resources for biocatalyst growth and desired compound synthesis. This mini-review discusses synthetic biology strategies for dynamically controlling expression of genes to enable dual-phase fermentations in which growth and production are separated into dedicated phases. Emphasis is placed on practical examples which can be reliably scaled to commercial production with the current state of technology.

Structural and functional insights into asymmetric enzymatic dehydration of alkenols.

The asymmetric dehydration of alcohols is an important process for the direct synthesis of alkenes. We report the structure and substrate specificity of the bifunctional linalool dehydratase isomerase (LinD) from the bacterium Castellaniella defragrans that catalyzes in nature the hydration of β-myrcene to linalool and the subsequent isomerization to geraniol. Enzymatic kinetic resolutions of truncated and elongated aromatic and aliphatic tertiary alcohols (C5-C15) that contain a specific signature motif demonstrate the broad substrate specificity of LinD.

Development of a commercial scale process for production of 1,4-butanediol from sugar.

A sustainable bioprocess for the production of 1,4-butanediol (BDO) from carbohydrate feedstocks was developed. BDO is a chemical intermediate that goes into a variety of products including automotive parts, electronics, and apparel, and is currently manufactured commercially through energy-intensive petrochemical processes using fossil raw materials. This review highlights the development of an Escherichia coli strain and an overall process that successfully performed at commercial scale for direct production of bio-BDO from dextrose.

Identification of metabolic engineering targets for the enhancement of 1,4-butanediol production in recombinant E. coli using large-scale kinetic models.

Rational metabolic engineering methods are increasingly employed in designing the commercially viable processes for the production of chemicals relevant to pharmaceutical, biotechnology, and food and beverage industries. With the growing availability of omics data and of methodologies capable to integrate the available data into models, mathematical modeling and computational analysis are becoming important in designing recombinant cellular organisms and optimizing cell performance with respect to desired criteria. In this contribution, we used the computational framework ORACLE (Optimization and Risk Analysis of Complex Living Entities) to analyze the physiology of recombinant Escherichia coli producing 1,4-butanediol (BDO) and to identify potential strategies for improved production of BDO.

Biotechnology for Chemical Production: Challenges and Opportunities.

Biotechnology offers a new sustainable approach to manufacturing chemicals, enabling the replacement of petroleum-based raw materials with renewable biobased feedstocks, thereby reducing greenhouse gas (GHG) emissions, toxic byproducts, and the safety risks associated with traditional petrochemical processing. Development of such bioprocesses is enabled by recent advances in genomics, molecular biology, and systems biology, and will continue to accelerate as access to these tools becomes faster and cheaper.

An integrated biotechnology platform for developing sustainable chemical processes.

Genomatica has established an integrated computational/experimental metabolic engineering platform to design, create, and optimize novel high performance organisms and bioprocesses. Here we present our platform and its use to develop E. coli strains for production of the industrial chemical 1,4-butanediol (BDO) from sugars.

From the first drop to the first truckload: commercialization of microbial processes for renewable chemicals.

Fermentation of carbohydrate substrates by microorganisms represents an attractive route for the manufacture of industrial chemicals from renewable resources. The technology to manipulate metabolism of bacteria and yeast, including the introduction of heterologous chemical pathways, has accelerated research in this field. However, the public literature contains very few examples of strains achieving the production metrics required for commercialization.

Metabolic engineering of Escherichia coli for direct production of 1,4-butanediol.

1,4-Butanediol (BDO) is an important commodity chemical used to manufacture over 2.5 million tons annually of valuable polymers, and it is currently produced exclusively through feedstocks derived from oil and natural gas. Herein we report what are to our knowledge the first direct biocatalytic routes to BDO from renewable carbohydrate feedstocks, leading to a strain of Escherichia coli capable of producing 18 g l(-1) of this highly reduced, non-natural chemical.

Dynamic modeling of Escherichia coli metabolic and regulatory systems for amino-acid production.

Our aim is to construct a practical dynamic-simulation system that can model the metabolic and regulatory processes involved in the production of primary metabolites, such as amino acids. We have simulated the production of glutamate by transient batch-cultivation using a model of Escherichia coli central metabolism. Kinetic data were used to produce both the metabolic parts of the model, including the phosphotransferase system, glycolysis, the pentose-phosphate pathway, the tricarboxylic acid cycle, the glyoxylate shunt, and the anaplerotic pathways, and the regulatory parts of the model, including regulation by transcription factors, cyclic AMP receptor protein (CRP), making large colonies protein (Mlc), catabolite repressor/activator (Cra), pyruvate dehydrogenase complex repressor (PdhR), and acetate operon repressor (IclR).

Absolute metabolite concentrations and implied enzyme active site occupancy in Escherichia coli.

Absolute metabolite concentrations are critical to a quantitative understanding of cellular metabolism, as concentrations impact both the free energies and rates of metabolic reactions. Here we use LC-MS/MS to quantify more than 100 metabolite concentrations in aerobic, exponentially growing Escherichia coli with glucose, glycerol or acetate as the carbon source. The total observed intracellular metabolite pool was approximately 300 mM.

Elucidation of an alternate isoleucine biosynthesis pathway in Geobacter sulfurreducens.

