Role of methylotrophic bacteria in managing abiotic stresses for enhancing agricultural production

Abiotic stresses are a significant factor that considerably limits plant growth and productivity.Methylotrophs are an essential group of bacteria that utilize volatile carbon compounds,are prolific colonizers of different plant parts,and play a vital role in plant growth promotion(PGP)under stress conditions.Numerous rhizospheric and phyllosphere methylotrophs have been reported to exhibit PGP activities with superior stress-tolerating capacity against drought,heavy metal,salinity,high and low temperatures,solar ultraviolet radiation,and other harsh environmental conditions.Methylotrophs promote plant growth through N2 fixation,phosphate solubilization,production of phytohormones(i.e.,auxins,gibberellins,and cytokinins),1-aminocyclopropane-1-carboxylic acid(ACC)deaminase activity,siderophore,ammonia production,and secondary metabolites.The production of these compounds by methylotrophs protects a plant against adverse environmental conditions and influences its productivity.This review discusses the role methylotrophs play in managing various abiotic stresses,how they help mitigate these stresses,and how they improve agricultural productivity.

Advances in engineering methylotrophic yeast for biosynthesis of valuable chemicals from methanol

Methylotrophic yeasts and bacteria, which can use methanol as carbon and energy source, have beenwildly used as microbial cell factories for biomanufacturing. Due to their robustness in industrial harshconditions, methylotrophic yeasts such as Pichia pastoris have been explored as a cell factory forproduction of proteins and high-value chemicals. Methanol utilization pathway （MUT） is highlyregulated for efficient methanol utilization, and the downstream pathways need extensively constructedand optimized toward target metabolite biosynthesis. Here, we present an overview of methanolmetabolism and regulation in methylotrophic yeasts, among which we focus on the regulation of keygenes involved in methanol metabolism. Besides, the recent progresses in construction and optimizationof downstream biosynthetic pathways for production of high value chemicals, such as polyketides, fattyacids and isoprenoids, are further summarized. Finally, we discuss the current challenges and feasiblestrategies toward constructing efficient methylotrophic cell factories may promote wide applications inthe future.

Efficient fatty acid synthesis from methanol in methylotrophic yeast

Methanol is an attractive C1 feedstock with high abundance and low cost in bio-manufacturing.However,the metabolic construction of cell factories to utilize methanol for chemicals production remains a challenge due to the toxic intermediates and complicated metabolic pathways.The group of Zhou rescued methylotrophic yeast from cell death and achieved high-level production of free fatty acids from methanol through a combination of adaptive laboratory evolution,rational metabolic engineering and multi-omics analysis.

Constructing efficient bacterial cell factories to enable one-carbon utilization based on quantitative biology:A review

Developing methylotrophic cell factories that can efficiently catalyze organic one-carbon(C1)feedstocks derived from electrocatalytic reduction of carbon dioxide into bio-based chemicals and biofuels is of strategic significance for building a carbon-neutral,sustainable economic and industrial system.With the rapid advancement of RNA sequencing technology and mass spectrometer analysis,researchers have used these quantitative microbiology methods extensively,especially isotope-based metabolic flux analysis,to study the metabolic processes initiating from C1 feedstocks in natural C1-utilizing bacteria and synthetic C1 bacteria.This paper reviews the use of advanced quantitative analysis in recent years to understand the metabolic network and basic principles in the metabolism of natural C1-utilizing bacteria grown on methane,methanol,or formate.The acquired knowledge serves as a guide to rewire the central methylotrophic metabolism of natural C1-utilizing bacteria to improve the carbon conversion efficiency,and to engineer non-C1-utilizing bacteria into synthetic strains that can use C1 feedstocks as the sole carbon and energy source.These progresses ultimately enhance the design and construction of highly efficient C1-based cell factories to synthesize diverse high value-added products.The integration of quantitative biology and synthetic biology will advance the iterative cycle of understand–design–build–testing–learning to enhance C1-based biomanufacturing in the future.

Methanol-based biomanufacturing of fuels and chemicals using native and synthetic methylotrophs

Methanol has recently gained significant attention as a potential carbon substrate for the production of fuels and chemicals,owing to its high degree of reduction,abundance,and low price.Native methylotrophic yeasts and bacteria have been investigated for the production of fuels and chemicals.Alternatively,synthetic methylotrophic strains are also being developed by reconstructing methanol utilization pathways in model microorganisms,such as Escherichia coli.Owing to the complex metabolic pathways,limited availability of genetic tools,and methanol/formaldehyde toxicity,the high-level production of target products for industrial applications are still under development to satisfy commercial feasibility.This article reviews the production of biofuels and chemicals by native and synthetic methylotrophic microorganisms.It also highlights the advantages and limitations of both types of methylotrophs and provides an overview of ways to improve their efficiency for the production of fuels and chemicals from methanol.

