Metabolic Engineering Tools Research Articles

Development of Artificial Riboswitches for Monitoring of Naringenin In Vivo.

Microbial strains are considered promising hosts for production of flavonoids because of their rapid growth rate and suitability for large-scale manufacturing. However, productivity and titer of current recombinant strains still do not meet the requirements of industrial processes. Genetically encoded biosensors have been applied for high-throughput screening or dynamic regulation of biosynthetic pathways for enhancing the performance of microbial strains that produce valuable chemicals. Currently, few protein sensor-regulators for flavonoids exist. Unlike the protein-based trans-regulating controllers, riboswitches can respond to their ligands faster and eliminate off-target effects. Here, we developed artificial riboswitches that activate gene expression in response to naringenin, an important flavonoid. RNA aptamers for naringenin were developed using SELEX and cloned upstream of a dual selectable marker gene to construct a riboswitch library. Two in vivo selection routes were applied separately to the library by supplementing naringenin at two different concentrations during enrichments to modulate the operational ranges of the riboswitches. The selected riboswitches were responsive to naringenin and activated gene expression up to 2.91-fold. Operational ranges of the riboswitches were distinguished on the basis of their selection route. Additionally, the specificity of the riboswitches was assessed, and their applicability as dynamic regulators was confirmed. Collectively, the naringenin riboswitches reported in this work will be valuable tools in metabolic engineering of microorganisms for the production of flavonoids.

A WRKY transcription factor from Withania somnifera regulates triterpenoid withanolide accumulation and biotic stress tolerance through modulation of phytosterol and defense pathways.

Withania somnifera produces pharmacologically important triterpenoid withanolides that are derived via phytosterol pathway; however, their biosynthesis and regulation remain to be elucidated. A jasmonate- and salicin-inducible WRKY transcription factor from W.somnifera (WsWRKY1) exhibiting correlation with withaferin A accumulation was functionally characterized employing virus-induced gene silencing and overexpression studies combined with transcript and metabolite analyses, and chromatin immunoprecipitation assay. WsWRKY1 silencing resulted in stunted plant growth, reduced transcripts of phytosterol pathway genes with corresponding reduction in phytosterols and withanolides in W.somnifera. Its overexpression elevated the biosynthesis of triterpenoids in W.somnifera (phytosterols and withanolides), as well as tobacco and tomato (phytosterols). Moreover, WsWRKY1 binds to W-box sequences in promoters of W.somnifera genes encoding squalene synthase and squalene epoxidase, indicating its direct regulation of triterpenoid pathway. Furthermore, while WsWRKY1 silencing in W.somnifera compromised the tolerance to bacterial growth, fungal infection, and insect feeding, its overexpression in tobacco led to improved biotic stress tolerance. Together these findings demonstrate that WsWRKY1 has a positive regulatory role on phytosterol and withanolides biosynthesis, and defense against biotic stress, highlighting its importance as a metabolic engineering tool for simultaneous improvement of triterpenoid biosynthesis and plant defense.

Assessing and Resolving Model Misspecifications in Metabolic Flux Analysis.

Metabolic flux analysis (MFA) is an indispensable tool in metabolic engineering. The simplest variant of MFA relies on an overdetermined stoichiometric model of the cell’s metabolism under the pseudo-steady state assumption to evaluate the intracellular flux distribution. Despite its long history, the issue of model error in overdetermined MFA, particularly misspecifications of the stoichiometric matrix, has not received much attention. We evaluated the performance of statistical tests from linear least square regressions, namely Ramsey’s Regression Equation Specification Error Test (RESET), the F-test, and the Lagrange multiplier test, in detecting model misspecifications in the overdetermined MFA, particularly missing reactions. We further proposed an iterative procedure using the F-test to correct such an issue. Using Chinese hamster ovary and random metabolic networks, we demonstrated that: (1) a statistically significant regression does not guarantee high accuracy of the flux estimates; (2) the removal of a reaction with a low flux magnitude can cause disproportionately large biases in the flux estimates; (3) the F-test could efficiently detect missing reactions; and (4) the proposed iterative procedure could robustly resolve the omission of reactions. Our work demonstrated that statistical analysis and tests could be used to systematically assess, detect, and resolve model misspecifications in the overdetermined MFA.

