A major facilitator superfamily transporter MdtH in Escherichia coli is involved in anthocyanin biosynthesis and secretion.

Anthocyanin cyanidin 3-O-glucoside (C3G) is a natural pigment widely used in food and nutraceutical industries. Its microbial synthesis in Escherichia coli is a promising and efficient way toward large-scale production. The current production titer is low partly due to the accumulation of C3G inside the producing microbes; thus, it is important to explore native transporters responsible for anthocyanin secretion. Currently, there has been only one native E. coli transporter identified with C3G-transporting capability, and its overexpression has a very limited effect on the promotion of extracellular C3G production. In this study, we report the identification and verification of an efficient intrinsic C3G efflux transporter MdtH in E. coli through transcriptomic analysis and genetic/biochemical studies. MdtH could bind C3G with high affinity, and its overexpression increased the extracellular C3G biosynthesis in E. coli by 110%. Our study provides a new regulation target for microbial biosynthesis of C3G and other anthocyanins. IMPORTANCE: Cyanidin 3-O-glucoside (C3G) is a natural colorant with health-promoting activities and is, hence, widely used in food, cosmetic, and nutraceutical industries. Its market supply is currently dependent on extraction from plants. As an alternative, C3G can be produced by the microbe Escherichia coli in a green and sustainable way. However, a large portion of this compound is retained inside the cell of E. coli, thus complicating the purification process and limiting the high-level production. We have identified and verified an efficient native transporter named MdtH in E. coli that can export C3G to the cultivation medium. Overexpression of MdtH could improve extracellular C3G production by 110% without modifications of the metabolic pathway genes or enzymes. This study reveals a new regulation target for C3G production in bacteria and provides guidance to the microbial biosynthesis of related compounds.

Simultaneous glucose and xylose utilization by an Escherichia coli catabolite repression mutant.

As advances are made toward the industrial feasibility of mass-producing biofuels and commodity chemicals with sugar-fermenting microbes, high feedstock costs continue to inhibit commercial application. Hydrolyzed lignocellulosic biomass represents an ideal feedstock for these purposes as it is cheap and prevalent. However, many microbes, including Escherichia coli, struggle to efficiently utilize this mixture of hexose and pentose sugars due to the regulation of the carbon catabolite repression (CCR) system. CCR causes a sequential utilization of sugars, rather than simultaneous utilization, resulting in reduced carbon yield and complex process implications in fed-batch fermentation. A mutant of the gene encoding the cyclic AMP receptor protein, crp*, has been shown to disable CCR and improve the co-utilization of mixed sugar substrates. Here, we present the strain construction and characterization of a site-specific crp* chromosomal mutant in E. coli BL21 star (DE3). The crp* mutant strain demonstrates simultaneous consumption of glucose and xylose, suggesting a deregulated CCR system. The proteomics further showed that glucose was routed to the C5 carbon utilization pathways to support both de novo nucleotide synthesis and energy production in the crp* mutant strain. Metabolite analyses further show that overflow metabolism contributes to the slower growth in the crp* mutant. This highly characterized strain can be particularly beneficial for chemical production by simultaneously utilizing both C5 and C6 substrates from lignocellulosic biomass.IMPORTANCEAs the need for renewable biofuel and biochemical production processes continues to grow, there is an associated need for microbial technology capable of utilizing cheap, widely available, and renewable carbon substrates. This work details the construction and characterization of the first B-lineage Escherichia coli strain with mutated cyclic AMP receptor protein, Crp*, which deregulates the carbon catabolite repression (CCR) system and enables the co-utilization of multiple sugar sources in the growth medium. In this study, we focus our analysis on glucose and xylose utilization as these two sugars are the primary components in lignocellulosic biomass hydrolysate, a promising renewable carbon feedstock for industrial bioprocesses. This strain is valuable to the field as it enables the use of mixed sugar sources in traditional fed-batch based approaches, whereas the wild-type carbon catabolite repression system leads to biphasic growth and possible buildup of non-preferential sugars, reducing process efficiency at scale.

