Biosynthesis and biotechnological production of flavanones: current state and perspectives

Abstract Polyphenols produced in a wide variety of flowering and fruit-bearing plants have the potential to be valuable fine chemicals for the treatment of an assortment of human maladies. One of the major constituents within this chemical class are flavonoids, among which flavanones, as the precursor to all flavonoid structures, are the most prevalent. We review the current status of flavanone production technology using microorganisms, with focus on heterologous protein expression. Such processes appear as attractive production alternatives for commercial synthesis of these high-value chemicals as traditional chemical, and plant cell cultures have significant drawbacks. Other issues of importance, including fermentation configurations and economics, are also considered

Development of Non-Natural Flavanones as Antimicrobial Agents

With growing concerns over multidrug resistance microorganisms, particularly strains of bacteria and fungi, evolving to become resistant to the antimicrobial agents used against them, the identification of new molecular targets becomes paramount for novel treatment options. Recently, the use of new treatments containing multiple active ingredients has been shown to increase the effectiveness of existing molecules for some infections, often with these added compounds enabling the transport of a toxic molecule into the infecting species. Flavonoids are among the most abundant plant secondary metabolites and have been shown to have natural abilities as microbial deterrents and anti-infection agents in plants. Combining these ideas we first sought to investigate the potency of natural flavonoids in the presence of efflux pump inhibitors to limit Escherichia coli growth. Then we used the natural flavonoid scaffold to synthesize non-natural flavanone molecules and further evaluate their antimicrobial efficacy on Escherichia coli, Bacillus subtilis and the fungal pathogens Cryptococcus neoformans and Aspergillus fumigatus. Of those screened, we identified the synthetic molecule 4-chloro-flavanone as the most potent antimicrobial compound with a MIC value of 70 µg/mL in E. coli when combined with the inhibitor Phe-Arg-ß-naphthylamide, and MICs of 30 µg/mL in S. cerevesiae and 30 µg/mL in C. neoformans when used alone. Through this study we have demonstrated that combinatorial synthesis of non-natural flavonones can identify novel antimicrobial agents with activity against bacteria and fungi but with minimal toxicity to human cells

Metabolic engineering of C. glutamicum for amino acid production improvement

Thesis (Ph.D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, February 2001.Includes bibliographical references (leaves 183-210).A central goal in metabolic engineering is the design of more productive biological systems by genetically modifying metabolic pathways. In this thesis we report such an optimization in the bacterial strain Corynebacterium glutamicum that is employed for the fermentative production of various amino acids such as lysine. The main goal of the research presented here was the application of metabolic and genetic engineering tools in order to investigate the role of the pyruvate node in cellular physiology. This was achieved by integrating the tools of bioinformatics, recombinant DNA technology, enzymology and classical bioengineering in the context of control and genetically engineered strains of C. glutamicum. First, the main anaplerotic pathway responsible for replenishing oxaloacetate, namely pyruvate carboxylase was targeted. After fruitless attempts to establish an in vitro enzymatic activity for this enzyme, our efforts were directed towards its gene identification. This was achieved by designing PCR primers corresponding to homologous regions among pyruvate carboxylases from other organisms. Utilizing these primers, a PCR fragment was isolated corresponding to part of the gene of the C. glutamicum pyruvate carboxylase. The sequence of the complete gene was finally obtained by screening a C. glutamicum cosmid library. In order to investigate the physiological effect that this enzyme has on lysine production, recombinant strains and deletion mutants were generated. The presence of the gene of pyruvate carboxylase in a multicopy plasmid is not sufficient to yield a significant overexpresssion of this enzyme in C. glutamicum. Contrary to our expectations, overexpression of pyruvate carboxylase has a negative effect on lysine production but improves significantly the growth properties of C. glutamicum. A metabolic model was developed according to which pyruvate carboxylase overexpression increases the carbon flux that enters the TCA cycle, thus the higher growth. However due to the presence of a rate-limiting step in the lysine biosynthesis pathway this increased carbon flux does not translate into higher lysine production. The role of aspartokinase, the first step in lysine biosynthesis, was explored as such a potential bottleneck. Its overexpression proves to increase the amount of lysine produced, however it leads to a lower growth and finally a lower productivity. Since pyruvate carboxylase and aspartokinase have opposite effects on cell physiology, the combination of the overexpression of these two enzymes was finally studied. By this simultaneous overexpression, we achieved to create a C. glutamicum recombinant strain with similar growth as that of the control but higher lysine production and productivity. In the context of exploring the physiological role of pyruvate carboxylase, a biotinylated enzyme, two other enzyme that utilize biotin were also investigated namely acetyl-CoA-carboxylase and biotin ligase. The first enzyme was purified to completion and its N-terminal as well internal amino acid sequences were obtained. A cosmid from the C. glutamicum cosmid library was identified that most likely contains the gene of the latter enzyme. In summary, in the present work we have achieved to prove unequivocally the presence of pyruvate carboxylase in C. glutamicum. We have also achieved to characterize the second biotinylated enzyme in this organism, namely acetyl-CoAcarboxylase. The physiological effect of both pyruvate carboxylase and aspartokinase was established and a metabolic model was developed based on these experimental results. This model finally led us to the construction of a new recombinant strain with improved lysine productivity. As such, this work stands as one of the few examples of a primary metabolite production improvement using metabolic engineering techniques.by Mattheos A.G. Kofas.Ph.D

