ORCID Profile
0000-0002-5538-7201
Current Organisation
King Abdullah University of Science and Technology (KAUST)
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Publisher: Elsevier BV
Date: 11-2016
DOI: 10.1016/J.YMBEN.2016.07.013
Abstract: The heterologous expression of terpene synthases in microbial hosts has opened numerous possibilities for bioproduction of desirable metabolites. Photosynthetic microbial hosts present a sustainable alternative to traditional fermentative systems, using freely available (sun)light and carbon dioxide as inputs for bio-production. Here, we report the expression of a patchoulol synthase from Pogostemon cablin Benth in the model green microalga Chlamydomonas reinhardtii. The sesquiterpenoid patchoulol was produced from the alga and was used as a marker of sesquiterpenoid production capacity. A novel strategy for gene loading was employed and patchoulol was produced up to 922±242µgg
Publisher: Oxford University Press (OUP)
Date: 21-06-2018
DOI: 10.1093/NAR/GKY532
Publisher: Frontiers Media SA
Date: 23-09-2022
DOI: 10.3389/FBIOE.2022.979607
Abstract: Fluorescent proteins (FPs) are powerful reporters with a broad range of applications in gene expression and subcellular localization. High-throughput screening is often required to identify in idual transformed cell lines in organisms that favor non-homologous-end-joining integration of transgenes into genomes, like in the model green microalga Chlamydomonas reinhardtii . Strategic transgene design, including genetic fusion of transgenes to FPs, and strain domestication have aided engineering efforts in this host but have not removed the need for screening large numbers of transformants to identify those with robust transgene expression levels. FPs facilitate transformant screening by providing a visual signal indicating transgene expression. However, limited combinations of FPs have been described in alga and inherent background fluorescence from cell pigments can hinder FP detection efforts depending on available infrastructure. Here, an updated set of algal nuclear genome-domesticated plasmid parts for seven FPs and six epitope tags were generated and tested in C. reinhardtii . Strategic filter selection was found to enable detection of up to five independent FPs signals from cyan to far-red separately from inherent chlorophyll fluorescence in live algae at the agar plate-level and also in protein electrophoresis gels. This work presents technical advances for algal engineering that can assist reporter detection efforts in other photosynthetic host cells or organisms with inherent background fluorescence.
Publisher: Elsevier BV
Date: 09-2018
DOI: 10.1016/J.YMBEN.2018.07.005
Abstract: Photosynthetic microalgae harbor enormous potential as light-driven green-cell factories for sustainable bio-production of a range of natural and heterologous products such as isoprenoids. Their capacity for photosynthesis and rapid low-input growth with (sun)light and CO
Publisher: Elsevier BV
Date: 08-2013
DOI: 10.1016/J.JBIOTEC.2012.10.010
Abstract: Microalgae are erse photosynthetic microbes which offer the potential for production of a number of high value products (HVP) such as pigments, oils, and bio-active compounds. Fast growth rates, ease of photo-autotrophic cultivation, unique metabolic properties and continuing progress in algal transgenics have raised interest in the use of microalgae systems for recombinant protein (RP) production. This work demonstrates the development of an advanced RP production and secretion system for the green unicellular model alga Chlamydomonas reinhardtii. We generated a versatile expression vector that employs the secretion signal of the native extracellular C. reinhardtii carbonic anhydrase for efficient RP secretion into the culture medium. Unique restriction sites were placed between the regulatory elements to allow fast and easy sub-cloning of sequences of interest. Positive transformants can rapidly be identified by high-throughput plate-level screens via a coupled Gaussia luciferase marker. The vector was tested in Chlamydomonas wild type CC-1883 (WT) and in the transgene expression transformant UVM4. Compared to the native secretion signal of the Gaussia luciferase, up to 84% higher RP production could be achieved. With this new expression system we could generate transformants that express up to 10 mg RP per liter culture without further optimization. The target RP is found exclusively in culture medium and can therefore easily be isolated and purified. We conclude that this new expression system will be a valuable tool for many heterologous protein expression applications from C. reinhardtii in the future.
