Unravelling and engineering an operon involved in the side-chain degradation of sterols in Mycolicibacterium neoaurum for the production of steroid synthons.

BACKGROUND: Harnessing engineered Mycolicibacteria to convert cheap phytosterols into valuable steroid synthons is a basic way in the industry for the production of steroid hormones. Thus, C-19 and C-22 steroids are the two main types of commercial synthons and the products of C17 side chain degradation of phytosterols. During the conversion process of sterols, C-19 and C-22 steroids are often produced together, although one may be the main product and the other a minor byproduct. This is a major drawback of the engineered Mycolicibacteria for industrial application, which could be attributed to the co-existence of androstene-4-ene-3,17-dione (AD) and 22-hydroxy-23,24-bisnorchol-4-ene-3-one (HBC) sub-pathways in the degradation of the sterol C17 side chain. Since the key mechanism underlying the HBC sub-pathway has not yet been clarified, the above shortcoming has not been resolved so far. RESULTS: The key gene involved in the putative HBC sub-pathway was excavated from the genome of M. neoaurum by comparative genomic analysis. Interestingly, an aldolase- encoding gene, atf1, was identified to be responsible for the first reaction of the HBC sub-pathway, and it exists as a conserved operon along with a DUF35-type gene chsH4, a reductase gene chsE6, and a transcriptional regulation gene kstR3 in the genome. Subsequently, atf1 and chsH4 were identified as the key genes involved in the HBC sub-pathway. Therefore, an updated strategy was proposed to develop engineered C-19 or C-22 steroid-producing strains by simultaneously modifying the AD and HBC sub-pathways. Taking the development of 4-HBC and 9-OHAD-producing strains as examples, the improved 4-HBC-producing strain achieved a 20.7 g/L production titer with a 92.5% molar yield and a 56.4% reduction in byproducts, and the improved 9-OHAD producing strain achieved a 19.87 g/L production titer with a 94.6% molar yield and a 43.7% reduction in byproduct production. CONCLUSIONS: The excellent performances of these strains demonstrated that the primary operon involved in the HBC sub-pathway improves the industrial strains in the conversion of phytosterols to steroid synthons.

Improved cryptic plasmids in probiotic Escherichia coli Nissle 1917 for antibiotic-free pathway engineering.

The engineered probiotic Escherichia coli Nissle 1917 (EcN) is expected to be employed in the diagnosis and treatment of various diseases. However, the introduced plasmids typically require antibiotics to maintain genetic stability, and the cryptic plasmids in EcN are usually eliminated to avoid plasmid incompatibility which may change the inherent probiotic characteristics. Here, we provided a simple design to minimize the genetic change of probiotics by eliminating native plasmids and reintroducing the recombinants carrying functional genes. Specific insertion sites in the vectors showed significant differences in the expression of fluorescence proteins. Selected integration sites were applied in the de novo synthesis of salicylic acid, leading to a titer of 142.0 ± 6.0 mg/L in a shake flask with good production stability. Additionally, the design successfully realized the biosynthesis of ergothioneine (45 mg/L) by one-step construction. This work expands the application scope of native cryptic plasmids to the easy construction of functional pathways. KEY POINTS: • Cryptic plasmids of EcN were designed to express exogenous genes • Insertion sites with different expression intensities in cryptic plasmids were provided • Target products were stably produced by engineering cryptic plasmids.

Driving the conversion of phytosterol to 9α-hydroxy-4-androstene-3,17-dione in Mycolicibacterium neoaurum by engineering the supply and regeneration of flavin adenine dinucleotide.