Enhanced poly-γ-glutamic acid synthesis in Corynebacterium glutamicum by reconstituting PgsBCA complex and fermentation optimization.

Previously, a novel Corynebacterium glutamicum strain for the de novo biosynthesis of tailored poly-γ-glutamic acid (γ-PGA) has been constructed by our group. The strain was based on the γ-PGA synthetase complex, PgsBCA, which is the only polyprotein complex responsible for γ-PGA synthesis in Bacillus spp. In the present study, PgsBCA was reconstituted and overexpressed in C. glutamicum to further enhance γ-PGA synthesis. First, we confirmed that all the components (PgsB, PgsC, and PgsA) of γ-PGA synthetase derived from B. licheniformis are necessary for γ-PGA synthesis, and γ-PGA was detected only when PgsB, PgsC, and PgsA were expressed in combination in C. glutamicum. Next, the expression level of each pgsB, pgsC, and pgsA was tuned in order to explore the effect of expression of each of the γ-PGA synthetase subunits on γ-PGA production. Results showed that increasing the transcription levels of pgsB or pgsC and maintaining a medium-level transcription level of pgsA led to 35.44% and 76.53% increase in γ-PGA yield (γ-PGA yield-to-biomass), respectively. Notably, the expression level of pgsC had the greatest influence (accounting for 68.24%) on γ-PGA synthesis, followed by pgsB. Next, genes encoding for PgsC from four different sources (Bacillus subtilis, Bacillus anthracis, Bacillus methylotrophicus, and Bacillus amyloliquefaciens) were tested in order to identify the influence of PgsC-encoding orthologues on γ-PGA production, but results showed that in all cases the synthesis of γ-PGA was significantly inhibited. Similarly, we also explored the influence of gene orthologues encoding for PgsB on γ-PGA production, and found that the titer increased to 17.14 ± 0.62 g/L from 8.24 ± 0.10 g/L when PgsB derived from B. methylotrophicus replaced PgsB alone in PgsBCA from B. licheniformis. The resulting strain was chosen for further optimization, and we achieved a γ-PGA titer of 38.26 g/L in a 5 L fermentor by optimizing dissolved oxygen level. Subsequently, by supplementing glucose, γ-PGA titer increased to 50.2 g/L at 48 h. To the best of our knowledge, this study achieved the highest titer for de novo production of γ-PGA from glucose, without addition of L-glutamic acid, resulting in a novel strategy for enhancing γ-PGA production.

Engineering of redox partners and cofactor NADPH supply of CYP68JX for efficient steroid two-step ordered selective hydroxylation activity.

CYP68JX, a P450 hydroxylase, derived from Colletotrichum lini ST-1 is capable of biotransforming dehydroepiandrosterone (DHEA) to 3ß,7α,15α-trihydroxy-5-androstene-17-one (7α,15α-diOH-DHEA). Redox partners and cofactor supply are important factors affecting the catalytic activity of CYP68JX. In this study, the heterologous expression of CYP68JX in Saccharomyces cerevisiae BY4741 was realized resulting in a 17.1% target product yield. In order to increase the catalytic efficiency of CYP68JX in S. cerevisiae BY4741, a complete cytochrome P450 redox system was constructed. Through the combination of CYP68JX and heterologous CPRs, the yield of the target product 7α,15α-diOH-DHEA in CYP68JX recombinant system was increased to 37.8%. Furthermore, by adding NADPH coenzyme precursor tryptophan of 40 mmol/L and co-substrate fructose of 20 g/L during the conversion process, the catalytic efficiency of CYP68JX was further improved, the target product yield reached 57.9% which was 3.39-fold higher than initial yield. Overall, this study provides a reference for improving the catalytic activity of P450s.

Isobutanol production by combined in vivo and in vitro metabolic engineering.

