A hybrid RNA-protein biosensor for high-throughput screening of adenosylcobalamin biosynthesis

Genetically encoded circuits have been successfully utilized to assess and characterize target variants with desirable traits from large mutant libraries.Adenosylcobalamin is an essential coenzyme that is required in many intracellular physiological reactions and is widely used in the pharmaceutical and food industries.High-throughput screening techniques capable of detecting adenosylcobalamin productivity and selecting superior adenosylcobalamin biosynthesis strains are critical for the creation of an effective microbial cell factory for the production of adenosylcobalamin at an industrial level.In this study,we developed an RNA-protein hybrid biosensor whose input part was an endogenous RNA riboswitch to specifically respond to adenosylcobalamin,the inverter part was an orthogonal transcriptional repressor to obtain signal inversion,and the output part was a fluorescent protein to be easily detected.The hybrid biosensor could specifically and positively correlate adenosylcobalamin concentrations to green fluorescent protein expression levels in vivo.This study also improved the operating concentration and dynamic range of the hybrid biosensor by systematic optimization.An individual cell harboring the hybrid biosensor presented over 20-fold higher fluorescence intensity than the negative control.Then,using such a biosensor combined with fluorescence-activated cell sorting,we established a high-throughput screening platform for screening adenosylcobalamin overproducers.This study demonstrates that this platform has significant potential to quickly isolate high-productive strains to meet industrial demand and that the framework is acceptable for various metabolites.

Engineering Escherichia coli to improve tryptophan production via genetic manipulation of precursor and cofactor pathways

Optimizing the supply of biosynthetic precursors and cofactors is usually an effective metabolic strategy to improve the production of target compounds.Here,the combination of optimizing precursor synthesis and balancing cofactor metabolism was adopted to improve tryptophan production in Escherichia coli.First,glutamine synthesis was improved by expressing heterologous glutamine synthetase from Bacillus subtilis and Bacillus megaterium in the engineered Escherichia coli strain KW001,resulting in the best candidate strain TS-1.Then icd and gdhA were overexpressed in TS-1,which led to the accumulation of 1.060 g/L tryptophan.Subsequently,one more copy of prs was introduced on the chromosome to increase the flux of 5-phospho-α-D-ribose 1-diphosphate followed by the expression of mutated serA and thrA to increase the precursor supply of serine,resulting in the accumulation of 1.380 g/L tryptophan.Finally,to maintain cofactor balance,sthA and pntAB,encoding transhydrogenase,were overexpressed.With sufficient amounts of precursors and balanced cofactors,the engineered strain could produce 1.710 g/L tryptophan after 48 h of shake-flask fermentation,which was 2.76-times higher than the titer of the parent strain.Taken together,our results demonstrate that the combination of optimizing precursor supply and regulating cofactor metabolism is an effective approach for high-level production of tryptophan.Similar strategies could be applied to the production of other amino acids or related derivatives.

Melatonin biosynthesis pathways in nature and its production in engineered microorganisms

Melatonin is a biogenic amine that can be found in plants,animals and microorganism.The metabolic pathway of melatonin is different in various organisms,and biosynthetic endogenous melatonin acts as a molecular signal and antioxidant protection against external stress.Microbial synthesis pathways of melatonin are similar to those of animals but different from those of plants.At present,the method of using microorganism fermentation to produce melatonin is gradually prevailing,and exploring the biosynthetic pathway of melatonin to modify microorganism is becoming the mainstream,which has more advantages than traditional chemical synthesis.Here,we review recent advances in the synthesis,optimization of melatonin pathway.L-tryptophan is one of the two crucial precursors for the synthesis of melatonin,which can be produced through a four-step reaction.Enzymes involved in melatonin synthesis have low specificity and catalytic efficiency.Site-directed mutation,directed evolution or promotion of cofactor synthesis can enhance enzyme activity and increase the metabolic flow to promote microbial melatonin production.On the whole,the status and bottleneck of melatonin biosynthesis can be improved to a higher level,providing an effective reference for future microbial modification.