BACKGROUND: The conversion of phytosterols to steroid synthons by engineered Mycolicibacteria comprises one of the core steps in the commercial production of steroid hormones. This is a complex oxidative catabolic process, and taking the production of androstenones as example, it requires about 10 equivalent flavin adenine dinucleotide (FAD). As the high demand for FAD, the insufficient supply of FAD may be a common issue limiting the conversion process. RESULTS: We substantiated, using the production of 9α-hydroxy-4-androstene-3,17-dione (9-OHAD) as a model, that increasing intracellular FAD supply could effectively increase the conversion of phytosterols into 9-OHAD. Overexpressing ribB and ribC, two key genes involving in FAD synthesis, could significantly enhance the amount of intracellular FAD by 167.4% and the production of 9-OHAD by 25.6%. Subsequently, styrene monooxygenase NfStyA2B from Nocardia farcinica was employed to promote the cyclic regeneration of FAD by coupling the oxidation of nicotinamide adenine dinucleotide (NADH) to NAD+, and the production of 9-OHAD was further enhanced by 9.4%. However, the viable cell numbers decreased by 20.1%, which was attributed to sharply increased levels of H2O2 because of the regeneration of FAD from FADH2. Thus, we tried to resolve the conflict between FAD regeneration and cell growth by the overexpression of catalase and promotor replacement. Finally, a robust strain NF-P2 was obtained, which could produce 9.02 g/L 9-OHAD after adding 15 g/L phytosterols with productivity of 0.075 g/(L h), which was 66.7% higher than that produced by the original strain. CONCLUSIONS: This study highlighted that the cofactor engineering, including the supply and recycling of FAD and NAD+ in Mycolicibacterium, should be adopted as a parallel strategy with pathway engineering to improve the productivity of the industrial strains in the conversion of phytosterols into steroid synthons.

Boosting the epoxidation of squalene to produce triterpenoids in Saccharomyces cerevisiae.

BACKGROUND: Polycyclic triterpenoids (PTs) are common in plants, and have attracted considerable interest due to their remarkable biological activities. Currently, engineering the ergosterol synthesis pathway in Saccharomyces cerevisiae is a safe and cost-competitive way to produce triterpenoids. However, the strict regulation of ERG1 involved in the epoxidation of squalene limits the triterpenoid production. RESULTS: In this study, we found that the decrease in ERG7 protein level could dramatically boost the epoxidation of squalene by improving the protein stability of ERG1. We next explored the potential factors that affected the degradation process of ERG1 and confirmed that ERG7 was involved in the degradation process of ERG1. Subsequently, expression of four different triterpene cyclases utilizing either 2,3-oxidosqualene or 2,3:22,23-dioxidosqualene as the substrate in ERG7-degraded strains showed that the degradation of ERG7 to prompt the epoxidation of squalene could significantly increase triterpenoid production. To better display the potential of the strategy, we increased the supply of 2,3-oxidosqualene, optimized flux distribution between ergosterol synthesis pathway and ß-amyrin synthesis pathway, and modified the GAL-regulation system to separate the growth stage from the production stage. The best-performing strain ultimately produced 4216.6 ± 68.4 mg/L of ß-amyrin in a two-stage fed-fermentation (a 47-fold improvement over the initial strain). CONCLUSIONS: This study showed that deregulation of the native restriction in ergosterol pathway was an effective strategy to increase triterpenoid production in yeast, which provided a new insight into triterpenoids biosynthesis.

Enhancing the Activity of an Alcohol Dehydrogenase by Using "Aromatic Residue Scanning" at Potential Plasticity Sites.

An alcohol dehydrogenase LkADH was successfully engineered to exhibit improved activity and substrate tolerance for the production of (S)-2-chloro-1-(3,4-difluorophenyl)ethanol, an important precursor of ticagrelor. Five potential hotspots were identified for enzyme mutagenesis by using natural residue abundance as an indicator to evaluate their potential plasticity. A semi-rational strategy named "aromatic residue scanning" was applied to randomly mutate these five sites simultaneously by using tyrosine, tryptophan, and phenylalanine as "exploratory residues" to introduce steric hindrance or potential π-π interactions. The best variant Lk-S96Y/L199W identified with 17.2-fold improvement in catalytic efficiency could completely reduce up to 600 g/L (3.1 M) 2-chloro-1-(3,4-difluorophenyl)ethenone in 12 h with >99.5 % ee, giving the highest space-time yield ever reported. This study, therefore, offers a strategy for mutating alcohol dehydrogenase to reduce aromatic substrates and provides an efficient variant for the efficient synthesis of (S)-2-chloro-1-(3,4-difluorophenyl)ethanol.