The production of the biofuel, isobutanol, in E. coli faces limitations due to alcohol toxicity, product inhibition, product recovery, and long-term industrial feasibility. Here we demonstrate an approach of combining both in vivo with in vitro metabolic engineering to produce isobutanol. The in vivo production of α-ketoisovalerate (KIV) was conducted through CRISPR mediated integration of the KIV pathway in bicistronic design (BCD) in E. coli and inhibition of competitive valine pathway using CRISPRi technology. The subsequent in vitro conversion to isobutanol was carried out with engineered enzymes for 2-ketoacid decarboxylase (KIVD) and alcohol dehydrogenase (ADH). For the in vivo production of KIV and subsequent in vitro production of isobutanol, this two-step serial approach resulted in yields of 56% and 93%, productivities of 0.62 and 0.074 g L-1 h-1, and titers of 5.6 and 1.78 g L-1, respectively. Thus, this combined biosynthetic system can be used as a modular approach for producing important metabolites, like isobutanol, without the limitations associated with in vivo production using a consolidated bioprocess.

Parallel screening and cheminformatics modeling of flavonoid activated aptasensors.

A parallel screening of 27 different flavonoids and chalcones was conducted using 6 artificial naringenin-activated riboswitches (M1, M2, M3, O, L and H). A quantitative structure-property relationship approach was applied to understand the physicochemical properties of the flavonoid structures resulting in specificity differences relied on the fluorescence intensity of a green fluorescent protein reporter. Robust models of riboswitches M1, M2 and O that had good predictive power were constructed with descriptors selected for their high correlation. Increased electronegativity and hydrophilicity of the flavonoids structures were identified as two properties that increased binding affinity to RNA riboswitches. Hydroxyl groups at the C-3' and C-4' positions of the flavonoid molecule were strictly required for ligand-activation with riboswitches M1 and M2. Riboswitches O and L preferred multi-hydroxylated flavones as ligands. Substitutions on the A ring of the flavonoid molecule were not important in the molecular recognition process. O-glycosylated derivatives were not recognized by any of the riboswitches, presumably due to steric hindrances. Despite the challenges of detecting RNA conformational change after ligand binding, the resulting models elucidate important physicochemical features in the ligands for conformational structural studies of artificial aptamer complexes and for design of ligands having higher binding specificity.

N-glycolylated carbohydrates in nature.

N-glycolylated carbohydrates are amino sugars with an N-glycolyl amide group. These glycans have not been well studied due to their surprising rarity in nature in comparison with N-acetylated carbohydrates. Recently, however, there has been increasing interest in N-glycolylated sugars because the non-human sialic acid N-glycolylneuraminic acid (Neu5Gc), apparently the only source of all N-glycolylated sugars in deuterostomes, appears to be involved in xenosialitis (inflammation associated with consumption of Neu5Gc-rich red meats). Xenosialitis has been implicated in cancers as well as other diseases including atherosclerosis. Furthermore, metabolites of Neu5Gc have been shown to be incorporated into glycosaminoglycans (GAGs), resulting in N-glycolylated GAGs. These N-glycolylated GAGs have important potential applications, such as dating the loss of the Neu5Gc-generating CMAH gene in humans and being explored as a xenosialitis biomarker and/or estimate of the body burden of diet-derived Neu5Gc, to understand the risks associated with the consumption of red meats. This review explores N-glycolylated carbohydrates, how they are metabolized to N-glycolylglucosamine and N-glycolylgalactosamine, and how these metabolites can be incorporated into N-glycolylated GAGs in human tissues. We also discuss other sources of N-glycolylated sugars, such as recombinant production from microorganisms using metabolic engineering as well as chemical synthesis.

Biosynthesis of eriodictyol from tyrosine by Corynebacterium glutamicum.

