Efficient production of natural sunscreens shinorine, porphyra-334, and mycosporine-2-glycine in Saccharomyces cerevisiae.

Mycosporine-like amino acids (MAAs) are promising natural sunscreens mainly produced in marine organisms. Until now, metabolic engineering efforts to produce MAAs in heterologous hosts have mainly focused on shinorine production, and the low production levels are still not suitable for industrial applications. In this study, we successfully developed Saccharomyces cerevisiae strains that can efficiently produce various disubstituted MAAs, including shinorine, porphyra-334, and mycosporine-2-glycine (M2G), which are formed by conjugating serine, threonine, and glycine to mycosporine-glycine (MG), respectively. We first generated an MG-producing strain by multiple integration of the biosynthetic genes from cyanobacteria and applying metabolic engineering strategies to increase sedoheptulose-7-phosphate pool, a substrate for MG production. Next, five mysD genes from cyanobacteria, which encode D-Ala-D-Ala ligase homologues that conjugate an amino acid to MG, were introduced into the MG-producing strain to determine the substrate preference of each MysD enzyme. MysDs from Lyngbya sp., Nostoclinckia, and Euhalothece sp. showed high specificity toward serine, threonine, and glycine, resulting in efficient production of shinorine, porphyra-334, and M2G, respectively. This is the first report on the production of porphyra-334 and M2G in S. cerevisiae. Furthermore, we identified that the substrate specificity of MysD was determined by the omega loop region of 43-45 amino acids predicted based on its structural homology to a D-Ala-D-Ala ligase from Thermus thermophilus involved in peptidoglycan biosynthesis. The substrate specificities of two MysD enzymes were interchangeable by swapping the omega loop region. Using the engineered strain expressing mysD from Lyngbya sp. or N. linckia, up to 1.53 g/L shinorine or 1.21 g/L porphyra-334 was produced by fed-batch fermentation in a 5-L bioreactor, the highest titer reported so far. These results suggest that S. cerevisiae is a promising host for industrial production of different types of MAAs, providing a sustainable and eco-friendly alternative for the development of natural sunscreens.

Identifying Key Residues in Lysine Decarboxylase for Soluble Expression Using Consensus Design Soluble Mutant Screening (ConsenSing).

Although recent advances in deep learning approaches for protein engineering have enabled quick prediction of hot spot residues improving protein solubility, the predictions do not always correspond to an actual increase in solubility under experimental conditions. Therefore, developing methods that rapidly confirm the linkage between computational predictions and empirical results is essential to the success of improving protein solubility of target proteins. Here, we present a simple hybrid approach to computationally predict hot spots possibly improving protein solubility by sequence-based analysis and empirically explore valuable mutants using split GFP as a reporter system. Our approach, Consensus design Soluble Mutant Screening (ConsenSing), utilizes consensus sequence prediction to find hot spots for improvement of protein solubility and constructs a mutant library using Darwin assembly to cover all possible mutations in one pot but still keeps the library as compact as possible. This approach allowed us to identify multiple mutants of Escherichia coli lysine decarboxylase, LdcC, with substantial increases in soluble expression. Further investigation led us to pinpoint a single critical residue for the soluble expression of LdcC and unveiled its mechanism for such improvement. Our approach demonstrated that following a protein's natural evolutionary path provides insights to improve protein solubility and/or increase protein expression by a single residue mutation, which can significantly change the profile of protein solubility.

Enhancing biosynthesis of 2'-Fucosyllactose in Escherichia coli through engineering lactose operon for lactose transport and α -1,2-Fucosyltransferase for solubility.

2'-Fucosyllactose (2'-FL) is the most abundant oligosaccharide in human milk and one of the most actively studied human milk oligosaccharides (HMOs). When 2'-FL is produced through biological production using a microorganism, like Escherichia coli, d-lactose is often externally fed as an acceptor substrate for fucosyltransferase (FT). When d-glucose is used as a carbon source for the cell growth and d-lactose is transported by lactose permease (LacY) in lac operon, d-lactose transport is under the control of catabolite repression (CR), limiting the supply of d-lactose for FT reaction in the cell, hence decreasing the production of 2'-FL. In this study, a remarkable increase of 2'-FL production was achieved by relieving the CR from the lac operon of the host E. coli BL21 and introducing adequate site-specific mutations into α-1,2-FT (FutC) for enhancement of catalytic activity and solubility. For the host engineering, the native lac promoter (Plac ) was substituted for tac promoter (Ptac ), so that the lac operon could be turned on, but not subjected to CR by high d-glucose concentration. Next, for protein engineering of FutC, family multiple sequence analysis for conserved amino acid sequences and protein-ligand substrate docking analysis led us to find several mutation sites, which could increase the solubility of FutC and its activity. As a result, a combination of four mutation sites (F40S/Q150H/C151R/Q239S) was identified as the best candidate, and the quadruple mutant of FutC enhanced 2'-FL titer by 2.4-fold. When the above-mentioned E. coli mutant host transformed with the quadruple mutant of futC was subjected to fed-batch culture, 40 g l-1 of 2'-FL titer was achieved with the productivity of 0.55 g l-1 h-1 and the specific 2'-FL yield of 1.0 g g-1 dry cell weight.

