Iterative genome editing of Escherichia coli for 3-hydroxypropionic acid production.

Synthetic biology requires strategies for the targeted, efficient, and combinatorial engineering of biological sub-systems at the molecular level. Here, we report the use of the iterative CRISPR EnAbled Trackable genome Engineering (iCREATE) method for the rapid construction of combinatorially modified genomes. We coupled this genome engineering strategy with high-throughput phenotypic screening and selections to recursively engineer multiple traits in Escherichia coli for improved production of the platform chemical 3-hydroxypropionic acid (3HP). Specifically, we engineered i) central carbon metabolism, ii) 3HP synthesis, and (iii) 3HP tolerance through design, construction and testing of ~ 162,000 mutations across 115 genes spanning global regulators, transcription factors, and enzymes involved in 3HP synthesis and tolerance. The iCREATE process required ~ 1 month to perform 13 rounds of combinatorial genome modifications with targeted gene knockouts, expression modification by ribosomal binding site (RBS) engineering, and genome-level site-saturation mutagenesis. Specific mutants conferring increased 3HP titer, yield, and productivity were identified and then combined to produce 3HP at a yield and concentration ~ 60-fold higher than the wild-type strain.

Combinatorial engineering of mevalonate pathway for improved amorpha-4,11-diene production in budding yeast.

Combinatorial genome integration of mevalonate pathway genes was performed with the aim of optimizing the metabolic flux for improved production of terpenoids in budding yeast. In the present study, we developed a novel δ-integration platform to achieve multiple genome integrations through modulating the concentration of antibiotics. By exploiting carotenoid biosynthesis as screening module, we successfully created a library of yeast colonies appeared with various intensities of orange color. As proof-of-concept that carotenoid overproducers could serve to boost the titer of other terpenoids, we further tested engineered strains for the production of amorpha-4,11-diene, an important precursor for antimalarial drug. However, we experienced some limitations of the carotenoid-based screening approach as it was only effective in detecting a small range of pathway activity improvement and further increasing mevalonate pathway activity led to a decreased orange color. By far, we were only able to obtain one mutant strain yielded more than 13-fold amorpha-4,11-diene over parental strains, which was approximately 64 mg/L of caryophyllene equivalents. Further qPCR studies confirmed that erg10, erg13, thmg1 and erg12 involved in mevalonate pathway were overexpressed in this mutant strain. We envision the current δ-integration platform would form the basis of a generalized technique for multiple gene integrations in yeast-a method that would be of significant interest to the metabolic engineering community.

GOLDBAR: A Framework for Combinatorial Biological Design.

With the rise of new DNA part libraries and technologies for assembling DNA, synthetic biologists are increasingly constructing and screening combinatorial libraries to optimize their biological designs. As combinatorial libraries are used to generate data on design performance, new rules for composing biological designs will emerge. Most formal frameworks for combinatorial design, however, do not yet support formal comparison of design composition, which is needed to facilitate automated analysis and machine learning in massive biological design spaces. To address this need, we introduce a combinatorial design framework called GOLDBAR. Compared with existing frameworks, GOLDBAR enables synthetic biologists to intersect and merge the rules for entire classes of biological designs to extract common design motifs and infer new ones. Here, we demonstrate the application of GOLDBAR to refine/validate design spaces for TetR-homologue transcriptional logic circuits, verify the assembly of a partial nif gene cluster, and infer novel gene clusters for the biosynthesis of rebeccamycin. We also discuss how GOLDBAR could be used to facilitate grammar-based machine learning in synthetic biology.

Modular Metabolic Engineering and Synthetic Coculture Strategies for the Production of Aromatic Compounds in Yeast.

Microbial-derived aromatics provide a sustainable and renewable alternative to petroleum-derived chemicals. In this study, we used the model yeast Saccharomyces cerevisiae to produce aromatic molecules by exploiting the concept of modularity in synthetic biology. Three different modular approaches were investigated for the production of the valuable fragrance raspberry ketone (RK), found in raspberry fruits and mostly produced from petrochemicals. The first strategy used was modular cloning, which enabled the generation of combinatorial libraries of promoters to optimize the expression level of the genes involved in the synthesis pathway of RK. The second strategy was modular pathway engineering and involved the creation of four modules, one for product formation: RK synthesis module (Mod. RK); and three for precursor synthesis: aromatic amino acid synthesis module (Mod. Aro), p-coumaric acid synthesis module (Mod. p-CA), and malonyl-CoA synthesis module (Mod. M-CoA). The production of RK by combinations of the expression of these modules was studied, and the best engineered strain produced 63.5 mg/L RK from glucose, which is the highest production described in yeast, and 2.1 mg RK/g glucose, which is the highest yield reported in any organism without p-coumaric acid supplementation. The third strategy was the use of modular cocultures to explore the effects of division of labor on RK production. Two two-member communities and one three-member community were created, and their production capacity was highly dependent on the structure of the synthetic community, the inoculation ratio, and the culture media. In certain conditions, the cocultures outperformed their monoculture controls for RK production, although this was not the norm. Interestingly, the cocultures showed up to 7.5-fold increase and 308.4 mg/L of 4-hydroxy benzalacetone, the direct precursor of RK, which can be used for the semi-synthesis of RK. This study illustrates the utility of modularity in synthetic biology tools and their applications to the synthesis of products of industrial interest.