Publisher: Elsevier BV
Date: 06-2023
Publisher: Cold Spring Harbor Laboratory
Date: 06-10-2023
Publisher: Springer Science and Business Media LLC
Date: 15-01-2015
DOI: 10.1007/S00253-014-6354-7
Abstract: We present a versatile vector toolkit for nuclear transgene expression in the model green microalga Chlamydomonas reinhardtii. The vector was designed in a modular fashion which allows quick replacement of regulatory elements and genes of interest. The current toolkit comprises two antibiotic resistance markers (paromomycin and hygromycin B), five codon-optimized light emission reporters, including the Gaussia princeps luciferase, as well as bright cyan, green, yellow, and red fluorescent protein variants. The system has demonstrated robust functional flexibility with signal options to target the protein of interest to the cytoplasm, the nucleus, cellular microbodies, the chloroplast, mitochondria, or via the endoplasmic reticulum-Golgi apparatus secretory pathway into the culture medium. Successful fluorescent reporter protein fusion to C. reinhardtii Rubisco small subunit 1 was accomplished with this system. Localization of the fluorescently tagged protein was observed in the chloroplast pyrenoid via live cell fluorescence microscopy, the first report of heterologous protein localization to this cellular structure. The functionalities of the vector toolkit, the in idual modular elements, as well as several combinations thereof are demonstrated in this manuscript. Due to its strategic design, this vector system can quickly be adapted to in idual tasks and should therefore be of great use to address specific scientific questions requiring nuclear recombinant protein expression in C. reinhardtii.
Publisher: Elsevier BV
Date: 04-2021
Publisher: Springer Science and Business Media LLC
Date: 12-2013
Abstract: Microalgae are gaining importance as sustainable production hosts in the fields of biotechnology and bioenergy. A robust biomass accumulating strain of the genus Monoraphidium (SAG 48.87) was investigated in this work as a potential feedstock for biofuel production. The genome was sequenced, annotated, and key enzymes for triacylglycerol formation were elucidated. Monoraphidium neglectum was identified as an oleaginous species with favourable growth characteristics as well as a high potential for crude oil production, based on neutral lipid contents of approximately 21% (dry weight) under nitrogen starvation, composed of predominantly C18:1 and C16:0 fatty acids. Further characterization revealed growth in a relatively wide pH range and salt concentrations of up to 1.0% NaCl, in which the cells exhibited larger structures. This first full genome sequencing of a member of the Selenastraceae revealed a diploid, approximately 68 Mbp genome with a G + C content of 64.7%. The circular chloroplast genome was assembled to a 135,362 bp single contig, containing 67 protein-coding genes. The assembly of the mitochondrial genome resulted in two contigs with an approximate total size of 94 kb, the largest known mitochondrial genome within algae. 16,761 protein-coding genes were assigned to the nuclear genome. Comparison of gene sets with respect to functional categories revealed a higher gene number assigned to the category “carbohydrate metabolic process” and in “fatty acid biosynthetic process” in M. neglectum when compared to Chlamydomonas reinhardtii and Nannochloropsis gaditana , indicating a higher metabolic ersity for applications in carbohydrate conversions of biotechnological relevance. The genome of M. neglectum, as well as the metabolic reconstruction of crucial lipid pathways, provides new insights into the ersity of the lipid metabolism in microalgae. The results of this work provide a platform to encourage the development of this strain for biotechnological applications and production concepts.
Publisher: Elsevier BV
Date: 02-2020
DOI: 10.1016/J.COPBIO.2019.09.019
Abstract: Microalgae are rapidly growing, low-input requiring, sun light-utilizing microorganisms capable of converting carbon dioxide into various natural products, a major portion of which are hydrocarbons. Their cellular compartmentalization and photosynthetic apparatus depend on robust turnover of two hydrocarbon classes, isoprenoids and acyl-lipids. This review summarizes the current understanding of algal hydrocarbon metabolism, including carbon partitioning capacities, the localization and size of precursor pools, environmental effects on flux distribution, and limiting factors towards efficient (heterologous) hydrocarbon production. Questions and challenges regarding our knowledge of algal hydrocarbon metabolism as well as guidelines for systematic engineering are presented. Recent engineering achievements indicate fundamental plasticity in the (heterologous) hydrocarbon metabolism of green algae while highlighting their potential as renewable sources of these products.
Publisher: American Chemical Society (ACS)
Date: 30-08-2018
DOI: 10.1021/ACSSYNBIO.8B00251
Abstract: Microalgae are regarded as promising organisms to develop innovative concepts based on their photosynthetic capacity that offers more sustainable production than heterotrophic hosts. However, to realize their potential as green cell factories, a major challenge is to make microalgae easier to engineer. A promising approach for rapid and predictable genetic manipulation is to use standardized synthetic biology tools and workflows. To this end we have developed a Modular Cloning toolkit for the green microalga Chlamydomonas reinhardtii. It is based on Golden Gate cloning with standard syntax, and comprises 119 openly distributed genetic parts, most of which have been functionally validated in several strains. It contains promoters, UTRs, terminators, tags, reporters, antibiotic resistance genes, and introns cloned in various positions to allow maximum modularity. The toolkit enables rapid building of engineered cells for both fundamental research and algal biotechnology. This work will make Chlamydomonas the next chassis for sustainable synthetic biology.