Application of Random Mutagenesis and Synthetic FadR Promoter for de novo Production of ω-Hydroxy Fatty Acid in Yarrowia lipolytica.

As a means to develop oleaginous biorefinery, Yarrowia lipolytica was utilized to produce ω-hydroxy palmitic acid from glucose using evolutionary metabolic engineering and synthetic FadR promoters for cytochrome P450 (CYP) expression. First, a base strain was constructed to produce free fatty acids (FFAs) from glucose using metabolic engineering strategies. Subsequently, through ethyl methanesulfonate (EMS)-induced random mutagenesis and fluorescence-activated cell sorting (FACS) screening, improved FFA overproducers were screened. Additionally, synthetic promoters containing bacterial FadR binding sequences for CYP expression were designed to respond to the surge of the concentration of FFAs to activate the ω-hydroxylating pathway, resulting in increased transcriptional activity by 14 times from the third day of culture compared to the first day. Then, endogenous alk5 was screened and expressed using the synthetic FadR promoter in the developed strain for the production of ω-hydroxy palmitic acid. By implementing the synthetic FadR promoter, cell growth and production phases could be efficiently decoupled. Finally, in batch fermentation, we demonstrated de novo production of 160 mg/L of ω-hydroxy palmitic acid using FmeN3-TR1-alk5 in nitrogen-limited media. This study presents an excellent example of the production of ω-hydroxy fatty acids using synthetic promoters with bacterial transcriptional regulator (i.e., FadR) binding sequences in oleaginous yeasts.

Multiplex Gene Disruption by Targeted Base Editing of Yarrowia lipolytica Genome Using Cytidine Deaminase Combined with the CRISPR/Cas9 System.

The oleaginous yeast Yarrowia lipolytica has a tendency to use the non-homologous end joining repair (NHEJ) over the homology directed recombination as double-strand breaks (DSB) repair system, making it difficult to edit the genome using homologous recombination. A recently developed Target-AID (activation-induced cytidine deaminase) base editor, designed to recruit cytidine deaminase (CDA) to the target DNA locus via the CRISPR/Cas9 system, can directly induce C to T mutation without DSB and donor DNA. In this study, this system is adopted in Y. lipolytica for multiplex gene disruption. Target-specific gRNA(s) and a fusion protein consisting of a nickase Cas9, pmCDA1, and uracil DNA glycosylase inhibitor are expressed from a single plasmid to disrupt target genes by introducing a stop codon via C to T mutation within the mutational window. Deletion of the KU70 gene involved in the NHEJ prevents the generation of indels by base excision repair following cytidine deamination, increasing the accuracy of genome editing. Using this Target-AID system with optimized expression levels of the base editor, single gene disruption and simultaneous double gene disruption are achieved with the efficiencies up to 94% and 31%, respectively, demonstrating this base editing system as a convenient genome editing tool in Y. lipolytica.

In silico identification of metabolic engineering strategies for improved lipid production in Yarrowia lipolytica by genome-scale metabolic modeling.

BACKGROUND: Yarrowia lipolytica, an oleaginous yeast, is a promising platform strain for production of biofuels and oleochemicals as it can accumulate a high level of lipids in response to nitrogen limitation. Accordingly, many metabolic engineering efforts have been made to develop engineered strains of Y. lipolytica with higher lipid yields. Genome-scale model of metabolism (GEM) is a powerful tool for identifying novel genetic designs for metabolic engineering. Several GEMs for Y. lipolytica have recently been developed; however, not many applications of the GEMs have been reported for actual metabolic engineering of Y. lipolytica. The major obstacle impeding the application of Y. lipolytica GEMs is the lack of proper methods for predicting phenotypes of the cells in the nitrogen-limited condition, or more specifically in the stationary phase of a batch culture. RESULTS: In this study, we showed that environmental version of minimization of metabolic adjustment (eMOMA) can be used for predicting metabolic flux distribution of Y. lipolytica under the nitrogen-limited condition and identifying metabolic engineering strategies to improve lipid production in Y. lipolytica. Several well-characterized overexpression targets, such as diglyceride acyltransferase, acetyl-CoA carboxylase, and stearoyl-CoA desaturase, were successfully rediscovered by our eMOMA-based design method, showing the relevance of prediction results. Interestingly, the eMOMA-based design method also suggested non-intuitive knockout targets, and we experimentally validated the prediction with a mutant lacking YALI0F30745g, one of the predicted targets involved in one-carbon/methionine metabolism. The mutant accumulated 45% more lipids compared to the wild-type. CONCLUSION: This study demonstrated that eMOMA is a powerful computational method for understanding and engineering the metabolism of Y. lipolytica and potentially other oleaginous microorganisms.

