Multi-engineering of Microbial Cytochrome P450 Enzymes

Selective oxidation of unactivated C–H bonds remains a central challenge in synthetic chemistry. Cytochrome P450 enzymes, a superfamily of ubiquitous hemoproteins, represent the nature’s primary solutions to overcome this challenge. As promising biocatalysts for practical applications in pharmaceutical, biotechnological and chemical industries, P450 enzymes have attracted a wealth of attention due to their great versatility in catalyzing diverse oxidative reactions (e.g., the sp3 C–H hydroxylation and the sp2 C=C epoxidation) on structurally complex and heavily functionalized substrates in regio- and/or stereoselective manners. However, wild type P450 enzymes usually show suboptimal activity, low stability, and narrow substrate spectra, which have significantly limited their broader applications. A typical P450 reaction system includes a P450 enzyme as the central catalyst, a substrate to be oxidized, redox partner proteins for electron transfer, NAD(P)H as the electron donor, and O2 as the oxidant. In the past five years, we have made significant progresses on enzyme engineering, substrate engineering, redox partner engineering, and electron donor engineering for a number of microbial P450 enzymes. These multi-engineering efforts have generated useful engineered P450 catalytic systems for bio-production of pharmaceuticals, chemical intermediates, and biofuel molecules. Please click Additional Files below to see the full abstract

Biochemical, Structural, and Bioengineering Studies of Cytochrome P450 Enzymes Involved in Biosynthesis of Secondary Metabolites.

The superfamily of cytochrome P450 monooxygenases is involved in diverse oxidative processes including xenobiotic catabolism, steroid synthesis, and biosynthetic tailoring of diverse natural products. During the past decade, the synthetic potential of biosynthetic P450 enzymes from microorganisms has gained special attention due to their non-membrane bound nature, considerable catalytic efficiency, and high regio- and stereoselectivity. However, current barriers to their application in synthetic chemistry include their instability, inherent dependence on separate redox partners, and narrow substrate spectra. As these hurdles have been gradually overcome, it is likely that these biosynthetic P450s will find expanded use in the production of chemicals, fragrances, pharmaceutical compounds, biofuels, and application in bioremediation. My dissertation research has focused on the bacterial cytochrome P450 PikC from the pikromycin macrolide antibiotic biosynthetic pathway in Streptomyces venezuelae. The inherent substrate flexibility and hydroxylation pattern of PikC suggests its unique oxidative mechanism and synthetic potential. Starting from the rystal structures of PikC, we not only elucidated the structural basis for its substrate flexibility, but also discovered a unique desosamine sugar anchoring functionality of this enzyme. These observations directly inspired a substrate engineering strategy that utilizes the desosamine anchor to deliver diverse structures into the PikC active site for selective oxidation. Using this approach, the substrate spectrum of PikC has been significantly broadened. Specifically, by using an engineered PikCD50N-RhFRED with self-sufficiency and significantly higher catalytic efficiency, a series of carbocyclic rings linked to the desosamine glycoside were effectively hydroxylated in a regioselective manner. Associated analysis of co-crystal structures of PikC with selected unnatural desosaminyl substrates provided significant insights into the mechanism of its oxidative selectivity control. Taken together, these results offer an applicable enzymatic solution of a central challenge in synthetic chemistry - the selective oxidation of an unactivated sp3 C-H bond. Moreover, a number of other biosynthetic P450 enzymes, two O-methyltransferases, an FAD-dependent oxidase, and a type III polyketide synthase were also studied during the course of my dissertation research. Together, these studies provide new insights into biosynthesis of secondary metabolites and how these enzymes can be adapted for biotechnological use.Ph.D.Medicinal ChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/64678/1/listened_1.pd

Can Internet construction promote enterprise upgrading?

