Precise microbiome engineering using natural and synthetic bacteriophages targeting an artificial bacterial consortium.

In natural microbiomes, microorganisms interact with each other and exhibit diverse functions. Microbiome engineering, which enables bacterial knockdown, is a promising method to elucidate the functions of targeted bacteria in microbiomes. However, few methods to selectively kill target microorganisms in the microbiome without affecting the growth of nontarget microorganisms are available. In this study, we focused on the host-specific lytic ability of virulent phages and validated their potency for precise microbiome engineering. In an artificial microbiome consisting of Escherichia coli, Pseudomonas putida, Bacillus subtilis, and Lactiplantibacillus plantarum, the addition of bacteriophages infecting their respective host strains specifically reduced the number of these bacteria more than 102 orders. Remarkably, the reduction in target bacteria did not affect the growth of nontarget bacteria, indicating that bacteriophages were effective tools for precise microbiome engineering. Moreover, a virulent derivative of the λ phage was synthesized from prophage DNA in the genome of λ lysogen by in vivo DNA assembly and phage-rebooting techniques, and E. coli-targeted microbiome engineering was achieved. These results propose a novel approach for precise microbiome engineering using bacteriophages, in which virulent phages are synthesized from prophage DNA in lysogenic strains without isolating phages from environmental samples.

Effects of small heat shock proteins from thermotolerant bacteria on the stress resistance of Escherichia coli to temperature, pH, and hyperosmolarity.

Small heat shock proteins (HSPs), such as HSP20, represent cellular thermal resistance mechanisms, to avoid protein aggregation at elevated temperatures. Recombinantly expressed HSP20s serve as a molecular tool for improving the tolerance of living cells to various physical and chemical stressors. Here, we aimed to heterologously express 18 HSP20s from 12 thermotolerant bacteria in Escherichia coli and evaluate their effects on various physical and chemical cellular stresses. Seventeen HSP20s were successfully expressed as soluble proteins. Recombinant E. coli cells were subjected to heat, cold, acidic, alkaline, and hyperosmolar stress to evaluate the effects of HSP20 proteins on stress resistance. Notably, the overexpression of 15 HSP20s enhanced the stress resistance of E. coli compared to that of the control strain. In particular, HSPs from Tepidimonas sediminis and Oceanithermus profundus improved the stress tolerance of E. coli under all tested conditions. In addition, E. coli harboring HSP20 from T. sediminis retained cell viability even after heat treatment at 52 °C for 5 days. To our knowledge, this is the first report of E. coli tolerance to prolonged (> 100 h) high-temperature stress. These findings indicate the potential of thermotolerant HSPs as molecular tools for improving stress tolerance in E. coli.

Valorization of agro-industrial waste from the cassava industry as esterified cellulose butyrate for polyhydroxybutyrate-based biocomposites.

The aim of this study was to utilize cassava pulp to prepare biocomposites comprising microcrystalline cellulose from cassava pulp (CP-MCC) as a filler and polyhydroxybutyrate (PHB) synthesized in-house by Cupriavidus necator strain A-04. The CP-MCC was extracted from fresh cassava pulp. Next, the CP-MCC surface was modified with butyryl chloride (esterified to CP-MCC butyrate) to improve dissolution and compatibility with the PHB. FTIR results confirmed that the esterified CP-MCC butyrate had aliphatic chains replacing the hydroxyl groups; this substitution increased the solubilities in acetone, chloroform, and tetrahydrofuran. Biocomposite films were prepared by varying the composition of esterified CP-MCC butyrate as a filler in the PHB matrix at 0, 5, 10, 15, 20 and 100 wt%. The results for the 95:5 and 90:10 CP-MCC butyrate biocomposite films showed that esterification led to improvements in the thermal properties and increased tensile strengths and elongations at break. All prepared biocomposite films maintained full biodegradability.

Bio-coatings in permeated cultivation systems: Unprecedented impacts on microalgal monoculture growth and organic matter yield.

