Engineering light-energy conversion into nonphotosynthetic hosts

University of Minnesota Ph.D. dissertation. December 2013. Major: Biochemistry, Molecular Bio, and Biophysics. Advisor: Claudia Schmidt-Dannert. 1 computer file (PDF); vii, 139 pages.Over billions of years photosynthetic organisms have refined the molecular machinery required for the capture and conversion of light into chemical energy. To date, much research has been devoted into harnessing this unique trait from photosynthetic organisms and utilizing them for ecologically clean production of valuable resources, such as alternatives to fossil fuels or commodity chemicals. Unfortunately, photosynthetic organisms are not always ideal host for the production of desired chemicals and are frequently difficult to engineer. In order to bypass those hurdles, this work focused on introducing the machinery responsible for the light-energy conversion into a nonphotosynthetic host. The supplementation of a heterologous host with the energy captured via the light-energy conversion could alleviate some of the host's metabolic burden and allow for greater yields of desired compounds. In order to achieve our goals, we set out to engineer functional expression of the bacterial reaction center from R. sphaeroides as well as the enzymes required for the production of bacteriochlorophyll into E. coli. For the first time we were able to demonstrate the expression of the reaction center complex as well as its primarily polar localization with E. coli cells. Furthermore, we characterized two previously poorly understood enzymes involved in the production bacteriochlorophyll, the 8-vinyl reductase (BciA) and the Mg protoporphyrin monomethylester cyclase (BchE). In the case of BciA, we showed that unexpectedly the BciA from R. sphaeroides was not functional when expressed in E. coli, unlike the BciA from C. tepidum. At the beginning of this work, BchE was the only enzyme involved in the biosynthesis of bacteriochlorophyll that has not been heterologously expressed and had no published biochemical or biophysical data. Through our efforts, we were able to demonstrate that BchE contained an oxygen sensitive 4Fe-4S cluster able to interact with SAM, the predicted co-factor. Additionally, for the first time, we showed the interaction of BchE with several intermediates of the bacteriochlorophyll biosynthetic pathways. Complementary to our efforts, we also produced a set of protein expression vectors for use in R. sphaeroides. R. sphaeroides is a photosynthetic organism which has been used extensively for the production of value added compounds and has the potential to be used for the production of membrane proteins. The novel vectors are BioBrickTM compatible and contain DsRed as a reporter protein driven by the photosynthetic puf promoter. We demonstrated that by selecting which section of the promoter was utilized in combination with various culture conditions, final reporter levels could be modulated. Reporter levels ranged from virtually undetectable to higher than what is present in E. coli when expression is driven from a constitutive lac promoter from the same vector backbone

PufQ regulates porphyrin flux at the haem/bacteriochlorophyll branchpoint of tetrapyrrole biosynthesis via interactions with ferrochelatase

Facultative phototrophs such as Rhodobacter sphaeroides can switch between heterotrophic and photosynthetic growth. This transition is governed by oxygen tension and involves the large-scale production of bacteriochlorophyll, which shares a biosynthetic pathway with haem up to protoporphyrin IX. Here, the pathways diverge with the insertion of Fe(2+) or Mg(2+) into protoporphyrin by ferrochelatase or magnesium chelatase, respectively. Tight regulation of this branchpoint is essential, but the mechanisms for switching between respiratory and photosynthetic growth are poorly understood. We show that PufQ governs the haem/bacteriochlorophyll switch; pufQ is found within the oxygen-regulated pufQBALMX operon encoding the reaction centre-light harvesting photosystem complex. A pufQ deletion strain synthesises low levels of bacteriochlorophyll and accumulates the biosynthetic precursor coproporphyrinogen III; a suppressor mutant of this strain harbours a mutation in the hemH gene encoding ferrochelatase, substantially reducing ferrochelatase activity. FLAG-immunoprecipitation experiments retrieve a ferrochelatase-PufQ-carotenoid complex, proposed to regulate the haem/bacteriochlorophyll branchpoint by directing porphyrin flux towards bacteriochlorophyll production under oxygen-limiting conditions. The co-location of pufQ and the photosystem genes in the same operon ensures that switching of tetrapyrrole metabolism towards bacteriochlorophyll is coordinated with the production of reaction centre and light harvesting polypeptides. This article is protected by copyright. All rights reserved

A paralog of a bacteriochlorophyll biosynthesis enzyme catalyzes the formation of 1,2-dihydro-carotenoids in green sulfur bacteria