Metabolic engineering of microalgae: a review and future prospects

Microalgae are widely distributed in nature, which can produce lipids, pigments, polysaccharides, as well as other high-value products through cell metabolism. However, low product content and high cost of cell harvesting negatively impact the practical application of microalgae. To utilize microalgal resources more effectively, it is necessary to modify the microalgae strains with genetic modification technologies for producing specific metabolites efficiently. Genome editing, metabolic engineering and synthetic biology tools are being developed for accelerating the production of important metabolites to meet the growing demands of energy, food and pharmaceutical industries using microalgae. The recent progress of related studies is summarize in this review, with future prospects provided.

Synthetic Biology and Metabolic Engineering Approaches and Its Impact on Non-Conventional Yeast and Biofuel Production

The increasing fossil fuel scarcity has led to an urgent need to develop alternative fuels. One of the most promising alternatives to petroleum for the production of fuels is microbial production. Yeasts are highly efficient producer of bioethanol with several superior traits over bacterial counterparts. Tools of synthetic biology has revolutionised the field of microbial cell factories especially in the case of ethanol and fatty acid production. Era of yeast synthetic biology began with the well-studied industrial work horse Saccharomyces cerevisiae. Despite many highly beneficial traits like ethanol tolerance, thermotolerance, inhibitor tolerance, genetic diversity, non-conventional yeast has for synthetic biology, it currently lags behind Sachharomyces cerevisiae in the number of synthetic networks that have been described. Currently synthetic biology is slowly widening to the non-conventional yeasts like Hansenula polymorpha, Kluyveromyces lactis, Pichia pastoris and Yarrowia lipolytica. Here we review basic synthetic biology tools that we can apply to non-conventional yeasts. Moreover we discuss how metabolic engineering and synthetic biology tools can be applied in nonconventional yeasts for improved biofuel production.

Selection Finder (SelFi): A computational metabolic engineering tool to enable directed evolution of enzymes

Directed evolution of enzymes consists of an iterative process of creating mutant libraries and choosing desired phenotypes through screening or selection until the enzymatic activity reaches a desired goal. The biggest challenge in directed enzyme evolution is identifying high-throughput screens or selections to isolate the variant(s) with the desired property. We present in this paper a computational metabolic engineering framework, Selection Finder (SelFi), to construct a selection pathway from a desired enzymatic product to a cellular host and to couple the pathway with cell survival. We applied SelFi to construct selection pathways for four enzymes and their desired enzymatic products xylitol, D-ribulose-1,5-bisphosphate, methanol, and aniline. Two of the selection pathways identified by SelFi were previously experimentally validated for engineering Xylose Reductase and RuBisCO. Importantly, SelFi advances directed evolution of enzymes as there is currently no known generalized strategies or computational techniques for identifying high-throughput selections for engineering enzymes.

Deconstruction of complex protein signaling switches: a roadmap toward engineering higher-order gene regulators.

The control of gene expression is an important tool for metabolic engineering, the design of synthetic gene networks, gene-function analysis, and protein manufacturing. The most successful approaches to date are based on modulating messenger RNA (mRNA) synthesis via their inducible coupling to transcriptional effectors, which requires biosensing functionality. A hallmark of biological sensing is the conversion of an exogenous signal, usually a small molecule or environmental cue such as a protein-ligand interaction, into a useful output or response. One of the most utilized regulatory proteins is the lactose repressor (LacI). In this review we will (1) explore the mechanochemical structure-function relationship of LacI; (2) discuss how the physical attributes of LacI can be leveraged to identify and understand other regulatory proteins; (3) investigate the designability (tunability) of LacI; (4) discuss the potential of the modular design of novel regulatory proteins, fashioned after the topology and mechanochemical properties of LacI. WIREs Nanomed Nanobiotechnol 2017, 9:e1461. doi: 10.1002/wnan.1461 For further resources related to this article, please visit the WIREs website.