Bio-coating, a recent and promising approach in attached microalgal cultivation systems, has garnered attention due to its efficiency in enhancing immobilized algal growth, particularly in submerged cultivation systems. However, when the cells are cultured on thin solid microporous substrates that physically separate them from the nutrient medium, it remains unclear whether the applied bio-coatings still have a significant impact on algal growth or the subsequent rates of algal organic matter (AOM) release. Therefore, this current work investigated the role of bio-coatings on the microalgal monoculture growth of one freshwater species, Chlorella vulgaris ESP 31, and one marine species, Cylindrotheca fusiformis on a hydrophilic substrate, polyvinylidene fluoride membrane in a permeated cultivation system. Wide range of bio-coating sources were adapted, with the result demonstrating that bacteria-derived coating promoted algal growth by as high as 140% when compared with the control group for both species. Interestingly, two distinct adaptation mechanisms were observed between the species, with only C. fusiformis demonstrating a positive correlation between cell growth and AOM productivity, particularly in its extracellularly bound fractions. It is worth noting that despite this specific fraction exhibiting the lowest content among all; it displayed significant relevance in terms of AOM productivity. High extracellular protein-to-polysaccharide ratio (>5.7 fold) quantified on bacterial intracellular exudate-coated membranes indirectly revealed an underlying symbiotic microalgal-bacterial interaction. This is the first study showing how bio-coating influenced AOM yield without any physical interaction between microalgae and bacteria. It further confirms the practical benefits of bio-coating in attached cultivation systems.

Chitin- and Streptavidin-Mediated Affinity Purification Systems: A Screening Platform for Enzyme Discovery.

Affinity purification of recombinant proteins is an essential technique in biotechnology. However, current affinity purification methods are very cost-intensive, and this imposes limits on versatile use of affinity purification for obtaining purified proteins for a variety of applications. To overcome this problem, we developed a new affinity purification system which we call CSAP (chitin- and streptavidin-mediated affinity purification) for low-cost purification of Strep-tag II fusion proteins. The CSAP system is designed to utilize commercially available chitin powder as a chromatography matrix, thereby significantly improving the cost-efficiency of protein affinity purification. We investigated the CSAP system for protein screening in 96-well format as a demonstration. Through the screening of 96 types of purified hemoproteins, several proteins capable of the catalytic diastereodivergent synthesis of cyclopropanes were identified as candidates for an abiotic carbene transfer reaction.

Cell-Free Production and Regeneration of Cofactors.

Cofactors, such as adenosine triphosphate, nicotinamide adenine dinucleotide, and coenzyme A, are involved in nearly 50% of enzymatic reactions and widely used in biocatalytic production of useful chemicals. Although commercial production of cofactors has been mostly dependent on extraction from microbial cells, this approach has a theoretical limitation to achieve a high-titer, high-yield production of cofactors owing to the tight regulation of cofactor biosynthesis in living cells. Besides the cofactor production, their regeneration is also a key challenge to enable continuous use of costly cofactors and improve the feasibility of enzymatic chemical manufacturing. Construction and implementation of enzyme cascades for cofactor biosynthesis and regeneration in a cell-free environment can be a promising approach to these challenges. In this chapter, we present the available tools for cell-free cofactor production and regeneration, the pros and cons, and how they can contribute to promote the industrial application of enzymes.

Current advances in alteration of fatty acid profile in Rhodotorula toruloides: a mini-review.

Microbial lipids are considered promising and environmentally friendly substitutes for fossil fuels and plant-derived oils. They alleviate the depletion of limited petroleum storage and the decrement of arable lands resulting from the greenhouse effect. Microbial lipids derived from oleaginous yeasts provide fatty acid profiles similar to plant-derived oils, which are considered as sustainable and alternative feedstocks for use in the biofuel, cosmetics, and food industries. Rhodotorula toruloides is an intriguing oleaginous yeast strain that can accumulate more than 70% of its dry biomass as lipid content. It can utilize a wide range of substrates, including low-cost sugars and industrial waste. It is also robust against various industrial inhibitors. However, precise control of the fatty acid profile of the lipids produced by R. toruloides is essential for broadening its biotechnological applications. This mini-review describes recent progress in identifying fatty synthesis pathways and consolidated strategies used for specific fatty acid-rich lipid production via metabolic engineering, strain domestication. In addition, this mini-review summarized the effects of culture conditions on fatty acid profiles in R. toruloides. The perspectives and constraints of harnessing R. toruloides for tailored lipid production are also discussed in this mini-review.