Publisher: Cold Spring Harbor Laboratory
Date: 03-2022
DOI: 10.1101/2022.02.28.482248
Abstract: Chlamydomonas reinhardtii has emerged as a powerful green cell factory for metabolic engineering of sustainable products created from the photosynthetic lifestyle of this microalga. Advances in nuclear genome and transgene expression engineering are allowing robust engineering strategies to be demonstrated in this host. However, commonly used lab strains are not equipped with features to enable their broader implementation in non-sterile conditions and high-cell density concepts. Here, we use combinatorial chloroplast and nuclear genome engineering to complement the C. reinhardtii strain UVM4 with publicly available genetic tools to enable the use of inorganic phosphite and nitrate as a sole the source phosphorous and nitrogen, respectively. We present recipes to create phosphite-buffered media solutions that enable high cell density algal cultivation. We then combine previously reported engineering strategies to produce the heterologous sesquiterpenoid patchoulol to high titers from our engineered green cell factories and show these products are possible to produce in under non-sterile conditions. Our work presents a straightforward means to generate C. reinhardtii strains for broader application in bio-processes for the sustainable generation of products.
Publisher: Elsevier BV
Date: 09-2018
DOI: 10.1016/J.YMBEN.2018.08.008
Abstract: Liquid fuels sourced from fossil sources are the dominant energy form for mobile transport today. The consumption of fossil fuels is still increasing, resulting in a continued search for more sustainable methods to renew our supply of liquid fuel. Photosynthetic microorganisms naturally accumulate hydrocarbons that could serve as a replacement for fossil fuel, however productivities remain low. We report successful introduction of five synthetic metabolic pathways in two green cell factories, prokaryotic cyanobacteria and eukaryotic algae. Heterologous thioesterase expression enabled high-yield conversion of native fatty acyl-acyl carrier protein (ACP) into free fatty acids (FFA) in Synechocystis sp. PCC 6803 but not in Chlamydomonas reinhardtii where the polar lipid fraction instead was enhanced. Despite no increase in measurable FFA in Chlamydomonas, genetic recoding and over-production of the native fatty acid photodecarboxylase (FAP) resulted in increased accumulation of 7-heptadecene. Implementation of a carboxylic acid reductase (CAR) and aldehyde deformylating oxygenase (ADO) dependent synthetic pathway in Synechocystis resulted in the accumulation of fatty alcohols and a decrease in the native saturated alkanes. In contrast, the replacement of CAR and ADO with Pseudomonas mendocina UndB (so named as it is responsible for 1-undecene biosynthesis in Pseudomonas) or Chlorella variabilis FAP resulted in high-yield conversion of thioesterase-liberated FFAs into corresponding alkenes and alkanes, respectively. At best, the engineering resulted in an increase in hydrocarbon accumulation of 8- (from 1 to 8.5 mg/g cell dry weight) and 19-fold (from 4 to 77 mg/g cell dry weight) for Chlamydomonas and Synechocystis, respectively. In conclusion, reconstitution of the eukaryotic algae pathway in the prokaryotic cyanobacteria host generated the most effective system, highlighting opportunities for mix-and-match synthetic metabolism. These studies describe functioning synthetic metabolic pathways for hydrocarbon fuel synthesis in photosynthetic microorganisms for the first time, moving us closer to the commercial implementation of photobiocatalytic systems that directly convert CO
Publisher: Public Library of Science (PLoS)
Date: 30-07-2020
Publisher: Frontiers Media SA
Date: 13-05-2022
DOI: 10.3389/FMICB.2022.885840
Abstract: Chlamydomonas reinhardtii has emerged as a powerful green cell factory for metabolic engineering of sustainable products created from the photosynthetic lifestyle of this microalga. Advances in nuclear genome modification and transgene expression are allowing robust engineering strategies to be demonstrated in this host. However, commonly used lab strains are not equipped with features to enable their broader implementation in non-sterile conditions and high-cell density concepts. Here, we used combinatorial chloroplast and nuclear genome engineering to augment the metabolism of the C. reinhardtii strain UVM4 with publicly available genetic tools to enable the use of inorganic phosphite and nitrate as sole sources of phosphorous and nitrogen, respectively. We present recipes to create phosphite-buffered media solutions that enable high cell density algal cultivation. We then combined previously reported engineering strategies to produce the heterologous sesquiterpenoid patchoulol to high titers from our engineered green cell factories and show these products are possible to produce in non-sterile conditions. Our work presents a straightforward means to generate C. reinhardtii strains for broader application in bio-processes for the sustainable generation of products from green microalgae.