Competitive growth experiments with a high-lipid Chlamydomonas reinhardtii mutant strain and its wild-type to predict industrial and ecological risks

Key microalgal species are currently being exploited as biomanufacturing platforms using mass cultivation systems. The opportunities to enhance productivity levels or produce non-native compounds are increasing as genetic manipulation and metabolic engineering tools are rapidly advancing. Regardless of the end product, there are both environmental and industrial risks associated to open pond cultivation of mutant microalgal strains. A mutant escape could be detrimental to local biodiversity and increase the risk of algal blooms. Similarly, if the cultivation pond is invaded by a wild-type (WT) microalgae or the mutant reverts to WT phenotypes, productivity could be impacted. To investigate these potential risks, a response surface methodology was applied to determine the competitive outcome of two Chlamydomonas reinhardtii strains, a WT (CC-124) and a high-lipid accumulating mutant (CC-4333), grown in mixotrophic conditions, with differing levels of nitrogen and initial WT to mutant ratios. Results of the growth experiments show that mutant cells have double the exponential growth rate of the WT in monoculture. However, due to a slower transition from lag phase to exponential phase, mutant cells are outcompeted by the WT in every co-culture treatment. This suggests that, under the conditions tested, outdoor cultivation of the C. reinhardtii cell wall-deficient mutant strains does not carry a significant environmental risk to its WT in an escape scenario. Furthermore, lipid results show the mutant strain accumulates over 200% more TAGs per cell, at 50 mg L−1 NH4Cl, compared to the WT, therefore, the fragility of the mutant strain could impact on overall industrial productivity.

Development and implementation of rapid metabolic engineering tools for chemical and fuel production in Geobacillus thermoglucosidasius NCIMB 11955

BackgroundThe thermophile Geobacillus thermoglucosidasius has considerable attraction as a chassis for the production of chemicals and fuels. It utilises a wide range of sugars and oligosaccharides typical of those derived from lignocellulose and grows at elevated temperatures. The latter improves the rate of feed conversion, reduces fermentation cooling costs and minimises the risks of contamination. Full exploitation of its potential has been hindered by a dearth of effective gene tools.ResultsHere we designed and tested a collection of vectors (pMTL60000 series) in G. thermoglucosidasius NCIMB 11955 equivalent to the widely used clostridial pMTL80000 modular plasmid series. By combining a temperature-sensitive replicon and a heterologous pyrE gene from Geobacillus kaustophilus as a counter-selection marker, a highly effective and rapid gene knock-out/knock-in system was established. Its use required the initial creation of uracil auxotroph through deletion of pyrE using allele-coupled exchange (ACE) and selection for resistance to 5-fluoroorotic acid. The turnaround time for the construction of further mutants in this pyrE minus strain was typically 5 days. Following the creation of the desired mutant, the pyrE allele was restored to wild type, within 3 days, using ACE and selection for uracil prototrophy. Concomitant with this process, cargo DNA (pheB) could be readily integrated at the pyrE locus. The system’s utility was demonstrated through the generation in just 30 days of three independently engineered strains equivalent to a previously constructed ethanol production strain, TM242. This involved the creation of two in-frame deletions (ldh and pfl) and the replacement of a promoter region of a third gene (pdh) with an up-regulated variant. In no case did the production of ethanol match that of TM242. Genome sequencing of the parental strain, TM242, and constructed mutant derivatives suggested that NCIMB 11955 is prone to the emergence of random mutations which can dramatically affect phenotype.ConclusionsThe procedures and principles developed for clostridia, based on the use of pyrE alleles and ACE, may be readily deployed in G. thermoglucosidasius. Marker-less, in-frame deletion mutants can be rapidly generated in 5 days. However, ancillary mutations frequently arise, which can influence phenotype. This observation emphasises the need for improved screening and selection procedures at each step of the engineering processes, based on the generation of multiple, independent strains and whole-genome sequencing.