A review on microalgal-bacterial co-culture: The multifaceted role of beneficial bacteria towards enhancement of microalgal metabolite production.

Mass microalgal-bacterial co-cultures have come to the fore of applied physiological research, in particularly for the optimization of high-value metabolite from microalgae. These co-cultures rely on the existence of a phycosphere which harbors unique cross-kingdom associations that are a prerequisite for the cooperative interactions. However, detailed mechanisms underpinning the beneficial bacterial effects onto microalgal growth and metabolic production are rather limited at the moment. Hence, the main purpose of this review is to shed light on how bacteria fuels microalgal metabolism or vice versa during mutualistic interactions, building upon the phycosphere which is a hotspot for chemical exchange. Nutrients exchange and signal transduction between two not only increase the algal productivity, but also facilitate in the degradation of bio-products and elevate the host defense ability. Main chemical mediators such as photosynthetic oxygen, N-acyl-homoserine lactone, siderophore and vitamin B12 were identified to elucidate beneficial cascading effects from the bacteria towards microalgal metabolites. In terms of applications, the enhancement of soluble microalgal metabolites is often associated with bacteria-mediated cell autolysis while bacterial bio-flocculants can aid in microalgal biomass harvesting. In addition, this review goes in depth into the discussion on enzyme-based communication via metabolic engineering such as gene modification, cellular metabolic pathway fine-tuning, over expression of target enzymes, and diversion of flux toward key metabolites. Furthermore, possible challenges and recommendations aimed at stimulating microalgal metabolite production are outlined. As more evidence emerges regarding the multifaceted role of beneficial bacteria, it will be crucial to incorporate these findings into the development of algal biotechnology.

Green composites made of polyhydroxybutyrate and long-chain fatty acid esterified microcrystalline cellulose from pineapple leaf.

Pineapple leaf fibres are an abundant agricultural waste product that contains 26.9% cellulose. The objective of this study was to prepare fully degradable green biocomposites made of polyhydroxybutyrate (PHB) and microcrystalline cellulose from pineapple leaf fibres (PALF-MCC). To improve compatibility with PHB, the PALF-MCC was surface modified using lauroyl chloride as an esterifying agent. The influence of the esterified PALF-MCC laurate content and changes in the film surface morphology on biocomposite properties was studied. The thermal properties obtained by differential scanning calorimetry revealed a decrease in crystallinity for all biocomposites, with 100 wt% PHB displaying the highest values, whereas 100 wt% esterified PALF-MCC laurate showed no crystallinity. The addition of esterified PALF-MCC laurate increased the degradation temperature. The maximum tensile strength and elongation at break were exhibited when adding 5% of PALF-MCC. The results demonstrated that adding esterified PALF-MCC laurate as a filler in the biocomposite film could retain a pleasant value of tensile strength and elastic modulus whereas a slight increase in elongation can help to enhance flexibility. For soil burial testing, PHB/ esterified PALF-MCC laurate films with 5-20% (w/w) PALF-MCC laurate ester had higher degradation than films consisting of 100% PHB or 100% esterified PALF-MCC laurate. PHB and esterified PALF-MCC laurate derived from pineapple agricultural wastes are particularly suitable for the production of relatively low-cost biocomposite films that are 100% compostable in soil.

Characterization of thermostable serine hydroxymethyltransferase for ß-hydroxy amino acids synthesis.

