Rachit Jain

Microbial production of antioxidant food ingredients via metabolic engineering

Antioxidants are biological molecules with the ability to protect vital metabolites from harmful oxidation. Due to this fascinating role, their beneficial effects on human health are of paramount importance. Traditional approaches using solvent-based extraction from food/non-food sources and chemical synthesis are often expensive, exhaustive, and detrimental to the environment. With the advent of metabolic engineering tools, the successful reconstitution of heterologous pathways in Escherichia coli and other microorganisms provides a more exciting and amenable alternative to meet the increasing demand of natural antioxidants. In this review, we elucidate the recent progress in metabolic engineering efforts for the microbial production of antioxidant food ingredients - polyphenols, carotenoids, and antioxidant vitamins.

Dehydratase mediated 1-propanol production in metabolically engineered Escherichia coli

BackgroundWith the increasing consumption of fossil fuels, the question of meeting the global energy demand is of great importance in the near future. As an effective solution, production of higher alcohols from renewable sources by microorganisms has been proposed to address both energy crisis and environmental concerns. Higher alcohols contain more than two carbon atoms and have better physiochemical properties than ethanol as fuel substitutes.ResultsWe designed a novel 1-propanol metabolic pathway by expanding the well-known 1,2-propanediol pathway with two more enzymatic steps catalyzed by a 1,2-propanediol dehydratase and an alcohol dehydrogenase. In order to engineer the pathway into E. coli, we evaluated the activities of eight different methylglyoxal synthases which play crucial roles in shunting carbon flux from glycolysis towards 1-propanol biosynthesis, as well as two secondary alcohol dehydrogenases of different origins that reduce both methylglyoxal and hydroxyacetone. It is evident from our results that the most active enzymes are the methylglyoxal synthase from Bacillus subtilis and the secondary alcohol dehydrogenase from Klebsiella pneumoniae, encoded by mgsA and budC respectively. With the expression of these two genes and the E. coliydjG encoding methylglyoxal reductase, we achieved the production of 1,2-propanediol at 0.8 g/L in shake flask experiments. We then characterized the catalytic efficiency of three different diol dehydratases on 1,2-propanediol and identified the optimal one as the 1,2-propanediol dehydratase from Klebsiella oxytoca, encoded by the operon ppdABC. Co-expressing this enzyme with the above 1,2-propanediol pathway in wild type E. coli resulted in the production of 1-propanol at a titer of 0.25 g/L.ConclusionsWe have successfully established a new pathway for 1-propanol production by shunting the carbon flux from glycolysis. To our knowledge, it is the first time that this pathway has been utilized to produce 1-propanol in E. coli. The work presented here forms a basis for further improvement in production. We speculate that dragging more carbon flux towards methylglyoxal by manipulating glycolytic pathway and eliminating competing pathways such as lactate generation can further enhance the production of 1-propanol.

Systematically Engineering Escherichia coli for Enhanced Production of 1,2-Propanediol and 1-Propanol

The biological production of high value commodity 1,2-propanediol has been established by engineering the glycolysis pathway. However, the simultaneous achievement of high titer and high yield has not been reported yet, as all efforts in increasing the titer have resulted in low yields. In this work, we overcome this limitation by employing an optimal minimal set of enzymes, channeling the carbon flux into the 1,2-propanediol pathway, increasing NADH availability, and improving the anaerobic growth of the engineered Escherichia coli strain by developing a cell adaptation method. These efforts lead to 1,2-propanediol production at a titer of 5.13 g/L with a yield of 0.48 g/g glucose in 20 mL shake flask studies. On this basis, we pursue the enhancement of 1-propanol production from the 1,2-propanediol platform. By constructing a fusion diol dehydratase and developing a dual strain process, we achieve a 1-propanol titer of 2.91 g/L in 20 mL shake flask studies. To summarize, we report the production of 1,2-propanediol at enhanced titer and enhanced yield simultaneously in E. coli for the first time. Furthermore, we establish an efficient system for the production of biofuel 1-propanol biologically.