TowardGenome-Based Metabolic Engineering in Bacteria.

Prokaryotes modified stably on the genome are of great importance for production of fine and commodity chemicals. Traditional methods for genome engineering have long suffered from imprecision and low efficiencies, making construction of suitable high-producer strains laborious. Here, we review the recent advances in discovery and refinement of molecular precision engineering tools for genome-based metabolic engineering in bacteria for chemical production, with focus on the λ-Red recombineering and the clustered regularly interspaced short palindromic repeats/Cas9 nuclease systems. In conjunction, they enable the integration of in vitro-synthesized DNA segments into specified locations on the chromosomeand allow for enrichment of rare mutants by elimination of unmodified wild-type cells. Combination with concurrently developing improvements in important accessory technologies such as DNA synthesis, high-throughput screening methods, regulatory element design, and metabolic pathway optimization tools has resulted in novel efficient microbial producer strainsand given access to new metabolic products. These new tools have made and will likely continue to make a big impact on the bioengineering strategies that transform the chemical industry.

Unique attributes of cyanobacterial metabolism revealed by improved genome-scale metabolic modeling and essential gene analysis.

The model cyanobacterium, Synechococcus elongatus PCC 7942, is a genetically tractable obligate phototroph that is being developed for the bioproduction of high-value chemicals. Genome-scale models (GEMs) have been successfully used to assess and engineer cellular metabolism; however, GEMs of phototrophic metabolism have been limited by the lack of experimental datasets for model validation and the challenges of incorporating photon uptake. Here, we develop a GEM of metabolism in S. elongatus using random barcode transposon site sequencing (RB-TnSeq) essential gene and physiological data specific to photoautotrophic metabolism. The model explicitly describes photon absorption and accounts for shading, resulting in the characteristic linear growth curve of photoautotrophs. GEM predictions of gene essentiality were compared with data obtained from recent dense-transposon mutagenesis experiments. This dataset allowed major improvements to the accuracy of the model. Furthermore, discrepancies between GEM predictions and the in vivo dataset revealed biological characteristics, such as the importance of a truncated, linear TCA pathway, low flux toward amino acid synthesis from photorespiration, and knowledge gaps within nucleotide metabolism. Coupling of strong experimental support and photoautotrophic modeling methods thus resulted in a highly accurate model of S. elongatus metabolism that highlights previously unknown areas of S. elongatus biology.

Developing a set of strong intronic promoters for robust metabolic engineering in oleaginous Rhodotorula (Rhodosporidium) yeast species.

BackgroundRed yeast species in the Rhodotorula/Rhodosporidium genus are outstanding producers of triacylglyceride and cell biomass. Metabolic engineering is expected to further enhance the productivity and versatility of these hosts for the production of biobased chemicals and fuels. Promoters with strong activity during oil-accumulation stage are critical tools for metabolic engineering of these oleaginous yeasts.ResultsThe upstream DNA sequences of 6 genes involved in lipid biosynthesis or accumulation in Rhodotorula toruloides were studied by luciferase reporter assay. The promoter of perilipin/lipid droplet protein 1 gene (LDP1) displayed much stronger activity (4–11 folds) than that of glyceraldehyde-3-phosphate dehydrogenase gene (GPD1), one of the strongest promoters known in yeasts. Depending on the stage of cultivation, promoter of acetyl-CoA carboxylase gene (ACC1) and fatty acid synthase β subunit gene (FAS1) exhibited intermediate strength, displaying 50–160 and 20–90% levels of GPD1 promoter, respectively. Interestingly, introns significantly modulated promoter strength at high frequency. The incorporation of intron 1 and 2 of LDP1 (LDP1in promoter) enhanced its promoter activity by 1.6–3.0 folds. Similarly, the strength of ACC1 promoter was enhanced by 1.5–3.2 folds if containing intron 1. The intron 1 sequences of ACL1 and FAS1 also played significant regulatory roles. When driven by the intronic promoters of ACC1 and LDP1 (ACC1in and LDP1in promoter, respectively), the reporter gene expression were up-regulated by nitrogen starvation, independent of de novo oil biosynthesis and accumulation. As a proof of principle, overexpression of the endogenous acyl-CoA-dependent diacylglycerol acyltransferase 1 gene (DGA1) by LDP1in promoter was significantly more efficient than GPD1 promoter in enhancing lipid accumulation.ConclusionIntronic sequences play an important role in regulating gene expression in R. toruloides. Three intronic promoters, LDP1in, ACC1in and FAS1in, are excellent promoters for metabolic engineering in the oleaginous and carotenogenic yeast, R. toruloides. Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0600-x) contains supplementary material, which is available to authorized users.