BACKGROUND: Eriodictyol is a bioactive flavonoid compound that shows potential applications in medicine development and food processing. Microbial synthesis of eriodictyol has been attracting increasing attention due to several benefits. In this study, we employed a GRAS strain Corynebacterium glutamicum as the host to produce eriodictyol directly from tyrosine. RESULTS: We firstly optimized the biosynthetic module of naringenin, the upstream intermediate for eriodictyol production, through screening of different gene orthologues. Next, to improve the level of the precursor malonyl-CoA necessary for naringenin production, we introduced matB and matC from Rhizobium trifolii into C. glutamicum to convert extracellular malonate to intracellular malonyl-CoA. This combinatorial engineering resulted in around 35-fold increase in naringenin production from tyrosine compared to the initial recombinant C. glutamicum. Subsequently, the hpaBC genes from E. coli encoding 4-hydroxyphenylacetate 3-hydroxylase were expressed in C. glutamicum to synthesize eriodictyol from naringenin. Further optimization of the biotransformation process parameters led to the production of 14.10 mg/L eriodictyol. CONCLUSIONS: The biosynthesis of the ortho-hydroxylated flavonoid eriodictyol in C. glutamicum was achieved for the first time via functional expression of E. coli hpaBC, providing a baseline strain for biosynthesis of other complex flavonoids. Our study demonstrates the potential application of C. glutamicum as a host microbe for the biosynthesis of value-added natural compounds from tyrosine.

De novo Biosynthesis of Salvianolic Acid B in Saccharomyces cerevisiae Engineered with the Rosmarinic Acid Biosynthetic Pathway.

Salvianolic acid B (SAB), also named lithospermic acid B, belongs to a class of water-soluble phenolic acids, originating from plants such as Salvia miltiorrhiza. SAB exhibits a variety of biological activities and has been clinically used to treat cardio- and cerebrovascular diseases and also has great potential as a health care product and medicine for other disorders. However, its biosynthetic pathway has not been completely elucidated. Here, we report the de novo biosynthesis of SAB in Saccharomyces cerevisiae engineered with the heterologous rosmarinic acid (RA) biosynthetic pathway. The created pathway contains seven genes divided into three modules on separate plasmids, pRS424-FjTAL-Sm4CL2, pRS425-SmTAT-SmHPPR or pRS425-SmTAT-CbHPPR, and pRS426-SmRAS-CbCYP-CbCPR. These three modules were cotransformed into S. cerevisiae, resulting in the recombinant strains YW-44 and YW-45. Incubation of the recombinant strains in a basic medium without supplementing any substrates yielded 34 and 30 µg/L of SAB. The findings in this study indicate that the created heterologous RA pathway cooperates with the native metabolism of S. cerevisiae to enable the de novo biosynthesis of SAB. This provides a novel insight into a biosynthesis mechanism of SAB and also lays the foundation for the production of SAB using microbial cell factories.

Dual regulation of lipid droplet-triacylglycerol metabolism and ERG9 expression for improved ß-carotene production in Saccharomyces cerevisiae.

BACKGROUND: The limitation of storage space, product cytotoxicity and the competition for precursor are the major challenges for efficiently overproducing carotenoid in engineered non-carotenogenic microorganisms. In this work, to improve ß-carotene accumulation in Saccharomyces cerevisiae, a strategy that simultaneous increases cell storage capability and strengthens metabolic flux to carotenoid pathway was developed using exogenous oleic acid (OA) combined with metabolic engineering approaches. RESULTS: The direct separation of lipid droplets (LDs), quantitative analysis and genes disruption trial indicated that LDs are major storage locations of ß-carotene in S. cerevisiae. However, due to the competition for precursor between ß-carotene and LDs-triacylglycerol biosynthesis, enlarging storage space by engineering LDs related genes has minor promotion on ß-carotene accumulation. Adding 2 mM OA significantly improved LDs-triacylglycerol metabolism and resulted in 36.4% increase in ß-carotene content. The transcriptome analysis was adopted to mine OA-repressible promoters and IZH1 promoter was used to replace native ERG9 promoter to dynamically down-regulate ERG9 expression, which diverted the metabolic flux to ß-carotene pathway and achieved additional 31.7% increase in ß-carotene content without adversely affecting cell growth. By inducing an extra constitutive ß-carotene synthesis pathway for further conversion precursor farnesol to ß-carotene, the final strain produced 11.4 mg/g DCW and 142 mg/L of ß-carotene, which is 107.3% and 49.5% increase respectively over the parent strain. CONCLUSIONS: This strategy can be applied in the overproduction of other heterogeneous FPP-derived hydrophobic compounds with similar synthesis and storage mechanisms in S. cerevisiae.