ß-hydroxy amino acids, such as serine, threonine, and phenylserine, are important compounds for medical purposes. To date, there has been only limited exploration of thermostable serine hydroxylmethyltransferase (SHMT) for the synthesis of these amino acids, despite the great potential that thermostable enzymes may offer for commercial use due to their high stability and catalytic efficiencies. ITBSHMT_1 (ITB serine hydroxylmethyltransferase clone number 1) from thermophilic and methanol-tolerant bacteria Pseudoxanthomonas taiwanensis AL17 was successfully cloned. Biocomputational analysis revealed that ITBSHMT_1 contains Pyridoxal-3'-phosphate and tetrahydrofolatebinding residues. Structural comparisons show that ITBSHMT_1 has 5 additional residues VSRQG on loop near PLP-binding site as novel structural feature which distinguish this enzyme with other characterized SHMTs. In silico mutation revealed that the fragment might have very essential role in maintaining of PLP binding on structure of ITBSHMT_1. Recombinant protein was produced in Escherichia coli Rosetta 2(DE3) in soluble form and purified using NiNTA affinity chromatography. The purified protein demonstrated the best activity at 80 °C and pH 7.5 based on the retro aldol cleavage of phenylserine. Activity decreased significantly in the presence of 3 mM transition metal ions but increased in the presence of 30 mM ß-mercaptoethanol. ITBSHMT_1 demonstrated Vmax, Km, Kcat, and Kcat/Km at 242 U/mg, 23.26 mM, 186/s, and 8/(mM.s), respectively. The aldol condensation reaction showed the enzyme's best activity at 80 °C for serine, threonine, or phenylserine, with serine synthesis showing the highest specific activity. Biocomputational analysis revealed that high intramolecular interaction within the 3D structure of ITBSHMT_1 might be correlated with the enzyme's high thermal stability. The above data suggest that ITBSHMT_1 is a potential and novel enzyme for the production of various ß-hydroxy amino acids.

Subtractive modification of bacterial consortium using antisense peptide nucleic acids.

Microbiome engineering is an emerging research field that aims to design an artificial microbiome and modulate its function. In particular, subtractive modification of the microbiome allows us to create an artificial microbiome without the microorganism of interest and to evaluate its functions and interactions with other constituent bacteria. However, few techniques that can specifically remove only a single species from a large number of microorganisms and can be applied universally to a variety of microorganisms have been developed. Antisense peptide nucleic acid (PNA) is a potent designable antimicrobial agent that can be delivered into microbial cells by conjugating with a cell-penetrating peptide (CPP). Here, we tested the efficacy of the conjugate of CPP and PNA (CPP-PNA) as microbiome modifiers. The addition of CPP-PNA specifically inhibited the growth of Escherichia coli and Pseudomonas putida in an artificial bacterial consortium comprising E. coli, P. putida, Pseudomonas fluorescens, and Lactiplantibacillus plantarum. Moreover, the growth inhibition of P. putida promoted the growth of P. fluorescens and inhibited the growth of L. plantarum. These results indicate that CPP-PNA can be used not only for precise microbiome engineering but also for analyzing the growth relationships among constituent microorganisms in the microbiome.

One-Step Preparation of Cell-Free ATP Regeneration Module Based on Non-Oxidative Glycolysis Using Thermophilic Enzymes.

Adenosine triphosphate (ATP) is an essential cofactor for energy-dependent enzymatic reactions that occur during in vitro biochemical conversion. Recently, an enzyme cascade based on non-oxidative glycolysis, which uses starch and orthophosphate as energy and phosphate sources, respectively, for the regeneration of ATP from adenosine diphosphate, has been developed (Wei et al., ChemCatChem 2018, 10, 5597-5601). However, the 12 enzymes required for this system hampered its practical usability and further testing potential. Here, we addressed this issue by constructing co-expression vectors for the simultaneous gene expression of the 12 enzymes in a single expression strain. All enzymes were sourced from (hyper)thermophiles, which enabled a one-step purification via a heat-treatment process. We showed that the combination of the two enabled the ATP regeneration system to function in a single recombinant Escherichia coli strain. Additionally, this work provides a strategy to rationally design and control proteins expression levels in the co-expression vectors.

l-Lactate oxidase-mediated removal of l-lactic acid derived from fermentation medium for the production of optically pure D-lactic acid.

