A Phosphite-Based Sscreening Platform for Identification of Enzymes Favoring Nonnatural Cofactors

Abstract BackgroundEnzymes with dedicated cofactor preference are essential for advanced biocatalysis and biomanufacturing. However, directed evolution of an enzyme to switch its cofactor preference is often hindered by the lack of efficient and affordable method for screening as the cofactor per se or the substrate can be prohibitively expensive. Here, we developed a growth-based selection platform to identify nonnatural cofactor-dependent oxidoreductase mutants.ResultsThe growth-based selection platform was designed by coupling with nonnatural cofactor-dependent phosphite dehydrogenase (Pdh) mediated the conversion of non-metabolizable phosphite into phosphate in the culture media. Thus, Pdh variant that strongly favors nicotinamide cytosine dinucleotide (NCD), a NAD analogue, the feasibility of this strategy was successfully demonstrated using derived NCD-active malic enzyme as well as for the directed evolution of NCD synthetase in Escherichia coli.ConclusionsHere, we built a phosphite-based screening platform for identification of enzymes favoring nonnatural cofactor NCD. In the future, once Pdh variants favoring other biomimetic or nonnatural cofactors are available this selection platform may be readily redesigned to attain new enzyme variants with anticipated cofactor preference, providing opportunities to further expand the chemical space of redox cofactors in chemical biology and synthetic biology.

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Emergence of NADP+-reducing enzymes in Escherichia coli central metabolism via adaptive evolution

10.1101/2021.02.08.430375 ◽

2021 ◽

Author(s):

Madeleine Bouzon ◽

Volker Döring ◽

Ivan Dubois ◽

Anne Berger ◽

Gabriele M. M. Stoffel ◽

...

Keyword(s):

Escherichia Coli ◽

Enzyme Activity ◽

Directed Evolution ◽

Adaptive Evolution ◽

Malic Enzyme ◽

Mass Action ◽

Single Mutation ◽

Central Metabolism ◽

Dihydrolipoamide Dehydrogenase ◽

Cofactor Specificity

AbstractThe nicotinamide cofactor specificity of enzymes plays a key role in regulating metabolic processes and attaining cellular homeostasis. Multiple studies have used enzyme engineering tools or a directed evolution approach to switch the cofactor preference of specific oxidoreductases. However, whole-cell adaptation towards the emergence of novel cofactor regeneration routes was not previously explored. To address this challenge, we used an Escherichia coli NADPH-auxotroph strain. We continuously cultivated this strain under selective conditions. After 500-1100 generations of adaptive evolution using different carbon sources, we isolated several strains capable of growing without an external NADPH source. Most isolated strains were found to harbor a mutated NAD-dependent malic enzyme (MaeA). A single mutation in MaeA was found to switch cofactor specificity while lowering enzyme activity. Most mutated MaeA variants also harbored a second mutation that restored the catalytic efficiency of the enzyme. Remarkably, the best MaeA variants identified this way displayed overall superior kinetics relative to the wildtype variant with NAD+. In other evolved strains, the dihydrolipoamide dehydrogenase (Lpd) was mutated to accept NADP+ thus enabling the pyruvate dehydrogenase and 2-ketoglutarate dehydrogenase complexes to regenerate NADPH. Interestingly, no other central metabolism oxidoreductase seems to evolve towards reducing NADP+, which we attribute to several biochemical constraints such as unfavorable thermodynamics. This study demonstrates the potential and biochemical limits of evolving oxidoreductases within the cellular context towards changing cofactor specificity, further showing that long-term adaptive evolution can optimize enzyme activity beyond what is achievable via rational design or directed evolution using small libraries.ImportanceIn the cell, NAD(H) and NADP(H) cofactors have different functions. The former mainly accepts electrons from catabolic reactions and carries them to respiration, while the latter provides reducing power for anabolism. Correspondingly, the ratio of the reduced to the oxidized form differs for NAD (low) and NADP (high), reflecting their distinct roles. We challenged the flexibility of E. coli’s central metabolism in multiple adaptive evolution experiments using an NADPH-auxotroph strain. We found several mutations in two enzymes, changing the cofactor preference of malic enzyme and dihydrolipoamide dehydrogenase. Upon deletion of their corresponding genes we performed additional evolution experiments which did not lead to the emergence of any additional mutants. We attribute this restricted number of mutational targets to intrinsic thermodynamic barriers: The high ratio of NADPH to NADP+ limits metabolic redox reactions which can regenerate NADPH, mainly by mass action constraints.