Construction, properties, and application of the pCB5 plasmid, a novel conjugative shuttle vector with a Cupriavidus basilensis origin of replication.

Cupriavidus basilensis is a species with diverse metabolic capabilities, including degradation of xenobiotics and heavy metal resistance. Although the genomes of several strains of this species have been sequenced, no plasmid has yet been constructed for genetic engineering in this species. In this study, we identified a novel plasmid, designated pWS, from C. basilensis WS with a copy number of 1-3 per cell and a length of 2150bp. pWS contained three protein-coding genes, among which only rep was required for plasmid replication. Rep showed no homology with known plasmid replication initiators. Unlike most plasmids, pWS did not have a cis-acting replication origin outside the region of rep. The minimal replicon of pWS was stable in C. basilensis WS without selection. A conjugative C. basilensis/Escherichia coli shuttle vector, pCB5, was constructed using the minimal replicon of pWS. Interestingly, the copy number of pCB5 was flexible and could be manipulated. Enhancing the expression level of Rep in pCB5 by either doubling the promoter or coding region of rep resulted in doubling of the plasmid copy number. Moreover, replacing the native promoter of rep with the lac promoter increased the copy number by over fivefold. Finally, using two different β-galactosidase reporting systems constructed with pCB5, we successfully demonstrated the different regulatory patterns of bph and dmp operons during diphenyl ether (DE) degradation in C. basilensis WS. Thus, this shuttle vector provided an efficient tool for DNA cloning and metabolic engineering in C. basilensis.

An auto-inducible Escherichia coli strain obtained by adaptive laboratory evolution for fatty acid synthesis from ionic liquid-treated bamboo hydrolysate

Adaptive laboratory evolution (ALE) is a useful metabolic engineering strategy, which allows the selection of the microorganisms with beneficial phenotype through accumulative beneficial mutations among genetic variations occurrencely. Following ALE strategy, a rational constructed Escherichia coli strain DQ101 for fatty acids synthesis was adaptively evolved for 90days with increasing [C4mim]Cl concentration from 1% to 7% (w/v). The evolved strain DQ102 reached a final OD600 of 4.93 at the end of the 24h culture with 7% (w/v) ionic liquid. DQ102/pDQTES with a thioesterase ‘TesA overexpression could produce 1.12g/L fatty acid with a productivity of 0.023g/L-h from ionic liquid-treated bamboo hydrolysate. With another β-hydroxyacyl-ACP dehydratases (fabZ) overexpression, DQ102/pDQTESZ could reach a higher concentration of 2.29g/L with a productivity of 0.048g/L-h. These results indicated that ALE could be implemented as a useful tool for metabolic engineering and production of bio-fuels, as well as commodity and specialty chemicals.