Rational engineering of diol dehydratase enables 1,4-butanediol biosynthesis from xylose.

Establishing novel synthetic routes for microbial production of chemicals often requires overcoming pathway bottlenecks by tailoring enzymes to enhance bio-catalysis or even achieve non-native catalysis. Diol dehydratases have been extensively studied for their interactions with C2 and C3 diols. However, attempts on utilizing these insights to enable catalysis on non-native substrates with more than two hydroxyl groups have been plagued with low efficiencies. Here, we rationally engineered the Klebsiella oxytoca diol dehydratase to enable and enhance catalytic activity toward a non-native C4 triol, 1,2,4-butanetriol. We analyzed dehydratases interaction with 1,2-propanediol and glycerol, which led us to develop rationally conceived hypotheses. An in silico approach was then developed to identify and screen candidate mutants with desired activity. This led to an engineered diol dehydratase with nearly 5 fold higher catalytic activity toward 1,2,4-butanetriol than the wild type as determined by in vitro assays. Based on this result, we then expanded the 1,2,4-butanetriol pathway to establish a novel 1,4-butanediol production platform. We engineered Escherichia colis xylose catabolism to enhance the biosynthesis of 1,2,4-butanetriol from 224mg/L to 1506mg/L. By introducing the complete pathway in the engineered strain we achieve de novo biosynthesis of 1,4-butanediol at 209mg/L from xylose. This work expands the repertoire of substrates catalyzed by diol dehydratases and serves as an elucidation to establish novel biosynthetic pathways involving dehydratase based biocatalysis.

Aerobic biosynthesis of hydrocinnamic acids in Escherichia coli with a strictly oxygen-sensitive enoate reductase

3-Phenylpropionic acid (3PPA) and 3-(4-hydroxyphenyl) propionic acid (HPPA) are important commodity aromatic acids widely used in food, pharmaceutical and chemical industries. Currently, 3PPA and HPPA are mainly manufactured through chemical synthesis, which contains multiple steps involving toxic solvents and catalysts harmful to environment. Therefore, replacement of such existing petroleum-derived approaches with simple and environmentally friendly biological processes is highly desirable for manufacture of these chemicals. Here, for the first time we demonstrated the de novo biosynthesis of 3PPA and HPPA using simple carbon sources in E. coli by extending the cinnamic acids biosynthesis pathways through biological hydrogenation. We first screened 11 2-enoate reductases (ER) from nine microorganisms, leading to efficient conversion of cinnamic acid and p-coumaric acid to 3PPA and HPPA, respectively. Surprisingly, we found a strictly oxygen-sensitive Clostridia ER capable of functioning efficiently in E. coli even under aerobic conditions. On this basis, reconstitution of the full pathways led to the de novo production of 3PPA and HPPA and the accumulation of the intermediates (cinnamic acid and p-coumaric acid) with cell toxicity. To address this problem, different expression strategies were attempted to optimize individual enzyme׳s expression level and minimize intermediates accumulation. Finally, the titers of 3PPA and HPPA reached 366.77mg/L and 225.10mg/L in shake flasks, respectively. This study not only demonstrated the potential of microbial approach as an alternative to chemical process, but also proved the possibility of using oxygen-sensitive enzymes under aerobic conditions.

Establishing a novel biosynthetic pathway for the production of 3,4-dihydroxybutyric acid from xylose in Escherichia coli