Engineering Escherichia coli for l-homoserine production.

l-homoserine, a nonprotein amino acid, is used to synthesize many active substances in the industry. Here, to develop a robust l-homoserine-producing strain, Escherichia coli W3110 was used as a chassis to be engineered. Based on a previous construct with blocked competing routes for l-homoserine synthesis, five genes were overexpressed by promoter replacement strategy to increase the l-homoserine production, including enhancement of precursors for l-homoserine synthesis (ppc, thrA, and asd), reinforcement of the NADPH supply (pntAB) and efflux transporters (rhtA) to improve the l-homoserine production. However, the plasmid losing was to blame for the wildly fluctuating fermentation performance of engineered strains, ranging between 2.1 and 6.2 g/L. Then, a hok/sok toxin/antitoxin system was introduced into the free plasmid expression cassette to maintain the genetic stability of the episomal plasmid; consequently, the plasmid-losing rate sharply decreased, resulting in the engineered strain SHL17, which exhibited excellent stability in l-homoserine production, with 6.3 g/L in shake flasks and 44.4 g/L in a 5-L fermenter without antibiotic addition. This work verified the effective use of the hok/sok toxin/antitoxin system combined with promoter engineering to improve the genetic stability of E. coli episomal plasmids without antibiotics.

Multiplexed site-specific genome engineering in Mycolicibacterium neoaurum by Att/Int system.

Genomic integration of genes and pathway-sized DNA cassettes is often an indispensable way to construct robust and productive microbial cell factories. For some uncommon microbial hosts, such as Mycolicibacterium and Mycobacterium species, however, it is a challenge. Here, we present a multiplexed integrase-assisted site-specific recombination (miSSR) method to precisely and iteratively integrate genes/pathways with controllable copies in the chromosomes of Mycolicibacteria for the purpose of developing cell factories. First, a single-step multi-copy integration method was established in M. neoaurum by a combination application of mycobacteriophage L5 integrase and two-step allelic exchange strategy, the efficiencies of which were ∼100% for no more than three-copy integration events and decreased sharply to ∼20% for five-copy integration events. Second, the R4, Bxb1 and ΦC31 bacteriophage Att/Int systems were selected to extend the available integration toolbox for multiplexed gene integration events. Third, a reconstructed mycolicibacterial Xer recombinases (Xer-cise) system was employed to recycle the selection marker of gene recombination to facilitate the iterative gene manipulation. As a proof of concept, the biosynthetic pathway of ergothioneine (EGT) in Mycolicibacterium neoaurum ATCC 25795 was achieved by remodeling its metabolic pathway with a miSSR system. With six copies of the biosynthetic gene clusters (BGCs) of EGT and pentose phosphate isomerase (PRT), the titer of EGT in the resulting strain in a 30 mL shake flask within 5 days was enhanced to 66 mg/L, which was 3.77 times of that in the wild strain. The improvements indicated that the miSSR system was an effective, flexible, and convenient tool to engineer the genomes of Mycolicibacteria as well as other strains in the Mycobacteriaceae due to their proximate evolutionary relationships.

CRISPR-Cas Assisted Shotgun Mutagenesis Method for Evolutionary Genome Engineering.

Genome mutagenesis drives the evolution of organisms. Here, we developed a CRISPR-Cas assisted random mutation (CARM) technique for whole-genome mutagenesis. The method leverages an entirely random gRNA library and SpCas9-NG to randomly damage genomes in a controllable shotgunlike manner that then triggers diverse and abundant mutations via low-fidelity repair. As a proof of principle, CARM was applied to evolve the capacity of Saccharomyces cerevisiae BY4741 to produce ß-carotene. After seven rounds of iterative evolution over two months, a ß-carotene hyperproducing strain, C7-143, was isolated with a 10.5-fold increase in ß-carotene production and 857 diverse genomic mutations that comprised indels, duplications, inversions, and chromosomal rearrangements. Transcriptomic analysis revealed that the expression of 2541 genes of strain C7-143 was significantly altered, suggesting that the metabolic landscape of the strain was deeply reconstructed. In addition, CARM was applied to evolve industrially relevant S. cerevisiae CEN.PK2-1C for S-adenosyl-L-methionine production, which was increased 2.28 times after just one round. Thus, CARM can contribute to increasing genetic diversity to identify new phenotypes that could further be investigated by reverse engineering.

One-pot biosynthesis of 7ß-hydroxyandrost-4-ene-3,17-dione from phytosterols by cofactor regeneration system in engineered mycolicibacterium neoaurum.