High-Throughput Screening of Coenzyme Preference Change of Thermophilic 6-Phosphogluconate Dehydrogenase from NADP+ to NAD+

Coenzyme engineering that changes NAD(P) selectivity of redox enzymes is an important tool in metabolic engineering, synthetic biology, and biocatalysis. Here we developed a high throughput screening method to identify mutants of 6-phosphogluconate dehydrogenase (6PGDH) from a thermophilic bacterium Moorella thermoacetica with reversed coenzyme selectivity from NADP+ to NAD+. Colonies of a 6PGDH mutant library growing on the agar plates were treated by heat to minimize the background noise, that is, the deactivation of intracellular dehydrogenases, degradation of inherent NAD(P)H, and disruption of cell membrane. The melted agarose solution containing a redox dye tetranitroblue tetrazolium (TNBT), phenazine methosulfate (PMS), NAD+, and 6-phosphogluconate was carefully poured on colonies, forming a second semi-solid layer. More active 6PGDH mutants were examined via an enzyme-linked TNBT-PMS colorimetric assay. Positive mutants were recovered by direct extraction of plasmid from dead cell colonies followed by plasmid transformation into E. coli TOP10. By utilizing this double-layer screening method, six positive mutants were obtained from two-round saturation mutagenesis. The best mutant 6PGDH A30D/R31I/T32I exhibited a 4,278-fold reversal of coenzyme selectivity from NADP+ to NAD+. This screening method could be widely used to detect numerous redox enzymes, particularly for thermophilic ones, which can generate NAD(P)H reacted with the redox dye TNBT.

Affibody Scaffolds Improve Sesquiterpene Production in Saccharomyces cerevisiae.

Enzyme fusions have been widely used as a tool in metabolic engineering to increase pathway efficiency by reducing substrate loss and accumulation of toxic intermediates. Alternatively, enzymes can be colocalized through attachment to a synthetic scaffold via noncovalent interactions. Here we describe the use of affibodies for enzyme tagging and scaffolding. The scaffolding is based on the recognition of affibodies to their anti-idiotypic partners in vivo, and was first employed for colocalization of farnesyl diphosphate synthase and farnesene synthase in S.cerevisiae. Different parameters were modulated to improve the system, and the enzyme:scaffold ratio was most critical for its functionality. Ultimately, the yield of farnesene on glucose YSFar could be improved by 135% in fed-batch cultivations using a 2-site affibody scaffold. The scaffolding strategy was then extended to a three-enzyme polyhydroxybutyrate (PHB) pathway, heterologously expressed in E.coli. Within a narrow range of enzyme and scaffold induction, the affibody tagging and scaffolding increased PHB production 7-fold. This work demonstrates how the versatile affibody can be used for metabolic engineering purposes.

Efficient gene knockdown in Clostridium acetobutylicum by synthetic small regulatory RNAs.

Clostridium is considered a promising microbial host for the production of valuable industrial chemicals. However, Clostridium is notorious for the difficulty of genetic manipulations, and consequently metabolic engineering. Thus, much effort has been exerted to develop novel tools for genetic and metabolic engineering of Clostridium strains. Here, we report the development of a synthetic small regulatory RNA (sRNA)-based system for controlled gene expression in Clostridium acetobutylicum, consisting of a target recognition site, MicC sRNA scaffold, and an RNA chaperone Hfq. To examine the functional operation of sRNA system in C. acetobutylicum, expression control was first examined with the Evoglow fluorescent protein as a model protein. Initially, a C. acetobutylicum protein annotated as Hfq was combined with the synthetic sRNA based on the Escherichia coli MicC scaffold to knockdown Evoglow expression. However, C. acetobutylicum Hfq did not bind to E. coli MicC, while MicC scaffold-based synthetic sRNA itself was able to knockdown the expression of Evoglow. When E. coli hfq gene was introduced, the knockdown efficiency assessed by measuring fluorescence intensity, could be much enhanced. Then, this E. coli MicC scaffold-Hfq system was used to knock down adhE1 gene expression in C. acetobutylicum. Knocking down the adhE1 gene expression using the synthetic sRNA led to a 40% decrease in butanol production (2.5 g/L), compared to that (4.5 g/L) produced by the wild-type strain harboring an empty vector. The sRNA system was further extended to knock down the pta gene expression in the buk mutant C. acetobutylicum strain PJC4BK for enhanced butanol production. The PJC4BK (pPta-HfqEco ) strain, which has the pta gene expression knocked down, was able to produce 16.9 g/L of butanol, which is higher than that (14.9 g/L) produced by the PJC4BK strain, mainly due to reduced acetic acid production. Fed-batch culture of PJC4BK (pPta-HfqEco ) strain coupled with in situ gas stripping produced 105.5 g of total solvents (70.7 g butanol, 20.5 g acetone, and 14.3 g ethanol), demonstrating that the sRNA-based engineered C. acetobutylicum strain can be cultured without instability. The synthetic sRNA system reported in this study will be useful for more efficient development of engineered C. acetobutylicum strains capable of producing valuable chemicals and fuels. Biotechnol. Bioeng. 2017;114: 374-383. © 2016 Wiley Periodicals, Inc.

