Membrane transporter identification and modulation via adaptive laboratory evolution.

Membrane transport proteins are potential targets for medical and biotechnological applications. However, more than 30% of reported membrane transporter families are either poorly characterized or lack adequate functional annotation. Here, adaptive laboratory evolution was leveraged to identify membrane transporters for a set of four amino acids as well as specific mutations that modulate the activities of these transporters. Specifically, Escherichia coli was adaptively evolved under increasing concentrations of L-histidine, L-phenylalanine, L-threonine, and L-methionine separately with multiple replicate evolutions. Evolved populations and isolated clones displayed growth rates comparable to the unstressed ancestral strain at elevated concentrations (four-to six-fold increases) of the targeted amino acids. Whole genome sequencing of the evolved strains revealed a diverse number of key mutations, including SNPs, small deletions, and copy number variants targeting the transporters leuE for histidine, yddG for phenylalanine, yedA for methionine, and brnQ and rhtC for threonine. Reverse engineering of the mutations in the ancestral strain established mutation causality of the specific mutations for the tolerant phenotypes. The functional roles of yedA and brnQ in the transport of methionine and threonine, respectively, are novel assignments and their functional roles were validated using a flow cytometry cellular accumulation assay. To demonstrate how the identified transporters can be leveraged for production, an L-phenylalanine overproduction strain was shown to be a superior producer when the identified yddG exporter was overexpressed. Overall, the results revealed the striking efficiency of laboratory evolution to identify transporters and specific mutational mechanisms to modulate their activities, thereby demonstrating promising applicability in transporter discovery efforts and strain engineering.

Sphingomonas lacus sp. nov., an astaxanthin-dideoxyglycoside-producing species isolated from soil near a pond.

Taxonomic studies were performed on an astaxanthin-dideoxyglycoside-producing strain, designated PB304(T), isolated from soil near a pond in Daejeon city, South Korea. Cells of strain PB304(T) were Gram-staining-negative, strictly aerobic, orange-coloured and motile, and occurred as single or paired short chains. PB304(T) did not contain bacteriochlorophyll a. 16S rRNA gene sequence analysis revealed that strain PB304(T) was closely related to 'Sphingomonas humi' KCTC 12341 (98.7%), Sphingomonas kaistensis KCTC 12344(T)(97.9%), Sphingomonas astaxanthinifaciens DSM 22298(T) (97.6%) and Sphingomonas ginsengisoli KCTC 12630(T) (97.5%). Analysis of pufLM gene sequences revealed strain PB304(T) to be closely related to 'S. humi' KCTC 12341 (88.1%). The major cellular fatty acids were C16 : 0, summed feature 4 (comprising iso-C15 : 0 2-OH and/or C16 : 1ω7c), and summed feature 7 (comprising C18  : 1ω7c/ω9t/ω12t). Ubiquinone 10 (Q-10) was the sole quinone identified, and the major pigment was astaxanthin dideoxyglycoside. The major polar lipids were sphingoglycolipid, phosphatidylcholine, phosphatidylglycerol, diphosphatidylglycerol and phosphatidylethanolamine. The polyamine was spermidine. The DNA-DNA relatedness values of strain PB304(T) with respect to its closest phylogenetic neighbours were 57.1% for 'S. humi' KCTC 12341, 51.2% for Sphingomonas kaistensis KCTC 12334T, 50.6% for Sphingomonas astaxanthinifaciens DSM 22298(T) and 50.2% for Sphingomonas ginsengisoli KCTC 12630(T). The DNA G+C content of strain PB304(T) was 66.6 mol%. On the basis of the phenotypic, chemotaxonomic and phylogenetic data, strain PB304T is concluded to represent a novel species of the genus Sphingomonas, for which the name Sphingomonas lacus is proposed. The type strain is PB304(T) ( = KCTC 32458(T) = CECT 8383(T)).

Proposed cytotoxic mechanisms of the saffron carotenoids crocin and crocetin on cancer cell lines.