BACKGROUND: 7ß-hydroxylated steroids (7ß-OHSt) possess significant activities in anti-inflammatory and neuroprotection, and some of them have been widely used in clinics. However, the production of 7ß-OHSt is still a challenge due to the lack of cheap 7ß-hydroxy precursor and the difficulty in regio- and stereo-selectively hydroxylation at the inert C7 site of steroids in industry. The conversion of phytosterols by Mycolicibacterium species to the commercial precursor, androst-4-ene-3,17-dione (AD), is one of the basic ways to produce different steroids. This study presents a way to produce a basic 7ß-hydroxy precursor, 7ß-hydroxyandrost-4-ene-3,17-dione (7ß-OH-AD) in Mycolicibacterium, for 7ß-OHSt synthesis. RESULTS: A mutant of P450-BM3, mP450-BM3, was mutated and engineered into an AD producing strain for the efficient production of 7ß-OH-AD. The enzyme activity of mP450-BM3 was then increased by 1.38 times through protein engineering and the yield of 7ß-OH-AD was increased from 34.24 mg L- 1 to 66.25 mg L- 1. To further enhance the performance of 7ß-OH-AD producing strain, the regeneration of nicotinamide adenine dinucleotide phosphate (NADPH) for the activity of mP450-BM3-0 was optimized by introducing an NAD kinase (NADK) and a glucose-6-phosphate dehydrogenase (G6PDH). Finally, the engineered strain could produce 164.52 mg L- 1 7ß-OH-AD in the cofactor recycling and regeneration system. CONCLUSIONS: This was the first report on the one-pot biosynthesis of 7ß-OH-AD from the conversion of cheap phytosterols by an engineered microorganism, and the yield was significantly increased through the mutation of mP450-BM3 combined with overexpression of NADK and G6PDH. The present strategy may be developed as a basic industrial pathway for the commercial production of high value products from cheap raw materials.

Production of sesquiterpene patchoulol in mitochondrion-engineered Saccharomyces cerevisiae.

Patchoulol is a natural sesquiterpene, which is widely used in perfumes and cosmetics. In the work, the mitochondria of S. cerevisiae were engineered for patchoulol production. The patchoulol titer of mitochondria-compartmentalized strain (1.79 mg/L) was 2.71-fold higher than that of control strain (0.66 mg/L) using genome-integrated patchoulol synthase, indicating that mitochondria compartmentation resulted in higher concentration of FPP (farnesyl pyrophosphate) precursor for patchoulol production. Moreover, when fused FPP synthase and patchoulol synthase was overexpressed in the strain with a mitochondria-localized DMAPP (dimethylallyl diphosphate) pathway, the production of patchoulol increased significantly to 19.24 mg/L, indicating more precursors were provided for patchoulol production. Nevertheless, the introduction of excess foreign proteins into mitochondria might cause a certain stress on mitochondria and showed a negative effect on the growth of yeast cells, which could hinder the expression of foreign pathways and reduce the patchoulol production. In conclusion, mitochondria-engineered yeast cells showed important potential for the enhanced biosynthesis of patchoulol, and further engineering could be considered based on the present work.

CRISPR-Cas12a assisted precise genome editing of Mycolicibacterium neoaurum.

Efficient and convenient genetic manipulation of mycobacteria, important microorganisms in human healthcare and the pharmaceutical industry, is limited. In this study, using a model strain Mycolicibacterium neoaurum ATCC 25795, the classical bacterium for the production of valuable steroidal pharmaceuticals, a genome editing system employing CRISPR-Cas12a to achieve efficient and precise genetic manipulation has been developed. Targeted genome mutations could be easily achieved by the CRISPR-Cas12a system without exogenous donor templates, assisted by innate non-homologous end-joining (NHEJ). CRISPR-Cas12a enabled rapid one-step genomic DNA fragment deletions of 1 kb, 5 kb, 10 kb, 15 kb, 20 kb and 24 kb with efficiencies of 70 %, 30 %, 30 %, 20 %, 20 % and 10 %, respectively. Combined with the pNIL/pGOAL system, CRISPR-Cas12a successfully integrated the gene of interest into the targeted genomic site by single crossover and double crossovers with efficiencies of 100 % and 9 %, respectively, using a two-plasmid system. The robust CRISPR systems developed demonstrated strong potential for precise genome editing in M. neoaurum, including targeted deletion of DNA sequences of various lengths and integration of targeted genes into desired sites in the genome.

Improving the biotransformation efficiency of soybean phytosterols in Mycolicibacterium neoaurum by the combined deletion of fbpC3 and embC in cell envelope synthesis.

