| Alkanes are the simplest of organic compounds,consisting of only two elements,carbon and hydrogen,and are the main components of petrol,diesel and jet fuels,with excellent chemical and physical properties.Alkanes are mainly derived from the fractional distillation of fossil fuels such as oil and natural gas.With the rapid development of the global economy,the expanding demand for fossil fuels and the serious ecological problems caused by their use,such as the greenhouse effect,there is an urgent need to develop an alternative production process.With the rapid development of green energy,the use of microorganisms to produce alkanes has gradually become a hot topic of international attention and an important development direction.Microbial alkane production,as a renewable and clean energy source,is an ideal alternative to fossil fuels and does not cause environmental pollution after combustion.In recent years,it has been discovered that microorganisms primarily utilize the alkane synthesis pathway,known as aar-ado,derived from cyanobacteria.The enzymes AAR and ADO synthesized in this pathway can sequentially convert the intermediate metabolites of the fatty acid metabolic pathway,acyl-ACP,into fatty aldehydes and alkanes.In recent years,there have been studies on heterologous expression of these two enzymes,while knockout or overexpression of host bacteria fatty acid metabolism pathway related genes to improve the yield of alkanes.However,the yield of microbial synthesis of alkanes is still very low,which is difficult to meet the industrial demand.In addition,many current studies only focus on the modification of chassis cells,but due to the lack of high-throughput screening methods,few studies attempt to improve the production of alkanes by modifying the rate-limiting enzymes AAR and ADO for alkanes synthesis.In the process of using microbial cells to produce compounds,real-time and in situ monitoring of key metabolites plays a crucial role.Therefore,whole-cell biosensors capable of perceiving cellular metabolic network processes and target metabolite concentrations have great potential in the actual production of related compounds.Biological sensors mainly consist of responsive elements composed of biological active materials such as tissues,nucleic acids,and proteins,as well as reporting elements composed of transducers and converters.Compared to traditional mass spectrometry and isotope tracing methods,wholecell biosensors can detect metabolites in real-time and rapidly convert metabolite concentration information into easily detectable output signals,such as resistance,fluorescence,and growth phenotypes.Currently,whole-cell biosensors have been widely applied in the quantitative determination of metabolites,high-throughput screening,adaptive evolution,and metabolic regulation.Until now,two sets of alkane-responsive transcriptional regulatory systems(Alk R-Palk M in Acinetobacter baylyi ADP1 and Alk S-Palk B in Pseudomonas oleovorans)have beendocumented,and biosensors developed based on them have been widely used in environmental alkane detection.It is known that alkane biosensors consisting of Alk R-Palk M and report genes have natively wide alkane detection spectra(C8-C36)in Acinetobacter.However,this regulatory system is located on the chromosome of Acinetobacter sp.and could not work in Escherichia coli,which limits its practical application in biosensor design.The Alk S-Palk B regulatory system is better established and more widely used,but it has an inherent flaw in that it only responds to alkanes with carbon chain lengths less than C12 in E.coli.Yet,the carbon chain lengths of alkanes produced in E.coli were mainly C13-C17 when introducing AAR and ADO,which severely restricts the application of Alk S-Palk B in detecting the endogenously produced long-chain alkanes.Hence,the development of biosensors that can respond to long-chain alkanes is of great significance for monitoring the metabolic flow of alkanes and improving the yield of long-chain alkanes.In this study,we utilized the wild-type alkane-responsive regulatory subsystem Alk S-Palk B as the responsive element and green fluorescent protein as the reporting element to construct an alkane biosensor for detecting and screening high-yield alkane mutant strains.Initial testing revealed that the constructed wild-type sensor only responded to short-chain alkanes(C10 and below)and did not respond to long-chain alkanes(C15,C17,etc.)produced by microorganisms.Subsequently,through site-directed mutagenesis and directed evolution techniques,we modified the transcription factor Alk S and developed the biosensor ep S4-156,capable of responding to long-chain alkanes.The evolved sensor exhibited a 36-fold increase in fluorescence response intensity for C15 and a 9-fold increase for C17.Further optimization and evaluation of the selected biosensors demonstrated that the evolved alkane biosensor exhibited a good dynamic response range to long-chain alkanes and could monitor intracellular alkane production in real-time.Subsequently,using ep S4-156 as a screening tool and fluorescence protein expression level as a screening indicator,we conducted highthroughput flow cytometry screening experiments for alkane-producing strains.The selected mutants showed a 13-fold increase in alkane production.This study demonstrates the feasibility of using the development of whole-cell biosensors as a tool to improve the production of alkanes by modifying the rate-limiting enzymes through directed evolution techniques.It provides important references for the construction of microbial cell factories and industrial production,and holds significant implications for the development and utilization of new energy sources and the protection of the ecological environment. |
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