3-Hydroxy-γ-butyrolactone (3HBL) is an attractive building block owing to its broad applications in pharmaceutical industry. Currently, 3HBL is commercially produced by chemical routes using petro-derived carbohydrates, which involves hazardous materials and harsh processing conditions. Only one biosynthetic pathway has been reported for synthesis of 3HBL and its hydrolyzed form 3,4-dihydroxybutyric acid (3,4-DHBA) using glucose and glycolic acid as the substrates and coenzyme A as the activator, which involves multiple steps (>10 steps) and suffers from low productivity and yield. Here we established a novel five-step biosynthetic pathway for 3,4-DHBA generation from D-xylose based on the non-phosphorylative D-xylose metabolism, which led to efficient production of 3,4-DHBA in Escherichia coli. Pathway optimization by incorporation of efficient enzymes for each step and host strain engineering by knocking out competing pathways enabled 1.27g/L 3,4-DHBA produced in shake flasks, which is the highest titer reported so far. The novel pathway established in engineered E. coli strain demonstrates a new route for 3,4-DHBA biosynthesis from xylose, and this engineered pathway has great potential for industrial biomanufacturing of 3,4-DHBA and 3HBL.

Enhanced photo‐bioelectrochemical energy conversion by genetically engineered cyanobacteria

Photosynthetic energy conversion using natural systems is increasingly being investigated in the recent years. Photosynthetic microorganisms, such as cyanobacteria, exhibit light-dependent electrogenic characteristics in photo-bioelectrochemical cells (PBEC) that generate substantial photocurrents, yet the current densities are lower than their photovoltaic counterparts. Recently, we demonstrated that a cyanobacterium named Nostoc sp. employed in PBEC could generate up to 35 mW m(-2) even in a non-engineered PBEC. With the insights obtained from our previous research, a novel and successful attempt has been made in the current study to genetically engineer the cyanobacteria to further enhance its extracellular electron transfer. The cyanobacterium Synechococcus elongatus PCC 7942 was genetically engineered to express a non-native redox protein called outer membrane cytochrome S (OmcS). OmcS is predominantly responsible for metal reducing abilities of exoelectrogens such as Geobacter sp. The engineered S. elongatus exhibited higher extracellular electron transfer ability resulting in approximately ninefold higher photocurrent generation on the anode of a PBEC than the corresponding wild-type cyanobacterium. This work highlights the scope for enhancing photocurrent generation in cyanobacteria, thereby benefiting faster advancement of the photosynthetic microbial fuel cell technology.

Engineering the Autotroph Methanococcus maripaludis for Geraniol Production

The rapid autotrophic growth of the methanogenic archaeon Methanococcus maripaludis on H2 and CO2 makes it an attractive microbial chassis to inexpensively produce biochemicals. To explore this potential, a synthetic gene encoding geraniol synthase (GES) derived from Ocimum basilicum was cloned into a M. maripaludis expression vector under selection for puromycin resistance. Recombinant expression of GES in M. maripaludis during autotrophic growth on H2/CO2 or formate yielded geraniol at 2.8 and 4.0 mg g(-1) of dry weight, respectively. The yield of geraniol decreased 2-3-fold when organic carbon sources were added to stimulate heterotrophic growth. In the absence of puromycin, geraniol production during autotrophic growth on formate increased to 4.6 mg g(-1) of dry weight. A conceptual model centered on the autotrophic acetyl coenzyme A biosynthetic pathway identified strategies to divert more autotrophic carbon flux to geraniol production.

Establishing a synergetic carbon utilization mechanism for non-catabolic use of glucose in microbial synthesis of trehalose

In nature glucose is a common carbon and energy source for catabolic use and also a building unit of polysaccharides and glycosylated compounds. The presence of strong glucose catabolic pathways in microorganism rapidly decomposes glucose into smaller metabolites and challenges non-catabolic utilization of glucose as C6 building unit or precursor. To address this dilemma, we design a synergetic carbon utilization mechanism (SynCar), in which glucose catabolism is inactivated and a second carbon source (e.g. glycerol) is employed to maintain cell growth and rationally strengthen PEP driving force for glucose uptake and non-catabolic utilization. Remarkably, a trehalose biosynthesis model developed for proof-of-concept indicates that SynCar leads to 131% and 200% improvement in trehalose titer and yield, respectively. The conversion rate of glucose to trehalose reaches 91% of the theoretical maximum. This work demonstrates the broad applicability of SynCar in the biosynthesis of molecules derived from non-catabolic glucose.