Biotransformation of soybean phytosterols into 9α-hydroxy-4-androstene-3,17-dione (9-OHAD) by mycobacteria is the core step in the synthesis of adrenocortical hormone. However, the low permeability of the dense cell envelope largely inhibits the overall conversion efficiency of phytosterols. The antigen 85 (Ag85) complex encoded by fbpA, fbpB, and fbpC was proposed as the key factor in the combined catalysis of mycoloyl for producing mycolyl-arabinogalactan (m-AG) and trehalose dimycolate (TDM) in mycobacterial cell envelope. Herein, we confirmed that fbpC3 was essential for the biotransformation of trehalose monomycolate (TMM) to TDM in Mycolicibacterium neoaurum. The deficiency of this gene raised the cell permeability, thereby enhancing the steroid uptake and utilization. The 9-OHAD yield in the fbpC3-deficient 9-OHAD-producing strain was increased by 21.3%. Moreover, the combined deletion of fbpC3 and embC further increased the 9-OHAD yield compared to the single deletion of fbpC3. Finally, after 96 h of bioconversion in industrial resting cells, the 9-OHAD yield of 11.2 g/L was achieved from 20 g/L phytosterols and the productivity reached 0.116 g/L/h. In summary, this study suggested the critical role of the fbpC3 gene in the synthesis of TDM in M. neoaurum and verified the feasibility of improving the bioconversion efficiency of phytosterols through the cell envelope engineering strategy.

Engineering Saccharomyces cerevisiae for Hyperproduction of ß-Amyrin by Mitigating the Inhibition Effect of Squalene on ß-Amyrin Synthase.

The study aims to enhance ß-amyrin production in Saccharomyces cerevisiae by peroxisome compartmentalization. First, overaccumulated squalene was determined as a key limiting factor for the production of ß-amyrin since it could inhibit the activity of ß-amyrin synthase GgbAs1. Second, to mitigate the inhibition effect, the enhanced squalene synthesis pathway was compartmentalized into peroxisomes to insulate overaccumulated squalene from GgbAs1, and thus the specific titer of ß-amyrin reached 57.8 mg/g dry cell weight (DCW), which was 2.6-fold higher than that of the cytosol engineering strain. Third, by combining peroxisome compartmentalization with the "push-pull-restrain" strategy (ERG1 and GgbAs1 overexpression and ERG7 weakening), the production of ß-amyrin was further increased to 81.0 mg/g DCW (347.0 mg/L). Finally, through fed-batch fermentation in a 5 L fermenter, the titer of ß-amyrin reached 2.6 g/L, which is the highest reported to date. The study provides a new perspective to engineering yeasts as a platform for triterpene production.

Structure-guided engineering of a flavin-containing monooxygenase for the efficient production of indirubin.

Indirubin is a bisindole compound for the treatment of chronic myelocytic leukemia. Here, we presented a structure-guided method to improve the activity of a flavin-containing monooxygenase (bFMO) for the efficient production of indirubin in Escherichia coli. A flexible loop interlocked with the active pocket through a helix and the substrate tunnel rather than the active pocket in bFMO were identified to be two reconfigurable structures to improve its activity, resulting in K223R and N291T mutants with enhanced catalytic activity by 2.5- and 2.0-fold, respectively. A combined modification at the two regions (K223R/D317S) achieved a 6.6-fold improvement in catalytic efficiency (kcat/Km) due to enhancing π-π stacking interactions stabilization. Finally, an engineered E. coli strain was constructed by metabolic engineering, which could produce 860.7 mg/L (18 mg/L/h) indirubin, the highest yield ever reported. This work provides new insight into the redesign of FMOs to boost their activities and an efficient approach to produce indirubin.

Metabolic compartmentalization in yeast mitochondria: Burden and solution for squalene overproduction.