BACKGROUND: There has been an increasing demand for optically pure d-lactic and l-lactic acid for the production of stereocomplex-type polylactic acid. The d-lactic acid production from lignocellulosic biomass is important owing to its great abundance in nature. Corn steep liquor (CSL) is a cheap nitrogen source used for industrial fermentation, though it contains a significant amount of l-lactic acid, which decreases the optical purity of d-lactic acid produced. METHOD AND RESULTS: To remove l-lactic acid derived from the CSL-based medium, l-lactate oxidase (LoxL) from Enterococcus sp. NBRC 3427 was expressed in an engineered Lactiplantibacillus plantarum (formally called Lactobacillus plantarum) strain KOLP7, which exclusively produces d-lactic acid from both hexose and pentose sugars. When the resulting strain was applied for d-lactic acid fermentation from the mixed sugars consisting of the major constituent sugars of lignocellulose (35 g L-1 glucose, 10 g L-1 xylose, and 5 g L-1 arabinose) using the medium containing 10 g L-1 CSL, it completely removed l-lactic acid derived from CSL (0.52 g L-1 ) and produced 41.7 g L-1 of d-lactic acid. The l-lactic acid concentration was below the detection limit, and improvement in the optical purity of d-lactic acid was observed (from 98.2% to > 99.99%) by the overexpression of LoxL. CONCLUSION AND IMPLICATIONS: The LoxL-mediated consumption of l-lactic acid would enable the production of optically pure d-lactic acid in any medium contaminated by l-lactic acid.

Improvement of production yield of l-cysteine through in vitro metabolic pathway with thermophilic enzymes.

The demand for the amino acid l-cysteine is increasing in the food, cosmetic, and pharmaceutical industries. Conventionally, the commercial production of l-cysteine is achieved by its extraction from the acid hydrolysate of hair and feathers. However, this production method is associated with the release of environmentally hazardous wastewater. Additionally, l-cysteine produced from animal sources cannot be halal-certified, which limits the market size. Although recent studies have developed an alternative commercial l-cysteine production method based on microbial fermentation, the production yield was insufficient owing to the cytotoxicity of l-cysteine against the host cells. In a previous study, we had developed an in vitrol-cysteine production method with a combination of 11 thermophilic enzymes, which yielded 10.5 mM l-cysteine from 20 mM glucose. In this study, we performed re-screening for enzymes catalyzing the rate-limiting steps of the in vitro pathway. Subsequently, the genes encoding enzymes necessary for the in vitro synthesis of l-cysteine were assembled in an expression vector and co-expressed in a single strain. To prevent the synthesis of hydrogen peroxide (H2O2), which is a byproduct and inhibits the enzyme activity, the redox balance in this biosynthetic pathway was maintained by replacing the H2O2-forming NADH oxidase with another enzymatic reaction in which pyruvate was used as a sacrificial substrate. The re-designed in vitro synthetic pathway resulted in the production of 28.2 mM l-cysteine from 20 mM glucose with a molar yield of 70.5%.

Heterologous gene expression and characterization of two serine hydroxymethyltransferases from Thermoplasma acidophilum.

Serine hydroxymethyltransferase (SHMT) and threonine aldolase are classified as fold type I pyridoxal-5'-phosphate-dependent enzymes and engaged in glycine biosynthesis from serine and threonine, respectively. The acidothermophilic archaeon Thermoplasma acidophilum possesses two distinct SHMT genes, while there is no gene encoding threonine aldolase in its genome. In the present study, the two SHMT genes (Ta0811 and Ta1509) were heterologously expressed in Escherichia coli and Thermococcus kodakarensis, respectively, and biochemical properties of their products were investigated. Ta1509 protein exhibited dual activities to catalyze tetrahydrofolate (THF)-dependent serine cleavage and THF-independent threonine cleavage, similar to other SHMTs reported to date. In contrast, the Ta0811 protein lacks amino acid residues involved in the THF-binding motif and catalyzes only the THF-independent cleavage of threonine. Kinetic analysis revealed that the threonine-cleavage activity of the Ta0811 protein was 3.5 times higher than the serine-cleavage activity of Ta1509 protein. In addition, mRNA expression of Ta0811 gene in T. acidophilum was approximately 20 times more abundant than that of Ta1509. These observations suggest that retroaldol cleavage of threonine, mediated by the Ta0811 protein, has a major role in glycine biosynthesis in T. acidophilum.