Methods for the Development of Recombinant Microorganisms for the Production of Natural Products.

Metabolic engineering strives to develop microbial strains that are capable of producing a target chemical in a biological organism. There are still many challenges to overcome in order to achieve titers, yields, and productivities necessary for industrial production. The use of recombinant microorganisms to meet these needs is the next step for metabolic engineers. In this chapter, we aim to provide insight on both the applications of metabolic engineering for natural product biosynthesis as well as optimization methods.

Multi-level rebalancing of the naringenin pathway using riboswitch-guided high-throughput screening.

Recombinant microbes have emerged as promising alternatives to natural sources of naringenin-a key molecular scaffold for flavonoids. In recombinant strains, expression levels of the pathway genes should be optimized at both transcription and the translation stages to precisely allocate cellular resources and maximize metabolite production. However, the optimization of the expression levels of naringenin generally relies on evaluating a small number of variants from libraries constructed by varying transcription efficiency only. In this study, we introduce a systematic strategy for the multi-level optimization of biosynthetic pathways. We constructed a multi-level combinatorial library covering both transcription and translation stages using synthetic T7 promoter variants and computationally designed 5'-untranslated regions. Furthermore, we identified improved strains through high-throughput screening based on a synthetic naringenin riboswitch. The most-optimized strain obtained using this approach exhibited a 3-fold increase in naringenin production, compared with the parental strain in which only the transcription efficiency was modulated. Furthermore, in a fed-batch bioreactor, the optimized strain produced 260.2 mg/L naringenin, which is the highest concentration reported to date using glycerol and p-coumaric acid as substrates. Collectively, this work provides an efficient strategy for the expression optimization of the biosynthetic pathways.

Improved glucose and xylose co-utilization by overexpression of xylose isomerase and/or xylulokinase genes in oleaginous fungus Mucor circinelloides.

Most of the oleaginous microorganisms cannot assimilate xylose in the presence of glucose, which is the major bottleneck in the bioconversion of lignocellulose to biodiesel. Our present study revealed that overexpression of xylose isomerase (XI) gene xylA or xylulokinase (XK) gene xks1 increased the xylose consumption by 25 to 37% and enhanced the lipid content by 8 to 28% during co-fermentation of glucose and xylose. In xylA overexpressing strain Mc-XI, the activity of XI was 1.8-fold higher and the mRNA level of xylA at 24 h and 48 h was 11- and 13-fold higher than that of the control, respectively. In xks1 overexpressing strain Mc-XK, the mRNA level of xks1 was 4- to 11-fold of that of the control strain and the highest XK activity of 950 nmol min-1 mg-1 at 72 h which was 2-fold higher than that of the control. Additionally, expression of a translational fusion of xylA and xks1 further enhanced the xylose utilization rate by 45%. Our results indicated that overexpression of xylA and/or xks1 is a promising strategy to improve the xylose and glucose co-utilization, alleviate the glucose repression, and produce lipid from lignocellulosic biomass in the oleaginous fungus M. circinelloides. KEY POINTS: • Overexpressing xylA or xks1 increased the xylose consumption and the lipid content. • The xylose isomerase activity and the xylA mRNA level were enhanced in strain Mc-XI. • Co-expression of xylA and xks1 further enhanced the xylose utilization rate by 45%.

Abiotic-biotic hybrid for CO2 biomethanation: From electrochemical to photochemical process.

Converting CO2 into sustainable fuels (e.g., CH4) has great significance to solve carbon emission and energy crisis. Generally, CO2 methanation needs abundant of energy input to overcome the eight-electron-transfer barrier. Abiotic-biotic hybrid system represents one of the cutting-edge technologies that use renewable electric/solar energy to realize eight-electron-transfer CO2 biomethanation. However, the incompatible abiotic-biotic hybrid can result in low efficiency of electron transfer and CO2 biomethanation. Herein, we present the comprehensive review to highlight how to design abiotic-biotic hybrid for electric/solar-driven CO2 biomethanation. We primarily introduce the CO2 biomethanation mechanism, and further summarize state-of-the-art electrochemical and photochemical CO2 biomethanation in hybrid systems. We also propose excellent synthetic biology strategies, which are useful to design tunable methanogenic microorganisms or enzymes when cooperating with electrode/semiconductor in hybrid systems. This review provides theoretical guidance of abiotic-biotic hybrid and also shows the bright future of sustainable fuel production in the form of CO2 biomethanation.

