The fluorescent protein iLOV as a reporter for screening of high-yield production of antimicrobial peptides in Pichia pastoris

The methylotrophic yeast Pichia pastoris is commonly used for the production of recombinant proteins at scale. The identification of an optimally overexpressing strain following transformation can be time and reagent consuming. Fluorescent reporters like GFP have been used to assist identification of superior producers, but their relatively big size, maturation requirements and narrow temperature range restrict their applications. Here, we introduce the use of iLOV, a flavin-based fluorescent protein, as a fluorescent marker to identify P. pastoris high-yielding strains easily and rapidly. The use of this fluorescent protein as a fusion partner is exemplified by the production of the antimicrobial peptide NI01, a difficult target to overexpress in its native form. iLOV fluorescence correlated well with protein expression level and copy number of the chromosomally integrated gene. An easy and simple medium-throughput plate-based screen directly following transformation is demonstrated for low complexity screening, while a high-throughput method using fluorescence-activated cell sorting (FACS) allowed for comprehensive library screening. Both codon optimization of the iLOV_NI01 fusion cassettes and different integration strategies into the P. pastoris genome were tested to produce and isolate a high-yielding strain. Checking the genetic stability, process reproducibility and following the purification of the active native peptide are eased by visualization of and efficient cleavage from the iLOV reporter. We show that this system can be used for expression and screening of several different antimicrobial peptides recombinantly produced in P. pastoris

Understanding and improving the fluorescent reporter iLOV for applications in biotechnology

Abstract not currently available

A Metabolically Integrated Lossen Rearrangement in Escherichia coli

Biocompatible chemistry enables abiotic reactions to be interfaced with metabolic pathways in living microorganisms. This includes both native and de novo biosynthetic processes to access abiotic feedstocks, intermediates and products in vivo. Herein we report a biocompatible Lossen rearrangement that is catalysed by phosphate in the bacterium Escherichia coli for the chemical transformation of activated acyl hydroxamates to primary amines in living cells. Using a para-carboxylated substrate, the biocompatible reaction can be used to generate the metabolite para-aminobenzoic acid (PABA) to rescue ∆pabA/B and ∆aroC auxotrophy and enable cell growth. The Lossen substrate can also be synthesised from polyethylene terephthalate (PET) and applied to whole-cell biocatalytic reactions and fermentations generating industrial small molecule products – including the analgesic and antipyretic drug paracetamol – paving the way for a general strategy to addict E. coli and other industrial chassis strains to PET plastic waste as a bioremediation strategy and for the upcycling of plastic waste using engineered biology. Together, this work showcases how non-enzymatic biocompatible reactions can be interfaced and cooperate with microbial metabolism to expand the available toolbox of metabolic chemistry for small molecule synthesis in native and engineered cellular systems

Two photon spectroscopy and microscopy of the fluorescent flavoprotein, iLOV

LOV-domains are ubiquitous photosensory proteins that are commonly re-engineered to serve as powerful and versatile fluorescent proteins and optogenetic tools. The photoactive, flavin chromophore, however, is excited using short wavelengths of light in the blue and UV regions, which have limited penetration into biological samples and can cause photodamage. Here, we have used non-linear spectroscopy and microscopy of the fluorescent protein, iLOV, to reveal that functional variants of LOV can be activated to great effect by two non-resonant photons of lower energy, near infrared light, not only in solution but also in biological samples. The two photon cross section of iLOV has a significantly blue-shifted S0 → S1 transition compared with the one photon absorption spectrum, suggesting preferential population of excited vibronic states. It is highly likely, therefore, that the two photon absorption wavelength of engineered, LOV-based tools is tuneable. We also demonstrate for the first time two photon imaging using iLOV in human epithelial kidney cells. Consequently, two photon absorption by engineered, flavin-based bio-molecular tools can enable non-invasive activation with high depth resolution and the potential for not only improved image clarity but also enhanced spatiotemporal control for optogenetic applications