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The Influence of Glycosylation on the Catalytic and Fibrinolytic Properties of Pro-Urokinase

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646314 ◽

1992 ◽

Vol 68 (05) ◽

pp. 539-544 ◽

Cited By ~ 22

Author(s):

Catherine Lenich ◽

Ralph Pannell ◽

Jack Henkin ◽

Victor Gurewich

Keyword(s):

Escherichia Coli ◽

Mammalian Cell ◽

Human Gene ◽

Culture Media ◽

Mammalian Cell Culture ◽

Glycosylation Site ◽

Clot Lysis ◽

Cell Culture Media ◽

E Coli ◽

The Uk

SummaryWe previously found that human pro-UK expressed in Escherichia coli is more active in fibrinolysis than recombinant human pro-UK obtained from mammalian cell culture media. To determine whether this difference is related to the lack of glycosylation of the E. coli product, we compared the activity of E. coli-derived pro-UK [(-)pro-UK] with that of a glycosylated pro-UK [(+)pro-UK] and of a mutant of pro-UK missing the glycosylation site at Asn-302 [(-) (302) pro-UK]. The latter two pro-UKs were obtained by expression of the human gene in a mammalian cell. The nonglycosylated pro-UKs were activated by plasmin more efficiently (≈2-fold) and were more active in clot lysis (1.5-fold) than the (+)pro-UK. Similarly, the nonglycosylated two-chain derivatives (UKs) were more active against plasminogen and were more rapidly inactivated by plasma inhibitors than the (+)UK.These findings indicate that glycosylation at Asn-302 influences the activity of pro-UK/UK and could be the major factor responsible for the enhanced activity of E. coli-derived pro-UK.

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Microbe Engineering of Guaia-6,10(14)-diene as a Building Block for the Semisynthetic Production of Plant-Derived (−)-Englerin A

10.26434/chemrxiv.10333625.v1 ◽

2019 ◽

Author(s):

Thomas Siemon ◽

Zhangqian Wang ◽

Guangkai Bian ◽

Tobias Seitz ◽

Ziling Ye ◽

...

Keyword(s):

Escherichia Coli ◽

Saccharomyces Cerevisiae ◽

Synthetic Biology ◽

Chemical Synthesis ◽

Building Block ◽

Transient Receptor Potential ◽

Receptor Potential ◽

Economical Method ◽

Englerin A ◽

Transient Receptor

Herein, we report the semisynthetic production of the potent transient receptor potential canonical (TRPC) channel agonist (−)-englerin A (EA), using guaia-6,10(14)-diene as the starting material. Guaia-6,10(14)-diene was systematically engineered in Escherichia coli and Saccharomyces cerevisiae using the CRISPR/Cas9 system and produced with high titers. This provided us the opportunity to execute a concise chemical synthesis of EA and the two related guaianes (−)-oxyphyllol and (+)-orientalol E. The potentially scalable approach combines the advantages of synthetic biology and chemical synthesis and provides an efficient and economical method for producing EA as well as its analogs.

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Microbe Engineering of Guaia-6,10(14)-diene as a Building Block for the Semisynthetic Production of Plant-Derived (−)-Englerin A

10.26434/chemrxiv.10333625 ◽

2019 ◽

Author(s):

Thomas Siemon ◽

Zhangqian Wang ◽

Guangkai Bian ◽

Tobias Seitz ◽

Ziling Ye ◽

...