A general post-translational tool for metabolic engineering.

A general post-translational tool for metabolic engineering.

CRISPR interference as a titratable, trans-acting regulatory tool for metabolic engineering in the cyanobacterium Synechococcus sp. strain PCC 7002

Trans-acting regulators provide novel opportunities to study essential genes and regulate metabolic pathways. We have adapted the clustered regularly interspersed palindromic repeats (CRISPR) system from Streptococcus pyogenes to repress genes in trans in the cyanobacterium Synechococcus sp. strain PCC 7002 (hereafter PCC 7002). With this approach, termed CRISPR interference (CRISPRi), transcription of a specific target sequence is repressed by a catalytically inactive Cas9 protein recruited to the target DNA by base-pair interactions with a single guide RNA that is complementary to the target sequence. We adapted this system for PCC 7002 and achieved conditional and titratable repression of a heterologous reporter gene, yellow fluorescent protein. Next, we demonstrated the utility of finely tuning native gene expression by downregulating the abundance of phycobillisomes. In addition, we created a conditional auxotroph by repressing synthesis of the carboxysome, an essential component of the carbon concentrating mechanism cyanobacteria use to fix atmospheric CO2. Lastly, we demonstrated a novel strategy for increasing central carbon flux by conditionally downregulating a key node in nitrogen assimilation. The resulting cells produced 2-fold more lactate than a baseline engineered cell line, representing the highest photosynthetically generated productivity to date. This work is the first example of titratable repression in cyanobacteria using CRISPRi, enabling dynamic regulation of essential processes and manipulation of flux through central carbon metabolism. This tool facilitates the study of essential genes of unknown function and enables groundbreaking metabolic engineering capability, by providing a straightforward approach to redirect metabolism and carbon flux in the production of high-value chemicals.

Combinatorial metabolic pathway assembly in the yeast genome with RNA-guided Cas9.

The yeast Saccharomyces cerevisiae is an important industrial platform for the production of grain and cellulosic ethanol, isobutanol, butanediol, isoprenoids, and other chemicals. The construction of a successful production strain usually involves multiple gene knockouts and chromosomal integration of expression cassettes to redirect the metabolic fluxes for the conversion of sugars and other feed stocks into the desired product. RNA-guided Cas9 based genome editing has been demonstrated in many prokaryotic and eukaryotic hosts including S. cerevisiae, in which it has been additionally exploited as a tool for metabolic engineering. To extend the utilization of RNA-guided Cas9 as a metabolic pathway building tool, we demonstrated the direct assembly and chromosomal integration of up to 17 overlapping DNA fragments encoding the beta-carotene biosynthetic pathway. Furthermore, we generated a combinatorial strain library for the beta-carotene biosynthetic pathway, directly integrated into the yeast genome to create a diverse library of strains. This enabled the screening of combinatorial libraries in stable chromosomally integrated strains for rapid improvements of product titers. This combinatorial approach for pathway assembly will significantly accelerate the current speed of metabolic engineering for S. cerevisiae as an industrial platform, and increase the number of strains that can be simultaneously evaluated for enzyme screening, expression optimization and protein engineering to achieve the titer, rate and yield necessary for the commercialization of new industrial fermentation products.