Recent Advances in the Recombinant Biosynthesis of Polyphenols

Plants are the source of various natural compounds with pharmaceutical and nutraceutical importance which have shown numerous health benefits with relatively fewer side effects. However, extraction of these compounds from native producers cannot meet the ever-increasing demands of the growing population due to, among other things, the limited production of the active compound(s). Their production depends upon the metabolic demands of the plant and is also subjected to environmental conditions, abundance of crop species and seasonal variations. Moreover, their extraction from plants requires complex downstream processing and can also lead to the extinction of many useful plant varieties. Microbial engineering is one of the alternative approaches which can meet the global demand for natural products in an eco-friendly manner. Metabolic engineering of microbes or pathway reconstruction using synthetic biology tools and novel enzymes lead to the generation of a diversity of compounds (like flavonoids, stilbenes, anthocyanins etc.) and their natural and non-natural derivatives. Strain and pathway optimization, pathway regulation and tolerance engineering have produced microbial cell factories into which the metabolic pathway of plants can be introduced for the production of compounds of interest on an industrial scale in an economical and eco-friendly way. While microbial production of phytochemicals needs to further increase product titer if it is ever to become a commercial success. The present review covers the advancements made for the improvement of microbial cell factories in order to increase the product titer of recombinant polyphenolic compounds

Engineering of Artificial Plant Cytochrome P450 Enzymes for Synthesis of Isoflavones by Escherichia coli▿

Engineered microbes are becoming increasingly important as recombinant production platforms. However, the nonfunctionality of membrane-bound cytochrome P450 enzymes precludes the use of industrially relevant prokaryotes such as Escherichia coli for high-level in vivo synthesis of many functional plant-derived compounds. We describe the design of a series of artificial isoflavone synthases that allowed the robust production of plant estrogen pharmaceuticals by E. coli. Through this methodology, a plant P450 construct was assembled to mimic the architecture of a self-sufficient bacterial P450 and contained tailor-made membrane recognition signals. The specific in vivo production catalyzed by one identified chimera was up to 20-fold higher than that achieved by the native enzyme expressed in a eukaryotic host and up to 10-fold higher than production by plants. This novel biological device is a strategy for the utilization of laboratory bacteria to robustly manufacture high-value plant P450 products

Biosynthesis of Natural Flavanones in Saccharomyces cerevisiae

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

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

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

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

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)