We investigated the cytotoxic activities of crocin and crocetin, 2 major carotenoids isolated from the stigma of Crocus sativus (saffron), on 5 human cancer cell lines and proposed their possible anticancer mechanisms. Crocetin, a glycosylated carotenoid, showed approximately 5- to 18-fold higher cytotoxicity than crocin, a carboxylic carotenoid (IC50 of 0.16-0.61 mmol/L for crocetin vs. 2.0-5.5 mmol/L for crocin). This suggests that structural differences account for the different efficacies between them. Fluorescence-activated cell sorting (FACS) analysis showed that crocetin induced a significant level of cellular reactive oxygen species (ROS) in HeLa cells, whereas crocin did not. This ROS induction supported the cytotoxicity of crocetin, but not of crocin. A significant activation of nuclear factor erythroid 2-related factor 2 (Nrf2) was observed in both HeLa cells treated with crocin and crocetin: a 3.0-fold increase by 1 mmol/L crocetin and a 1.6-fold increase by 0.8 mmol/L crocin compared to the control. Furthermore, both crocetin and crocin reduced the protein expression of lactate dehydrogenase A (LDHA), one of the targets for chemoprevention in cancer cells, by 34.2% and 10.5%, respectively, compared to the control in HeLa cells. These findings suggest that crocetin and crocin have different mechanisms for their observed cytotoxicity in cancer cell lines.

The astaxanthin dideoxyglycoside biosynthesis pathway in Sphingomonas sp. PB304.

A major carotenoid in Sphingomonas sp. PB304, originally isolated from a river in Daejon City, South Korea, was identified as astaxanthin dideoxyglycoside. Gene clusters encoding the astaxanthin dideoxyglycoside biosynthetic enzymes were identified by screening Sphingomonas sp. PB304 fosmid libraries using degenerate probes that harbor highly conserved sequences from the Sphigomonas elodea-derived crtI and Nostoc sp. PCC 7120-dervied crtW genes. Selected positive gene clusters were fully sequenced and annotated, revealing genes encoding six putative carotenogenic enzymes: phytoene synthase (CrtB), phytoene desaturase (CrtI), lycopene cyclase (CrtY), carotene hydroxylase (CrtZ), carotene ketolase (CrtW), and glycosyltransferase (CrtX). All of the carotenogenic enzymes, except for CrtX, were functional in the recombinant host Escherichia coli expressing synthetic carotenogenic modules from Pantoea agglomerans. CrtX did not take up UDP-glucose or GDP-fucose as sugar substrates during the in vitro reaction. Although no direct experimental evidence was obtained for the function of Sphingomonas sp. PB304 CrtX, it can be categorized as a putative deoxyglycosyltransferase based on the presence of astaxanthin dideoxyglycoside in Sphingomonas sp. PB304, a putative corresponding gene in the carotenoid biosynthetic gene cluster, and high amino acid sequence homology to the existing glycosyltransferases. Therefore, we propose that astaxanthin dideoxyglycoside can be synthesized in Sphingomonas sp. PB304 via sequential reactions of six pathway enzymes, including CrtX on the phytoene intermediate.

Carotenoid production from n-alkanes with a broad range of chain lengths by the novel species Gordonia ajoucoccus A2(T).

A novel diesel-degrading bacterial strain, A2(T), was isolated from soil that was heavily contaminated with oil. Based on phenotypic, phylogenetic, and DNA analyses, strain A2(T) was identified as a novel species of the genus Gordonia and named Gordonia ajoucoccus A2(T) (KCTC 11900BP and CECT8382). G. ajoucoccus A2(T) is able to synthesize carotenoids and produces mainly γ-carotene and keto-γ-carotene. G. ajoucoccus A2(T) is also capable of assimilating n-alkanes with a broad range of chain lengths (C6, C8-C25). Batch culture of G. ajoucoccus A2(T) in a bioreactor containing 1 % (v/v) hexadecane or 1 % (v/v) commercial diesel yielded 25 mg L⁻¹ and 2.6 mg L⁻¹ of carotenoids, respectively. Gas chromatography/mass spectrometry (GC-MS) analysis of hexadecane and hexane degradation metabolites suggested that G. ajoucoccus A2(T) may possess a terminal oxidation pathway that allows it to utilize n-alkanes and hexane as carbon and energy sources. G. ajoucoccus A2(T) could therefore serve as a good model system for understanding microbial n-alkane degradation pathways. Additionally, the metabolic capabilities of G. ajoucoccus A2(T) suggest potential biotechnological applications, such as the bioproduction of carotenoids from industrial discharge or other sources of n-alkanes.

Functional expression and extension of staphylococcal staphyloxanthin biosynthetic pathway in Escherichia coli.