Chlorobaculum tepidum, a green sulfur bacterium, utilizes chlorobactene as its major carotenoid, and this organism also accumulates a reduced form of this monocyclic pigment, 1',2'-dihydrochlorobactene. The protein catalyzing this reduction is the last unidentified enzyme in the biosynthetic pathways for all of the green sulfur bacterial pigments used for photosynthesis. The genome of Chlorobaculum tepidum contains two paralogous genes encoding members of the FixC family of flavoproteins: bchP, that has been shown to encode an enzyme of bacteriochlorophyll biosynthesis; and bchO, for which a function has not been assigned. Here we demonstrate that a bchO mutant is unable to synthesize 1',2'-dihydrochlorobactene, and when bchO is heterologously expressed in a neurosporene-producing mutant of the purple bacterium, Rhodobactersphaeroides, the encoded protein is able to catalyze the formation of 1,2-dihydroneurosporene, the major carotenoid of the only other organism reported to synthesize 1,2-dihydrocarotenoids, Blastochloris viridis Identification of this enzyme completes the pathways for the synthesis of photosynthetic pigments in Chlorobiaceae, and accordingly and consistent with its role in carotenoid biosynthesis, we propose to rename the gene, cruI Notably, the absence of cruI in Blastochloris viridis indicates that a second 1,2-carotenoid reductase, which is structurally unrelated to CruI (BchO), must exist in nature. The evolution of this carotenoid reductase in green sulfur bacteria is discussed herein

Regulation of Contractility by Adenosine A1 and A2A Receptors in the Murine Heart: Role of Protein Phosphatase 2A: A Dissertation

Adenosine is a nucleoside that plays an important role in the regulation of contractility in the heart. Adenosine receptors are G-protein coupled and those implicated in regulation of contractility are presumed to act via modulating the activity of adenylyl cyclase and cAMP content of cardiomyocytes. Adenosine A1 receptors (A1R) reduce the contractile response of the myocardium to β-adrenergic stimulation. This is known as anti adrenergic action. The A2A adenosine receptor (A2AR) has the opposite effect of increasing contractile responsiveness of the myocardium. The A2AR also appears to attenuate the effects of A1R. The effects of these receptors have been primarily studied in the rat heart and with the utilization of cardiomyocyte preparations. With the increasing use of receptor knockout murine models and murine models of various pathological states, it is of importance to comprehensively study the effects of adenosine receptors on regulation of contractility in the murine heart. The following studies examine the adenosinergic regulation of myocardial contractility in isolated murine hearts. In addition, adenosinergic control of contractility is examined in hearts isolated from A2AR knockout animals. Responses to adenosinergic stimulation in murine isolated hearts are found to be comparable to those observed in the rat, with A1R exhibiting an anti adrenergic action and A2AR conversely enhancing contractility. A significant part of the A2AR effect was found to occur via inhibition of the A1R antiadrenergic action. A part of the anti adrenergic action of A1R has previously been shown to be the result of protein phosphatase 2A activation and localization to membranes. Additional experiments in the present study examine the effect of adenosinergic signaling on PP2A in myocardial extracts from wild type and A2AR knockout hearts. A2AR activation was found to decrease the activity of PP2A and enhance localization of the active enzyme to the cytosol; away from its presumed sites of action. In the A2AR knockout the response to A1R activation was enhanced compared with the wild type and basal PP2A activity was reduced. It is concluded that A2AR modulation of PP2A activity may account for the attenuation of the A1R effect by A2AR observed in the contractile studies.MD/Ph

Leveraging modern DNA assembly techniques for rapid, markerless genome modification

Abstract The ability to alter the genomic material of a prokaryotic cell is necessary for experiments designed to define the biology of the organism. In addition, the production of biomolecules may be significantly improved by application of engineered prokaryotic host cells. Furthermore, in the age of synthetic biology, speed and efficiency are key factors when choosing a method for genome alteration. To address these needs, we have developed a method for modification of the Escherichia coli genome named FAST-GE for Fast Assembly-mediated Scarless Targeted Genome Editing. Traditional cloning steps such as plasmid transformation, propagation and isolation were eliminated. Instead, we developed a DNA assembly-based approach for generating scarless strain modifications, which may include point mutations, deletions and gene replacements, within 48 h after the receipt of polymerase chain reaction primers. The protocol uses established, but optimized, genome modification components such as I-SceI endonuclease to improve recombination efficiency and SacB as a counter-selection mechanism. All DNA-encoded components are assembled into a single allele-exchange vector named pDEL. We were able to rapidly modify the genomes of both E. coli B and K-12 strains with high efficiency. In principle, the method may be applied to other prokaryotic organisms capable of circular dsDNA uptake and homologous recombination.</jats:p