This paper investigates the impact of Internet infrastructure construction on enterprise transformation and upgrading and the underlying mechanisms using a progressive double difference model based on a quasi-natural experiment of the Chinese government’s "broadband China" policy by matching A-share listed companies and city panel data from 2008–2019 in Shanghai and Shenzhen. The conclusions show that the "broadband China" policy can significantly promote the transformation and upgrading of enterprises in pilot cities. However, its effect shows a diminishing marginal contribution, and the policy is more effective for traditional manufacturing industries during the implementation period. Enhancing human capital and reducing internal transaction costs are two important channels for Internet infrastructure construction to help enterprises transform and upgrade. Combined with the life cycle theory, we find that the "broadband China" policy has the most significant impact on changing and upgrading enterprises in the growth and maturity stages, especially those in the manufacturing industry, but not those in the maturity and decline stages of the service industry. Finally, a series of robustness tests using Monte Carlo simulation, entropy balance method, and instrumental variables method, excluding other factors, show that the findings are still robust

Unusual acylation of chloramphenicol in \u3ci\u3eLysobacter enzymogenes,\u3c/i\u3e a biocontrol agent with intrinsic resistance to multiple antibiotics

Background: The environmental gliding bacteria Lysobacter are emerging as a new group of biocontrol agents due to their prolific production of lytic enzymes and potent antibiotic natural products. These bacteria are intrinsically resistant to many antibiotics, but the mechanisms behind the antibiotic resistance have not been investigated. Results: Previously, we have used chloramphenicol acetyltransferase gene (cat) as a selection marker in genetic manipulation of natural product biosynthetic genes in Lysobacter, because chloramphenicol is one of the two common antibiotics that Lysobacter are susceptible to. Here, we found L. enzymogenes, the most studied species of this genus, could still grow in the presence of a low concentration of chloramphenicol. Three chloramphenicol derivatives (1–3) with an unusual acylation pattern were identified in a cat-containing mutant of L. enzymogenes and in the wild type. The compounds included chloramphenicol 3\u27-isobutyrate (1), a new compound chloramphenicol 1\u27- isobutyrate (2), and a rare chloramphenicol 3\u27-isovalerate (3). Furthermore, a mutation of a global regulator gene (clp) or a Gcn5-related N-acetyltransferase (GNAT) gene in L. enzymogenes led to nearly no growth in media containing chloramphenicol, whereas a complementation of clp restored the chloramphenicol acylation as well as antibiotic HSAF production in the clp mutant. Conclusions: The results indicated that L. enzymogenes contains a pool of unusual acyl donors for enzymatic modification of chloramphenicol that confers the resistance, which may involve the Clp-GNAT regulatory system. Because Lysobacter are ubiquitous inhabitants of soil and water, the finding may have important implications in understanding microbial competitions and bioactive natural product regulation

Gene targeting for O -methyltransferase genes, mycE and mycF , on the chromosome of Micromonospora griseorubida producing mycinamicin with a disruption cassette containing the bacteriophage φC31 attB attachment site

Mycinamicin, a 16-membered macrolide antibiotic produced by Micromonospora griseorubida , comprises a macrolactone and two deoxysugars: desosamine and mycinose. Mycinose is synthesized through two modification steps: the methylation of 6-deoxyallose in mycinamicin VI and of javose in mycinamicin III. To confirm the role of mycE and mycF genes in mycinamicin biosynthesis in M. griseorubida , disruption mutants of mycE and mycF were constructed by disruption plasmids containing attB in the disruption cassette FRT -neo-oriT- FRT -attB for the integration of φC31-derivative vector plasmids; the disruption mutants were complemented through the integration of pSET152 derivatives containing intact mycE or mycF into the artificially inserted attB site. These disruption mutants did not produce mycinamicin II, but mainly accumulated mycinamicins VI and III, indicating that MycE and MycF methylated the C2″-OH group of 6-deoxyallose in mycinamicin VI and the C3″-OH group of C2″-methylated 6-deoxyallose in mycinamicin III, respectively. The complemented strains of mycE and mycF recovered the mycinamicin II productivity.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/79258/1/j.1574-6968.2010.01899.x.pd

The PstI/RsaI and DraI polymorphisms of CYP2E1 and head and neck cancer risk: a meta-analysis based on 21 case-control studies

<p>Abstract</p> <p>Background</p> <p><it>CYP2E1 </it>encodes a member of the cytochrome P450 superfamily of enzymes which play a central role in activating and detoxifying many carcinogens and endogenous compounds thought to be involved in the development of cancer. The PstI/RsaI and DraI polymorphism are two of the most commonly studied polymorphisms of the gene for their association with risk of head and neck cancer, but the results are conflicting.</p> <p>Methods</p> <p>We performed a meta-analysis using 21 eligible case-control studies with a total of 4,951 patients and 6,071 controls to summarize the data on the association between the <it>CYP2E1 </it>PstI/RsaI and DraI polymorphism and head and neck cancer risk, especially by interacting with smoking or alcohol.</p> <p>Results</p> <p>Compared with the wild genotype, the OR was 1.96 (95% CI: 1.33-2.90) for PstI/RsaI and 1.56 (95% CI: 1.06-2.27) for DraI polymorphism respectively. When stratified according to ethnicity, the OR increased in the Asians for both polymorphisms (OR = 2.04, 95% CI: 1.32-3.15 for PstI/RsaI; OR = 2.04, 95% CI: 1.27-3.29 for DraI), suggesting that the risk is more pronounced in Asians.</p> <p>Conclusion</p> <p>Our meta-analysis suggests that individuals with the homozygote genotypes of PstI/RsaI or DraI polymorphism might be associated with an increased risk of head and neck cancer, especially in Asians.</p