The biosynthetic pathway for staphyloxanthin, a C(30) carotenoid biosynthesized by Staphylococcus aureus, has previously been proposed to consist of five enzymes (CrtO, CrtP, CrtQ, CrtM, and CrtN). Here, we report a missing sixth enzyme, 4,4'-diaponeurosporen-aldehyde dehydrogenase (AldH), in the staphyloxanthin biosynthetic pathway and describe the functional expression of the complete staphyloxanthin biosynthetic pathway in Escherichia coli. When we expressed the five known pathway enzymes through artificial synthetic operons and the wild-type operon (crtOPQMN) in E. coli, carotenoid aldehyde intermediates such as 4,4'-diaponeurosporen-4-al accumulated without being converted into staphyloxanthin or other intermediates. We identified an aldH gene located 670 kilobase pairs from the known staphyloxanthin gene cluster in the S. aureus genome and an aldH gene in the non-staphyloxanthin-producing Staphylococcus carnosus genome. These two putative enzymes catalyzed the missing oxidation reaction to convert 4,4'-diaponeurosporen-4-al into 4,4'-diaponeurosporenoic acid in E. coli. Deletion of the aldH gene in S. aureus abolished staphyloxanthin biosynthesis and caused accumulation of 4,4'-diaponeurosporen-4-al, confirming the role of AldH in staphyloxanthin biosynthesis. When the complete staphyloxanthin biosynthetic pathway was expressed using an artificial synthetic operon in E. coli, staphyloxanthin-like compounds, which contained altered fatty acid acyl chains, and novel carotenoid compounds were produced, indicating functional expression and coordination of the six staphyloxanthin pathway enzymes.

New insight into the cleavage reaction of Nostoc sp. strain PCC 7120 carotenoid cleavage dioxygenase in natural and nonnatural carotenoids.

Carotenoid cleavage dioxygenases (CCDs) are enzymes that catalyze the oxidative cleavage of carotenoids at a specific double bond to generate apocarotenoids. In this study, we investigated the activity and substrate preferences of NSC3, a CCD of Nostoc sp. strain PCC 7120, in vivo and in vitro using natural and nonnatural carotenoid structures. NSC3 cleaved ß-apo-8'-carotenal at 3 positions, C-13 C-14, C-15 C-15', and C-13' C-14', revealing a unique cleavage pattern. NSC3 cleaves the natural structure of carotenoids 4,4'-diaponeurosporene, 4,4'-diaponeurosporen-4'-al, 4,4'-diaponeurosporen-4'-oic acid, 4,4'-diapotorulene, and 4,4'-diapotorulen-4'-al to generate novel cleavage products (apo-14'-diaponeurosporenal, apo-13'-diaponeurosporenal, apo-10'-diaponeurosporenal, apo-14'-diapotorulenal, and apo-10'-diapotorulenal, respectively). The study of carotenoids with natural or nonnatural structures produced by using synthetic modules could provide information valuable for understanding the cleavage reactions or substrate preferences of other CCDs in vivo and in vitro.

Heterologous carotenoid-biosynthetic enzymes: functional complementation and effects on carotenoid profiles in Escherichia coli.

A limited number of carotenoid pathway genes from microbial sources have been studied for analyzing the pathway complementation in the heterologous host Escherichia coli. In order to systematically investigate the functionality of carotenoid pathway enzymes in E. coli, the pathway genes of carotenogenic microorganisms (Brevibacterium linens, Corynebacterium glutamicum, Rhodobacter sphaeroides, Rhodobacter capsulatus, Rhodopirellula baltica, and Pantoea ananatis) were modified to form synthetic expression modules and then were complemented with Pantoea agglomerans pathway enzymes (CrtE, CrtB, CrtI, CrtY, and CrtZ). The carotenogenic pathway enzymes in the synthetic modules showed unusual activities when complemented with E. coli. For example, the expression of heterologous CrtEs of B. linens, C. glutamicum, and R. baltica influenced P. agglomerans CrtI to convert its substrate phytoene into a rare product-3,4,3',4'-tetradehydrolycopene-along with lycopene, which was an expected product, indicating that CrtE, the first enzyme in the carotenoid biosynthesis pathway, can influence carotenoid profiles. In addition, CrtIs of R. sphaeroides and R. capsulatus converted phytoene into an unusual lycopene as well as into neurosporene. Thus, this study shows that the functional complementation of pathway enzymes from different sources is a useful methodology for diversifying biosynthesis as nature does.

Understanding Functional Redundancy and Promiscuity of Multidrug Transporters in E. coli under Lipophilic Cation Stress.

