https://escholarship.org/content/qt5mz1s1pz/qt5mz1s1pz.pdf?t=p9sqjd

Escherichia coli as a host for metabolic engineering.

Recent advances in systems and synthetic biology approaches for developing novel cell-factories in non-conventional yeasts.

Exploitation of plant growth promoting (PGP) rhizobacteria (PGPR) as crop inoculants could propel sustainable intensification of agriculture to feed our rapidly growing population. However, field performance of PGPR is typically inconsistent due to suboptimal rhizosphere colonisation and persistence in foreign soils, promiscuous host-specificity, and in some cases, the existence of undesirable genetic regulation that has evolved to repress PGP traits. While the genetics underlying these problems remain largely unresolved, molecular mechanisms of PGP have been elucidated in rigorous detail. Engineering and subsequent transfer of PGP traits into selected efficacious rhizobacterial isolates or entire bacterial rhizosphere communities now offers a powerful strategy to generate improved PGPR that are tailored for agricultural use. Through harnessing of synthetic plant-to-bacteria signalling, attempts are currently underway to establish exclusive coupling of plant-bacteria interactions in the field, which will be crucial to optimise efficacy and establish biocontainment of engineered PGPR. This review explores the many ecological and biotechnical facets of this research.

Engineering rhizobacteria for sustainable agriculture.

Microbial production of short and medium chain esters: Enzymes, pathways, and applications.

Since the industrial revolution, the rapid growth and development of global industries have depended largely upon the utilization of coal-derived chemicals, and more recently, the utilization of petroleum-based chemicals. These developments have followed a linear economy model (produce, consume, and dispose). As the world is facing a serious threat from the climate change crisis, a more sustainable solution for manufacturing, i.e., circular economy in which waste from the same or different industries can be used as feedstocks or resources for production offers an attractive industrial/business model. In nature, biological systems, i.e., microorganisms routinely use their enzymes and metabolic pathways to convert organic and inorganic wastes to synthesize biochemicals and energy required for their growth. Therefore, an understanding of how selected enzymes convert biobased feedstocks into special (bio)chemicals serves as an important basis from which to build on for applications in biocatalysis, metabolic engineering, and synthetic biology to enable biobased processes that are greener and cleaner for the environment. This review article highlights the current state of knowledge regarding the enzymatic reactions used in converting biobased wastes (lignocellulosic biomass, sugar, phenolic acid, triglyceride, fatty acid, and glycerol) and greenhouse gases (CO2 and CH4) into value-added products and discusses the current progress made in their metabolic engineering. The commercial aspects and life cycle assessment of products from enzymatic and metabolic engineering are also discussed. Continued development in the field of metabolic engineering would offer diversified solutions which are sustainable and renewable for manufacturing valuable chemicals.

Enzymes, In Vivo Biocatalysis, and Metabolic Engineering for Enabling a Circular Economy and Sustainability.

Rose-like odor 2-phenylethanol (2-PE) and its more fruit-like ester 2-phenylethyl acetate (2-PEAc) are two important aromatic compounds and have wide applications. In the past, 2-PE and 2-PEAc were mainly produced from l-phenylalanine. In this study, Escherichia coli was engineered to de novo biosynthesis of 2-PE and 2-PEAc from glucose: first, overexpression of deregulated 3-deoxy-d-arabinoheptulosonate-7-phosphate synthase aroG fbr and chorismate mutase/prephenate dehydratase pheA fbr for increasing phenylpyruvate production in E. coli, subsequently, heterologous expression of decarboxylase kdc and overexpression of reductase yjgB for the conversion of phenylpyruvate to 2-PE, with the engineered strain DG01 producing 578 mg/L 2-PE, and, finally, heterologous expression of an aminotransferase aro8 to redirect the metabolic flux to phenylpyruvate. 2-PE (1016 mg/L) was accumulated in the engineered strain DG02. Alcohol acetyltransferase ATF1 from Saccharomyces cerevisiae can esterify a wide variety of alcohols, including 2-PE. We have further demonstrated the biosynthesis of 2-PEAc from glucose by overexpressing atf1 for the subsequent conversion of 2-PE to 2-PEAc. The engineered strain DG03 produced 687 mg/L 2-PEAc.