Production of 12-hydroxy dodecanoic acid methyl ester using a signal peptide sequence-optimized transporter AlkL and a novel monooxygenase.

In this study, a signal peptide of AlkL was replaced with other signal peptides to improve the soluble expression and thereby facilitate the transport of dodecanoic acid methyl ester (DAME) substrate into the E. coli. Consequently, AlkL with signal peptide FadL (AlkLf) showed higher transport activity toward DAME. Furthermore, the promoter optimization for the efficient heterologous expression of the transporter AlkLf and alkane monooxygenase (AlkBGT) system was conducted and resulted in increased ω-oxygenation activity of AlkBGT system. Moreover, bioinformatic studies led to the identification of novel monooxygenase from Pseudomonas pelagia (Pel), which exhibited 20% higher activity towards DAME as substrate compared to AlkB. Finally, the construction of a chimeric transporter and the expression of newly identified monooxygenase enabled the production of 44.8 ±â€¯7.5 mM of 12-hydroxy dodecanoic acid methyl ester (HADME) and 31.8 ±â€¯1.7 mM of dodecanedioic acid monomethyl ester (DDAME) in a two-phase reaction system.

Rewiring FadR regulon for the selective production of ω-hydroxy palmitic acid from glucose in Escherichia coli.

ω-Hydroxy palmitic acid (ω-HPA) is a valuable compound for an ingredient of artificially synthesized ceramides and an additive for lubricants and adhesives. Production of such a fatty acid derivative is limited by chemical catalysis, but plausible by biocatalysis. However, its low productivity issue, including formations of unsaturated fatty acid (UFA) byproducts in host cells, remains as a hurdle toward industrial biological processes. In this study, to achieve selective and high-level production of ω-HPA from glucose in Escherichia coli, FadR, a native transcriptional regulator of fatty acid metabolism, and its regulon were engineered. First, FadR was co-expressed with a thioesterase with a specificity toward palmitic acid production to enhance palmitic acid production yield, but a considerable quantity of UFAs was also produced. In order to avoid the UFA production caused by fadR overexpression, FadR regulon was rewired by i) mutating FadR consensus binding sites of fabA or fabB, ii) integrating fabZ into fabI operon, and iii) enhancing the strength of fabI promoter. This approach led to dramatic increases in both proportion (48.3-83.0%) and titer (377.8 mg/L to 675.8 mg/L) of palmitic acid, mainly due to the decrease in UFA synthesis. Introducing a fatty acid ω-hydroxylase, CYP153A35, into the engineered strain resulted in a highly selective production of ω-HPA (83.5 mg/L) accounting for 87.5% of total ω-hydroxy fatty acids. Furthermore, strategies, such as i) enhancement in CYP153A35 activity, ii) expression of a fatty acid transporter, iii) supplementation of triton X-100, and iv) separation of the ω-HPA synthetic pathway into two strains for a co-culture system, were applied and resulted in 401.0 mg/L of ω-HPA production. For such selective productions of palmitic acid and ω-HPA, the rewiring of FadR regulation in E. coli is a promising strategy to develop an industrial process with economical downstream processing.

Exploiting transcriptomic data for metabolic engineering: toward a systematic strain design.

Transcriptomics is now recognized as a primary tool for metabolic engineering as it can be used for identifying new strain designs by diagnosing current states of microbial cells. This review summarizes current application of transcriptomic data for strain design. Along with a few successful examples, limitations of conventionally used differentially expressed gene-based strain design approaches have been discussed, which have been major reasons why transcriptomic data are considerably underutilized. Recently, integrative network-based approaches interpreting transcriptomic data in the context of biological networks were invented to provide complimentary solutions for metabolic engineering by overcoming the limitations of conventional approaches. Here, we highlight recent pioneering studies in which integrative network-based methods have been used for providing novel strain designs.

Semi-rational engineering of CYP153A35 to enhance ω-hydroxylation activity toward palmitic acid.

CYP153A35 from Gordonia alkanivorans was recently characterized as fatty acid ω-hydroxylase. To enhance the catalytic activity of CYP153A35 toward palmitic acid, site-directed saturation mutagenesis was attempted using a semi-rational approach that combined structure-based computational analysis and subsequent saturation mutagenesis. Using colorimetric high-throughput screening (HTS) method based on O-demethylation activity of P450, CYP153A35 D131S and D131F mutants were selected. The best mutant, D131S, having a single mutation on BC-loop, showed 13- and 17-fold improvement in total turnover number (TTN) and catalytic efficiency (k cat/K M) toward palmitic acid compared to wild-type, respectively. However, in whole-cell reaction, D131S mutant showed only 50% improvement in ω-hydroxylated palmitic acid yield compared to the wild type. Docking simulation studies explained that the effect of D131S mutation on the catalytic activity would be mainly caused by the binding pose of fatty acids in the substrate access tunnel of the enzyme. This effect of D131S mutation on the catalytic activity is synergistic with that of the mutations in the active site previously reported.