Publisher: Elsevier BV
Date: 12-2015
DOI: 10.1016/J.JBIOTEC.2015.05.001
Abstract: Production of recombinant proteins with microalgae represents an alternative platform over plant- or bacterial-based expression systems for certain target proteins. Secretion of recombinant proteins allows accumulation of the target product physically separate from the valuable algal biomass. To date, there has been little investigation into the dynamics of recombinant protein secretion from microalgal hosts-the culture parameters that encourage secreted product accumulation and stability, while encouraging biomass production. In this work, the efficiency of recombinant protein production was optimized by adjusting cultivation parameters for a strain of Chlamydomonas reinhardtii previously engineered to secrete a functional recombinant Lolium perenne ice binding protein (LpIBP), which has applications as a frozen food texturing and cryopreservation additive, into its culture medium. Three media and several cultivation styles were investigated for effects on secreted LpIBP titres and culture growth. A combination of acetate and carbon dioxide feeding with illumination resulted in the highest overall biomass and recombinant protein titres up to 10mgL(-1) in the culture medium. Pure photoautotrophic production was possible using two media types, with recombinant protein accumulation in all cultivations correlating to culture cell density. Two different cultivation systems were used for scale-up to 10L cultivations, one of which produced yields of secreted recombinant protein up to 12mgL(-1) within six cultivation days. Functional ice recrystallization inhibition (IRI) of the LpIBP from total concentrated extracellular protein extracts was demonstrated in a sucrose solution used as a simplified ice cream model. IRI lasted up to 7 days, demonstrating the potential of secreted products from microalgae for use as food additives.
Publisher: MDPI AG
Date: 17-04-2018
DOI: 10.3390/GENES9040219
Publisher: Springer Science and Business Media LLC
Date: 15-09-2013
DOI: 10.1007/S00253-013-5226-X
Abstract: A Lolium perenne ice-binding protein (LpIBP) demonstrates superior ice recrystallization inhibition (IRI) activity and has proposed applications in cryopreservation, food texturing, as well as in being a "green" gas hydrate inhibitor. Recombinant production of LpIBP has been previously conducted in bacterial and yeast systems for studies of protein characterization, but large-scale applications have been hitherto limited due to high production costs. In this work, a codon-optimized LpIBP was recombinantly expressed and secreted in a novel one-step vector system from the nuclear genome of the green microalga Chlamydomonas reinhardtii. Both mixotrophic and photoautotrophic growth regimes supported LpIBP expression, indicating the feasibility of low-cost production using minimal medium, carbon dioxide, and light energy as input. In addition, multiple growth and bioproduct extraction cycles were performed by repetitive batch cultivation trials, demonstrating the potential for semi-continuous production and biomass harvesting. Concentrations of recombinant protein reached in this proof of concept approach were sufficient to demonstrate IRI activity in culture media without additional purification or concentration, with activity further verified by thermal hysteresis and morphology assays. The incorporation of the recombinant LpIBP into a model gas hydrate offers the promise that algal production may eventually find application as a "green" hydrate inhibitor.
Publisher: Elsevier BV
Date: 06-2016
Publisher: Springer Science and Business Media LLC
Date: 14-09-2022
DOI: 10.1186/S12934-022-01910-5
Abstract: Eukaryotic algae have recently emerged as hosts for metabolic engineering efforts to generate heterologous isoprenoids. Isoprenoid metabolic architectures, flux, subcellular localization, and transport dynamics have not yet been fully elucidated in algal hosts. In this study, we investigated the accessibility of different isoprenoid precursor pools for C 15 sesquiterpenoid generation in the cytoplasm and chloroplast of Chlamydomonas reinhardtii using the Abies grandis bisabolene synthase ( Ag BS) as a reporter. The abundance of the C 15 sesquiterpene precursor farnesyl pyrophosphate (FPP) was not increased in the cytosol by co-expression and fusion of Ag BS with different FPP synthases (FPPSs), indicating limited C 5 precursor availability in the cytoplasm. However, FPP was shown to be available in the plastid stroma, where bisabolene titers could be improved several-fold by FPPSs. Sesquiterpene production was greatest when Ag BS-FPPS fusions were directed to the plastid and could further be improved by increasing the gene dosage. During scale-up cultivation with different carbon sources and light regimes, specific sesquiterpene productivities from the plastid were highest with CO 2 as the only carbon source and light:dark illumination cycles. Potential prenyl unit transporters are proposed based on bioinformatic analyses, which may be in part responsible for our observations. Our findings indicate that the algal chloroplast can be harnessed in addition to the cytosol to exploit the full potential of algae as green cell factories for non-native sesquiterpenoid generation. Identification of a prenyl transporter may be leveraged for further extending this capacity.