The central metabolic model for Geobacter sulfurreducens included a single pathway for the biosynthesis of isoleucine that was analogous to that of Escherichia coli, in which the isoleucine precursor 2-oxobutanoate is generated from threonine. 13C labeling studies performed in G. sulfurreducens indicated that this pathway accounted for a minor fraction of isoleucine biosynthesis and that the majority of isoleucine was instead derived from acetyl-coenzyme A and pyruvate, possibly via the citramalate pathway.

Metabolic flux elucidation for large-scale models using 13C labeled isotopes.

A key consideration in metabolic engineering is the determination of fluxes of the metabolites within the cell. This determination provides an unambiguous description of metabolism before and/or after engineering interventions. Here, we present a computational framework that combines a constraint-based modeling framework with isotopic label tracing on a large scale.

Determination of metabolic flux changes during fed-batch cultivation from measurements of intracellular amino acids by LC-MS/MS.

Metabolic flux analysis using (13)C-labeled substrates is a well-developed method for investigating cellular behavior in steady-state culture condition. To extend its application, in particular to typical industrial conditions, such as batch and fed-batch cultivations, a novel method of (13)C metabolic flux analysis is proposed. An isotopomer balancing model was developed to elucidate flux distributions in the central metabolism and all amino acids synthetic pathways.

Theoretical analysis of amino acid-producing Escherichia coli using a stoichiometric model and multivariate linear regression.

This work demonstrates a novel computational approach combining flux balance modeling with statistical methods to identify correlations among fluxes in a metabolic network, providing insight as to how the fluxes should be redirected to achieve maximum product yield. The procedure is demonstrated using the example of amino acid production from an industrial Escherichia coli production strain and a hypothetical engineered strain overexpressing two heterologous genes. Regression analysis based on a random sampling of 5,000 points within the feasible solution space of the E.

Flux analysis uncovers key role of functional redundancy in formaldehyde metabolism.

Genome-scale analysis of predicted metabolic pathways has revealed the common occurrence of apparent redundancy for specific functional units, or metabolic modules. In many cases, mutation analysis does not resolve function, and instead, direct experimental analysis of metabolic flux under changing conditions is necessary. In order to use genome sequences to build models of cellular function, it is important to define function for such apparently redundant systems.

Genetic characterization of the carotenoid biosynthetic pathway in Methylobacterium extorquens AM1 and isolation of a colorless mutant.

Genomic searches were used to reconstruct the putative carotenoid biosynthesis pathway in the pink-pigmented facultative methylotroph Methylobacterium extorquens AM1. Four genes for putative phytoene desaturases were identified. A colorless mutant was obtained by transposon mutagenesis, and the insertion was shown to be in one of the putative phytoene desaturase genes.

Deciphering environmental signal integration in sigma54-dependent promoters with a simple mathematical model.

A mathematical model was developed to describe the physiological co-regulation of two Pseudomonas sigma54-dependent promoter/regulator systems, Pu/XylR and Po/DmpR of Pseudomonas strains mt2 and CF600, respectively. Five ordinary differential equations and six algebraic equations were developed to describe the following processes of transcription initiation: binding of the activator protein to the upstream activating sequence, union of the sigma factor with the core polymerase, formation of the open complex, and escape of the transcription machinery from the promoter region. In addition, growth-phase control of the integration host factor (IHF), sigma-70 regulation during stationary phase, and the contribution of (p)ppGpp to both sigma factor selectivity and promoter escape were hypothesized.

Quantification of central metabolic fluxes in the facultative methylotroph methylobacterium extorquens AM1 using 13C-label tracing and mass spectrometry.

The metabolic fluxes of central carbon metabolism were measured in chemostat-grown cultures of Methylobacterium extorquens AM1 with methanol as the sole organic carbon and energy source and growth-limiting substrate. Label tracing experiments were carried out using 70% (13)C-methanol in the feed, and the steady-state mass isotopomer distributions of amino acids derived from total cell protein were measured by gas chromatography coupled to mass spectrometry. Fluxes were calculated from the isotopomer distribution data using an isotopomer balance model and evolutionary error minimization algorithm.

Reconstruction of C(3) and C(4) metabolism in Methylobacterium extorquens AM1 using transposon mutagenesis.

The growth of Methylobacterium extorquens AM1 on C(1) compounds has been well-studied, but little is known about how this methylotroph grows on multicarbon compounds. A Tn5 transposon mutagenesis procedure was performed to identify genes involved in the growth of M. extorquens AM1 on succinate and pyruvate.

Stoichiometric model for evaluating the metabolic capabilities of the facultative methylotroph Methylobacterium extorquens AM1, with application to reconstruction of C(3) and C(4) metabolism.

A stoichiometric model of central metabolism was developed based on new information regarding metabolism in this bacterium to evaluate the steady-state growth capabilities of the serine cycle facultative methylotroph Methylobacterium extorquens AM1 during growth on methanol, succinate, and pyruvate. The model incorporates 20 reversible and 47 irreversible reactions, 65 intracellular metabolites, and experimentally-determined biomass composition. The flux space for this underdetermined system of equations was defined by finding the elementary modes, and constraints based on experimental observations were applied to determine which of these elementary modes give a reasonable description of the flux distribution for each growth substrate.