Complete biosynthesis of a sulfated chondroitin in Escherichia coli.

Sulfated glycosaminoglycans (GAGs) are a class of important biologics that are currently manufactured by extraction from animal tissues. Although such methods are unsustainable and prone to contamination, animal-free production methods have not emerged as competitive alternatives due to complexities in scale-up, requirement for multiple stages and cost of co-factors and purification. Here, we demonstrate the development of single microbial cell factories capable of complete, one-step biosynthesis of chondroitin sulfate (CS), a type of GAG. We engineer E. coli to produce all three required components for CS production-chondroitin, sulfate donor and sulfotransferase. In this way, we achieve intracellular CS production of ~27 µg/g dry-cell-weight with about 96% of the disaccharides sulfated. We further explore four different factors that can affect the sulfation levels of this microbial product. Overall, this is a demonstration of simple, one-step microbial production of a sulfated GAG and marks an important step in the animal-free production of these molecules.

The three NADH dehydrogenases of Pseudomonas aeruginosa: Their roles in energy metabolism and links to virulence.

Pseudomonas aeruginosa is a ubiquitous opportunistic pathogen which relies on a highly adaptable metabolism to achieve broad pathogenesis. In one example of this flexibility, to catalyze the NADH:quinone oxidoreductase step of the respiratory chain, P. aeruginosa has three different enzymes: NUO, NQR and NDH2, all of which carry out the same redox function but have different energy conservation and ion transport properties. In order to better understand the roles of these enzymes, we constructed two series of mutants: (i) three single deletion mutants, each of which lacks one NADH dehydrogenase and (ii) three double deletion mutants, each of which retains only one of the three enzymes. All of the mutants grew approximately as well as wild type, when tested in rich and minimal medium and in a range of pH and [Na+] conditions, except that the strain with only NUO (ΔnqrFΔndh) has an extended lag phase. During exponential phase, the NADH dehydrogenases contribute to total wild-type activity in the following order: NQR > NDH2 > NUO. Some mutants, including the strain without NQR (ΔnqrF) had increased biofilm formation, pyocyanin production, and killed more efficiently in both macrophage and mouse infection models. Consistent with this, ΔnqrF showed increased transcription of genes involved in pyocyanin production.

Metabolic engineering of E. coli for pyocyanin production.

Pyocyanin is a secondary metabolite from Pseudomonas aeruginosa that belongs to the class of phenazines, which are aromatic nitrogenous compounds with numerous biological functions. Besides its antifungal and antimicrobial activities, pyocyanin is a remarkable redox-active molecule with potential applications ranging from the pharma industry to the development of microbial fuel cells. Nevertheless, pyocyanin production has been restricted to P. aeruginosa strains, limiting its practical applicability. In this study, the pyocyanin biosynthetic pathway was engineered for the first time for high level production of this compound in a heterologous host. Escherichia coli cells harboring the nine-gene pathway divided into two plasmids were able to produce and secrete pyocyanin at higher levels than some Pseudomonas aeruginosa strains. The influence of culture and induction parameters were evaluated, and the optimized conditions led to an increase of 3.5-fold on pyocyanin accumulation. Pathway balancing was achieved by testing a set of plasmids with different copy numbers to optimize the expression levels of pyocyanin biosynthetic genes, resulting in a fourfold difference in product titer among the engineered strains. Further improvements were achieved by co-expression of Vitreoscilla hemoglobin Vhb, which relieved oxygen limitations and led to a final titer of 18.8 mg/L pyocyanin. These results show promise to use E. coli for phenazines production, and the engineered strain developed here has the potential to be used in electro-fermentation systems where pyocyanin plays a role as electron-shuttle.

Wall teichoic acids: physiology and applications.