Publisher: Elsevier BV
Date: 2018
DOI: 10.1016/J.YMBEN.2017.12.010
Abstract: Photosynthetic microbial hosts such as cyanobacteria and eukaryotic microalgae have recently emerged as alternative engineering platforms for the sustainable light-driven bio-production of terpenoids. Many desirable compounds with numerous applications can be produced in microorganisms by heterologous expression of terpene synthases. However, success of green microbial systems has been h ered by issues such as insufficient enzyme expression titers and low flux to desired terpenoid products from carbon fixed during photosynthesis. This work demonstrates how the green microalga Chlamydomonas reinhardtii can be engineered to produce the sesquiterpene biodiesel precursor (E)-α-bisabolene. Through strategic genetic engineering, substantial enhancements of productivity were achieved by coordinated tuning of the isoprenoid metabolism, combining serial enzyme loading for terpene synthase overexpression and amiRNA-based repression of competing pathways. Up to 10.3 ± 0.7mg bisabolene·g
Publisher: American Chemical Society (ACS)
Date: 08-10-2018
DOI: 10.1021/ACSSYNBIO.8B00226
Abstract: Light-driven recombinant protein (RP) production in eukaryotic microalgae offers a sustainable alternative to other established cell-culture systems. RP production via secretion into the culture medium enables simple product separation from the cells adding a layer of process value in addition to the algal biomass, which can be separately harvested. For the model microalga Chlamydomonas reinhardtii, a broad range of molecular tools have been established to enable heterologous gene expression however, low RP production levels and unreliable purification from secretion concepts have been reported. Domesticated C. reinhardtii strains used for genetic engineering are often cell-wall deficient. These strains nevertheless secrete cell-wall components such as insoluble (hydroxy)proline-rich glycoproteins into the culture media, which hinder downstream purification processes. Here, we attempted to overcome limitations in secretion titers and improve protein purification by combining fusion partners that enhance RP secretion and enable alternative aqueous two-phase (ATPS) RP extraction from the culture medium. Protein fusions were strategically designed to contain a stably folded peptide, which enhanced secretion capacities and gave insights into (some) regulatory mechanisms responsible for this process in the algal host. The elevated protein titers mediated by this fusion were then successfully applied in combination with a fungal hydrophobin tag, which enabled protein purification from the complex microalgal extracellular environment by ATPS, to yield functional recombinant human epidermal growth factor (hEGF) from the algal host.
Publisher: Cold Spring Harbor Laboratory
Date: 13-01-2023
DOI: 10.1101/2023.01.12.523746
Abstract: 1. Isoprene is a clear, colorless, volatile 5-carbon hydrocarbon that is one monomer of all cellular isoprenoids and a platform chemical with multiple applications in industry. Many plants have evolved isoprene synthases (IspSs) with the capacity to liberate isoprene from dimethylallyl pyrophosphate (DMAPP) as part of cellular protection mechanisms. Isoprene is hydrophobic and volatile, rapidly leaves plant tissues and is one of the main carbon emission sources from vegetation globally. The universality of isoprenoid metabolism allows volatile isoprene production from microbes expressing heterologous IspSs. Here, we compared heterologous overexpression from the nuclear genome and localization into the plastid of four plant terpene synthases (TPs) in the green microalga Chlamydomonas reinhardtii . Using sealed vial mixotrophic cultivation, direct quantification of isoprene production was achieved from the headspace of living cultures, with the highest isoprene production observed in algae expressing the Ipomoea batatas IspS. Perturbations of the downstream carotenoid pathway through keto carotenoid biosynthesis enhanced isoprene titers, which could be further enhanced by increasing flux towards DMAPP through heterologous co-expression of a yeast isopentenyl-PP delta isomerase. Multiplexed controlled-environment testing revealed that cultivation temperature, rather than illumination intensity, was the main factor affecting isoprene yield from the engineered alga. This is the first report of heterologous isoprene production from a eukaryotic alga and sets a foundation for further exploration of carbon conversion to this commodity chemical.
Location: Saudi Arabia
No related grants have been discovered for Kyle Lauersen.