Strategies for Poly(3-hydroxybutyrate) Production Using a Cold-Shock Promoter in Escherichia coli.

The present study attempted to increase poly(3-hydroxybutyrate) (PHB) production by improving expression of PHB biosynthesis operon derived from Cupriavidus necator strain A-04 using various types of promoters. The intact PHB biosynthesis operon of C. necator A-04, an alkaline tolerant strain isolated in Thailand with a high degree of 16S rRNA sequence similarity with C. necator H16, was subcloned into pGEX-6P-1, pColdI, pColdTF, pBAD/Thio-TOPO, and pUC19 (native promoter) and transformed into Escherichia coli JM109. While the phaC A-04 gene was insoluble in most expression systems tested, it became soluble when it was expressed as a fusion protein with trigger factor (TF), a ribosome associated bacterial chaperone, under the control of a cold shock promoter. Careful optimization indicates that the cold-shock cspA promoter enhanced phaCA-04 protein expression and the chaperone function of TF play critical roles in increasing soluble phaCA-04 protein. Induction strategies and parameters in flask experiments were optimized to obtain high expression of soluble PhaCA-04 protein with high YP/S and PHB productivity. Soluble phaCA-04 was purified through immobilized metal affinity chromatography (IMAC). The results demonstrated that the soluble phaCA-04 from pColdTF-phaCAB A-04 was expressed at a level of as high as 47.4 ± 2.4% of total protein and pColdTF-phaCAB A-04 enhanced soluble protein formation to approximately 3.09-4.1 times higher than that from pColdI-phaCAB A-04 by both conventional method and short induction method developed in this study. Cultivation in a 5-L fermenter led to PHB production of 89.8 ± 2.3% PHB content, a YP/S value of 0.38 g PHB/g glucose and a productivity of 0.43 g PHB/(L.h) using pColdTF-phaCAB A-04. The PHB film exhibited high optical transparency and possessed Mw 5.79 × 105 Da, Mn 1.86 × 105 Da, and PDI 3.11 with normal melting temperature and mechanical properties.

Polyhydroxybutyrate (PHB) Production Using an Arabinose-Inducible Expression System in Comparison With Cold Shock Inducible Expression System in Escherichia coli.

Cupriavidus necator strain A-04 has shown 16S rRNA gene identity to the well-known industrial strain C. necator H16. Nevertheless, the cell characteristics and polyhydroxyalkanoate (PHA) production ability of C. necator strain A-04 were different from those of C. necator H16. This study aimed to express PHA biosynthesis genes of C. necator strain A-04 in Escherichia coli via an arabinose-inducible expression system. In this study, the PHA biosynthesis operon of C. necator strain A-04, consisting of three genes encoding acetyl-CoA acetyltransferase (phaA A-04, 1182 bp, 40.6 kDa), acetoacetyl-CoA reductase (phaB A-04, 741 bp, 26.4 kDa) and PHB synthase Class I (phaC A-04, 1770 bp), was identified. Sequence analysis of the phaA A-04, phaB A-04, and phaCA-04 genes revealed that phaC A-04 was 99% similar to phaC H16 from C. necator H16. The difference in amino acid residue situated at position 122 of phaC A-04 was proline, whereas that of C. necator H16 was leucine. The intact phaCAB A-04 operon was cloned into the arabinose-inducible araBAD promoter and transformed into E. coli strains Top 10, JM109 and XL-1 blue. The results showed that optimal conditions obtained from shaken flask experiments yielded 6.1 ± 1.1 g/L cell dry mass (CDM), a PHB content of 93.3 ± 0.9% (w/w) and a productivity of 0.24 g/(L⋅h), whereas the wild-type C. necator strain A-04 accumulated 78% (w/w) PHB with a productivity of 0.09 g/(L⋅h). Finally, for the scaled-up studies, fed-batch cultivations by pH-stat control in a 5-L fermenter of E. coli strains XL1-Blue harboring pBAD/Thio-TOPO-phaCAB A-04 and pColdTF-phaCAB A-04 in MR or LB medium, leading to a PHB production of 31.4 ± 0.9 g/L at 54 h with a PHB content of 83.0 ± 3.8% (w/w), a CDM of 37.8 ± 1.2 g/L, a Y P/S value of 0.39 g PHB/g glucose and a productivity of 0.6 g PHB/(L⋅h) using pColdTF-phaCAB A-04 in MR medium. In addition, PHB production was 29.0 ± 1.1 g/L with 60.2 ± 2.3% PHB content in the CDM of 53.1 ± 1.0 g/L, a Y P/S value of 0.21 g PHB/g glucose and a productivity of 0.4 g PHB/(L⋅h) using pBAD/Thio-TOPO-phaCAB A-04 in LB medium. Thus, a relatively high PHB concentration and productivity were achieved, which demonstrated the possibility of industrial production of PHB.