Assembly of lipase and P450 fatty acid decarboxylase to constitute a novel biosynthetic pathway for production of 1-alkenes from renewable triacylglycerols and oils

<p> Background: Biogenic hydrocarbons (biohydrocarbons) are broadly accepted to be the ideal &#39;drop-in&#39; biofuel alternative to petroleum-based fuels due to their highly similar chemical composition and physical characteristics. The biological production of aliphatic hydrocarbons is largely dependent on engineering of the complicated enzymatic network surrounding fatty acid biosynthesis.</p

Crystal structures of γ-glutamylmethylamide synthetase provide insight into bacterial metabolism of oceanic monomethylamine

Monomethylamine (MMA) is an important climate-active oceanic trace gas and ubiquitous in the oceans. The γ-glutamylmethylamide synthetase (GmaS) catalyzes the conversion of MMA to γ-glutamylmethylamide (GMA), the first step in MMA metabolism in many marine bacteria. The gmaS gene occurs in ~23% of microbial genomes in the surface ocean and is a validated biomarker to detect MMA-utilizing bacteria. However, the catalytic mechanism of GmaS has not been studied due to the lack of structural information. Here, the GmaS from Rhodovulum sp. 12E13 (RhGmaS) was characterized, and the crystal structures of apo-RhGmaS and RhGmaS with different ligands in five states were solved. Based on structural and biochemical analyses, the catalytic mechanism of RhGmaS was explained. ATP is first bound in RhGmaS, leading to a conformational change of a flexible loop (Lys287-Ile305), which is essential for the subsequent binding of glutamate. During the catalysis of RhGmaS, the residue Arg312 participates in polarizing the γ-phosphate of ATP and in stabilizing the γ-glutamyl phosphate intermediate; Asp177 is responsible for the deprotonation of MMA, assisting the attack of MMA on γ-glutamyl phosphate to produce a tetrahedral intermediate; and Glu186 acts as a catalytic base to abstract a proton from the tetrahedral intermediate to finally generate GMA. Sequence analysis suggested that the catalytic mechanism of RhGmaS proposed in this study has universal significance in bacteria containing GmaS. Our results provide novel insights into MMA metabolism, contributing to a better understanding of MMA catabolism in global carbon and nitrogen cycles

Genetic Association of Olanzapine Treatment Response in Han Chinese Schizophrenia Patients

Olanzapine, a second-generation antipsychotic medication, plays a critical role in current treatment of schizophrenia (SCZ). It has been observed that the olanzapine responses in schizophrenia treatment are different across individuals. However, prediction of this individual-specific olanzapine response requires in-depth knowledge of biomarkers of drug response. Here, we performed an integrative investigation on 238 Han Chinese SCZ patients to identify predictive biomarkers that were associated with the efficacy of olanzapine treatment. This study applied HaloPlex technology to sequence 143 genes from 79 Han Chinese SCZ patients. Our result suggested that there were 12 single nucleotide polymorphisms (SNPs) had significant association with olanzapine response in Han Chinese SCZ patients. Using MassARRAY platform, we tested that if these 12 SNPs were also statistically significant in 159 other SCZ patients (independent cohort) and the combined 238 SCZ patients (composed of two tested cohorts). The result of this analysis showed that 2 SNPs were significantly associated with the olanzapine response in both independent cohorts (rs324026, P = 0.023; rs12610827, P = 0.043) and combined SCZ patient population (rs324026, adjust P = 0.014; rs12610827, adjust P = 0.012). Our study provides systematic analyses of genetic variants associated with olanzapine responses of Han Chinese SCZ patients. The discovery of these novel biomarkers of olanzapine-response will facilitate to advance future olanzapine treatment specific for Han Chinese SCZ patients