Genome-based rational engineering of Actinoplanes deccanensis for improving fidaxomicin production and genetic stability.

Microbial fermentation is currently still the major way to produce structural complicated clinical drugs. Yet, the low productivity and genetic instability of producing strains remain the bottlenecks in microbial pharmaceutical industry. Fidaxomicin is a microbial drug against the Clostridium difficile infection. Here, a genome-based combinatorial engineering strategy was established to improve both fidaxomicin production and the genetic stability of Actinoplanes deccanensis YP-1. Guided by genomic analysis, several genetic instability-associated elements were cumulatively deleted, generating a more genetically stable mutant. Further rational engineering approaches including elimination of a pigment pathway, duplication of the fidaxomicin gene cluster, overexpression of a positive regulator and optimization of the fermentation medium, led to an overall 27-folds improvement in fidaxomicin production. Taken together, the genome-based rational combinatorial engineering strategy was efficient to enhance the fidaxomicin production and ameliorate the genetic stability of YP-1, it can also be widely used in other industrial actinomycetes for strain improvement.

Engineering of E. coli inherent fatty acid biosynthesis capacity to increase octanoic acid production.

BACKGROUND: As a versatile platform chemical, construction of microbial catalysts for free octanoic acid production from biorenewable feedstocks is a promising alternative to existing petroleum-based methods. However, the bio-production strategy has been restricted by the low capacity of E. coli inherent fatty acid biosynthesis. In this study, a combination of integrated computational and experimental approach was performed to manipulate the E. coli existing metabolic network, with the objective of improving bio-octanoic acid production. RESULTS: First, a customized OptForce methodology was run to predict a set of four genetic interventions required for production of octanoic acid at 90% of the theoretical yield. Subsequently, all the ten candidate proteins associated with the predicted interventions were regulated individually, as well as in contrast to the combination of interventions as suggested by the OptForce strategy. Among these enzymes, increased production of 3-hydroxy-acyl-ACP dehydratase (FabZ) resulted in the highest increase (+ 45%) in octanoic acid titer. But importantly, the combinatorial application of FabZ with the other interventions as suggested by OptForce further improved octanoic acid production, resulting in a high octanoic acid-producing E. coli strain +fabZ ΔfadE ΔfumAC ΔackA (TE10) (+ 61%). Optimization of TE10 expression, medium pH, and C:N ratio resulted in the identified strain producing 500 mg/L of C8 and 805 mg/L of total FAs, an 82 and 155% increase relative to wild-type MG1655 (TE10) in shake flasks. The best engineered strain produced with high selectivity (> 70%) and extracellularly (> 90%) up to 1 g/L free octanoic acid in minimal medium fed-batch culture. CONCLUSIONS: This work demonstrates the effectiveness of integration of computational strain design and experimental characterization as a starting point in rewiring metabolism for octanoic acid production. This result in conjunction with the results of other studies using OptForce in strain design demonstrates that this strategy may be also applicable to engineering E. coli for other customized bioproducts.

Synthetic Biology Approaches to New Bisindoles.

Bisindoles are a class of natural products derived from oxidative dimerization of tryptophan, and many of these molecules have potential use as anticancer agents. The recent isolation of new bisindoles and their corresponding gene clusters has greatly expanded the repertoire of biosynthetic genes available to synthetic biologists. This chapter describes methods to exploit the biosynthetic pathways leading to bisindoles, using cladoniamides as a representative example. Specifically, we describe how to identify and heterologously express gene clusters and how to manipulate pathways in order to generate new bisindoles. We also discuss methods for cultivating, extracting, purifying, and characterizing these new metabolites.

Expansion of bisindole biosynthetic pathways by combinatorial construction.

Cladoniamides are indolotryptoline natural products that derive from indolocarbazole precursors. Here, we present a microbial platform to artificially redirect the cladoniamide pathway to generate unnatural bisindoles for drug discovery. Specifically, we target glycosyltransferase, halogenase, and oxidoreductase genes from the phylogenetically related indolocarbazole rebeccamycin and staurosporine pathways. We generate a series of novel compounds, reveal details about the substrate specificities of a number of enzymes, and set the stage for future efforts to develop new catalysts and compounds by engineering of bisindole genes. The strategy for structural diversification we use here could furthermore be applied to other natural product families with known biosynthetic genes.

Combinatorial engineering for heterologous gene expression.

Tools for strain engineering with predictable outcome are of crucial importance for the nascent field of synthetic biology. The success of combining different DNA biological parts is often restricted by poorly understood factors deriving from the complexity of the systems. We have previously identified variants for different regulatory elements of the expression cassette XylS/Pm. When such elements are combined they act in a manner consistent with their individual behavior, as long as they affect different functions, such as transcription and translation. Interestingly, sequence context does not seem to influence the final outcome significantly. Expression of reporter gene bla could be increased up to 75 times at the protein level by combining three variants in one cassette. For other tested reporter genes similar results were obtained, except that the stimulatory effect was quantitatively less. Combination of individually characterized DNA parts thus stands as suitable method to achieve a desired phenotype.