Keyword(s):

Escherichia Coli ◽

Saccharomyces Cerevisiae ◽

Synthetic Biology ◽

Chemical Synthesis ◽

Building Block ◽

Transient Receptor Potential ◽

Receptor Potential ◽

Economical Method ◽

Englerin A ◽

Transient Receptor

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Creating enzymes and self-sufficient cells for biosynthesis of the non-natural cofactor nicotinamide cytosine dinucleotide

Nature Communications ◽

10.1038/s41467-021-22357-z ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Xueying Wang ◽

Yanbin Feng ◽

Xiaojia Guo ◽

Qian Wang ◽

Siyang Ning ◽

...

Keyword(s):

Biological Sciences ◽

Escherichia Coli ◽

Nicotinamide Adenine Dinucleotide ◽

Chemical Biology ◽

Reduced Form ◽

Redox Transformations ◽

Nicotinamide Mononucleotide ◽

Self Sufficiency ◽

Binding Pockets ◽

Pathway Regulation

AbstractNicotinamide adenine dinucleotide (NAD) and its reduced form are indispensable cofactors in life. Diverse NAD mimics have been developed for applications in chemical and biological sciences. Nicotinamide cytosine dinucleotide (NCD) has emerged as a non-natural cofactor to mediate redox transformations, while cells are fed with chemically synthesized NCD. Here, we create NCD synthetase (NcdS) by reprograming the substrate binding pockets of nicotinic acid mononucleotide (NaMN) adenylyltransferase to favor cytidine triphosphate and nicotinamide mononucleotide over their regular substrates ATP and NaMN, respectively. Overexpression of NcdS alone in the model host Escherichia coli facilitated intracellular production of NCD, and higher NCD levels up to 5.0 mM were achieved upon further pathway regulation. Finally, the non-natural cofactor self-sufficiency was confirmed by mediating an NCD-linked metabolic circuit to convert L-malate into D-lactate. NcdS together with NCD-linked enzymes offer unique tools and opportunities for intriguing studies in chemical biology and synthetic biology.

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Human NAD(+)-dependent mitochondrial malic enzyme. cDNA cloning, primary structure, and expression in Escherichia coli.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)49948-6 ◽

1991 ◽

Vol 266 (5) ◽

pp. 3016-3021

Author(s):

G Loeber ◽

A A Infante ◽

I Maurer-Fogy ◽

E Krystek ◽

M B Dworkin

Keyword(s):

Escherichia Coli ◽

Cdna Cloning ◽

Primary Structure ◽

Malic Enzyme ◽

Expression In Escherichia Coli ◽

Mitochondrial Malic Enzyme

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Improving cell-free glycoprotein synthesis by characterizing and enriching native membrane vesicles

Nature Communications ◽

10.1038/s41467-021-22329-3 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Jasmine M. Hershewe ◽

Katherine F. Warfel ◽

Shaelyn M. Iyer ◽

Justin A. Peruzzi ◽

Claretta J. Sullivan ◽

...

Keyword(s):

Gene Expression ◽

Escherichia Coli ◽

Synthetic Biology ◽

Membrane Protein ◽

Membrane Vesicles ◽

Processing Methods ◽

Glycoprotein Synthesis ◽

On Demand ◽

Free Gene ◽

Membrane Bound

AbstractCell-free gene expression (CFE) systems from crude cellular extracts have attracted much attention for biomanufacturing and synthetic biology. However, activating membrane-dependent functionality of cell-derived vesicles in bacterial CFE systems has been limited. Here, we address this limitation by characterizing native membrane vesicles in Escherichia coli-based CFE extracts and describing methods to enrich vesicles with heterologous, membrane-bound machinery. As a model, we focus on bacterial glycoengineering. We first use multiple, orthogonal techniques to characterize vesicles and show how extract processing methods can be used to increase concentrations of membrane vesicles in CFE systems. Then, we show that extracts enriched in vesicle number also display enhanced concentrations of heterologous membrane protein cargo. Finally, we apply our methods to enrich membrane-bound oligosaccharyltransferases and lipid-linked oligosaccharides for improving cell-free N-linked and O-linked glycoprotein synthesis. We anticipate that these methods will facilitate on-demand glycoprotein production and enable new CFE systems with membrane-associated activities.