Genome editing by miniature CRISPR/Cas12f1 enzyme in Escherichia coli.

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system is a valuable genome editing tool for microorganisms. However, the commonly used Cas9 nuclease derived from Streptococcus pyogenes (SpCas9) is not applicable to many industrially relevant bacteria, due to its cytotoxicity and large size (1368 amino acids [aa]). We developed an alternative genome editing system using a miniature Cas12f1 nuclease (529 aa) derived from an uncultured archaeon, Un1Cas12f1. When editing four dispensable genes in Escherichia coli MG1655 and BW25113, the CRISPR/Un1Cas12f1 system showed higher efficiency (63%-100%) than the CRISPR/SpCas9 system (50%-79%). The CRISPR/Un1Cas12f1 genome editing system is expected to be applied to the genome editing of a wide variety of bacteria.

In Vitro Production of Coenzyme A Using Thermophilic Enzymes.

Coenzyme A (CoA) is an essential cofactor present in all domains of life and is involved in numerous metabolic pathways, including fatty acid metabolism, pyruvate oxidation through the tricarboxylic acid (TCA) cycle, and the production of secondary metabolites. This characteristic makes CoA a commercially valuable compound in the pharmaceutical, cosmetic, and clinical industries. However, CoA is difficult to accumulate in living cells at a high level, since it is consumed in multiple metabolic pathways, hampering its manufacturing by typical cell cultivation and extraction approaches. The feedback inhibition by CoA to a biosynthetic enzyme, pantothenate kinase (PanK), is also a serious obstacle for the high-titer production of CoA. To overcome this challenge, in vitro production of CoA, in which the CoA biosynthetic pathway was reconstructed outside cells using recombinant thermophilic enzymes, was performed. The in vitro pathway was designed to be insensitive to the feedback inhibition of CoA using CoA-insensitive type III PanK from the thermophilic bacterium Thermus thermophilus. Furthermore, a statistical approach using design of experiments (DOE) was employed to rationally determine the enzyme loading ratio to maximize the CoA production rate. Consequently, 0.94 mM CoA could be produced from 2 mM d-pantetheine through the designed pathway. We hypothesized that the insufficient conversion yield is attributed to the high Km value of T. thermophilus PanK toward ATP. Based on these observations, possible CoA regulation mechanisms in T. thermophilus and approaches to improve the feasibility of CoA production through the in vitro pathway have been investigated. IMPORTANCE The biosynthesis of coenzyme A (CoA) in bacteria and eukaryotes is regulated by feedback inhibition targeting type I and type II pantothenate kinase (PanK). Type III PanK is found only in bacteria and is generally insensitive to CoA. Previously, type III PanK from the hyperthermophilic bacterium Thermotoga maritima was shown to defy this typical characteristic and instead shows inhibition toward CoA. In the present study, phylogenetic analysis combined with functional analysis of type III PanK from thermophiles revealed that the CoA-sensitive behavior of type III PanK from T. maritima is uncommon. We cloned type III PanKs from Thermus thermophilus and Geobacillus sp. strain 30 and showed that neither enzyme's activities were inhibited by CoA. Furthermore, we utilized type III PanK for a one-pot cascade reaction to produce CoA.