Harnessing mitochondria is considered as a promising method for biosynthesis of terpenes due to the adequate supply of acetyl-CoA and redox equivalents in mitochondria. However, mitochondrial engineering often causes serious metabolic burden indicated by poor cell growth. Here, we systematically analyzed the metabolic burden caused by the compartmentalization of the MVA pathway in yeast mitochondria for squalene synthesis. The phosphorylated intermediates of the MVA pathway, especially mevalonate-5-P and mevalonate-5-PP, conferred serious toxicity within mitochondria, which significantly compromised its possible advantages for squalene synthesis and was difficult to be significantly improved by routine pathway optimization. These phosphorylated intermediates were converted into ATP analogues, which strongly inhibited ATP-related cell function, such as mitochondrial oxidative respiration. Fortunately, the introduction of a partial MVA pathway from acetyl-CoA to mevalonate in mitochondria as well as the augmentation of the synthesis of mevalonate in cytosol could significantly promote the growth of yeasts. Accordingly, a combinatorial strategy of cytoplasmic and mitochondrial engineering was proposed to alleviate the metabolic burden caused by the compartmentalized MVA pathway in mitochondria and improve cell growth. The strategy also displayed the superimposed effect of cytoplasmic engineering and mitochondrial engineering on squalene production. Through a two-stage fermentation process, the squalene titer reached 21.1 g/L with a specific squalene titer of 437.1 mg/g dcw, which was the highest at present. This provides new insight into the production of squalene and other terpenes in yeasts based on the advantages of mitochondrial engineering.

The Inhibitory Effect of Cyclodextrin on Oxygen Bioavailability Is a Key Factor for the Metabolic Flux Redistribution Toward Steroid Alcohols in Phytosterol Resting Cells Bioconversion.

In the present work, we tried to identify the mechanism why by which the steroid alcohols accumulated when hydroxypropyl-ß-cyclodextrin (HP-ß-CD) was present to enhance the sterol conversion rate. Compared with the bioconversion system without HP-ß-CD, the reaction rate was greatly improved in presence of HP-ß-CD, but the steroid alcohols largely accumulated concurrently. In a reaction system with an enhanced reaction rate, the higher intracellular NADH/NAD+ level was detected, and the production of steroid alcohols increased also. Mycobacterium neoaurum mutants with higher KshA activity (3-ketosteroid 9α-hydrolase, a monooxygenase hydroxylating the nucleus at C-9 at the expense of NAD(P)H consumption) reduced the steroid alcohol production, and in the meantime, the NADH/NAD+ level was decreased consequently. Further research found that oxygen availability was seriously inhibited by the cyclodextrin in a reaction system. These results indicated that NADH formed in the bioconversion was not properly regenerated via the respiratory chain because of the poor oxygen bioavailability. The inhibitory effect of cyclodextrin on oxygen bioavailability is a key factor for the metabolic flux redistribution toward steroid alcohols in phytosterol resting cells bioconversion.

High-Level Production of Sesquiterpene Patchoulol in Saccharomyces cerevisiae.

Patchoulol is a tricyclic sesquiterpene widely used in perfumes and cosmetics. Herein, comprehensive engineering strategies were employed to construct an efficient yeast strain for patchoulol production. First, a platform strain was constructed via pathway modification. Second, three off-pathway genes were deleted, which led to significant physiological changes in yeast. Further, strengthening of the ergosterol pathway, enhancement of the energy supply, and a decrease in intracellular reactive oxygen species were implemented to improve the physiological status of yeast, demonstrating a new promotive relationship between ergosterol biosynthesis and synthesis of patchoulol. Moreover, patchoulol synthase was improved through protein modification and Mg2+ addition, reaching a final titer of 141.5 mg/L in a shake flask. Finally, a two-stage fermentation with dodecane addition was employed to achieve the highest production (1632.0 mg/L, 87.0 mg/g dry cell weight, 233.1 mg/L/d) ever reported for patchoulol in a 5 L bioreactor. This work lays a foundation for green and efficient patchoulol production.

Improving the biotransformation of phytosterols to 9α-hydroxy-4-androstene-3,17-dione by deleting embC associated with the assembly of cell envelope in Mycobacterium neoaurum.

The conversion of low value-added phytosterols into 9α-hydroxy-4-androstene-3,17-dione (9-OHAD) by mycobacteria is an important step in the steroid pharmaceutical industry. However, the highly dense cell envelope with extremely low permeability largely affects the overall transformation efficiency. Here, we preliminarily located the key gene embC required for the synthesis of lipoarabinomannan from lipomannan in Mycobacterium neoaurum. The genetic manipulation of embC indicated that it might be the only functional enzyme catalyzing the above synthesis process. The deficiency of lipoarabinomannan led to a significantly increased cell permeability, which in turn caused the enhanced uptake capacity of cells. The sterol substrate conversion efficiency of mycobacterial cells was increased by about 52.4 % after 72-h conversion. Ultimately, the absence of embC increased the productivity from 0.0927 g/L/h to 0.1031 g/L/h, as confirmed by a resting cell system. This study verified the feasibility of improving the efficiency of the microbial conversion system through the cell envelope engineering strategy.