Multidrug transporters (MDTs) are major contributors to microbial drug resistance and are further utilized for improving host phenotypes in biotechnological applications. Therefore, the identification of these MDTs and the understanding of their mechanisms of action in vivo are of great importance. However, their promiscuity and functional redundancy represent a major challenge towards their identification. Here, a multistep tolerance adaptive laboratory evolution (TALE) approach was leveraged to achieve this goal. Specifically, a wild-type E. coli K-12-MG1655 and its cognate knockout individual mutants ΔemrE, ΔtolC, and ΔacrB were evolved separately under increasing concentrations of two lipophilic cations, tetraphenylphosphonium (TPP+), and methyltriphenylphosphonium (MTPP+). The evolved strains showed a significant increase in MIC values of both cations and an apparent cross-cation resistance. Sequencing of all evolved mutants highlighted diverse mutational mechanisms that affect the activity of nine MDTs including acrB, mdtK, mdfA, acrE, emrD, tolC, acrA, mdtL, and mdtP. Besides regulatory mutations, several structural mutations were recognized in the proximal binding domain of acrB and the permeation pathways of both mdtK and mdfA. These details can aid in the rational design of MDT inhibitors to efficiently combat efflux-based drug resistance. Additionally, the TALE approach can be scaled to different microbes and molecules of medical and biotechnological relevance.

Identification and Engineering of Transporters for Efficient Melatonin Production in Escherichia coli.

Transporter discovery and engineering play an important role in cell factory development. Decreasing the intracellular concentration of the product reduces product inhibition and/or toxicity. Lowering intracellular concentrations is especially beneficial for achieving a robust strain at high titers. However, the identification of transporters for xenobiotic chemicals in the host strain is challenging. Here we present a high-throughput workflow to discover Escherichia coli transporters responsible for the efflux of the inhibitory xenobiotic compound melatonin. We took advantage of the Keio collection and screened about 400 transporter knockouts in the presence of a high concentration of melatonin. We found five transporters that when knocked out showed decreased tolerance to melatonin, indicating they are exporters of melatonin. We overexpressed these five genes individually in the production strain and found that one of them, yhjV, encoding a transporter with unknown substrates, resulted in a 27% titer increase in cultivation mimicking fed-batch fermentation. This study demonstrates how microbial cell factories can be improved through transporter identification and engineering. Further, these results lay the foundation for the scale-up of melatonin production in E. coli.

Escherichia coli Data-Driven Strain Design Using Aggregated Adaptive Laboratory Evolution Mutational Data.

Microbes are being engineered for an increasingly large and diverse set of applications. However, the designing of microbial genomes remains challenging due to the general complexity of biological systems. Adaptive Laboratory Evolution (ALE) leverages nature's problem-solving processes to generate optimized genotypes currently inaccessible to rational methods. The large amount of public ALE data now represents a new opportunity for data-driven strain design. This study describes how novel strain designs, or genome sequences not yet observed in ALE experiments or published designs, can be extracted from aggregated ALE data and demonstrates this by designing, building, and testing three novel Escherichia coli strains with fitnesses comparable to ALE mutants. These designs were achieved through a meta-analysis of aggregated ALE mutations data (63 Escherichia coli K-12 MG1655 based ALE experiments, described by 93 unique environmental conditions, 357 independent evolutions, and 13 957 observed mutations), which additionally revealed global ALE mutation trends that inform on ALE-derived strain design principles. Such informative trends anticipate ALE-derived strain designs as largely gene-centric, as opposed to noncoding, and composed of a relatively small number of beneficial variants (approximately 6). These results demonstrate how strain design efforts can be enhanced by the meta-analysis of aggregated ALE data.

Redesign, reconstruction, and directed extension of the Brevibacterium linens C40 carotenoid pathway in Escherichia coli.

In this study, the carotenoid biosynthetic pathways of Brevibacterium linens DSMZ 20426 were reconstructed, redesigned, and extended with additional carotenoid-modifying enzymes of other sources in a heterologous host Escherichia coli. The modular lycopene pathway synthesized an unexpected carotenoid structure, 3,4-didehydrolycopene, as well as lycopene. Extension of the novel 3,4-didehydrolycopene pathway with the mutant Pantoea lycopene cyclase CrtY(2) and the Rhodobacter spheroidene monooxygenase CrtA generated monocyclic torulene and acyclic oxocarotenoids, respectively. The reconstructed beta-carotene pathway synthesized an unexpected 7,8-dihydro-beta-carotene in addition to beta-carotene. Extension of the beta-carotene pathway with the B. linens beta-ring desaturase CrtU and Pantoea beta-carotene hydroxylase CrtZ generated asymmetric carotenoid agelaxanthin A, which had one aromatic ring at the one end of carotene backbone and one hydroxyl group at the other end, as well as aromatic carotenoid isorenieratene and dihydroxy carotenoid zeaxanthin. These results demonstrate that reconstruction of the biosynthetic pathways and extension with promiscuous enzymes in a heterologous host holds promise as a rational strategy for generating structurally diverse compounds that are hardly accessible in nature.

Directed Metabolic Pathway Evolution Enables Functional Pterin-Dependent Aromatic-Amino-Acid Hydroxylation in Escherichia coli.