Engineering the native methylotrophs for the bioconversion of methanol to value-added chemicals:current status and future perspectives

Methanol is becoming an attractive fermentation feedstock for large-scale bioproduction of chemicals,due to its natural abundance and mature production technology.Native methylotrophs,which can utilize methanol as the only source of carbon and energy,are ideal hosts for methanol bioconversion due to their high methanol utili-zation rate and have been extensively employed in the production of value-added chemicals from methanol.Here,we review the natural methanol utilization pathways in native methylotrophs,describing the available synthetic biology tools developed for engineering native methylotrophs,and discuss the strategies for improving their methanol utilization efficiency.Finally,the representative examples of engineering the native methylotrophs to produce value-added products from methanol are summarized.Furthermore,we also discuss the major challenges and possible solutions for the application of native methylotrophs in methanol-based biomanufacturing.

Metabolic engineering strategies for microbial utilization of methanol

The increasing shortage of fossil resources and environmental pollution has renewed interest in the synthesis of value-added biochemicals from methanol.However,most of native or synthetic methylotrophs are unable to assimilate methanol at a sufficient rate to produce biochemicals.Thus,the performance of methylotrophs still needs to be optimized to meet the demands of industrial applications.In this review,we provide an in-depth discussion on the properties of natural and synthetic methylotrophs,and summarize the natural and synthetic methanol assimilation pathways.Further,we discuss metabolic engineering strategies for enabling microbial utilization of methanol for the bioproduction of value-added chemicals.Finally,we highlight the potential of microbial engineering for methanol assimilation and offer guidance for achieving a low-carbon footprint for the biosynthesis of chemicals.

Individual stages of bacterial dichloromethane degradation mapped by carbon and chlorine stable isotope analysis

The fractionation of carbon and chlorine stable isotopes of dichloromethane(CH_2Cl_2,DCM)upon dechlorination by cells of the aerobic methylotroph Methylobacterium extorquens DM4 and by purified DCM dehalogenases of the glutathione S-transferase family was analyzed.Isotope effects for individual steps of the multi-stage DCM degradation process,including transfer across the cell wall from the aqueous medium to the cell cytoplasm,dehalogenase binding,and catalytic reaction,were considered.The observed carbon and chlorine isotope fractionation accompanying DCM consumption by cell supensions and enzymes was mainly determined by the breaking of C\Cl bonds,and not by inflow of DCM into cells.Chlorine isotope effects of DCM dehalogenation were initially masked in high density cultures,presumably due to inverse isotope effects of non-specific DCM oxidation under conditions of oxygen excess.Glutathione cofactor supply remarkably affected the correlation of variations of DCM carbon and chlorine stable isotopes(Δδ^(13)C/Δδ^(37)Cl),increasing corresponding ratio from 7.2–8.6 to 9.6–10.5 under conditions of glutathione deficiency.This suggests that enzymatic reaction of DCM with glutathione thiolate may involve stepwise breaking and making of bonds with the carbon atom of DCM,unlike the uncatalyzed reaction,which is a one-stage process,as shown by quantum-chemical modeling.

Microbial stabilisation and kinetic enhancement of marine methane hydrates in both deionised-and sea-water

The large quantity of marine methane hydrates has driven substantial interest in methane-gas-fuel potential,especially with the qualified success of Shensu(2017)and Nankai-Trough(2014&17)production trials via depressurisation(blighted ultimately by sanding out),building on an earlier Malik-2008 trial for permafrost-bound hydrate.In particular,obviating deep-water-drilling approaches,such as the MeBO production rig(without such a drill bit),together with blowout preventers,constitutes a tantalising cost-saving measure.Tailored means of addressing sand production by customised gravel packs,wellbore screens and slotted liners with from-seafloor drilling will be expected to lead to future production-trial success.However,despite these exciting engineering advances and a few marinemimicking laboratory studies of methane-hydrate kinetics and stabilisation from microbial perspectives,relatively little is known about the thermogenic or microbial origin of marine hydrates,nor their possible formation kinetics or potential stabilisation by microbial sources as an exponent of Gaia's hypothesis,or within the context of“Gaia's breath”as regards global methane‘exhalations’.Here,for the first time,we elucidate the methylotrophic-microbial basis for kinetic enhancement and stabilisation of marine-hydrate formation in both deionised-and sea-water,identifying the key protein at play,which has some similarity to porins in other methylotrophic communities.In so doing,we suggest such phenomena in marine hydrates as evidence of Gaia's hypothesis.