Efficient ethanol production from brown macroalgae sugars by a synthetic yeast platform.

The increasing demands placed on natural resources for fuel and food production require that we explore the use of efficient, sustainable feedstocks such as brown macroalgae. The full potential of brown macroalgae as feedstocks for commercial-scale fuel ethanol production, however, requires extensive re-engineering of the alginate and mannitol catabolic pathways in the standard industrial microbe Saccharomyces cerevisiae. Here we present the discovery of an alginate monomer (4-deoxy-L-erythro-5-hexoseulose uronate, or DEHU) transporter from the alginolytic eukaryote Asteromyces cruciatus. The genomic integration and overexpression of the gene encoding this transporter, together with the necessary bacterial alginate and deregulated native mannitol catabolism genes, conferred the ability of an S. cerevisiae strain to efficiently metabolize DEHU and mannitol. When this platform was further adapted to grow on mannitol and DEHU under anaerobic conditions, it was capable of ethanol fermentation from mannitol and DEHU, achieving titres of 4.6% (v/v) (36.2 g l(-1)) and yields up to 83% of the maximum theoretical yield from consumed sugars. These results show that all major sugars in brown macroalgae can be used as feedstocks for biofuels and value-added renewable chemicals in a manner that is comparable to traditional arable-land-based feedstocks.

Rational, combinatorial, and genomic approaches for engineering L-tyrosine production in Escherichia coli.

Although microbial metabolic engineering has traditionally relied on rational and knowledge-driven techniques, significant improvements in strain performance can be further obtained through the use of combinatorial approaches exploiting phenotypic diversification and screening. Here, we demonstrate the combined use of global transcriptional machinery engineering and a high-throughput L-tyrosine screen towards improving L-tyrosine production in Escherichia coli. This methodology succeeded in generating three strains from two separate mutagenesis libraries (rpoA and rpoD) exhibiting up to a 114% increase in L-tyrosine titer over a rationally engineered parental strain with an already high capacity for production. Subsequent strain characterization through transcriptional analysis and whole genome sequencing allowed complete phenotype reconstruction from well-defined mutations and point to important roles for both the acid stress resistance pathway and the stringent response of E. coli in imparting this phenotype. As such, this study presents one of the first examples in which cell-wide measurements have helped to elucidate the genetic and biochemical underpinnings of an engineered cellular property, leading to the total restoration of metabolite overproduction from specific chromosomal mutations.

Optimization of a heterologous pathway for the production of flavonoids from glucose.

The development of efficient microbial processes for the production of flavonoids has been a metabolic engineering goal for the past several years, primarily due to the purported health-promoting effects of these compounds. Although significant strides have been made recently in improving strain titers and yields, current fermentation strategies suffer from two major drawbacks-(1) the requirement for expensive phenylpropanoic precursors supplemented into the media and (2) the need for two separate media formulations for biomass/protein generation and flavonoid production. In this study, we detail the construction of a series of strains capable of bypassing both of these problems. A four-step heterologous pathway consisting of the enzymes tyrosine ammonia lyase (TAL), 4-coumarate:CoA ligase (4CL), chalcone synthase (CHS), and chalcone isomerase (CHI) was assembled within two engineered l-tyrosine Escherichia coli overproducers in order to enable the production of the main flavonoid precursor naringenin directly from glucose. During the course of this investigation, we discovered that extensive optimization of both enzyme sources and relative gene expression levels was required to achieve high quantities of both p-coumaric acid and naringenin accumulation. Once this metabolic balance was achieved, however, such strains were found to be capable of producing 29 mg/l naringenin from glucose and up to 84 mg/l naringenin with the addition of the fatty acid enzyme inhibitor, cerulenin. These results were obtained through cultivation of E. coli in a single minimal medium formulation without additional precursor supplementation, thus paving the way for the development of a simple and economical process for the microbial production of flavonoids directly from glucose.

Combinatorial engineering of microbes for optimizing cellular phenotype.

Although random mutagenesis and screening and evolutionary engineering have long been the gold standards for strain improvement in industry, the development of more sophisticated recombinant DNA tools has led to the introduction of alternate methods for engineering strain diversity. Here, we summarize several combinatorial cell optimization methods developed in recent years, many of which are more amenable to phenotypic transfer and more efficient in probing greater dimensions of the available phenotypic space. They include tools that enable the fine-tuning of pathway expression (synthetic promoter libraries, tunable intergenic regions (TIGRs)), methods for generating randomized knockout and overexpression libraries, and more global techniques (artificial transcription factor engineering, global transcription machinery engineering, ribosome engineering, and genome shuffling) for eliciting complex, multigenic cellular properties.

Mutagenesis of the bacterial RNA polymerase alpha subunit for improvement of complex phenotypes.

Combinatorial or random methods for strain engineering have been extensively used for the improvement of multigenic phenotypes and other traits for which the underlying mechanism is not fully understood. Although the preferred method has traditionally been mutagenesis and selection, our laboratory has successfully used mutant transcription factors, which direct the RNA polymerase (RNAP) during transcription, to engineer complex phenotypes in microbial cells. Here, we show that it is also possible to impart new phenotypes by altering the RNAP core enzyme itself, in particular through mutagenesis of the alpha subunit of the bacterial polymerase. We present the use of this tool for improving tolerance of Escherichia coli to butanol and other solvents and for increasing the titers of two commercially relevant products, L-tyrosine and hyaluronic acid. In addition, we explore the underlying physiological changes that give rise to the solvent-tolerant mutant.

Targeted Mass Spectrometry-Based Proteomics Tools for Strain Optimization.