Metabolic Engineering of Escherichia coli for Production of 2-Phenylethanol and 2-Phenylethyl Acetate from Glucose.

In order to meet the need of consumer preferences for natural flavor compounds, microbial synthesis method has become a very attractive alternative to the chemical production. The 2-phenylethanol (2-PE) and its ester 2-phenylethylacetate (2-PEAc) are two extremely important flavor compounds with a rose-like odor. In recent years, Escherichia coli and yeast have been metabolically engineered to produce 2-PE. However, a metabolic engineering approach for 2-PEAc production is rare. Here, we designed and expressed a 2-PEAc biosynthetic pathway in E. coli. This pathway comprised four steps: aminotransferase (ARO8) for transamination of L-phenylalanine to phenylpyruvate, 2-keto acid decarboxylase KDC for the decarboxylation of the phenylpyruvate to phenylacetaldehyde, aldehyde reductase YjgB for the reduction of phenylacetaldehyde to 2-PE, alcohol acetyltransferase ATF1 for the esterification of 2-PE to 2-PEAc. Using the engineered E. coli strain for shake flasks cultivation with 1 g/L L-phenylalanine, we achieved co-production of 268 mg/L 2-PEAc and 277 mg/L 2-PE. Our results suggest that approximately 65% of L-phenylalanine was utilized toward 2-PEAc and 2-PE biosynthesis and thus demonstrate potential industrial applicability of this microbial platform.

https://www.onlinelibrary.wiley.com/doi/pdf/10.1002/mbo3.486

Metabolic engineering of Escherichia coli for production of 2-Phenylethylacetate from L-phenylalanine

Microbial production of biodiesel from renewable feedstock has attracted intensive attention. Biodiesel is known to be produced from short-chain alcohols and fatty acyl-CoAs through the expression of wax ester synthase/fatty acyl-CoA: diacylglycerol acyltransferase that catalyzes the esterification of short-chain alcohols and fatty acyl-CoAs. Here, we engineered Escherichia coli to produce various fatty alcohol acetate esters, which depend on the expression of Saccharomyces cerevisiae alcohol acetyltransferase ATF1 that catalyzes the esterification of fatty alcohols and acetyl-CoA. The fatty acid biosynthetic pathways generate fatty acyl-ACPs, fatty acyl-CoAs, or fatty acids, which can be converted to fatty alcohols by fatty acyl-CoA reductase, fatty acyl-ACP reductase, or carboxylic acid reductase, respectively. This study showed the biosynthesis of biodiesel from three fatty acid biosynthetic pathway intermediates.

Metabolic engineering of Escherichia coli for production of biodiesel from fatty alcohols and acetyl-CoA

De novo biosynthesis of 2-phenylethanol in engineered Pichia pastoris

Indole-3-acetic acid (IAA) is considered the most common and important naturally occurring auxin in plants and a major regulator of plant growth and development. In this study, an aldehyde dehydrogenase AldH from Escherichia coli was found to convert indole-3-acetylaldehyde into IAA. Then we established an artificial pathway in engineered E. coli for microbial production of IAA from glucose. The overall pathway includes the upstream pathway from glucose to L-tryptophan and the downstream pathway from L-tryptophan to IAA. To our knowledge, this is the first report on the biosynthesis of IAA directly from a renewable carbon source. The study described here shows the way for the development of a beneficial microbe for biosynthesis of auxin and promoting plant growth in the future.

Xun Li

Papers

Metabolic Engineering of Escherichia coli for Production of 2-Phenylethanol and 2-Phenylethyl Acetate from Glucose.

Metabolic engineering of Escherichia coli for production of 2-Phenylethylacetate from L-phenylalanine

Metabolic engineering of Escherichia coli for production of biodiesel from fatty alcohols and acetyl-CoA

De novo biosynthesis of 2-phenylethanol in engineered Pichia pastoris

De Novo Biosynthesis of Indole-3-acetic Acid in Engineered Escherichia coli.