Mattheos A. G. Koffas

Modular optimization of multi-gene pathways for fatty acids production in E. coli

Microbial fatty acid-derived fuels have emerged as promising alternatives to petroleum-based transportation fuels. Here we report a modular engineering approach that systematically removed metabolic pathway bottlenecks and led to significant titre improvements in a multi-gene fatty acid metabolic pathway. On the basis of central pathway architecture, E. coli fatty acid biosynthesis was re-cast into three modules: the upstream acetyl coenzyme A formation module; the intermediary acetyl-CoA activation module; and the downstream fatty acid synthase module. Combinatorial optimization of transcriptional levels of these three modules led to the identification of conditions that balance the supply of acetyl-CoA and consumption of malonyl-CoA/ACP. Refining protein translation efficiency by customizing ribosome binding sites for both the upstream acetyl coenzyme A formation and fatty acid synthase modules enabled further production improvement. Fed-batch cultivation of the engineered strain resulted in a final fatty acid production of 8.6 g l(-1). The modular engineering strategies demonstrate a generalized approach to engineering cell factories for valuable metabolites production.

Improving fatty acids production by engineering dynamic pathway regulation and metabolic control

Significance One important synthetic chemistry reaction endowed by nature is the decarboxylative carbon condensation reaction using malonyl-CoA as carbon donor. Previous metabolic engineering efforts centered on the malonyl-CoA–dependent pathway have resulted in the production of many value-added compounds. Here we mimicked the native biological systems and used a dynamic regulatory network to optimize production titers and yield. The naturally existing transcriptional regulator FapR was rewired to dynamically control gene expressions involved in the supply and consumption of malonyl-CoA. Applying this metabolic control allowed the engineered cell to dynamically regulate pathway expression and compensated the metabolic activity of critical enzymes. The synthetic malonyl-CoA switch engineered in this study opens up new venues for dynamic pathway optimization and efficient production of malonyl-CoA–derived compounds. Global energy demand and environmental concerns have stimulated increasing efforts to produce carbon-neutral fuels directly from renewable resources. Microbially derived aliphatic hydrocarbons, the petroleum-replica fuels, have emerged as promising alternatives to meet this goal. However, engineering metabolic pathways with high productivity and yield requires dynamic redistribution of cellular resources and optimal control of pathway expression. Here we report a genetically encoded metabolic switch that enables dynamic regulation of fatty acids (FA) biosynthesis in Escherichia coli. The engineered strains were able to dynamically compensate the critical enzymes involved in the supply and consumption of malonyl-CoA and efficiently redirect carbon flux toward FA biosynthesis. Implementation of this metabolic control resulted in an oscillatory malonyl-CoA pattern and a balanced metabolism between cell growth and product formation, yielding 15.7- and 2.1-fold improvement in FA titer compared with the wild-type strain and the strain carrying the uncontrolled metabolic pathway. This study provides a new paradigm in metabolic engineering to control and optimize metabolic pathways facilitating the high-yield production of other malonyl-CoA–derived compounds.

Genome-scale metabolic network modeling results in minimal interventions that cooperatively force carbon flux towards malonyl-CoA

Malonyl-coenzyme A is an important precursor metabolite for the biosynthesis of polyketides, flavonoids and biofuels. However, malonyl-CoA naturally synthesized in microorganisms is consumed for the production of fatty acids and phospholipids leaving only a small amount available for the production of other metabolic targets in recombinant biosynthesis. Here we present an integrated computational and experimental approach aimed at improving the intracellular availability of malonyl-CoA in Escherichia coli. We used a customized version of the recently developed OptForce methodology to predict a minimal set of genetic interventions that guarantee a prespecified yield of malonyl-CoA in E. coli strain BL21 Star™. In order to validate the model predictions, we have successfully constructed an E. coli recombinant strain that exhibits a 4-fold increase in the levels of intracellular malonyl-CoA compared to the wild type strain. Furthermore, we demonstrate the potential of this E. coli strain for the production of plant-specific secondary metabolites naringenin (474mg/L) with the highest yield ever achieved in a lab-scale fermentation process. Combined effect of the genetic interventions was found to be synergistic based on a developed analysis method that correlates genetic modification to cell phenotype, specifically the identified knockout targets (ΔfumC and ΔsucC) and overexpression targets (ACC, PGK, GAPD and PDH) can cooperatively force carbon flux towards malonyl-CoA. The presented strategy can also be readily expanded for the production of other malonyl-CoA-derived compounds like polyketides and biofuels.

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.

Engineering Central Metabolic Pathways for High-Level Flavonoid Production in Escherichia coli

ABSTRACT The identification of optimal genotypes that result in improved production of recombinant metabolites remains an engineering conundrum. In the present work, various strategies to reengineer central metabolism in Escherichia coli were explored for robust synthesis of flavanones, the common precursors of plant flavonoid secondary metabolites. Augmentation of the intracellular malonyl coenzyme A (malonyl-CoA) pool through the coordinated overexpression of four acetyl-CoA carboxylase (ACC) subunits from Photorhabdus luminescens (PlACC) under a constitutive promoter resulted in an increase in flavanone production up to 576%. Exploration of macromolecule complexes to optimize metabolic efficiency demonstrated that auxiliary expression of PlACC with biotin ligase from the same species (BirAPl) further elevated flavanone synthesis up to 1,166%. However, the coexpression of PlACC with Escherichia coli BirA (BirAEc) caused a marked decrease in flavanone production. Activity improvement was reconstituted with the coexpression of PlACC with a chimeric BirA consisting of the N terminus of BirAEc and the C terminus of BirAPl. In another approach, high levels of flavanone synthesis were achieved through the amplification of acetate assimilation pathways combined with the overexpression of ACC. Overall, the metabolic engineering of central metabolic pathways described in the present work increased the production of pinocembrin, naringenin, and eriodictyol in 36 h up to 1,379%, 183%, and 373%, respectively, over production with the strains expressing only the flavonoid pathway, which corresponded to 429 mg/liter, 119 mg/liter, and 52 mg/liter, respectively.