Functional expression of a novel methanol-stable esterase from Geobacillus subterraneus DSM13552 for biocatalytic synthesis of cinnamyl acetate in a solvent-free system.

BACKGROUND: Esterases are widely distributed in nature and have important applications in medical, industrial and physiological. Recently, the increased demand for flavor esters has prompted the search of catalysts like lipases and esterases. Esterases from thermophiles also show thermal stability at elevated temperatures and have become enzymes of special interest in biotechnological applications. Although most of esterases catalyzed reactions are carried out in toxic and inflammable organic solvents, the solvent-free system owning many advantages such as low cost and easy downstream processing. RESULTS: The gene estGSU753 from Geobacillus subterraneus DSM13552 was cloned, sequenced and overexpressed into Escherichia coli BL21 (DE3). The novel gene has an open reading frame of 753 bp and encodes 250-amino-acid esterase (EstGSU753). The sequence analysis showed that the protein contains a catalytic triad formed by Ser97, Asp196 and His226, and the Ser of the active site is located in the conserved motif Gly95-X-Ser97-X-Gly99 included in most esterases and lipases. The protein catalyzed the hydrolysis of pNP-esters of different acyl chain lengths, and the enzyme specific activity was 70 U/mg with the optimum substrate pNP-caprylate. The optimum pH and temperature of the recombinant enzyme were 8.0 and 60 °C respectively. The resulting EstGSU753 showed remarkable stability against methanol. After the incubation at 50% methanol for 9 days, EstGSU753 retained 50% of its original activity. Even incubation at 90% methanol for 35 h, EstGSU753 retained 50% of its original activity. Also, the preliminary study of the transesterification shows the potential value in synthesis of short-chain flavor esters in a solvent-free system, and more than 99% conversion was obtained in 6 h (substrate: cinnamyl alcohol, 1.0 M). CONCLUSIONS: This is the first report of esterase gene cloning from Geobacillus subterraneus with detailed enzymatic properties. This methanol-stable esterase showed potential value in industrial applications especially in the perfume industry.

Enhancing the bioconversion of phytosterols to steroidal intermediates by the deficiency of kasB in the cell wall synthesis of Mycobacterium neoaurum.

BACKGROUND: The bioconversion of phytosterols into high value-added steroidal intermediates, including the 9α-hydroxy-4-androstene-3,17-dione (9-OHAD) and 22-hydroxy-23,24-bisnorchol-4-ene-3-one (4-HBC), is the cornerstone in steroid pharmaceutical industry. However, the low transportation efficiency of hydrophobic substrates into mycobacterial cells severely limits the transformation. In this study, a robust and stable modification of the cell wall in M. neoaurum strain strikingly enhanced the cell permeability for the high production of steroids. RESULTS: The deletion of the nonessential kasB, encoding a ß-ketoacyl-acyl carrier protein synthase, led to a disturbed proportion of mycolic acids (MAs), which is one of the most important components in the cell wall of Mycobacterium neoaurum ATCC 25795. The determination of cell permeability displayed about two times improvement in the kasB-deficient strain than that of the wild type M. neoaurum. Thus, the deficiency of kasB in the 9-OHAD-producing strain resulted in a significant increase of 137.7% in the yield of 9α-hydroxy-4-androstene-3,17-dione (9-OHAD). Ultimately, the 9-OHAD productivity in an industrial used resting cell system was reached 0.1135 g/L/h (10.9 g/L 9-OHAD from 20 g/L phytosterol) and the conversion time was shortened by 33%. In addition, a similar self-enhancement effect (34.5%) was realized in the 22-hydroxy-23,24-bisnorchol-4-ene-3-one (4-HBC) producing strain. CONCLUSIONS: The modification of kasB resulted in a meaningful change in the cell wall mycolic acids. Deletion of the kasB gene remarkably improved the cell permeability, leading to a self-enhancement of the steroidal intermediate conversion. The results showed a high efficiency and feasibility of this construction strategy.