Tetrahydrobiopterin-dependent hydroxylation of aromatic amino acids is the first step in the biosynthesis of many neuroactive compounds in humans. A fundamental challenge in building these pathways in Escherichia coli is the provision of the non-native hydroxylase cofactor, tetrahydrobiopterin. To solve this, we designed a genetic selection that relies on the tyrosine synthesis activity of phenylalanine hydroxylase. Using adaptive laboratory evolution, we demonstrate the use of this selection to discover: (1) a minimum set of heterologous enzymes and a host folE (T198I) mutation for achieving this type of hydroxylation chemistry in whole cells, (2) functional complementation of tetrahydrobiopterin by indigenous cofactors, and (3) a tryptophan hydroxylase mutation for improving protein abundance. Thus, the goal of having functional aromatic-amino-acid hydroxylation in E. coli was achieved through directed metabolic pathway evolution.

Standardized Cloning and Curing of Plasmids.

Plasmids are highly useful tools for studying living cells and for heterologous expression of genes and pathways in cell factories. Standardized tools and operating procedures for handling such DNA vectors are core principles in synthetic biology. Here, we describe protocols for molecular cloning and exchange of genetic parts in the Standard European Vectors Architecture (SEVA) vector system. Additionally, to facilitate rapid testing and iterative bioengineering using different vector designs, we provide a one-step protocol for a universal CRISPR-Cas9-based plasmid curing system (pFREE) and demonstrate the application of this system to cure SEVA constructs (all vectors are available at SEVA/Addgene).

SEVA Linkers: A Versatile and Automatable DNA Backbone Exchange Standard for Synthetic Biology.

DNA vectors serve to maintain and select recombinant DNA in cell factories, and as design complexity increases, there is a greater need for well-characterized parts and methods for their assembly. Standards in synthetic biology are top priority, but standardizing molecular cloning contrasts flexibility, and different researchers prefer and master different molecular technologies. Here, we describe a new, highly versatile and automatable standard "SEVA linkers" for vector exchange. SEVA linkers enable backbone swapping with 20 combinations of classical enzymatic restriction/ligation, Gibson isothermal assembly, uracil excision cloning, and a nicking enzyme-based methodology we term SEVA cloning. SEVA cloning is a simplistic one-tube protocol for backbone swapping directly from plasmid stock solutions. We demonstrate the different performance of 30 plasmid backbones for small molecule and protein production and obtain more than 10-fold improvement from a four-gene biosynthetic pathway and 430-fold improvement with a difficult-to-express membrane protein. The standardized linkers and protocols add to the Standard European Vectors Architecture (SEVA) resource and are freely available to the synthetic biology community.

Accurate DNA Assembly and Genome Engineering with Optimized Uracil Excision Cloning.

Simple and reliable DNA editing by uracil excision (a.k.a. USER cloning) has been described by several research groups, but the optimal design of cohesive DNA ends for multigene assembly remains elusive. Here, we use two model constructs based on expression of gfp and a four-gene pathway that produces ß-carotene to optimize assembly junctions and the uracil excision protocol. By combining uracil excision cloning with a genomic integration technology, we demonstrate that up to six DNA fragments can be assembled in a one-tube reaction for direct genome integration with high accuracy, greatly facilitating the advanced engineering of robust cell factories.

Construction of homologous and heterologous synthetic sucrose utilizing modules and their application for carotenoid production in recombinant Escherichia coli.

Sucrose is one of the most promising carbon sources for industrial fermentation. We expressed synthetic modules expressing genes of the PEP-PTS and non-PTS pathways in Escherichia coli K12 for comparison. We selected PEP-PTS pathway genes of Lactobacillus plantarum and Staphylococcus xylosus and non-PTS pathway genes of sucrose-utilizing (Scr(+)) E. coli EC3132. Switchable Scr(+) modules expressing E. coli EC3132 non-PTS genes conferred better sucrose-utilizing ability on Scr(-)E. coli K12 than E. coli EC3132. Scr(+) modules expressing S. xylosus PEP-PTS genes conferred a sucrose-utilizing ability on E. coli K12. Among L. plantarum PEP-PTS genes, SacA(LP) and SacK(LP) were functional in E. coli K12. CscA(EC)-CscB(EC)-CscK(EC) (non-PEP-PTS module) or ScrA(SX)-SacA(LP)-SacK(LP) (PEP-PTS module) was introduced to a diapolycopene-producing E. coli strain. In both Scr(+)E. coli K12, the sucrose-utilizing ability of the modules was not affected by diapolycopene formation, indicating that the modular Scr(+) systems could be employed for developing sustainable bioprocesses using sucrose.