Systems biology for understanding and engineering of heterotrophic oleaginous microorganisms.

Heterotrophic oleaginous microorganisms continue to draw interest as they can accumulate a large amount of lipids which is a promising feedstock for the production of biofuels and oleochemicals. Nutrient limitation, especially nitrogen limitation, is known to effectively trigger the lipid production in these microorganisms. For the aim of developing improved strains, the mechanisms behind the lipid production have been studied for a long time. Nowadays, system-level understanding of their metabolism and associated metabolic switches is attainable with modern systems biology tools. This work reviews the systems biology studies, based on (i) top-down, large-scale 'omics' tools, and (ii) bottom-up, mathematical modeling methods, on the heterotrophic oleaginous microorganisms with an emphasis on further application to metabolic engineering.

Production of ω-hydroxy palmitic acid using CYP153A35 and comparison of cytochrome P450 electron transfer system in vivo.

Bacterial cytochrome P450 enzymes in cytochrome P450 (CYP)153 family were recently reported as fatty acid ω-hydroxylase. Among them, CYP153As from Marinobacter aquaeolei VT8 (CYP153A33), Alcanivorax borkumensis SK2 (CYP153A13), and Gordonia alkanivorans (CYP153A35) were selected, and their specific activities and product yields of ω-hydroxy palmitic acid based on whole cell reactions toward palmitic acid were compared. Using CamAB as redox partner, CYP153A35 and CYP153A13 showed the highest product yields of ω-hydroxy palmitic acid in whole cell and in vitro reactions, respectively. Artificial self-sufficient CYP153A35-BMR was constructed by fusing it to the reductase domain of CYP102A1 (i.e., BM3) from Bacillus megaterium, and its catalytic activity was compared with CYP153A35 and CamAB systems. Unexpectedly, the system with CamAB resulted in a 1.5-fold higher yield of ω-hydroxy palmitic acid than that using A35-BMR in whole cell reactions, whereas the electron coupling efficiency of CYP153A35-BM3 reductase was 4-fold higher than that of CYP153A35 and CamAB system. Furthermore, various CamAB expression systems according to gene arrangements of the three proteins and promoter strength in their gene expression were compared in terms of product yields and productivities. Tricistronic expression of the three proteins in the order of putidaredoxin (CamB), CYP153A35, and putidaredoxin reductase (CamA), i.e., A35-AB2, showed the highest product yield from 5 mM palmitic acid for 9 h in batch reaction owing to the concentration of CamB, which is the rate-limiting factor for the activity of CYP153A35. However, in fed-batch reaction, A35-AB1, which expressed the three proteins individually using three T7 promoters, resulted with the highest product yield of 17.0 mM (4.6 g/L) ω-hydroxy palmitic acid from 20 mM (5.1 g/L) palmitic acid for 30 h.

fadD deletion and fadL overexpression in Escherichia coli increase hydroxy long-chain fatty acid productivity.

A major problem of long-chain fatty acid (LCFA) hydroxylation using Escherichia coli is that FadD (long-chain fatty acyl-CoA synthetase), which is necessary for exogenous LCFA transport, also initiates cellular consumption of LCFA. In this study, an effective method to prevent the cellular consumption of LCFA without impairing its transport is proposed. The main idea is that a heterologous enzyme which consumes LCFA can replace FadD in LCFA transport. For the model heterologous enzyme, CYP153A from Marinobacter aquaeolei, which converts palmitic acid into ω-hydroxy palmitic acid, was expressed in E. coli. When fadD was deleted from an E. coli strain, CYP153A indeed maintained the ability to transport LCFA. A disadvantage of fadD deletion mutant is the fact that FadD deficiency downregulates the transcription of fadL (outer membrane LCFA transporter) via FadR (fatty acid metabolism regulator protein), was solved by fadL overexpression from a plasmid. In addition, the overexpression of fadL was able to offset catabolite repression on fadL, allowing glucose to be used as the primary carbon source. In conclusion, the strain with fadD deletion and fadL overexpression showed 5.5-fold increase in productivity compared to the wild-type strain, converting 2.6 g/L (10.0 mM) of palmitic acid into 2.4 g/L (8.8 mM) of ω-hydroxy palmitic acid in a shake flask. This simple genetic manipulation can be applied to any LCFA hydroxylation using E. coli.