Squalene is a precursor of thousands of bioactive triterpenoids and also has industrial value as a lubricant, health-promoting agent, and/or drop-in biofuel. To establish an efficient Escherichia coli-based system for squalene production, we tested two different squalene synthases and their mutants in combination with precursor pathways. By co-expressing a chimeric mevalonate pathway with human or Thermosynechococcus squalene synthase, E. coli accumulated squalene up to 230 mg/L or 55 mg/g-DCW in flask culture. We also determined that a significant truncation of squalene synthase at the C-terminus retains partial cellular activity. The squalene-producing strain described herein represents a convenient platform for gene discovery and the construction of the pathway toward natural and non-natural hopanoids/steroids.

Synthetic biology aspires to construct natural and non-natural pathways to useful compounds. However, pathways that rely on multiple promiscuous enzymes may branch, which might preclude selective production of the target compound. Here, we describe the assembly of a six-enzyme pathway in Escherichia coli for the synthesis of C50-astaxanthin, a non-natural purple carotenoid. We show that by judicious matching of engineered size-selectivity variants of the first two enzymes in the pathway, farnesyl diphosphate synthase (FDS) and carotenoid synthase (CrtM), branching and the production of non-target compounds can be suppressed, enriching the proportion of C50 backbones produced. We then further extend the C50 pathway using evolved or wild-type downstream enzymes. Despite not containing any substrate- or product-specific enzymes, the resulting pathway detectably produces only C50 carotenoids, including ∼90% C50-astaxanthin. Using this approach, highly selective pathways can be engineered without developing absolutely specific enzymes.

Terpene synthases catalyze the formation of a variety of terpene chemical structures. Systematic mutagenesis studies have been effective in providing insights into the characteristic and complex mechanisms of C-C bond formations and in exploring the enzymatic potential for inventing new chemical structures. In addition, there is growing demand to increase terpene synthase activity in heterologous hosts, given the maturation of metabolic engineering and host breeding for terpenoid synthesis. We have developed a simple screening method for the cellular activities of terpene synthases by scoring their substrate consumption based on the color loss of the cell harboring carotenoid pathways. We demonstrate that this method can be used to detect activities of various terpene synthase or prenyltransferase genes in a high-throughput manner, irrespective of the product type, enabling the mutation analysis and directed evolution of terpene synthases. We also report the possibility for substrate-specific screening system of terpene synthases by taking advantage of the substrate-size specificity of C30 and C40 carotenoid pathways.

Capsanthin, a characteristic red carotenoid found in the fruits of red pepper (Capsicum annuum), is widely consumed as a food and a functional coloring additive. An enzyme catalyzing capsanthin synthesis was identified as capsanthin/capsorubin synthase (CCS) in the 1990s, but no microbial production of capsanthin has been reported. We report here the first successful attempt to biosynthesize capsanthin in Escherichia coli by carotenoid-pathway engineering. Our initial attempt to coexpress eight enzyme genes required for capsanthin biosynthesis did not detect the desired product. The dual activity of CCS as a lycopene β-cyclase as well as a capsanthin/capsorubin synthase likely complicated the task. We demonstrated that a particularly high expression level of the CCS gene and the minimization of byproducts by regulating the seven upstream carotenogenic genes were crucial for capsanthin formation in E. coli. Our results provide a platform for further study of CCS activity and capsanthin production in microorganisms.

a b s t r a c tThe first committed steps of steroid/hopanoid pathways involve squalene synthase (SQS). Here, we report the Escherichia coli production of diaponeurosporene and diapolycopene, yellow C 30 carotenoid pigments, by expressing human SQS and Staphylococcus aureus dehydrosqualene (C 30 carotenoid) desaturase (CrtN). We suggest that the carotenoid pigments are synthesized mainly via the desaturation of squalene rather than the direct synthesis of dehydrosqualene through the nonreductive condensation of prenyl diphosphate precursors, indicating the possible existence of a ''squalene route'' and a ''lycopersene route'' for C 30 and C 40 carotenoids, respectively. Additionally, this finding yields a new method of colorimetric screening for the cellular activity of squalene synthases, which are major targets for cholesterol-lowering drugs.

LuxR family transcriptional regulators are the core components of quorum sensing in Gram-negative bacteria and exert their effects through binding to the signaling molecules acyl-homoserine lactones (acyl-HSLs). The function of the LuxR homologs is remarkably plastic, and naturally occurring acyl-HSLs are structurally diverse. To investigate the molecular basis of the functional plasticity of Vibrio fischeri LuxR, we directed the evolution of LuxR toward three different specificities in the laboratory. We found an orthogonal pair of LuxR mutants specific either to 3-oxo-hexanoyl homoserine lactone or to 3-oxo-octanoyl homoserine lactone. Interestingly, the majority of the specificity changes did not arise from modulating the recognition event but rather from changing the efficiency of the transition from the inactive form to the active form upon signal binding. This finding explains how quorum sensing systems can rapidly diverge in nature and in the laboratory and how signal orthogonality and mutual inhibition frequently occur among closely related diverging systems.

Tumor-selective contrast agents have the potential to aid in the diagnosis and treatment of cancer using noninvasive imaging modalities such as magnetic resonance imaging (MRI). Such contrast agents can consist of magnetic nanoparticles incorporating functionalities that respond to cues specific to tumor environments. Genetically engineering magnetotactic bacteria to display peptides has been investigated as a means to produce contrast agents that combine the robust image contrast effects of magnetosomes with the transgenic-targeting peptides displayed on their surface. This work reports the first use of magnetic nanoparticles that display genetically encoded pH low insertion peptide (pHLIP), a long peptide intended to enhance MRI contrast by targeting the extracellular acidity associated with the tumors. To demonstrate the modularity of this versatile platform to incorporate diverse targeting ligands by genetic engineering, we also incorporated the cyclic αv integrin-binding peptide iRGD into separate magnetosomes. Specifically, we investigate their potential for enhanced binding and tumor imaging both in vitro and in vivo. Our experiments indicate that these tailored magnetosomes retain their magnetic properties, making them well suited as T2 contrast agents, while exhibiting an increased binding compared to the binding in wild-type magnetosomes.

a b s t r a c tSqualene synthase (SQS) catalyzes the first step of sterol/hopanoid biosynthesis in various organisms. It has been long recognized that SQSs share a common ancestor with carotenoid synthases, but it is not known how these enzymes selectively produce their own product. In this study, SQSs from yeast, human, and bacteria were independently subjected to directed evolution for the production of the C 30 carotenoid backbone, dehydrosqualene. This was accomplished via high-throughput screening with Pantoea ananatis phytoene desaturase, which can selectively convert dehydrosqualene into yellow carotenoid pigments. Genetic analysis of the resultant mutants revealed various mutations that could effectively convert SQS into a ''dehydrosqualene synthase.'' All of these mutations are clustered around the residues that have been proposed to be important for NADPH binding.