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Engineering Escherichia coli for production of geraniol by systematic synthetic biology approaches and laboratory-evolved fusion tags

Metabolic Engineering ◽

10.1016/j.ymben.2021.04.008 ◽

2021 ◽

Vol 66 ◽

pp. 60-67

Author(s):

Xun Wang ◽

Jiaming Chen ◽

Jia Zhang ◽

Yujunjie Zhou ◽

Yu Zhang ◽

...

Keyword(s):

Escherichia Coli ◽

Synthetic Biology

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CRIMoClo plasmids for modular assembly and orthogonal chromosomal integration of synthetic circuits in Escherichia coli

Journal of Biological Engineering ◽

10.1186/s13036-019-0218-8 ◽

2019 ◽

Vol 13 (1) ◽

Cited By ~ 1

Author(s):

Stefano Vecchione ◽

Georg Fritz

Keyword(s):

Escherichia Coli ◽

Synthetic Biology ◽

Integrated Circuits ◽

High Efficiency ◽

Genetic Circuit ◽

Dna Assembly ◽

Chromosomal Integration ◽

Golden Gate ◽

Genetic Circuits ◽

E Coli

Abstract Background Synthetic biology heavily depends on rapid and simple techniques for DNA engineering, such as Ligase Cycling Reaction (LCR), Gibson assembly and Golden Gate assembly, all of which allow for fast, multi-fragment DNA assembly. A major enhancement of Golden Gate assembly is represented by the Modular Cloning (MoClo) system that allows for simple library propagation and combinatorial construction of genetic circuits from reusable parts. Yet, one limitation of the MoClo system is that all circuits are assembled in low- and medium copy plasmids, while a rapid route to chromosomal integration is lacking. To overcome this bottleneck, here we took advantage of the conditional-replication, integration, and modular (CRIM) plasmids, which can be integrated in single copies into the chromosome of Escherichia coli and related bacteria by site-specific recombination at different phage attachment (att) sites. Results By combining the modularity of the MoClo system with the CRIM plasmids features we created a set of 32 novel CRIMoClo plasmids and benchmarked their suitability for synthetic biology applications. Using CRIMoClo plasmids we assembled and integrated a given genetic circuit into four selected phage attachment sites. Analyzing the behavior of these circuits we found essentially identical expression levels, indicating orthogonality of the loci. Using CRIMoClo plasmids and four different reporter systems, we illustrated a framework that allows for a fast and reliable sequential integration at the four selected att sites. Taking advantage of four resistance cassettes the procedure did not require recombination events between each round of integration. Finally, we assembled and genomically integrated synthetic ECF σ factor/anti-σ switches with high efficiency, showing that the growth defects observed for circuits encoded on medium-copy plasmids were alleviated. Conclusions The CRIMoClo system enables the generation of genetic circuits from reusable, MoClo-compatible parts and their integration into 4 orthogonal att sites into the genome of E. coli. Utilizing four different resistance modules the CRIMoClo system allows for easy, fast, and reliable multiple integrations. Moreover, utilizing CRIMoClo plasmids and MoClo reusable parts, we efficiently integrated and alleviated the toxicity of plasmid-borne circuits. Finally, since CRIMoClo framework allows for high flexibility, it is possible to utilize plasmid-borne and chromosomally integrated circuits simultaneously. This increases our ability to permute multiple genetic modules and allows for an easier design of complex synthetic metabolic pathways in E. coli.

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