The goal of strain optimization is to create high-performance strains producing compounds of interest at a high titer, yield, and volumetric productivity. The effectiveness of strain optimization relies on methodologies used to aid optimization of native or novel pathways within cells. Many different factors, including mRNA abundance, protein abundance, and enzyme activity/stability, will contribute to the strain performance, which is not often evident by simply monitoring product titers. To this end, targeted proteomics tools utilizing LC-MS-MS (liquid chromatography coupled with tandem mass spectrometry) have recently been developed and can monitor protein levels at great sensitivities. Here, we describe all relevant aspects when developing proteomics tools for strain optimization.

Melanin-based high-throughput screen for L-tyrosine production in Escherichia coli.

We present the development of a simple, high-throughput screen for identifying bacterial strains capable of L-tyrosine production. Through the introduction of a heterologous gene encoding a tyrosinase, we were able to link L-tyrosine production in Escherichia coli with the synthesis of the black and diffusible pigment melanin. Although melanin was initially produced only at low levels in morpholinepropanesulfonic acid (MOPS) minimal medium, phosphate supplementation was found to be sufficient for increasing both the rates of synthesis and the final titers of melanin. Furthermore, a strong linear correlation between extracellular L-tyrosine content and melanin formation was observed by use of this new medium formulation. A selection strategy that utilizes these findings has been developed and has been shown to be effective in screening large combinatorial libraries in the search for L-tyrosine-overproducing strains.

Engineering complex biological systems in bacteria through recombinase-assisted genome engineering.

Here we describe an advanced paradigm for the design, construction and stable implementation of complex biological systems in microbial organisms. This engineering strategy was previously applied to the development of an Escherichia coli-based platform, which enabled the use of brown macroalgae as a feedstock for the production of biofuels and renewable chemicals. In this approach, functional genetic modules are first designed in silico and constructed on a bacterial artificial chromosome (BAC) by using a recombineering-based inchworm extension technique. Stable integration into the recipient chromosome is then mediated through the use of recombinase-assisted genome engineering (RAGE). The flexibility, simplicity and speed of this method enable a comprehensive optimization of several different parameters, including module configuration, strain background, integration locus, gene copy number and intermodule compatibility. This paradigm therefore has the potential to markedly expedite most strain-engineering endeavors. Once a biological system has been designed and constructed on a BAC, its implementation and optimization in a recipient host can be carried out in as little as 1 week.

Multivariate modular metabolic engineering for pathway and strain optimization.

Despite the potential in utilizing microbial fermentation for chemical production, the field of industrial biotechnology still lacks a standard, universally applicable principle for strain optimization. A key challenge has been in finding and applying effective ways to address metabolic flux imbalances. Strategies based on rational design require significant a priori knowledge and often fail to take a holistic view of cellular metabolism. Combinatorial approaches enable more global searches but require a high-throughput screen. Here, we present the recent advances and promises of a novel approach to metabolic pathway and strain optimization called multivariate modular metabolic engineering (MMME). In this technique, key enzymes are organized into distinct modules and simultaneously varied based on expression to balance flux through a pathway. Because of its simplicity and broad applicability, MMME has the potential to systematize and revolutionize the field of metabolic engineering and industrial biotechnology.

Implementation of stable and complex biological systems through recombinase-assisted genome engineering.

Evaluating the performance of engineered biological systems with high accuracy and precision is nearly impossible with the use of plasmids due to phenotypic noise generated by genetic instability and natural population dynamics. Minimizing this uncertainty therefore requires a paradigm shift towards engineering at the genomic level. Here, we introduce an advanced design principle for the stable installment and implementation of complex biological systems through recombinase-assisted genome engineering (RAGE). We apply this concept to the development of a robust strain of Escherichia coli capable of producing ethanol directly from brown macroalgae. RAGE significantly expedites the optimal implementation of a 34 kb heterologous pathway for alginate metabolism based on genetic background, integration locus, copy number and compatibility with two other pathway modules (alginate degradation and ethanol production). The resulting strain achieves a ~40% higher titre than its plasmid-based counterpart and enables substantial improvements in titre (~330%) and productivity (~1,200%) after 50 generations.

An engineered microbial platform for direct biofuel production from brown macroalgae.

Prospecting macroalgae (seaweeds) as feedstocks for bioconversion into biofuels and commodity chemical compounds is limited primarily by the availability of tractable microorganisms that can metabolize alginate polysaccharides. Here, we present the discovery of a 36-kilo-base pair DNA fragment from Vibrio splendidus encoding enzymes for alginate transport and metabolism. The genomic integration of this ensemble, together with an engineered system for extracellular alginate depolymerization, generated a microbial platform that can simultaneously degrade, uptake, and metabolize alginate. When further engineered for ethanol synthesis, this platform enables bioethanol production directly from macroalgae via a consolidated process, achieving a titer of 4.7% volume/volume and a yield of 0.281 weight ethanol/weight dry macroalgae (equivalent to ~80% of the maximum theoretical yield from the sugar composition in macroalgae).

Perspectives of biotechnological production of L-tyrosine and its applications.

The aromatic amino acid L-tyrosine is used as a dietary supplement and has promise as a valuable precursor compound for various industrial and pharmaceutical applications. In contrast to chemical production, biotechnological methods can produce L-tyrosine from biomass feedstocks under environmentally friendly and near carbon-free conditions. In this minireview, various strategies for synthesizing L-tyrosine by employing biocatalysts are discussed, including initial approaches as well as more recent advances. Whereas early attempts to engineer L-tyrosine-excreting microbes were based on auxotrophic and antimetabolite-resistant mutants, recombinant deoxyribonucleic acid technology and a vastly increasing knowledge of bacterial physiology allowed recently for more targeted genetic manipulations and strain improvements. As an alternative route, L-tyrosine can also be obtained from the conversion of phenol, pyruvate, and ammonia or phenol and serine in reactions catalyzed by the enzyme tyrosine phenol lyase.