Strain improvement of recombinant Escherichia coli for efficient production of plant flavonoids.

Plant flavonoid polyphenols continue to find increasing pharmaceutical and nutraceutical applications; however their isolation, especially of pure compounds, from plant material remains an underlying challenge. In the past Escherichia coli, one of the most well-characterized microorganisms, has been utilized as a recombinant host for protein expression and heterologous biosynthesis of small molecules. However, in many cases the expressed protein activities and biosynthetic efficiency are greatly limited by the host cellular properties, such as precursor and cofactor availability and protein or product tolerance. In the present work, we developed E. coli strains capable of high-level flavonoid synthesis through traditional metabolic engineering techniques. In addition to grafting the plant biosynthetic pathways, the methods included engineering of an alternative carbon assimilation pathway and the inhibition of competitive reaction pathways in order to increase intracellular flavonoid backbone precursors and cofactors. With this strategy, we report the production of plant-specific flavanones up to 700 mg/L and anthocyanins up to 113 mg/L from phenylpropanoic acid and flavan-3-ol precursors, respectively. These results demonstrated the efficient and scalable production of plant flavonoids from E. coli for pharmaceutical and nutraceutical applications.

High-yield resveratrol production in engineered Escherichia coli.

ABSTRACT Plant polyphenols have been the subject of several recent scientific investigations since many of the molecules in this class have been found to be highly active in the human body, with a plethora of health-promoting activities against a variety of diseases, including heart disease, diabetes, and cancer, and with even the potential to slow aging. Further development of these potent natural therapeutics hinges on the formation of robust industrial production platforms designed using specifically selected as well as engineered protein sources along with the construction of optimal expression platforms. In this work, we first report the investigation of various stilbene synthases from an array of plant species considering structure-activity relationships, their expression efficiency in microorganisms, and their ability to synthesize resveratrol. Second, we looked into the construct environment of recombinantly expressed stilbene synthases, including different promoters, construct designs, and host strains, to create an Escherichia coli strain capable of producing superior resveratrol titers sufficient for commercial usage. Further improvement of metabolic capabilities of the recombinant strain aimed at improving the intracellular malonyl-coenzyme A pool, a resveratrol precursor, resulted in a final improved titer of 2.3 g/liter resveratrol.

Biosynthesis of Natural Flavanones in Saccharomyces cerevisiae

ABSTRACT A four-step flavanone biosynthetic pathway was constructed and introduced into Saccharomyces cerevisiae. The recombinant yeast strain was fed with phenylpropanoid acids and produced the flavanones naringenin and pinocembrin 62 and 22 times more efficiently compared to previously reported recombinant prokaryotic strains. Microbial biosynthesis of the flavanone eriodictyol was also achieved.

Increased Malonyl Coenzyme A Biosynthesis by Tuning the Escherichia coli Metabolic Network and Its Application to Flavanone Production

ABSTRACT Identification of genetic targets able to bring about changes to the metabolite profiles of microorganisms continues to be a challenging task. We have independently developed a cipher of evolutionary design (CiED) to identify genetic perturbations, such as gene deletions and other network modifications, that result in optimal phenotypes for the production of end products, such as recombinant natural products. Coupled to an evolutionary search, our method demonstrates the utility of a purely stoichiometric network to predict improved Escherichia coli genotypes that more effectively channel carbon flux toward malonyl coenzyme A (CoA) and other cofactors in an effort to generate recombinant strains with enhanced flavonoid production capacity. The engineered E. coli strains were constructed first by the targeted deletion of native genes predicted by CiED and then second by incorporating selected overexpressions, including those of genes required for the coexpression of the plant-derived flavanones, acetate assimilation, acetyl-CoA carboxylase, and the biosynthesis of coenzyme A. As a result, the specific flavanone production from our optimally engineered strains was increased by over 660% for naringenin (15 to 100 mg/liter/optical density unit [OD]) and by over 420% for eriodictyol (13 to 55 mg/liter/OD).

ePathBrick: A Synthetic Biology Platform for Engineering Metabolic Pathways in E. coli

Harnessing cell factories for producing biofuel and pharmaceutical molecules has stimulated efforts to develop novel synthetic biology tools customized for modular pathway engineering and optimization. Here we report the development of a set of vectors compatible with BioBrick standards and its application in metabolic engineering. The engineered ePathBrick vectors comprise four compatible restriction enzyme sites allocated on strategic positions so that different regulatory control signals can be reused and manipulation of expression cassette can be streamlined. Specifically, these vectors allow for fine-tuning gene expression by integrating multiple transcriptional activation or repression signals into the operator region. At the same time, ePathBrick vectors support the modular assembly of pathway components and combinatorial generation of pathway diversities with three distinct configurations. We also demonstrated the functionality of a seven-gene pathway (~9 Kb) assembled on one single ePathBrick vector. The ePathBrick vectors presented here provide a versatile platform for rapid design and optimization of metabolic pathways in E. coli.