Wall teichoic acids (WTAs) are charged glycopolymers containing phosphodiester-linked polyol units and represent one of the major components of Gram-positive cell envelope. WTAs have important physiological functions in cell division, gene transfer, surface adhesion, drug resistance and biofilm formation, and are critical virulence factors and vital determinants in mediating cell interaction with and tolerance to environmental factors. Here, we first briefly introduce WTA structure, biosynthesis and its regulation, and then summarize in detail four major physiological roles played by WTAs, i.e. WTA-mediated resistance to antimicrobials, virulence to mammalian cells, interaction with bacteriolytic enzymes and regulation of cell metabolism. We also review the applications of WTAs in these fields that are closely related to the human society, including antibacterial drug discovery targeting WTA biosynthesis, development of vaccines and antibodies regarding WTA-mediated pathogenicity, specific and sensitive detection of pathogens in food using WTAs as a surface epitope and regulation of WTA-related pathways for efficient microbial production of useful compounds. We also point out major problems remaining in these fields, and discuss some possible directions in the future exploration of WTA physiology and applications.

Improved soluble expression and use of recombinant human renalase.

Electrochemical bioreactor systems have enjoyed significant attention in the past few decades, particularly because of their applications to biobatteries, artificial photosynthetic systems, and microbial electrosynthesis. A key opportunity with electrochemical bioreactors is the ability to employ cofactor regeneration strategies critical in oxidative and reductive enzymatic and cell-based biotransformations. Electrochemical cofactor regeneration presents several advantages over other current cofactor regeneration systems, such as chemoenzymatic multi-enzyme reactions, because there is no need for a sacrificial substrate and a recycling enzyme. Additionally, process monitoring is simpler and downstream processing is less costly. However, the direct electrochemical reduction of NAD(P)+ on a cathode may produce adventitious side products, including isomers of NAD(P)H that can act as potent competitive inhibitors to NAD(P)H-requiring enzymes such as dehydrogenases. To overcome this limitation, we examined how nature addresses the adventitious formation of isomers of NAD(P)H. Specifically, renalases are enzymes that catalyze the oxidation of 1,2- and 1,6-NAD(P)H to NAD(P)+, yielding an effective recycling of unproductive NAD(P)H isomers. We designed several mutants of recombinant human renalase isoform 1 (rhRen1), expressed them in E. coli BL21(DE3) to enhance protein solubility, and evaluated the activity profiles of the renalase variants against NAD(P)H isomers. The potential for rhRen1 to be employed in engineering applications was then assessed in view of the enzyme's stability upon immobilization. Finally, comparative modeling was performed to assess the underlying reasons for the enhanced solubility and activity of the mutant enzymes.

Novel Prokaryotic CRISPR-Cas12a-Based Tool for Programmable Transcriptional Activation and Repression.

Transcriptional perturbation using inactivated CRISPR-nucleases (dCas) is a common method in eukaryotic organisms. While rare examples of dCas9-based tools for prokaryotes have been described, multiplexing approaches are limited due to the used effector nuclease. For the first time, a dCas12a derived tool for the targeted activation and repression of genes was developed. Therefore, a previously described SoxS activator domain was linked to dCas12a to enable the programmable activation of gene expression. A proof of principle of transcriptional regulation was demonstrated on the basis of fluorescence reporter assays using the alternative host organism Paenibacillus polymyxa as well as Escherichia coli. Single target and multiplex CRISPR interference targeting the exopolysaccharide biosynthesis of P. polymyxa was shown to emulate polymer compositions of gene knockouts. The simultaneous expression of 11 gRNAs targeting multiple lactate dehydrogenases and a butanediol dehydrogenase resulted in decreased lactate formation, as well as an increased butanediol production in microaerobic fermentation processes. Even though Cas12a is more restricted in terms of its genomic target sequences compared to Cas9, its ability to efficiently process its own guide RNAs in vivo makes it a promising tool to orchestrate sophisticated genetic reprogramming of bacterial cells or to screen for engineering targets in the genome. The developed tool will accelerate metabolic engineering efforts in the alternative host organism P. polymyxa and might be also applied for other bacterial cell factories.